Key Important Research for Covid-19 vaccines

A collection of key important research for Covid-19 vaccines and articles to help understand the implications of this pandemic. The references are complete with citation, authors, year published, link and key findings.

This page will constantly evolve as more studies are published so be sure to check back often.

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1. Historical Studies

Olsen et al; 1992; “Monoclonal Antibodies to the Spike Protein of Feline Infectious Peritonitis Virus Mediate Antibody-Dependent Enhancement of Infection of Feline Macrophages” (https://journals.asm.org/doi/epdf/10.1128/jvi.66.2.956-965.1992?src=getftr)

“Antibody-dependent enhancement of virusinfection is a process whereby virus-antibody complexes initiate infection of cells via Fcr eceptor-mediated endocytosis.We sought to investigate antibody-dependent enhance-ment of feline infectious peritonitis virus infection of primary feline peritoneal macrophages in vitro.Enhancement of infection was assessed, after indirect immunofluorescent-antibody labelling of infected cells,by determining the ratio between the number of cells infected in the presence and absence of virus-specific antibody. Infection enhancement was initially demonstrated by using heat-inactivated, virus-specific feline anti serum. Functional compatibility between murine immunoglobulin molecules and feline Fc receptors was demonstrated by using murine anti-sheepery throcyte serum and anantibody-coated sheepery throcytephagocytosis assay. Thirty-seven urine monoclonal ntibodies specific for the nucleocapsid, membrane, or spike proteins of feline infectious peritonitis virus or transmissible gastroenteritis virus were assayed for their ability to enhance the infectivity of feline infectious peritonitis virus. Infection enhancement was mediated by a subset of spike protein-specific onoclonalantibodies. A distinct correlation was seen between the ability of a monoclonal antibody to cause virus neutralization in a routine cell culture neutralization assay and its ability to mediate infection enhancement of macrophages. Infection enhancement was shown to be Fc receptor mediated by blockade of antibody-Fc receptor interaction using staphylococcal proteinA. Our results are consistent with the hypothesis that antibody-dependent enhancement of feline infectious peritonitis virus infectivity is mediated by antibody directed against specific sites on the spike protein.”

Tseng et al; 2012; “Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus” (https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0035421)

Results

All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing responses. Significant reductions of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All mice exhibited histopathologic changes in lungs two days after challenge including all animals vaccinated (Balb/C and C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathology seen in animals given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathology with prominent eosinophil infiltration, confirmed with special eosinophil stains. The pathologic changes seen in all control groups lacked the eosinophil prominence.

Conclusions

These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However, challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathology suggesting hypersensitivity to SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated

Wolff et al; 1990; “Direct Gene Transfer into Mouse Muscle in Vivo” (https://www.science.org/doi/10.1126/science.1690918)

RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and β-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining for β-galactosidase activity was localized to muscle cells following injection of the β-galactosidase DNA vector. After injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.

Sorenson et al; 2020; “Biovacc-19: A Candidate Vaccine for Covid-19 (SARS-CoV-2) Developed from Analysis of its General Method of Action for Infectivity” (https://www.cambridge.org/core/journals/qrb-discovery/article/biovacc19-a-candidate-jjab-for-covid19-sarscov2-developed-from-analysis-of-its-general-method-of-action-for-infectivity/DBBC0FA6E3763B0067CAAD8F3363E527)

This study presents the background, rationale and method of action of Biovacc-19, a candidate vaccine for corona virus disease 2019 (Covid-19), now in advanced preclinical development, which has already passed the first acute toxicity testing. Unlike conventionally developed vaccines, Biovacc-19’s method of operation is upon nonhuman-like (NHL) epitopes in 21.6% of the composition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)’s spike protein, which displays distinct distributed charge including the presence of a charged furin-like cleavage site. The logic of the design of the vaccine is explained, which starts with empirical analysis of the aetiology of SARS-CoV-2. Mistaken assumptions about SARS-CoV-2’s aetiology risk creating ineffective or actively harmful vaccines, including the risk of antibody-dependent enhancement. Such problems in vaccine design are illustrated from past experience in the human immunodeficiency viruses domain. We propose that the dual effect general method of action of this chimeric virus’s spike, including receptor binding domain, includes membrane components other than the angiotensin-converting enzyme 2 receptor, which explains clinical evidence of its infectivity and pathogenicity.

We show the nonreceptor dependent phagocytic general method of action to be specifically related to cumulative charge from insertions placed on the SARS-CoV-2 spike surface in positions to bind efficiently by salt bridge formations; and from blasting the spike we display the NHL epitopes from which Biovacc-19 has been down-selected.

The possibility of inducing autoimmune responses or antibody-dependent enhancements, needs to be carefully guarded against because there is published evidence that an HIV candidate vaccine has actually enhanced infectivity (Duerr et al., Reference Duerr, Huang, Buchbinder, Coombs, Sanchez, Del Rio, Casapia, Santiago, Gilbert, Corey and Robertson2012): ‘Vaccinations were halted; participants were unblinded. In post hoc analyses, more HIV infections occurred in vaccines versus placebo recipients in men who had Ad5-neutralizing antibodies and/or were uncircumcised. Follow-up was extended to assess relative risk of HIV acquisition in vaccines versus placebo recipients over time’. Such antibody-dependent enhancement (ADE) has been observed for coronaviruses in animal models, allowing them to enter cells expressing Fc𝛾R. ADE is not fully understood: however, it is suggested that antibody-dependent enhancements may come as a result of amino acid variability and antigenic drift (Negro et al., Reference Negro2020; Ricke et al., Reference Ricke and Malone2020).

Sui et al; 2014; “Effects of Human Anti-Spike Protein Receptor Binding Domain Antibodies on Severe Acute Respiratory Syndrome Coronavirus Neutralization Escape and Fitness” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248992/)

“We also investigated the neutralization escape profiles of these nAbs and evaluated their effects on receptor binding and virus fitness in vitro and in mice. We found that some nAbs had great potency and breadth in neutralizing multiple viral strains, including neutralization escape viruses derived from other nAbs; however, no single nAb or combination of two blocked neutralization escape. Interestingly, in mice the neutralization escape mutant viruses showed either attenuation (Urbani background) or increased virulence (GD03 background) consistent with the different binding affinities between their RBDs and the mouse ACE2. We conclude that using either single nAbs or dual nAb combinations to target a SARS-CoV RBD epitope that shows plasticity may have limitations for preventing neutralization escape during in vivo immunotherapy. However, RBD-directed nAbs may be useful for providing broad neutralization and prevention of escape variants when combined with other nAbs that target a second conserved epitope with less plasticity and more structural constraint.”

Li et al; 2021; “The impact of receptor-binding domain natural mutations on antibody recognition of SARS-CoV-2” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985591/)

To evaluate the impact of RBD natural mutations on the binding efficacy of anti-SARS-CoV-2 antibodies, we expressed and purified 41 representative RBD variants, which included mutations of the three most frequently-mutated residues (S477, N439, T478), as well as all the variants emerged during the first 4 months of SARS-CoV-2 outbreak. All RBDs expressed well (Supplementary Fig. S1) and retained the capability to bind ACE2 (Supplementary Fig. S2–S4). Then, we measured the binding ability of the RBD mutants to a panel of 8 antibodies, developed by us and other groups that recognize a diverse set of epitopes on SARS-CoV-2 RBD^6–9. According to the binding epitopes, these antibodies could be divided into two groups: those who recognize epitopes within the ACE2-RBD binding interface and could compete with ACE2 for RBD binding (ACE2-competing group), and those could not (ACE2 non-competing group). Surprisingly, we found that all of the ACE2-competing antibodies exhibited negligible binding to at least one of the mutant RBDs as measured by ELISA (Fig. ​(Fig.1c)1c) and bio-layer interferometry (Fig. ​(Fig.1d).1d). For instance, 414-1, a potent SARS-CoV-2 neutralizing antibody isolated from a COVID-19 recovered patient⁷, exhibited no binding to the RBD mutants L452R and N501Y, and evidently reduced binding to nine other RBD mutants. In contrast, the antibodies S309⁸ and n3063⁶ that engage epitopes distinct from the receptor-binding motif showed exceptional breadth, with no escape mutants observed. The antibodies n3130⁶ and CR3022⁹ were reported to target cryptic epitopes located in the spike trimeric interface, and retained their binding affinities towards most of the RBD variants (Fig. ​(Fig.1c).1c). Taken together, these results indicate that a single natural mutation on the SARS-CoV-2 RBD was able to completely abolish antibody binding. Besides, it seems that the natural RBD mutants had a higher tendency to escape the binding of ACE2-competing antibodies than non-competing antibodies (Fig. 1e and f), although further studies on more extensive panels of antibodies are required to confirm this finding.

Next, we evaluated the binding breadth of a combination of two antibodies recognizing distinct epitopes. Notably, the combination of ACE2-competing antibody 414-1 with non-competing antibody n3130 resulted in full coverage of SARS-CoV-2 RBD variants (Fig. ​(Fig.1c).1c). The mixture exhibited strong binding to most of the RBD variants, and slightly reduced binding only to one double mutant (K448R/H519P). To confirm whether the reduction in RBD binding potency correlates with reduced SARS-CoV-2 neutralization, we also measured the neutralizing activity of the antibodies against SARS-CoV-2 pseudoviruses bearing RBD mutations. As expected, 414-1 and n3130 did not show effective neutralization against pseudoviruses with their corresponding escape mutations (N501Y and E516Q, respectively), while the mixture of the two antibodies broadly neutralized all the tested viruses (Fig. ​(Fig.1g).1g). All the RBD variants pseudoviruses still possessed the infectivity of target cells (Supplementary Fig. S5). In addition, plasma from convalescent COVID-19 patients were also measured for their binding and neutralization activities. Similarly, all plasma samples had superior breadth in binding to naturally mutated RBDs and neutralizing multiple viral variants (Supplementary Figs. S6, S7). Considering that a number of neutralizing antibodies are being developed to treat COVID-19, our results suggest that some of these antibodies should be used in combinations to increase the neutralization breadth and reduce the possibility that an escape mutant is fixed in the treated host population. Nevertheless, the low frequencies of RBD mutants identified here revealed that they are more likely from randomized mutation, and may not represent the result of fitness selection.

Collectively, these results confirmed the capability of SARS-CoV-2 to escape from antibodies, especially the ACE2-competing antibodies, by acquiring resistance mutations in RBD. Such escape mutations can occur within the binding epitopes of antibodies, or regions away from antibody epitopes but may affect immunogenicity of RBD and render antibodies ineffective (Fig. ​(Fig.1f).1f). Notably, the ACE2-competing antibodies constitute the majority of anti-RBD antibodies elicited by SARS-CoV-2 infection or vaccination. Therefore, our findings highlight the importance of continuously monitoring RBD natural mutations and evaluating their impact on antibody recognition of SARS-CoV-2, which may help guide the development and implementation of therapeutic antibodies and vaccines against SARS-CoV-2.

Zhang et al; 2024; “COVID-19 antibody responses in individuals with natural immunity and with vaccination-induced immunity: a systematic review and meta-analysis” (https://systematicreviewsjournal.biomedcentral.com/articles/10.1186/s13643-024-02597-y)

The meta-analysis showed that within the first week after COVID-19 symptom onset/diagnosis or vaccination, antibody response rates in vaccinated individuals were lower than those in infected patients (p < 0.01), but no significant difference was observed from the second week to the sixth month. IgG, IgA, and IgM positivity rates increased during the first 3 weeks; thereafter, IgG positivity rates were maintained at a relatively high level, while the IgM seroconversion rate dropped.

Conclusions

Antibody production following vaccination might not occur as quickly or strongly as after natural infection, and the IgM antibody response was less persistent than the IgG response.

Yang et al; 2005; “Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses” (https://www.pnas.org/doi/full/10.1073/pnas.0409065102)

“Molecular characterization of the severe acute respiratory syndrome coronavirus has revealed genetic diversity among isolates. The spike (S) glycoprotein, the major target for vaccine and immune therapy, shows up to 17 substitutions in its 1,255-aa sequence; however, the biologic significance of these changes is unknown. Here, the functional effects of S mutations have been determined by analyzing their affinity for a viral receptor, human angiotensin-converting enzyme 2 (hACE-2), and their sensitivity to Ab neutralization with viral pseudotypes. Although minor differences among eight strains transmitted during human outbreaks in early 2003 were found, substantial functional changes were detected in S derived from a case in late 2003 from Guangdong province [S(GD03T0013)] and from two palm civets, S(SZ3) and S(SZ16). S(GD03T0013) depended less on the hACE-2 receptor and was markedly resistant to Ab inhibition. Unexpectedly, Abs that neutralized most human S glycoproteins enhanced entry mediated by the civet virus S glycoproteins. The mechanism of enhancement involved the interaction of Abs with conformational epitopes in the hACE-2-binding domain. Finally, improved immunogens and mAbs that minimize this complication have been defined. These data show that the entry of severe acute respiratory syndrome coronaviruses can be enhanced by Abs, and they underscore the need to address the evolving diversity of this newly emerged virus for vaccines and immune therapies.”

Alberer et al; 2013/17; “Safety and immunogenicity of a mRNA rabies vaccine in healthy adults: an open-label, non-randomised, prospective, first-in-human phase 1 clinical trial” (https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(17)31665-3/fulltext)

“Between Oct 21, 2013, and Jan 11, 2016, we enrolled and vaccinated 101 participants with 306 doses of mRNA (80–640 μg) by needle-syringe (18 intradermally and 24 intramuscularly) or needle-free devices (46 intradermally and 13 intramuscularly). In the 7 days post vaccination, 60 (94%) of 64 intradermally vaccinated participants and 36 (97%) of 37 intramuscularly vaccinated participants reported solicited injection site reactions, and 50 (78%) of 64 intradermally vaccinated participants and 29 (78%) of 37 intramuscularly vaccinated participants reported solicited systemic adverse events, including ten grade 3 events. One unexpected, possibly related, serious adverse reaction that occurred 7 days after a 640 μg intramuscular dose resolved without sequelae. mRNA vaccination by needle-free intradermal or intramuscular device injection induced virus neutralising antibody titres of 0·5 IU/mL or more across dose levels and schedules in 32 (71%) of 45 participants given 80 μg or 160 μg CV7201 doses intradermally and six (46%) of 13 participants given 200 μg or 400 μg CV7201 doses intramuscularly. 1 year later, eight (57%) of 14 participants boosted with an 80 μg needle-free intradermal dose of CV7201 achieved titres of 0·5 IU/mL or more. Conversely, intradermal or intramuscular needle-syringe injection was ineffective, with only one participant (who received 320 μg intradermally) showing a detectable immune response.“

Bahl et al; 2017; “Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses” (https://www.sciencedirect.com/science/article/pii/S1525001617301569)

“Given this innovative vaccine platform, we examined the biodistribution of the mRNA vaccines for both routes of administration. Male CD-1 mice received 6 μg formulated H10 mRNA either IM or ID. Following IM administration, the maximum concentration (C_max) of the injection site muscle was 5,680 ng/mL, and the level declined with an estimated t_1/2 of 18.8 hr (Table 1). Proximal lymph nodes had the second highest concentration at 2,120 ng/mL (t_max of 8 hr with a relatively long t_1/2 of 25.4 hr), suggesting that H10 mRNA distributes from the injection site to systemic circulation through the lymphatic system. The spleen and liver had a mean C_max of 86.9 ng/mL (area under the curve [AUC]_0–264 of 2,270 ng.hr/mL) and 47.2 ng/mL (AUC_0–264 of 276 ng.hr/mL), respectively. In the remaining tissues and plasma, H10 mRNA was found at 100- to 1,000-fold lower levels.

H10 mRNA Immunogenicity and Safety in Humans

The majority of adverse events (AEs) were mild (107/163 events; 66%) or moderate (52/163 events; 32%), using the Center for Biologics Evaluation and Research (CBER) severity scale.³⁸ AEs were comparable in frequency, nature, and severity to unadjuvanted and adjuvanted H1N1 influenza vaccines.³⁹ Twenty-three subjects who received 100 μg H10 IM reported 163 reactogenicity events with no idiosyncratic or persistent AEs observed. The majority of events were injection site pain, myalgia, headache, fatigue, and chills/common-cold-like symptoms (Table S2). Only four events (2.5%), reported by three subjects (13% of exposed subjects), were categorized as severe and included injection site erythema (1.2%), injection site induration (0.6%), and chills/common cold (0.6%) (Table 2; Table S2). No serious AE occurred and all events were expected and reversible. Overall, this reactogenicity profile is similar to that of a monovalent AS03-adjuvanted H1N1 vaccine, and it is comparable to that of meningococcal conjugate vaccine in healthy adults (19–55 years).40, 41“

2. Spike / modRNA / Lipid Nanoparticles

Bitounis et al; 2024; “Strategies to reduce the risks of mRNA drug and vaccine toxicity” (https://pubmed.ncbi.nlm.nih.gov/38263456/)

mRNA formulated with lipid nanoparticles is a transformative technology that has enabled the rapid development and administration of billions of coronavirus disease 2019 (COVID-19) vaccine doses worldwide. However, avoiding unacceptable toxicity with mRNA drugs and vaccines presents challenges. Lipid nanoparticle structural components, production methods, route of administration and proteins produced from complexed mRNAs all present toxicity concerns. Here, we discuss these concerns, specifically how cell tropism and tissue distribution of mRNA and lipid nanoparticles can lead to toxicity, and their possible reactogenicity. We focus on adverse events from mRNA applications for protein replacement and gene editing therapies as well as vaccines, tracing common biochemical and cellular pathways. The potential and limitations of existing models and tools used to screen for on-target efficacy and de-risk off-target toxicity, including in vivo and next-generation in vitro models, are also discussed.

Szebeni J., 2025; “The Unique Features and Collateral Immune Effects of mRNA-Based COVID-19 Vaccines: Potential Plausible Causes of Adverse Events and Complications” (https://www.preprints.org/manuscript/202501.1462/v1)

The lipid nanoparticle (LNP)-enclosed mRNA-containing COVID-19 vaccines were used successfully to vaccinate billions of people during the COVID-19 pandemic. However, as with all medicines, Comirnaty and Spikevax can also cause adverse events (AEs) and complications. Although such events occur in less than 0.5% of vaccinated individuals, the huge scale of global vaccination means that the number of “vaccine injuries” could reach millions worldwide. These AEs have a uniquely broad spectrum affecting multiple organs, with most cases being associated with inflammatory and autoimmune processes. Yet, the clinical significance of AEs is debated, and their cellular and molecular mechanisms are poorly understood. The present review surveys the distinctive structural and functional features of these vaccines linking them to secondary immune effects causing unusual AEs/complications. The discussed unique mRNA-LNP properties include the ribosomal synthesis of the spike protein (SP) fundamentally transforming antigen processing and presentation; the multiple chemical modifications of the mRNA, increasing its stability and translation efficacy; toxicity of the SP casing multiorgan damage; immune stimulation and mRNA transfection by LNP; reduced stability of vaccine nanoparticles in water; immune reactivity and immunogenicity of PEG on the LNP surface; stabilization of the SP by enrichment with proline; and contaminations of the vaccine with plasmid DNA and inorganic elements or complexes. The considered collateral immune effects that may theoretically underlie the AEs are: diversification of the processing and presentation of the SP, innate immune activation, T-cell and antibody-mediated cytotoxicities, dissemination of virus/vaccine hybrid exosomes, somatic hypermutation, reverse transcription, insertion mutagenesis and frameshift mutation.

2a. Spike Protein

Parry et al; 2023; “‘Spikeopathy’: COVID-19 Spike Protein Is Pathogenic, from Both Virus and Vaccine mRNA” (https://pubmed.ncbi.nlm.nih.gov/37626783/)

This first paper explores peer-reviewed data counter to the ‘safe and effective’ narrative attached to these new technologies. Spike protein pathogenicity, termed ‘spikeopathy’, whether from the SARS-CoV-2 virus or produced by vaccine gene codes, akin to a ‘synthetic virus’, is increasingly understood in terms of molecular biology and pathophysiology. Pharmacokinetic transfection through body tissues distant from the injection site by lipid-nanoparticles or viral-vector carriers means that ‘spikeopathy’ can affect many organs. The inflammatory properties of the nanoparticles used to ferry mRNA; N1-methylpseudouridine employed to prolong synthetic mRNA function; the widespread biodistribution of the mRNA and DNA codes and translated spike proteins, and autoimmunity via human production of foreign proteins, contribute to harmful effects. This paper reviews autoimmune, cardiovascular, neurological, potential oncological effects, and autopsy evidence for spikeopathy. With many gene-based therapeutic technologies planned, a re-evaluation is necessary and timely.

Halma et al; 2023; “Strategies for the Management of Spike Protein-Related Pathology” (https://pubmed.ncbi.nlm.nih.gov/37317282/)

Mechanisms of Harm

As mentioned previously, while it was expected that the LNP-encapsulated synthetic mRNAs would remain at the injection site and rapidly degrade, there is substantial evidence that they enter the bloodstream [60], deposit in other tissues [61], and even in the breast milk of lactating mothers [62]. The S1 subunit of the spike protein can damage the endothelial lining of blood vessels [63,64,65]. Vaccine particles in the bloodstream can cause a significant inflammatory response in blood vessels [66].

Several hypotheses for the mechanisms of long COVID-19 exist, including immune dysregulation, auto-immunity, endothelial dysfunction, activation of coagulation, and latent viral persistence [67,68], though this review focuses on the elements common to both COVID-19 infection and vaccine injury. Cardiovascular complications, particularly microthrombus formation, feature both in the etiologies of long COVID-19 [69,70] as well as COVID-19 vaccine injury [71].

The SARS-CoV-2 (infection or vaccine produced) spike protein can bind to the ACE2 receptor on platelets, leading to their activation [72], and it can cause fibrinogen-resistant blood clots [73]. Spike protein fragments can also be amyloidogenic on their own [74]. Several reports demonstrate elevated troponin levels in cardiac symptoms following the COVID-19 vaccine [75].

Ontologically, both infection and vaccination express the spike protein, though some subtle differences exist between the vaccine-generated and the infection-generated spike protein. Importantly, the spike protein encoded by vaccines is static and does not undergo evolution, whereas the spike protein produced by infection evolves as the virus evolves [76,77]. There is one exception to this, and that is when the vaccine is updated, as it is in the bivalent boosters of Pfizer and Moderna, which express the spike protein of both the B.1.1.529 (omicron) BA.5 sublineage and the ancestral WA1/2020 strain [78]. The other important distinction between vaccine spike and infection spike is the stabilized pre-fusion state in the vaccine spike, which results in an increased ACE2 binding affinity compared to spike proteins generated via SARS-CoV-2 infection [79]. The difference in the circulating (in the population) SARS-CoV-2 spike protein to the spike protein (either vaccine or infection generated) of one’s initial immune imprinting has important implications for immune escape [77,80] and immune-mediated damage [81]. Immune escape is demonstrated in population studies showing waning vaccine efficacy [82].

In 2021, a comprehensive investigation revealed consistent pathophysiological alterations after vaccination with COVID-19 vaccines, including alterations of immune cell gene expression [83].

A number of factors are associated with an increased risk of adverse events; these include:

• Genetics: first-degree relatives of people who have suffered a vaccine injury appear to be at a very high risk of vaccine injury. People with a methylenetetrahydrofolate reductase (MTHFR) gene mutation [93] and those with Ehlers-Danlos type syndromes, may be at an increased risk of injury. Increased homocysteine levels have been linked to worse outcomes in patients with COVID-19 [94,95]. Increased homocysteine levels may potentiate the microvascular injury and thrombotic complications associated with spike protein-related vaccine injury [96,97].
• mRNA load and quantity of spike protein produced: this may be linked to specific vaccine lots that contain a higher concentration of mRNA due to variances in manufacturing quality, as well as heterogeneity within the vial [98].
• Type and batch of vaccine: variances in the levels of adverse reactions were observed, depending on the manufacturer of the vaccine [91].
• Number of vaccines given: the risk of antibody enhancement (ADE) increases with each exposure to the virus or a vaccine. A negative inverse correlation of dosages given, as well as effectiveness, was also observed [99].
• Sex: the majority of vaccine-injured people are female [100], and vaccines historically have sex-specific effects [101].
• Underlying nutritional status and comorbidities: certain preexisting conditions may likely have primed the immune system to be more reactive after vaccination [102]. This includes those with preexisting autoimmune disorders [103].

Bellavite et al; 2023; “Immune Response and Molecular Mechanisms of Cardiovascular Adverse Effects of Spike Proteins from SARS-CoV-2 and mRNA Vaccines” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9953067/)

Whether these vaccines fulfil the definition of “vaccine” or should instead be regarded as pro-pharmacologic drugs is a matter of debate [24]. However, for the sake of practicality, we shall not discuss here the name that better suits these immunostimulatory gene-based pro-drugs falling in the category of immunological-genetic product and will rather focus on their mechanisms of action. Here we will discuss how the mRNA-based vaccine elicits the immune response along with serious side effects on the cardiovascular system, whose severity depends on the distribution in the body of the Spike protein and the extent of the immune response elicited by the vaccine.

The problem of the benefit/risk ratio of anti-COVID-19 vaccines is extremely complex for several reasons, including: (a) The disease severity is very different depending on age, gender and general health condition of the person. (b) The efficacy of vaccines wanes over time and changes according to the variants. (c) Pharmacovigilance data are obtained mainly through passive detection systems that are inadequate. The argument as to whether the risks of vaccination may in some circumstances outweigh the benefits of defence against disease is not within the scope of this paper, which focuses instead on the molecular mechanisms of adverse events following vaccination.

Essentials of mRNA Vaccines Design and Functioning

Taken together, the vaccine mRNAs driving the Spike protein synthesis have been engineered in a manner that challenges the cellular stress response for the recognition of exogenous nucleic acids and proteins, and this is likely to impact the distribution of the mRNAs coding for the Spike protein and of the protein itself, which may then explain the biological and pathophysiological effects in organs distant from the site of injection. Indeed, the true biodistribution and the half-life of the vaccine mRNA in humans are currently unknown. Normally, mRNA is very fragile and is quickly degraded (within a few days). It was initially thought that vaccine mRNA would remain localized in the site of injection and be degraded within a few days, as is normal mRNA. However, real-world observations contradict this prediction. The S-protein has been detected in the plasma of mRNA-1273 COVID-19 vaccinees at 15 days following injection [59]. Both mRNA and S protein have been found in axillary lymph nodes after 60 days [60]. Very recently, Spike-mRNA has been detected in the blood of vaccinated individuals 15 and up to 28 days after COVID-19 vaccination [61,62]. Thus, it is likely that mRNA-LNPs remain in circulation for extended periods of time, retaining their ability to induce S protein expression in encountered cells. Updated bivalent mRNA vaccines that include the coding sequence for the Omicron BA.4/BA.5 variant were made available in September 2022, and studies on their efficacy and safety are still ongoing. Based on two pre-print studies, not yet peer-reviewed, the bivalent mRNA vaccine shows modest protection [63] and a higher rate of adverse events compared to the monovalent mRNA vaccine [64].

Immunization Pathways of the SARS-CoV-2 and mRNA Vaccines

mRNA COVID-19 vaccines are meant to induce B lymphocytes capable of producing antibodies against the (viral) S protein for preventing SARS-CoV-2 entry into the cells as well as T lymphocytes capable of killing the virus-infected cells (in the lung, kidney, etc.) expressing the S antigen on the membrane. However, the pathway for eliciting the immune response to the S protein coded by mRNA vaccines presents many peculiarities that need to be elucidated.

In the specific case of mRNA-driven antigen delivered via LNP, the following peculiarities should be considered: 1. The LNPs may fuse with the membrane of any cell they encounter and therein release the payload [83]. This implies that the mRNA may direct the synthesis of the Spike protein not exclusively in muscle cells but also in APCs and other somatic cells. 2. The mRNA is provided with a leader sequence, which directs the synthesis of the Spike protein in endoplasmic reticulum-associated ribosomes. The membrane bound S protein would then travel through the Golgi complex (here it will be split into S1 and S2 by furin) and then be exposed on the plasma membrane via insertional exocytosis [14]. Transfected cells could free the S protein and/or its fragments following T cell killing, and S1 (which is non-covalently bound to S2) could be shed from the membrane [14,22]. Consistently, high levels of soluble Spike proteins are found in the circulation of vaccinees with myocarditis [84]. The soluble Spike can be subsequently endocytosed by APCs and B lymphocytes. The transfected cells may release exosomes expressing the S protein on the membrane, which also contribute to immunostimulation of APCs in distant organs [66].

However, in the case of the COVID-19 vaccination with LNP loaded with the modified mRNA we face unpredicted outcomes, since the mRNA transfection could aspecifically occur in any cell, including APCs, endothelial cells, and parenchymal cells of distant organs, wherein the mRNA would then direct the persistent synthesis of the modified (stabilized in open conformation) S protein. The processing route of the S protein will determine the fate of the transfected cells.

In case of LNP transfection of parenchymal cells (ideally only the muscle cells at the injection site), the exposure on the membrane of the S protein would predictably trigger the CD8+ T lymphocyte cytotoxicity, much like what would happen to virus-infected cells. Yet, at variance from natural infection with SARS-CoV-2, in the transfected cells the S protein may (in part) not be processed, and be exposed on the membrane not in the context of the MHC class I. This eventuality could deceive the immune cells, which could consider the protein as a self.

Hence, the mRNA vaccine “theory” neglects the possibility that any cell producing the Spike protein and displaying it on its membrane (associated or not with MHC-I) will be attacked and destroyed by CD8+T cells. The severity of the consequences for the host following the vaccination will depend on the type and number of cells affected and the tissue where the reaction occurs. For example, myocarditis is considered an adverse reaction to mRNA vaccination [85,86]. The facts that this event is more frequent after the second dose and it occurs a few days after the inoculation [27], suggest an immune-mediated mechanism analogous to an auto-immune reaction. To conclude, the Spike protein acts in a peculiar way, not simply as an immunogen, but as a disease-causing agent.

LNP Biodistribution and Spike Detection

In the dossier submitted for mRNA-1273 authorization to the FDA, the vaccine producer (Moderna) claimed that immune reaction to the Spike would occur “in situ”, i.e., at the point of injection [77]. However, the few biodistribution studies carried out [88] showed that in mice and rats challenged with LNP labelled with radioactive probe or luciferase the signal is detected in various tissues, with the injection site, the spleen and the liver being the most enriched ones [89]. The technical dossier presented for the registration of Pfizer anti-COVID-19 vaccine reports that within 48 h from injection, LNP redistributed mainly to the liver, adrenal glands, spleen, and ovaries.

Subsequent studies have shown the presence of vaccine-derived Spike proteins in the blood [59,90]. Since receptors for Spike are ubiquitously expressed in a variety of tissues and organs, it is likely that this protein performs activities that clearly go beyond its intended function as simple “antigen” [91,92]. Studies in laboratory animals have shown that Spike proteins may also cross the blood-brain barrier, which may account for neurological symptoms of the disease as well as of the vaccine [93].

Furthermore, immunohistochemical staining of axillary lymph node biopsies shows that vaccine Spike proteins were still present up to 60 days after the second dose of mRNA vaccines [60]. These authors found the Spike protein also in plasma in the first few days after vaccination (mean concentration of 47 pg/mL), yet the measurement of the Spike in blood after boosts was affected by the presence of specific antibodies. Circulating exosomes containing the Spike protein were found on day 14 after vaccination, and they increased after the booster dose, lasting up to four months [66]. While it has been suggested that these vesicles expressing the Spike protein on the membrane have the function of stimulating the immune response, it is not known whether they may interact with cells expressing ACE2. Vaccine-derived mRNA and Spike protein have been detected in the germinal centre of secondary lymphoid tissues two months after vaccination, suggesting sustained induction of protein synthesis [60]. Recently, circulating Spike proteins were detected in the blood of subjects hospitalized for myocarditis after mRNA vaccination [84]. Remarkably, the concentration of Spike protein (mean 33.9 ± 22.4 pg/mL) was significantly higher in symptomatic vaccinees than in asymptomatic ones, and it was measurable until three weeks after vaccination [84].

The Spike protein was detected by immunohistochemistry in the vessel wall of the brain and heart of a 76-year-old patient deceased three weeks after receiving his third COVID-19 vaccination [94]. Since no nucleocapsid (N) protein was detected, the authors suggest that the pathology was caused by vaccination and not by SARS-CoV-2 virus infection.

Another troubling study on the Pfizer vaccine presents evidence of the possible permanence of the message inside the cell in the form of DNA [97]. According to this study, the rapid entry of mRNA into human liver cells would be followed by “reverse transcription” to the DNA within a few hours [97]. Whether the DNA reverse transcribed from BNT162b2 mRNA is integrated into the cell genome has not been proved, yet the finding raises the concern that the integrity of genomic DNA could be affected, underscoring possible genotoxic side effects. Furthermore, if the mRNA message is retro-transcribed in DNA, which is more stable, the synthesis of the Spike proteins may persist for long time.

Sass E.; 2024; “SARS-CoV2 spike protein pathogenicity research collection” (https://zenodo.org/records/14269255)

Originally part of the outer coat of the SARS-CoV2 virus, where it functions as a “key” to “unlock” (infect) cells, spike proteins are also produced in large amounts by the mRNA “vaccines,” triggering a short-lived immune response in the form of antibodies. However, a growing body of evidence has shown thatthe spike protein is harmful by itself, independent of the rest of the virus.

The following (I. Alphabetical List) collects over 250 peer-reviewed scientific studies confirming that the spike protein is highly pathogenic on its own; most in vitro studies cited here used recombinant spike proteins or spike proteins in pseudoviral vectors, and produced pathological effects not reliant on the SARS-CoV2 viral machinery.

The second section (II. Categories) organizes the research into broad categories including affected tissues and organ systems, mechanisms, and evidence from clinical pathology. Because these areas overlap, many articles appear more than once in the second section.

Wucher et al; 2024; “mRNA “vaccine” biodistribution, persistence, and adjuvant toxicity library” (https://zenodo.org/records/14559625)

Originally part of the outer coat of the SARS-CoV2 virus, where it functions as a “key” to “unlock” (infect) cells, spike proteins are also produced in large amounts by the mRNA “vaccines,” triggering a short-lived immune response in the form of antibodies. However, a growing body of evidence has shown thatthe spike protein is harmful by itself (see: “Spike protein pathogenicity research library,” https://zenodo.org/records/14559644). Furthermore, research has demonstrated that:

1)        Both the “vaccine” mRNA encoding for the spike protein antigen and the spike protein itself can penetrate distant tissues, causing systemic harms.

2)        “Vaccine” mRNA and the spike protein antigen persist in the tissues of human vaccine recipients and animal test subjects far longer than claimed by public health officials, while viral spike proteins have been shown to persist even longer.

3)        The ionizable lipid nanoparticles (LNPs) used in the experimental mRNA injections are highly inflammatory on their own, including their polyethylene glycol (PEG) component, an established cause of anaphylaxis (an extreme allergic reaction).

The following research collection presents over 100 peer-reviewed studies (n=130) documenting I) the wide distribution and II) persistence of “vaccine” mRNA and the encoded spike protein, as well as III) the potential harms of the LNP delivery system (some studies with overlapping findings appear in more than one category). Taken together with evidence of the spike protein’s pathogenicity (https://zenodo.org/records/14559644), these findings suggest that the mRNA “vaccines” can distribute harmful, long-lasting spike protein uncontrollably throughout the body, causing injuries and death by various means.

Please note that a small number of studies in section I) investigate the ability of viral spike protein resulting from infection to cross important physiological barriers on its own, while some studies in section II) demonstrate the long persistence of viral-derived spike protein in the absence of viable virus, bolstering concerns about the identical “vaccine” spike.

Hatfill S., & Sass E.,; 2024; “COVID “vaccine” immune imprinting library” (https://zenodo.org/records/14632346)

Immune imprinting, dubbed “original antigenic sin” by Thomas Francis Jr., occurs when memory B lymphocytes produced in response to an initial viral infection dominate subsequent responses to related viruses, producing antibodies geared to the original exposure. Long-term immune memory has many advantages, but immune imprinting can be harmful if it interferes with immune response to later infections. 

The following collection of over 100 peer-reviewed papers (n=131) suggests that COVID “vaccines” imprinted the immune systems of recipients through exposure to the “wild type” spike protein from the original Wuhan strain, shaping their response to subsequent variants in potentially harmful ways. Immune imprinting impaired responses to new variants by skewing B cell production of antibodies toward the “ancestral” spike protein at the expense of new antibodies specifically tailored to the variants’ heavily mutated spike. Additionally, by imprinting a single antigen – the spike protein – on recipients’ immune systems, the “vaccines” prevented them from forming antibodies to other, less mutation-prone parts of the virus, such as proteins from the virus nucleocapsid (Ahmed MIM et al., Delgado JF et al., Paula NM et al., Smith CP et al., Yao D et al). Further findings point to “deep immunological imprinting” or “hybrid immune damping,” in which “vaccination” combined with infection alters later immune response unpredictably (Aguilar-Bretones M et al., Gao B et al., Hornsby H et al., Ju B et al., Reynolds CJ et al., Wang Q et al.).

This collection originated with Dr. Steven Hatfill’s contribution to TOXIC SHOT: Facing the Dangers of the COVID “Vaccines” (Chapter 5: Debunking CDC’s Bad Science).

Boros et al; 2024; “Long-lasting, biochemically modified mRNA, and its frameshifted recombinant spike proteins in human tissues and circulation after COVID-19 vaccination” (https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1002/prp2.1218)

According to the CDC, both Pfizer and Moderna COVID-19 vaccines contain nucleoside-modified messenger RNA (mRNA) encoding the viral spike glycoprotein of severe acute respiratory syndrome caused by corona virus (SARS-CoV-2), administered via intramuscular injections. Despite their worldwide use, very little is known about how nucleoside modifications in mRNA sequences affect their breakdown, transcription and protein synthesis. It was hoped that resident and circulating immune cells attracted to the injection site make copies of the spike protein while the injected mRNA degrades within a few days. It was also originally estimated that recombinant spike proteins generated by mRNA vaccines would persist in the body for a few weeks. In reality, clinical studies now report that modified SARS-CoV-2 mRNA routinely persist up to a month from injection and can be detected in cardiac and skeletal muscle at sites of inflammation and fibrosis, while the recombinant spike protein may persist a little over half a year in blood. Vaccination with 1-methylΨ (pseudouridine enriched) mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in major histocompatibility complex-diverse people. The translation of 1-methylΨ mRNA using liquid chromatography tandem mass spectrometry identified nine peptides derived from the mRNA +1 frame. These products impact on off-target host T cell immunity that include increased production of new B cell antigens with far reaching clinical consequences. As an example, a highly significant increase in heart muscle 18-flourodeoxyglucose uptake was detected in vaccinated patients up to half a year (180 days).

Scholkmann and May; 2023; “COVID-19, post-acute COVID-19 syndrome (PACS, “long COVID”) and post-COVID-19 vaccination syndrome (PCVS, “post-COVIDvac-syndrome”): Similarities and differences” (https://pubmed.ncbi.nlm.nih.gov/37192595/)

Worldwide there have been over 760 million confirmed coronavirus disease 2019 (COVID-19) cases, and over 13 billion COVID-19 vaccine doses have been administered as of April 2023, according to the World Health Organization. An infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can lead to an acute disease, i.e. COVID-19, but also to a post-acute COVID-19 syndrome (PACS, “long COVID”). Currently, the side effects of COVID-19 vaccines are increasingly being noted and studied. Here, we summarise the currently available indications and discuss our conclusions that (i) these side effects have specific similarities and differences to acute COVID-19 and PACS, that (ii) a new term should be used to refer to these side effects (post-COVID-19 vaccination syndrome, PCVS, colloquially “post-COVIDvac-syndrome”), and that (iii) there is a need to distinguish between acute COVID-19 vaccination syndrome (ACVS) and post-acute COVID-19 vaccination syndrome (PACVS) – in analogy to acute COVID-19 and PACS (“long COVID”). Moreover, we address mixed forms of disease caused by natural SARS-CoV-2 infection and COVID-19 vaccination. We explain why it is important for medical diagnosis, care and research to use the new terms (PCVS, ACVS and PACVS) in order to avoid confusion and misinterpretation of the underlying causes of disease and to enable optimal medical therapy. We do not recommend to use the term “Post-Vac-Syndrome” as it is imprecise. The article also serves to address the current problem of “medical gaslighting” in relation to PACS and PCVS by raising awareness among the medical professionals and supplying appropriate terminology for disease.

Li et al; 2024; “SARS-CoV-2 spike-induced syncytia are senescent and contribute to exacerbated heart failure” (https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012291)

Here, we found that the senescent outcome of SARS-2-S induced syncytia exacerbated heart failure progression. We first demonstrated that syncytium formation in cells expressing SARS-2-S delivered by DNA plasmid or LNP-mRNA exhibits a senescence-like phenotype. Extracellular vesicles containing SARS-2-S (S-EVs) also confer a potent ability to form senescent syncytia without de novo synthesis of SARS-2-S. However, it is important to note that currently approved COVID-19 mRNA vaccines do not induce syncytium formation or cellular senescence. Mechanistically, SARS-2-S syncytia provoke the formation of functional MAVS aggregates, which regulate the senescence fate of SARS-2-S syncytia by TNFα. We further demonstrate that senescent SARS-2-S syncytia exhibit shrinked morphology, leading to the activation of WNK1 and impaired cardiac metabolism. In pre-existing heart failure mice, the WNK1 inhibitor WNK463, anti-syncytial drug niclosamide, and senolytic dasatinib protect the heart from exacerbated heart failure triggered by SARS-2-S. Our findings thus suggest a potential mechanism for COVID-19-mediated cardiac pathology and recommend the application of WNK1 inhibitor for therapy especially in individuals with post-acute sequelae of COVID-19.

Author summary

In this paper, we directly linked SARS-2-S-triggered syncytium formation in the absence of infection with the ensuing induction of cellular senescence and its pathophysiological contribution to heart failure. We propose that both SARS-2-S expression and SARS-2-S protein internalization were sufficient to induce senescence in nonsenescent ACE2-expressing cells. This is important because of the persistent existence of SARS-2-S or extracellular vesicles containing SARS-2-S during the post-acute stages of SARS-CoV-2 infection in human subjects. In searching for the underlying molecular mechanisms determining syncytial fate, the formation of functional MAVS aggregates dependent on RIG-I was observed at an early stage during fusion and regulated the anti-death to senescence fate of SARS-2-S syncytia through the TNFα-TNFR2 axis. We also found impaired cardiac metabolism in SARS-2-S syncytia induced by condensed WNK1. However, syncytium formation or cellular senescence observed with the wild-type fusogenic S protein does not occur with the spike proteins produced by currently approved COVID-19 mRNA vaccines. Importantly, SARS-2-S-exacerbated heart failure could be largely rescued by WNK1 inhibitor, anti-syncytial drug or senolytic agent. Together, we suggest that rescuing metabolism dysfunction in senescent SARS-2-S syncytia should be taken into consideration in individuals with post-acute sequelae of COVID-19 (PASC).

Demongeot et al; 2023; “mRNA COVID-19 Vaccines—Facts and Hypotheses on Fragmentation and Encapsulation” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9864138/)

Discussion: we discuss two aspects related to mRNA vaccine: (i) the plausibility of mRNA fragmentation, and (ii) the role of liposomal nanoparticles (LNPs) used in the vaccine and their impact on mRNA biodistribution. Conclusion: we insist on the need to develop lipid nanoparticles allowing personalized administration of vaccines and avoiding adverse effects due to mRNA fragmentation and inefficient biodistribution. Hence, we recommend (i) adapting the mRNA of vaccines to the least mutated virus proteins and (ii) personalizing its administration to the categories of chronic patients at risk most likely to suffer from adverse effects.

2.6. mRNA and Poly(A) Tail Size

Polyadenylation, which consists of the addition of a Poly(A) tail, plays several essential roles during the different stages of the life of a messenger RNA [39]. It intervenes in particular in the protection against degradation, in the nucleo-cytoplasmic transport (passage of mRNA from the nucleus to the cytoplasm) and in the recruitment of the ribosome, to allow its translation into protein. Polyadenylation is also involved in excision of the last exon and plays a role in mRNA stability. Indeed, the Poly(A) tail modulates mRNA degradation in the cytosol. Whether for the mRNA vaccine from Pfizer/BioNtech^® laboratories or that of Moderna^®, the two mRNA sequences end in a Poly(A) tail. The purpose of this addition is to increase the stability of the mRNA in biological medium and also to allow the recruitment of the ribosome, in order to initiate an efficient translation.

After translation, the mRNA can be reused several times, but when this happens, it also loses part of the Adenines of its Poly(A) tail, as enzymatic degradation begins there, which only ensures a transient protection against this degradation [40]. When this tail is too degraded, the mRNA is no longer functional and is destroyed. The poly(A) tail stabilizes mRNA and boosts protein translation, and the length of the poly(A) tail is proportional to translation efficiency. It is a critical factor in determining the longevity of mRNA molecules. [41] Mammalian cell mRNA molecules contain Poly(A) tails that are approximately 250 nucleotides in length, which gradually decrease in direction 3′ to 5′, during their life in the cytoplasm. Poly(A) tails of around 100 nucleotides have been shown to be ideal for mRNA-based therapies, since tail size modulates the 3′-exonucleolytic degradation of mRNA [42].

Deadenylation does not permanently inactivate the mRNA, because it can eventually be polyadenylated again. However, more often than not, when a certain number of “A” residues remaining on the mRNA is reached (on the order of 30 to 60 in mammals), the mRNA engages in rapid and irreversible degradation. For example, a long-tailed Poly(A) mRNA (such as hemoglobin mRNA) will be minimally degraded and therefore highly translated, allowing sustained production of the corresponding protein.

5. Biodistribution of mRNA Vaccine

During vaccination by the intramuscular route, the liposome can, due to its chemical properties, cross the capillary barrier and disseminate itself in all the tissues [117,118]. By this route, the liposome reaches the general circulation, without having to pass through the portal system and the liver. Absorption will be more or less complete and more or less rapid depending on the physicochemical properties of the liposome: injected into the arm, it does not remain locally, if it is not targeting, and diffuses very quickly (between 15 min and 4 h maximum throughout the body) and can, therefore, be incorporated into any cell of any organ. Before the COVID-19 epidemic, only one drug using encapsulated mRNA was approved by the FDA, followed by the European Commission in August 2018, the Patisiran^®, using LNPs encapsulating siRNAs (anti- senses). This treatment required more than 15 years of development, it ensures a prolonged release of siRNA in the tumor region, thanks to the biodegradable polymer PLGA [119]. We can see that this technology is very recent in humans and is mainly used in oncology, for patients with a vital prognosis. It is significantly different from current mRNA vaccine technology, with a distinct purpose.

6.1. Differences between “Classic” Vaccines and New mRNA Vaccines Encapsulated in a Nanoparticle Liposome

There is a major difference between the usual vaccines imagined by Pasteur and the new mRNA and DNA vaccines. In the usual vaccines, we inject either a recombinant protein (technology used by Sanofi^® and GSK^®), or an adenovirus (technology used by Johnson & Johnson^® and AstraZeneca^®), or an inactivated virus (technology used by Valneva^® and Sinopharm^®). Thus, the antigen is directly in contact with the tissues at the injection site and is quickly taken up by the immune system. Because of this rapid management, antigens, even cytotoxic ones, do not have time to spread to all the tissues, which limits the adverse effects related to the toxicity of the surface proteins of the pathogenic agent. Similarly, for new DNA vaccines, the adenovirus quickly recognized by the immune system does not have time to spread to all tissues. However, thrombocytopenia-like thromboses have been consistently reported following administration of adenoviral gene transfer vectors in animal models [120,121,122].

Vaccine-induced thrombotic thrombocytopenia (VITT) has been reported following vaccination with Astra Zeneca^® AZD1222 (the estimated rate of thrombocytopenia syndrome within 14 days after the first dose is equal to 8.1 per million of vaccinated people compared to 2.3 per million of vaccinated people at the second dose of AZD1222) and with the Johnson & Johnson^® Ad26 [123]. In the case of mRNA vaccines, host own cells make the antigen (here the Spike protein). The synthetic mRNA is encapsulated in a nanoparticle liposome (LNP), which does not have antigenic proteins on its surface, nor molecules targeting particular cells. LNPs, peptides and especially polymers, seem to be at the center of attention, as evidenced by the large and growing number of publications that have cited them for the past fifteen years, in the field of polymer chemistry research. Size, charge and surface of these nano vectors are important parameters to obtain a targeted delivery, with a minimal quantity of vectors [124].

Huynh Van Tin et al; 2024; “Spike Protein of SARS-CoV-2 Activates Cardiac Fibrogenesis through NLRP3 Inflammasomes and NF-κB Signaling” (https://www.mdpi.com/2073-4409/13/16/1331 )

S1 protein enhanced CFs migration and the expressions of collagen 1, α-smooth muscle actin, transforming growth factor β1 (TGF-β1), phosphorylated SMAD2/3, interleukin 1β (IL-1β), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). S1 increased ROS production but did not affect mitochondrial calcium content and cell morphology. Treatment with an anti-ACE2 neutralizing antibody attenuated the effects of S1 on collagen 1 and TGF-β1 expressions. Moreover, NLRP3 (MCC950) and NF-kB inhibitors, but not the TLR4 inhibitor TAK-242, prevented the S1-enhanced CFs migration and overexpression of collagen 1, TGF-β1, and IL-1β.

Conclusion: S1 activates human CFs by priming NLRP3 inflammasomes through NF-κB signaling in an ACE2-dependent manner.

Boros et al; 2024; “Long-lasting, biochemically modified mRNA, and its frameshifted recombinant spike proteins in human tissues and circulation after COVID-19 vaccination” (https://bpspubs.onlinelibrary.wiley.com/doi/10.1002/prp2.1218)

According to the CDC, both Pfizer and Moderna COVID-19 vaccines contain nucleoside-modified messenger RNA (mRNA) encoding the viral spike glycoprotein of severe acute respiratory syndrome caused by corona virus (SARS-CoV-2), administered via intramuscular injections. Despite their worldwide use, very little is known about how nucleoside modifications in mRNA sequences affect their breakdown, transcription and protein synthesis. It was hoped that resident and circulating immune cells attracted to the injection site make copies of the spike protein while the injected mRNA degrades within a few days. It was also originally estimated that recombinant spike proteins generated by mRNA vaccines would persist in the body for a few weeks. In reality, clinical studies now report that modified SARS-CoV-2 mRNA routinely persist up to a month from injection and can be detected in cardiac and skeletal muscle at sites of inflammation and fibrosis, while the recombinant spike protein may persist a little over half a year in blood. Vaccination with 1-methylΨ (pseudouridine enriched) mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in major histocompatibility complex-diverse people. The translation of 1-methylΨ mRNA using liquid chromatography tandem mass spectrometry identified nine peptides derived from the mRNA +1 frame. These products impact on off-target host T cell immunity that include increased production of new B cell antigens with far reaching clinical consequences. As an example, a highly significant increase in heart muscle 18-flourodeoxyglucose uptake was detected in vaccinated patients up to half a year (180 days). This review article focuses on medical biochemistry, proteomics and deutenomics principles that explain the persisting spike phenomenon in circulation with organ-related functional damage even in asymptomatic individuals. Proline and hydroxyproline residues emerge as prominent deuterium (heavy hydrogen) binding sites in structural proteins with robust isotopic stability that resists not only enzymatic breakdown, but virtually all (non)-enzymatic cleavage mechanisms known in chemistry.

Quay S.,; 2024; “The thrombo-inflammation and neuropathology sequence motifs of the SARS-CoV-2 spike protein appear to have been engineered into the virus” (https://zenodo.org/records/14559752)

A landmark paper[1] entitled, “Fibrin drives thrombo-inflammation and neuropathology in COVID-19,” was published in August 2024 that concluded the mechanism of the thrombotic and neurological symptoms following a SARS-CoV-2 infection, often called “long COVID,” is attributable to the binding of fibrin to discrete portions of the spike protein, specifically three N-terminal domains. This paper is a high impact publication with >110,000 views, placing it in the 99^th percentile of articles published contemporaneously.

Here I examine the regions of the spike protein that bind to fibrin, fibrinogen, or both. The N-terminus of the spike protein contains the three strongest binding peptides and surprisingly, these regions are also the three insertions in the protein sequence that are unique to SARS-CoV-2 and not found in natural sarbecoviruses. All pre-pandemic sarbecoviruses have either a partial deletion in these regions or have protein amino acid substitutions that are non-conserved and therefore would not support fibrin binding.

In addition, the three inserts also correspond to regions of the spike protein that have been shown previously to have high sequence homology with the HIV gp120 protein. GP120 is an HIV surface protein that binds to a host cell surface receptor on CD4+ T-cells and facilitates cell entry to begin infections.  In comparing the immunological and clinical presentation of HIV and COVID-19 patients, the commonality of D-dimer production, CD4+ lymphopenia, neurotropism, and IL-10 expression strongly suggests that these protein sequence homologies are clinically relevant.

A conclusion that the pathophysiology of long COVID is based on the insertion of spike protein motifs with sequence homology that mimic the HIV gp120 protein motif properties, and that these SARS-CoV-2 motifs are not found in the sarbecovirus subgenus strongly suggests that these inserts were design features in the synthetic assembly of SARS-CoV-2.

2b. Off-target protein manufacture

Patel et al; 2023; “Characterization of BNT162b2 mRNA to Evaluate Risk of Off-Target Antigen Translation” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9836996/)

mRNA fragment species from BNT162b2 mRNA were isolated and characterized. The translational viability of intact and fragmented mRNA species was further explored using orthogonal expression systems to understand the risk of truncated spike protein or off-target antigen translation. The study demonstrates that mRNA fragments are primarily derived from premature transcriptional termination during manufacturing, and only full-length mRNA transcripts are viable for expression of the SARS-CoV-2 spike protein antigen

Mulroney et al; 2023; “N¹-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting” (https://www.nature.com/articles/s41586-023-06800-3)

We then compared IFNγ ELISpot responses to predicted +1 frameshifted SARS-CoV-2 spike protein products in 21 individuals vaccinated with BNT162b2 and compared these responses to those of 20 individuals vaccinated with ChAdOx1 nCoV-19, none of whom reported undue effects as a result of vaccination. We detected a significantly higher IFNγ response to +1 frameshifted antigen in the BNT162b2 vaccine group, compared to ChAdOx1 nCoV-19 (Fig. 2d). There was no association between T cell responses to +1 frameshifted antigen and age, sex or HLA subtype (Supplementary Table 1 and Extended Data Figs. 2 and 3). Both ChAdOx1 nCoV-19 and BNT162b2 vaccination produced ELISpot responses to in-frame SARS-CoV-2 spike, but responses to +1 frameshifted products were observed only in individuals vaccinated with BNT162b2 (Fig. 2e,f). During SARS-CoV-2 viral replication, a programmed −1 ribosomal frameshift occurs naturally during translation of open reading frame (ORF) 1a and ORF1b (ref. ²³). It is not feasible that these data are a consequence of natural SARS-CoV-2 infection for the following, non-exhaustive, reasons. First, no frameshifting activity is known to occur during SARS-CoV-2 spike subgenomic mRNA translation (which would be a major discovery in its own right). Second, −1 frameshifting (and not +1 frameshifting) is restricted to a single programmed site in ORF1a and ORF1b (ref. ²³). Third, +1 frameshifted peptides are predicted from the BNT162b2 mRNA sequence, and not the S gene sequence from wild virus (Extended Data Fig. 4). Instead, these data suggest that vaccination with 1-methylΨ mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in both major histocompatibility complex (MHC)-diverse people and MHC-uniform mice.

To provide further mechanistic insight into +1 ribosome frameshifting during translation of 1-methylΨ mRNA, and identify potential frameshift sites or sequences, we translated 1-methylΨ Fluc+1FS mRNA, purified the major putative +1 frameshifted polypeptide and carried out liquid chromatography tandem mass spectrometry (LC–MS/MS) of tryptic digests. From this single polypeptide, we identified six in-frame peptides and nine peptides derived from the mRNA +1 frame (Fig. 3a and Extended Data Table 1). All in-frame peptides were mapped to the N-terminal region, whereas +1 frameshifted peptides were mapped downstream (Fig. 3a). We then repeated this analysis using a different protease and identified a junction peptide spanning the main frame and the +1 frame (Fig. 3b). These data demonstrated that the elongated polypeptide was indeed a chimeric polypeptide consisting of in-frame N-terminal residues and +1 frameshifted C-terminal residues. As expected, shorter frameshifted products were also produced from translation of 1-methylΨ mRNA encoding full-length Fluc (Extended Data Fig. 5).

Wiseman et al; 2023; “Ribosomal frameshifting and misreading of mRNA in COVID-19 vaccines produces “off-target” proteins and immune responses eliciting safety concerns: Comment on UK study by Mulroney et al” (https://www.researchgate.net/publication/376265782_Ribosomal_frameshifting_and_misreading_of_mRNA_in_COVID-19_vaccines_produces_off-target_proteins_and_immune_responses_eliciting_safety_concerns_Comment_on_UK_study_by_Mulroney_et_al)

We comment on the study by Mulroney et al.(1) entitled: “N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting.” The study found evidence in mice and humans for the misreading of the modRNA contained within the Pfizer COVID-19 vaccine to inadvertently produce “off-target” proteins capable of eliciting “off-target” immune responses. The authors propose that these novel proteins are the result of ribosomal frameshifting occasioned by the substitution of N1-methyl pseudouridine. The authors state that the “error prone” code is a safety concern with a “huge potential to be harmful” and that “it is essential that these therapeutics are designed to be free from unintended side-effects.” The findings reveal a developmental and regulatory failure to ask fundamental questions that could affect the safety and effectiveness of these products. According to WHO guidelines for mRNA vaccines, (2) manufacturers should provide details of “unexpected ORFs”(Open Reading Frames). The formation of these off-target proteins is not disclosed in the package insert for COMIRNATY. The finding that unintended proteins may be produced as a result of vaccination is sufficient cause for regulators to conduct full risk assessments of past or future harms that may have ensued. Given that this study was conducted under the auspices of the United Kingdom Government, we must assume UK regulators, manufacturers, and international regulatory agencies, including FDA, were apprised of the data many months ago. We await their account of what steps they have taken to investigate why the formation of off-target proteins was not discovered sooner, what toxic effects they may have caused and what steps they are taken to prevent harm in the future and to inform the public of these findings.

Pardi et al; 2018; “Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses” (https://pubmed.ncbi.nlm.nih.gov/29739835/)

T follicular helper (Tfh) cells are required to develop germinal center (GC) responses and drive immunoglobulin class switch, affinity maturation, and long-term B cell memory. In this study, we characterize a recently developed vaccine platform, nucleoside-modified, purified mRNA encapsulated in lipid nanoparticles (mRNA-LNPs), that induces high levels of Tfh and GC B cells. Intradermal vaccination with nucleoside-modified mRNA-LNPs encoding various viral surface antigens elicited polyfunctional, antigen-specific, CD4+ T cell responses and potent neutralizing antibody responses in mice and nonhuman primates. Importantly, the strong antigen-specific Tfh cell response and high numbers of GC B cells and plasma cells were associated with long-lived and high-affinity neutralizing antibodies and durable protection. Comparative studies demonstrated that nucleoside-modified mRNA-LNP vaccines outperformed adjuvanted protein and inactivated virus vaccines and pathogen infection. The incorporation of noninflammatory, modified nucleosides in the mRNA is required for the production of large amounts of antigen and for robust immune responses.

Palacios- Castrillo R., ; 2023; “Design Flaws Unveiled: The Risk of Autoimmunity from Defective RNA Reading Frames in Pfizer’s ModRNA COVID-19 Vaccine” (https://www.preprints.org/manuscript/202312.1502/v1)

Recently, Mulroney,et.al., demonstrated that Pfizer’s ModRNA Covid vaccine, but not AstraZeneca Covid vaccine, produce aberrant foreign proteins in vivo due to reading frame shift in ribosomes. This phenomenon could elucidate the notable surge in autoimmune diseases following the administration of ModRNA vaccines compared to Influenza vaccines between 2020 and 2023, as found in the VAERS database. These findings bear significant implications for the future application of mRNA technology, emphasizing the necessity of modifying the design of the mRNA sequence to mitigate these defects. Remarkably, Mulroney and collaborators achieved a reduction in frame shift proteins by introducing additional edits to the mRNA sequences. This adjustment is pivotal for rectifying the flawed design of the ModRNA employed by Pfizer. In light of these findings, many advocate for the immediate recall and further investigation of these products. The notion of a “future mRNA-based therapy” should be postponed indefinitely.

2c. Spike Protein Interactions

Igyarto and Qin; 2024; “The mRNA-LNP vaccines – the good, the bad and the ugly?” (https://pubmed.ncbi.nlm.nih.gov/38390323/)

The mRNA-LNP vaccine has received much attention during the COVID-19 pandemic since it served as the basis of the most widely used SARS-CoV-2 vaccines in Western countries. Based on early clinical trial data, these vaccines were deemed safe and effective for all demographics. However, the latest data raise serious concerns about the safety and effectiveness of these vaccines. Here, we review some of the safety and efficacy concerns identified to date. We also discuss the potential mechanism of observed adverse events related to the use of these vaccines and whether they can be mitigated by alterations of this vaccine mechanism approach.

The nucleoside modifications and removal of dsRNA using HPLC were initially introduced by Karikó et al., to circumvent the activation of innate immune sensors, a characteristic of unmodified mRNAs, and the production of inflammatory cytokines, such as type I interferon, that would limit protein translation from the mRNA (4, 8, 9). The long in vivo half-life was desirable because the mRNA technology was initially meant to replace or deliver a therapeutic protein (10). Nevertheless, the nucleoside modification was touted as a breakthrough discovery that allowed the human use of mRNA-based vaccines (11). The importance of the inflammatory components in the mRNA-LNP platform is highlighted by the fact that highly purified mRNA (no detectable dsRNA) combined with lipid nanoparticles (LNPs) that do not contain the inflammation-inducing ionizable lipid is unable to induce innate and adaptive immune responses in vivo, while the LNP containing the ionizable lipid mixed with protein or mRNA supported similar adaptive immune responses (12, 13). The contaminating dsRNA in the mRNA-LNP vaccines (7), in combination with the highly inflammatory nature of the LNPs (12, 14), might essentially obviate the need for nucleoside modification from the immune sensing perspective. Thus, the critical component that transformed the mRNA into an immune response-inducing vaccine is the inflammatory LNP – initially thought to be an inert carrier/delivery vehicle for mRNA (10) – and not the nucleoside modifications.

Different levels of contaminants between vaccine lots, besides storage, transportation, and clinical handling, might explain a recent finding from Denmark that different lots of the mRNA Pfizer vaccines induced distinct levels of adverse events. Some lots caused almost no side effects, while others were associated with a medium or very high incidence of adverse events (all suspected side effects, -serious and -related deaths) (15). It would be important to determine how the same vaccine lots behaved across different countries and demographics to define whether these findings can be generalized. While contaminants likely contribute to the inflammatory nature of this platform and induction of adverse events, the LNPs’ ionizable lipid component of the mRNA-LNP vaccine is highly inflammatory (12), and as we already discussed above, it is key for the reactogenicity and immunogenicity of this platform (12, 13). Thus, hypothetically, another potential explanation for the distinct lots triggering different levels of adverse events could be that the amounts of mRNA-LNP or the mRNA : LNP ratio differed between lots (16). To assess different possibilities, it would be essential to determine the level of adaptive immune responses triggered by the different vaccine lots and if any properly stored vaccine leftovers are still available to test them for impurities and the levels of therapeutic agents. In summary, these findings highlight the need for a strict assessment of purity criteria and allowable limits for this novel vaccine class.

Concerning assumptions made regarding the mRNA-LNP platform

Different experts and officials have made several assumption-based public statements regarding the mRNA-LNP vaccines. One of the most publicized ones was that the vaccine mRNA cannot be reverse transcribed into DNA; thus, there is no risk of insertion into the human genome (17–20). New DNA insertion into the human genome would be a serious concern if it happens on the level of stem cells of the reproductive system. In support of their statement, a modified version of Francis Crick’s central dogma of molecular biology (21) has often been cited that the information flow in eukaryotic cells is unidirectional, from DNA to RNA to protein. While the information flow in eukaryotic cells, in general, indeed is DNA to RNA to protein, in particular instances, RNA can be reverse transcribed into DNA. This process is mediated by reverse transcriptase, enzymes that are naturally associated with retroviruses. However, eukaryotic cells, including human cells, use reverse transcription-like processes to replicate telomeres and retrotransposons (22–25). With the Pfizer mRNA-LNP vaccine, it has been shown experimentally that the vaccine mRNA can be reverse-transcribed into DNA in an immortalized human hepatocyte cell line. Exposure to the mRNA-LNP vaccine also correlated with an increase in overall LINE-1 retrotransposon expression levels and localization to the nucleus (26). It has been proposed that the sequence features of the vaccines’ mRNA meet all known requirements for retroposition using LINE-1 (25). Whether these have any in vivo relevance remains to be determined (27). Spike protein localization to the nucleus was previously reported (28, 29). In support of studies revealing nuclear localization of the spike protein, a recent preprint study reported that the spike protein of the SARS-CoV-2, unlike other SARS viruses, contains a nuclear localization signal (NLS). The NLS allowed the transport of the spike protein to the nucleus, and it seems that the spike protein also shuttled the spike mRNA to the nucleus (30). A study also showed that SARS-CoV-2 RNA can be reverse-transcribed and integrated into the genome of cultured human cells, a process potentially mediated by LINE-1 elements, and can be expressed in patient-derived tissues. The authors propose that these findings could also possibly explain why some patients test PCR positive for SARS-CoV-2 even after clearance of the virus (31). These results, however, have been criticized as not reproducible (32, 33), infrequent and artefactual (34). While, to our knowledge, similar studies have not been performed with COVID-19 mRNA vaccines that code for full-length pre-fusion fixed form of SARS-CoV-2 spike protein, comparable transport of spike protein/mRNA to the nucleus could be expected. Because the mRNA can enter the nucleus, where it might be reverse-transcribed into DNA, this increases its potential to integrate into the genome. Furthermore, the mRNA-LNP diffuses throughout the body and can accumulate in both the testes and ovaries (5, 6) and is reported to alter the menstrual cycle in women (35, 36). Therefore, it could potentially be reaching the stem cells of the reproductive organs. These findings highlight the need to take these data and concerns seriously and conduct specific experiments to address them (25).

Another often touted feature of the vaccine mRNA is that it is degraded in vivo in hours or a few days, thus further limiting its potential to disrupt normal cell biology (17–20). This assumption likely arose since unmodified mRNAs have, in general, short in vivo half-life (37). However, human lymph node biopsies taken at different time points post-exposure to the mRNA-LNP revealed detectable levels of vaccine mRNA and spike proteins up to eight weeks (38). Circulating vaccine mRNA and spike protein have been detected in the plasma from a few weeks to several months post-vaccination (39–42). A recent post-mortem study also found vaccine mRNA in the lymph nodes of most subjects 30 days post-exposure and less frequently in their heart tissue but not in the liver and spleen (43). Thus, in light of these, we should admit to our limited understanding when it comes to how different modifications to the mRNA (5’ and 3’ modifications, the use of unique nucleotides, etc.) affect its in vivo half-life in the human body because no specific studies have been conducted to address this.

Safety

Careful analysis and re-analysis of the Pfizer and Moderna clinical trial data led by Dr. Doshi, an expert in clinical trials, revealed excess risk of severe adverse events (SAEs) of special interest with both the Pfizer and Moderna COVID-19 vaccines. Combined, there was a 16% higher risk of SAEs in mRNA vaccine recipients (47, 48). This concurs with a recent admission from the German Health Minister to ~1 in 10,000 severe/permanent damage events (49). The Countermeasures Injury Compensation Program(CICP) data show that out of a total of 13,406 CICP claims ever filed, 12,854 were COVID-19 countermeasure claims, out of which 9,682 allege injuries/deaths from COVID-19 vaccines (50). Peer-reviewed case reports, including but not limited to severe inflammatory/autoimmune events of bone marrow (51–55), liver (56–58), skin (59–64), cardiovascular- (65–77), musculoskeletal- (76, 78, 79), endocrine- (80–84), and nervous system (66, 84–87), etc. that sometimes had been fatal, have been steadily increasing. However, the incidence of SAEs is hard to judge based on the number of these reports alone (88). To supplement the case report data, we performed a limited analysis of the publicly available data from the Vaccine Adverse Event Reporting System (VAERS), co-managed by the Centers for Disease Control and Prevention (CDC) and FDA (89). According to VAERS, all death reports and selected serious events of interest are investigated (89). The analyses mirror the diverse adverse effects of these therapeutics reported in the literature and might provide some information on incidence ( Figure 1 ). Compared to all other non-COVID-19 vaccines combined, the incidence of adverse events is far higher for the mRNA-LNP-based COVID-19 vaccines per million doses administered ( Figure 1A ). Deaths and SAEs also often occurred soon after injection ( Figure 1B ), making it more likely to be the consequence of the vaccine and not just a random event. Nevertheless, it is essential to emphasize that one cannot establish causation simply by looking at VAERS reports. The causative relationship between the mRNA-LNP vaccine and SAEs has only been officially recognized by the regulatory agencies for peri- and myocarditis affecting primarily young males (90). While most symptomatic patients might be young males, recent, in vivo physiological tests, such as heart glucose uptake, showed a 40% increase in asymptomatic vaccinated patients irrespective of gender and demographics (91), potentially suggesting a much broader impact. These findings are also supported by a postmortem study, in some of which an autopsy revealed heart inflammation and the presence of vaccine RNA (43). Whether the increase in heart attacks and death in young people (92, 93) might be linked to these vaccines remains to be determined.

Efficacy

The effectiveness of these therapeutics in preventing infections and limiting the spreading of the virus has been highly eroded from the early reports (94), and nowadays, their efficacy is mainly limited to potentially decreasing the disease severity and death in susceptible people (95). Excess inflammation caused by an overreacting immune system (cytokine storm) is one of the major pathological features in patients with severe COVID-19 (96). Thus, hypothetically, if exposure to the mRNA-LNP vaccine leads to a dampened systemic inflammatory response, that may explain why vaccination also reduced disease severity in the case of the delta and omicron variants, in which case the antibodies induced by the original vaccines were not (97), or minimally neutralizing (98). In support of this hypothesis, Dr. Netea’s group reported dampened transcriptional reactivity of the immune cells and decreased type I interferon responses in vaccinated individuals to secondary viral stimulation (97), while our group described inhibition of adaptive immune responses and alteration in innate immune fitness in mice with this platform (99). The immune-tolerant environment induced by these vaccines is further supported by recent studies that have discovered a correlation between an increased number of prior mRNA vaccine doses and a higher risk of catching COVID-19 (100–102). Thus, these data suggest that these vaccines’ efficacy in decreasing disease severity and death might lie with their previously undiscovered immune suppressive characteristics. These findings further highlight the need for rigorous pre-clinical studies to limit potential unexpected consequences for novel platforms.

2d. N1-methyl pseudouridine

Mauger et al; 2019; “mRNA structure regulates protein expression through changes in functional half-life” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883848/)

While counterintuitive, the increase in protein output is consistent with previous work in cell-free lysates, showing that mRNAs containing m¹Ψ slow ribosome elongation, increase total protein output, and improve initiation through decreased phosphorylation of the initiation factor, eIF2α (35). Our data help explain the first 2 observations, in that m¹Ψ stabilizes mRNA secondary structure leading to greater protein output. If eIF2α dephosphorylation were the primary driver of the change in translation, we would have expected to see a strong correlation between translation efficiency and total protein output. However, we found very little correlation between translation efficiency, which should closely correlate to initiation rate and total protein output (Fig. 5E).

Kariko et al; 2005; “Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA” (https://www.cell.com/immunity/fulltext/S1074-7613(05)00211-6)

We show that RNA signals through human TLR3, TLR7, and TLR8, but incorporation of modified nucleosides m5C, m6A, m5U, s2U, or pseudouridine ablates activity. Dendritic cells (DCs) exposed to such modified RNA express significantly less cytokines and activation markers than those treated with unmodified RNA. DCs and TLR-expressing cells are potently activated by bacterial and mitochondrial RNA, but not by mammalian total RNA, which is abundant in modified nucleosides. We conclude that nucleoside modifications suppress the potential of RNA to activate DCs. The innate immune system may therefore detect RNA lacking nucleoside modification as a means of selectively responding to bacteria or necrotic tissue.

Andries et al; 2015; “N¹-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice” (https://www.sciencedirect.com/science/article/abs/pii/S0168365915300948?via%3Dihub)

Messenger RNA as a therapeutic modality is becoming increasingly popular in the field of gene therapy. The realization that nucleobase modifications can greatly enhance the properties of mRNA by reducing the immunogenicity and increasing the stability of the RNA molecule (the Kariko paradigm) has been pivotal for this revolution. Here we find that mRNAs containing the N¹-methylpseudouridine (m1Ψ) modification alone and/or in combination with 5-methylcytidine (m5C) outperformed the current state-of-the-art pseudouridine (Ψ) and/or m5C/Ψ-modified mRNA platform by providing up to ~ 44-fold (when comparing double modified mRNAs) or ~ 13-fold (when comparing single modified mRNAs) higher reporter gene expression upon transfection into cell lines or mice, respectively. We show that (m5C/)m1Ψ-modified mRNA resulted in reduced intracellular innate immunogenicity and improved cellular viability compared to (m5C/)Ψ-modified mRNA upon in vitro transfection. The enhanced capability of (m5C/)m1Ψ-modified mRNA to express proteins may at least partially be due to the increased ability of the mRNA to evade activation of endosomal Toll-like receptor 3 (TLR3) and downstream innate immune signaling. We believe that the (m5C/)m1Ψ-mRNA platform presented here may serve as a new standard in the field of modified mRNA-based therapeutics.

Rubio-Casillas et al; 2024; “Review: N1-methyl-pseudouridine (m1Ψ): Friend or foe of cancer?” (https://pubmed.ncbi.nlm.nih.gov/38583833/) Full paper:

The potential of these vaccines in preventing admission to hospitals and serious illness in people with comorbidities has recently been called into question due to the vaccines’ rapidly waning immunity. Mounting evidence indicates that these vaccines, like many others, do not generate sterilizing immunity, leaving people vulnerable to recurrent infections. Additionally, it has been discovered that the mRNA vaccines inhibit essential immunological pathways, thus impairing early interferon signaling. Within the framework of COVID-19 vaccination, this inhibition ensures an appropriate spike protein synthesis and a reduced immune activation. Evidence is provided that adding 100 % of N1-methyl-pseudouridine (m1Ψ) to the mRNA vaccine in a melanoma model stimulated cancer growth and metastasis, while non-modified mRNA vaccines induced opposite results, thus suggesting that COVID-19 mRNA vaccines could aid cancer development. Based on this compelling evidence, we suggest that future clinical trials for cancers or infectious diseases should not use mRNA vaccines with a 100 % m1Ψ modification, but rather ones with the lower percentage of m1Ψ modification to avoid immune suppression.

3. Biodistribution

Schadlich et al; 2012; “Accumulation of nanocarriers in the ovary: A neglected toxicity risk?” (https://www.sciencedirect.com/science/article/abs/pii/S0168365912000892?via%3Dihub)

Several nanocarrier systems are frequently used in modern pharmaceutical therapies. Within this study a potential toxicity risk of all nanoscaled drug delivery systems was found. An accumulation of several structurally different nanocarriers but not of soluble polymers was detected in rodent ovaries after intravenous (i.v.) administration. Studies in different mouse species and Wistar rats were conducted and a high local accumulation of nanoparticles, nanocapsules and nanoemulsions in specific locations of the ovaries was found in all animals. We characterised the enrichment by in vivo and ex vivo multispectral fluorescence imaging and confocal laser scanning microscopy. The findings of this study emphasise the role of early and comprehensive in vivo studies in pharmaceutical research. Nanocarrier accumulation in the ovaries may also comprise an important toxicity issue in humans but the results might as well open a new field of targeted ovarian therapies.

Wang et al; 2018; “Potential adverse effects of nanoparticles on the reproductive system” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294055/)

In medicine, nanoparticles (NPs) can be used as nanoscopic drug carriers and for nanoimaging technologies. Thus, substantial attention has been paid to the potential risks of NPs. Previous studies have shown that numerous types of NPs are able to pass certain biological barriers and exert toxic effects on crucial organs, such as the brain, liver, and kidney. Only recently, attention has been directed toward the reproductive toxicity of nanomaterials. NPs can pass through the blood–testis barrier, placental barrier, and epithelial barrier, which protect reproductive tissues, and then accumulate in reproductive organs. NP accumulation damages organs (testis, epididymis, ovary, and uterus) by destroying Sertoli cells, Leydig cells, and germ cells, causing reproductive organ dysfunction that adversely affects sperm quality, quantity, morphology, and motility or reduces the number of mature oocytes and disrupts primary and secondary follicular development. In addition, NPs can disrupt the levels of secreted hormones, causing changes in sexual behavior. However, the current review primarily examines toxicological phenomena. The molecular mechanisms involved in NP toxicity to the reproductive system are not fully understood, but possible mechanisms include oxidative stress, apoptosis, inflammation, and genotoxicity. Previous studies have shown that NPs can increase inflammation, oxidative stress, and apoptosis and induce ROS, causing damage at the molecular and genetic levels which results in cytotoxicity.

Fertig et al; 2024; “Beyond the injection site: identifying the cellular targets of mRNA vaccines” (https://www.cellidentity.org/wp-content/uploads/2024/06/JoCI_003-Reviews_003_Fertig_MRNa.pdf)

Vaccines against COVID-19 based on the mRNA technology have broken many records, from the speed of development and production, to the number of doses administered and have overall proven safe, with only very rare reported adverse events. The accelerated rollout and the permissive regulatory framework had the major caveat that manufacturers did not provide biodistribution and pharmacokinetics data for their products in humans, despite this being essential for interpreting both the dynamics of the immune response and any potential toxic effects. Thankfully, in the past two years, the scientific community has attempted to fill the gaps, which will undoubtedly help in fine-tuning the next generation of mRNA vaccines.

Here we review existing data on the biodistribution and pharmacokinetics of the commercially available mRNA vaccine platforms, focusing on human studies, where available. We structure this review by tissue type and we discuss potential correlations between vaccine mRNA uptake and pathogenic effects, if applicable. We find that many studies have focused on the heart, due to the medical and social impact of myocarditis, especially in adolescents. We conclude by observing critical data is still missing for many organs and we suggest potential avenues for future research.

Luo et al; 2025; “Nanocarrier imaging at single-cell resolution across entire mouse bodies with deep learning” (https://www.nature.com/articles/s41587-024-02528-1)

Here we present Single Cell Precision Nanocarrier Identification (SCP-Nano), an integrated experimental and deep learning pipeline to comprehensively quantify the targeting of nanocarriers throughout the whole mouse body at single-cell resolution. SCP-Nano reveals the tissue distribution patterns of lipid nanoparticles (LNPs) after different injection routes at doses as low as 0.0005 mg kg⁻¹—far below the detection limits of conventional whole body imaging techniques. We demonstrate that intramuscularly injected LNPs carrying SARS-CoV-2 spike mRNA reach heart tissue, leading to proteome changes, suggesting immune activation and blood vessel damage. SCP-Nano generalizes to various types of nanocarriers, including liposomes, polyplexes, DNA origami and adeno-associated viruses (AAVs), revealing that an AAV2 variant transduces adipocytes throughout the body.

Pateev et al; 2023; “Biodistribution of RNA Vaccines and of Their Products: Evidence from Human and Animal Studies” (https://pubmed.ncbi.nlm.nih.gov/38255166/)

Despite its effectiveness in the fight against COVID-19, rare adverse effects of the vaccination have been shown in some studies, including vascular microcirculation disorders and autoimmune and allergic reactions. The biodistribution of mRNA vaccines remains one of the most poorly investigated topics. This mini-review discussed the results of recent experimental studies on humans and rodents regarding the biodistribution of mRNA vaccines, their constituents (mRNA and lipid nanoparticles), and their encoded antigens. We focused on the dynamics of the biodistribution of mRNA vaccine products and on the possibility of crossing the blood-brain and blood-placental barriers as well as transmission to infants through breast milk.

Based on the Pfizer report and other studies of biodistribution of reporter proteins, one can conclude that after intramuscular injection of mRNA-LNP, the luminescent signal from luciferase is detected mostly in the muscle (site of injection) and the liver. For example, 3 h after intramuscular injection of 5 μg of mRNA-loaded LNPs to BALB/c mice, robust luciferase expression was found at the injection site and a weak signal was detected in the liver [19]. Similar results were obtained on BALB/c mice, which were injected intramuscularly with 5-10 µg luciferase mRNA-LNP. 6 h after immunization robust luminescence signal was detected by the IVIS method at the site of injection and in the liver [20,21]. Further analysis of ex vivo luminescence in the organs of immunized mice showed the presence of the reporter protein signal in the spleen as well. In the Ripoll et al. study [22] in BALB/c mice, it was also shown that the maximum bioluminescence signal during intramuscular administration of FLuc-LNP occurs at 6 h at the injection site and disappears within 4 days regardless of the composition of the LNP.

There are two reasons for such distribution at the injection site and in the liver. Firstly, the bioluminescence intensity in vivo is dependent on the photon flux per cell, the number of cells, and the migration of photons through tissue [23]. In other words, the larger the organ and the closer it is to the surface, the more pronounced the intensity of bioluminescence will be. Secondly, four-component LNPs composed of ionizable lipids, SM-102 (Moderna) or ALC-0315 (Pfizer/BioNTech/Acuitas), are typically adsorbed to apolipoprotein E (ApoE) in the bloodstream and are taken up by hepatocytes that express high levels of low-density lipoprotein receptors (LDLRs) [24].

Additionally, the kinetics and pattern of biodistribution of the signal of reporter protein significantly depend on the immunization route. It was shown that the duration and intensity of luminescence after the administration of mRNA-LNP in mice decreased in the following order: intradermal > intramuscular > intraperitoneal and subcutaneous > intravenous > intratracheal [25]. Furthermore, high accumulation of the reporter protein occurs in the liver with almost all immunization routes, but in the case of subcutaneous and intradermal routes luciferase expression was observed only at the injection site. Qiu et al. [26] demonstrated that biodistribution depends on the route of administration of a mRNA-based drug, using the example of the administration of luciferase RNA formulated in LNPs to C57Bl/6J mice. Intravenous injection led to the detection of fluorescent proteins in the liver, spleen, lungs, and lymph nodes; the maximum bioluminescence signal occurred 6 h after injection and then slowly decreased. With subcutaneous administration, luciferase expression was observed only in the lymph nodes, the maximum bioluminescence was observed after 24 h and decreased rapidly.

Di et al; 2022; “Biodistribution and Non-linear Gene Expression of mRNA LNPs Affected by Delivery Route and Particle Size” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8791091/)

Results

Some intramuscularly injected LNPs were found circulating in the system, resulting in accumulation in the liver and spleen, especially when the LNP sizes were relatively small. Bigger LNPs were more likely to remain at the injection site. Transgene expression in the liver was found most prominent compared with other organs and tissues.

Conclusions

Biomolecules such as mRNAs encapsulated in locally injected LNPs can reach other organs and tissues via systemic circulation. Gene expression levels are affected by the LNP biodistribution and pharmacokinetics (PK), which are further influenced by the particle size and injection route. As transfection efficiency varies in different organs, the LNP exposure and mRNA expression are not linearly correlated.

Rong et al; 2023; “SARS-CoV-2 Spike Protein Accumulation in the Skull-Meninges-Brain Axis: Potential Implications for Long-Term Neurological Complications in post-COVID-19” (https://www.biorxiv.org/content/10.1101/2023.04.04.535604v1)

In this study, we utilized mouse models and human post-mortem tissues to investigate the presence and distribution of the SARS-CoV-2 spike protein in the skull-meninges-brain axis. Our results revealed the accumulation of the spike protein in the skull marrow, brain meninges, and brain parenchyma. The injection of the spike protein alone caused cell death in the brain, highlighting a direct effect on brain tissue. Furthermore, we observed the presence of spike protein in the skull of deceased long after their COVID-19 infection, suggesting that the spike’s persistence may contribute to long-term neurological symptoms. The spike protein was associated with neutrophil-related pathways and dysregulation of the proteins involved in the PI3K-AKT as well as complement and coagulation pathway. Overall, our findings suggest that SARS-CoV-2 spike protein trafficking from CNS borders into the brain parenchyma and identified differentially regulated pathways may present insights into mechanisms underlying immediate and long-term consequences of SARS-CoV-2 and present diagnostic and therapeutic opportunities.

Rhea et al; 2021; “The S1 protein of SARS-CoV-2 crosses the blood–brain barrier in mice” (https://www.nature.com/articles/s41593-020-00771-8)

We show that intravenously injected radioiodinated S1 (I-S1) readily crossed the blood–brain barrier in male mice, was taken up by brain regions and entered the parenchymal brain space. I-S1 was also taken up by the lung, spleen, kidney and liver. Intranasally administered I-S1 also entered the brain, although at levels roughly ten times lower than after intravenous administration. APOE genotype and sex did not affect whole-brain I-S1 uptake but had variable effects on uptake by the olfactory bulb, liver, spleen and kidney. I-S1 uptake in the hippocampus and olfactory bulb was reduced by lipopolysaccharide-induced inflammation. Mechanistic studies indicated that I-S1 crosses the blood–brain barrier by adsorptive transcytosis and that murine angiotensin-converting enzyme 2 is involved in brain and lung uptake, but not in kidney, liver or spleen uptake.

Lin et al; 2024; “Transplacental Transmission of the COVID-19 Vaccine mRNA: Evidence from
Placental, Maternal and Cord Blood Analyses Post-Vaccination” (https://www.ajog.org/article/S0002-9378(24)00063-2/pdf)

Our findings suggest that the vaccine mRNA is not localized to the injection site and can spread systemically to the placenta and umbilical cord blood. The detection of the spike protein in the placental tissue indicates the bioactivity of the vaccine mRNA reaching the placenta. Notably, the vaccine mRNA was largely fragmented in the cord blood and, to a lesser extent, in the placenta.

To our knowledge, these two cases demonstrate, for the first time, the ability of the COVID-19 vaccine mRNA to penetrate the fetal-placental barrier and reach the intrauterine environment.

Ogata et al; 2022;“Circulating Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccine Antigen Detected in the Plasma of mRNA-1273 Vaccine Recipients” (https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciab465/6279075)

S1 antigen was detected as early as day 1 postvaccination, and peak levels were detected on average 5 days after the first injection (Figure 1A). The mean S1 peak level was 68 pg/mL ± 21 pg/mL. S1 in all participants declined and became undetectable by day 14. No antigen was detected at day zero for 12 of 13 participants, as expected. However, one individual presented detectable S1 on day zero, possibly due to assay cross-reactivity with other human coronaviruses or asymptomatic infection at the time of vaccination. Spike protein was detectable in 3 of 13 participants an average of 15 days after the first injection. The mean spike peak level was 62 pg/mL ± 13 pg/mL. After the second vaccine dose, no S1 or spike was detectable, and both antigens remained undetectable through day 56. For one individual (participant 8), spike was detected at day 29, 1 day after the second injection and was undetectable 2 days later.

Kent et al; 2024; “Blood Distribution of SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine in Humans” (https://pubs.acs.org/doi/10.1021/acsnano.4c11652)

Lipid nanoparticle mRNA vaccines are an exciting but new technology used in humans. There is limited understanding of factors that influence their biodistribution and immunogenicity. Antibodies to polyethylene glycol (PEG), which is on the surface of the lipid nanoparticle, are detectable in humans and boosted by human mRNA vaccination. We hypothesized that PEG-specific antibodies could increase the clearance of mRNA vaccines. We developed methods to quantify both the mRNA and ionizable lipid in frequent serial blood samples from 19 subjects receiving Moderna SPIKEVAX mRNA booster immunization. Both the mRNA and ionizable lipid peaked in blood 1-2 days post vaccination (median peak level 0.19 and 3.22 ng mL^–1, respectively). The mRNA was detectable out to 14-28 days post-vaccination in most subjects. We measured the proportion of mRNA that was relatively intact in blood over time and found the decay kinetics of the intact mRNA and ionizable lipid were identical, suggesting the intact lipid nanoparticle recirculates in blood. However, mRNA and ionizable lipid decay rates did not correlate with baseline levels of PEG-specific nor spike-specific antibodies. The magnitude of mRNA and ionizable lipid detected in blood did correlate with the boost in PEG antibodies. Further, the ability of subject’s monocytes to phagocytose lipid nanoparticles had an inverse relationship with the rise in PEG antibodies. This suggests circulation of mRNA lipid nanoparticle vaccines into the blood and their ability to be cleared by phagocytes influence PEG immunogenicity of mRNA vaccines. Overall, this work defines the pharmacokinetics of lipid nanoparticle mRNA vaccine components in human blood after intramuscular injection and the factors that influence this.

Fertig et al 2022; “Vaccine mRNA Can Be Detected in Blood at 15 Days Post-Vaccination” (https://www.mdpi.com/2227-9059/10/7/1538)

3.2. Vaccine mRNA Remains in Circulation for at Least 15 Days

We used qPCR to investigate the presence and persistence of synthetic mRNA in blood after one, two and three doses of BNT162b2, assuming that the biodistribution of the vaccine is similar after each dose. We also aimed to probe whether vaccine mRNA was associated with circulating WBCs, either as a result of cell–LNP interactions at the injection site and in draining LNs or of random collisions in circulation. Consequently, we separated the plasma and cellular fractions and analysed them independently for all individuals.

Template copy numbers decreased with the day of sampling. In plasma, mRNA was immediately detectable at just hours following vaccination, remained detectable when sampled at 6 and 15 days (Figure 1A, green), but was below the limit of quantification (LoQ) for one sample at 27 days. Samples from negative controls did not amplify. For subject B3, we observed a similar trend for plasma: vaccine-associated mRNA became detectable immediately after vaccination and remained significantly above the LoQ at day 14 (Figure 1B, green). Interestingly, vaccine mRNA was detected in the cellular fraction up to day 6 in some samples from our cohort, whereas for B3, it was only detectable at one day after vaccination (Figure 1A,B, orange). It has to be noted that the likelihood of detecting vaccine mRNA in the cellular fraction decreased significantly at 24 h from injection, which was in contrast to plasma in which mRNA remained consistently detectable up to day 15 (Figure 1C).

Castruita et al; 2023; “SARS-CoV-2 spike mRNA vaccine sequences circulate in blood up to 28 days after COVID-19 vaccination” (https://onlinelibrary.wiley.com/doi/epdf/10.1111/apm.13294?s=09)

De novoassembly of human-depleted RNA-seqreads from two patient samples produced contigs of>1000 nt with closest homology to bat coronavirusand 67% homology to the SARS-CoV-2 referencegenome (NC_045512.2). Translation of the contignucleotide sequences to amino acid sequencesrevealed 100% identity to parts of the spike proteinof SARS-CoV-2. From the literature we found thesequences of the two commercial SARS-CoV-2mRNA vaccines which were used as references tomap reads from all the samples[16,25]. This led tothe identification of an additional eight sampleswith reads that matched the mRNA vaccinesequences. Both mRNA vaccine sequences havebeen modified and are only~70% identical to thespike reference genome on a nucleotide level, mak-ing them distinct from circulating infectious SARS-CoV-2 sequences. Thus, of the 108 patient samples,10 samples (9.3%) had partial or up to fullsequences of the vaccine mRNA sequence (Fig.1),identified from one to 28 days postvaccination.There was~100% identity between the detectedmRNA nucleotide sequences found in plasma andthe specific mRNA vaccine given. The 10 sampleshad a median of 5.5 million raw read pairs avail-able (see TableS1). Breadth and depth of coverageof the vaccine mRNA sequences ranged from com-pleteness and>20 000, respectively, to short frag-ments with a depth of coverage of 100 (Fig.1).None of the negative or the HCV-positive controlshad SARS-CoV-2 matching reads.

Krauson et al; 2023 “Duration of SARS-CoV-2 mRNA vaccine persistence and factors associated with cardiac involvement in recently vaccinated patients” (https://www.nature.com/articles/s41541-023-00742-7)

At the start of the COVID-19 pandemic, the BNT162b2 (BioNTech-Pfizer) and mRNA-1273 (Moderna) mRNA vaccines were expediently designed and mass produced. Both vaccines produce the full-length SARS-CoV-2 spike protein for gain of immunity and have greatly reduced mortality and morbidity from SARS-CoV-2 infection. The distribution and duration of SARS-CoV-2 mRNA vaccine persistence in human tissues is unclear. Here, we developed specific RT-qPCR-based assays to detect each mRNA vaccine and screened lymph nodes, liver, spleen, and myocardium from recently vaccinated deceased patients. Vaccine was detected in the axillary lymph nodes in the majority of patients dying within 30 days of vaccination, but not in patients dying more than 30 days from vaccination. Vaccine was not detected in the mediastinal lymph nodes, spleen, or liver. Vaccine was detected in the myocardium in a subset of patients vaccinated within 30 days of death. Cardiac ventricles in which vaccine was detected had healing myocardial injury at the time of vaccination and had more myocardial macrophages than the cardiac ventricles in which vaccine was not detected. These results suggest that SARS-CoV-2 mRNA vaccines routinely persist up to 30 days from vaccination and can be detected in the heart.

Roltgen et al; 2022; “Immune imprinting, breadth of variant recognition, and germinal center response in human SARS-CoV-2 infection and vaccination” (https://www.cell.com/cell/fulltext/S0092-8674(22)00076-9)

We performed in situ hybridization with control and SARS-CoV-2 vaccine mRNA-specific RNAScope probes in the core needle biopsies of the ipsilateral axillary LNs that were collected 7–60 days after the second dose of mRNA-1273 or BNT162b2 vaccination and detected vaccine mRNA collected in the GCs of LNs on days 7, 16, and 37 postvaccination, with lower but still appreciable specific signal at day 60 (Figures 7A–7E). Only rare foci of vaccine mRNA were seen outside of GCs. Axillary LN core needle biopsies of nonvaccinees (n = 3) and COVID-19 patient specimens were negative for vaccine probe hybridization. Immunohistochemical staining for spike antigen in mRNA-vaccinated patient LNs varied between individuals but showed abundant spike protein in GCs 16 days post-second dose, with spike antigen still present as late as 60 days post-second dose.

Bansai et al; 2021 “Cutting Edge: Circulating Exosomes with COVID Spike Protein Are Induced by BNT162b2 (Pfizer–BioNTech) Vaccination prior to Development of Antibodies: A Novel Mechanism for Immune Activation by mRNA Vaccines” ( https://www.jimmunol.org/content/207/10/2405.long)

Circulating exosomes isolated from vaccinated individuals contained SARS-CoV-2 spike protein Ag S2

We analyzed plasma from vaccinated healthy individuals at days 0, 7, and 14 after the first dose of the vaccine and day 14 of the second dose for the presence of exosomes carrying the SARS-CoV-2 spike protein (Fig. 2B, 2C). The results demonstrated the presence of SARS-CoV-2 spike Ag S2 on exosomes at day 14 of dose 1. There is a significant increase in the concentration of the spike protein at day 14 of dose 2, with a p value of 0.0299. The amount of SARS-CoV-2 spike protein in exosomes after 4 mo of both doses of vaccine was significantly decreased compared with day 14 after the second dose, with the p value of 0.0078. Kinetics of Ab development to the spike protein and exosomes with spike protein for each healthy individual at different time points (day 14 after first and second dose and 4 mo after second dose) are shown in (Fig. 2D. Both the kinetics of Ab development and the amount of SARS-CoV-2 spike protein exosomes are in agreement with each other, as both are increased following the second booster dose at day 14 (Fig. 2E). There is a decrease in Ab levels to the SARS-CoV-2 spike protein and the amount of SARS-CoV-2 spike protein in exosomes in each healthy individual from day14 of second booster dose to 4 mo of the second booster dose (Fig. 2D, 2E). Data for each individual for this subset is given as Supplemental Fig. 1

Brogna et al; 2023; “Detection of recombinant Spike protein in the blood of individuals vaccinated against SARS-CoV-2: Possible molecular mechanisms” (https://onlinelibrary.wiley.com/doi/epdf/10.1002/prca.202300048 PubMed https://pubmed.ncbi.nlm.nih.gov/37650258/)

Results: The specific PP-Spike fragment was found in 50% of the biological samples analyzed, and its presence was independent of the SARS-CoV-2 IgG antibody titer. The minimum and maximum time at which PP-Spike was detected after vaccination was 69 and 187 days, respectively.

Conclusions and clinical relevance: The presented method allows to evaluate the half-life of the Spike protein molecule “PP” and to consider the risks or benefits in continuing to administer additional booster doses of the SARS-CoV-2 mRNA vaccine. This approach is of valuable support to complement antibody level monitoring and represents the first proteomic detection of recombinant Spike in vaccinated subjects.

Patterson et al; 2024; “Persistence of S1 Spike Protein in CD16+ Monocytes up to 245 Days in SARS-CoV-2 Negative Post COVID-19 Vaccination Individuals with Post-Acute Sequalae of COVID-19 (PASC)-Like Symptoms” (https://www.medrxiv.org/content/10.1101/2024.03.24.24304286v1)

We studied 50 individuals who received one of the approved COVID-19 vaccines and who experienced new onset PASC-like symptoms along with 45 individuals post-vaccination without symptoms as controls. We performed multiplex cytokine/chemokine profiling with machine learning as well as SARS-CoV-2 S1 protein detection on CD16+ monocyte subsets using flow cytometry and mass spectrometry. We determined that post-vaccination individuals with PASC- like symptoms had similar symptoms to PASC patients. When analyzing their immune profile, Post-vaccination individuals had statistically significant elevations of sCD40L (p<0.001), CCL5 (p=0.017), IL-6 (p=0.043), and IL-8 (p=0.022). Machine learning characterized these individuals as PASC using previously developed algorithms. Of the S1 positive post-vaccination patients, we demonstrated by liquid chromatography/ mass spectrometry that these CD16+ cells from post-vaccination patients from all 4 vaccine manufacturers contained S1, S1 mutant and S2 peptide sequences. Post-COVID vaccination individuals with PASC-like symptoms exhibit markers of platelet activation and pro-inflammatory cytokine production, which may be driven by the persistence of SARS-CoV-2 S1 proteins in intermediate and non-classical monocytes. The data from this study also cannot make any inferences on epidemiology and prevalence for persistent post-COVID vaccine symptoms.

Paterson et al; 2022; “Persistence of SARS CoV-2 S1 Protein in CD16+ Monocytes in Post-Acute Sequelae of COVID-19 (PASC) Up to 15 Months Post-Infection” ( https://pubmed.ncbi.nlm.nih.gov/35082777/ Published https://europepmc.org/article/PPR/PPR362323)

Since the reports by our group and others found that monocyte subsets can be infected by HIV, HCV, Zika virus and Dengue fever virus (10–12), we screened peripheral blood mononuclear cells (PBMCs) from PASC individuals, as well as acute severe COVID-19 as controls, for SARS-CoV-2 RNA ( Table 1 ). Using the highly sensitive, quantitative digital droplet PCR (ddPCR), we found that 36% (4 of 11) of severe COVID-19 patients’ PBMCs contained SARS-CoV-2 RNA compared to 4% (1/26) of PASC patients’ PBMCs. The one PASC patient that was RNA positive was 15 months post infection.

Bhattacharjee et al; 2025; “Immunological and Antigenic Signatures Associated with Chronic Illnesses after COVID-19 Vaccination” (https://www.medrxiv.org/content/10.1101/2025.02.18.25322379v1)

COVID-19 vaccines have prevented millions of COVID-19 deaths. Yet, a small fraction of the population reports a chronic debilitating condition after COVID-19 vaccination, often referred to as post- vaccination syndrome (PVS). To explore potential pathobiological features associated with PVS, we conducted a decentralized, cross-sectional study involving 42 PVS participants and 22 healthy controls enrolled in the Yale LISTEN study. Compared with controls, PVS participants exhibited differences in immune profiles, including reduced circulating memory and effector CD4 T cells (type 1 and type 2) and an increase in TNFα+ CD8 T cells. PVS participants also had lower anti-spike antibody titers, primarily due to fewer vaccine doses. Serological evidence of recent Epstein-Barr virus (EBV) reactivation was observed more frequently in PVS participants. Further, individuals with PVS exhibited elevated levels of circulating spike protein compared to healthy controls. These findings reveal potential immune differences in individuals with PVS that merit further investigation to better understand this condition and inform future research into diagnostic and therapeutic approaches.”

Increase in circulating SARS-CoV-2 Spike protein in participants with PVS
It has been reported that the BNT162b2 or mRNA-1273 derived S proteins circulate in the plasma of those vaccinated as early as one day after the vaccine and interactions of the circulating protein16. Hence, we next sought to investigate whether the S1 subunit of the SARS-CoV-2 S protein could be detected in the plasma. For this, we used an anti-S1 Successive Proximity Extension Amplification Reaction (SPEAR) immunoassay. This method can detect S1 levels as low as 5.64 fM. We conducted a one-sided
Kolmogorov–Smirnov test with 1000 permutations to see if the participants with PVS had higher circulating S1. The results indicated that participants with PVS had significantly higher circulating S1 levels compared with the control group (p = 0.01). However, circulating S1 was found in only a subset of participants with PVS at varying concentrations while the control group mostly exhibited a bimodal distribution of zero and non-zero values (Fig. 5A, Table S2). Detectable S1 was found in participants’
plasma ranging from 26 to 709 days from the most recent known exposure (Figure 5B).

To fully account for the width of this dataset, we included all non-detectable values in the analysis and applied a generalized regression model accounting for zero-inflation. We found that both PVS-I and PVS+I groups displayed significantly elevated S1 levels than the Control-I group (p= <0.01 and p= 0.02, respectively) (Figure 5C).

Immune signatures in PVS subgroups based on the presence of circulating S1 protein

To gain a clearer understanding of the variability of circulating S1 protein levels, we first compiled a structured timeline that summarizes the self-reported infection dates, vaccine numbers (including types and administration dates), and the number of days between the latest known exposure and the collection of biospecimens. This timeline was organized for both the PVS-I and PVS+I groups (Figure 5G). Notably, we observed that the highest levels of detectable S1 in the PVS-I group were the furthest away from the last known exposure and ranging between greater than 600-700 days (NI-1 & NI-5;391 Figure 5G). This suggested that prolonged antigen persistence might be associated with
PVS in a subgroup of patients. Further, most of the PVS+I group participants experienced breakthrough SARS-CoV-2 infections with the exception of two cases, indicating that PVS symptoms started prior to infection (Figure. 5G)

4. Original Trials

Mead et al; 2024; “COVID-19 mRNA Vaccines: Lessons Learned from the Registrational Trials and Global Vaccination Campaign” (https://www.cureus.com/articles/203052-covid-19-mrna-vaccines-lessons-learned-from-the-registrational-trials-and-global-vaccination-campaign#!/)

Published reports from the original randomized phase 3 trials concluded that the COVID-19 mRNA vaccines could greatly reduce COVID-19 symptoms. In the interim, problems with the methods, execution, and reporting of these pivotal trials have emerged. Re-analysis of the Pfizer trial data identified statistically significant increases in serious adverse events (SAEs) in the vaccine group. Numerous SAEs were identified following the Emergency Use Authorization (EUA), including death, cancer, cardiac events, and various autoimmune, hematological, reproductive, and neurological disorders. Furthermore, these products never underwent adequate safety and toxicological testing in accordance with previously established scientific standards. Among the other major topics addressed in this narrative review are the published analyses of serious harms to humans, quality control issues and process-related impurities, mechanisms underlying adverse events (AEs), the immunologic basis for vaccine inefficacy, and concerning mortality trends based on the registrational trial data. The risk-benefit imbalance substantiated by the evidence to date contraindicates further booster injections and suggests that, at a minimum, the mRNA injections should be removed from the childhood immunization program until proper safety and toxicological studies are conducted. Federal agency approval of the COVID-19 mRNA vaccines on a blanket-coverage population-wide basis had no support from an honest assessment of all relevant registrational data and commensurate consideration of risks versus benefits. Given the extensive, well-documented SAEs and unacceptably high harm-to-reward ratio, we urge governments to endorse a global moratorium on the modified mRNA products until all relevant questions pertaining to causality, residual DNA, and aberrant protein production are answered.

Fraiman et al; 2022; “Serious Adverse Events of Special Interest Following mRNA Vaccination in Randomized Trials” (https://papers.ssrn.com/sol3/papers.cfm?abstract_id=4125239)

Results: Pfizer and Moderna mRNA COVID-19 vaccines were associated with an increased risk of serious adverse events of special interest, with an absolute risk increase of 10.1 and 15.1 per 10,000 vaccinated over placebo baselines of 17.6 and 42.2 (95% CI -0.4 to 20.6 and -3.6 to 33.8), respectively. Combined, the mRNA vaccines were associated with an absolute risk increase of serious adverse events of special interest of 12.5 per 10,000 (95% CI 2.1 to 22.9). The excess risk of serious adverse events of special interest surpassed the risk reduction for COVID-19 hospitalization relative to the placebo group in both Pfizer and Moderna trials (2.3 and 6.4 per 10,000 participants, respectively).

Discussion: The excess risk of serious adverse events found in our study points to the need for formal harm-benefit analyses, particularly those that are stratified according to risk of serious COVID-19 outcomes such as hospitalization or death.

Michels et al; 2023; “Forensic analysis of the 38 subject deaths in the 6-Month Interim Report of the Pfizer/BioNTech BNT162b2 mRNA Vaccine Clinical Trial” (https://ijvtpr.com/index.php/IJVTPR/article/view/86)

The analysis reported here is unique in that it is the first study of the original data from the Pfizer/BioNTech BNT162b2 mRNA vaccine clinical trial (CA4591001) to be carried out by a group unaffiliated with the trial sponsor. Our study is a forensic analysis of the 38 trial subjects who died between July 27, 2020, the start of Phase 2/3 of the clinical trial, and March 13, 2021, the data end date of their 6-Month Interim Report. Phase 2/3 of the trial involved 44,060 subjects who were equally distributed into two groups and received Dose 1 of either the BNT162b2 mRNA vaccinated or the Placebo control (0.9% normal saline). At Week 20, when the BNT162b2 mRNA vaccine received Emergency Use Authorization from the U.S. FDA, subjects in the placebo arm were given the option to be BNT162b2 vaccinated. All but a few accepted. Surprisingly, a comparison of the number of subject deaths per week during the 33 Weeks of this study found no significant difference between the number of deaths in the vaccinated versus placebo arms for the first 20 weeks of the trial, the placebo-controlled portion of the trial. After Week 20, as subjects in the Placebo were unblinded and vaccinated, deaths among this still unvaccinated cohort of this group slowed and eventually plateaued. Deaths in the BNT162b2 vaccinated subjects continued at the same rate. Our analysis revealed inconsistencies between the subject data listed in the 6-Month Interim Report and publications authored by Pfizer/BioNTech trial site administrators. Most importantly, we found evidence of an over 3.7-fold increase in number of deaths due to cardiovascular events in BNT162b2 vaccinated subjects compared to Placebo controls. This significant adverse event signal was not reported by Pfizer/BioNTech. Potential sources of these data inconsistencies are identified.

5. Efficacy

Lind et al; 2023; “Evidence of leaky protection following COVID-19 vaccination and SARS-CoV-2 infection in an incarcerated population” (https://www.nature.com/articles/s41467-023-40750-8)

During both the Delta and Omicron periods, we found that neither prior infection, nor vaccination, nor hybrid immunity provided significant levels of protection against SARS-CoV-2 infection following cell exposure events and that the levels of protection were significantly smaller following cell exposure events than following events without documented exposures. Further, despite having a limited sample during the period of Delta predominance, we observed similar gradients in the level of protection offered by prior infection, vaccination, and hybrid immunity against symptomatic infection. These findings provide empirical evidence that, while accounting for factors thought to be associated with vaccine acceptance and infection, the protection offered by prior infection, vaccination, and hybrid immunity, appears to be leaky. They suggest that there may be an additional mechanism, based on the intensity of the infectious exposure, which may explain observed, partial levels of immunity conferred by infection and vaccination, in addition to factors such as variant-specific immune escape, waning immunity and reduced effectiveness in specific subpopulations, such as older people^28,29,30.

Neil et al; 2025; “The extent and impact of vaccine status miscategorisation on covid-19 vaccine efficacy studies” (https://www.medrxiv.org/content/10.1101/2024.03.09.24304015v3)

It is recognised that many studies reporting high efficacy for Covid-19 vaccines suffer from various miscategorisation biases. Systematic review identified thirty-eight studies that suffered from one particular and serious form of bias called miscategorisation bias, whereby study participants who have been vaccinated are categorised as unvaccinated up to and until some arbitrarily defined time after vaccination occurred. Simulation demonstrates that this miscategorisation bias artificially boosts vaccine efficacy and infection rates even when a vaccine has zero or negative efficacy. Furthermore, simulation demonstrates that repeated boosters, given every few months, are needed to maintain this misleading impression of efficacy. Given this, any claims of Covid-19 vaccine efficacy based on these studies are likely to be a statistical illusion or are exaggerated.

Conclusions

Our review reveals that a serious form of bias, miscategorisation, is pervasive throughout the many research studies that aim to measure Covid-19 vaccine efficacy. The effect of this bias is to artificially inflate vaccine efficacy and present the misleading impression that these vaccines are effective and that the non-vaccinated suffer from higher Covid-19 infection rates compared to the vaccinated.

We presented a simulation model to demonstrate the effects of this bias and show it artificially boosts vaccine efficacy in all cases, and with the application of repeated ‘booster’ vaccinations, the efficacy of repeated Covid-19 vaccines could be maintained at artificial levels in perpetuity should boosting be continued indefinitely. This effect occurs with a both a zero-efficacy (placebo) vaccine and a negative-efficacy vaccine that increases, rather than reduces, infection rates in those vaccinated.

This miscategorisation is guaranteed to lead to initially very high efficacy claims (usually above 90%) during peak vaccine rollout even if the vaccine were a placebo or worse. Efficacy then falls toward zero a few weeks later. This pattern of high initial efficacy, tapering off after 3 months is also consistently observed in real-world studies, and is often used as justification for additional, booster vaccinations to maintain efficacy. The corresponding Covid-19 infection rate is also likewise artificially elevated in the unvaccinated cohort compared to the vaccinated cohort. These issues apply to other measures of vaccination effectiveness related to mortality and morbidity.

Thus, we conclude that any claims of Covid-19 vaccine efficacy based on these studies are likely to be a statistical illusion or are exaggerated.

Fung et al; 2023; “Sources of bias in observational studies of covid-19 vaccine effectiveness” (https://onlinelibrary.wiley.com/doi/10.1111/jep.13839)

Our analysis shows that real-world conditions such as non-randomised vaccination, crossovers, and trends in background infection rates introduce strong, complex biases into these observational datasets. Our contribution is to size up three important biases, the magnitude of which surprised us and may surprise you. We conclude that “real-world” studies using methodologies popular in early 2021 overstate vaccine effectiveness. Our finding highlights how difficult it is to conduct high-quality observational studies during a pandemic.

CASE-COUNTING WINDOW BIAS

In randomised trials, applying the “fully vaccinated” case counting window to both vaccine and placebo arms is easy. But in cohort studies, the case-counting window is only applied to the vaccinated group. Because unvaccinated people do not take placebo shots, counting 14 days after the second shot is simply inoperable. This asymmetry, in which the case-counting window nullifies cases in the vaccinated group but not in the unvaccinated group, biases estimates. As a result, a completely ineffective vaccine can appear substantially effective—48% effective in the example shown in Table 1. (The placebo data in Table 1 comes from the Pfizer Phase III randomised trial, and is the assumed case counts for the unvaccinated group in a counterfactual observational study occurring simultaneously; this setup illustrates the potential size of a case-counting window bias in a real-world setting as well as why this bias does not exist in a randomised trial.).

AGE BIAS

In trials, randomisation helps ensure statistically identical age distributions in vaccinated and unvaccinated groups, so that the average vaccine efficacy estimate is unbiased, even if vaccine efficacy and/or infection rates differ across age groups (see Figure 2A). However, unlike trials, in real life, vaccination status is not randomly assigned (see Figure 2B). While vaccination rates are high in many countries, the vaccinated remain, on average, older and less healthy than the unvaccinated because vaccines were prioritised for those older and at higher risk. Individuals also self-select for vaccination regardless of policy.

Because covid-19 related risks (of infection, disease, and complications) also vary by age, this can confound the estimate of vaccine effectiveness. To illustrate this, consider the REACT-1 study.¹⁸ This study conducts PCR testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on a random sample of England’s population once a month. In June–July 2021 (the most recent data available), SARS-CoV-2 positivity rates varied considerably by age (from 1.7 to 15.6 positives per 1000 individuals), with higher rates among people under 25 years of age (see Figure 2C). REACT-1 also reports vaccination status. As seen in Figure 2B, almost half of the unvaccinated group is aged between 5 and 12, while the most common age group in the vaccinated was 45–54 years old. While details differ, age bias is present in all observational data sets.

To understand the impact of age bias, consider a hypothetical vaccine with zero efficacy. The vaccinated and unvaccinated groups’ case rates should be statistically identical if the vaccine were completely ineffective (Figure 2D). But age bias in observational data alters the age-weighted case rates in both the vaccinated and the unvaccinated groups, resulting in different infection rates by vaccination status. Since older people recorded lower infection rates, the age-weighted case rate of the (older) vaccinated group registered at 5.5 per 1000 while the corresponding value for the (younger) unvaccinated group was 11.2 per 1000 (Figure 2C). The resultant vaccine effectiveness, which is the relative ratio of these case rates, reflects the interaction between differential age distributions and the correlation of covid-19 incidence with age. The vaccine effectiveness appears as 51% even though the vaccine is completely ineffective by assumption. (Note that the direction of the age bias would reverse if older age groups had suffered higher case rates during the study period.).

BACKGROUND INFECTION RATE BIAS

From December 2020, the speedy dissemination of vaccines, particularly in wealthier nations (Figure 1), coincided with a period of plunging infection rates. However, accurately determining the contribution of vaccines to this decline is far from straightforward. Indeed, the considerable variation in case decline by country, such as the time lag observed in Israel—by far the quickest to reach 50% vaccinated relative to the UK and the United States—defies simple explanation (Figure 1, timepoint “B”). The sharp drop in infections complicates estimating vaccine effectiveness from observational data in a manner similar to age bias. The risk of virus exposure was considerably higher in January than in April. Thus exposure time was not balanced between unvaccinated and vaccinated individuals. Exposure time for the unvaccinated group was heavily weighted towards the early months of 2021 while the inverse pattern was observed in the vaccinated group. This imbalance is inescapable in the real world due to the timing of vaccination rollout.

In addition, unlike trials, individuals in “real-world” studies do not stay in a single analysis subgroup throughout the study period: each person is unvaccinated on the first day of the study until the day of vaccination (or the end of the study should the person remain unvaccinated). Instead of crudely categorising individuals as either “vaccinated” or “unvaccinated,” many observational studies split each person’s exposure time into an unvaccinated period followed by a vaccinated period if the individual got vaccinated.^4–6 This technique is essential in contexts where the vast majority of the population becomes vaccinated, to avoid losing a comparison population. However, this procedure injects a strong bias into the analysis subgroups because the unvaccinated exposure time is heavily skewed to the early period in a study while the exposure time for vaccinated people skews towards the end of the study period.

For a hypothetical vaccine with zero efficacy, the case rates for vaccinated and unvaccinated should be equal during each week of the study period. Indeed in RCTs, changes in background infection rate do not bias estimates of vaccine efficacy because by design, vaccine and placebo arms follow a synchronised dosing schedule that ensures exposure (at-risk) time is balanced, even in the context of changing infection rates.

But background infection rate bias can cause estimates of vaccine efficacy in “real world” studies to vary widely from 0%. For example, using infection rate data from an actual observational study of Danish nursing home residents,²⁰ where infection rates rapidly declined simultaneous with vaccine rollout (from 12 per 1000 residents in December 2020, to almost 0 during the last 2 weeks of the study),²⁰ vaccine effectiveness of a hypothetically ineffective vaccine appears as 67%, an illusion chiefly created because unvaccinated people were preferentially exposed to the earlier weeks of higher background infection rates (Figure 3). We note that the direction of this bias would reverse if the background infection rate were to have steadily risen during the study period (i.e., vaccinating into a wave rather than out of one).

Riedmann et al; 2025 (pre-print); “Estimates of underlying health biases in SARS-CoV-2 vaccination recipients: a nationwide study in previously-infected adults” (https://www.medrxiv.org/content/10.1101/2025.02.19.25322515v1)

Results Overall, 4,324,485 individuals (median age (IQR): 46 (33-59) years; 52.56% female) were eligible and 2.23 non-COVID-19 deaths occurred per 100,000 person days. Group differences in non-COVID-19 mortality risk were most prominent in the early periods (e.g., in Q4 2021, adjusted HRs (95% CI) in vaccinated versus unvaccinated were 0.69 (0.59 – 0.81), 0.65 (0.58 – 0.74), and 0.56 (0.48 – 0.66) for 1-, 2-, and 3-vaccinations, respectively) and decreased thereafter. Matched analyses for the first two weeks after vaccination showed HRs below 0.5 for vaccinated versus unvaccinated individuals irrespective of vaccination numbers. Similar findings were retrieved for non-COVID-19, all-cause, and cancer deaths. Overall, COVID-19 deaths were significantly reduced in vaccinated individuals.

Conclusions HVE for SARS-CoV-2 vaccines was strong early after vaccination and diminished over time. HVE should be considered when estimating VE.

Eythorsson et al; 2022; “Rate of SARS-CoV-2 Reinfection During an Omicron Wave in Iceland” (https://jamanetwork.com/journals/jamanetworkopen/article-abstract/2794886)

The probability of reinfection increased with time from the initial infection (odds ratio of 18 months vs 3 months, 1.56; 95% CI, 1.18-2.08) (Figure) and was higher among persons who had received 2 or more doses compared with 1 dose or less of vaccine (odds ratio, 1.42; 95% CI, 1.13-1.78). Defining reinfection after 30 or more days or 90 or more days did not qualitatively change the results.

Shrestha et al; 2023; “Effectiveness of the Coronavirus Disease 2019 Bivalent Vaccine” (https://academic.oup.com/ofid/article/10/6/ofad209/7131292?login=false)

A possible explanation for a lower-than-expected vaccine effectiveness is that a substantial proportion of the population may have had prior asymptomatic Omicron variant infection. About a third of SARS-CoV-2 infections have been estimated to be asymptomatic in studies performed in different places at different times [17–19]. If so, protection from the bivalent vaccine may have been masked because those with prior Omicron variant infection may have already been somewhat protected against COVID-19 by virtue of natural immunity. A seroprevalence study conducted by the CDC found that by February 2022, 64% of the 18–64-year age-group population and 75% of children and adolescents had serologic evidence of prior SARS-CoV-2 infection [20], with almost half of the positive serologic results attributed to infections occurring between December 2021 and February 2022, which would have predominantly been Omicron BA.1/BA.2-lineage infections. With such a large proportion of the population expected to have already been previously exposed to the Omicron variant of SARS-CoV-2, it is possible that a substantial proportion of individuals may be unlikely to derive any meaningful benefit from a bivalent vaccine.

The association of increased risk of COVID-19 with more prior vaccine doses was unexpected. A simplistic explanation might be that those who received more doses were more likely to be individuals at higher risk of COVID-19. A small proportion of individuals may have fit this description. However, the majority of participants in this study were young, and all were eligible to have received ≥3 doses of vaccine by the study start date, which they had every opportunity to do. Therefore, those who received <3 doses (46% of individuals in the study) were not ineligible to receive the vaccine but rather chose not to follow the CDC’s recommendations on remaining updated with COVID-19 vaccination, and one could reasonably expect these individuals to have been more likely to exhibit risk-taking behavior. Despite this, their risk of acquiring COVID-19 was lower than that that of participants those who received more prior vaccine doses.

Ours is not the only study to find a possible association with more prior vaccine doses and higher risk of COVID-19. During an Omicron wave in Iceland, individuals who had previously received ≥2 doses were found to have a higher odds of reinfection than those who had received <2 doses, in an unadjusted analysis [21]. A large study found, in an adjusted analysis, that those who had an Omicron variant infection after previously receiving 3 doses of vaccine had a higher risk of reinfection than those who had an Omicron variant infection after previously receiving 2 doses [22]. Another study found, in multivariable analysis, that receipt of 2 or 3 doses of am mRNA vaccine following prior COVID-19 was associated with a higher risk of reinfection than receipt of a single dose [7]. Immune imprinting from prior exposure to different antigens in a prior vaccine [22, 23] and class switch toward noninflammatory spike-specific immunoglobulin G4 antibodies after repeated SARS-CoV-2 mRNA vaccination [24] have been suggested as possible mechanisms whereby prior vaccine may provide less protection than expected. We still have a lot to learn about protection from COVID-19 vaccination, and in addition to vaccine effectiveness, it is important to examine whether multiple vaccine doses given over time may not be having the beneficial effect that is generally assumed.

Shrestha et al; 2023; “Risk of Coronavirus Disease 2019 (COVID-19) among those up-to-date and not up-to-date on COVID-19 vaccination by US CDC criteria” (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0293449)

Risk of COVID-19 Based on Vaccination Status and Prior Infection

The risk of COVID-19 was lower in the “not up-to-date” state than in the “up-to-date” state, with respect to COVID-19 vaccination (Figure 1). When stratified by tertiles of propensity to get tested for COVID-19, the “not up-to-date” state was not associated with a higher risk of COVID-19 than the “up-to-date” state in any tertile (Figure 2).

DISCUSSION

This study found that not being “up-to-date” on COVID-19 vaccination, using the current CDC definition, was associated with a lower risk of COVID-19 than being “up-to-date”, while the XBB lineages were the dominant circulating strains of SARS-CoV-2.

There are two reasons why not being “up-to-date” on COVID-19 vaccination by the CDC definition was associated with a lower risk of COVID-19. The first is that the bivalent vaccine was somewhat effective against strains that were more similar to the strains on the basis of which the bivalent vaccine was developed, but is not effective against the XBB lineages of the Omicron variant [2]. The second is that the CDC definition does not consider the protective effect of immunity acquired from prior infection. Because the COVID-19 bivalent vaccine provided some protection against the BA.4/BA.5 and BQ lineages [2], those “not-up-to-date” were more likely than those “up-to-date” to have acquired a BA.4/BA.5 or BQ lineage infection when those lineages were the dominant circulating strains. It is now well-known that SARS-CoV-2 infection provides more robust protection than vaccination [4,11,12]. Therefore it is not surprising that not being “up-to-date” according to the CDC definition was associated with a higher risk of prior BA.4/BA.5 or BQ lineage infection, and therefore a lower risk of COVID-19, than being “up-to-date”, while the XBB lineages were dominant.

The strengths of our study include its large sample size, and its conduct in a healthcare system that devoted resources to have an accurate accounting of who had COVID-19, when COVID-19 was diagnosed, who received a COVID-19 vaccine, and when. The study methodology, treating vaccination status as a time-dependent covariate, allowed for determining vaccine effectiveness in real time. Adjusting for the propensity to get tested for COVID-19 should have mitigated against concern that individuals who bothered to remain up-to-date on COVID-19 vaccination may have been more likely to get tested for COVID-19 when they had symptoms.

Covid-19 Forecasting Team; 2023; “Past SARS-CoV-2 infection protection against re-infection: a systematic review and meta-analysis” (https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(22)02465-5/fulltext)

Protection from past infection against re-infection from pre-omicron variants was very high and remained high even after 40 weeks. Protection was substantially lower for the omicron BA.1 variant and declined more rapidly over time than protection against previous variants. Protection from severe disease was high for all variants. The immunity conferred by past infection should be weighed alongside protection from vaccination when assessing future disease burden from COVID-19, providing guidance on when individuals should be vaccinated, and designing policies that mandate vaccination for workers or restrict access, on the basis of immune status, to settings where the risk of transmission is high, such as travel and high-occupancy indoor settings.

Andrews et al; 2022; “Duration of Protection against Mild and Severe Disease by Covid-19 Vaccines” (https://www.nejm.org/doi/full/10.1056/NEJMoa2115481)

Results

Vaccine effectiveness against symptomatic Covid-19 with the delta variant peaked in the early weeks after receipt of the second dose and then decreased by 20 weeks to 44.3% (95% confidence interval [CI], 43.2 to 45.4) with the ChAdOx1-S vaccine and to 66.3% (95% CI, 65.7 to 66.9) with the BNT162b2 vaccine. Waning of vaccine effectiveness was greater in persons 65 years of age or older than in those 40 to 64 years of age. At 20 weeks or more after vaccination, vaccine effectiveness decreased less against both hospitalization, to 80.0% (95% CI, 76.8 to 82.7) with the ChAdOx1-S vaccine and 91.7% (95% CI, 90.2 to 93.0) with the BNT162b2 vaccine, and death, to 84.8% (95% CI, 76.2 to 90.3) and 91.9% (95% CI, 88.5 to 94.3), respectively. Greater waning in vaccine effectiveness against hospitalization was observed in persons 65 years of age or older in a clinically extremely vulnerable group and in persons 40 to 64 years of age with underlying medical conditions than in healthy adults.

Conclusions

We observed limited waning in vaccine effectiveness against Covid-19–related hospitalization and death at 20 weeks or more after vaccination with two doses of the ChAdOx1-S or BNT162b2 vaccine. Waning was greater in older adults and in those in a clinical risk group.

Chemaitelly et al; 2023; “Long-term COVID-19 booster effectiveness by infection history and clinical vulnerability and immune imprinting: a retrospective population-based cohort study” (https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(23)00058-0/fulltext)

Among people vaccinated with BNT162b2, booster effectiveness was 30·4% (95% CI 27·5–33·3) against infection and 74·0% (37·2–89·2) against severe, critical, or fatal COVID-19 (table 2; appendix pp 18–19). Among people vaccinated with mRNA-1273, booster effectiveness was 9·1% (5·0–13·0%) against infection. Effectiveness against severe, critical, or fatal COVID-19 could not be estimated because of too few severe cases. The p values for the interaction terms for all subgroup analyses were <0·01.

Booster effectiveness against infection was highest at 61·4% (95% CI 60·2–62·6) in the first month after the start of follow-up, but waned gradually thereafter and was modest at only 15·5% (8·3–22·2) by the sixth month of follow-up (figure 3). In the seventh month and thereafter, coincident with follow-up time during which BA.4/BA.5²⁷ and BA.2.75*²⁸ dominated incidence, effectiveness was progressively negative although with relatively wide 95% CIs. Adjusting the month-by-month aHRs in a sensitivity analysis for differences in testing rate showed similar results (appendix p 20).

Chemaitelly et al; 2022; “Long-term COVID-19 booster effectiveness by infection history and clinical vulnerability and immune imprinting” (https://www.medrxiv.org/content/10.1101/2022.11.14.22282103v1.full)

Results Booster effectiveness relative to primary series was 41.1% (95% CI: 40.0-42.1%) against infection and 80.5% (95% CI: 55.7-91.4%) against severe, critical, or fatal COVID-19, over one-year follow-up after the booster. Among persons clinically vulnerable to severe COVID-19, effectiveness was 49.7% (95% CI: 47.8-51.6%) against infection and 84.2% (95% CI: 58.8-93.9%) against severe, critical, or fatal COVID-19. Effectiveness against infection was highest at 57.1% (95% CI: 55.9-58.3%) in the first month after the booster but waned thereafter and was modest at only 14.4% (95% CI: 7.3-20.9%) by the sixth month. In the seventh month and thereafter, coincident with BA.4/BA.5 and BA.2.75* subvariant incidence, effectiveness was progressively negative reaching -20.3% (95% CI: -55.0-29.0%) after one year of follow-up. Similar levels and patterns of protection were observed irrespective of prior infection status, clinical vulnerability, or type of vaccine (BNT162b2 versus mRNA-1273).

Conclusions Boosters reduced infection and severe COVID-19, particularly among those clinically vulnerable to severe COVID-19. However, protection against infection waned after the booster, and eventually suggested an imprinting effect of compromised protection relative to the primary series. However, imprinting effects are unlikely to negate the overall public health value of booster vaccinations.

Ioannou et al.; 2025; “Effectiveness of the 2023-to-2024 XBB.1.5 COVID-19 Vaccines Over Long-Term Follow-up: A Target Trial Emulation” (https://www.acpjournals.org/doi/10.7326/ANNALS-24-01015)

Results:

Participants (91.3% male; mean age, 69.9 years) included 587 137 pairs of vaccinated and matched unvaccinated persons. Over a mean follow-up of 176 days (range, 118 to 211 days), VE was −3.26% (95% CI, −6.78% to −0.22%) against documented SARS-CoV-2 infection, 16.64% (CI, 6.47% to 25.77%) against SARS-CoV-2–associated hospitalization, and 26.61% (CI, 5.53% to 42.32%) against SARS-CoV-2–associated death. When estimated at 60, 90, and 120 days, respectively, VE against documented infection (14.21%, 7.29%, and 3.15%), hospitalization (37.57%, 30.84%, and 25.25%), or death (54.24%, 44.33%, and 30.25%) showed substantial waning.

Conclusion:

COVID-19 vaccines targeting the XBB.1.5 variant of Omicron were not effective in preventing infection and had relatively low VE against hospitalization and death, which declined rapidly over time.

Gazit et al; 2022; “Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Naturally Acquired Immunity versus Vaccine-induced Immunity, Reinfections versus Breakthrough Infections: A Retrospective Cohort Study” (https://academic.oup.com/cid/article/75/1/e545/6563799?login=false)

Results – SARS-CoV-2-naive vaccinees had a 13.06-fold (95% confidence interval [CI], 8.08–21.11) increased risk for breakthrough infection with the Delta variant compared to unvaccinated-previously-infected individuals, when the first event (infection or vaccination) occurred during January and February of 2021. The increased risk was significant for symptomatic disease as well. When allowing the infection to occur at any time between March 2020 and February 2021, evidence of waning naturally acquired immunity was demonstrated, although SARS-CoV-2 naive vaccinees still had a 5.96-fold (95% CI: 4.85–7.33) increased risk for breakthrough infection and a 7.13-fold (95% CI: 5.51–9.21) increased risk for symptomatic disease.

Conclusions – Naturally acquired immunity confers stronger protection against infection and symptomatic disease caused by the Delta variant of SARS-CoV-2, compared to the BNT162b2 2-dose vaccine-indued immunity.

Chalupka et al; 2023; “Effectiveness of a fourth SARS-CoV-2 vaccine dose in previously infected individuals from Austria” (https://onlinelibrary.wiley.com/doi/full/10.1111/eci.14136)

Results

Among 3,986,312 previously infected individuals, 281,291 (7,1%) had four and 1,545,242 (38.8%) had three vaccinations at baseline. We recorded 69 COVID-19 deaths and 89,056 SARS-CoV-2 infections. rVE for four versus three vaccine doses was −24% (95% CI: −120 to 30) against COVID-19 deaths, and 17% (95% CI: 14–19) against SARS-CoV-2 infections. This latter effect rapidly diminished over time and infection risk with four vaccinations was higher compared to less vaccinated individuals during extended follow-up until June 2023. Adjusted HR (95% CI) for all-cause mortality for four versus three vaccinations was 0.79 (0.74–0.85).

Discussion

In previously infected individuals, a fourth vaccination was not associated with COVID-19 death risk, but with transiently reduced risk of SARS-CoV-2 infections and reversal of this effect in longer follow-up. All-cause mortality data suggest healthy vaccinee bias.

Menegale et al; 2023; “Evaluation of Waning of SARS-CoV-2 Vaccine–Induced Immunity – A Systematic Review and Meta-analysis” (https://jamanetwork.com/journals/jamanetworkopen/fullarticle/2804451)

Findings  This systematic review and meta-analysis of secondary data from 40 studies found that the estimated vaccine effectiveness against both laboratory-confirmed Omicron infection and symptomatic disease was lower than 20% at 6 months from the administration of the primary vaccination cycle and less than 30% at 9 months from the administration of a booster dose. Compared with the Delta variant, a more prominent and quicker waning of protection was found.

Meaning  These findings suggest that the effectiveness of COVID-19 vaccines against Omicron rapidly wanes over time.

Tamandjou et al; 2023; “Effectiveness of second booster compared to first booster and protection conferred by previous SARS-CoV-2 infection against symptomatic Omicron BA.2 and BA.4/5 in France” (https://www.sciencedirect.com/science/article/pii/S0264410X23003158)

Compared to people without previous infection, the more recent was the previous infection, the higher was the protection against symptomatic infection (Fig. 2). For instance, the adjusted protection associated with a 61–112 days old previous infection, which occurred during the Omicron BA.2 dominant period, was 96% [95%–96%] whereas a 321–467 days old previous infection from the Delta-predominant period was associated with protection of 62% [58%–66%]. Note that comparing the protection induced by the different dominant variants of previous infections can only be done when data are available at similar time intervals since previous infection. For instance, five months (≥150 days) following a previous infection, protection was higher if the previous infection occurred during the BA.2 dominant period compared to during the BA.1 period (Fig. 2, Table S1): 91% [89%–92%] vs 79% [77%–80%], respectively.

Xu et al; 2022; “Effectiveness of COVID-19 Vaccines over 13 Months Covering the Period of the Emergence of the Omicron Variant in the Swedish Population” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9782222/)

VE during a 13-Month Follow-Up Period

The initial analysis was performed for the entire follow-up (13 months). After two doses of any vaccine, VE against COVID-19 infection peaked at week three at 72.0% (95%CI 71.0–73.0%) but then dropped quickly to 19.5% (18.8–20.2%) by weeks 14–17 and showed no protection from week 18 (Figure 1a, Table S6). VE against hospitalization was above 82% from weeks one to 25 and peaked above 90% at weeks five to six (Figure 1b, Table S6). VE after two doses against severe COVID-19 outcomes (ICU admission and death) was even higher and more durable (Figure 1c,d, Table S7). Figure 2 shows unrestricted spline curves illustrating smoothed trends for all COVID-19 outcomes. The smoothed trends corresponded well with the time interval estimates shown in Figure 1. For the later period (e.g., after week 40), the curves are less stable as the last two knots are at the 95th and 99th percentiles in a time period with fewer data. Therefore, VE estimates from both time interval analysis and spline analysis during the later period should be interpreted with appropriate caution.

Feldstein et al; 2024; “Protection From COVID-19 Vaccination and Prior SARS-CoV-2 Infection Among Children Aged 6 Months–4 Years, United States, September 2022–April 2023” (https://academic.oup.com/jpids/advance-article-abstract/doi/10.1093/jpids/piae121/7917119?redirectedFrom=fulltext&login=false)

To understand how coronavirus disease 2019 vaccines impact infection risk in children <5 years, we assessed risk of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection from September 2022 to April 2023 in 3 cohort studies. There was no difference in risk by vaccination status. While vaccines reduce severe disease, they may not reduce SARS-CoV-2 infections in naïve young children.

Neto et al; 2024; “Effectiveness of the fourth dose of COVID-19 vaccines against severe COVID-19 among adults 40 years or older in Brazil: a population-based cohort study” (https://www.thelancet.com/journals/lanam/article/PIIS2667-193X(24)00082-6/fulltext)

Findings

46,693,484 individuals were included in Cohort A and 6,763,016 in Cohort B. 45% of them were aged between 40 and 60 years old, and 48% between 60 and 79 years old. In Cohort A, the most common previous series was a ChAdOx1 two-dose followed by BNT162b2 (44%), and a CoronaVac two-dose followed by a BNT162b2 (36%). Among those fourth dose vaccinated, 36.9% received ChAdOx1, 32.7% Ad26.COV2.S, 25.8% BNT162b2, and 4.7% CoronaVac. In Cohort B, among those who received an adenovirus fourth dose, 53.7% received ChAdOx1 and 46.3% received Ad26.COV2.S. The estimated rVE for the primary outcome of four doses compared to three doses was 44.1% (95% CI 42.3–46.0), with some waning during follow-up (rVE 7–60 days 46.8% [95% CI 44.4–49.1], rVE after 120 days 33.8% [95% CI 18.0–46.6]). Among fourth dose vaccinated individuals, mRNA-based vaccinated individuals had lower hazards for hospitalization or death compared to adenovirus-vaccinated individuals (HR 0.81, 95% CI 0.75–0.87). After 120 days, no difference in hazards between groups was observed (HR 1.35, 95% CI 0.93–1.97). Similar findings were observed for hospitalization and death separately, except no evidence for differences between fourth dose brands for death in Cohort B.

Note: For the above Neto study, calculations can be drawn for the Absolute Risk Reduction provided for the shots. These are:

The Number Needed to Vaccinate (NNV) to prevent one case of COVID-19 hospitalization or death after a fourth dose compared to three doses.

Extracting Key Data

•   Incidence in Control Group (Three Doses):
•   15,691 COVID-19 hospitalizations or deaths out of 23,346,742 individuals.
•   Incidence rate  = 0.000672  (or 0.0672%).
•   Relative Vaccine Efficacy (rVE) of Fourth Dose:
•   rVE = 44.1% (or 0.441 as a decimal).

Interpretation

Based on the provided data within the study, approximately 3,375 individuals would need to receive a fourth dose of the vaccine to prevent one additional case of COVID-19 hospitalization or death compared to those who have received only three doses.

This calculation uses the average relative vaccine efficacy over the entire study period. Variations in effectiveness over time (e.g., waning immunity) or among subgroups (e.g., different age groups) could result in different NNV values.

Given the ARR it is calculated:

ARR = 0.000296352

We convert this to a percentage:

ARR\ (\%) = 0.000296352 \times 100 = 0.0296352\%

Interpretation

The Absolute Risk Reduction (ARR) for the fourth dose compared to three doses is approximately 0.0296%.

Dan-Yu Lin et al; 2023; “Effectiveness of Bivalent Boosters against Severe Omicron Infection” (https://www.nejm.org/doi/full/10.1056/NEJMc2215471)

Booster effectiveness peaked at approximately 4 weeks and waned afterward. For all participants 12 years of age or older, vaccine effectiveness against severe infection resulting in hospitalization over days 15 to 99 after receipt of one monovalent booster dose was 25.2% (95% confidence interval [CI], –0.2 to 44.2), and the corresponding vaccine effectiveness for one bivalent booster dose was 58.7% (95% CI, 43.7 to 69.8); the difference in vaccine effectiveness against this outcome between the bivalent booster and the monovalent booster was 33.5 percentage points (95% CI, 2.9 to 62.1). Vaccine effectiveness against severe infection resulting in hospitalization or death was 24.9% (95% CI, 1.4 to 42.8) for one monovalent booster dose and 61.8% (95% CI, 48.2 to 71.8) for one bivalent booster dose; the difference in vaccine effectiveness against this outcome between the bivalent booster and the monovalent booster was 36.9 percentage points (95% CI, 12.6 to 64.3) (Fig. S3 and Table 1). We obtained similar vaccine effectiveness estimates when the analysis was restricted to participants who were 18 years of age or older or 65 years of age or older, to participants who received an mRNA vaccine as their primary vaccine, or to previously uninfected participants (Table 1). In addition, estimates of vaccine effectiveness were similar for the Moderna and Pfizer–BioNTech boosters and similar among the first, second, and third booster doses (Table 1).

Echevarria et al; 2024; “Incidence and risk factors of SARS-CoV-2 breakthrough infection in the early Omicron variant era among vaccinated and boosted individuals in Chicago” (https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0302338)

Methods and findings

A retrospective clinical cohort study was performed utilizing the Northwestern Medicine Enterprise Data Warehouse. Our study population was identified as fully vaccinated adults with at least one booster. The primary risk factor of interest was the number of co-morbidities. The primary outcome was the incidence and time to the first positive SARS-CoV-2 molecular test in the Omicron predominant era. Multivariable Cox modeling analyses to determine the hazard of SARS-CoV-2 infection were stratified by calendar time (Period 1: January 1 –June 30, 2022; Period 2: July 1 –December 31, 2022) due to violations in the proportional hazards assumption. In total, 133,191 patients were analyzed. During Period 1, having 3+ comorbidities was associated with increased hazard for breakthrough (HR = 1.16 CI 1.08–1.26). During Period 2 of the study, having 2 comorbidities (HR = 1.45 95% CI 1.26–1.67) and having 3+ comorbidities (HR 1.73, 95% CI 1.51–1.97) were associated with increased hazard for Omicron breakthrough. Older age was associated with decreased hazard in Period 1 of follow-up. Interaction terms for calendar time indicated significant changes in hazard for many factors between the first and second halves of the follow-up period.

Conclusions

Omicron breakthrough is common with significantly higher risk for our most vulnerable patients with multiple co-morbidities. Age plays an important role in breakthrough infection with the highest incidence among young adults, which may be due to age-related behavioral factors. These findings reflect real-world differences in immunity and exposure risk behaviors for populations vulnerable to COVID-19.

6. Adverse Events

David Allen; 2024; “The correlation between Australian Excess Deaths by State and Booster Vaccinations” (https://esmed.org/MRA/mra/article/view/5485)

The study explores the relationship by Australia State between COVID Booster Vaccinations and excess deaths. There is evidence of a very strong correlation in ordinary least squares regression analysis. Cross-validation tests support the strength of the regression relationship. The results suggest that it would be worthwhile to explore these associations in greater depth as it is an important public health issue.

This paper presents the results of an OLS regression of the number of excess deaths, by State and Territory in Australia, as reported in official sources in 2023, on recorded excess booster vaccine doses by State and Territory. The results are quite striking and suggest the existence of a strong regression relationship with significant coefficients and an Adjusted R-squared of 71 percent. Simple cross-validation tests suggest that the regression results are robust, despite the very limited sample size available in this cross-sectional regression. These results match those of a recent study by Allen (2023) on the OECD country experience of excess deaths[1]. They suggest that this topic deserves greater scrutiny given that it is an important public health policy issue with considerable cost implications.

Raw et al; 2021; “Previous COVID-19 infection, but not Long-COVID, is associated with increased adverse events following BNT162b2/Pfizer vaccination” (https://www.journalofinfection.com/article/S0163-4453(21)00277-2/fulltext)

This study of healthcare workers demonstrated that prior COVID-19, but not Long-COVID, was associated with increased risk of AEs following BNT162b2/Pfizer vaccination, although there was no relationship with duration since COVID-19 illness. Women and younger individuals were also more likely to report AEs. Our study adds to other reports supporting the wider understanding of AEs following COVID-19 vaccination [1,2,3,4]. Importantly, given hesitancy surrounding recently developed COVID-19 vaccines [[6]], our findings may help inform those with previous COVID-19 of increased susceptibility to certain AEs. Our study also adds weight to the question of whether a second dose of mRNA vaccine is necessary in those with previous COVID-19, assuming effective immunity is established after the first dose [[1],[2],[8],[9]]. This is relevant, given that Tre-Hardy’s and other studies have reported worse AEs following second doses of vaccine [[1],[3]].

Faksova et al; 2024; “COVID-19 vaccines and adverse events of special interest: A multinational Global Vaccine Data Network (GVDN) cohort study of 99 million vaccinated individuals” (https://www.sciencedirect.com/science/article/pii/S0264410X24001270)

Results

Participants included 99,068,901 vaccinated individuals. In total, 183,559,462 doses of BNT162b2, 36,178,442 doses of mRNA-1273, and 23,093,399 doses of ChAdOx1 were administered across participating sites in the study period. Risk periods following homologous vaccination schedules contributed 23,168,335 person-years of follow-up. OE ratios with LBCI > 1.5 were observed for Guillain-Barré syndrome (2.49, 95 % CI: 2.15, 2.87) and cerebral venous sinus thrombosis (3.23, 95 % CI: 2.51, 4.09) following the first dose of ChAdOx1 vaccine. Acute disseminated encephalomyelitis showed an OE ratio of 3.78 (95 % CI: 1.52, 7.78) following the first dose of mRNA-1273 vaccine. The OE ratios for myocarditis and pericarditis following BNT162b2, mRNA-1273, and ChAdOx1 were significantly increased with LBCIs > 1.5.

Conclusion

This multi-country analysis confirmed pre-established safety signals for myocarditis, pericarditis, Guillain-Barré syndrome, and cerebral venous sinus thrombosis. Other potential safety signals that require further investigation were identified.

Salmaggi et al; 2025; “Impact of COVID-19 disease and COVID-19 vaccinations on hospital admissions for neurological diseases in the Lombardia over-12 population. Data from a self-controlled case series analysis” (https://pubmed.ncbi.nlm.nih.gov/39560882/)

Methods: Linkable administrative health databases from the Lombardia region were used. By using the adapted self controlled case series (SCCS) method for event dependent exposures, we estimated the relative incidence of different neurological diseases following pre-specified windows at risk after vaccination and after COVID-19 infection in the over-12 population of Lombardia. Follow-up time before vaccination (Pre-Vax period) was compared with follow-up time 0-28 days (high-risk period) from the day of vaccination as well as for COVID infection. The SCCS model was fitted using a conditional Poisson regression model to estimate the relative incidences (RI) and their 95% Confidence Intervals (CI).

Results: The 28-day post-vaccination period was associated with a significant increase in the occurrence of ischemic stroke, cerebral haemorrhage, TIAs and myelitis (IRR 1.44, 1.50, 1.67 and 2.65 respectively). When the risk conferred by COVID19 infection was assessed in the same cohort, significant IRR were greater in the occurrence of ischemic stroke, cerebral haemorrhage, and TIAs (IRR 5.6, 3.62, 6.83) and includes also Multiple Sclerosis, neuromyelitis, and polymyositis (5.25, 8.81, 5.67).

Raethke et al; 2024; “Frequency and timing of adverse reactions to COVID-19 vaccines; A multi-country cohort event monitoring study” (https://www.sciencedirect.com/science/article/pii/S0264410X24002731)

A total of 29,837 participants completed at least the baseline and the first follow-up questionnaire for 1st and 2nd vaccination and 7,250 participants for the booster. The percentage of participants who reported at least one ADR is 74.32% (95%CI 73.82–74.81). Solicited ADRs, including injection site reactions, are very common across vaccination moments. Potential predictors for these reactions are the brand of vaccine used, the patient’s age, sex and prior SARS-CoV-2 infection. The percentage of serious ADRs in the study is low for 1st and 2nd vaccination (0.24%, 95%CI 0.19––0.31) and booster (0.26%, 95%CI 0.15, 0.41). The TTO was 14 h (median) for dose 1 and slightly longer for dose 2 and booster dose. TTR is generally also within a few days. The effect of L2FU on estimations of frequency is limited.

Authors found that, expressed as a ratio:

• For the first and second vaccinations, approximately 1 person in 417 experiences a serious ADR.
• For the booster vaccination, approximately 1 person in 385 experiences a serious ADR.

Platschek, B., & Boege, F., 2024; “The Post-Acute COVID-19-Vaccination Syndrome in the Light of Pharmacovigilance” (https://pubmed.ncbi.nlm.nih.gov/39772040/)

Results: (i) PACVS is distinguished from normal vaccination reactions solely by prolonged duration. (ii) Symptom duration is poorly monitored by post-authorisation pharmacovigilance. (iii) PACVS-specific signals were faithfully recorded by pharmacovigilance systems but have not prompted appropriate reactions of health authorities. (iv) The most widely applied SARS-CoV-2 mRNA-vaccine has been modified after roll-out without renewed phase III evaluation; the modification has increased DNA contaminations suspected to extend the spectrum of adverse events. (v) Crossing of pharmacovigilance data with corresponding estimates of applied vaccine doses suggest a PACVS prevalence of 0.003% in the general population. In contrast, occupational surveillance studies suggest a PACVS prevalence of 0.9% in young and middle-aged persons. Conclusions: (a) Denial of official recognition of PACVS is unjustified. (b) PACVS seems to target preferentially young and middle-aged persons. (c) Without official disease recognition, access to public healthcare and welfare services is made difficult for PACVS-affected persons, which creates considerable socio-economic problems. (d) Without official disease recognition, development and evaluation of PACVS therapies is impaired.

Rogers et al; 2024; “COVID-19 Vaccines: A Risk Factor for Cerebral Thrombotic Syndromes” (https://ijirms.in/index.php/ijirms/article/view/1982)

Results: There are 5137 cerebral thromboembolism AEs reported in the 3 years (36 months) after COVID-19 vaccines compared to 52 AEs for the influenza vaccines over the past 34 years (408 months) and 282 AEs for all other vaccines (excluding COVID-19) over the past 34 years (408 months). The PRR’s are significant when comparing AEs by time from COVID-19 vaccines to that of the influenza vaccines (p < 0.0001) or to that of all other vaccines (p < 0.0001). The CTE AEs PRR by time (95% confidence intervals) for the COVID-19 vaccine AEs vs influenza AEs is 1120 (95% confidence interval (723-1730), p < 0.0001) and for COVID-19 vaccines vs all others is 207 (95% confidence interval (144-296), p < 0.0001). Cerebral venous thromboembolism AEs are female predominant with a female/male odds ratio of 1.63 (95% confidence interval (1.52-1.74), p < 0.0001). Conversely, cerebral arterial thromboembolism has a nonsignificant male preponderance. Cerebral venous thromboembolism is far more common than cerebral arterial thromboembolism over 36 months with an odds ratio (OR) of 14.8 (95% confidence interval 14.0-15.5, p < 0.0001). Atrial fibrillation, the most common identifiable cause of cerebral arterial thromboembolism, occurs far more commonly after the COVID-19 as compared to all other vaccines with a PRR of 123 (95% CI 88.3-172, p < 0.0001). Conclusions: There is an alarming breach in the safety signal threshold concerning cerebral thrombosis AEs after COVID-19 vaccines compared to that of the influenza vaccines and even when compared to that of all other vaccines. An immediate global moratorium on the use of COVID-19 vaccines is necessary with an absolute contraindication in women of reproductive age.

Seung-Won Jung et al; 2024; “Long-term risk of autoimmune diseases after mRNA-based SARS-CoV2 vaccination in a Korean, nationwide, population-based cohort study” (https://www.nature.com/articles/s41467-024-50656-8)

In this nationwide, population-based cohort study involving 9,258,803 individuals, we aim to determine whether the incidence of AI-CTDs is associated with mRNA vaccination. The study spans over 1 year of observation and further analyses the risk of AI-CTDs by stratifying demographics and vaccination profiles and treating booster vaccination as time-varying covariate. We report that the risk of developing most AI-CTDs did not increase following mRNA vaccination, except for systemic lupus erythematosus with a 1.16-fold risk in vaccinated individuals relative to controls. Comparable results were reported in the stratified analyses for age, sex, mRNA vaccine type, and prior history of non-mRNA vaccination. However, a booster vaccination was associated with an increased risk of some AI-CTDs including alopecia areata, psoriasis, and rheumatoid arthritis. Overall, we conclude that mRNA-based vaccinations are not associated with an increased risk of most AI-CTDs, although further research is needed regarding its potential association with certain conditions.

McMillan et al; 2023; “Fatal Post COVID mRNA-Vaccine Associated Cerebral Ischemia” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10091442/)

Case Summary

24 hrs after receiving her first dose of the Moderna COVID-19 vaccine, a 30-year-old female developed severe headache. Three weeks later she was admitted with subacute headache and confusion. Imaging initially showed scattered cortical thrombosis with an elevated opening pressure on lumbar puncture. An external ventricular drain was placed, but she continued to have elevated intracranial pressure. Ultimately, she required a hemicraniectomy, but intractable cerebral edema resulted in her death. Pathology was consistent with thrombosis and associated inflammatory response.

Conclusion

Though correlational, her medical team surmised that the mRNA vaccine may have contributed to this presentation. The side effects of COVID-19 infection and vaccination are still incompletely understood. Though complications are rare, clinicians should be aware of presentations like this one.

Hulscher et al; 2024; “A Systematic REVIEW of Autopsy findings in deaths after covid-19 vaccination” (https://www.sciencedirect.com/science/article/pii/S0379073824001968)

Highlights

• We found that 73.9% of deaths were directly due to or significantly contributed to by COVID-19 vaccination.
• Our data suggest a high likelihood of a causal link between COVID-19 vaccination and death.
• These findings indicate the urgent need to elucidate the pathophysiologic mechanisms of death with the goal of risk stratification and avoidance of death for the large numbers of individuals who have taken or will receive one or more COVID-19 vaccines in the future.
• This review helps provide the medical and forensic community a better understanding of COVID-19 vaccine fatal adverse events.

Results

We initially identified 678 studies and, after screening for our inclusion criteria, included 44 papers that contained 325 autopsy cases and one necropsy case. The mean age of death was 70.4 years. The most implicated organ system among cases was the cardiovascular (49%), followed by hematological (17%), respiratory (11%), and multiple organ systems (7%). Three or more organ systems were affected in 21 cases. The mean time from vaccination to death was 14.3 days. Most deaths occurred within a week from last vaccine administration. A total of 240 deaths (73.9%) were independently adjudicated as directly due to or significantly contributed to by COVID-19 vaccination, of which the primary causes of death include sudden cardiac death (35%), pulmonary embolism (12.5%), myocardial infarction (12%), VITT (7.9%), myocarditis (7.1%), multisystem inflammatory syndrome (4.6%), and cerebral hemorrhage (3.8%).

Conclusions

The consistency seen among cases in this review with known COVID-19 vaccine mechanisms of injury and death, coupled with autopsy confirmation by physician adjudication, suggests there is a high likelihood of a causal link between COVID-19 vaccines and death. Further urgent investigation is required for the purpose of clarifying our findings.

Al-Rousan & Al-Najjar; 2024; “Evaluation of the effects of MERCK, MODERNA, PFIZER/BioNTech, and JANSSEN COVID-19 vaccines on vaccinated people: A metadata analysis” (https://www.sciencedirect.com/science/article/pii/S2352914824001205)

Results

The overall mortality rate among the vaccinated population is noteworthy. Notably, 40 different mild to severe symptoms were reported among vaccinated individuals. The research highlights the 10 most common symptoms experienced after vaccination. Females under 60 years of age constitute the majority of the dataset.

Conclusions

The vaccination-related mortality rate stands at approximately 3 % of those who received the vaccine, with the majority of cases occurring among individuals under the age of 60, who were not hospitalized and had received their initial vaccine dose.

6a. Cardiac research

Nakahara et al; 2023; “Assessment of Myocardial ¹⁸F-FDG Uptake at PET/CT in Asymptomatic SARS-CoV-2-vaccinated and Nonvaccinated Patients” (https://pubmed.ncbi.nlm.nih.gov/37724969/)

This retrospective study included patients who underwent ¹⁸F-FDG PET/CT for indications unrelated to myocarditis during the period before (11/1/2020 – 2/16/2021) and after (2/17/20121 – 3/31/2022) SARS-CoV-2 vaccines were available. Myocardial and axillary FDG uptake were quantitatively assessed using maximum standardized uptake value (SUVmax). SUVmax values in all patients and in patients stratified by sex (male/female), age (<40, 41-60, >60 years), and time interval between vaccination and PET/CT were compared using Mann-Whitney U test or Kruskal-Wallis test with post ad -hoc Dwass, Steel, Critchlow-Fligner multiple comparison analysis. Results The study included 303 nonvaccinated patients (mean age, 52.9 years ± 14.9 [SD]; 157 females) and 700 vaccinated patients (mean age, 56.8 years ± 13.7 [SD]; 344 females). Vaccinated patients had overall higher myocardial FDG uptake compared to nonvaccinated patients (median SUVmax, 4.8 [IQR: 3.0-8.5] vs median SUVmax, 3.3 [IQR: 2.5-6.2]; P < .0001). Myocardial SUVmax was higher in vaccinated patients regardless of sex (median range, 4.7-4.9 [IQR: 2.9-8.6]) or patient age (median range, 4.7-5.6 [IQR: 2.9-8.6]) compared to corresponding nonvaccinated groups (sex median range, 3.2-3.9 [IQR: 2.4-7.2]; age median range, 3.3-3.3 [IQR: 2.3-6.1]; P range, <.001-.015). Furthermore, increased myocardial FDG uptake was observed in patients imaged 1-30, 31-60, 61-120, and 121-180 days after their second vaccination (median SUVmax range, 4.6-5.1 [IQR: 2.9-8.6]) and increased ipsilateral axillary uptake was observed in patients imaged 1-30, 31-60, 61-120 days after their 2nd vaccination (median SUVmax range, 1.5-2.0 [IQR: 1.2-3.4]) compared to the nonvaccinated patients (P range, <.001-<.001). Conclusion Compared to nonvaccinated patients, asymptomatic patients who received their 2nd vaccination 1-180 days prior to imaging showed increased myocardial FDG uptake on PET/CT.

McGonagle and Giryes; 2024; “An immunology model for accelerated coronary atherosclerosis and unexplained sudden death in the COVID-19 era“

For the first time in the history of immunology the immune system has simultaneously faced an attack from without (natural infection) and near simultaneously vaccine mediated immune activation from within. Unfortunately, much evidence points towards inflammation in the vicinity of “widow-maker” coronary artery territories for several microbes and even vaccines. With particular respect to coronavirus is the potential impact of spike protein itself on coronary artery and cardiovascular biology that is a further potentially relevant factor [118]. 

The perception that there is a fear of investigating the current excess deaths is probably driven by a reticence towards uncovering a negative link with coronavirus vaccinations that might be perceived to undermine general societal confidence or the public health drive to “eradicate the virus.” 

The coronary artery vasa vasorum resides in the visceral pericardium or epicardial tissue that is adipose rich and is physiologically a liquid at body temperature thus contributing to shock absorption and likely contributes to normal coronary arteries homeostasis in adults. The epicardial adipose tissue (EAT) contains several types of immune cells including macrophages and B cells, and these cells play an important role in vascular development, homeostasis and repair, remodelling and immunosurveillance. Cardiac yolk sac derived macrophages (CC chemokine receptor type 2 negative (CCR2-)) populate the heart during embryogenesis and persists throughout life and are essential for the coronary artery maturation. The EAT resident macrophage population is capable of induction of high levels of multiple pro-inflammatory cytokines. One of the unique features of epicardial adipose tissue that is contiguous with the myocardium without muscle facia and share the same microcirculation. In pathological conditions, epicardial adipose tissue can become pro-atherogenic and pro-arrhythmogenic due to increased pro-inflammatory macrophage activity. The arrhythmogenic potential is likely secondary to the loss of fatty insulation immediately adjacent to the sinoatrial and atrioventricular nodes. 

Beyond direct triggering of atherosclerotic disease, epicardial tissue disruption of electrochemical homeostasis of the conducting tissue could also predispose to various atrial and ventricular arrhythmia. Accordingly, the presence of a pandemic with a widely circulating virus and potentially the simultaneous boosting of coronavirus immunity provides a credible mechanism for unexplained cardiovascular disease that is not evidently an acute vasculitis pathology, nor immediately evident as an immunologically driven process but rather manifests as a late atherosclerotic sequela. 

Lee et al; 2024; “Cardiac and Neurological Complications Post COVID-19 Vaccination: A Systematic Review of Case Reports and Case Series” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11209191/)

Following mass vaccinations for the control of the COVID-19 epidemic, a spectrum of cardiac and neurological disorders was reported among vaccinated individuals. This study examined the range of complications documented and factors related to their occurrence. Three electronic databases were searched for case reports and case series with descriptions of cardiac and/or neurological complications in COVID-19 vaccine recipients. A total of 698 vaccinees were included in this review, of which 259 (37.1%) had cardiac and 439 (62.9%) had neurological complications. Inflammatory conditions were the commonest among the cardiac complications; while polyneuropathy, demyelinating diseases and cerebrovascular disorders were the more common neurological complications. The mean age of those with cardiac complications (33.8 years) was much younger than those with neurological complications (49.7 years). There was no notable difference in the gender distribution between these two groups of vaccine recipients. mRNA vaccines (all brands) were associated with almost 90.0% of the cardiac complications, whereas viral vector vaccines were associated with slightly over half (52.6%) of the neurological complications. With regard to the dose, cardiac complications were more common after the second (69.1%), whereas neurological complications were more common after the first dose (63.6%). The majority of the cases had an uncomplicated clinical course. Nevertheless, 5.9% of cases with neurological complications and 2.5% of those with cardiac complications were fatal, underscoring the significance of the consistent surveillance and vigilant monitoring of vaccinated individuals to mitigate these occurrences.

Baumeier et al; 2024; “Intramyocardial Inflammation after COVID-19 Vaccination: An Endomyocardial Biopsy-Proven Case Series” (https://pubmed.ncbi.nlm.nih.gov/35805941/)

Myocarditis in response to COVID-19 vaccination has been reported since early 2021. In particular, young male individuals have been identified to exhibit an increased risk of myocardial inflammation following the administration of mRNA-based vaccines. Even though the first epidemiological analyses and numerous case reports investigated potential relationships, endomyocardial biopsy (EMB)-proven cases are limited. Here, we present a comprehensive histopathological analysis of EMBs from 15 patients with reduced ejection fraction (LVEF = 30 (14-39)%) and the clinical suspicion of myocarditis following vaccination with Comirnaty^® (Pfizer-BioNTech) (n = 11), Vaxzevria^® (AstraZenica) (n = 2) and Janssen^® (Johnson & Johnson) (n = 2). Immunohistochemical EMB analyses reveal myocardial inflammation in 14 of 15 patients, with the histopathological diagnosis of active myocarditis according the Dallas criteria (n = 2), severe giant cell myocarditis (n = 2) and inflammatory cardiomyopathy (n = 10). Importantly, infectious causes have been excluded in all patients. The SARS-CoV-2 spike protein has been detected sparsely on cardiomyocytes of nine patients, and differential analysis of inflammatory markers such as CD4⁺ and CD8⁺ T cells suggests that the inflammatory response triggered by the vaccine may be of autoimmunological origin. Although a definitive causal relationship between COVID-19 vaccination and the occurrence of myocardial inflammation cannot be demonstrated in this study, data suggest a temporal connection. The expression of SARS-CoV-2 spike protein within the heart and the dominance of CD4⁺ lymphocytic infiltrates indicate an autoimmunological response to the vaccination.

Schreckenberg et al; 2024; “Cardiac side effects of RNA-based SARS-CoV-2 vaccines: Hidden cardiotoxic effects of mRNA-1273 and BNT162b2 on ventricular myocyte function and structure” (https://pubmed.ncbi.nlm.nih.gov/37828636/)

Background and purpose: To protect against SARS-CoV-2 infection, the first mRNA-based vaccines, Spikevax (mRNA-1273, Moderna) and Comirnaty (BNT162b2, Pfizer/Biontech), were approved in 2020. The structure and assembly of the immunogen-in both cases, the SARS-CoV-2 spike (S) glycoprotein-are determined by a messenger RNA sequence that is translated by endogenous ribosomes. Cardiac side-effects, which for the most part can be classified by their clinical symptoms as myo- and/or pericarditis, can be caused by both mRNA-1273 and BNT162b2.

Experimental approach: As persuasive theories for the underlying pathomechanisms have yet to be developed, this study investigated the effect of mRNA-1273 and BNT162b2 on the function, structure, and viability of isolated adult rat cardiomyocytes over a 72 h period.

Key results: In the first 24 h after application, both mRNA-1273 and BNT162b2 caused neither functional disturbances nor morphological abnormalities. After 48 h, expression of the encoded spike protein was detected in ventricular cardiomyocytes for both mRNAs. At this point in time, mRNA-1273 induced arrhythmic as well as completely irregular contractions associated with irregular as well as localized calcium transients, which provide indications of significant dysfunction of the cardiac ryanodine receptor (RyR2). In contrast, BNT162b2 increased cardiomyocyte contraction via significantly increased protein kinase A (PKA) activity at the cellular level.

Conclusion and implications: Here, we demonstrated for the first time, that in isolated cardiomyocytes, both mRNA-1273 and BNT162b2 induce specific dysfunctions that correlate pathophysiologically to cardiomyopathy. Both RyR2 impairment and sustained PKA activation may significantly increase the risk of acute cardiac events.

McCullough and Hulscher; 2024; “Risk Stratification for Future Cardiac Arrest after COVID-19 Vaccination” (https://www.preprints.org/manuscript/202408.0821/v1)

Unheralded cardiac arrest among previously healthy young people without antecedent illness, months or years after COVID-19 vaccination, highlights the urgent need for risk stratification. The most likely underlying pathophysiology is subclinical myopericarditis and reentrant ventricular tachycardia or spontaneous ventricular fibrillation that is commonly precipitated after a surge in catecholamines during exercise or the waking hours of terminal sleep. Small patches of inflammation and/or edema can be missed on cardiac imaging and autopsy, and the heart can appear grossly normal. This paper reviews evidence linking COVID-19 vaccines to cardiac arrest where unfortunately the majority of victims have had no antecedent clinical evaluation. We propose a comprehensive strategy for evaluating cardiovascular risk post-vaccination, incorporating detailed patient history, antibody testing, and cardiac diagnostics in the best attempt to detect abnormalities before sudden cardiac death. This approach aims to identify individuals at higher risk of cardiac events after COVID-19 vaccination and guide appropriate clinical management.

Hulscher et al; 2024; “Autopsy findings in cases of fatal COVID-19 vaccine-induced myocarditis” (https://onlinelibrary.wiley.com/doi/10.1002/ehf2.14680)

All autopsy studies that include COVID-19 vaccine-induced myocarditis as a possible cause of death were included. Causality in each case was assessed by three independent physicians with cardiac pathology experience and expertise. We initially identified 1691 studies and, after screening for our inclusion criteria, included 14 papers that contained 28 autopsy cases. The cardiovascular system was the only organ system affected in 26 cases. In two cases, myocarditis was characterized as a consequence from multisystem inflammatory syndrome. The mean age of death was 44.4 years old. The mean and median number of days from last COVID-19 vaccination until death were 6.2 and 3 days, respectively. We established that all 28 deaths were most likely causally linked to COVID-19 vaccination by independent review of the clinical information presented in each paper. The temporal relationship, internal and external consistency seen among cases in this review with known COVID-19 vaccine-induced myocarditis, its pathobiological mechanisms, and related excess death, complemented with autopsy confirmation, independent adjudication, and application of the Bradford Hill criteria to the overall epidemiology of vaccine myocarditis, suggests that there is a high likelihood of a causal link between COVID-19 vaccines and death from myocarditis.

Hviid et al; 2024; “Booster vaccination with SARS-CoV-2 mRNA vaccines and myocarditis in adolescents and young adults: a Nordic cohort study” (https://academic.oup.com/eurheartj/advance-article/doi/10.1093/eurheartj/ehae056/7608548?login=false)

A total of 8.9 million residents were followed for 12 271 861 person-years and 1533 cases of myocarditis were identified. In 12-to-39-year-old males, the 28-day acute risk period following the third dose of BNT162b2 or mRNA-1273 was associated with an increased incidence rate of myocarditis compared to the post-acute risk period 28 days or more after the second dose [IRR 2.08 (95% CI 1.31–3.33) and 8.89 (2.26–35.03), respectively]. For females, the corresponding IRR was only estimable for BNT162b2, 3.99 (0.41–38.64). The corresponding absolute risks following the third dose of BNT162b2 and mRNA-1273 in males were 0.86 (95% CI 0.53–1.32) and 1.95 (0.53–4.99) myocarditis events within 28 days per 100 000 individuals vaccinated, respectively. In females, the corresponding absolute risks following the third dose of BNT162b2 were 0.15 (0.04–0.39) events per 100 000 individuals vaccinated. No deaths occurred within 30 days of vaccine-related cases.

Krauson et al; 2023; “Duration of SARS-CoV-2 mRNA vaccine persistence and factors associated with cardiac involvement in recently vaccinated patients” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10533894/)

Detection of BNT162b2 and mRNA-1273 vaccines in human tissues

In the bilateral axillary lymph node samples, vaccine was detected in 2 (33%) of the 6 available samples from patients vaccinated with mRNA-1273 and in 6 (46%) of the 13 patients vaccinated with BNT162b2 (Figs. ​(Figs.1b,1b, c, ​c,2).2). Overall, for both vaccines, vaccine was detected in 8 (73%) of the 11 available axillary lymph nodes samples from the 12 patients dying within 30 days of vaccination compared with no detection of vaccine in any of the axillary lymph nodes samples from the 8 patients dying after 30 days from vaccination (P = 0.003, Fisher exact test). For none of the patients was vaccine detected in the liver, spleen, or mediastinal lymph nodes (each n = 19). For the 20 cardiac left ventricle and 20 cardiac right ventricle samples, vaccine was detected in 2 samples of left ventricle and 2 samples of right ventricle from a total of three patients. All three of these patients had been vaccinated with BNT162b2 within 30 days of death. All samples positive for vaccine were validated for sequences outside of the dsDNA control sequence (Supplementary Tables 3–4). Since SARS-CoV-2 can involve tissues throughout the body including the heart in the setting of severe respiratory tract infection^22,23, as a further control, all tissue samples with detectable vaccine mRNA were screened for the SARS-CoV-2 virus E gene (Fig. ​(Fig.1d)1d) and were found to be negative for the virus. Vaccine was not detected by RT-qPCR in any of the tissues from the 5 non-vaccinated control patients. Immunohistochemistry for the spike protein in the axillary lymph nodes, left ventricle, right ventricle and liver performed on all 20 vaccinated patients showed only non-specific staining (not shown).

SARS-CoV-2 mRNA vaccine in the heart is associated with myocardial injury

To gain insight into why some of the patients dying within 30 days of vaccination had detectable vaccine in the heart, the 3 patients with vaccine in the heart were compared with the 9 patients dying within 30 days of vaccination without vaccine in the heart (Table ​(Table1).1). There was no significant difference in age, sex, body mass index (BMI), time since vaccination, or basic cardiac risk factors. While all three of the patients with vaccine in the heart had received BNT162b2, this was not statistically significant. Upon evaluating the histologic slides, none of the patients had myocarditis. However, all three (100%) of the patients with vaccine in the heart had healing myocardial injury which initiated before or at the time of the most recent vaccine injection compared with only 2 (22%) of the 9 patients without vaccine in the heart (Fig. ​(Fig.3a).3a). Considering the specific location of the myocardial injury (left ventricle vs right ventricle) in these patients, vaccine was detected in 4 (57%) of 7 ventricles with myocardial injury at the time of vaccination compared with none (0%) of 17 ventricles without myocardial injury at the time of vaccination (P = 0.003, Fisher exact test, Fig. ​Fig.3b).3b). Healing myocardial injury is associated with macrophage infiltration of the myocardium. Those patients with vaccine in the heart had more macrophages in the myocardium than those patients dying within 30 days of vaccination without vaccine in the heart (Fig. 3c, d, Supplementary Fig. 1, P = 0.0003, t test).

Buergin et al; 2023; “Sex-specific differences in myocardial injury incidence after COVID-19 mRNA-1273 booster vaccination” (https://onlinelibrary.wiley.com/doi/full/10.1002/ejhf.2978)

First, our findings confirmed the study hypothesis. mRNA-1273 booster vaccination-associated elevation of markers of myocardial injury occurred in about one out of 35 persons (2.8%), a greater incidence than estimated in meta-analyses of hospitalized cases with myocarditis (estimated incidence 0.0035%) after the second vaccination.^{14, 15} Elevated hs-cTnT was independent of previous COVID-19 infection or the interval since the last vaccine dose. Among the overall group of participants, hs-cTnT concentration on day 3 after mRNA-1273 booster vaccination as a continuous variable, was significantly higher compared to a well-matched control cohort. Second, all cases were mild with only a transient and short period of myocardial injury (maximum hs-cTnT concentration 35 ng/L). No patient showed ECG changes and no patient developed MACE within 30 days. Potentially, such outcomes were averted by the safety net provided by early detection and early implementation of preventive measures for deterioration including avoidance of strenuous exercise.

Yun et al; 2024; “The impact of COVID-19 status and vaccine type following the first dose on acute heart disease: A nationwide retrospective cohort study in South Korea” (https://pubmed.ncbi.nlm.nih.gov/39444354/)

We analysed heart disease risk, including acute cardiac injury, acute myocarditis, acute pericarditis, cardiac arrest, and cardiac arrhythmia, in relation to vaccine type and COVID-19 within 21 days after the first vaccination date, employing Cox proportional hazards models with time-varying covariates. This study included 3,350,855 participants. The results revealed higher heart disease risk in individuals receiving mRNA vaccines than other types (adjusted HR, 1.48; 95% CI, 1.35-1.62). Individuals infected by SARS-CoV-2 also exhibited significantly higher heart disease risk than those uninfected (adjusted HR, 3.56; 95% CI, 1.15-11.04). We found no significant interaction effect between vaccine type and COVID-19 status on the risk of acute heart disease. Notably, however, younger individuals who received mRNA vaccines had a higher heart disease risk compared to older individuals. These results may suggest the need to consider alternative vaccine options for the younger population.

Blasco et al; 2024; “Association of SARS-CoV-2 immunoserology and vaccination status with myocardial infarction severity and outcome” (https://pubmed.ncbi.nlm.nih.gov/39244425/)

Results: Total sample of 949 patients, 656 with ST-segment elevation MI (STEMI) and 293 with non-ST-segment elevation MI (NSTEMI). Mean age was 64 (SD 13) years, 80 % men. Pre-admission vaccination status was: ≥ 1 dose, 53 % of patients; complete vaccination, 49 %; first booster dose, 25 %. The majority (84 %) of vaccines administered were mRNA-based. Six months after MI, 92 (9.7 %) patients had a major adverse cardiac event (MACE) and 50 died; 11 % of patients had severe heart failure or cardiogenic shock (Killip III-IV) after STEMI. Vaccinated patients with STEMI and positive serology (Pos/Vax group) had a higher risk of Killip III-IV on admission: OR 2.63 (1.27-5.44), p = 0.010. SARS-CoV-2 S-specific IgG titers were highest in this group (median > 2080 AU/mL, [IQR 1560- >2080] vs 91 [32-198] in the unvaccinated group). In the overall sample, a higher incidence of 6-month MACE was not demonstrated (OR 1.89 [0.98-3.61], p = 0.055).

Conclusions: The combination of vaccination and natural SARS-CoV2 infection was associated with the development of severe heart failure and cardiogenic shock in patients with STEMI, possibly related to an increased serological response.

Jain et al; 2024; “Cardiac manifestations and outcomes of COVID-19 vaccine-associated myocarditis in the young in the USA: longitudinal results from the Myocarditis After COVID Vaccination (MACiV) multicenter study” (https://www.thelancet.com/journals/eclinm/article/PIIS2589-5370(24)00388-2/fulltext)

Findings Patients with C-VAM were predominantly white (67%) adolescent males (91%, 15.7 ± 2.8 years). Their initial clinical course was more likely to be mild (80% vs. 23%, p < 0.001) and cardiac dysfunction was less common (17% vs. 68%, p < 0.0001), compared to MIS-C. In contrast, LGE on CMR was more prevalent in C-VAM (82% vs. 16%, p < 0.001). The probability of LGE was higher in males (OR 3.28 [95% CI: 0.99, 10.6, p = 0.052]), in older patients (>15 years, OR 2.74 [95% CI: 1.28, 5.83, p = 0.009]) and when C-VAM occurred after the first or second dose as compared to the third dose of mRNA vaccine. Mid-term clinical outcomes of C-VAM at a median follow-up of 178 days (IQR 114–285 days) were reassuring. No cardiac deaths or heart transplantations were reported until the time of submission of this report. LGE persisted in 60% of the patients at follow up.

Interpretation Myocardial injury at initial presentation and its persistence at follow up, despite a mild initial course and favorable mid-term clinical outcome, warrants continued clinical surveillance and long-term studies in affected patients with C-VAM.

Schreckenberg et al; 2024; “Cardiac side effects of RNA-based SARS-CoV-2 vaccines: Hidden cardiotoxic effects of mRNA-1273 and BNT162b2 on ventricular myocyte function and structure” (https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.16262)

Key Results

In the first 24 h after application, both mRNA-1273 and BNT162b2 caused neither functional disturbances nor morphological abnormalities. After 48 h, expression of the encoded spike protein was detected in ventricular cardiomyocytes for both mRNAs. At this point in time, mRNA-1273 induced arrhythmic as well as completely irregular contractions associated with irregular as well as localized calcium transients, which provide indications of significant dysfunction of the cardiac ryanodine receptor (RyR2). In contrast, BNT162b2 increased cardiomyocyte contraction via significantly increased protein kinase A (PKA) activity at the cellular level.

Conclusion and Implications

Here, we demonstrated for the first time, that in isolated cardiomyocytes, both mRNA-1273 and BNT162b2 induce specific dysfunctions that correlate pathophysiologically to cardiomyopathy. Both RyR2 impairment and sustained PKA activation may significantly increase the risk of acute cardiac events.

Barmada et al; 2023; “Cytokinopathy with aberrant cytotoxic lymphocytes and profibrotic myeloid response in SARS-CoV-2 mRNA vaccine-associated myocarditis” (https://pubmed.ncbi.nlm.nih.gov/37146127/)

Rare immune-mediated cardiac tissue inflammation can occur after vaccination, including after SARS-CoV-2 mRNA vaccines. However, the underlying immune cellular and molecular mechanisms driving this pathology remain poorly understood. Here, we investigated a cohort of patients who developed myocarditis and/or pericarditis with elevated troponin, B-type natriuretic peptide, and C-reactive protein levels as well as cardiac imaging abnormalities shortly after SARS-CoV-2 mRNA vaccination. Contrary to early hypotheses, patients did not demonstrate features of hypersensitivity myocarditis, nor did they have exaggerated SARS-CoV-2-specific or neutralizing antibody responses consistent with a hyperimmune humoral mechanism.

We additionally found no evidence of cardiac-targeted autoantibodies. Instead, unbiased systematic immune serum profiling revealed elevations in circulating interleukins (IL-1β, IL-1RA, and IL-15), chemokines (CCL4, CXCL1, and CXCL10), and matrix metalloproteases (MMP1, MMP8, MMP9, and TIMP1). Subsequent deep immune profiling using single-cell RNA and repertoire sequencing of peripheral blood mononuclear cells during acute disease revealed expansion of activated CXCR3⁺ cytotoxic T cells and NK cells, both phenotypically resembling cytokine-driven killer cells. In addition, patients displayed signatures of inflammatory and profibrotic CCR2⁺ CD163⁺ monocytes, coupled with elevated serum-soluble CD163, that may be linked to the late gadolinium enhancement on cardiac MRI, which can persist for months after vaccination.

Together, our results demonstrate up-regulation in inflammatory cytokines and corresponding lymphocytes with tissue-damaging capabilities, suggesting a cytokine-dependent pathology, which may further be accompanied by myeloid cell-associated cardiac fibrosis. These findings likely rule out some previously proposed mechanisms of mRNA vaccine–associated myopericarditis and point to new ones with relevance to vaccine development and clinical care.

Koizumi and Ono; 2025; “Cardiac Multiple Micro-Scars: An Autopsy Study” (https://www.jacc.org/doi/10.1016/j.jaccas.2024.103083)

Case Summary

Multiple micro-scars (MMS) found in the myocardium of 3 patients who died of unexplained cardiac arrest were presented at our clinicopathology conference. Upon review of the clinical record, patients with MMS before death had arrhythmia (ie, atrial fibrillation and nonsustained ventricular tachycardia, including new onset). Interestingly, MMS were found in the left ventricle, the junction of the pulmonary vein and left atrium, and the right ventricle and right atrium. All 3 patients had histories of COVID-19 booster vaccination, and 1 of the 3 patients had a history of COVID-19.

Discussion

For patients with unexplained cardiac arrest complicated with arrhythmia, cardiac MMS is given as the differential background disease.

6b. Myocarditis / Pericarditis research

Li et al; 2022; “Intravenous Injection of Coronavirus Disease 2019 (COVID-19) mRNA Vaccine Can Induce Acute Myopericarditis in Mouse Model” (https://academic.oup.com/cid/article/74/11/1933/6353927?login=false)

Results

Although significant weight loss and higher serum cytokine/chemokine levels were found in IM group at 1–2 days post-injection (dpi), only IV group developed histopathological changes of myopericarditis as evidenced by cardiomyocyte degeneration, apoptosis, and necrosis with adjacent inflammatory cell infiltration and calcific deposits on visceral pericardium, although evidence of coronary artery or other cardiac pathologies was absent. Serum troponin level was significantly higher in IV group. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike antigen expression by immunostaining was occasionally found in infiltrating immune cells of the heart or injection site, in cardiomyocytes and intracardiac vascular endothelial cells, but not skeletal myocytes. The histological changes of myopericarditis after the first IV-priming dose persisted for 2 weeks and were markedly aggravated by a second IM- or IV-booster dose. Cardiac tissue mRNA expression of interleukin (IL)-1β, interferon (IFN)-β, IL-6, and tumor necrosis factor (TNF)-α increased significantly from 1 dpi to 2 dpi in the IV group but not the IM group, compatible with presence of myopericarditis in the IV group. Ballooning degeneration of hepatocytes was consistently found in the IV group. All other organs appeared normal.

Conclusions

This study provided in vivo evidence that inadvertent intravenous injection of COVID-19 mRNA vaccines may induce myopericarditis. Brief withdrawal of syringe plunger to exclude blood aspiration may be one possible way to reduce such risk.

Block et al; 2022; “Cardiac Complications After SARS-CoV-2 Infection and mRNA COVID-19 Vaccination — PCORnet, United States, January 2021–January 2022” (https://www.cdc.gov/mmwr/volumes/71/wr/mm7114e1.htm)

Block et al. reported myocarditis incidences per 100,000 for males aged 12–17 years as follows:

• Second vaccine dose: 22.0–35.9 cases
• Post-infection: 50.1–64.9 cases

While infection poses a higher absolute risk, the risk from the second vaccine dose (approximately 1 in 2,800 to 1 in 4,500) remains significant, particularly given the lower overall risk of severe disease in this demographic.

For males aged 18–29 years:

• Second vaccine dose: 6.5–15.0 cases per 100,000
• Post-infection: 55.3–100.6 cases per 100,000

Complementary studies like Patone et al. (2022) and Oster et al. (2022) estimate myocarditis rates after the second dose in males aged 16–29 to be 1 in 5,000 to 1 in 6,000, depending on the vaccine product. Together, these findings highlight the need for careful risk-benefit analysis in young males, balancing the myocarditis risk from vaccination against the higher risks posed by infection.

Oster et al; 2022; “Myocarditis Cases Reported After mRNA-Based COVID-19 Vaccination in the US From December 2020 to August 2021” (https://jamanetwork.com/journals/jama/fullarticle/2788346)

Results  Among 192 405 448 persons receiving a total of 354 100 845 mRNA-based COVID-19 vaccines during the study period, there were 1991 reports of myocarditis to VAERS and 1626 of these reports met the case definition of myocarditis. Of those with myocarditis, the median age was 21 years (IQR, 16-31 years) and the median time to symptom onset was 2 days (IQR, 1-3 days). Males comprised 82% of the myocarditis cases for whom sex was reported. The crude reporting rates for cases of myocarditis within 7 days after COVID-19 vaccination exceeded the expected rates of myocarditis across multiple age and sex strata. The rates of myocarditis were highest after the second vaccination dose in adolescent males aged 12 to 15 years (70.7 per million doses of the BNT162b2 vaccine), in adolescent males aged 16 to 17 years (105.9 per million doses of the BNT162b2 vaccine), and in young men aged 18 to 24 years (52.4 and 56.3 per million doses of the BNT162b2 vaccine and the mRNA-1273 vaccine, respectively). There were 826 cases of myocarditis among those younger than 30 years of age who had detailed clinical information available; of these cases, 792 of 809 (98%) had elevated troponin levels, 569 of 794 (72%) had abnormal electrocardiogram results, and 223 of 312 (72%) had abnormal cardiac magnetic resonance imaging results. Approximately 96% of persons (784/813) were hospitalized and 87% (577/661) of these had resolution of presenting symptoms by hospital discharge. The most common treatment was nonsteroidal anti-inflammatory drugs (589/676; 87%).

Mansanguan et al; 2022; “Cardiovascular Manifestation of the BNT162b2 mRNA COVID-19 Vaccine in Adolescents” (https://www.mdpi.com/2414-6366/7/8/196)

This prospective cohort study enrolled students aged 13–18 years from two schools, who received the second dose of the BNT162b2 mRNA COVID-19 vaccine. Data including demographics, symptoms, vital signs, ECG, echocardiography, and cardiac enzymes were collected at baseline, Day 3, Day 7, and Day 14 (optional) using case record forms. We enrolled 314 participants; of these, 13 participants were lost to follow-up, leaving 301 participants for analysis. The most common cardiovascular signs and symptoms were tachycardia (7.64%), shortness of breath (6.64%), palpitation (4.32%), chest pain (4.32%), and hypertension (3.99%). One participant could have more than one sign and/or symptom. Seven participants (2.33%) exhibited at least one elevated cardiac biomarker or positive lab assessments. Cardiovascular manifestations were found in 29.24% of patients, ranging from tachycardia or palpitation to myopericarditis. Myopericarditis was confirmed in one patient after vaccination. Two patients had suspected pericarditis and four patients had suspected subclinical myocarditis. In conclusion, Cardiovascular manifestation in adolescents after BNT162b2 mRNA COVID-19 vaccination included tachycardia, palpitation, and myopericarditis.

Chiu et al; 2023; “Changes of ECG parameters after BNT162b2 vaccine in the senior high school students” (https://pubmed.ncbi.nlm.nih.gov/36602621/)

Among 7934 eligible students, 4928 (62.1%) were included in the study. The male/female ratio was 4576/352. In total, 763 students (17.1%) had at least one cardiac symptom after the second vaccine dose, mostly chest pain and palpitations. The depolarization and repolarization parameters (QRS duration and QT interval) decreased significantly after the vaccine with increasing heart rate. Abnormal ECGs were obtained in 51 (1.0%) of the students, of which 1 was diagnosed with mild myocarditis and another 4 were judged to have significant arrhythmia. None of the patients needed to be admitted to hospital and all of these symptoms improved spontaneously. Using these five students as a positive outcome, the sensitivity and specificity of this screening method were 100% and 99.1%, respectively.

Conclusion: Cardiac symptoms are common after the second dose of BNT162b2 vaccine, but the incidences of significant arrhythmias and myocarditis are only 0.1%. The serial ECG screening method has high sensitivity and specificity for significant cardiac adverse effect but cost effect needs further discussed.

What is Known: • The incidence of cardiac adverse effects was reported to be as high as 1.5 per 10 000 persons after the second dose BNT162b2 COVID-19 vaccine in the young male population based on the reporting system.

What is New: • Through this mass ECG screening study after the second dose of BNT162b2 vaccine we found: (1) The depolarization and repolarization parameters (QRS duration and QT interval) decreased significantly after the vaccine with increasing heart rate; (2) the incidence of post-vaccine myocarditis and significant arrhythmia are 0.02% and 0.08%; (3) The serial ECG screening method has high sensitivity and specificity for significant cardiac adverse effect.

Jain et al; 2023; “Cardiac manifestations and outcomes of COVID-19 vaccine-associated myocarditis in the young in the USA: longitudinal results from the Myocarditis After COVID Vaccination (MACiV) multicenter study” (https://www.thelancet.com/action/showPdf?pii=S2589-5370%2824%2900388-2)

Findings Patients with C-VAM were predominantly white (67%) adolescent males (91%, 15.7 ± 2.8 years). Their initial clinical course was more likely to be mild (80% vs. 23%, p < 0.001) and cardiac dysfunction was less common (17% vs. 68%, p < 0.0001), compared to MIS-C. In contrast, LGE on CMR was more prevalent in C-VAM (82% vs. 16%, p < 0.001). The probability of LGE was higher in males (OR 3.28 [95% CI: 0.99, 10.6, p = 0.052]), in older patients (>15 years, OR 2.74 [95% CI: 1.28, 5.83, p = 0.009]) and when C-VAM occurred after the first or second dose as compared to the third dose of mRNA vaccine. Mid-term clinical outcomes of C-VAM at a median follow-up of 178 days (IQR 114–285 days) were reassuring. No cardiac deaths or heart transplant tions were reported until the time of submission of this report. LGE persisted in 60% of the patients at follow up

Krug et al; 2022; “BNT162b2 Vaccine-Associated Myo/Pericarditis in Adolescents: A Stratified Risk-Benefit Analysis” (BNT162b2 Vaccine‐Associated Myo/Pericarditis in Adolescents: A Stratified Risk‐Benefit Analysis – Krug – 2022 – European Journal of Clinical Investigation – Wiley Online Library)

Results

Cases of myo/pericarditis (n = 253) included 129 after dose 1 and 124 after dose 2; 86.9% were hospitalized. Incidence per million after dose two in male patients aged 12–15 and 16–17 was 162.2 and 93.0, respectively. Weighing post-vaccination myo/pericarditis against COVID-19 hospitalization during delta, our risk-benefit analysis suggests that among 12–17-year-olds, two-dose vaccination was uniformly favourable only in nonimmune girls with a comorbidity. In boys with prior infection and no comorbidities, even one dose carried more risk than benefit according to international estimates. In the setting of omicron, one dose may be protective in nonimmune children, but dose two does not appear to confer additional benefit at a population level.

Conclusions

Our findings strongly support individualized paediatric COVID-19 vaccination strategies which weigh protection against severe disease vs. risks of vaccine-associated myo/pericarditis. Research is needed into the nature and implications of this adverse effect as well as immunization strategies which reduce harms in this overall low-risk cohort.

Rose et al; 2024; “Determinants of COVID-19 vaccine-induced myocarditis” (https://journals.sagepub.com/doi/10.1177/20420986241226566)

Results:

We found the number of myocarditis reports in VAERS after COVID-19 vaccination in 2021 was 223 times higher than the average of all vaccines combined for the past 30 years. This represented a 2500% increase in the absolute number of reports in the first year of the campaign when comparing historical values prior to 2021. Demographic data revealed that myocarditis occurred most in youths (50%) and males (69%). A total of 76% of cases resulted in emergency care and hospitalization. Of the total myocarditis reports, 92 individuals died (3%). Myocarditis was more likely after dose 2 (p < 0.00001) and individuals less than 30 years of age were more likely than individuals older than 30 to acquire myocarditis (p < 0.00001).

Conclusion:

COVID-19 vaccination is strongly associated with a serious adverse safety signal of myocarditis, particularly in children and young adults resulting in hospitalization and death. Further investigation into the underlying mechanisms of COVID-19 vaccine-induced myocarditis is imperative to create effective mitigation strategies and ensure the safety of COVID-19 vaccination programs across populations.

Buergin et al; 2023; “Sex-specific differences in myocardial injury incidence after COVID-19 mRNA-1273 booster vaccination” (https://onlinelibrary.wiley.com/doi/10.1002/ejhf.2978)

Methods and results

Hospital employees scheduled to undergo mRNA-1273 booster vaccination were assessed for mRNA-1273 vaccination-associated myocardial injury, defined as acute dynamic increase in high-sensitivity cardiac troponin T (hs-cTnT) concentration above the sex-specific upper limit of normal on day 3 (48–96 h) after vaccination without evidence of an alternative cause. To explore possible mechanisms, antibodies against interleukin-1 receptor antagonist (IL-1RA), the SARS-CoV-2-nucleoprotein (NP) and -spike (S1) proteins and an array of 14 inflammatory cytokines were quantified. Among 777 participants (median age 37 years, 69.5% women), 40 participants (5.1%; 95% confidence interval [CI] 3.7–7.0%) had elevated hs-cTnT concentration on day 3 and mRNA-1273 vaccine-associated myocardial injury was adjudicated in 22 participants (2.8% [95% CI 1.7–4.3%]). Twenty cases occurred in women (3.7% [95% CI 2.3–5.7%]), two in men (0.8% [95% CI 0.1–3.0%]). Hs-cTnT elevations were mild and only temporary. No patient had electrocardiographic changes, and none developed major adverse cardiac events within 30 days (0% [95% CI 0–0.4%]). In the overall booster cohort, hs-cTnT concentrations (day 3; median 5, interquartile range [IQR] 4–6 ng/L) were significantly higher compared to matched controls (n = 777, median 3 [IQR 3–5] ng/L, p < 0.001). Cases had comparable systemic reactogenicity, concentrations of anti-IL-1RA, anti-NP, anti-S1, and markers quantifying systemic inflammation, but lower concentrations of interferon (IFN)-λ1 (IL-29) and granulocyte-macrophage colony-stimulating factor (GM-CSF) versus persons without vaccine-associated myocardial injury.

Takeda et al; 2024; “SARS-CoV-2 mRNA vaccine-related myocarditis and pericarditis: An analysis of the Japanese Adverse Drug Event Report database” (https://pubmed.ncbi.nlm.nih.gov/39103148/)

Results: The total number of reports was 880,999 (myocarditis: 1846; pericarditis: 761). The adverse events associated with the vaccines included myocarditis (919 cases) and pericarditis (321 cases), with the ROR [95 % CIs] being significant for both (myocarditis: 30.51 [27.82-33.45], pericarditis: 21.99 [19.03-25.40]). Furthermore, the ROR [95 % CIs] of BNT162b2 and mRNA-1273 were 15.64 [14.15-17.28] and 54.23 [48.13-61.10], respectively, for myocarditis, and 15.78 [13.52-18.42] and 27.03 [21.58-33.87], respectively, for pericarditis. Furthermore, most cases were ≤30 years or male. The period from vaccination to onset was ≤8 days, corresponding to early failure type based on analysis using the Weibull distribution. Outcomes were recovery or remission for most cases; however, they were severe or caused death in some cases.

Conclusion: In the Japanese population, SARS-CoV-2 mRNA vaccination was significantly associated with the onset of myocarditis/pericarditis. The influencing factors included age of ≤30 years and male.

Mele et al; 2021; “Myocarditis in COVID-19 patients: current problems” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823176/)

Overall, on the basis of the reports above, although myocarditis has been clearly documented at EBM or autopsy in some patients with COVID-19, there is no current evidence of myocarditis directly produced by the SARS-CoV-2 in humans. The associated lymphocytic myocarditis observed in patients with COVID-19 has been related to the generalized inflammatory reaction induced by cytokines [22]. The presence of virus particles in cardiac macrophages has been interpreted as the result of a viremic phase or, alternatively, the migration of infected alveolar macrophages in extra-pulmonary tissues [24]. As far as the eosinophilic myocarditis is concerned, it has been considered the possibility of an independent idiopathic eosinophilic myocarditis in which the stress of the COVID-19 contributed to the cardiac decompensation [34]. In the genesis of myocardial inflammatory findings at autopsy of patients with COVID-19 who died after potentially cardiotoxic anti-viral therapies, a drug-induced myocarditis should also be considered [35, 36].

Tuvali et al; 2021; “The Incidence of Myocarditis and Pericarditis in Post COVID-19 Unvaccinated Patients—A Large Population-Based Study” (https://pmc.ncbi.nlm.nih.gov/articles/PMC9025013/)

Post COVID-19 infection was not associated with either myocarditis (aHR 1.08; 95% CI 0.45 to 2.56) or pericarditis (aHR 0.53; 95% CI 0.25 to 1.13). We did not observe an increased incidence of neither pericarditis nor myocarditis in adult patients recovering from COVID-19 infection.

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Balbona et al; 2024; “Case of Myocarditis, Pericarditis, and Fatal Aortic Dissection Following COVID-19
mRNA Vaccination” (https://www.opastpublishers.com/open-access-articles/case-of-myocarditis-pericarditis-and-fatal-aortic-dissection-following-covid19-mrna-vaccination.pdf)

We present a case study of a 34-year-old male who was in good health prior to his COVID-19 mRNA vaccination. Sixteen days after his first dose, he experienced acute inflammation, sudden thoracic aortic dissection, and pericardial tamponade, rapidly leading to his death. Studies suggest that young males, in particular, appear to be at increased risk of adverse cardiac events following COVID-19 mRNA vaccination. Although the incidence of such complications are believed to be low, we propose that information gaps exist in the criteria and findings that inform both public health agencies and the public on incidence rates of even severe myocarditis and cardiac adverse events following COVID-19 vaccination. This view is shared within many COVID-19 vaccine myocarditis studies and is evident within the findings of this case of Myocarditis, Pericarditis, and Fatal Aortic Dissection presented here.

Jeon et al; 2024; “SARS-CoV2 mRNA vaccine intravenous administration induces myocarditis in chronic inflammation” (https://pmc.ncbi.nlm.nih.gov/articles/PMC11469607/)

The current COVID-19 mRNA vaccines were developed and applied for pandemic-emergent conditions. These vaccines use a small piece of the virus’s genetic material (mRNA) to stimulate an immune response against COVID-19. However, their potential effects on individuals with chronic inflammatory conditions and vaccination routes remain questionable. Therefore, we investigated the effects of mRNA vaccines in a mouse model of chronic inflammation, focusing on their cardiac toxicity and immunogenicity dependent on the injection route. mRNA vaccine intravenous administration with or without chronic inflammation exacerbated cardiac pericarditis and myocarditis; immunization induced mild inflammation and inflammatory cytokine IL-1beta and IL-6 production in the heart. Further, IV mRNA vaccination induced cardiac damage in LPS chronic inflammation, particularly serum troponin I (TnI), which dramatically increased. IV vaccine administration may induce more cardiotoxicity in chronic inflammation. These findings highlight the need for further research to understand the underlying mechanisms of mRNA vaccines with chronic inflammatory conditions dependent on injection routes.

Stowe et al; 2023; “Risk of myocarditis and pericarditis after a COVID-19 mRNA vaccine booster and after COVID-19 in those with and without prior SARS-CoV-2 infection: A self-controlled case series analysis in England” (https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1004245)

We conducted a self-controlled case series analysis of hospital admissions for myocarditis or pericarditis in England between 22 February 2021 and 6 February 2022 in the 50 million individuals eligible to receive the adenovirus-vectored vaccine (ChAdOx1-S) for priming or an mRNA vaccine (BNT162b2 or mRNA-1273) for priming or boosting. Myocarditis and pericarditis admissions were extracted from the Secondary Uses Service (SUS) database in England and vaccination histories from the National Immunisation Management System (NIMS); prior infections were obtained from the UK Health Security Agency’s Second-Generation Surveillance Systems. The relative incidence (RI) of admission within 0 to 6 and 7 to 14 days of vaccination compared with periods outside these risk windows stratified by age, dose, and prior SARS-CoV-2 infection for individuals aged 12 to 101 years was estimated. The RI within 27 days of an infection was assessed in the same model. There were 2,284 admissions for myocarditis and 1,651 for pericarditis in the study period. Elevated RIs were only observed in 16- to 39-year-olds 0 to 6 days postvaccination, mainly in males for myocarditis. Both mRNA vaccines showed elevated RIs after first, second, and third doses with the highest RIs after a second dose 5.34 (95% confidence interval (CI) [3.81, 7.48]; p < 0.001) for BNT162b2 and 56.48 (95% CI [33.95, 93.97]; p < 0.001) for mRNA-1273 compared with 4.38 (95% CI [2.59, 7.38]; p < 0.001) and 7.88 (95% CI [4.02, 15.44]; p < 0.001), respectively, after a third dose. For ChAdOx1-S, an elevated RI was only observed after a first dose, RI 5.23 (95% CI [2.48, 11.01]; p < 0.001). An elevated risk of admission for pericarditis was only observed 0 to 6 days after a second dose of mRNA-1273 vaccine in 16 to 39 year olds, RI 4.84 (95% CI [1.62, 14.01]; p = 0.004). RIs were lower in those with a prior SARS-CoV-2 infection than in those without, 2.47 (95% CI [1.32,4.63]; p = 0.005) versus 4.45 (95% [3.12, 6.34]; p = 0.001) after a second BNT162b2 dose, and 19.07 (95% CI [8.62, 42.19]; p < 0.001) versus 37.2 (95% CI [22.18, 62.38]; p < 0.001) for mRNA-1273 (myocarditis and pericarditis outcomes combined). RIs 1 to 27 days postinfection were elevated in all ages and were marginally lower for breakthrough infections, 2.33 (95% CI [1.96, 2.76]; p < 0.001) compared with 3.32 (95% CI [2.54, 4.33]; p < 0.001) in vaccine-naïve individuals respectively.

Conclusions

We observed an increased risk of myocarditis within the first week after priming and booster doses of mRNA vaccines, predominantly in males under 40 years with the highest risks after a second dose. The risk difference between the second and the third doses was particularly marked for the mRNA-1273 vaccine that contains half the amount of mRNA when used for boosting than priming. The lower risk in those with prior SARS-CoV-2 infection, and lack of an enhanced effect post-booster, does not suggest a spike-directed immune mechanism. Research to understand the mechanism of vaccine-associated myocarditis and to document the risk with bivalent mRNA vaccines is warranted.

Mevorach et al; 2021; “Myocarditis after BNT162b2 mRNA Vaccine against Covid-19 in Israel” (https://www.nejm.org/doi/full/10.1056/NEJMoa2109730)

Results

Among 304 persons with symptoms of myocarditis, 21 had received an alternative diagnosis. Of the remaining 283 cases, 142 occurred after receipt of the BNT162b2 vaccine; of these cases, 136 diagnoses were definitive or probable. The clinical presentation was judged to be mild in 129 recipients (95%); one fulminant case was fatal. The overall risk difference between the first and second doses was 1.76 per 100,000 persons (95% confidence interval [CI], 1.33 to 2.19), with the largest difference among male recipients between the ages of 16 and 19 years (difference, 13.73 per 100,000 persons; 95% CI, 8.11 to 19.46). As compared with the expected incidence based on historical data, the standardized incidence ratio was 5.34 (95% CI, 4.48 to 6.40) and was highest after the second dose in male recipients between the ages of 16 and 19 years (13.60; 95% CI, 9.30 to 19.20). The rate ratio 30 days after the second vaccine dose in fully vaccinated recipients, as compared with unvaccinated persons, was 2.35 (95% CI, 1.10 to 5.02); the rate ratio was again highest in male recipients between the ages of 16 and 19 years (8.96; 95% CI, 4.50 to 17.83), with a ratio of 1 in 6637.

Conclusions

The incidence of myocarditis, although low, increased after the receipt of the BNT162b2 vaccine, particularly after the second dose among young male recipients. The clinical presentation of myocarditis after vaccination was usually mild.

Patone et al; 2022; “Risk of Myocarditis After Sequential Doses of COVID-19 Vaccine and SARS-CoV-2 Infection by Age and Sex” (https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.122.059970)

Results:

In 42 842 345 people receiving at least 1 dose of vaccine, 21 242 629 received 3 doses, and 5 934 153 had SARS-CoV-2 infection before or after vaccination. Myocarditis occurred in 2861 (0.007%) people, with 617 events 1 to 28 days after vaccination. Risk of myocarditis was increased in the 1 to 28 days after a first dose of ChAdOx1 (incidence rate ratio, 1.33 [95% CI, 1.09–1.62]) and a first, second, and booster dose of BNT162b2 (1.52 [95% CI, 1.24–1.85]; 1.57 [95% CI, 1.28–1.92], and 1.72 [95% CI, 1.33–2.22], respectively) but was lower than the risks after a positive SARS-CoV-2 test before or after vaccination (11.14 [95% CI, 8.64–14.36] and 5.97 [95% CI, 4.54–7.87], respectively). The risk of myocarditis was higher 1 to 28 days after a second dose of mRNA-1273 (11.76 [95% CI, 7.25–19.08]) and persisted after a booster dose (2.64 [95% CI, 1.25–5.58]). Associations were stronger in men younger than 40 years for all vaccines. In men younger than 40 years old, the number of excess myocarditis events per million people was higher after a second dose of mRNA-1273 than after a positive SARS-CoV-2 test (97 [95% CI, 91–99] versus 16 [95% CI, 12–18]). In women younger than 40 years, the number of excess events per million was similar after a second dose of mRNA-1273 and a positive test (7 [95% CI, 1–9] versus 8 [95% CI, 6–8]).

Elevated Myocarditis Risk After COVID-19 Vaccination – Editor take

The above 2022 study by Patone et al. found that the risk of myocarditis was significantly elevated following mRNA COVID-19 vaccination, particularly after the second dose of Moderna’s mRNA-1273 vaccine. The study, which analyzed data from over 42 million people, showed that in men under 40, the risk of myocarditis after the second Moderna dose was 97 excess cases per million—over six times higher than the myocarditis risk after a COVID-19 infection (16 per million). The risk remained elevated even after a booster dose, contradicting claims that vaccine-induced myocarditis is both rare and mild.

Public health officials repeatedly claimed that COVID-19 infection carried a greater risk of myocarditis than vaccination, but the data from this large-scale study directly refutes that narrative for young men. With the highest myocarditis rates occurring after repeated exposure to vaccine-induced spike protein, these findings call into question the safety of continued booster campaigns, particularly for lower-risk demographics. Despite these clear safety signals, governments and regulatory agencies failed to properly reassess vaccine recommendations, continuing to push a one-size-fits-all approach even when risks outweighed benefits for younger populations.

Alami et al; 2023; “Risk of myocarditis and pericarditis in mRNA COVID-19-vaccinated and unvaccinated populations: a systematic review and meta-analysis” (https://bmjopen.bmj.com/content/13/6/e065687)

Study selection Epidemiological studies of individuals of any age who received at least one dose of an mRNA COVID-19 vaccine, reported a risk of myo/pericarditis and compared the risk of myo/pericarditis to individuals who did not receive any dose of an mRNA COVID-19 vaccine.

Data extraction and synthesis Two reviewers independently conducted screening and data extraction. The rate of myo/pericarditis among vaccinated and unvaccinated groups was recorded, and the rate ratios were calculated. Additionally, the total number of individuals, case ascertainment criteria, percentage of males and history of SARS-CoV-2 infection were extracted for each study. Meta-analysis was done using a random-effects model.

Results Seven studies met the inclusion criteria, of which six were included in the quantitative synthesis. Our meta-analysis indicates that within 30-day follow-up period, vaccinated individuals were twice as likely to develop myo/pericarditis in the absence of SARS-CoV-2 infection compared to unvaccinated individuals, with a rate ratio of 2.05 (95% CI 1.49–2.82).

Conclusion Although the absolute number of observed myo/pericarditis cases remains quite low, a higher risk was detected in those who received mRNA COVID-19 vaccinations compared with unvaccinated individuals in the absence of SARS-CoV-2 infection. Given the effectiveness of mRNA COVID-19 vaccines in preventing severe illnesses, hospitalisations and deaths, future research should focus on accurately determining the rates of myo/pericarditis linked to mRNA COVID-19 vaccines, understanding the biological mechanisms behind these rare cardiac events and identifying those most at risk.

Massari et al; 2022; “Postmarketing active surveillance of myocarditis and pericarditis following vaccination with COVID-19 mRNA vaccines in persons aged 12 to 39 years in Italy: A multi-database, self-controlled case series study” (https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1004056)

We conducted a self-controlled case series study (SCCS) using national data on COVID-19 vaccination linked to emergency care/hospital discharge databases. The outcome was the first diagnosis of myocarditis/pericarditis between 27 December 2020 and 30 September 2021. Exposure risk period (0 to 21 days from the vaccination day, subdivided in 3 equal intervals) for first and second dose was compared with baseline period. The SCCS model, adapted to event-dependent exposures, was fitted using unbiased estimating equations to estimate relative incidences (RIs) and excess of cases (EC) per 100,000 vaccinated by dose, age, sex, and vaccine product. Calendar period was included as time-varying confounder in the model. During the study period 2,861,809 persons aged 12 to 39 years received mRNA vaccines (2,405,759 BNT162b2; 456,050 mRNA-1273); 441 participants developed myocarditis/pericarditis (346 BNT162b2; 95 mRNA-1273). Within the 21-day risk interval, 114 myocarditis/pericarditis events occurred, the RI was 1.99 (1.30 to 3.05) after second dose of BNT162b2 and 2.22 (1.00 to 4.91) and 2.63 (1.21 to 5.71) after first and second dose of mRNA-1273. During the [0 to 7) days risk period, an increased risk of myocarditis/pericarditis was observed after first dose of mRNA-1273, with RI of 6.55 (2.73 to 15.72), and after second dose of BNT162b2 and mRNA-1273, with RIs of 3.39 (2.02 to 5.68) and 7.59 (3.26 to 17.65). The number of EC for second dose of mRNA-1273 was 5.5 per 100,000 vaccinated (3.0 to 7.9). The highest risk was observed in males, at [0 to 7) days after first and second dose of mRNA-1273 with RI of 12.28 (4.09 to 36.83) and RI of 11.91 (3.88 to 36.53); the number of EC after the second dose of mRNA-1273 was 8.8 (4.9 to 12.9). Among those aged 12 to 17 years, the RI was of 5.74 (1.52 to 21.72) after second dose of BNT162b2; for this age group, the number of events was insufficient for estimating RIs after mRNA-1273. Among those aged 18 to 29 years, the RIs were 7.58 (2.62 to 21.94) after first dose of mRNA-1273 and 4.02 (1.81 to 8.91) and 9.58 (3.32 to 27.58) after second dose of BNT162b2 and mRNA-1273; the numbers of EC were 3.4 (1.1 to 6.0) and 8.6 (4.4 to 12.6) after first and second dose of mRNA-1273. The main study limitations were that the outcome was not validated through review of clinical records, and there was an absence of information on the length of hospitalization and, thus, the severity of the outcome.

6c. Cancer research

Kyriakopoulos et al; 2024; “Oncogenesis and autoimmunity as a result of mRNA COVID-19 vaccination” (https://www.authorea.com/users/455597/articles/737938-oncogenesis-and-autoimmunity-as-a-result-of-mrna-covid-19-vaccination)

When an antigen stimulates the immune system, specific T regulatory (Treg) and T effector (Teff) subpopulations develop from naïve T cells. The Treg cell population will produce the memory Treg (mTreg) cells against that specific antigen. An inappropriate homeostatic balance among Teff, Treg and mTreg cells can direct the immune system toward either cancer or autoimmunity. When cancer is present, Treg cells suppress anti-tumor immunity, and, when cancer is absent, Treg cells play the beneficial role of preventing the development of autoimmunity. In this review, we analyze Treg responses after SARS-CoV-2 mRNA vaccination and find distinct pathological responses under differing conditions. In cancer patients, the degree of disease progression depends on the cancer status at the time of vaccination and the type of cancer treatment they receive concurrently. We hypothesize that migration of circulating dendritic cells and mTreg cells back to the thymus accelerates thymic involution, a direct cause of immunosenescence. In summary, the Treg responses produced after mRNA vaccination and the subsequent mRNA-encoded SARS-CoV-2 spike protein expression may lead to a harmful influence on the immune system of vaccinees, and subsequent accelerated development of cancer and autoimmune disease. These mechanisms are consistent with both epidemiological findings and case reports.

Zhang & El-Deiry; 2024; “SARS-CoV-2 spike S2 subunit inhibits p53 activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2 proteins in cancer cells” (https://www.biorxiv.org/content/10.1101/2024.04.12.589252v1)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 infection has led to worsened outcomes for patients with cancer. SARS-CoV-2 spike protein mediates host cell infection and cell-cell fusion that causes stabilization of tumor suppressor p53 protein. In-silico analysis previously suggested that SARS-CoV-2 spike interacts with p53 directly but this putative interaction has not been demonstrated in cells. We examined the interaction between SARS-CoV-2 spike, p53 and MDM2 (E3 ligase, which mediates p53 degradation) in cancer cells using an immunoprecipitation assay. We observed that SARS-CoV-2 spike protein interrupts p53-MDM2 protein interaction but did not detect SARS-CoV-2 spike bound with p53 protein in the cancer cells. We further observed that SARS-CoV-2 spike suppresses p53 transcriptional activity in cancer cells including after nutlin exposure of wild-type p53-, spike S2-expressing tumor cells and inhibits chemotherapy-induced p53 gene activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2. The suppressive effect of SARS-CoV-2 spike on p53-dependent gene activation provides a potential molecular mechanism by which SARS-CoV-2 infection may impact tumorigenesis, tumor progression and chemotherapy sensitivity. In fact, cisplatin-treated tumor cells expressing spike S2 were found to have increased cell viability as compared to control cells. Further observations on gamma-H2AX expression in spike S2-expressing cells treated with cisplatin may indicate altered DNA damage sensing in the DNA damage response pathway.

7. Transmission

Singanayagam et al; 2020; “Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020” (https://pmc.ncbi.nlm.nih.gov/articles/PMC7427302)

Severe acute respiratory syndrome coronavirus 2 viral load in the upper respiratory tract peaks around symptom onset and infectious virus persists for 10 days in mild-to-moderate coronavirus disease (n = 324 samples analysed). RT-PCR cycle threshold (Ct) values correlate strongly with cultivable virus. Probability of culturing virus declines to 8% in samples with Ct > 35 and to 6% 10 days after onset; it is similar in asymptomatic and symptomatic persons. Asymptomatic persons represent a source of transmissible virus.

Since the emergence of coronavirus disease (COVID-19) at the end of 2019, rapid tracing and isolation of confirmed cases and close contacts with restrictions on social movement have played an important role in controlling onward spread of the virus. Understanding the duration of infectiousness in persons who test positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to developing evidence-based public health policies on isolation, contact tracing and return to work. Virus detection by reverse transcription-PCR (RT-PCR) from respiratory samples is widely used to diagnose and monitor SARS-CoV-2 infection and, increasingly, to infer infectivity of an individual. However, RT-PCR does not distinguish between infectious and non-infectious virus. Propagating virus from clinical samples confirms the presence of infectious virus but is not widely available, requires biosafety level 3 facilities, and the results are not timely to inform public health actions. The aim of this work was to understand how RT-PCR detection relates to cultivable virus, which can be used as a proxy for infectiousness and can inform and support decisions on infection control.

Bleier et al; 2020; “COVID-19 Vaccines May Not Prevent Nasal SARS-CoV-2 Infection and Asymptomatic Transmission” (https://journals.sagepub.com/doi/full/10.1177/0194599820982633)

The divergence in immune response between mucosal and systemic vaccination is derived from the fact that human mucosal surfaces contain a localized immune system composed largely of mucosa-associated lymphoid tissue, which contributes up to 80% of all immunocytes within the body. Given that it operates within a highly heterogenous and contaminated milieu, the mucosa-associated lymphoid tissue tends to be highly compartmentalized and, to some extent, functions independently of the systemic immune system. Consequently, following local antigen exposure, activated mucosal-derived B and T cells may only selectively populate the mucosa of origin through immunocyte and mucosa–specific receptor interactions. This site specificity of the mucosal immune response thereby significantly impedes the efficacy of systemic vaccination on local nasal respiratory mucosal immunity.

Russell et al; 2020; “Mucosal Immunity in COVID-19: A Neglected but Critical Aspect of SARS-CoV-2 Infection” (https://pubmed.ncbi.nlm.nih.gov/33329607/)

Most attention has been given to virus-neutralizing antibodies, especially circulating antibodies (13–15). However, these can only be effective in the prevention of infection or disease if they reach the mucosal surfaces where the virus is present, and it should be noted that circulating IgA, even in polymeric form, is not effectively transported into secretions (16). While plasma-derived IgG occurs in the URT and especially the lower respiratory tract (LRT), IgG is inflammatory in its mode of action, by the induction of such effector mechanisms as complement activation and the engagement of phagocytes such as macrophages and neutrophils as well as natural killer (NK) cells. The serious pathology of COVID-19 occurs in the terminal airways of the lungs, where circulating IgG is the dominant immunoglobulin. The resulting intense inflammation involves multiple molecular and cellular factors, including cells recruited by virus-induced chemo-attractants (17). The cellular arm of the adaptive immune response, including CD4+ and cytotoxic CD8+ T cells, is also delivered via the circulation and can reach the alveoli. However, cytotoxic cells by their nature cannot prevent infection: they destroy already infected cells and thereby curtail further propagation of the infection.

Almost all efforts at vaccine development against COVID-19 focus on systemic injection, which predominantly induces circulatory IgG antibodies and, potentially, cytotoxic T cells (18). These routes are poorly effective at generating mucosal immune responses, which can only be induced by mucosal routes of immunization, including through the NALT in the URT. Mucosal immune responses are partly compartmentalized, as the distribution of the responses depends on the actual route of induction (7, 19). For example, the enteric route predominantly generates responses in the gastro-intestinal tract, whereas the nasal route predominantly generates responses in the respiratory tract and salivary glands (7). The reasons for these differential distributions lie in the imprinting of the T and B cells induced in the respective inductive sites, the gut-associated lymphoid tissues (GALT, such as the intestinal Peyer’s patches) or NALT, with “homing” receptors including specific integrins and chemokine receptors specific for the target tissues (20). In practical terms this means that intranasal immunization should be an effective means of generating predominantly SIgA antibody responses in the URT and LRT, where SARS-CoV-2 could be neutralized and eliminated without inflammatory consequences. In addition, it implies that assaying IgA antibodies in nasal secretions or saliva should be a more informative way of assessing effective immune responses against SARS-CoV-2, whether induced by the natural infection or by intranasal immunization.

Havervall et al; 2022; “Anti-Spike Mucosal IgA Protection against SARS-CoV-2 Omicron Infection” (https://pubmed.ncbi.nlm.nih.gov/36103621/)

We analyzed the kinetics of mucosal antibody responses after omicron breakthrough infection. Levels of spike-specific, receptor-binding domain–specific, and nucleocapsid-specific mucosal IgA increased over time after infection, in both previously infected participants and previously uninfected participants (Figure 1D and Fig. S3). This finding is in contrast to findings in recent studies by our group⁴ and Reynolds et al.,⁵ which showed omicron-induced boosting of systemic spike-specific IgG responses predominantly in participants who had not previously been infected. Levels of wild-type spike-specific mucosal IgA were not correlated with levels of wild-type spike-specific mucosal or serum IgG (Fig. S4A and S4B). However, a strong correlation was seen between levels of spike-specific serum and mucosal IgG (Spearman’s r=0.7, P<0.001) (Fig. S4C), a finding that corroborates an IgG “spillover” from the circulation to the mucosa.¹

Taken together, these findings suggest that wild-type SARS-CoV-2 spike-specific mucosal IgA is protective against omicron infection. Further studies are warranted to determine whether vaccines that induce a combination of mucosal and systemic immune responses would confer stronger protection than intramuscular vaccines.

Franco-Paredes; 2022; “Transmissibility of SARS-CoV-2 among fully vaccinated individuals“ (https://www.thelancet.com/journals/laninf/article/PIIS1473-30992100768-4/fulltext)

Vaccine effectiveness studies have conclusively demonstrated the benefit of COVID-19 vaccines in reducing individual symptomatic and severe disease, resulting in reduced hospitalisations and intensive care unit admissions.¹ However, the impact of vaccination on transmissibility of SARS-CoV-2 needs to be elucidated. A prospective cohort study in the UK by Anika Singanayagam and colleagues² regarding community transmission of SARS-CoV-2 among unvaccinated and vaccinated individuals provides important information that needs to be considered in reassessing vaccination policies. This study showed that the impact of vaccination on community transmission of circulating variants of SARS-CoV-2 appeared to be not significantly different from the impact among unvaccinated people.^2, 3 The scientific rationale for mandatory vaccination in the USA relies on the premise that vaccination prevents transmission to others, resulting in a “pandemic of the unvaccinated”.⁴ Yet, the demonstration of COVID-19 breakthrough infections among fully vaccinated health-care workers (HCW) in Israel, who in turn may transmit this infection to their patients,⁵ requires a reassessment of compulsory vaccination policies leading to the job dismissal of unvaccinated HCW in the USA. Indeed, there is growing evidence that peak viral titres in the upper airways of the lungs and culturable virus are similar in vaccinated and unvaccinated individuals.^2,3,5–7 A recent investigation by the US Centers for Disease Control and Prevention of an outbreak of COVID-19 in a prison in Texas showed the equal presence of infectious virus in the nasopharynx of vaccinated and unvaccinated individuals.⁶ Similarly, researchers in California observed no major differences between vaccinated and unvaccinated individuals in terms of SARS-CoV-2 viral loads in the nasopharynx, even in those with proven asymptomatic infection.⁷

Thus, the current evidence suggests that current mandatory vaccination policies might need to be reconsidered, and that vaccination status should not replace mitigation practices such as mask wearing, physical distancing, and contact-tracing investigations, even within highly vaccinated populations.

Lane et al; 2023; “Quantity of SARS-CoV-2 RNA copies exhaled per minute during natural breathing over the course of COVID-19 infection” (https://www.medrxiv.org/content/10.1101/2023.09.06.23295138v1.full)

Vaccinated and unvaccinated individuals exhaled similar levels of SARS-CoV-2 RNA

Whether vaccinated individuals who experience breakthrough infection with COVID-19 shed lower levels of virus on breath is unknown. Our data set included 57 samples collected by 11 vaccinated participants with breakthrough infections (Table 1). We found that vaccinated and unvaccinated participants exhaled similar numbers of SARS-CoV-2 RNA copies (Fig 2C), when accounting for age, sex, presence of co-morbidities, days since symptom onset and symptom severity (Supplementary Appendix, Wilcoxon rank sum test, P = 0.31, z = 1.02) (Fig 3A). Viral RNA levels in 57 samples collected by vaccinated individuals on days 1 to 16 from symptom onset ranged from 0 to 549 exhaled copies per minute (%95 CI: [17, 78]) while viral loads for unvaccinated individuals on days 1 to 16 ranged from 0 to 876 exhaled copies per minute (%95 CI: [25, 55]).

Galbadage et al; 2020; “Does COVID-19 Spread Through Droplets Alone?” (https://www.frontiersin.org/articles/10.3389/fpubh.2020.00163/full)

Acharya et al; 2022; “Viral Load Among Vaccinated and Unvaccinated, Asymptomatic and Symptomatic Persons Infected With the SARS-CoV-2 Delta Variant” (https://academic.oup.com/ofid/article/9/5/ofac135/6550312?login=false)

We found no significant difference in cycle threshold values between vaccinated and unvaccinated persons infected with severe acute respiratory syndrome coronavirus 2 Delta, overall or stratified by symptoms. Given the substantial proportion of asymptomatic vaccine breakthrough cases with high viral levels, interventions, including masking and testing, should be considered in settings with elevated coronavirus disease 2019 transmission.

Woodbridge et al; 2022; “Viral load dynamics of SARS-CoV-2 Delta and Omicron variants following multiple vaccine doses and previous infection“ (https://www.nature.com/articles/s41467-022-33096-0)

This study indicates that overall the presumed vaccination-related immunity to SARS-CoV-2 has only a negligible long term (>70-days) effect on Ct value, a common surrogate for VL and infectiousness. The combination of vaccine waning and vaccine evasion are most likely the drivers of this finding. In lieu of several prominent publications describing vaccine effectiveness in prevention morbidity and hospitalization for Omicron18,19,20, this study mandates reevaluating the role of current vaccination campaigns in harnessing the potential infectivity of COVID-19 at a time scale >2 months.

Sheikh-Mohamed et al; 2022; “Systemic and mucosal IgA responses are variably induced in response to SARS-CoV-2 mRNA vaccination and are associated with protection against subsequent infection” (https://www.nature.com/articles/s41385-022-00511-0)

Although SARS-CoV-2 infects the upper respiratory tract, we know little about the amount, type, and kinetics of antibodies (Ab) generated in the oral cavity in response to COVID-19 vaccination. We collected serum and saliva samples from participants receiving two doses of mRNA COVID-19 vaccines and measured the level of anti-SARS-CoV-2 Ab. We detected anti-Spike and anti-Receptor Binding Domain (RBD) IgG and IgA, as well as anti-Spike/RBD associated secretory component in the saliva of most participants after dose 1. Administration of a second dose of mRNA boosted the IgG but not the IgA response, with only 30% of participants remaining positive for IgA at this timepoint. At 6 months post-dose 2, these participants exhibited diminished anti-Spike/RBD IgG levels, although secretory component-associated anti-Spike Ab were more stable. Examining two prospective cohorts we found that participants who experienced breakthrough infections with SARS-CoV-2 variants had lower levels of vaccine-induced serum anti-Spike/RBD IgA at 2–4 weeks post-dose 2 compared to participants who did not experience an infection, whereas IgG levels were comparable between groups. These data suggest that COVID-19 vaccines that elicit a durable IgA response may have utility in preventing infection.

Participants who experience a breakthrough infection have lower levels of vaccination-induced anti-Spike IgA

Correlates of protection against SARS-CoV-2 breakthrough infection are ill-described and have only been examined for IgG¹⁰. Given that we have detected anti-Spike/RBD IgA in the serum and saliva of mRNA vaccinated participants, we examined whether IgA levels may be associated with protection against breakthrough infection. On 20th April 2021, an outbreak of P.1 lineage SARS-CoV-2 (Gamma variant) was declared in a Toronto LTCH home¹¹. The residents, who were all doubly vaccinated with mRNA-1273, were part of a separate serum antibody study following LTCH residents that included sampling of blood, but not saliva, at 2–4 weeks post-dose 2 (received on February 2, 2021). Since anti-Spike and anti-RBD IgA levels correlate with each other at 2–4 weeks post-dose 2 (Supplementary Fig. 2), we also measured serum anti-Spike IgA at this time point as a proxy of the salivary IgA response. In the context of this isolated outbreak where n = 5 residents were infected we did not see a significant difference between exposed infected vs. exposed uninfected participants in terms of levels of anti-Spike and anti-RBD IgG at 2–4 weeks post-dose 2 (Fig. 4A). We noted a trend of reduced anti-Spike and anti-RBD IgA levels in exposed infected vs. exposed uninfected participants that reached significance but did not survive a post hoc multiple test correction (Fig. 4B).

Given the number of breakthrough infections in this isolated outbreak was small, we tested a replication cohort. We therefore measured serum antibody levels at 2–4 weeks post-dose 2 in a larger case–control cohort of healthcare workers (HCW) who had received 2 doses of BNT162b2 at the Sheba Medical Center in Ramat Gan, Israel, with the majority of cases infected with the Alpha SARS-CoV-2 variant¹⁰. We found that anti-Spike/RBD IgG levels were modestly but not significantly lower in the serum of cases versus controls (Fig. 4C). In contrast, anti-Spike and anti-RBD IgA levels were both significantly lower in cases vs. controls, and this held up to a multiple comparison test (Fig. 4D). Using the more well-powered Sheba cohort, if we convert these values to the BAU/ml using the 20/136 WHO serum standard (Supplementary Table 7), the median level of IgA that is associated with breakthrough cases is 152.78 BAU/ml and 162.31 BAU/ml for anti-Spike and anti-RBD respectively compared to uninfected controls whose median IgA levels were 471.36 BAU/ml and 495.68 BAU/ml for anti-Spike and anti-RBD respectively.

Boucau et al; 2022; “Duration of Shedding of Culturable Virus in SARS-CoV-2 Omicron (BA.1) Infection” (https://www.nejm.org/doi/full/10.1056/NEJMc2202092)

In this longitudinal cohort of participants, most of whom had symptomatic, nonsevere Covid-19 infection, the viral decay kinetics were similar with omicron infection and delta infection. Although vaccination has been shown to reduce the incidence of infection and the severity of disease, we did not find large differences in the median duration of viral shedding among participants who were unvaccinated, those who were vaccinated but not boosted, and those who were vaccinated and boosted.

Lyngse et al; 2022; “Household transmission of the SARS-CoV-2 Omicron variant in Denmark” (https://www.nature.com/articles/s41467-022-33328-3)

Among 26,675 households (8,568 with the Omicron VOC), we identified 14,140 secondary infections within a 1–7-day follow-up period. The secondary attack rate was 29% and 21% in households infected with Omicron and Delta, respectively. For Omicron, the odds of infection were 1.10 (95%-CI: 1.00-1.21) times higher for unvaccinated, 2.38 (95%-CI: 2.23-2.54) times higher for fully vaccinated and 3.20 (95%-CI: 2.67-3.83) times higher for booster-vaccinated contacts compared to Delta. We conclude that the transition from Delta to Omicron VOC was primarily driven by immune evasiveness and to a lesser extent an inherent increase in the basic transmissibility of the Omicron variant.

White et al; 2021; “Incident SARS-CoV-2 Infection among mRNA-Vaccinated and Unvaccinated Nursing Home Residents” (https://www.nejm.org/doi/full/10.1056/NEJMc2104849)

Administration of covid-19 mRNA vaccine to Nursing Home patients did NOT lower total SARSCOV2 infections, barely lowered symptomatic infections (-0.39%), vs. unvaxxed, when analyzed appropriately, i.e., incl ALL infections!

1240/18,242 =6.8% with ANY incident SARS-CoV-2 infections among the vaccinated, & 270/3,990 also=6.8% incident SARSCOV2 infections among the unvaccinated by including infections which accrue days 0-14 Vaccinated with at least one dose=335/18,242=0.0184 → (1.84%) with SYMPTOMATIC incident SARS-CoV-2 infections among the vaccinated, & 90/3,990=0.0223 = (2.23%), a mere 0.39% absolute reduction, & a Number Needed to Vaccinate of 256 to prevent 1 SYMPTOMATIC SARS-CoV-2 infection

Supplementary Table S3

Nursing home staff vaccination rate showed NO association with resident infection rate. Yet despite this hospitals across the country fired unvaccinated nurses claiming they placed patients at risk.

Rolfes et al; 2023; “Reduced risk of SARS-CoV-2 infection among household contacts with recent vaccination and past COVID-19 infection: results from two multi-site case-ascertained household transmission studies” (https://www.medrxiv.org/content/10.1101/2023.10.20.23297317v1)

Findings There were 1,532 contacts from 905 households included in this analysis. Of these, 67% were enrolled May–November 2022, when Omicron BA.4/5 predominated. Most contacts (89%) had some immunity to SARS-CoV-2 at the time of household exposure: 8% had immunity from prior infection alone, 51% from vaccination alone, and 29% had hybrid immunity. Sixty percent of contacts tested SARS-CoV-2-positive during follow-up. The risk of SARS-CoV-2 infection was not significantly reduced by vaccination but was reduced among those with prior infection considering such immunity separately (adjusted relative risk 0.83; 95% confidence interval: 0.77, 0.90); however, when accounting for both sources of immunity, only contacts with vaccination and prior infection had significantly reduced risk of infection (aRR: 0.81, 95% CI: 0.70, 0.93). The risk of infection was lower when the last immunizing event (vaccination or infection) occurred ≤6 months before COVID-19 affected the household (aRR: 0.69, 95% CI: 0.57, 0.83).

Chau et al; 2021; “Transmission of SARS-CoV-2 Delta Variant Among Vaccinated Healthcare Workers, Vietnam” (https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3897733)

Between 11th–25th June 2021 (week 7–8 after dose 2), 69 healthcare workers were tested positive for SARS-CoV-2. 62 participated in the clinical study. 49 were (pre)symptomatic with one requiring oxygen supplementation. All recovered uneventfully. 23 complete-genome sequences were obtained. They all belonged to the Delta variant, and were phylogenetically distinct from the contemporary Delta variant sequences obtained from community transmission cases, suggestive of ongoing transmission between the workers. Viral loads of breakthrough Delta variant infection cases were 251 times higher than those of cases infected with old strains detected between March-April 2020. Time from diagnosis to PCR negative was 8–33 days (median: 21). Neutralizing antibody levels after vaccination and at diagnosis of the cases were lower than those in the matched uninfected controls. There was no correlation between vaccine-induced neutralizing antibody levels and viral loads or the development of symptoms.

Yoshimura et al; 2024; “Insufficient anti-spike RBD IgA responses after triple vaccination with intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2” (https://pubmed.ncbi.nlm.nih.gov/38187240/)

Results: The seropositivity of anti-RBD IgA at 1 M after the second vaccine (2D-1M) and after the third dose (3D-1M) was 65.4% and 87.4%, respectively, and wanes quickly. The boosting effect on anti-RBD Ab titers following breakthrough infections was more notable for anti-RBD IgA than for IgG. There were partial cause-relationships between the lower anti-RBD IgA or IgG at pre-breakthrough sera and the breakthrough infection.

Conclusions: Parenterally administered COVID-19 vaccines do not generate sufficient mucosal-type IgA responses despite strong systemic IgG responses to SARS-CoV-2. These results demonstrate the necessity and importance of reevaluating vaccine design and scheduling to efficiently increase oral or respiratory mucosal immunity against SARS-CoV-2.

Focosi et al; 2024; “Mucosal Vaccines, Sterilizing Immunity, and the Future of SARS-CoV-2 Virulence” (https://pubmed.ncbi.nlm.nih.gov/35215783/)

Sterilizing immunity after vaccination is desirable to prevent the spread of infection from vaccinees, which can be especially dangerous in hospital settings while managing frail patients. Sterilizing immunity requires neutralizing antibodies at the site of infection, which for respiratory viruses such as SARS-CoV-2 implies the occurrence of neutralizing IgA in mucosal secretions. Systemic vaccination by intramuscular delivery induces no or low-titer neutralizing IgA against vaccine antigens. Mucosal priming or boosting, is needed to provide sterilizing immunity. On the other side of the coin, sterilizing immunity, by zeroing interhuman transmission, could confine SARS-CoV-2 in animal reservoirs, preventing spontaneous attenuation of virulence in humans as presumably happened with the endemic coronaviruses. We review here the pros and cons of each vaccination strategy, the current mucosal SARS-CoV-2 vaccines under development, and their implications for public health.

Lasrado et al; 2024; “SARS-CoV-2 XBB.1.5 mRNA booster vaccination elicits limited mucosal immunity” (https://pubmed.ncbi.nlm.nih.gov/39441905/)

Intramuscularly administered COVID-19 vaccines induce robust serum neutralizing antibodies (NAbs), but their ability to boost mucosal immune responses remains to be determined. In this study, we show that the XBB.1.5 messenger RNA (mRNA) boosters result in increased serum neutralization to multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in humans, including the dominant circulating variant JN.1. In contrast, we found that the XBB.1.5 mRNA booster did not augment mucosal NAbs or mucosal IgA responses, although acute SARS-CoV-2 XBB infection substantially increased mucosal antibody responses. These data demonstrate that current XBB.1.5 mRNA boosters substantially enhance peripheral antibody responses but do not robustly increase mucosal antibody responses. Our data highlight a separation between the peripheral and mucosal immune systems in humans and emphasize the importance of developing next-generation vaccines to augment mucosal immunity to protect against respiratory virus infections.

Tang et al; 2024; “COVID-19 mRNA vaccines induce robust levels of IgG but limited amounts of IgA within the oronasopharynx of young children” (https://pubmed.ncbi.nlm.nih.gov/39253950/)

Methods: We measured the levels of antibodies against SARS-CoV-2 from a cohort of children under 5 years of age (N=24) undergoing SARS-CoV-2 mRNA vaccination (serially collected, matched serum and saliva samples) or in a convenience sample of children under 5 years of age presenting to pediatric emergency department (nasal swabs, N=103). Further, we assessed salivary and nasal samples for the ability to induce SARS-CoV-2 spike-mediated neutrophil extracellular traps (NET) formation.

Results: Longitudinal analysis of post-vaccine responses in saliva revealed the induction of SARS-CoV-2 specific IgG but not IgA. Similarly, SARS-CoV-2 specific IgA was only observed in nasal samples obtained from previously infected children with or without vaccination, but not in vaccinated children without a history of infection. In addition, oronasopharyngeal samples obtained from children with prior infection were able to trigger enhanced spike-mediated NET formation, and IgA played a key role in driving this process.

Conclusions: Despite the induction of specific IgG in the oronasal mucosa, current intramuscular vaccines have limited ability to generate mucosal IgA in young children. These results confirm the independence of mucosal IgA responses from systemic humoral responses following mRNA vaccination and suggest potential future vaccination strategies for enhancing mucosal protection in this young age group.

Althaus et al; 2024; “How effective is the BNT162b2 mRNA vaccine against SARS-CoV-2 transmission and infection? A national programme analysis in Monaco, July 2021 to September 2022” (https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-024-03444-6)

Results

In households, the SAR was 55% (95% CI 54–57) and 50% (48–51) among unvaccinated and vaccinated contacts, respectively. The SAR was 32% (28–36) and 12% (10–13) in workplaces, and 7% (6–9) and 6% (3–10) in schools, among unvaccinated and vaccinated contacts respectively. In household, the aHR was lower in contacts than in index cases (aHR 0.68 [0.55–0.83] and 0.93 [0.74–1.1] for delta; aHR 0.73 [0.66–0.81] and 0.89 [0.80–0.99] for omicron BA.1&2, respectively). Vaccination had no significant effect on either direct or indirect aVE for omicron BA.4&5. The direct aVE in contacts was 32% (17, 45) and 27% (19, 34), and for index cases the indirect aVE was 7% (− 17, 26) and 11% (1, 20) for delta and omicron BA.1&2, respectively. The greatest aVE was in contacts with a previous SARS-CoV-2 infection and a single vaccine dose during the omicron BA.1&2 period (45% [27, 59]), while the lowest were found in contacts with either three vaccine doses (aVE − 24% [− 63, 6]) or one single dose and a previous SARS-CoV-2 infection (aVE − 36% [− 198, 38]) during the omicron BA.4&5 period.

Conclusions

Protection conferred by the BNT162b2 mRNA vaccine against transmission and infection was low for delta and omicron BA.1&2, regardless of the number of vaccine doses and previous SARS-CoV-2 infection. There was no significant vaccine effect for omicron BA.4&5. Health authorities carrying out vaccination campaigns should bear in mind that the current generation of COVID-19 vaccines may not represent an effective tool in protecting individuals from either transmitting or acquiring SARS-CoV-2 infection.

8. Viral evolution – Selection pressure

Kambarani et al; 2023; “Molecular evolution and adaptation of SARS-CoV-2 omicron XBB sub-lineage Spike protein under African selection pressure” (https://www.biorxiv.org/content/10.1101/2023.08.16.553557v1)

We analysed a sub-lineage of Omicron, designated XBB, that showed structural and functional changes in the S protein in response to the African selection pressures. We used molecular modelling to compare the S protein structures of Omicron and XBB and found that XBB had a reduced receptor-binding domain (RBD) due to the loss of some β-sheets, which may increase its affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. We also used Fast Unconstrained Bayesian AppRoximation (FUBAR) and Recombination Detection Program 4 (RDP 4) to perform selection and recombination analysis of the S protein sequences of Omicron and XBB and detected signals of positive selection and recombination in the N-terminal domain (NTD) of the S1 subunit, which contains antibody-binding epitopes, and the RBD, which is involved in viral entry.

Halma; 2024; “Alterations of SARS-CoV-2 Evolutionary Dynamics by Pharmaceutical Factors” (https://journals.lww.com/idi/fulltext/2024/01000/alterations_of_sars_cov_2_evolutionary_dynamics_by.5.aspx)

The outbreak of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has been influenced by the human response to the virus. These responses have undoubtedly impacted the evolutionary dynamics of the virus in ways distinct from a scenario lacking a widespread response. Two important pharmaceutical interventions, vaccination and the utilization of medications, particularly molnupiravir, known to have mutagenic properties, were the focus of this article. The impact of molnupiravir on human health was evaluated through 3 mechanisms: viral resistance, mutagenesis of SARS-CoV-2, and mutagenesis occurring in patients undergoing treatment with molnupiravir. These mechanisms, as well as the impact of vaccination, have inadvertently given rise to unforeseen challenges in the management of the COVID-19 crisis. Taking a systems view in future pandemic responses, and taking into account the evolution of the pandemic virus, may be critical to ending the pandemic at an earlier date.

Al-Khatib et al; 2022; “Comparative analysis of within-host diversity among vaccinated COVID-19 patients infected with different SARS-CoV-2 variants” (https://www.cell.com/iscience/fulltext/S2589-0042(22)01710-2)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a rapidly evolving RNA virus that mutates within hosts and exists as viral quasispecies. Here, we evaluated the within-host diversity among vaccinated and unvaccinated individuals (n = 379) infected with different SARS-CoV-2 Variants of Concern. The majority of samples harbored less than 14 intra-host single-nucleotide variants (iSNVs). A deep analysis revealed a significantly higher intra-host diversity in Omicron samples than in other variants (p value < 0.05). Vaccination status and type had a limited impact on intra-host diversity except for Beta-B.1.315 and Delta-B.1.617.2 vaccinees, who exhibited higher diversity than unvaccinated individuals (p values: <0.0001 and <0.0021, respectively). Three immune-escape mutations were identified: S255F in Delta and R346K and T376A in Omicron-B.1.1.529. The latter 2 mutations were fixed in BA.1 and BA.2 genomes, respectively. Overall, the relatively higher intra-host diversity among vaccinated individuals and the detection of immune-escape mutations, despite being rare, suggest a potential vaccine-induced immune pressure in vaccinated individuals.

Duerr et al; 2023; “Selective adaptation of SARS-CoV-2 Omicron under booster vaccine pressure: a multicentre observational study” (https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(23)00409-7/fulltext)

Findings.

The study of >5400 SARS-CoV-2 infections between July 2021 and August 2022 in metropolitan New York portrayed the evolutionary transition from Delta to Omicron BA.1-BA.5 variants. Booster vaccinations were implemented during the Delta wave, yet booster breakthrough infections and SARS-CoV-2 re-infections were almost exclusive to Omicron. In adjusted logistic regression analyses, BA.1, BA.2, and BA.5 had a significant growth advantage over co-occurring lineages in the boosted population, unlike BA.2.12.1 or BA.4. Selection pressure by booster shots translated into diffuse adaptive evolution in Delta spike, contrasting with strong, receptor-binding motif-focused adaptive evolution in BA.2-BA.5 spike (Fisher Exact tests; non-synonymous/synonymous mutation rates per site). Convergent evolution has become common in Omicron, engaging spike positions crucial for immune escape, receptor binding, or cleavage.

Interpretation.

Booster shots are required to cope with gaps in immunity. Their discriminative immune pressure contributes to their effectiveness but also requires monitoring of selective viral adaptation processes. Omicron BA.2 and BA.5 had a selective advantage under booster vaccination pressure, contributing to the evolution of BA.2 and BA.5 sublineages and recombinant forms that predominate in 2023.

Jena et al; 2024; “Impact of vaccination on SARS-CoV-2 evolution and immune escape variants” (https://www.sciencedirect.com/science/article/abs/pii/S0264410X24008132)

Here, we have analyzed 2295 whole-genome sequences of SARS-CoV-2 collected from vaccinated and unvaccinated cases to evaluate the impact of vaccines on virus diversity within hosts. Our comparative analysis revealed a significant higher incidence of intra-host single nucleotides variants (iSNVs) in vaccinated cases compared to unvaccinated ones (p value<0.0001). Furthermore, we have found that specific mutational processes, including APOBEC (C > T) mediated and ADAR1 (A > G) mediated mutations, were found more prevalent in vaccinated cases. Vaccinated cases exhibited higher accumulation of nonsynonymous mutation than unvaccinated cases. Fixed iSNVs were predominantly located in the ORF1ab and spike genes, several key omicron defining immune escape variants S477N, Q493R, Q498R, Y505H, L452R, and N501Y were identified in the RBD domain of spike gene in vaccinated cases. Our findings suggest that vaccine plays an important role in the evolution of the virus genome. The virus genome acquires random mutations due to error-prone replication of the virus, host modification through APOBEC and ADAR1 mediated editing mechanism, and oxidative stress. These mutations become fixed in the viral population due to the selective pressure imposed by vaccination.

López-Cortés et al; 2022; “The Spike Protein of SARS-CoV-2 Is Adapting Because of Selective Pressures” (https://pubmed.ncbi.nlm.nih.gov/35746472/)

Here, we applied a neutral substitution evolution test to the spike (S) protein of Omicron’s protein and compared it to the others’ variant of concern (VOC) neutral evolution. We carried out comparisons among the interactions between the S proteins from the VOCs (Alpha, Beta, Gamma, Delta and Omicron) and the receptor ACE2. The shared amino acids among all the ACE2 binding S proteins remain constant, indicating that these amino acids are essential for the accurate binding to the receptor. The complexes of the RBD for every variant with the receptor were used to identify the amino acids involved in the protein-protein interaction (PPI). The RBD of Omicron establishes 82 contacts, compared to the 74 of the Wuhan original viral protein. Hence, the mean number of contacts per residue is higher, making the contact thermodynamically more stable. The RBDs of the VOCs are similar in sequence and structure; however, Omicron’s RBD presents the largest deviation from the structure by 1.11 Å RMSD, caused by a set of mutations near the glycosylation N343. The chemical properties and structure near the glycosylation N343 of the Omicron S protein are different from the original protein, which provoke reduced recognition by the neutralizing antibodies. Our results hint that selective pressures are induced by mass vaccination throughout the world and by the persistence of recurrent infections in immunosuppressed individuals, who did not eliminate the infection and ended up facilitating the selection of viruses whose characteristics are different from the previous VOCs, less pathogenic but with higher transmissibility.

Wilks et al; 2023; “Mapping SARS-CoV-2 antigenic relationships and serological responses” (https://pubmed.ncbi.nlm.nih.gov/37797027/)

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, multiple variants escaping preexisting immunity emerged, causing reinfections of previously exposed individuals. Here, we used antigenic cartography to analyze patterns of cross-reactivity among 21 variants and 15 groups of human sera obtained after primary infection with 10 different variants or after messenger RNA (mRNA)-1273 or mRNA-1273.351 vaccination. We found antigenic differences among pre-Omicron variants caused by substitutions at spike-protein positions 417, 452, 484, and 501. Quantifying changes in response breadth over time and with additional vaccine doses, our results show the largest increase between 4 weeks and >3 months after a second dose. We found changes in immunodominance of different spike regions, depending on the variant an individual was first exposed to, with implications for variant risk assessment and vaccine-strain selection.

Cobey et al; 2021; “Concerns about SARS-CoV-2 evolution should not hold back efforts to expand vaccination” (https://www.nature.com/articles/s41577-021-00544-9)

When vaccines are in limited supply, expanding the number of people who receive some vaccine, such as by halving doses or increasing the interval between doses, can reduce disease and mortality compared with concentrating available vaccine doses in a subset of the population. A corollary of such dose-sparing strategies is that the vaccinated individuals may have less protective immunity. Concerns have been raised that expanding the fraction of the population with partial immunity to SARS-CoV-2 could increase selection for vaccine-escape variants, ultimately undermining vaccine effectiveness. We argue that, although this is possible, preliminary evidence instead suggests such strategies should slow the rate of viral escape from vaccine or naturally induced immunity. As long as vaccination provides some protection against escape variants, the corresponding reduction in prevalence and incidence should reduce the rate at which new variants are generated and the speed of adaptation. Because there is little evidence of efficient immune selection of SARS-CoV-2 during typical infections, these population-level effects are likely to dominate vaccine-induced evolution.

9. Immune System

Yang et al; 2006; “Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients” (https://www.sciencedirect.com/science/article/pii/S1521661606007352?via%3Dihub#aep-section-id32)

In the current study, we found that antigen-specific memory T cells were capable of secreting high levels of IFN-γ by ELISA assay upon stimulation in vitro with a pool of SARS-CoV S peptides. These memory T cells persisted for more than 1 year after recovery in peripheral blood of SARS individuals. These data were further confirmed by IFN-γ ELISpot assay showing a high frequency of SARS-CoV S-specific IFN-γ-producing T cells in recovered SARS patients. Our results are consistent with the report that two newly identified HLA-A*0201-restricted CD8⁺ T-cell epitopes from SARS-CoV S protein, S1203 and S978, elicit memory T-cell responses in PBMCs from HLA-A*0201⁺ recovered SARS patients up to 3 months post-infection [13], [14].

Our results also demonstrated that the majority of memory CD8⁺ T cells exhibited effector memory phenotype of CD62L⁻. This observation is in agreement with recent studies showing that only central memory CD8⁺ T cells produce IL-2 following a short-peptide stimulation in lymphocytic choriomeningitis virus (LCMV)-infected mouse model [19]. Generally, the expression of CD62L is associated with the expression of CCR7 [20]. Surprisingly, SARS-CoV S-specific memory CD4⁺ T cells expressed a mixed CCR7⁺ CD62L⁻ phenotype that differs from phenotypes of classical CCR7⁺ CD62L⁺ T_CM and CCR7⁻ CD62L⁻ T_EM cells. Some groups have also found memory CD4⁺ or CD8⁺ T cells expressing a mixed CCR7⁺ CD62L⁻ phenotype in mouse model [21], [22]. These studies suggest that the expression of CCR7 and CD62L in memory CD4⁺ or CD8⁺ T cells may overlap only partially.

In conclusion, SARS-CoV infection elicits a cellular response mediated by both CD4⁺ and CD8⁺ T cells to the S protein. SARS-CoV S-specific memory CD4⁺ and CD8⁺ T cells are persistent for more than 1 year in peripheral blood of recovered SARS patients. This study provided an opportunity to delineate the memory T-cell response in survivors of a disease with high morbidity and mortality. The conclusions implicate a target population of T cells as a correlate for protection for effective vaccines.

Majdoubi et al; 2021; “A majority of uninfected adults show preexisting antibody reactivity against SARS-CoV-2” (https://pubmed.ncbi.nlm.nih.gov/33720905/)

Preexisting cross-reactivity to SARS-CoV-2 occurs in the absence of prior viral exposure. However, this has been difficult to quantify at the population level due to a lack of reliably defined seroreactivity thresholds. Using an orthogonal antibody testing approach, we estimated that about 0.6% of nontriaged adults from the greater Vancouver, Canada, area between May 17 and June 19, 2020, showed clear evidence of a prior SARS-CoV-2 infection, after adjusting for false-positive and false-negative test results. Using a highly sensitive multiplex assay and positive/negative thresholds established in infants in whom maternal antibodies have waned, we determined that more than 90% of uninfected adults showed antibody reactivity against the spike protein, receptor-binding domain (RBD), N-terminal domain (NTD), or the nucleocapsid (N) protein from SARS-CoV-2. This seroreactivity was evenly distributed across age and sex, correlated with circulating coronaviruses’ reactivity, and was partially outcompeted by soluble circulating coronaviruses’ spike. Using a custom SARS-CoV-2 peptide mapping array, we found that this antibody reactivity broadly mapped to spike and to conserved nonstructural viral proteins. We conclude that most adults display preexisting antibody cross-reactivity against SARS-CoV-2, which further supports investigation of how this may impact the clinical severity of COVID-19 or SARS-CoV-2 vaccine responses.

Murray et al; 2022; “The impact of pre-existing cross-reactive immunity on SARS-CoV-2 infection and vaccine responses” (https://www.nature.com/articles/s41577-022-00809-x)

Pre-existing cross-reactive immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins in infection-naive subjects have been described by several studies. In particular, regions of high homology between SARS-CoV-2 and common cold coronaviruses have been highlighted as a likely source of this cross-reactivity. However, the role of such cross-reactive responses in the outcome of SARS-CoV-2 infection and vaccination is currently unclear. Here, we review evidence regarding the impact of pre-existing humoral and T cell immune responses to outcomes of SARS-CoV-2 infection and vaccination. Furthermore, we discuss the importance of conserved coronavirus epitopes for the rational design of pan-coronavirus vaccines and consider cross-reactivity of immune responses to ancestral SARS-CoV-2 and SARS-CoV-2 variants, as well as their impact on COVID-19 vaccination.

Several studies have shown immune reactivity to SARS-CoV-2 epitopes or antigen in samples from individuals that were collected before the COVID-19 pandemic^{4,26,27,28,49}, providing definitive evidence that SARS-CoV-2 cross-reactive immune responses may be derived from non-SARS-CoV-2 antigens. Moreover, epitopes of cross-reactive T and B cells have been predicted with computational tools and identified using conventional laboratory assays^28,50,51,52. These are located in the regions of high homology between SARS-CoV-2 and HCoVs such as the nucleocapsid protein⁴, the spike protein S2 region^27,49,53 and NSPs found in ORF1^{4,27,28,39,49,53,54,55} (Fig. 2 and Supplementary Table 1). However, it is feasible that cross-reactivity, facilitated by receptor-binding degeneracy or epitope structural similarity⁵³, is not limited to CCCs and that other common infectious agents may provide a source of epitopes with high homology to SARS-CoV-2 epitopes.

Cross-reactive antibodies and B cells

A number of studies have detected cross-reactive antibodies in samples taken from SARS-CoV-2-unexposed individuals before or early in the pandemic^{45,49,56,57,58,59}. In unexposed children, adolescents and adults, a high seroprevalence was found against SARS-CoV-2 total spike and nucleocapsid proteins but not against the RBD or the S1 subunits of the spike protein, suggesting that responses were mainly targeting S2 in these cohorts^49,59. Cross-reactive antibodies binding within the final 743 amino acids of the spike protein were mapped to multiple epitopes within the S2 region of the spike protein, which were largely well conserved between SARS-CoV-2 and other HCoVs⁴⁹ (Table 1). Similar findings were made in an analysis of 196 overlapping peptides covering the SARS-CoV-2 spike protein, demonstrating that antibodies from serum samples taken before the pandemic can bind SARS-CoV-2 S2 regions with high sequence homology to other HCoVs.

Similarly, SARS-CoV-2-reactive memory B cell populations have been investigated for cross-reactivity with HCoVs⁵⁷. In COVID-19 convalescent donors, up to 4% of immunoglobulin G (IgG)⁺ memory B cells bound to both the SARS-CoV-2 spike protein and either HKU1 or NL63, demonstrating cross-reactivity of the B cell receptor to epitopes of both SARS-CoV-2 and HCoVs⁵⁷. Monoclonal antibodies generated from these cross-reactive B cells also showed cross-reactive binding to several other HCoVs, including OC43, MERS-CoV-2 and SARS-CoV⁵⁷. Pre-existing cross-reactive memory B cells have also been shown to expand in the early response to SARS-CoV-2 infection⁶⁰. These cross-reactive memory B cells had already undergone significant hypermutation before the initial SARS-CoV-2 infection, but their frequency decreased over time (between 3 and 6 months after SARS-CoV-2 infection). This may suggest that, although cross-reactive memory B cells form part of the initial response to SARS-CoV-2 infection, they are not selected for among the overall memory B cell pool, which forms after infection⁶⁰. Notably, however, some cross-reactive memory B cells that are available for rechallenge may not be detectable by conventional flow cytometry staining in peripheral blood, and techniques such as tetramer enrichment may be required to detect rare cross-reactive memory B cell populations⁶¹.

Cross-reactive T cells

Several studies have also identified cross-reactive T cells to SARS-CoV-2 in blood samples from SARS-CoV-2-unexposed individuals^{4,27,28,30,31,49,58,62}. Compared with antibody epitopes, and of relevance to vaccine design, a greater number of epitopes for cross-reactive T cells were identified in SARS-CoV-2 proteins other than spike (Supplementary Table 1). For example, responses to the structural nucleocapsid protein and non-structural ORF1 proteins (targeting mainly NSP7 and NSP13) were detected in 19 of 37 SARS-CoV-2-unexposed donors using an interferon-γ ELISpot assay⁴. These T cell epitopes mapped to a region of the nucleocapsid protein (N_101–120) that was highly conserved between SARS-CoV-2 and HCoVs and contained a T cell epitope that had been identified in individuals who had recovered from SARS-CoV infection 17 years earlier (Supplementary Table 1). This epitope has undergone considerable subsequent analysis and is suggested to be an important target for cross-reactive CD8⁺ T cells^63,64. A study by Swadling et al.³⁹, using samples taken before the COVID-19 pandemic and samples from SARS-CoV-2-exposed but uninfected health-care workers, identified epitopes of cross-reactive T cells in the non-structural components NSP7, NSP12 and NSP13 of the replication transcription complex of SARS-CoV-2. Bioinformatic analysis in this study found significantly higher sequence homology between SARS-CoV-2 and all other HCoVs in the replication transcription complex compared with structural proteins³⁹. Cross-reactive T cells from three out of five seronegative health-care workers showed a higher response to the corresponding HKU1 epitope when compared with the homologous SARS-CoV-2 epitope, suggesting that SARS-CoV-2 cross-reactivity is a consequence of previous HKU1 infection in these individuals³⁹.

Cohort studies of antibody and B cell cross-reactivity

Several studies have explored the potential effect of cross-reactive antibodies and T cells on COVID-19 severity. Although cross-reactive antibodies against SARS-CoV-2 spike protein domains can be detected in pre-pandemic samples and may neutralize SARS-CoV-2 infection in vitro (SARS-CoV-2 spike pseudotypes and authentic SARS-CoV-2)^5,49, there is an ongoing debate as to whether these influence COVID-19 clinical outcomes. In a large case–control study, levels of CCC-specific antibodies were found to be elevated in response to SARS-CoV-2 infection; however, the baseline CCC antibody titre (measured before SARS-CoV-2 infection) was not associated with protection against infection or predictive of disease severity⁸⁴. This finding was supported by a second study, which similarly showed that SARS-CoV-2 cross-reactive antibodies were non-neutralizing and did not protect against SARS-CoV-2 infection or hospitalization⁵⁹.

Cohort studies of T cell cross-reactivity

Several studies have investigated the impact of SARS-CoV-2 cross-reactive T cells on COVID-19 severity and SARS-CoV-2 infection, with a growing body of evidence showing that these have a beneficial role^31,39,62. Cross-reactive T cells, which are potentially capable of preventing SARS-CoV-2 infection before seroconversion, have been detected in two independent studies. The first demonstrated that SARS-CoV-2 cross-reactive T cells found in health-care workers who were highly exposed to SARS-CoV-2 but found to be both seronegative and PCR-negative for COVID-19 were associated with levels of an innate immune marker (IFNα-inducible protein 27; IFI27) that can be used as a surrogate marker for early SARS-CoV-2 infection³⁹. The authors propose that T cell responses to NSP12, a protein with high sequence homology to CCCs, were linked with ‘abortive’ infection, whereby SARS-CoV-2 infection was cleared before seroconversion or PCR positivity. Similarly, CD4⁺ T cells that produce IL-2 in response to a pool of SARS-CoV-2 peptides with high CCC homology (located primarily in regions outside the spike protein, including ORF1, 4, 6, 7 and 8, nucleocapsid protein and envelope protein) have been associated with protection from PCR-positive SARS-CoV-2 infection in people who were close-contacts with SARS-CoV-2-infected individuals⁹⁰.

Mahajan et al; 2021; “Immunodominant T-cell epitopes from the SARS-CoV-2 spike antigen reveal robust pre-existing T-cell immunity in unexposed individuals” (https://www.nature.com/articles/s41598-021-92521-4)

The COVID-19 pandemic has revealed a range of disease phenotypes in infected patients with asymptomatic, mild, or severe clinical outcomes, but the mechanisms that determine such variable outcomes remain unresolved. In this study, we identified immunodominant CD8 T-cell epitopes in the spike antigen using a novel TCR-binding algorithm. The predicted epitopes induced robust T-cell activation in unexposed donors demonstrating pre-existing CD4 and CD8 T-cell immunity to SARS-CoV-2 antigen. The T-cell reactivity to the predicted epitopes was higher than the Spike-S1 and S2 peptide pools in the unexposed donors. A key finding of our study is that pre-existing T-cell immunity to SARS-CoV-2 is contributed by TCRs that recognize common viral antigens such as Influenza and CMV, even though the viral epitopes lack sequence identity to the SARS-CoV-2 epitopes. This finding is in contrast to multiple published studies in which pre-existing T-cell immunity is suggested to arise from shared epitopes between SARS-CoV-2 and other common cold-causing coronaviruses. However, our findings suggest that SARS-CoV-2 reactive T-cells are likely to be present in many individuals because of prior exposure to flu and CMV viruses.

Joseph et al; 2023; “Humoral Immunity of Unvaccinated COVID-19 Recovered vs. Naïve BNT162b2 Vaccinated Individuals: A Prospective Longitudinal Study” (https://pubmed.ncbi.nlm.nih.gov/37512801/)

In this prospective longitudinal study, we followed unvaccinated COVID-19-recovered individuals (n = 130) and naïve, two-dose BNT162b2-vaccinated individuals (n = 372) who were age- and BMI-matched for six months during the first pandemic year. Anti-RBD-IgG, neutralizing antibodies (NAbs), and avidity were assessed monthly. For recovered patients, data on symptoms and the severity of the disease were collected. Anti-RBD-IgG and NAbs titers at peak were higher after vaccination vs. after infection, but the decline was steeper (peak log IgG: 3.08 vs. 1.81, peak log NAbs: 5.93 vs. 5.04, slopes: -0.54 vs. -0.26). Peak anti-RBD-IgG and NAbs were higher in recovered individuals with BMI > 30 and in older individuals compared to individuals with BMI < 30, younger population. Of the recovered, 42 (36%) experienced long-COVID symptoms. Avidity was initially higher in vaccinated individuals compared with recovered individuals, though with time, it increased in recovered individuals but not among vaccinated individuals. Here, we show that while the initial antibody titers, neutralization, and avidity are lower in SARS-CoV-2-recovered individuals, they persist for a longer duration.

Xiao C., & Chen G.; 2023; “Insufficient epitope specific T cell clones is responsible for impaired cellular immunity to inactivated SARS-CoV-2 vaccine in the elderly” (https://journals.aai.org/jimmunol/article/210/1_Supplement/252.09/263922/Insufficient-epitope-specific-T-cell-clones-is)

Aging is a critical risk factor for SARS-CoV-2 vaccine efficacy. Although low humoral immunity in the elderly was observed in mRNA and recombinant protein vaccination, the immune responses to inactivated vaccine in the elderly, and the underlying mechanisms of differences to young, if any, are still unclear. Here, we studied a cohort of 121 young (18–30 years old) and 48 old (60–85 years old) donors vaccinated with inactivated SARS-CoV-2 and demonstrate that the neutralizing antibody response is slower in the elderly, but eventually reached the similar level to day 7 post-vaccination in young. We identified a range of epitopes targeted by CD8 T cells that were underrepresented in immune responses in the old group, especially when measured with tetramers derived from the 13 major SARS-CoV-2 variants. Comparison of the transcriptomes of B, CD4 and CD8 T cells from pre- and post-vaccinated young and elderly revealed genes potentially responsible for low immune responses in elderly, functionally related to antigen processing and presentation. We further built an epitope specific TCR repertoire database using single-cell RNA and TCR sequencing. Statistical and machine-learning based analyses demonstrated that SARS-CoV-2 epitope specific CD8 T cell clones were significantly lower in the old than young, and failed to be boosted after vaccination in elderly. Comparison of BCRs and TCRs from pre- and post-vaccinated young and elderly individuals revealed inadequate receptor repertoire size and diversity might be responsible for low immune response in the elderly. Together, the altered immune cell function and the attenuated antigen specific TCR repertoire were responsible for the impaired CD8 T cell immune response in the elderly.

Shrestha et al; 2022; “Necessity of Coronavirus Disease 2019 (COVID-19) Vaccination in Persons Who Have Already Had COVID-19” (https://academic.oup.com/cid/article/75/1/e662/6507165?login=false)

Results

Among 52 238 employees, 4718 (9%) were previously infected and 36 922 (71%) were vaccinated by the study’s end. Cumulative incidence of COVID-19 was substantially higher throughout for those previously uninfected who remained unvaccinated than for all other groups, lower for the vaccinated than unvaccinated, and lower for those previously infected than those not. Incidence of COVID-19 increased dramatically in all groups after the Omicron variant emerged. In multivariable Cox proportional hazards regression, both prior COVID-19 and vaccination were independently associated with significantly lower risk of COVID-19. Among previously infected subjects, a lower risk of COVID-19 overall was not demonstrated, but vaccination was associated with a significantly lower risk of symptomatic COVID-19 in both pre-Omicron (HR, .60; 95% CI, .40–.90) and Omicron (HR, .36; 95% CI, .23–.57) phases.

Le Bert et al; 2020; “SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls” (https://www.nature.com/articles/s41586-020-2550-z)

Here we studied T cell responses against the structural (nucleocapsid (N) protein) and non-structural (NSP7 and NSP13 of ORF1) regions of SARS-CoV-2 in individuals convalescing from coronavirus disease 2019 (COVID-19) (n = 36). In all of these individuals, we found CD4 and CD8 T cells that recognized multiple regions of the N protein. Next, we showed that patients (n = 23) who recovered from SARS (the disease associated with SARS-CoV infection) possess long-lasting memory T cells that are reactive to the N protein of SARS-CoV 17 years after the outbreak of SARS in 2003; these T cells displayed robust cross-reactivity to the N protein of SARS-CoV-2. We also detected SARS-CoV-2-specific T cells in individuals with no history of SARS, COVID-19 or contact with individuals who had SARS and/or COVID-19 (n = 37). SARS-CoV-2-specific T cells in uninfected donors exhibited a different pattern of immunodominance, and frequently targeted NSP7 and NSP13 as well as the N protein. Epitope characterization of NSP7-specific T cells showed the recognition of protein fragments that are conserved among animal betacoronaviruses but have low homology to ‘common cold’ human-associated coronaviruses. Thus, infection with betacoronaviruses induces multi-specific and long-lasting T cell immunity against the structural N protein. Understanding how pre-existing N- and ORF1-specific T cells that are present in the general population affect the susceptibility to and pathogenesis of SARS-CoV-2 infection is important for the management of the current COVID-19 pandemic.

Le Bert et al; 2021; “Highly functional virus-specific cellular immune response in asymptomatic SARS-CoV-2 infection” (https://rupress.org/jem/article/218/5/e20202617/211835/Highly-functional-virus-specific-cellular-immune)

We longitudinally studied SARS-CoV-2–specific T cells in a cohort of asymptomatic (n = 85) and symptomatic (n = 75) COVID-19 patients after seroconversion. We quantified T cells reactive to structural proteins (M, NP, and Spike) using ELISpot and cytokine secretion in whole blood. Frequencies of SARS-CoV-2–specific T cells were similar between asymptomatic and symptomatic individuals, but the former showed an increased IFN-γ and IL-2 production. This was associated with a proportional secretion of IL-10 and proinflammatory cytokines (IL-6, TNF-α, and IL-1β) only in asymptomatic infection, while a disproportionate secretion of inflammatory cytokines was triggered by SARS-CoV-2–specific T cell activation in symptomatic individuals. Thus, asymptomatic SARS-CoV-2–infected individuals are not characterized by weak antiviral immunity; on the contrary, they mount a highly functional virus-specific cellular immune response.

Peluso et al; 2022; “Long-term immunologic effects of SARS-CoV-2 infection: leveraging translational research methodology to address emerging questions” (https://pubmed.ncbi.nlm.nih.gov/34780969/)

The current era of COVID-19 is characterized by emerging variants of concern, waning vaccine- and natural infection-induced immunity, debate over the timing and necessity of vaccine boosting, and the emergence of post-acute sequelae of SARS-CoV-2 infection. As a result, there is an ongoing need for research to promote understanding of the immunology of both natural infection and prevention, especially as SARS-CoV-2 immunology is a rapidly changing field, with new questions arising as the pandemic continues to grow in complexity. The next phase of COVID-19 immunology research will need focus on clearer characterization of the immune processes defining acute illness, development of a better understanding of the immunologic processes driving protracted symptoms and prolonged recovery (ie, post-acute sequelae of SARS-CoV-2 infection), and a growing focus on the impact of therapeutic and prophylactic interventions on the long-term consequences of SARS-CoV-2 infection. In this review, we address what is known about the long-term immune consequences of SARS-CoV-2 infection and propose how experience studying the translational immunology of other infections might inform the approach to some of the key questions that remain.

Most individuals with SARS-CoV-2 infection develop robust and persistent immunologic responses following natural infection, and as of the time of this review, many studies have characterized the humoral1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and cell-mediated^{19 ,} 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 immune responses during convalescence for periods of up to 1 year. While the magnitude of the immune response to natural infection is at least in part determined by the severity of the illness,^{3 , 6 , 7 , 15 , 28 , 30} the predictors of the duration of natural immunity are not fully understood and may be determined by a variety of clinical and measurement factors.^{15 , 18} Despite this complexity, there is general consensus that, in most cases, natural immunity persists for up to at least 8 months. Despite the observation that antibody levels may wane over time, several studies have now demonstrated persistence of virus-specific lymphocytes over 12 months following natural infection by various intracellular cytokine staining (ICS), activation induced marker (AIM), and EliSpot assays. These assays quantify T cell cytokine expression (ICS/EliSpot) or surface markers of T cell activation (AIM) following antigenic stimulation with various virus-specific peptide pools. For example, the percentage of virus-specific CD8 and CD4 T cells as measured by ICS or AIM range from approximately 0.01%–10% during this extended time period across multiple studies,^{25 , 26 , 28 , 29 , 31 , 32} with the median or mean percentage typically <1%. Spot forming cells/units in ELISpot assays tend to range from 10 to >1,000 in response to SARS-CoV-2 peptides, including HLA-restricted pools.^{27 , 34} These responses wane slowly over time in all assays depending on initial disease severity and various clinical factors but can typically be detected across a range of virus gene regions (eg, Spike, Nucleocapsid, Membrane).

Diani et al; 2022; “SARS-CoV-2—The Role of Natural Immunity: A Narrative Review” (https://pubmed.ncbi.nlm.nih.gov/36362500/)

Results: nearly 900 studies were collected, and 246 pertinent articles were included. It was highlighted that the vast majority of the individuals after suffering from COVID-19 develop a natural immunity both of cell-mediated and humoral type, which is effective over time and provides protection against both reinfection and serious illness. Vaccine-induced immunity was shown to decay faster than natural immunity. In general, the severity of the symptoms of reinfection is significantly lower than in the primary infection, with a lower degree of hospitalizations (0.06%) and an extremely low mortality.

A very recent retrospective and large study analyzed the entire Swedish population, demonstrating the presence of natural antibody and cellular immunity capable of protecting from hospitalization after about 20 months. In fact, natural immunity was associated with a 95% lower risk of SARS-CoV-2 reinfection and an 87% lower risk of COVID-19 hospitalization compared to the non-previously infected subjects, for up to 20 months [36]. To prevent one reinfection in the natural immunity cohort during the follow-up, 767 individuals needed to be vaccinated with two doses. In the same study, vaccination was shown to reduce the risk of contracting COVID-19 and hospitalization for up to 9 months, although the differences in absolute numbers, especially as to the hospitalization rate, were small.

Other data suggested that more than 90% of seroconverters make detectable neutralizing antibody responses. Furthermore, it was shown that these titers remain relatively stable for several months after COVID-19 infection [37,38,39]. An older study regarding SARS-CoV infection already showed the presence of SARS coronavirus-specific T cells in three SARS-recovered individuals at 9 and 11 years follow-up. It was also shown that all the detected T memory cell responses targeted the SARS-CoV structural proteins. Furthermore, these responses were found to persist up to 11 years post-infection [24].

The duration of the follow-up regarding the duration of the immunity after the SARS-CoV-2 infection is getting increasingly longer: the presence of CD4+ and CD8+ T-lymphocytes has been confirmed over time in subjects recovering from SARS-CoV-2 up to 18 months after infection, as reported in a few recent publications [56,57,58,59]; furthermore this T-lymphocyte-based immunity was shown to occur regardless of the severity of the clinical picture related to the infection itself [60,61]. Interestingly, no statistically significant differences between the effectiveness of the immune response to natural infection or to the hybrid stimulation (vaccination + natural infection) was documented after about 20 months [36]. This finding confirms the valid antiviral protection put in place by our immune system over time, after SARS-CoV-2 infection. In these patients the circulating memory of the T CD8+ leukocytes also includes cells with a memory phenotype which is similar to that of stem cells, with sustained polyfunctionality and proliferation capacity. Consequently, these immune cells are likely to play a crucial role in supporting an anamnestic response [62].

Inoue et al; 2024; “SARS-CoV-2-Specific Immune Responses in Vaccination and Infection during the Pandemic in 2020-2022” (https://pubmed.ncbi.nlm.nih.gov/38543812/)

To gain insight into how immunity develops against SARS-CoV-2 from 2020 to 2022, we analyzed the immune response of a small group of university staff and students who were either infected or vaccinated. We investigated the levels of receptor-binding domain (RBD)-specific and nucleocapsid (N)-specific IgG and IgA antibodies in serum and saliva samples taken early (around 10 days after infection or vaccination) and later (around 1 month later), as well as N-specific T-cell responses. One patient who had been infected in 2020 developed serum RBD and N-specific IgG antibodies, but declined eight months later, then mRNA vaccination in 2021 produced a higher level of anti-RBD IgG than natural infection. In the vaccination of naïve individuals, vaccines induced anti-RBD IgG, but it declined after six months. A third vaccination boosted the IgG level again, albeit to a lower level than after the second. In 2022, when the Omicron variant became dominant, familial transmission occurred among vaccinated people. In infected individuals, the levels of serum anti-RBD IgG antibodies increased later, while anti-N IgG peaked earlier. The N-specific activated T cells expressing IFN γ or CD107a were detected only early. Although SARS-CoV-2-specific salivary IgA was undetectable, two individuals showed a temporary peak in RBD- and N-specific IgA antibodies in their saliva on the second day after infection. Our study, despite having a small sample size, revealed that SARS-CoV-2 infection triggers the expected immune responses against acute viral infections. Moreover, our findings suggest that the temporary mucosal immune responses induced early during infection may provide better protection than the currently available intramuscular vaccines.

Rocío Morlanes Pallás; 2024; “Innate and adaptative immune mechanisms of COVID-19 vaccines. Serious adverse events associated with SARS-CoV-2 vaccination: A systematic review” (https://www.sciencedirect.com/science/article/abs/pii/S1576988724000037)

Results

The systematic review identified 2033 records which, after a screening process according to the inclusion criteria and the elimination of duplicated papers, works with methodological problems and works without open access, were reduced to 58 articles, of which 50 articles are human models and 2 are cellular models.

Conclusion

The results of this systematic review reveal the causal and temporal association of the various serious adverse events following administration of COVID-19 vaccines and the “peak effect” of COVID-19 vaccines is recognized.

Available data on innate immune activation in humans following mRNA-iLNP administration are limited, however, the studies published to date are consistent with previous reports in animals. They indicate a similar inflammatory signature in the serum of mice after vaccination with BNT162b2: CXCL10, CCL4, IL-6, interferon alpha and gamma (IFN-α, IFN-γ).⁵⁴ Li et al. (2022) highlight how vaccine-stimulated IFN-I and IFN-stimulated gene (ISG) signatures in various cell types including monocytes, macrophages, DCs, natural killer (NK) cells, peak at day 1 and return to baseline levels by day 7, whereas NK cells and T cells exhibit a continuous increase in expression of cell-cycle- and transcription-associated genes (analysed by single-cell transcriptional profiling in draining LNs). Six hours after administration of a second vaccine dose, serum IFN-γ is 8.6-fold higher, coming largely from CD4 + and CD8 +, compared with 6 h after the first dose, when most of the IFN-γ is derived from NK cells.^3,54 It is not clear which component of the mRNA-iLNP vaccine can induce type 1 IFNs. They state that IFN-γ signalling activates antigen-presenting cells, but, in this condition, plasmacytoid DCs produce IFN-α only when exposed to mRNA-iLNP and not empty LNPs, supporting the theory that the mRNA component may be responsible for inducing type I IFN.⁷⁵

A higher concentration of IFN-γ is generally observed, i.e., a general immune dysfunction following vaccination, consistent with reports of adverse effects following approved mRNA COVID-19 vaccinations, mainly for systemic reactions, and it is theorised that enhanced T cell and myeloid cell activation results in cross-talk between lymphocytes and myeloid cells that increases their responsiveness to subsequent vaccine encounters and/or COVID-19 infection.^{2,3,31,54,74,}[86], [87], [88], [89], [90], [91] It is suggested that mRNA-iLNP vaccines have persistent enhancing effects on trained immunity (myeloid cells).^3,60,91,92 There may be important species-dependent differences in inflammatory responses that need to be considered when testing vaccines in animal models, including reactogenicity, fever, and induction of other inflammatory cytokines stimulated by mRNA-iLNP. Human peripheral blood mononuclear cells (PBMCs) treated in vitro with m1ψ-modified mRNA-iLNPs formulated with SM-102 lipid release IL-β, IL-6, TNF, CCL5, vascular endothelial growth factor (VEGF-A), GM-CSF, and other molecules have been detected, and research is needed in this area.^3,15,55 Plant-Hately et al. (2022) note the stimulation of basophils in the generation and release of histamine within the vascular system.^50,57

The innate immune signalling pathways implicated in the iLNP adjuvant effect are: (1) oxidised phospholipids or metabolised and/or modified lipid products, e.g., oxidative impurities of ionisable lipid⁹³; (2) individual cholesterol and lipids, e.g., ionisable lipids^16,56,57; (3) the entire nanoparticle or other multimolecular structures; (4) endogenous molecules (apolipoprotein (ApoE) or complement proteins) that bind to iLNPs after inoculation with the mRNA vaccine are involved in both sensing mechanisms and receptor-mediated uptake by innate immune cells; (5) finally, the presence of a cellular receptor, such as a TLR, that specifically detects iLNPs.^3,13,15 Certain vaccine components such as LNPs and cationic liposomes are sensed by nucleotide binding domain, TLR2, TLR4, the stimulator of interferon genes (STING), and protein 3 (including the leucine-rich repeat pyrin domain-containing protein 3 [NLRP3]).^3,57,[94], [95], [96] In contrast, Li et al. (2022) and Pichmair et al. (2009) suggest that certain nucleic acid and microbial lipid sensors, including inflammasome mediators, are not required for a strong immune response to this vaccine.^54,97 It is therefore possible that SARS-CoV-2 vaccines composed of viral vectors (AstraZeneca’s Vaxzevria and Johnson & Johnson’s Janssen) and protein subunits (Novavax’s Nuvaxovid) deliver more attenuated inflammatory signals or use other mechanisms that contribute to innate immune activation, not just the mRNA encapsulated in lipid nanoparticles of Pfizer’s Comirnaty BioNTech, Moderna’s Spikevax, and Curevac. For example, Li et al. (2022) analyse the immunogenicity of the BNT162b2 vaccine (CD8 + T-cell and antibody response), it is not attenuated in the absence of TLR2, TLR4, TLR5, TLR7, protein 3 including leucine-enriched pyrin repeat domain (NLRP3), apoptosis-associated Speck-like protein also containing caspase (CARD/ASC), cyclic GMP-AMP synthetase (cGAS), or the stimulator of interferon genes (STING). They suggest the possibility that mRNA (modified with m1ψ and without dsRNA) or its degradation products may be sensed by the above sensors sending signals, supporting the theory of its function as an adjuvant.^{3,12,16,17,22,}[26], [27], [28]^{,30,31,33,34,36,37,39,}[41], [42], [43], [44]^,[46], [47], [48], [49], [50], [51], [52], [53], [54]^,57,98

Holder et al; 2024; “Sequence Matters: Primary COVID-19 Vaccination after Infection Elicits Similar Anti-spike Antibody Levels, but Stronger Antibody Dependent Cell-mediated Cytotoxicity than Breakthrough Infection” (https://journals.aai.org/jimmunol/article/doi/10.4049/jimmunol.2400250/267130/Sequence-Matters-Primary-COVID-19-Vaccination)

Infection before primary vaccination (herein termed “hybrid immunity”) engenders robust humoral immunity and broad Ab-dependent cell-mediated cytotoxicity (ADCC) across SARS-CoV-2 variants. We measured and compared plasma IgG and IgA against Wuhan-Hu-1 and Omicron (B.1.1.529) full-length spike (FLS) and receptor binding domain after three mRNA vaccines encoding Wuhan-Hu-1 spike (S) and after Omicron breakthrough infection. We also measured IgG binding to Wuhan-Hu-1 and Omicron S1, Wuhan-Hu-1 S2 and Wuhan-Hu-1 and Omicron cell-based S. We compared ADCC using human embryonic lung fibroblast (MRC-5) cells expressing Wuhan-Hu-1 or Omicron S. The effect of Omicron breakthrough infection on IgG anti-Wuhan-Hu-1 and Omicron FLS avidity was also considered. Despite Omicron breakthrough infection increasing IgG and IgA against FLS and receptor binding domain to levels similar to those seen with hybrid immunity, there was no boost to ADCC. Preferential recognition of Wuhan-Hu-1 persisted following Omicron breakthrough infection, which increased IgG avidity against Wuhan-Hu-1 FLS. Despite similar total anti-FLS IgG levels following breakthrough infection, 4-fold higher plasma concentrations were required to elicit ADCC comparable to that elicited by hybrid immunity. The greater capacity for hybrid immunity to elicit ADCC was associated with a differential IgG reactivity pattern against S1, S2, and linear determinants throughout FLS. Immunity against SARS-CoV-2 following Omicron breakthrough infection manifests significantly less ADCC capacity than hybrid immunity. Thus, the sequence of antigenic exposure by infection versus vaccination and other factors such as severity of infection affect antiviral functions of humoral immunity in the absence of overt quantitative differences in the humoral response.

9a. Immune Suppression

Channappanavar et al; 2016; “Dysregulated Type I Interferon and Inflammatory Monocyte-Macrophage Responses Cause Lethal Pneumonia in SARS-CoV-Infected Mice” (https://pubmed.ncbi.nlm.nih.gov/26867177/)

Using mice infected with SARS (severe acute respiratory syndrome)-CoV, we show that robust virus replication accompanied by delayed type I interferon (IFN-I) signaling orchestrates inflammatory responses and lung immunopathology with diminished survival. IFN-I remains detectable until after virus titers peak, but early IFN-I administration ameliorates immunopathology. This delayed IFN-I signaling promotes the accumulation of pathogenic inflammatory monocyte-macrophages (IMMs), resulting in elevated lung cytokine/chemokine levels, vascular leakage, and impaired virus-specific T cell responses. Genetic ablation of the IFN-αβ receptor (IFNAR) or IMM depletion protects mice from lethal infection, without affecting viral load. These results demonstrate that IFN-I and IMM promote lethal SARS-CoV infection and identify IFN-I and IMMs as potential therapeutic targets in patients infected with pathogenic coronavirus and perhaps other respiratory viruses.

Gimenez et al; 2025; “Monocytic reactive oxygen species-induced T cell apoptosis impairs cellular immune response to SARS-CoV-2 mRNA vaccine” (https://www.sciencedirect.com/science/article/abs/pii/S0091674925000119)

Results

In most vaccinees, we observed the presence of circulating RBD peaking on Day 14, and linked to an increase in AngII plasma levels with a peak on Day 28. This increase correlated with i) the ability of monocytes to produce ROS and to induce ROS-mediated DNA damage in neighboring cells, including PBMCs, ii) CD4⁺ and CD8⁺ T lymphocyte apoptosis, and iii) a poor response to S in vitro from both CD4⁺ and CD8⁺ T cells.

Clinical implications

We observed the same cascade of events triggered by the vaccinal antigen as by SARS-CoV-2 infection. This cascade may account for the suboptimal efficiency of mRNA SARS-CoV-2 vaccines in preventing the infection, the limited vaccine memory, and certain side-effects. In this model, AngII receptor antagonists and/or antioxidants might improve the performance of the SARS-CoV-2 vaccine.

Cai et al; 2024; “SARS-CoV-2 S protein disrupts the formation of ISGF3 complex through conserved S2 subunit to antagonize type I interferon response” (https://journals.asm.org/doi/10.1128/jvi.01516-24)

Here, we found that the spike (S) protein, the major vaccine antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), strongly suppresses host innate immunity by inhibiting interferon-stimulated gene (ISG) expression through both S1 and S2 subunits. Mechanistically, the S protein inhibited the formation of the classic interferon-stimulated gene factor 3 (ISGF3) complex composed of STAT1, STAT2, and IRF9 by competing with STAT2 for binding to IRF9, thereby impeding the transcription of ISGs. A strong interaction between S and the STAT1/STAT2 proteins further traps the ISGF3 complex in the endoplasmic reticulum and hinders the nuclear translocation of ISGF3. Notably, the interferon-inhibitory mechanism of the S protein was universal among SARS-CoV-2 variants and other human coronaviruses, including SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), and human coronavirus HKU1 (HCoV-HKU1), through the most evolutionarily conserved region of S2 subunit. Taken together, the findings of this study reveal a new mechanism by which the coronavirus S protein attenuates the host antiviral immune response and provides new insights into the proper design of coronavirus S-based vaccines to prevent immunosuppressive effects.

Sui et al; 2021; “SARS-CoV-2 Spike Protein Suppresses ACE2 and Type I Interferon Expression in Primary Cells From Macaque Lung Bronchoalveolar Lavage” (https://pubmed.ncbi.nlm.nih.gov/34149696/)

“[W]e provide evidence that SARS-CoV-2 spike protein S1 reduced the mRNA expression of ACE2 and type I interferons in primary cells of lung bronchoalveolar lavage (BAL) from naïve rhesus macaques. The expression levels of ACE2 and type I interferons were also found to be correlated with each other, consistent with the recent finding that ACE2 is an interferon-inducible gene. Furthermore, induction of ACE2 and type I interferons by poly I:C, an interferon inducer, was suppressed by S1 protein in primary cells of BAL. These observations suggest that the downregulation of ACE2 and type I interferons induced by S1 protein may directly contribute to SARS-CoV-2-associated lung diseases.”

Gao et al; 2022; “Extended SARS-CoV-2 RBD booster vaccination induces humoral and cellular immune tolerance in mice” (https://www.sciencedirect.com/science/article/pii/S2589004222017515)

“Multiple vaccine boosters after the conventional vaccination course significantly decreased RBD-specific antibody titers and serum neutralizing efficacy against the Delta and Omicron variants, and profoundly impaired CD4⁺ and CD8⁺T cell activation and increased PD-1 and LAG-3 expressions in these T cells. Mechanistically, we confirmed that extended vaccination with RBD boosters overturned the protective immune memories by promoting adaptive immune tolerance. Our findings demonstrate potential risks with the continuous use of SARS-CoV-2 vaccine boosters, providing immediate implications for the global COVID-19 vaccination enhancement strategies.”

Haralambieva et al; 2024; “Restricted Omicron-specific cross-variant memory B-cell immunity after a 3rd dose/booster of monovalent Wuhan-Hu-1-containing COVID-19 mRNA vaccine” https://pubmed.ncbi.nlm.nih.gov/38233288/

In this longitudinal study subjects receiving two or three doses of monovalent ancestral strain-containing COVID-19 mRNA vaccine were evaluated. In contrast to others, we observed significantly lower frequencies of MBCs reactive to the receptor-binding domain/RBD, the N-terminal domain/NTD, and the S1 of Omicron/BA.1, compared to Wuhan and Delta, even after a 3rd vaccine dose/booster. Our study is a proof of concept that MBC cross-reactivity to variants with greater sequence divergence from the vaccine strain may be overestimated and suggests that these variants may exhibit immune escape with reduced recognition by circulating pre-existing MBCs upon infection.

Tani et al; 2023; “Five doses of the mRNA vaccination potentially suppress ancestral-strain stimulated SARS-CoV2-specific cellular immunity: a cohort study from the Fukushima vaccination community survey, Japan” (https://pmc.ncbi.nlm.nih.gov/articles/PMC10469480)

In this study, we examined the long-term cellular and humoral immune responses following the fifth administration of the mRNA severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine in patients undergoing hemodialysis. To our knowledge, this is the first study to monitor long-term data on humoral and cellular immunity dynamics in high-risk populations after five doses of mRNA vaccination, including the bivalent mRNA vaccine. Whereas most patients maintained humoral immunity throughout the observation period, we observed reduced cellular immune reactivity as measured by the ancestral-strain-stimulated ELISpot assay in a subset of patients. Half of the individuals (50%; 14/28) maintained cellular immunity three months after the fifth dose, despite acquiring humoral immunity. The absence of a relationship between positive controls and T-Spot reactivity suggests that these immune alterations were specific to SARS-CoV-2. In multivariable analysis, participants aged ≥70 years showed a marginally significant lower likelihood of having reactive results. Notably, among the 14 individuals who received heterologous vaccines, 13 successfully acquired cellular immunity, supporting the effectiveness of this administration strategy.

Qin et al; 2022; “Pre-exposure to mRNA-LNP inhibits adaptive immune responses and alters innate immune fitness in an inheritable fashion” (https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010830)

The mRNA-LNP-based SARS-CoV-2 vaccine is highly inflammatory, and its synthetic ionizable lipid component responsible for the induction of inflammation has a long in vivo half-life. Since chronic inflammation can lead to immune exhaustion and non-responsiveness, we sought to determine the effects of pre-exposure to the mRNA-LNP on adaptive immune responses and innate immune fitness. We found that pre-exposure to mRNA-LNPs or LNP alone led to long-term inhibition of the adaptive immune response, which could be overcome using standard adjuvants. On the other hand, we report that after pre-exposure to mRNA-LNPs, the resistance of mice to heterologous infections with influenza virus increased while resistance to Candida albicans decreased. The diminished resistance to Candida albicans correlated with a general decrease in blood neutrophil percentages. Interestingly, mice pre-exposed to the mRNA-LNP platform can pass down the acquired immune traits to their offspring, providing better protection against influenza. In summary, the mRNA-LNP vaccine platform induces long-term unexpected immunological changes affecting both adaptive immune responses and heterologous protection against infections.

Segato et al., 2024; “T Cell Responses to BA.2.86 and JN.1 SARS-CoV-2 Variants in Elderly Subjects” (https://pubmed.ncbi.nlm.nih.gov/39772110/)

Elderly subjects showed reduced IgG levels against JN.1 compared with the ancestral strain. BA.2.86 stimulation resulted in lower IFN-γ levels in the elderly versus the COVID-19-naïve group. AIM analysis showed that among T cells, CD4+ were the most responsive, with a reduced proportion of JN.1-reactive CD4+ T cells compared with the ancestral strain in the SARS-CoV-2-unexposed group. Despite receiving the updated booster, the elderly group showed reduced CD4+ T cell reactivity to BA.2.86.

Conclusions: The XBB.1.5-containing vaccine induced lower CD4+ T cell responses against BA.2.86 in the elderly. CD4+ T cells from BNT16b2-vaccinated, COVID-19-naïve subjects recognized ancestral and BA.2.86 RBD strains while showing reduced responses to JN.1. These results emphasize the need for tailored vaccine strategies for emerging variants, particularly in vulnerable populations.

Esmailian et al; 2024; “Case report of secondary T-cell deficiency following the AstraZeneca COVID-19 vaccine” (https://www.jaci-global.org/article/S2772-8293(24)00135-8/fulltext)

We present a case of secondary T-cell deficiency particularly affecting CD4 T cells, along with the emergence of chronic spontaneous urticaria in a patient following COVID-19 vaccination. The condition was partially managed with omalizumab after initial first-line therapy proved ineffective.

The patient had no notable infections during the follow-up period, but he did exhibit impaired T-cell proliferation, with PHA stimulation showing a rate of 43.7% compared with 95.7% in the control (by a flow-based assay using intracellular Ki67 staining). Furthermore, his immunoglobulin profile demonstrated an IgG level of 10.9 g/L (reference range 7.5-15.6 g/L), IgM level of 1.5 g/L (reference range 0.4-3.0 g/L), and IgA level of 1.1 g/L (reference range 0.8-4.5 g/L)

Boretti; 2024; “mRNA vaccine boosters and impaired immune system response in immune compromised individuals: a narrative review” (https://link.springer.com/article/10.1007/s10238-023-01264-1)

Over the last 24 months, there has been growing evidence of a correlation between mRNA COVID-19 vaccine boosters and increased prevalence of COVID-19 infection and other pathologies. Recent works have added possible causation to correlation. mRNA vaccine boosters may impair immune system response in immune compromised individuals. Multiple doses of the mRNA COVID-19 vaccines may result in much higher levels of IgG 4 antibodies, or also impaired activation of CD

Uversky et al; 2023; “IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein” (https://www.mdpi.com/2076-393X/11/5/991) – ALSO RELEVANT TO CLASS SWITCHING SECTION

“As the immunity provided by these vaccines rapidly wanes, their ability to prevent hospitalization and severe disease in individuals with comorbidities has recently been questioned, and increasing evidence has shown that, as with many other vaccines, they do not produce sterilizing immunity, allowing people to suffer frequent re-infections. Additionally, recent investigations have found abnormally high levels of IgG4 in people who were administered two or more injections of the mRNA vaccines. HIV, Malaria, and Pertussis vaccines have also been reported to induce higher-than-normal IgG4 synthesis. Overall, there are three critical factors determining the class switch to IgG4 antibodies: excessive antigen concentration, repeated vaccination, and the type of vaccine used. It has been suggested that an increase in IgG4 levels could have a protecting role by preventing immune over-activation, similar to that occurring during successful allergen-specific immunotherapy by inhibiting IgE-induced effects. However, emerging evidence suggests that the reported increase in IgG4 levels detected after repeated vaccination with the mRNA vaccines may not be a protective mechanism; rather, it constitutes an immune tolerance mechanism to the spike protein that could promote unopposed SARS-CoV2 infection and replication by suppressing natural antiviral responses. Increased IgG4 synthesis due to repeated mRNA vaccination with high antigen concentrations may also cause autoimmune diseases, and promote cancer growth and autoimmune myocarditis in susceptible individuals.”

We propose a hypothetical immune tolerance mechanism induced by mRNA vaccines, which could have at least six negative unintended consequences:

(1) By ignoring the spike protein synthesized as a consequence of vaccination, the host immune system may become vulnerable to re-infection with the new Omicron subvariants, allowing for free replication of the virus once a re-infection takes place. In this situation, we suggest that even these less pathogenic Omicron subvariants could cause significant harm and even death in individuals with comorbidities and immuno-compromised conditions.

(2) mRNA and inactivated vaccines temporally impair interferon signaling [142,143], possibly causing immune suppression and leaving the individual in a vulnerable situation against any other pathogen. In addition, this immune suppression could allow the re-activation of latent viral, bacterial, or fungal infections and might also allow the uncontrolled growth of cancer cells [144].

(3) A tolerant immune system might allow SARS-CoV-2 persistence in the host and promote the establishment of a chronic infection, similar to that generated by the hepatitis B virus (HBV), the human immune deficiency virus (HIV), and the hepatitis C virus (HCV) [145].

(4) The combined immune suppression (produced by SARS-CoV-2 infection [15,16,17,18,19,20,21,22] and further enhanced by vaccination [142,143,144]) could explain a plethora of autoimmune conditions, such as cancers, re-infections, and deaths temporally associated with both. It is conceivable that the excess deaths reported in several highly COVID-19-vaccinated countries may be explained, in part, by this combined immunosuppressive effect.

(5) Repeated vaccination could also lead to auto-immunity: in 2009, the results of an important study went largely unnoticed. Researchers discovered that in mice that are otherwise not susceptible to spontaneous autoimmune disorders, repeated administration of the antigen promotes systemic autoimmunity. The development of CD4+ T cells that can induce autoantibodies (autoantibody-inducing CD4+ T cells, or aiCD4+ T cells), which had their T cell receptors (TCR) modified, was triggered by excessive stimulation of CD4+ T cells. The aiCD4+ T cell was generated by new genetic TCR modification rather than a cross-reaction. The excessively stimulated CD8+ T cells induced them to develop into cytotoxic T lymphocytes (CTL) that are specific for an antigen. These CTLs were able to mature further by antigen cross-presentation, so in that situation, they induced autoimmune tissue damage resembling systemic lupus erythematosus (SLE) [146]. According to the self-organized criticality theory, when the immune system of the host is continually overstimulated by antigen exposure at concentrations higher than the immune system’s self-organized criticality can tolerate, systemic autoimmunity inevitably occurs [147].

It has been proposed that the amount and duration of the spike protein produced are presumably affected by the higher mRNA concentrations in the mRNA-1273 vaccine (100 µg) compared to the BNT162b2 vaccine (30 µg) [31]. Thus, it is probable that the spike protein produced in response to mRNA vaccination is too high and lasts too long in the body. That could overwhelm the capacity of the immune system, leading to autoimmunity [146,147]. Indeed, several investigations have found that COVID-19 immunization is associated with the development of autoimmune responses [148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166].

(6) Increased IgG4 levels induced by repeated vaccination could lead to autoimmune myocarditis; it has been suggested that IgG4 antibodies can also cause an autoimmune reaction by impeding the immune system’s ability to be suppressed by regulatory T cells [102]. Patients using immune checkpoint inhibitors alone or in combination have been linked to occurrences of acute myocarditis [103,104,105,106,107], sometimes with lethal consequences [102]. As anti-PD-1 antibodies are class IgG4, and these antibodies are also induced by repeated vaccination, it is plausible to suggest that excessive vaccination could be associated with the occurrence of an increased number of myocarditis cases and sudden cardiac deaths.”

Seneff et al; 2022; “Innate immune suppression by SARS-CoV-2 mRNA vaccinations: The role of G-quadruplexes, exosomes, and MicroRNAs” (https://www.sciencedirect.com/science/article/pii/S027869152200206X?via%3Dihub)

“The mRNA SARS-CoV-2 vaccines were brought to market in response to the public health crises of Covid-19. The utilization of mRNA vaccines in the context of infectious disease has no precedent. The many alterations in the vaccine mRNA hide the mRNA from cellular defenses and promote a longer biological half-life and high production of spike protein. However, the immune response to the vaccine is very different from that to a SARS-CoV-2 infection. In this paper, we present evidence that vaccination induces a profound impairment in type I interferon signaling, which has diverse adverse consequences to human health. Immune cells that have taken up the vaccine nanoparticles release into circulation large numbers of exosomes containing spike protein along with critical microRNAs that induce a signaling response in recipient cells at distant sites. We also identify potential profound disturbances in regulatory control of protein synthesis and cancer surveillance. These disturbances potentially have a causal link to neurodegenerative disease, myocarditis, immune thrombocytopenia, Bell’s palsy, liver disease, impaired adaptive immunity, impaired DNA damage response and tumorigenesis.”

Schiepers et al; 2023; “Molecular fate-mapping of serum antibody responses to repeat immunization” (https://pubmed.ncbi.nlm.nih.gov/36646114/)

“We show that serum responses to sequential homologous boosting derive overwhelmingly from primary cohort B cells, while later induction of new antibody responses from naive B cells is strongly suppressed. Such ‘primary addiction’ decreases sharply as a function of antigenic distance, allowing reimmunization with divergent viral glycoproteins to produce de novo antibody responses targeting epitopes that are absent from the priming variant. Our findings have implications for the understanding of OAS and for the design and testing of vaccines against evolving pathogens.”

Lee et al; 2023; “Low humoral and cellular immune responses early after breakthrough infection may contribute to severe COVID-19” (https://pubmed.ncbi.nlm.nih.gov/37033936/)

Results: Anti-S1 IgG titers were significantly lower in the severe group than in the non-severe group within 1 week of symptom onset and higher in the non-severe group than in the control group. Compared with the control group, the cellular immune response tended to be diminished in breakthrough cases, particularly in the severe group. In multivariate analysis, advanced age and low anti-S1 IgG titer were associated with severe breakthrough COVID-19.

Conclusions: Severe breakthrough COVID-19 might be attributed by low humoral and cellular immune responses early after infection. In the vaccinated population, delayed humoral and cellular immune responses may contribute to severe breakthrough COVID-19.

9b. Antibody class switching

Irrgang et al; 2022; “Class switch toward noninflammatory, spike-specific IgG4 antibodies after repeated SARS-CoV-2 mRNA vaccination” (https://www.science.org/doi/10.1126/sciimmunol.ade2798)

Here, we report that several months after the second vaccination, SARS-CoV-2–specific antibodies were increasingly composed of noninflammatory IgG4, which were further boosted by a third mRNA vaccination and/or SARS-CoV-2 variant breakthrough infections. IgG4 antibodies among all spike-specific IgG antibodies rose, on average, from 0.04% shortly after the second vaccination to 19.27% late after the third vaccination. This induction of IgG4 antibodies was not observed after homologous or heterologous SARS-CoV-2 vaccination with adenoviral vectors. Single-cell sequencing and flow cytometry revealed substantial frequencies of IgG4-switched B cells within the spike-binding memory B cell population [median of 14.4%; interquartile range (IQR) of 6.7 to 18.1%] compared with the overall memory B cell repertoire (median of 1.3%; IQR of 0.9 to 2.2%) after three immunizations. This class switch was associated with a reduced capacity of the spike-specific antibodies to mediate antibody-dependent cellular phagocytosis and complement deposition. Because Fc-mediated effector functions are critical for antiviral immunity, these findings may have consequences for the choice and timing of vaccination regimens using mRNA vaccines, including future booster immunizations against SARS-CoV-2.

Impact of breakthrough infections on vaccine-induced antibody responses

The fact that individuals who experienced a breakthrough infection after being vaccinated three times with mRNA showed the highest IgG4 levels in cohort 1 (Fig. 1) suggested that infections with SARS-CoV-2 can also activate IgG4-switched memory B cells. To investigate this in more detail, we identified 12 persons from a study cohort of breakthrough infections (CoVaKo study) who were vaccinated two or three times with SARS-CoV-2 mRNA vaccines and who experienced a breakthrough infection 25 to 257 days after the second or 57 to 164 days after the third mRNA vaccination (table S4). Serum samples were collected on the day of study inclusion [visit (V) 1, typically within the first week] as well as 2 (V2) and 4 weeks (V4) after infection-confirming polymerase chain reaction.

In all individuals, we detected an anamnestic antibody response with an increase in spike-binding antibodies from V1 to V4 irrespective of IgG subclasses (fig. S8). Consistent with our previous findings, IgG4 levels were generally higher in individuals who received three compared with two mRNA vaccinations. In the cohort that had two mRNA vaccinations before breakthrough infection, only three individuals developed IgG4 antibodies that were above the lower limit of quantification. These three individuals experienced the infection with the largest time difference from the last vaccination at 95, 201, or 257 days after the second vaccination, whereas in the other nine patients, the infection took place between 25 and 78 days after the second mRNA shot. This supports the hypothesis that the switch to IgG4 is a consequence of ongoing GC maturation and that it takes several months until IgG4-switched memory B cells appear.

Buhre et al; 2023; “mRNA vaccines against SARS-CoV-2 induce comparably low long-term IgG Fc galactosylation and sialylation levels but increasing long-term IgG4 responses compared to an adenovirus-based vaccine” (https://www.frontiersin.org/articles/10.3389/fimmu.2022.1020844/full)

We show that the initially high mRNA vaccine-induced blood and salivary anti-S IgG levels, particularly IgG1, markedly decrease over time and approach the lower levels induced with the adenovirus-based vaccine. All three vaccines induced, contrary to the short-term anti-S IgG1 response with high sialylation and galactosylation levels, a long-term anti-S IgG1 response that was characterized by low sialylation and galactosylation with the latter being even below the corresponding total IgG1 galactosylation level. Instead, the mRNA, but not the adenovirus-based vaccines induced long-term IgG4 responses – the IgG subclass with inhibitory effector functions. Furthermore, salivary anti-S IgA levels were lower and decreased faster in naïve as compared to pre-infected vaccinees. Predictively, age correlated with lower long-term anti-S IgG titers for the mRNA vaccines. Furthermore, higher total IgG1 galactosylation, sialylation, and bisection levels correlated with higher long-term anti-S IgG1 sialylation, galactosylation, and bisection levels, respectively, for all vaccine combinations.

Uversky et al; 2023; “IgG4 Antibodies Induced by Repeated Vaccination May Generate Immune Tolerance to the SARS-CoV-2 Spike Protein” (https://www.mdpi.com/2076-393X/11/5/991)

As the immunity provided by these vaccines rapidly wanes, their ability to prevent hospitalization and severe disease in individuals with comorbidities has recently been questioned, and increasing evidence has shown that, as with many other vaccines, they do not produce sterilizing immunity, allowing people to suffer frequent re-infections. Additionally, recent investigations have found abnormally high levels of IgG4 in people who were administered two or more injections of the mRNA vaccines. HIV, Malaria, and Pertussis vaccines have also been reported to induce higher-than-normal IgG4 synthesis. Overall, there are three critical factors determining the class switch to IgG4 antibodies: excessive antigen concentration, repeated vaccination, and the type of vaccine used. It has been suggested that an increase in IgG4 levels could have a protecting role by preventing immune over-activation, similar to that occurring during successful allergen-specific immunotherapy by inhibiting IgE-induced effects. However, emerging evidence suggests that the reported increase in IgG4 levels detected after repeated vaccination with the mRNA vaccines may not be a protective mechanism; rather, it constitutes an immune tolerance mechanism to the spike protein that could promote unopposed SARS-CoV2 infection and replication by suppressing natural antiviral responses. Increased IgG4 synthesis due to repeated mRNA vaccination with high antigen concentrations may also cause autoimmune diseases, and promote cancer growth and autoimmune myocarditis in susceptible individuals.

3.2.1. Repeated Inoculation with COVID-19 Vaccines

Researchers have reported that quickly upon the administration of the first two mRNA vaccine doses, the pro-inflammatory subclasses IgG1 and IgG3 dominated the IgG response. Nevertheless, a few months following the second Pfizer vaccine shot, spike-specific antibodies were further enhanced by a third mRNA injection and/or new infections caused by the SARS-CoV-2 variant [30]. Of all IgG antibodies generated against the spike protein, the IgG4 increased the most, steadily from 0.04% immediately after the second vaccination to 19.27% late after the third one.

Such an increase in IgG4 levels was not observed in individuals who received either the same type or a different type of SARS-CoV-2 vaccine based on adenoviral vectors, proving that, in this study, the mRNA Pfizer vaccine was the only one to cause this response. Surprisingly, 7 months after the second inoculation, the IgG4 levels in the serum of approximately half of the vaccinees surpassed the lower limit of detection [30]. To determine if the increase in IgG4 antibody concentration was exclusive to the homologous mRNA vaccination schedule utilized, researchers studied sera from an independent group that evaluated the immune system’s capacity to react to immunization schedules that are similar and different, with the Pfizer and the adenoviral vector-based vaccine from AstraZeneca. Anti-spike IgG4 antibodies were again detected in 50% of the sera from the BNT-BNT group five to six months after the second vaccination but in only one of the 51 serum samples from the other two vaccine groups. Significantly, following the third booster immunization, a significant rise in IgG4 antibody levels was detected in virtually all vaccine recipients [30].

In this regard, it was recently demonstrated that following the traditional vaccination scheme, the serum-neutralizing effectiveness in mice against the Delta and Omicron variants of the COVID-19 Pfizer vaccine was dramatically diminished after numerous booster doses [112]. Repeated antigen stimulation reportedly caused CD8+ T cells to become exhausted. These boosters also significantly diminished CD4+ and CD8+ T cell responses and enhanced programmed cell death protein 1 (PD-1) and lymphocyte activation gene-3 (LAG-3) production in these T cells [112]. Prolonged vaccination decreased the normal development of the germinal center and hindered the generation of memory B cells specific for RBD. This research additionally revealed that prolonged RBD vaccine booster immunization increased the concentration of the immunosuppressive cytokine IL-10 as well as the proportion of CD25+Foxp3+CD4+ Treg cells. The conventional SARS-CoV-2 vaccine’s ability to provide immunological protection may be significantly impacted by over-vaccination. If this happens, either newly diagnosed COVID-19 cases or people who have already contracted the virus again may have a more severe case of the illness. This concept was proposed after seeing tolerance of both the humoral and cellular immune responses to prolonged booster immunization doses [112].

Raszek et al; 2024; “Exploring the possible link between the spike protein immunoglobulin G4 antibodies and cancer progression” (https://www.explorationpub.com/Journals/ei/Article/1003140)

Repeated inoculation with messenger RNA (mRNA) vaccines elicits immunoglobulin G4 (IgG4) antibody production. Such an increase in the concentration of specific and non-specific IgG4 antibodies allows the growth of some types of cancer by blocking the activation of effector immune cells. This work proposes the hypothesis that cancer growth may be indirectly promoted by increased concentrations of non-specific IgG4 antibodies by the following mechanisms: 1) IgG4 antibodies can bind to anti-tumor IgG1 antibodies and block their interaction with receptors located on effector cells, thus preventing the destruction of cancer cells, 2) IgG4 can interact with fragment crystallizable gamma receptor IIb (FcγRIIB) inhibitory receptors, thus reducing effector functions of innate immune cells, and 3) targeting of specific epitopes by IgG4 could be oncogenic by inducing the production of a microenvironment that can promote cancer development. This article reviews the supporting literature and suggests several experimental protocols to evaluate this hypothesis in the context of repeated inoculation with mRNA vaccines. Additionally, this work proposes some management options aimed at reducing the unfavorable molecular consequences that could mediate cancer development when encountering high concentrations of IgG4 antibodies.

Łysek-Gładysińska et al; 2023; “The Levels of Anti-SARS-CoV-2 Spike Protein IgG Antibodies Before and After the Third Dose of Vaccination Against COVID-19” (https://pubmed.ncbi.nlm.nih.gov/36660373/)

The IgG levels were significantly higher and less diverse after the same follow-up time from the second booster vaccination compared to the first booster. The antibody levels were positively correlated with female, healthcare workers, the elderly and participants with a negative COVID-19 history. Furthermore, the increase in IgG antibodies after the second booster vaccination correlated inversely with the baseline level of antibodies before the vaccination. The latest results showed that antibody levels dropped 1.5-fold after approx. 10 months from the second booster vaccination but still remained at a protective level. At the end of the observation period the level of IgG_4 among 75% of the participants was at least 1.5 times higher than IgG_1 value, and 25% of the participants had values at least 9.5 times higher than the IgG_1 value.

Akhtar et al; 2023; “Appearance of tolerance-induction and non-inflammatory SARS-CoV-2 spike-specific IgG4 antibodies after COVID-19 booster vaccinations” ( https://pubmed.ncbi.nlm.nih.gov/38173725/ )

Comprehensive IgG subclass analysis showed primary Covishield/mRNA vaccination generated predominantly IgG1 responses with limited IgG2/IgG3, Remarkably, IgG4 responses exhibited a distinct pattern. IgG4 remained undetectable initially but increased extensively six months after the second mRNA dose, eventually replacing IgG1 after the 3rd/4th mRNA doses. Conversely, initial Covishield recipients lack IgG4, surged post-second mRNA booster. Notably, mRNA-vaccinated individuals displayed earlier, robust IgG4 levels post first mRNA booster versus Covishield counterparts. IgG1 to IgG4 ratios decreased with increasing doses, most pronounced with four mRNA doses.

Valk et al; 2023; “Suppressed IgG4 class switching in dupilumab- and TNF inhibitor-treated patients after repeated SARS-CoV-2 mRNA vaccination” (https://onlinelibrary.wiley.com/doi/10.1111/all.16089)

Methods Antibody responses to the receptor-binding domain (RBD) of the spike protein upon repeated SARS-CoV-2 mRNA vaccination were measured in 604 individuals including patients with immune-mediated inflammatory diseases treated with TNFi and/or MTX, or dupilumab, as well as healthy controls and untreated patients.

Results We observed a substantial increase in the proportion of RBD-specific IgG4 antibodies (median 21%) in healthy/untreated controls after a third mRNA vaccination. This IgG4 skewing was absent when primary vaccination was adenoviral vector-based and was profoundly reduced in both dupilumab- and TNFi-treated patients (<1%), but only moderately in patients treated with MTX (7%).

Conclusion Our results imply a major role for both IL-4/IL-13 as well as TNF in IgG4 class switching. These novel findings advance our understanding of IgG4 class switch dynamics, and may benefit future mRNA vaccine strategies, humoral tolerance induction, as well as treatment of IgG4 pathologies.

Yoshimura et al; 2023; “The appearance of anti-spike receptor binding domain immunoglobulin G4 responses after repetitive immunization with messenger RNA-based COVID-19 vaccines” (https://www.sciencedirect.com/science/article/pii/S1201971223007890)

Results

The seropositivity of anti-RBD IgG4 after the vaccination was 6.76% at 1 month after the second dose, gradually increased to 50.5% at 6 months after the second dose, and reached 97.2% at 1 month after the third dose. The seropositivity and titers of anti-RBD IgG1/IgG3 quickly reached the maximum at 1 month after the second dose and declined afterward. The elevated anti-RBD IgG4 Ab levels observed after repeated vaccinations were unlikely to increase the risk of breakthrough infection.

Conclusions

Repeated vaccinations induce delayed but drastic increases in anti-RBD IgG4 responses. Further functional investigations are needed to reveal the magnitude of the high contribution of spike-specific IgG4 subclasses after repeated mRNA-based COVID-19 vaccinations.

Kalkeri et al; 2024; “Altered IgG4 Antibody Response to Repeated mRNA versus Recombinant Protein SARS-CoV-2 Vaccines” (https://www.journalofinfection.com/article/S0163-4453(24)00053-7/fulltext)

Highlights

• In agreement with recently published studies, repeated mRNA SARS-CoV-2 vaccination was associated with an increase in Spike-protein specific IgG4.
• By contrast, IgG4 class switch was not observed following four doses of Novavax protein-based SARS-CoV-2 vaccine.
• SARS-CoV-2 specific IgG3, an IgG subclass known to induce potent neutralization and Fc functions, was higher after Novavax homologous vaccination (>10x vs. mRNA).
• In contrast to the mRNA homologous regimens, Novavax homologous and heterologous vaccinations evoked stronger Fcγ-dependent effector activities.

Total anti-S IgG and IgG1 levels following three homologous doses of mRNA or NVX-CoV2373 were similar, although NVX-CoV2373 induced somewhat higher levels, and the fourth dose of NVX-CoV2373 led to increased responses in each group (Fig. 1A). Compared with recipients of prior mRNA vaccine, anti-S IgG3 levels were markedly higher (>10-fold) after three or four homologous doses of NVX-CoV2373. By contrast, much higher anti-S IgG4 levels (>75-fold) were observed following repeated mRNA vaccination, but not after three or four homologous doses of NVX-CoV2373 (Fig. 1A). The fourth dose of NVX-CoV2373 also appeared to enhance surrogate signals for ADCP, ADCC, and ADCD activities in recipients of prior mRNA vaccine, though the effect was greater after a fourth homologous dose of NVX-CoV2373 (Fig. 1B).

Gelderloos et al; 2024; “Repeated COVID-19 mRNA vaccination results in IgG4 class switching and decreased NK cell activation by S1-specific antibodies in older adults” (https://immunityageing.biomedcentral.com/articles/10.1186/s12979-024-00466-9)

Results

Spike S1-specific IgG subclass concentrations (expressed in arbitrary units per mL), antibody-dependent NK cell activation, complement deposition and monocyte phagocytosis were quantified in serum from older adults (n = 38–50, 65–83 years) at one month post-second, -third and -fifth vaccination. Subclass distribution in serum was compared to that in younger adults (n = 64, 18–47 years) at one month post-second and -third vaccination.

Compared to younger individuals, older adults showed increased levels of IgG2 and IgG4 at one month post-third vaccination (possibly related to factors other than age) and a further increase following a fifth dose. The capacity of specific serum antibodies to mediate NK cell activation and complement deposition relative to S1-specific total IgG concentrations decreased upon repeated vaccination. This decrease associated with an increased IgG4/IgG1 ratio.

Conclusions

In conclusion, these findings show that, like younger individuals, older adults produce antibodies with reduced functional capacity upon repeated COVID-19 mRNA vaccination. Additional research is needed to better understand the mechanisms underlying these responses and their potential implications for vaccine effectiveness. Such knowledge is vital for the future design of optimal vaccination strategies in the ageing population.

Marchese et al; 2024; “Mechanisms and Implications of IgG4 Responses to SARS-CoV-2 and Other Repeatedly Administered Vaccines” (https://www.journalofinfection.com/article/S0163-4453(24)00251-2/fulltext)

Highlights

•The predominant IgG subclasses induced by different vaccine platforms can vary

•Increased IgG4 can lead to reduced Fc-dependent effector functions

•IgG4 is disproportionately increased in response to some types of vaccines

•Mechanisms relate to dosing regimen, antigen exposure, and/or cytokine activation

•Clinical implications are uncertain, but IgG4 might reduce vaccine-specific immunity

Abstract

Vaccine-induced immunoglobulin G (IgG) profiles can vary with respect to the predominant subclasses that characterize the response. Among IgG subclasses, IgG4 is reported to have anti-inflammatory properties, but can also exhibit reduced capacity for virus neutralization and activation of Fc-dependent effector functions. Here, we review evidence that IgG4 subclass responses can be disproportionately increased in response to some types of vaccines targeting an array of diseases, including pertussis, HIV, malaria, and COVID-19. The basis for enhanced IgG4 induction by vaccines is poorly understood but may be associated with platform- or dose regimen–specific differences in antigen exposure and/or cytokine stimulation. The clinical implications of vaccine-induced IgG4 responses remain uncertain, though collective evidence suggests that proportional increases in IgG4 might reduce vaccine antigen-specific immunity. Additional work is needed to determine underlying mechanisms and to elucidate what role IgG4 may play in modifications of vaccine-induced immunity to disease.

Kiszel et al; 2023; “Class switch towards spike protein-specific IgG4 antibodies after SARS-CoV-2 mRNA vaccination depends on prior infection history” (https://www.nature.com/articles/s41598-023-40103-x)

We found that vector-based vaccines elicited lower total spike-specific IgG levels than mRNA vaccines. The pattern of spike-specific IgG subclasses in individuals infected before mRNA vaccinations resembled that of vector-vaccinated subjects or unvaccinated COVID-19 patients. However, the pattern of mRNA-vaccinated individuals without SARS-CoV-2 preinfection showed a markedly different pattern. In addition to IgG1 and IgG3 subclasses presented in all groups, a switch towards sdistal IgG subclasses (spike-specific IgG4 and IgG2) appeared almost exclusively in individuals who received only mRNA vaccines or were infected after mRNA vaccinations. In these subjects, the magnitude of the spike-specific IgG4 response was comparable to that of the spike-specific IgG1 response.

Lasrado et al; 2024; “Waning immunity and IgG4 responses following bivalent mRNA boosting” (https://pubmed.ncbi.nlm.nih.gov/38394195/)

Messenger RNA (mRNA) vaccines were highly effective against the ancestral SARS-CoV-2 strain, but the efficacy of bivalent mRNA boosters against XBB variants was substantially lower. Here, we show limited durability of neutralizing antibody (NAb) responses against XBB variants and isotype switching to immunoglobulin G4 (IgG4) responses following bivalent mRNA boosting. Bivalent mRNA boosting elicited modest XBB.1-, XBB.1.5-, and XBB.1.16-specific NAbs that waned rapidly within 3 months. In contrast, bivalent mRNA boosting induced more robust and sustained NAbs against the ancestral WA1/2020 strain, suggesting immune imprinting. Following bivalent mRNA boosting, serum antibody responses were primarily IgG2 and IgG4 responses with poor Fc functional activity. In contrast, a third monovalent mRNA immunization boosted all isotypes including IgG1 and IgG3 with robust Fc functional activity. These data show substantial immune imprinting for the ancestral spike and isotype switching to IgG4 responses following bivalent mRNA boosting, with important implications for future booster designs and boosting strategies.

Espino et al; 2024; “The Anti-SARS-CoV-2 IgG1 and IgG3 Antibody Isotypes with Limited Neutralizing Capacity against Omicron Elicited in a Latin Population a Switch toward IgG4 after Multiple Doses with the mRNA Pfizer-BioNTech Vaccine” (https://pubmed.ncbi.nlm.nih.gov/38399963/)

The aim of this study was to analyze the profiles of IgG subclasses in COVID-19 convalescent Puerto Rican subjects and compare these profiles with those of non-infected immunocompetent or immunocompromised subjects that received two or more doses of an mRNA vaccine. The most notable findings from this study are as follows:

(1) Convalescent subjects that were not hospitalized developed high and long-lasting antibody responses.

(2) Both IgG1 and IgG3 subclasses were more prevalent in the SARS-CoV-2-infected population, whereas IgG1 was more prevalent after vaccination.

(3) Individuals that were infected and then later received two doses of an mRNA vaccine exhibited a more robust neutralizing capacity against Omicron than those that were never infected and received two doses of an mRNA vaccine.

(4) A class switch toward the “anti-inflammatory” antibody isotype IgG4 was induced a few weeks after the third dose, which peaked abruptly and remained at high levels for a long period. Moreover, the high levels of IgG4 were concurrent with high neutralizing percentages against various VOCs including Omicron.

(5) Subjects with IBD also produced IgG4 antibodies after the third dose, although these antibody levels had a limited effect on the neutralizing capacity. Knowing that the mRNA vaccines do not prevent infections, the Omicron subvariants have been shown to be less pathogenic, and IgG4 levels have been associated with immunotolerance and numerous negative effects, the recommendations for the successive administration of booster vaccinations to people should be revised

Portilho et al; 2024; “An unexpected IgE anti-receptor binding domain response following natural infection and different types of SARS-CoV-2 vaccines” (https://pubmed.ncbi.nlm.nih.gov/39198569/)

High IgE levels are moderately followed by IgG4 isotype

IgE is typically induced by Th2 microenvironment, which may be propitiated by Alum¹⁹. However, we observed IgE in individuals immunized with ChAdOx1 and an enhancement after BNT162b2, both vaccines that do not use Alum as an adjuvant. Since IgE and IgG4 are both induced in strong Th2 environments and it has been previously reported that viral antigens may trigger both immunoglobulins^11,20, we tested the samples for IgG4 to better investigate Th2 antibodies (Fig. 5). While COVID-19 did not induce detectable levels of this IgG subclass, two vaccine doses increased its levels; but only after the booster IgG4 index was statistically higher than the pre-pandemic control, irrespective of previous SARS-CoV-2 infection (p < 0.001 for 2 doses + booster and for Cov + 2 doses + booster). When the vaccine schedules were studied separately, we confirmed that an mRNA booster was needed to achieve a higher IgG4 titer than control (p < 0.001 for ChAdOx1 + booster and p < 0.01 for CoronaVac + booster). Despite that, we found only a moderate correlation between IgE and IgG4 levels (r = 0.3408, p < 0.05).

Hartley et al; 2023; “Third dose COVID-19 mRNA vaccine enhances IgG4 isotype switching and recognition of Omicron subvariants by memory B cells after mRNA but not adenovirus priming” (https://www.biorxiv.org/content/10.1101/2023.09.15.557929v2)

Results Dose 3 boosters significantly increased ancestral RBD-specific plasma IgG and Bmem in both cohorts. Up to 80% of ancestral RBD-specific Bmem expressed IgG1⁺. IgG4⁺ Bmem were detectable after primary mRNA vaccination, and expanded significantly to 5-20% after dose 3, whereas heterologous boosting did not elicit IgG4⁺ Bmem. Recognition of Omicron BA.2 and BA.5 by ancestral RBD-specific plasma IgG increased from 20% to 60% after the 3rd dose in both cohorts. Reactivity of ancestral RBD-specific Bmem to Omicron BA.2 and BA.5 increased following a homologous booster from 40% to 60%, but not after a heterologous booster.

Conclusion A 3rd mRNA dose generates similarly robust serological and Bmem responses in homologous and heterologous vaccination groups. The expansion of IgG4⁺ Bmem after mRNA priming might result from the unique vaccine formulation or dosing schedule affecting the Bmem response duration and antibody maturation.

Martín Pérez et al; 2025; “Post-vaccination IgG4 and IgG2 class switch associates with increased risk of SARS-CoV-2 infections” (https://www.journalofinfection.com/article/S0163-4453(25)00067-2/fulltext)

Highlights

• IgG4 and IgG2 levels increase markedly after the third mRNA dose against SARS-CoV-2.
• Elevated IgG4 levels after booster vaccination associate with an increased risk of infections.
• Increased non-cytophilic to cytophilic antibody ratio correlates with reduced functionality.

Objectives

Repeated COVID-19 mRNA vaccinations increase SARS-CoV-2 IgG4 antibodies, indicating extensive IgG class switching following the first booster dose. This shift in IgG subclasses raises concerns due to the limited ability of IgG4 to mediate Fc-dependent effector functions.
Methods

To assess the impact of IgG4 induction on protective immunity, we analyzed longitudinal SARS-CoV-2 IgG subclasses, C1q and FcγR responses, and neutralizing activity in a well-characterized cohort of healthcare workers in Spain.
Results

Elevated IgG4 levels and higher ratios of non-cytophilic to cytophilic antibodies after booster vaccination were significantly associated with an increased risk of breakthrough infections (IgG4 HR[10-fold increase]=1.8, 95% CI=1.2–2.7; non-cytophilic to cytophilic ratio HR[10-fold increase]=1.5, 95% CI=1.1–1.9). Moreover, an increased non-cytophilic to cytophilic antibody ratio correlated with reduced functionality, including neutralization.
Conclusions

These findings suggest a potential association between IgG4 induction by mRNA vaccination and a higher risk of breakthrough infection, warranting further investigation into vaccination strategies to ensure sustained protection.

9c. Immunological Imprinting

Wheatley et al; 2021; “Immune imprinting and SARS-CoV-2 vaccine design” (https://pubmed.ncbi.nlm.nih.gov/34580004/)

Neutralizing antibodies correlate with protective immunity for first generation vaccines against SARS-CoV-2 (see Glossary) [1]. The viral Spike protein is the primary target for neutralizing antibodies and the principal antigen in most vaccines to date. SARS-CoV-2 infection can be prevented in animal models and humans with neutralizing antibodies [2]. Infection or immunization induce a remarkably robust antibody response in which such immunoglobulins bind a range of epitopes specific to Spike. However, most of these antibodies are nonneutralizing, with only a small fraction of recovered monoclonal antibodies
effectively neutralizing SARS-CoV-2 [3]. Although both nonneutralizing antibodies (including those with Fc-mediated functions) and T cells play an important role in control of many viral infections, including SARS-CoV-2, effective protection from SARS-CoV-2 appears primarily driven by neutralizing antibodies [1]. Several SARS-CoV-2 variants of concern (VOC) harboring mutations in the receptor binding domain (RBD) have emerged that efficiently escape the neutralizing antibody response raised by infection or by currently approved vaccines [4]. Existing vaccines have lower efficacy in preventing infection with newer or emerging escape variants that suffer neutralization deficits, most notably Beta and Delta variants. Thus, vaccine manufacturers are updating first generation vaccines by replacing the original Spike with the sequence from one or more new variants. This updated vaccine strategy is logical and rapid as it takes advantage of existing vaccine technologies. Early results suggest this may well be at least partially effective, with an updated mRNA booster vaccine (Moderna) expressing Beta Spike eliciting neutralization against this variant in preprimed individuals [5]. However, boosting with the Beta variant vaccine in the preprimed group resulted in better neutralization of the ancestral SARS-CoV-2 strain than the Beta strain. This suggests that immune imprinting of the response may have occurred. Immune imprinting is a phenomenon whereby initial exposure to one virus strain effectively primes B cell memory and limits
the development of memory B cells and neutralizing antibodies against new minor variant strains of the virus [6]. We hypothesize that repeatedly updating SARS-CoV-2 vaccines might not be fully effective because of limitations imposed by prior immune imprinting to ancestral SARS-CoV-2 strains. Although
it is hoped that immune imprinting will not be a major problem for SARS-CoV-2 infections, the possibility that immune imprinting will substantially reduce efficacy of future SARS-CoV-2 vaccines requires action now to both define the extent of the problem and begin to devise solutions.

Evans J., & Shan-Lu L.,; 2023; “Challenges and Prospects in Developing Future SARS-CoV-2 Vaccines: Overcoming Original Antigenic Sin and Inducing Broadly Neutralizing Antibodies” (https://journals.aai.org/jimmunol/article/211/10/1459/266281/Challenges-and-Prospects-in-Developing-Future-SARS)

The impacts of the COVID-19 pandemic led to the development of several effective SARS-CoV-2 vaccines. However, waning vaccine efficacy as well as the antigenic drift of SARS-CoV-2 variants has diminished vaccine efficacy against SARS-CoV-2 infection and may threaten public health. Increasing interest has been given to the development of a next generation of SARS-CoV-2 vaccines with increased breadth and effectiveness against SARS-CoV-2 infection. In this Brief Review, we discuss recent work on the development of these next-generation vaccines and on the nature of the immune response to SARS-CoV-2. We examine recent work to develop pan-coronavirus vaccines as well as to develop mucosal vaccines. We further discuss challenges associated with the development of novel vaccines including the need to overcome “original antigenic sin” and highlight areas requiring further investigation. We place this work in the context of SARS-CoV-2 evolution to inform how the implementation of future vaccine platforms may impact human health.

Paciello et al; 2024; “Antigenic sin and multiple breakthrough infections drive converging evolution of COVID-19 neutralizing responses” (https://pubmed.ncbi.nlm.nih.gov/39207904/)

We longitudinally analyze at the single-cell level almost 900 neutralizing human monoclonal antibodies (nAbs) isolated from vaccinated people and from individuals with hybrid and super hybrid immunity (SH), developed after three mRNA vaccine doses and two breakthrough infections. The most potent neutralization and Fc functions against highly mutated variants belong to the SH cohort. Repertoire analysis shows that the original Wuhan antigenic sin drives the convergent expansion of the same B cell germlines in vaccinated and SH cohorts. Only Omicron breakthrough infections expand previously unseen germ lines and generate broadly nAbs by restoring IGHV3-53/3-66 germ lines. Our analyses find that B cells initially expanded by the original antigenic sin continue to play a fundamental role in the evolution of the immune response toward an evolving virus.

Wooseob Kim; 2024; “Germinal Center Response to mRNA Vaccination and Impact of Immunological Imprinting on Subsequent Vaccination” (https://pubmed.ncbi.nlm.nih.gov/39246619/)

The emergence of the coronavirus disease 2019 pandemic has prompted rapid development and deployment of lipid nanoparticle encapsulated, mRNA-based vaccines. While these vaccines have demonstrated remarkable immunogenicity, concerns persist regarding their ability to confer durable protective immunity to continuously evolving severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. This review focuses on human B cell responses induced by SARS-CoV-2 mRNA vaccination, with particular emphasis on the crucial role of germinal center reactions in shaping enduring protective immunity. Additionally, we explored observations of immunological imprinting and dynamics of recalled pre-existing immunity following variants of concern-based booster vaccination. Insights from this review contribute to comprehensive understanding B cell responses to mRNA vaccination in humans, thereby refining vaccination strategies for optimal and sustained protection against evolving coronavirus variants.

B cell response to VOCs-based booster vaccination

The ongoing evolution of SARS-CoV-2 viruses, characterized by mutations in the spike protein, has endangered the efficacy of the initial two-dose of primary vaccination, leading to recommendations for additional booster shots (64,73). Multiple studies have suggested that administrating repeated doses of homologous boosters containing the ancestral Wuhan-hu-1 spike can substantially improve Ab responses against both the Wuhan-Hu-1 and VOCs (74,75,76). Concurrently, vaccines based on VOCs’ spike proteins have been developed to more effectively address these continuously evolving viruses (77,78,79,80,81). However, efforts to improve the effectiveness of vaccine by incorporating spike Ags from VOCs have encountered challenges due to the immunological imprinting induced by ancestral spike-based primary vaccination. A main concern is whether VOCs spike proteins-based vaccines can induce de novo Ab responses to VOC-specific, non-conserved epitopes.

Recent studies have compared immune responses in individuals who have received third doses of the monovalent Wuhan-Hu-1-based vaccine, a monovalent B.1.341 (Beta)-based vaccine (mRNA1273.351), a bivalent B.1.351/B.1.617.2 (Beta/Delta)-based vaccine (mRNA-1273.213), and an monovalent BA.1 (Omicron)-based vaccine (mRNA-1273.529) (32,82). Alsoussi et al. (32) focused on participants without prior COVID-19 infection history who had completed a two-dose primary vaccination. The primary objective was to assess the exclusive impact of booster vaccination on immune response against VOCs, considering both quantitative and qualitative aspects. Vaccine spike-specific plasmablasts were detected in blood samples of all participants. Their maturation was confirmed through increased SHM frequencies in their Ig heavy chain variable region gene (IGHV). Interestingly, plasmablasts identified after the booster vaccination exhibited significantly higher mutation frequencies than those after the primary two-dose vaccination series. This suggests that these plasmablasts might have originated from MBCs that have undergone affinity maturation after completion of the primary vaccination series (33,34). Meanwhile, administration of booster vaccination elicited a robust GC response specific to vaccine spike Ags in draining axillary lymph nodes, persisting for a minimum of eight wks post-vaccination. Consistent with B cell responses observed in conventional seasonal influenza vaccinations (26), it was observed that pre-existing MBCs participated and re-engaged in GC reactions after booster vaccination. The quantity of bone marrow-resident LLPCs was also notably increased following a booster vaccination. This phenomenon is a result of persistent GC reactions induced by the initial two vaccine doses combined with additional GC reactions induced by repeated Ag exposures from the booster vaccination.

Additionally, Alsoussi et al. (32) provides important insights regarding the extent and dynamics of immunological imprinting after VOCs-based vaccination. Even if any booster vaccines did not encode ancestral spike Ags, the majority of GC B cells and MBCs identified after bivalent beta/delta or monovalent omicron vaccination could recognize the ancestral spike protein. This implies that vaccine-responding B cells predominantly originate from pre-existing clonal lineage established by the primary vaccination. Given that the degree of immunological imprinting could be determined by the antigenic distance between prior and subsequent exposures (83,84,85), a high antigenic similarity between beta/delta spike and ancestral spike might have contributed to highly cross-reactive responses. As shown in the case of monovalent omicron vaccine, where spike Ags were antigenically distant from the ancestral spike, MBCs specific to omicron spike Ag were definitely observed. These B cells appeared to originate from de novo responses as they recognized omicron-specific epitopes with low SHM mutation frequencies. The immunological imprinting induced by ancestral spike-based vaccination was also reflected in serological responses, which are outcomes of B cell responses to subsequent exposures. Individuals who have received two doses of primary vaccination and encountered omicron infection still exhibit low levels of omicron-specific Ab responses (86,87,88). Moreover, a bivalent booster BA.1 (mRNA-1273.214) and a bivalent booster BA.4/BA.5 (mRNA-1273.222) demonstrated superior neutralizing activity to Wuhan-Hu-1 booster against Wuhan-Hu-1 as well as matched Omicron sublineages (89,90). Collectively, the abundant presence of serum Ab titer and MBCs that can recognize the ancestral spike protein following subsequent exposures serves as evidence of immunological imprinting. This phenomenon indicates that B cell responses to previously encountered Ags can maintain dominance even after exposure to antigenically related other Ags. Accordingly, the WHO Technical Advisory Group on COVID-19 Vaccine Composition recommended using monovalent Omicron XBB.1.5-based immunogen instead of Wuhan-Hu-1-based immunogen for future booster vaccine formulations due to antigenic divergence and the potential for immunological imprinting (91). This recommendation is supported by recent experimental evidence indicating that repeated boosting with omicron spike Ags has been shown to alleviate immunological imprinting (92). Taken together, these findings offer support to the idea that vaccination strategies with appropriately designed Ags are necessary to counter immune imprinting and activate infrequent naive B cells targeting novel variant epitopes.

Alvarez-Sierra et al; 2024; “The adaptive immune responses to SARS-CoV-2 as a recall response susceptible to immune imprinting” (https://www.medrxiv.org/content/10.1101/2024.08.11.24311358v1)

Analysis of the Ig isotype response trajectories to the Mpro, NP, and S structural proteins and the S RBD in this group of 191 patients and 44 controls revealed a pattern of recall response in 94.2 % of cases. The levels of antibodies correlated positively with the severity of the condition rather than a milder course. High-resolution flow cytometry of fresh PBMNCs showed trajectories of plasmablasts, B cell subsets, and cTfh, suggesting a recall response. The transcriptomic profile demonstrated that this group was directly comparable to other contemporary cohorts. Overall, these findings support the idea that the response to SARS-CoV-2 is, in most cases, a recall response, likely due to B and T memory cells to CCCV, and therefore susceptible to immune imprinting and antibody-dependent enhancement.

Niu et al; 2024; “Omicron-specific ultra-potent SARS-CoV-2 neutralizing antibodies targeting the N1/N2 loop of Spike N-terminal domain” (https://www.tandfonline.com/doi/full/10.1080/22221751.2024.2412990)

In this study, we find that most of NTD-targeting antibodies isolated from individuals with BA.5/BF.7 breakthrough infection (BTI) are ancestral (wildtype or WT)-reactive and non-neutralizing. Surprisingly, we identified five ultra-potent neutralizing antibodies (NAbs) that can only bind to Omicron but not WT NTD. Structural analysis revealed that they bind to a unique epitope on the N1/N2 loop of NTD and interact with the receptor-binding domain (RBD) via the light chain. These Omicron-specific NAbs achieve neutralization through ACE2 competition and blockage of ACE2-mediated S1 shedding. However, BA.2.86 and BA.2.87.1, which carry insertions or deletions on the N1/N2 loop, can evade these antibodies. Together, we provided a detailed map of the NTD-targeting antibody repertoire in the post-Omicron era, demonstrating their vulnerability to NTD mutations enabled by its evolutionary flexibility, despite their potent neutralization. These results revealed the function of the indels in the NTD of BA.2.86/JN.1 sublineage in evading neutralizing antibodies and highlighted the importance of considering the immunogenicity of NTD in vaccine design.

Carzaniga et al; 2024; “Serum antibody fingerprinting of SARS-CoV-2 variants in infected and vaccinated subjects by label-free microarray biosensor” (https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1323406/full)

Subjects vaccinated by WT antigen and convalescent unvaccinated subjects display RGU fingerprints with different features, as shown in Figure 3a-c. Further differences emerge from the analysis of the absolute quantification of Ig binding by GUIg. Figure 3d shows that, on average, vaccinated subjects display larger quantities of specific antibodies against WT spike protein and RBD than unvaccinated subjects do. Data exhibit a large subject-to-subject variation coherent with the wide range of IgG concentrations estimated by ELISA, 5 – 300 ng mL−1 (Supplementary Figure S3). Figure 3d also indicates that the antibodies binding WT-LtRBD are more that those binding WT-RBD (blue vs. orange columns and lines) for both vaccinated and unvaccinated subjects, suggesting that RBD developed in human cell lines, in which glycosylation is smaller, are slightly less prone to antibody recognition. Even more significant is the difference in binding to the full spike protein (grey column and line), much weaker for vaccinated subjects in comparison to convalescent ones. This difference is also shown in Figure 3e, where it appears that, for equal response to WT-RBD, unvaccinated convalescent subjects have on average a larger response to the full spike protein.

Finally, Figure 3f shows that anti-nucleocapsid Ig are only present in samples of convalescent subjects. This is expected since SARS-CoV-2 nucleocapsid protein is not contained in vaccine formulation (Ed: but needed for viral clearance, so samples show that the unvaccinated have copious amounts needed to clear the virus .. vaccinated do not). Two samples of vaccinated subjects displayed anti-nucleocapsid Ig: VX08 was in prolonged contacts with infected subjects after vaccination and VX13 was presumably infected before vaccination, since some symptoms were reported. As apparent from the vertical scales in panels 3f vs. 3d, the response to nucleocapsid is extremely variable among the subjects. Indeed, while positive response to nuclecapsid is a clear indication of a previous infection, undetectable levels of anti-nucleocapsid antibodies is not necessarily an indication of the absence of previous infections, as in the case of samples NV04 and NV10, negative to nucleocapsid despite their past infection, as confirmed by molecular testing.

Subjects vaccinated by WT antigen and convalescent unvaccinated subjects display RGU fingerprints with different features, as shown in Figure 3a-c. Further differences emerge from the analysis of the absolute quantification of Ig binding by GUIg. Figure 3d shows that, on average, vaccinated subjects display larger quantities of specific antibodies against WT spike protein and RBD than unvaccinated subjects do (Imprinting much???). Data exhibit a large subject-to-subject variation coherent with the wide range of IgG concentrations estimated by ELISA, 5 – 300 ng mL−1 (Supplementary Figure S3). Figure 3d also indicates that the antibodies binding WT-LtRBD are more that those binding WT-RBD (blue vs. orange columns and lines) for both vaccinated and unvaccinated subjects, suggesting that RBD developed in human cell lines, in which glycosylation is smaller, are slightly less prone to antibody recognition. Even more significant is the difference in binding to the full spike protein (grey column and line), much weaker for vaccinated subjects in comparison to convalescent ones. This difference is also shown in Figure 3e, where it appears that, for equal response to WT-RBD, unvaccinated convalescent subjects have on average a larger response to the full spike protein.

Finally, Figure 3f shows that anti-nucleocapsid Ig are only present in samples of convalescent subjects. This is expected since SARS-CoV-2 nucleocapsid protein is not contained in vaccine formulation (Ed: but needed for viral clearance, so samples show that the unvaccinated have copious amounts needed to clear the virus .. vaccinated do not). Two samples of vaccinated subjects displayed anti-nucleocapsid Ig: VX08 was in prolonged contacts with infected subjects after vaccination and VX13 was presumably infected before vaccination, since some symptoms were reported. As apparent from the vertical scales in panels 3f vs. 3d, the response to nucleocapsid is extremely variable among the subjects. Indeed, while positive response to nuclecapsid is a clear indication of a previous infection, undetectable levels of anti-nucleocapsid antibodies is not necessarily an indication of the absence of previous infections, as in the case of samples NV04 and NV10, negative to nucleocapsid despite their past infection, as confirmed by molecular testing.

Faraone et al; 2023; “Immune imprinting as a barrier to effective COVID-19 vaccines” (https://pubmed.ncbi.nlm.nih.gov/37992689/)

Immune imprinting can be induced either separately or concomitantly by one of two mechanisms: one is that the immune system favors a recalled response over a de novo one (“antigenic seniority”), and the other is that the de novo response is actively suppressed (“primary addiction”). Evidence for both phenomena has been found for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination.³ Antigenic seniority has been demonstrated by the fact that upon infection of vaccinated individuals with variants such as Alpha, Delta, or Omicron, the neutralizing antibody response remains biased toward wild-type spike. Additionally, infection with SARS-CoV-2 in unvaccinated individuals has been shown to favorably induce antibodies that cross-react with common cold betacoronaviruses HKU1 and OC43. Direct evidence for primary addiction has been demonstrated by experiments that tracked the induction of memory versus de novo B cell responses upon boosting with a BA.1 spike, which resulted in a dramatic suppression of a de novo response in favor of a response biased toward wild-type spike.³ These findings emphasize the strong influence of immune imprinting on the ability to mount effective responses against antigenically variable SARS-CoV-2 variants.

While the inclusion of the BA.4/5 spike in the bivalent formulations has helped boost the immune response toward Omicron subvariants, neutralizing antibody titers were nowhere near what was exhibited for wild-type/D614G spike, with all Omicron subvariants, including BA.4/5, still exhibiting dramatic reductions in neutralizing antibody titers.⁶ These were the first indicators of immune imprinting stimulated by the initial doses of monovalent vaccine. Since BA.4/5, there has been the emergence of more immune-evasive lineages including BQ.1, BQ.1.1, BA.2.75, and CH.1.1, as well as the recombinant subvariants XBB.1.5, XBB.1.16, XBB.2.3, EG.5, and FLip. In this issue of Cell Reports Medicine, Wang et al. sought to corroborate the idea of immune imprinting by demonstrating the role of breakthrough infection of Omicron BA.5 or BQ variants in helping overcome immune imprinting.¹ Breakthrough infection represents another means of boosting the immune response toward the pathogen in question, and it has been established that breakthrough infection with SARS-CoV-2 can stimulate a neutralizing antibody response biased toward the infecting variant.⁷

The data presented by Wang et al. demonstrate that immune imprinting is a critical issue in the current course of vaccines. They show that individuals who had three doses of monovalent vaccine with a bivalent booster containing BA.4/5 had little increase in titer toward Omicron subvariants, especially XBB.1.5 and XBB.1.16, relative to individuals who received three doses of monovalent vaccine with a monovalent booster lacking BA.4/5. In contrast, individuals who had received at least two doses of monovalent vaccine and experienced breakthrough infection with either BQ or, to a lesser extent, BA.5 exhibited more noticeable increases in titer toward Omicron XBB subvariants. Using antigenic mapping analysis, they discovered that breakthrough infection minimized antigenic distances between D614G, BA.5, and BQ.1.¹ The antigenic mapping method was originally developed to quantitatively measure the antigenic differences between influenza strains based on hemagglutination neutralization assay results.⁸ More recent results from other studies have also confirmed the concern of immune imprinting in COVID-19 vaccine by demonstrating impaired neutralizing antibody responses against the most recent XBB variants EG.5.1 and FLip.^9,10 Together, these works are crucial in informing vaccination strategies against SARS-CoV-2 and have led to the decision to roll out a monovalent XBB.1.5 mRNA vaccine in fall 2023. Based on the studies by Wang et al. and others, it has become clear that the exclusion of wild-type spike from the Omicron-lineage spike vaccine should help overcome the immune imprinting caused by the earlier vaccine course and better protect against the current Omicron subvariants in circulation.

Faraone et al; 2023; “Immune evasion and membrane fusion of SARS-CoV-2 XBB subvariants EG.5.1 and XBB.2.3” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10606793/)

Immune evasion by SARS-CoV-2 paired with immune imprinting from monovalent mRNA vaccines has resulted in attenuated neutralizing antibody responses against Omicron subvariants. In this study, we characterized two new XBB variants rising in circulation – EG.5.1 and XBB.2.3, for their neutralization and syncytia formation. We determined the neutralizing antibody titers in sera of individuals that received a bivalent mRNA vaccine booster, BA.4/5-wave infection, or XBB.1.5-wave infection. Bivalent vaccination-induced antibodies neutralized ancestral D614G efficiently, but to a much less extent, two new EG.5.1 and XBB.2.3 variants. In fact, the enhanced neutralization escape of EG.5.1 appeared to be driven by its key defining mutation XBB.1.5-F456L. Notably, infection by BA.4/5 or XBB.1.5 afforded little, if any, neutralization against EG.5.1, XBB.2.3 and previous XBB variants – especially in unvaccinated individuals, with average neutralizing antibody titers near the limit of detection. Additionally, we investigated the infectivity, fusion activity, and processing of variant spikes for EG.5.1 and XBB.2.3 in HEK293T-ACE2 and CaLu-3 cells but found no significant differences compared to earlier XBB variants. Overall, our findings highlight the continued immune evasion of new Omicron subvariants and, more importantly, the need to reformulate mRNA vaccines to include XBB spikes for better protection.

Collier et al; 2023; “Immunogenicity of BA.5 Bivalent mRNA Vaccine Boosters” (https://www.nejm.org/doi/full/10.1056/NEJMc2213948)

Our data indicate that both monovalent and bivalent mRNA boosters markedly increased antibody responses but did not substantially augment T-cell responses. Neutralizing antibody titers against the ancestral strain of SARS-CoV-2 were higher than titers against BA.5 after both monovalent and bivalent boosting. The median BA.5 neutralizing antibody titer was similar after monovalent and bivalent mRNA boosting, with a modest trend favoring the bivalent booster by a factor of 1.3. It is possible that larger studies may show a greater between-group difference, but any such comparative studies between monovalent and bivalent mRNA boosters would need to enroll the two cohorts within the same time frame and after the BA.5 surge, because negative results on nucleocapsid serologic analysis would not exclude all infected participants. These data are consistent with the modest benefits observed with a BA.1-containing bivalent mRNA booster.4 Our findings suggest that immune imprinting by previous antigenic exposure5 may pose a greater challenge than is currently appreciated for inducing robust immunity against SARS-CoV-2 variants.

Fujita et al; 2023; “Impact of Imprinted Immunity Induced by mRNA Vaccination in an Experimental Animal Model” (https://academic.oup.com/jid/article/228/8/1060/7209041?login=false)

We showed that the hamsters infected with a variety of SARS-CoV-2 Omicron subvariants exhibited specific humoral immunity against the SARS-CoV-2 variant infected. On the other hand, the infection of SARS-CoV-2 Omicron subvariants, particularly BQ.1.1 and XBB.1, in the hamsters that received 3 doses of monovalent mRNA vaccine did not induce specific humoral immunity against the SARS-CoV-2 variant infected. Consistent with findings in human samples [8, 12], these results suggest that breakthrough infections tend to boost the humoral immunity against the vaccine strain (ie, ancestral SARS-CoV-2) rather than the variant infected after vaccination. In particular, although the immunogenicity of XBB.1 was comparable to those of the other variants (Figure 1C), vaccination before XBB.1 infection failed to effectively induce antiviral humoral immunity against XBB.1 (Figure 1D). Because the antigenicity of XBB.1 is prominently different from that of ancestral SARS-CoV-2 [7, 8, 12], imprinted immunity can be observed when variants of SARS-CoV-2 breakthrough infections have different immunogenicity from the vaccine strain.

Degryse et al; 2024; “Antigenic imprinting dominates humoral responses to new variants of SARS-CoV-2 in a hamster model of COVID-19” (https://www.biorxiv.org/content/10.1101/2024.10.30.621174v1.full)

Next, we assessed the longevity of the humoral response induced by Comirnaty® XBB.1.5 using sera collected 22 weeks after vaccination (Figure 1b). Binding antibody titres decreased significantly (e.g. 4.9 log10 at 1:100 dilution, p < 0.001) compared to 7 weeks after the first vaccination; but remaining at relatively high levels compared to sham-vaccinated controls (Figure 2a). XBB.1.5 nAbs were still detectable after 22 weeks in 3/4 hamsters (GMT 32, 95% CI 0.4-2186). However, EG.5.1 nAbs had dropped to undetectable levels in 3/4 hamsters (GMT 2.0, 95% CI 0.2-18.2, Figure 2b). In addition, we evaluated whether nAbs induced by XBB.1.5 still covered the novel antigenically distant JN.1 variant, which had become the prevalent circulating strain among the American and European populations at the time these hamsters had been vaccinated for 22 weeks (in February 2024) [20, 21] (Figure 1a). Seven weeks after vaccination with Comirnaty® XBB.1.5, 3/4 hamsters had JN.1 nAbs, but at much lower levels than EG.5.1 nAbs (GMT 8.0, 95% CI 0.8-72.6). JN.1 nAbs had waned to undetectable levels in all hamsters by week 22 (Figure 2b).

Next, we infected the vaccinated hamsters intranasally with JN.1 (104 TCID50). A cohort of naïve hamsters were infected with B.1.1.7 and used as experimental benchmark for severe lung pathology [22]. Similar to its parental variant BA.2.86 [19], JN.1 infection did not evoke any change in body weight (data not shown) and caused a milder lung pathology compared to EG.5.1 with very low to undetectable peribronchial/perivascular inflammation at 4 dpi (Figure 2c). Recall responses in XBB.1.5-vaccinated hamsters after JN.1 infection were predominantly targeted against the cognate vaccine antigen; namely, a robust increase in XBB.1.5 nAbs in 4/4 hamsters (GMT 32.0, 95% CI 3.5-290.5). NAbs against XBB.1.5 descendent EG.5.1 could be detected at least in 2/4 animals at the highest serum concentration (GMT 4.0, 95% CI 0.3-51.1). By contrast, nAbs against JN.1 remained undetectable (Figure 2d). Taken together, these results suggest that, irrespective of the spike protein expressed by the challenge virus, B cells imprinted by vaccination towards a specific, possibly different, or increasingly mismatched, antigen are preferentially reactivated.

Despite the absence of JN.1-specific nAbs, viral RNA levels in throat swabs decreased significantly by day 4 post-infection, suggesting that other effector mechanisms, such as innate antiviral responses, cellular immunity, or Fc-mediated mechanisms, play a role in viral clearance in the upper airways (Figure 2e). Moreover, as observed before for EG.5.1 (Figure 1e), also in non-vaccinated animals, viral RNA levels in the upper airways showed a significantly decline 4 dpi. Neither infectious particles nor viral transcripts were detected in the lungs at 4 dpi (data not shown), confirming the reduced pathogenicity and propensity of BA.2.86 subvariants to replicate in Syrian hamsters [19].

Antigenic relatedness of the thus studied viral spike variants and resulting immune imprinting was further analyzed using antigenic cartography of nAb responses [16, 18]. In agreement with previous reports, we observed that EG.5.1 clustered closer to its parent XBB.1., while B.1.1.7 (VOC Alpha) and JN.1 (offspring of distinct Omicron sublineage BA.2.86) stand separated from XBB.1.5, each at opposite sides; indicative for a growing antigenic difference along the evolutionary trajectory (Figure 2f, squares). Fully as expected, sera from XBB.1.5 vaccinated animals clustered in close proximity to XBB.1.5, whereas YF-S0* sera grouped next to pre-Omicron VOC Alpha (B.1.1.7). Sera coordinates did not change significantly neither for the XBB.1.5-vaccinated animals (orange triangles) nor for the YF-S0*-vaccinated (blue triangles) after challenge with EG.5.1. In contrast, sera from XBB.1.5-vaccinated and subsequently JN.1-challenged hamsters relocated to XBB.1.5 antigen. Altogether these results corroborate that XBB.1.5 vaccine-induced nAbs remain predominant in their specificity of the humoral response to newer Omicron subvariants. Importantly, in turn this also indicates that significant cross-reactive neutralizing responses against pre-Omicron variants (e.g. historical VOC Alpha) are not anymore induced by descendants of Omicron (e.g. XBB.1.5, EG.5.1, BA.2.86, or JN.1). Formally, the herein studied early and late SARS-CoV-2 variants may be considered representatives of two distinct serotypes.

Wang et al; 2023; “Deep immunological imprinting due to the ancestral spike in the current bivalent COVID-19 vaccine” (https://www.cell.com/cell-reports-medicine/fulltext/S2666-3791(23)00435-4?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2666379123004354%3Fshowall%3Dtrue)

Utilizing binding immunoassays, pseudotyped virus neutralization assays, and antigenic mapping, we investigated antibody responses from 72 participants who received three monovalent mRNA vaccine doses followed by either a bivalent or monovalent booster, or who experienced breakthrough infections with the BA.5 or BQ subvariant after vaccinations with an original monovalent vaccine. Compared to a monovalent booster, the bivalent booster did not yield noticeably higher binding titers to D614G, BA.5, and BQ.1.1 spike proteins, nor higher virus-neutralizing titers against SARS-CoV-2 variants including the predominant XBB.1.5 and the emergent XBB.1.16. However, sera from breakthrough infection cohorts neutralized Omicron subvariants significantly better. Multiple analyses of these results, including antigenic mapping, made clear that inclusion of the ancestral spike prevents the broadening of antibodies to the BA.5 component in the bivalent vaccine, thereby defeating its intended goal. Our findings suggest that the ancestral spike in the current bivalent COVID-19 vaccine is the cause of deep immunological imprinting. Its removal from future vaccine compositions is therefore strongly recommended.

Chemaitelly et al; 2022; “Immune Imprinting and Protection against Repeat Reinfection with SARS-CoV-2” (https://www.nejm.org/doi/10.1056/NEJMc2211055)

During follow-up, 63 reinfections occurred in the double-primed cohort and 343 occurred in the omicron-primed cohort; none of the infections progressed to severe, critical, or fatal Covid-19 (Fig. S1). At 135 days after the start of follow-up, the cumulative incidence of reinfection was 1.1% (95% confidence interval [CI], 0.8 to 1.4) in the double-primed cohort and 2.1% (95% CI, 1.8 to 2.3) in the omicron-primed cohort (Figure 1A). In the comparison of the full matched double-primed cohort with the omicron-primed cohort, the adjusted hazard ratio for reinfection was 0.52 (95% CI, 0.40 to 0.68). In an analysis involving the subgroup of persons in the double-primed cohort whose primary infection was with the original virus or the alpha variant as compared with the omicron-primed cohort, the adjusted hazard ratio for infection was 0.59 (95% CI, 0.40 to 0.85) (Figure 1B).

In the first 70 days of follow-up, when infections were dominated by the BA.2 subvariant,^2,3 the adjusted hazard ratio for infection was 0.92 (95% CI, 0.51 to 1.65). However, the cumulative incidence curves diverged when the BA.4 and BA.5 subvariants were introduced and subsequently dominated⁴ (adjusted hazard ratio, 0.46; 95% CI, 0.34 to 0.62) (Figure 1A).

Omicron infection induces strong protection against a subsequent omicron infection.^2,4 In the present cohort study, an additional, earlier infection with non-omicron SARS-CoV-2 was found to strengthen this protection against a subsequent omicron infection. The earlier pre-omicron infection may have broadened the immune response against a future reinfection challenge.

Omicron infection induces strong protection against a subsequent omicron infection.^2,4 In the present cohort study, an additional, earlier infection with non-omicron SARS-CoV-2 was found to strengthen this protection against a subsequent omicron infection. The earlier pre-omicron infection may have broadened the immune response against a future reinfection challenge.

Wang et al; 2022 (pre-print); “Antibody responses to Omicron BA.4/BA.5 bivalent mRNA vaccine booster shot” (https://www.biorxiv.org/content/10.1101/2022.10.22.513349v1)

Boosting with a new bivalent mRNA vaccine targeting both BA.4/BA.5 and an ancestral SARS-CoV-2 strain did not elicit a discernibly superior virus-neutralizing antibody responses compared boosting with an original monovalent vaccine. These findings may be indicative of immunological imprinting⁵, although follow-up studies are needed to determine if the antibody responses will deviate in time, including the impact of a second bivalent booster.

Branche et al; 2023; “Immunogenicity of the BA.1 and BA.4/BA.5 Severe Acute Respiratory Syndrome Coronavirus 2 Bivalent Boosts: Preliminary Results From the COVAIL Randomized Clinical Trial” (https://academic.oup.com/cid/article-abstract/77/4/560/7111740?redirectedFrom=fulltext&login=false)

Titers against BQ.1.1 and XBB.1 were 8-22 times and 13-35 times lower than against BA.1 and D614G, respectively, with the Wildtype/Omicron BA.1 vaccine. Titers against BQ.1.1 and XBB.1 were 4-12 times and 8-22 times lower than against BA.4/BA.5 and D614G, respectively, with the Wildtype/Omicron BA.4/BA.5 vaccine. However, there was increasing neutralization escape with the late 2022 Omicron subvariants (BQ.1.1 and XBB.1). This escape is similar between the two bivalent vaccines as demonstrated by numerically similar GMTs with overlapping confidence intervals, even though BA.1 and BA.4/BA.5 spike sequences are known to have different mutations in the receptor binding domain.7 Our findings highlight ongoing concern that the breadth of antibody response from current updated vaccines is not optimal for the pace of virus evolution.

Schiepers et al; 2023; “Molecular fate-mapping of serum antibody responses to repeat immunization” (https://pubmed.ncbi.nlm.nih.gov/36646114/)

We show that serum responses to sequential homologous boosting derive overwhelmingly from primary cohort B cells, while later induction of new antibody responses from naive B cells is strongly suppressed. Such ‘primary addiction’ decreases sharply as a function of antigenic distance, allowing reimmunization with divergent viral glycoproteins to produce de novo antibody responses targeting epitopes that are absent from the priming variant. Our findings have implications for the understanding of OAS and for the design and testing of vaccines against evolving pathogens.

Jian et al; 2024; “Evolving antibody response to SARS-CoV-2 antigenic shift from XBB to JN.1” (https://pubmed.ncbi.nlm.nih.gov/39510125/)

Here, we provide a comprehensive analysis of the humoral immune response to XBB and JN.1 human exposure. We demonstrate the antigenic distinctiveness of XBB and JN.1 lineages in SARS-CoV-2-naive individuals, and JN.1 infection elicits superior plasma neutralization against its subvariants. We highlight KP.3’s strong immune evasion and receptor binding capability, supporting its foreseeable prevalence. Extensive analysis of the BCR repertoire, isolating ~2000 RBD-specific antibodies with their targeting epitopes characterized by deep mutational scanning (DMS), underscores the superiority of JN.1-elicited memory B cells ^4,5. Class 1 IGHV3-53/3-66-derived neutralizing antibodies (NAbs) contribute majorly within wildtype-reactive NAbs against JN.1. However, KP.2 and KP.3 evade a substantial subset, even those induced by JN.1, advocating for booster updates to KP.2/KP.3. JN.1-induced Omicron-specific antibodies also demonstrate high potency across Omicron. Escape hotspots of these NAbs have already been mutated, resulting in higher immune barrier to escape, considering probable recovery of escaped NAbs. Additionally, the prevalence of IGHV3-53/3-66-derived antibodies, and their capability of competing with all Omicron-specific NAbs suggests their inhibitory role on the activation of Omicron-specific naive B cells, potentially explaining the heavy immune imprinting in mRNA-vaccinated individuals ^6-8. These findings delineate the evolving antibody response to Omicron antigenic shift from XBB to JN.1, and highlight the importance of developing JN.1-lineage, especially KP.2/KP.3-based vaccine boosters.

Einhauser et al; 2024; “Longitudinal effects of SARS-CoV-2 breakthrough infection on
imprinting of neutralizing antibody responses” (https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(24)00474-2/fulltext)

Methods 173 vaccinated and 56 non-vaccinated individuals were enrolled after SARS-CoV-2 Alpha, Delta, or Omicron infection and visited four times within 6 months and nAbs were measured for D614G, Alpha, Delta, BA.1, BA.2, BA.5, BQ.1.1, XBB.1.5 and JN.1.
Findings Magnitude-breadth-analysis showed enhanced neutralization capacity in vaccinated individuals against multiple VOCs. Longitudinal analysis revealed sustained neutralization magnitude-breadth after antigenically distant Delta or Omicron breakthrough infection (BTI), with triple-vaccinated individuals showing significantly elevated titres and improved breadth. Antigenic mapping and antibody landscaping revealed initial boosting of vaccine-induced WT-specific responses after BTI, a shift in neutralization towards infecting VOCs at peak responses and an immune imprinted bias towards dominating WT immunity in the long-term. Despite that bias, machine-learning models confirmed a sustained shift of the immune-profiles following BTI.
Interpretation In summary, our longitudinal analysis revealed delayed and short lived nAb shifts towards the infecting VOC, but an immune imprinted bias towards long-term vaccine induced immunity after BTI

Uriu et al; 2024; “Antiviral humoral immunity induced by JN.1 monovalent mRNA vaccines against SARS-CoV-2 omicron subvariants including JN.1, KP.3.1.1, and XEC” (https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(24)00810-7/fulltext)

To validate the neutralising antibody response induced by JN.1 mRNA vaccines, sera were collected from individuals vaccinated with Pfizer–BioNTech JN.1 mRNA vaccine (n=15; appendix p 5) or Daiichi-Sankyo JN.1 mRNA vaccine (n=19; appendix p 5) before and 3–4 weeks after vaccination. We then performed a neutralisation assay using these sera and pseudoviruses. Both the Pfizer–BioNTech JN.1 vaccine (2·4-fold to 8·0-fold, p=0·0001; appendix p 5) and the Daiichi-Sankyo JN.1 vaccine (2·3-fold to 13-fold, p=0·0001; appendix p 5) boosted antiviral humoral immunity against all variants tested with statistical significance. The Pfizer–BioNTech mRNA vaccine encodes the full-length JN.1 spike (S), the Daiichi–Sankyo mRNA vaccine encodes the receptor-binding domain of JN.1 S. Our data suggest that the receptor-binding domain of JN.1 S can effectively induce antiviral humoral immunity against JN.1 subvariants and XEC comparable to the full-length JN.1 S. However, it should be considered that the sizes of our cohorts are relatively small (<20 donors per cohort). Additionally, it should be noted that donor characteristics of the two cohorts differ substantially in age, sex, underlying disease status, previous SARS-CoV-2 infection, and previous vaccination status (appendix p 4), which might affect the experimental results. Future investigations with larger cohorts will address this concern.

Moreover, as we reported last year, the neutralising antibody response induced by the XBB.1.5 monovalent mRNA vaccine against ancestral B.1.1 was stronger than that against a series of omicron subvariants.⁴ However, in the case of the JN.1 monovalent mRNA vaccine, here we showed that the 50% neutralisation titer against BA.5, a major omicron subvariant, is higher than that against B.1.1 (appendix p 5). These observations imply that immune imprinting has shifted from that biased toward pre-omicron to that biased toward omicron, depending on the alteration of time and number of immune stimuli (eg, infection or vaccination), as a previous study suggested.⁵

Zou et al; 2023; “Neutralization of BA.4–BA.5, BA.4.6, BA.2.75.2, BQ.1.1, and XBB.1 with Bivalent Vaccine” (https://www.nejm.org/doi/full/10.1056/NEJMc2214916)

Among all the participants, the fourth dose of monovalent BNT162b2 vaccine induced a geometric mean factor increase (i.e., an increase from the day of the fourth dose to 1 month after the fourth dose) in titers of 3.0 against WT; 2.9 against BA.4–BA.5; 2.3 against BA.4.6; 2.1 against BA.2.75.2; 1.8 against BQ.1.1; and 1.5 against XBB.1; the bivalent vaccine induced neutralizing geometric mean factor increases of 5.8, 13.0, 11.1, 6.7, 8.7, and 4.8, respectively (Fig. S1). In the participants without previous SARS-CoV-2 infection, the monovalent BNT162b2 vaccine induced neutralizing geometric mean factor increases of 4.4 against WT; 3.0 against BA.4–BA.5; 2.5 against BA.4.6; 2.0 against BA.2.75.2; 1.5 against BQ.1.1; and 1.3 against XBB.1; the bivalent vaccine induced neutralizing geometric mean factor increases of 9.9, 26.4, 22.2, 8.4, 12.6, and 4.7, respectively (Figure 1A). In the participants with previous SARS-CoV-2 infection, the monovalent BNT162b2 vaccine induced neutralizing geometric mean factor increases of 2.0 against WT; 2.8 against BA.4–BA.5; 2.1 against BA.4.6; 2.1 against BA.2.75.2; 2.2 against BQ.1.1; and 1.8 against XBB.1; the bivalent vaccine induced neutralizing geometric mean factor increases of 3.5, 6.7, 5.6, 5.3, 6.0, and 4.9, respectively (Figure 1B). Despite different intervals from dose 3 to dose 4, the neutralizing titers before the fourth dose were similar in the monovalent-vaccine group and the bivalent-vaccine group in all the participants, regardless of history of SARS-CoV-2 infection.

Sridhar et al; 2022; “VACCINE-INDUCED ANTIBODY DEPENDENT ENHANCEMENT IN COVID-19” (https://journal.chestnet.org/article/S0012-3692(22)01866-9/fulltext)

INTRODUCTION: Antibody dependent enhancement(ADE) after COVID vaccination can increase the severity of SARS-COV-2 infection. We present a case of acute respiratory failure where ADE secondary to COVID vaccination may have played a role in the patient’s fatal illness.

CASE PRESENTATION: A 74 year old female with asthma presented with two weeks of increasing shortness of breath treated with high flow nasal cannula and BiPAP. COVID-19 nasal swab PCR and respiratory bio-fire panel were negative, and chest X-ray showed diffuse opacities. A chest CT three days later demonstrated extensive bilateral reticular patchy ground-glass densities. Bacterial and fungal infectious work-up as well as SARS COV-2 PCR were persistently negative. The patient was intubated for respiratory failure on hospital day thirteen. Differential diagnoses included atypical pneumonia, cryptogenic organizing pneumonia, acute interstitial pneumonia and acute respiratory distress syndrome(ARDS) in the context of recent BNT162b2 mRNA COVID vaccination one week before admission. COVID-19 IgM and IgG were positive on hospital day thirteen. Despite extensive antibiotic and anti-fungal coverage, ICU stay was complicated by hypotension and acute kidney injury managed with continuous veno-venous hemofiltration. The patient developed refractory shock and died on hospital day twenty.

DISCUSSION: The case represents a patient with multi-system organ failure with serological evidence of SARS COV-2 IgM and IgG elevation who received BNT162b2 mRNA COVID vaccination one week before admission. It is possible that the patient developed ADE following COVID vaccination having recently acquired COVID-19 infection. Two proposed mechanisms for ADE include (a)sub-neutralizing antibodies binding to Fc-gamma receptor II a expressing phagocytic cells increasing viral entry into and replication within cells (1),(2) and (b)SARS COV-2 forms immune complexes between sub-neutralizing antibodies and the virus which bind to C1q receptor on airway epithelial cells activating immune cells with production of pro-inflammatory cytokines (1),(2) which can cause diffuse alveolar damage(DAD) in COVID-19(3). The patient’s autopsy revealed focal acute and proliferative phases of DAD and pulmonary emboli, all described in autopsies following COVID. In support of ADE was a history of vaccination with BNT162b2 mRNA pre hospitalization, possible infection with COVID-19 suggested by IgM and IgG antibodies and CT consistent with COVID-19 despite persistently negative SARS COV-2 PCR. Evidence against ADE includes negative SARS COV-2 PCR and it is unclear if observed IgM and IgG antibodies were produced against spike protein or nucleocapsid protein of the virus– the latter only produced in response to infection and not vaccination.

Lyons-Weiler; 2020; “Pathogenic priming likely contributes to serious and critical illness and mortality in COVID-19 via autoimmunity” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142689/)

Homology between human and viral proteins is an established factor in viral- or vaccine-induced autoimmunity. Failure of SARS and MERS vaccines in animal trials involved pathogenesis consistent with an immunological priming that could involve autoimmunity in lung tissues due to previous exposure to the SARS and MERS spike protein. Exposure pathogenesis to SARS-CoV-2 in COVID-19 likely will lead to similar outcomes. Immunogenic peptides in viruses or bacteria that match human proteins are good candidates for pathogenic priming peptides (similar to the more diffuse idea of “immune enhancement”). Here I provide an assessment of potential for human pathogenesis via autoimmunity via exposure, via infection or injection. SAR-CoV-2 spike proteins, and all other SARS-CoV-2 proteins, immunogenic epitopes in each SARS-CoV-2 protein were compared to human proteins in search of high local homologous matching. Only one immunogenic epitope in a SARS-CoV-2 had no homology to human proteins. If all of the parts of the epitopes that are homologous to human proteins are excluded from consideration due to risk of pathogenic priming, the remaining immunogenic parts of the epitopes may be still immunogenic and remain as potentially viable candidates for vaccine development. Mapping of the genes encoding human protein matches to pathways point to targets that could explain the observed presentation of symptoms in COVID-19 disease. It also strongly points to a large number of opportunities for expected disturbances in the immune system itself, targeting elements of MHC Class I and Class II antigen presentation, PD-1 signaling, cross-presentation of soluble exogenous antigens and the ER-Phagosome pathway. Translational consequences of these findings are explored.

Baker et al; 2024; “Antibody responses in blood and saliva post COVID-19 bivalent booster do not reveal an Omicron BA.4/BA.5- specific response” (https://pubmed.ncbi.nlm.nih.gov/38812500/)

Results: Our data provide a comprehensive analysis of the antibody response following a single dose of the bivalent boost over a 6-month period and support previous findings that the response induced after the bivalent boost does not create a strong BA.4/BA.5-specific antibody response.

Conclusion: We found no evidence of a specific anti-BA.4/BA.5 response developing over time, including in a sub-population of individuals who become infected after a single dose of the bivalent booster. Additionally, we present data that support the use of saliva samples as a reliable alternative to blood for antibody detection against specific SARS-CoV-2 antigens.

Song et al; 2024; “Finite immune imprinting on neutralizing antibody responses to Omicron subvariants by repeated vaccinations” (https://pubmed.ncbi.nlm.nih.gov/39117174/)

Methods: Individuals who received four-dose vaccinations with the Wuhan-hu-1 strain, individuals who were infected with the BA.5 variant alone without prior vaccination, and individuals who experienced a BA.5 breakthrough infection (BTI) following receiving 2-4 doses of the Wuhan-hu-1 vaccine were enrolled. Neutralizing antibodies against D614G, BA.5, XBB.1.5, EG.5.1, and BA.2.86 were detected using a pseudovirus-based neutralization assay. Antigenic cartography was used to analyze cross-reactivity patterns among D614G, BA.5, XBB.1.5, EG.5.1, and BA.2.86 and sera from individuals.

Results: The highest neutralizing antibody titers against D614G were observed in individuals who only received four-dose vaccination and those who experienced BA.5 BTI, which was also significantly higher than the antibody titers against XBB.1.5, EG.5.1, and BA.2.86. In contrast, only BA.5 infection elicited comparable neutralizing antibody titers against the tested variants. While neutralizing antibody titers against D614G or BA.5 were similar across the cohorts, the neutralizing capacity of antibodies against XBB.1.5, EG.5.1, and BA.2.86 was significantly reduced. BA.5 BTI following heterologous booster induced significantly higher neutralizing antibody titers against the variants, particularly against XBB.1.5 and EG.5.1, than uninfected vaccinated individuals, only BA.5 infected individuals, or those with BA.5 BTI after primary vaccination.

Conclusions: Our findings suggest that repeated vaccination with the Wuhan-hu-1 strain imprinted a neutralizing antibody response toward the Wuhan-hu-1 strain with limited effects on the antibody response to the Omicron subvariants.

Liang et al; 2024; “Imprinting of serum neutralizing antibodies by Wuhan-1 mRNA vaccines” (https://pubmed.ncbi.nlm.nih.gov/38749479/)

Here we characterized the serum antibody responses after mRNA vaccine boosting of mice and human clinical trial participants. In mice, a single dose of a preclinical version of mRNA-1273 vaccine encoding Wuhan-1 spike protein minimally imprinted serum responses elicited by Omicron boosters, enabling generation of type-specific antibodies. However, imprinting was observed in mice receiving an Omicron booster after two priming doses of mRNA-1273, an effect that was mitigated by a second booster dose of Omicron vaccine. In both SARS-CoV-2-infected and uninfected humans who received two Omicron-matched boosters after two or more doses of the prototype mRNA-1273 vaccine, spike-binding and neutralizing serum antibodies cross-reacted with Omicron variants as well as more distantly related sarbecoviruses. Because serum neutralizing responses against Omicron strains and other sarbecoviruses were abrogated after pre-clearing with Wuhan-1 spike protein, antibodies induced by XBB.1.5 boosting in humans focus on conserved epitopes targeted by the antecedent mRNA-1273 primary series. Thus, the antibody response to Omicron-based boosters in humans is imprinted by immunizations with historical mRNA-1273 vaccines, but this outcome may be beneficial as it drives expansion of cross-neutralizing antibodies that inhibit infection of emerging SARS-CoV-2 variants and distantly related sarbecoviruses.

Tortorici et al; 2024; “Persistent immune imprinting occurs after vaccination with the COVID-19 XBB.1.5 mRNA booster in humans” (https://pubmed.ncbi.nlm.nih.gov/38490197/)

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron breakthrough infections and bivalent COVID-19 vaccination primarily recall cross-reactive memory B cells induced by prior Wuhan-Hu-1 spike mRNA vaccination rather than priming Omicron-specific naive B cells. These findings indicate that immune imprinting occurs after repeated Wuhan-Hu-1 spike exposures, but whether it can be overcome remains unclear. To understand the persistence of immune imprinting, we investigated memory and plasma antibody responses after administration of the updated XBB.1.5 COVID-19 mRNA vaccine booster. We showed that the XBB.1.5 booster elicited neutralizing antibody responses against current variants that were dominated by recall of pre-existing memory B cells previously induced by the Wuhan-Hu-1 spike. Therefore, immune imprinting persists after multiple exposures to Omicron spikes through vaccination and infection, including post XBB.1.5 booster vaccination, which will need to be considered to guide future vaccination.

9d. Antibody Dependent Enhancement

Jaume et al; 2012; “SARS CoV subunit vaccine: antibody-mediated neutralisation and enhancement” (https://pubmed.ncbi.nlm.nih.gov/22311359/)

1. A SARS vaccine was produced based on recombinant native full-length Spike-protein trimers (triSpike) and efficient establishment of a vaccination procedure in rodents.

2. Antibody-mediated enhancement of SARS-CoV infection with anti-SARS-CoV Spike immune-serum was observed in vitro.

3. Antibody-mediated infection of SARS-CoV triggers entry into human haematopoietic cells via an FcγR-dependent and ACE2-, pH-, cysteine-protease-independent pathways.

4. The antibody-mediated enhancement phenomenon is not a mandatory component of the humoral immune response elicited by SARS vaccines, as pure neutralising antibody only could be obtained.

5. Occurrence of immune-mediated enhancement of SARS-CoV infection raises safety concerns regarding the use of SARS-CoV vaccine in humans and enables new ways to investigate SARS pathogenesis (tropism and immune response deregulation).

Wang et al; 2016; “Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins” (https://pubmed.ncbi.nlm.nih.gov/25073113/)

Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis.

Lee et al; 2020; “Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies” (https://www.nature.com/articles/s41564-020-00789-5)

Data from the study of SARS-CoV and other respiratory viruses suggest that anti-SARS-CoV-2 antibodies could exacerbate COVID-19 through antibody-dependent enhancement (ADE). Previous respiratory syncytial virus and dengue virus vaccine studies revealed human clinical safety risks related to ADE, resulting in failed vaccine trials. Here, we describe key ADE mechanisms and discuss mitigation strategies for SARS-CoV-2 vaccines and therapies in development. We also outline recently published data to evaluate the risks and opportunities for antibody-based protection against SARS-CoV-2.

Nakayama et al; 2023; “SARS-CoV-2 Related Antibody-Dependent Enhancement Phenomena In Vitro and In Vivo” (https://pubmed.ncbi.nlm.nih.gov/37110438/)

Antibody-dependent enhancement (ADE) is a phenomenon in which antibodies produced in the body after infection or vaccination may enhance subsequent viral infections in vitro and in vivo. Although rare, symptoms of viral diseases are also enhanced by ADE following infection or vaccination in vivo. This is thought to be due to the production of antibodies with low neutralizing activity that bind to the virus and facilitate viral entry, or antigen-antibody complexes that cause airway inflammation, or a predominance of T-helper 2 cells among the immune system cells which leads to excessive eosinophilic tissue infiltration. Notably, ADE of infection and ADE of disease are different phenomena that overlap. In this article, we will describe the three types of ADE: (1) Fc receptor (FcR)-dependent ADE of infection in macrophages, (2) FcR-independent ADE of infection in other cells, and (3) FcR-dependent ADE of cytokine production in macrophages. We will describe their relationship to vaccination and natural infection, and discuss the possible involvement of ADE phenomena in COVID-19 pathogenesis.

Is Vaccination Required for Vaccine Naïve but Infected Individuals to Establish Hybrid Immunity?

There has been some disagreement on whether vaccination is necessary for SARS-CoV-2 infected and recovered individuals [139]. The symptoms and problems are not limited to pneumonia but extend to dysfunction of other organs such as the heart and brain, and the impacts of long-COVID should be considered.

The US Department of Veterans Affairs national healthcare database was used to build a cohort of 443,588 individuals with a single SARS-CoV-2 infection, 40,947 with re-infection (two or more times) and 5,334,729 uninfected controls [140]. Compared with single infection, re-infection contributed additional risks of death (hazard ratio (HR) = 2.17) and hospitalization (HR = 3.32). The risks were evident in the acute phase but persisted in the post-acute phase at 6 months, with symptoms including pulmonary, cardiovascular, hematological, diabetes, gastrointestinal, kidney, mental health, musculoskeletal and neurological disorders [140]. It is not clear whether the secondary infection was more severe than the primary infection; however, if only 1% of re-infected cases become severe, this may result in a large burden on health services since the target population size is extremely large. Thus, continued vigilance to reduce the risk of re-infection may be important for people who have been previously infected.

The level of N protein is the benchmark factor that is considered when predicting disease severity [141]. Plasma N protein levels of 1000 ng/L or greater are associated with markedly higher risks of worsened pulmonary status at the acute phase (odds ratio = 5.06) and longer time required for hospital discharge (median, 7 vs. 4 days). Moreover, plasma N protein levels were higher in those who lacked anti-S antibodies [141]. Therefore, the neutralizing titer of anti-S antibody is critical for reducing levels of the N protein in vivo as well as the virus itself and the S protein, which are all involved in the pathogenesis of COVID-19.

There is an argument for vaccination with the disappeared wild Wuhan strain or the disappearing omicron BA.1 or BA.5 variants being sufficient to induce protective immunity against newly emerging omicron variants and future mutated viruses. However, vaccines composed of inactivated whole virion including the N protein are not recommended, as discussed in this review. It should be noted here that the antibody titers induced by in-activated whole virion were lower than for other vaccines [101], especially in the levels of IgA in the nasal epithelial lining fluid [142].

The four human IgG subclasses, IgG1 to IgG4, have distinct effector properties due to differences in FcR binding and complement activation. The human IgG4 normally exists in the serum at lower concentrations than IgG1, IgG2, or IgG3. A longitudinal analysis of the level of anti-S antibodies from each IgG subclass in recipients of the SARS-CoV-2 mRNA vaccine [143] revealed that IgG4 antibodies among all S-specific IgG antibodies increased from 0.04% shortly after the second vaccination to 19.27% after the third vaccination. Serum antibody effector activity, as assessed by antibody-dependent phagocytosis or complement deposition, was reduced after the third dose compared with after the second dose [143]. It should be noted that a third vaccination elicited superior neutralizing immunity to all the variants of concern [144]. Furthermore, the avidity was significantly increased after the third vaccination and superior neutralization of pseudo-typed virus was observed. In this study, antibody-mediated phagocytic activity and complement deposition related to ADE of disease were reduced in sera after the third vaccination. Because Fc-mediated effector function could be important for viral clearance, an increase in IgG4 subclasses might result in longer viral persistence in patients. However, it is also true that noninflammatory Fc-mediated effector functions reduce immunopathology, whereas viruses are efficiently neutralized via high-avidity antibody variable regions of IgG4 antibodies. In a cohort of vaccinees with breakthrough infections, no evidence of alteration in disease severity was found [143]. In addition, adverse effects related to antigen–antibody immune complex formation have not been observed in recipients of AstraZeneca’s Evusheld (AZD7442; tixagevimab and cilgavimab), in which the L234F/L235E/P331S modification reduces binding to FcγR and the C1q-like IgG4 isotype [145], indicating that the immunoglobulin class switch of IgG4 may control the balance between binding maturation and over-stimulation of inflammation without severe negative effect. Regardless of the immunoglobulin class switch, the somatic hypermutation and affinity maturation of the epitope binding region of antibody genes in memory B lymphocytes is important [146,147,148,149,150,151]. A broadly neutralizing antibody that has potent cross-reactivity against a wide variety of variants was reported in HIV infected individuals [152]. A long exposure interval is required to generate cross-neutralizing potency against different variants antigenically distant from the ancestral strain [153]. The ratio between protective anti-S antibody and detrimental anti-N antibody in each individual [154] may affect the clinical relevance with breakthrough infection or re-infection.

Xu et al; 2021; “Antibody dependent enhancement: Unavoidable problems in vaccine development” (https://pmc.ncbi.nlm.nih.gov/articles/PMC8438590/)

In some cases, antibodies can enhance virus entry and replication in cells. This phenomenon is called antibody-dependent infection enhancement (ADE). ADE not only promotes the virus to be recognized by the target cell and enters the target cell, but also affects the signal transmission in the target cell. Early formalin-inactivated virus vaccines such as aluminum adjuvants (RSV and measles) have been shown to induce ADE. Although there is no direct evidence that there is ADE in COVID-19, this potential risk is a huge challenge for prevention and vaccine development. This article focuses on the virus-induced ADE phenomenon and its molecular mechanism. It also summarizes various attempts in vaccine research and development to eliminate the ADE phenomenon, and proposes to avoid ADE in vaccine development from the perspective of antigens and adjuvants.

9e. DNA Contamination

Konig & Kirchner; 2024; “Methodological Considerations Regarding the Quantification of DNA Impurities in the COVID-19 mRNA Vaccine Comirnaty” (https://www.mdpi.com/2409-9279/7/3/41)

DNA impurities can impact the safety of genetically engineered pharmaceuticals; thus, a specific limit value must be set for them during marketing authorisation. This particularly applies to mRNA vaccines, as large quantities of DNA templates are used for their production. Furthermore, when quantifying the total DNA content in the final product, we must observe that, in addition to the mRNA active ingredient, DNA impurities are also encased in lipid nanoparticles and are therefore difficult to quantify. In fact, the manufacturer of the mRNA vaccine Comirnaty (BioNTech/Pfizer) only measures DNA impurities in the active substance by means of a quantitative polymerase chain reaction (qPCR), whose DNA target sequence is less than just 1% of the originally added DNA template. This means that no direct DNA quantification takes place, and compliance with the limit value for DNA contamination is only estimated from the qPCR data using mathematical extrapolation methods. However, it is also possible to dissolve the lipid nanoparticles with a detergent to directly measure DNA contamination in the final product by using fluorescence spectroscopic methods.

Conclusions

The available information and data indicate that the ready-to-use mRNA vaccine Comirnaty contains DNA impurities that exceed the permitted limit value by several hundred times and, in some cases, even more than 500 times, and that this went unnoticed because the DNA quantification carried out as part of batch testing only at the active substance level appears to be methodologically inadequate when using qPCR, as explained above. Because of the conditions during the production of the mRNA active substance of Comirnaty, the applied qPCR is designed so that a massive under-detection of DNA impurities appears to be the result. Here, we have to remember that qPCR is matchless if specific DNA sequences are being quantified, but this is not the case if the aim is the quantification of the total DNA content. However, DNA contamination in Comirnaty is about total DNA, regardless of the sequences that it contains. Accordingly, it can be assumed that a fluorescence spectrometric measurement of the total DNA in the end product, analogous to the quantification of the mRNA active ingredient, a process that is, in fact, carried out in the end product, is not associated with a risk of under-detecting DNA contaminations but rather provides reliable values and thus satisfies the required level of drug safety.

Against this background, experimental testing of the total DNA contained in the ready-to-use diluted vaccine Comirnaty^® via fluorescence spectrometric measurement, which is to be carried out by the authorities as part of the legal mandate for official batch testing, appears to be essential. Why this was systematically omitted by the European control laboratories according to the statements by the German Federal Government cited above should therefore be the subject of extensive expert discussions and reconsiderations.

Further, it should also be taken into account that DNA impurities in Comirnaty^® are apparently integrated into the lipid nanoparticles and are thus transported directly into the cells of a vaccinated person, just like the mRNA active ingredient. What this means for the safety risks, particularly the possible integration of this DNA into the human genome, i.e., the risk of insertional mutagenesis, should be a secondary focus of the discussion required, which must go far beyond what could have been considered years before the so unexpected introduction of mRNA pharmaceuticals into the global market.

Speicher et al; 2023; “DNA fragments detected in monovalent and bivalent Pfizer/BioNTech and Moderna modRNA COVID-19 vaccines from Ontario, Canada: Exploratory dose response relationship with serious adverse events.” (https://osf.io/preprints/osf/mjc97)

Results: Quantification cycle (Cq) values (1:10 dilution) for the plasmid origin of replication (ori) and spike sequences ranged from 18.44 – 24.87 and 18.03 – 23.83 and for Pfizer, and 22.52 – 24.53 and 25.24 – 30.10 for Moderna, respectively. These values correspond to 0.28 – 4.27 ng/dose and 0.22 – 2.43 ng/dose (Pfizer), and 0.01 -0.34 ng/dose and 0.25 – 0.78 ng/dose (Moderna), for ori and spike respectively measured by qPCR, and 1,896 – 3,720 ng/dose and 3,270 – 5,100 ng/dose measured by Qubit® fluorometry for Pfizer and Moderna, respectfully. The SV40 promoter-enhancer-ori was only detected in Pfizer vials with Cq scores ranging from 16.64 – 22.59. In an exploratory analysis, we found preliminary evidence of a dose response relationship of the amount of DNA per dose and the frequency of serious adverse events (SAEs). This relationship was different for the Pfizer and Moderna products. Size distribution analysis found mean and maximum DNA fragment lengths of 214 base pairs (bp) and 3.5 kb, respectively. The plasmid DNA is likely inside the LNPs and is protected from nucleases.
Conclusion: These data demonstrate the presence of billions to hundreds of billions of DNA molecules per dose in these vaccines. Using fluorometry, all vaccines exceed the guidelines for residual DNA set by FDA and WHO of 10 ng/dose by 188 – 509-fold. However, qPCR residual DNA content in all vaccines were below these guidelines emphasizing the importance of methodological clarity and consistency when interpreting quantitative guidelines. The preliminary evidence of a dose-response effect of residual DNA measured with qPCR and SAEs warrant confirmation and further investigation. Our findings extend existing concerns about vaccine safety and call into question the relevance of guidelines conceived before the introduction of efficient transfection using LNPs. With several obvious limitations, we urge that our work is replicated under forensic conditions and that guidelines be revised to account for highly efficient DNA transfection and cumulative dosing.

Haraguchi et al; 2022; “Transfected plasmid DNA is incorporated into the nucleus via nuclear envelope reformation at telophase” (https://www.nature.com/articles/s42003-022-03021-8)

This study elucidates the mechanism through which transfected DNA enters the nucleus for gene expression. To monitor the behavior of transfected DNA, we introduce plasmid bearing lacO repeats and RFP-coding sequences into cells expressing GFP-LacI and observe plasmid behavior and RFP expression in living cells. RFP expression appears only after mitosis. Electron microscopy reveals that plasmids are wrapped with nuclear envelope (NE)‒like membranes or associated with chromosomes at telophase. The depletion of BAF, which is involved in NE reformation, delays plasmid RFP expression. These results suggest that transfected DNA is incorporated into the nucleus during NE reformation at telophase.

Lim et al; 2023; “High spontaneous integration rates of end-modified linear DNAs upon mammalian cell transfection” (https://www.nature.com/articles/s41598-023-33862-0)

Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity. However, whether modified-end linear DNAs can also provide a safe, non-integrating gene transfer remains unanswered. Herein, we compare the genomic integration frequency upon transfection of cells with expression vectors in the forms of circular plasmid, unmodified linear DNA, CELiDs with thioester loops, and Streptavidin-conjugated blocked-end linear DNA. All of the forms of linear DNA resulted in a high fraction of the cells being stably transfected—between 10 and 20% of the initially transfected cells. These results indicate that blocking the ends of linear DNA is insufficient to prevent integration.

9f. Vaccinee antibody responses to variants – Immune evasion

Sui et al; 2014; “Effects of Human Anti-Spike Protein Receptor Binding Domain Antibodies on Severe Acute Respiratory Syndrome Coronavirus Neutralization Escape and Fitness” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4248992/) SEE ENTRY IN HISTORICAL RESEARCH SECTION

Israel et al; 2022; “Large-Scale Study of Antibody Titer Decay following BNT162b2 mRNA Vaccine or SARS-CoV-2 Infection” (https://www.mdpi.com/2076-393X/10/1/64)

Immune protection following either vaccination or infection with SARS-CoV-2 is thought to decrease over time. We designed a retrospective study, conducted at Leumit Health Services in Israel, to determine the kinetics of SARS-CoV-2 IgG antibodies following administration of two doses of BNT162b2 vaccine, or SARS-CoV-2 infection in unvaccinated individuals. Antibody titers were measured between 31 January 2021, and 31 July 2021 in two mutually exclusive groups: (i) vaccinated individuals who received two doses of BNT162b2 vaccine and had no history of previous infection with COVID-19 and (ii) SARS-CoV-2 convalescents who had not received the vaccine. A total of 2653 individuals fully vaccinated by two doses of vaccine during the study period and 4361 convalescent patients were included. Higher SARS-CoV-2 IgG antibody titers were observed in vaccinated individuals (median 1581 AU/mL IQR [533.8–5644.6]) after the second vaccination than in convalescent individuals (median 355.3 AU/mL IQR [141.2–998.7]; p < 0.001). In vaccinated subjects, antibody titers decreased by up to 38% each subsequent month while in convalescents they decreased by less than 5% per month. Six months after BNT162b2 vaccination 16.1% subjects had antibody levels below the seropositivity threshold of <50 AU/mL, while only 10.8% of convalescent patients were below <50 AU/mL threshold after 9 months from SARS-CoV-2 infection. This study demonstrates individuals who received the Pfizer-BioNTech mRNA vaccine have different kinetics of antibody levels compared to patients who had been infected with the SARS-CoV-2 virus, with higher initial levels but a much faster exponential decrease in the first group.

Chakraborty et al; 2022; “A Detailed Overview of Immune Escape, Antibody Escape, Partial Vaccine Escape of SARS-CoV-2 and Their Emerging Variants With Escape Mutations” (https://pmc.ncbi.nlm.nih.gov/articles/PMC8863680/)

Our review provides an overview of the emerging variants’ immune escape and vaccine escape ability. We have illustrated a broad view related to viral evolution, variants, and immune escape ability. Subsequently, different immune escape approaches of SARS-CoV-2 have been discussed. Different innate immune escape strategies adopted by the SARS-CoV-2 has been discussed like, IFN-I production dysregulation, cytokines related immune escape, immune escape associated with dendritic cell function and macrophages, natural killer cells and neutrophils related immune escape, PRRs associated immune evasion, and NLRP3 inflammasome associated immune evasion. Simultaneously we have discussed the significant mutations related to emerging variants and immune escape, such as mutations in the RBD region (N439K, L452R, E484K, N501Y, K444R) and other parts (D614G, P681R) of the S-glycoprotein. Mutations in other locations such as NSP1, NSP3, NSP6, ORF3, and ORF8 have also been discussed. Finally, we have illustrated the emerging variants’ partial vaccine (BioNTech/Pfizer mRNA/Oxford-AstraZeneca/BBIBP-CorV/ZF2001/Moderna mRNA/Johnson & Johnson vaccine) escape ability.

Follmann et al; 2022; “Antinucleocapsid Antibodies After SARS-CoV-2 Infection in the Blinded Phase of the Randomized, Placebo-Controlled mRNA-1273 COVID-19 Vaccine Efficacy Clinical Trial” (https://www.acpjournals.org/doi/10.7326/M22-1300)

Results:

Among 812 participants with PCR-confirmed COVID-19 illness during the blinded phase of the trial (through March 2021), seroconversion to anti-N Abs (median of 53 days after diagnosis) occurred in 21 of 52 mRNA-1273 vaccinees (40% [95% CI, 27% to 54%]) versus 605 of 648 placebo recipients (93% [CI, 92% to 95%]). Each 1-log increase in SARS-CoV-2 viral copies at diagnosis was associated with 90% higher odds of anti-N Ab seroconversion (odds ratio, 1.90 [CI, 1.59 to 2.28]).

Limitation:

The scope was restricted to mRNA-1273 vaccinees and the Elecsys assay, the sample size was small, data on Delta and Omicron infections were lacking, and the analysis did not address a prespecified objective of the trial.

Conclusion:

Vaccination status should be considered when interpreting seroprevalence and seropositivity data based solely on anti-N Ab testing.

Fernández Ciriza et al; 2024; “COVID-19 Vaccine Booster Dose Fails to Enhance Antibody Response to Omicron Variant in Reinfected Healthcare Workers” (https://www.mdpi.com/1999-4915/17/1/78)

We conducted a prospective cohort study to assess the durability and level of antibodies of 678 healthcare workers fully vaccinated against COVID-19. They were categorized based on their primary vaccination regimen. Blood samples were collected before the booster dose and 1 and 6 months after. Significant Anti-S-RBD differences were found between previously infected and naïve volunteers (p = 0.01). Considering the initial vaccine schedules, mRNA-based vaccines displayed significant higher antibody production and longer persistence among both infected and naïve participants. After the booster dose, participants primoinfected with the Omicron variant exhibited higher antibody concentrations than those who experienced reinfection, even after 6 months of follow-up (22,545 and 9460 U/mL, respectively). Moreover, these groups showed the most pronounced disparity in antibody titers ratios between infected and uninfected individuals. Overall, the booster dose failed to enhance humoral response in individuals reinfected with the Omicron variant after receiving it. Hybrid immunity and mRNA-based vaccine initial schedules showed higher levels and longer persistence of antibodies.

Nguyen et al; 2024; “SARS-CoV-2-specific plasma cells are not durably established in the bone marrow long-lived compartment after mRNA vaccination” (https://www.nature.com/articles/s41591-024-03278-y)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines are effective at protecting from severe disease, but the protective antibodies wane rapidly even though SARS-CoV-2-specific plasma cells can be found in the bone marrow (BM). Here, to explore this paradox, we enrolled 19 healthy adults at 2.5–33 months after receipt of a SARS-CoV-2 mRNA vaccine and measured influenza-, tetanus- or SARS-CoV-2-specific antibody-secreting cells (ASCs) in long-lived plasma cell (LLPC) and non-LLPC subsets within the BM. Only influenza- and tetanus-specific ASCs were readily detected in the LLPCs, whereas SARS-CoV-2 specificities were mostly absent. The ratios of non-LLPC:LLPC for influenza, tetanus and SARS-CoV-2 were 0.61, 0.44 and 29.07, respectively. In five patients with known PCR-proven history of recent infection and vaccination, SARS-CoV-2-specific ASCs were mostly absent from the LLPCs. We show similar results with measurement for secreted antibodies from BM ASC culture supernatant. While serum IgG titers specific for influenza and tetanus correlated with IgG LLPCs, serum IgG levels for SARS-CoV-2, which waned within 3–6 months after vaccination, were associated with IgG non-LLPCs. In all, our studies suggest that rapid waning of SARS-CoV-2-specific serum antibodies could be accounted for by the absence of BM LLPCs after these mRNA vaccines.

Vilela et al; 2024; “Longitudinal Immunological Analysis of Portuguese Healthcare Workers Across the COVID-19 Pandemic Reveals Differences in the Humoral Immune Response to Vaccines” (https://www.mdpi.com/2076-393X/12/12/1358)

Results: We observed that antibody titres peaked approximately one month after full vaccination and declined steadily thereafter. We also found that mRNA vaccination induces higher titres of antibodies than viral vector vaccination, and both generate greater antibody responses than mild or moderate COVID-19. Additionally, whilst the booster for the Oxford-AstraZeneca and Pfizer-BioNTech groups led to antibody levels higher than those at any previous sample collection point, the booster for the Post-COVID group (persons with a history of COVID-19 prior to vaccination) led to antibody levels lower than those attained one month after the second dose. Interpretation: Our results indicate that there are different kinetics of antibody production between individuals who received the Pfizer-BioNtech mRNA vaccine and those who received the Oxford-AstraZeneca vector vaccine, or individuals who had COVID-19 before being vaccinated. Additionally, we observed that exposure to either natural infection or vaccination modulates the response to subsequent vaccination. This is particularly evident after administration of the third dose to the Post-COVID group, where our findings point to a hindrance in vaccine boosting, probably due to unwanted feedback by high titres of pre-existing antibodies.

Favresse et al; 2024; “Durability of humoral and cellular immunity six months after the BNT162b2 bivalent booster” (https://pubmed.ncbi.nlm.nih.gov/38185981/)

Neutralizing antibodies against either the D614G strain, the delta variant, the BA.5 variant, or the XBB.1.5 subvariant were measured. The cellular response was assessed by measurement of the release of interferon gamma from T cells in response to an in vitro SARS-CoV-2 stimulation. A substantial waning of neutralizing antibodies was observed after 6 months (23.1-fold decrease), especially considering the XBB.1.5 subvariant. The estimated T_1/2 of neutralizing antibodies was 16.1 days (95% CI = 10.2-38.4 days). Although most participants still present a robust cellular response after 6 months (i.e., 95%), a significant decrease was also observed compared to the peak response (0.95 vs. 0.41 UI/L, p = 0.0083). A significant waning of the humoral and cellular response was observed after 6 months.

Sheehan et al; 2023; “Dynamics of Serum-Neutralizing Antibody Responses in Vaccinees through Multiple Doses of the BNT162b2 Vaccine” (https://www.mdpi.com/2076-393X/11/11/1720)

3.2. Booster Vaccination Induces IgG4-Switched Ag-Specific Responses in Serum

The three-dose vaccine series induced significant changes in RBD-reactive serum IgG subclass distribution. Following the second dose, IgG1 and IgG3 subclasses were predominant while IgG2 and IgG4 levels were negligible (Figure 2A). IgG1 and IgG3 levels declined by four months after the second dose, consistent with Ag-specific IgG titers. Booster vaccination restored RBD-reactive IgG1 levels along with significant increases in both α-RBD IgG2 and IgG4 titers. To confirm these findings, serum IgG subclass titers against the S ectodomain were measured, with similar profiles detected (Figure 2B). While RBD- and S-reactive IgG2 and IgG3 responses were not detected at six months post-boost, significant levels of Ag-specific IgG1 and IgG4 antibodies persisted at this late sampling point. These mRNA vaccine-driven changes in Ag-specific IgG subclass profiles suggest that repeated Ag delivery promoted ongoing class-switch recombination in memory B cells towards distal IgG2- and IgG4-encoding γ heavy chain genes.

3.3. Vaccination Elicits Potent but Transient Neutralizing Antibody Responses in Serum

Next, sera were evaluated in SARS-CoV-2 spike (Wuhan-Hu-1) pseudotyped virus inhibition assays to detect nAb responses. Mean serum 50% neutralization titer (NT₅₀) values increased significantly after two vaccine doses although with some heterogeneity (1:450 to 1:6500) but had decayed to pre-immune levels at four months (Figure 3A). A single vaccine dose did not establish substantial neutralization activity. Boosters rescued peak NT₅₀ values with a similarly wide distribution of neutralization activities but these declined to pre-immune levels by six months following the third vaccine dose.

Figure 3. Vaccination elicits potent but transient neutralizing antibody responses in serum. Neutralizing antibody responses were evaluated in SARS-CoV-2 spike pseudotyped virus inhibition assays (A) as described in Methods. Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparisons of mean NT₅₀ values at each sampling point: *** p = 0.003, **** p < 0.0001. Correlations between serum NT₅₀ values and RBD-reactive IgG or IgA levels were determined at post-vax time points 2A and 2B (B,C) and post-boosting at time points 3A and 3B (D,E); Pearson coefficients of correlation (r) are listed on each graph. ns, not significant.

Serum NT₅₀ values correlated strongly with peak α-RBD IgG titers but only moderately with α-RBD IgA levels (Figure 3B,C). Similarly, the decaying serum NT₅₀ values correlated strongly with RBD-reactive IgG and moderately with α-RBD IgA levels at these sampling points (Figure 3D,E). The rapid decay of serum-neutralizing activities suggests a transient character for vaccine-induced circulating nAb responses.

3.4. SARS-CoV-2 Variants Are Resistant to Vaccine-Induced Serum α-RBD Reactivity and Neutralizing Antibody Responses

To assess the impact of SARS-CoV-2 variants on vaccine-induced humoral immunity, sera collected at time points showing peak RBD-specific IgG titers (post-vax #2A and #3A) were rescreened in binding assays using recombinant RBD proteins from B.1.351 (Beta), B.1.617.2 (Delta), B.1.621 (Mu), or B.1.1.529 (Omicron) variants. Compared with Wuhan-Hu-1 RBD binding, vaccinee serum IgG reactivity declined significantly against each of the variant RBD proteins (Figure 4A,B). Notably, however, booster vaccination significantly improved RBD-reactive IgG titers against all variant RBD proteins (Figure 4C).

Figure 4. BNT162b2-induced serum IgG responses show impaired reactivity against variant SARS-CoV-2 spike RBD proteins that is improved by vaccine boosting. IgG levels against variant SARS-CoV-2 spike RBD proteins were measured by ELISA at time point 2A (A,C) and post-boosting at time point 3A (B,C) as described in Methods. Statistical significance was determined as in Figure 3. **** p < 0.0001. Reactivity for each variant at time points 2A and 3A was compared using paired t-tests. *** p = 0.0003, **** p < 0.0001.

Vaccinee sera were also screened in inhibition assays using variant spike pseudotyped viruses to evaluate the impact of SARS-CoV-2 evolution on BNT162b-induced nAb responses. Sera with peak nAb responses against Wuhan-Hu-1 (post-vax #2A and #3A) showed significantly reduced neutralization activity against all variant pseudoviruses (Figure 5A,B). Vaccine boosters enhanced serum neutralization activity against B.1.351 and B.1.1.529 spike pseudoviruses but activity against B.1.617.2 variant spike pseudovirus declined significantly following the third vaccine dose. Decreases observed in both serum IgG responses and neutralization activity against this panel of variants indicate that vaccine-elicited humoral responses were sensitive to ongoing SARS-CoV-2 evolution.

Figure 5. SARS-CoV-2 variants are resistant to BNT162b2-induced neutralizing antibody responses but neutralization activity is improved by vaccine boosting. Neutralizing antibody responses against SARS-CoV-2 variants at time points 2A (A,C) and post-boosting at time point 3A (B,C) were evaluated in SARS-CoV-2 spike pseudotyped virus inhibition assays as described in Methods. Statistical significance was determined as in Figure 3. **** p < 0.0001. Neutralization activity for each variant at time points 2A and 3A was compared using paired t tests. * p < 0.05, ** p < 0.01. ns, not significant.

Uriu et al; 2023; “Transmissibility, infectivity, and immune evasion of the SARS-CoV-2 BA.2.86 variant” (https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(23)00575-3/fulltext)

We then performed neutralisation assays using vaccine sera to assess the possibility that BA.2.86 evades the antiviral effect of vaccine-induced humoral immunity. The sera obtained from individuals vaccinated with third-dose monovalent, fourth-dose monovalent, BA.1 bivalent, and BA.5 bivalent mRNA vaccines exhibited very little or no antiviral effects against BA.2.86 (appendix p 10). Additionally, the three monoclonal antibodies (bebtelovimab, sotrovimab, and cilgavimab), which worked against the parental BA.2,⁶ did not exhibit antiviral effects against BA.2.86 (appendix p 10). Finally, a neutralisation assay using XBB breakthrough infection sera showed that the 50% neutralisation titre of XBB breakthrough infection sera against BA.2.86 was significantly (1·6-fold) lower than that against EG.5.1 (p<0·0001; appendix p 10). Altogether, these results suggest that BA.2.86 is one of the most highly immune evasive variants so far.

Yang et al; 2023; “Antigenicity and infectivity characterisation of SARS-CoV-2 BA.2.86” (https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(23)00573-X/fulltext)

First, we generated the pseudovirus of BA.2.86 and established its antigenic distance from B.1 (D614G), BA.5, BQ.1.1, and XBB using serum samples from mice that had received two doses of spike mRNA vaccines (figure A; appendix 1 p 8). BA.2.86 showed a high resistance to serum neutralisation across all vaccine groups (appendix 1 p 8). Antigenic cartography calculated on the basis of the pseudovirus neutralisation titres showing that BA.2.86 was antigenically distinct from wild-type, BA.2, BA.5, and XBB.1.5, suggesting a substantial antigenic drift, which indicates that BA.2.86 could strongly evade XBB-induced antibodies (figure A).

All involved participants received three doses of inactivated vaccines before having a XBB (XBB subvariants with S486P substitution) breakthrough infection. The first cohort (n=27) included individuals with single post-vaccination XBB breakthrough infection and the second cohort (n=54) comprised convalescents who had a XBB reinfection after BA.5 or BF.7 breakthrough infection (appendix 2). We found that BA.2.86 could induce significant antibody evasion of XBB-stimulated plasma (figure B). BA.2.86’s immune evasion capability even exceeded EG.5 and was similar to variants with the adjacent residue flipping mutation L455F and F456L (FLip variants) such as HK.3 (XBB.1.5, L455F, and F456L).

³ Notably, the relative activity against HK.3 and BA.2.86 varied from sample to sample, indicating a large antigenic distance despite a similar amount of evasion.

Qu et al; 2024; “Immune evasion, infectivity, and fusogenicity of SARS-CoV-2 BA.2.86 and FLip variants” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10872432/)

Neutralizing antibodies in 3-dose-vaccinated sera are unable to neutralize BA.2.86 similar to XBB variants

We next examined the nAb titers in 3-dose-mRNA-vaccinated HCWs (n = 15) that have received at 3 homologous doses of either Pfizer or Moderna monovalent mRNA vaccines (Table S1). Similar to XBB variants including XBB.1.5 and EG.5.1, BA.2.86 exhibited nAb titers around the limit of detection for the assay, i.e., the lowest fold of dilution, i.e., 40, with a 54.1-fold reduction compared to D614G (p < 0.0001) and a 20.1-fold reduction relative to its parental BA.2 (p < 0.0001), respectively (Figures 2C and ​and2D).2D). Notably, the FLip variant exhibited a more dramatic escape, with 138.0-fold and 51.4-fold reduced nAb titers relative to D614G and BA.2, respectively (p < 0.0001) (Figures 2C and ​and2D).2D). This extent of nAb escape by FLip was largely comparable to its parental variant XBB.1.5, with NT₅₀ values all falling below the limit of detection and due to both the XBB.1.5-L455F and XBB.1.5-F456L mutations (Figures 2C and ​and2D).2D). Overall, BA.2.86 and FLip variants exhibit marked escape of nAbs in 3-dose monovalent-vaccinated sera, with titers near or below the limit of detection.

XBB.1.5-wave breakthrough infections conferred almost no nAbs against BA.2.86 and FLip variants

The final cohort we investigated was individuals who became infected during the XBB.1.5 wave in Columbus, Ohio (n = 11). Nasal swabs were performed to confirm COVID-19-positive status of 8 individuals and sequencing identified XBB.1.5 as the infecting variant; 3 samples were not sequence confirmed but collected after February 2023 when XBB variants had become dominant in this area. Among these 11 samples, 8 were vaccinated, 3 of which received 3 doses of monovalent vaccine, 3 received at least 3 doses of monovalent and 1-dose bivalent booster, and 2 received 2 doses of monovalent vaccine (Table S1). Overall, the nAb titers against all variants in this cohort were much lower than in the bivalent- or monovalent-vaccinated cohorts, with NT₅₀ below the limit of detection for all XBB variants (Figures 2E and ​and2F).2F). Of note, BA.2.86 exhibited an average of NT₅₀ = 47, which was slightly above the limit of detection, i.e., NT₅₀ = 40. The nAb titers against FLip were the lowest among all the variants examined (Figures 2E and ​and2F).2F). Importantly, 3–5 of the 11 individuals who had received at least 3-dose mRNA vaccine (Table S1) exhibited nAb titers above the limit of detection for FLip or BA.2.86 (Figures 2E, ​,2F2F and S1B). In summary, while XBB.1.5-wave breakthrough infections confer limited, if any, neutralization against BA.2.86 and FLip, BA.2.86 still exhibits less nAb evasion compared to XBB variants in the XBB.1.5-convalescent cohort.

Molecular modeling revealed how mutations in BA.2.86 compromise S309 antibody neutralization

We performed homology modeling to understand the possible molecular and structural basis by which BA.2.86 exhibits distinct viral infectivity and evades S309 neutralization. Figure 6A shows a model of the BA.2.86 spike trimer, highlighting mutations that differ from the ancestral BA.2 variant. S309, classified as a class III mAb, targets the lateral segment of the RBD within the spike protein, especially residues 330–441. Among these residues, positions 339 and 356 are pivotal components of the S309-binding epitope. The replacement of the native glycine 339 residue with either aspartic acid (present in BA.2 and BA.4/5) or histidine (present in BA.2.86, XBB.1.5, and EG.5.1) creates steric hindrance effects that interfere binding with residues Y100 and L110 of the S309 antibody (Figure 6B). Simultaneously, the K356T mutation, which is also present in BA.2.86, disrupts the salt bridge interaction established with E108 of S309 (Figure 6B). Together, these dual mutations diminish the neutralization efficacy of the BA.2.86 variant by the S309 antibody.

Yahi N., Chahinian H. ,& Fantini J; 2021;“Infection-enhancing anti-SARS-CoV-2 antibodies recognize both the original Wuhan/D614G strain and Delta variants. A potential risk for mass vaccination?” (https://www.journalofinfection.com/article/S0163-4453(21)00392-3/abstract#articleInformation)

Highlights

• •Infection-enhancing antibodies have been detected in symptomatic Covid-19.
• •Antibody dependent enhancement (ADE) is a potential concern for vaccines.
• •Enhancing antibodies recognize both the Wuhan strain and delta variants.
• •ADE of delta variants is a potential risk for current vaccines.
• •Vaccine formulations lacking ADE epitope are suggested.

Abstract

Antibody dependent enhancement (ADE) of infection is a safety concern for vaccine strategies. In a recent publication, Li et al. (Cell 184 :4203–4219, 2021) have reported that infection-enhancing antibodies directed against the N-terminal domain (NTD) of the SARS-CoV-2 spike protein facilitate virus infection in vitro, but not in vivo. However, this study was performed with the original Wuhan/D614G strain. Since the Covid-19 pandemic is now dominated with Delta variants, we analyzed the interaction of facilitating antibodies with the NTD of these variants. Using molecular modeling approaches, we show that enhancing antibodies have a higher affinity for Delta variants than for Wuhan/D614G NTDs. We show that enhancing antibodies reinforce the binding of the spike trimer to the host cell membrane by clamping the NTD to lipid raft microdomains. This stabilizing mechanism may facilitate the conformational change that induces the demasking of the receptor binding domain. As the NTD is also targeted by neutralizing antibodies, our data suggest that the balance between neutralizing and facilitating antibodies in vaccinated individuals is in favor of neutralization for the original Wuhan/D614G strain. However, in the case of the Delta variant, neutralizing antibodies have a decreased affinity for the spike protein, whereas facilitating antibodies display a strikingly increased affinity. Thus, ADE may be a concern for people receiving vaccines based on the original Wuhan strain spike sequence (either mRNA or viral vectors). Under these circumstances, second generation vaccines with spike protein formulations lacking structurally-conserved ADE-related epitopes should be considered.

Liu et al; 2021; “An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies” (https://www.sciencedirect.com/science/article/pii/S0092867421006620)

Highlights

• •SARS-CoV-2 infectivity is enhanced by specific antibodies independent of the Fc receptor
• •The open RBD state is induced upon antibody binding to a specific site on the NTD
• •Divalent bridging of spikes is required to induce the RBD-up state
• •Infectivity-enhancing antibodies are detected in severe COVID-19 patients

Summary

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection

Lasrado et al; 2023; “Neutralization escape by SARS-CoV-2 Omicron subvariant BA.2.86” (https://www.researchgate.net/publication/374898140_Neutralization_escape_by_SARS-CoV-2_Omicron_subvariant_BA286)

Our data demonstrate that NAb responses to BA.2.86 were 5–13-fold lower than to BA.2 but were comparable or slightly higher than to XBB.1.5, XBB.1.16, EG.5, EG.5.1, and FL.1.5.1. These data suggest that
BA.2.86 evolved directly from the less resistant BA.2 variant, rather than from the current highly resistant circulating recombinant variants, which presumably were selected for increased NAb escape following
infection with XBB lineage viruses. Thus, BA.2.86 does not show increased antibody escape compared with current circulating variants.

Our findings are concordant with other studies from the U.S [4,5] but contrast with studies from Asia [6,7], which may reflect differences in population immunity due to different vaccine and variant exposures in
various regions of the world. XBC.1.6 is another highly mutated variant that is a BA.2/Delta recombinant (Figs. S1, S2) and similarly shows less NAb escape than XBB.1.5. Our data also show that NAb profiles at 6 months were comparable in participants who did or did not receive the bivalent mRNA boost,
consistent with its limited clinical durability [8,9]. However, NAb titers to multiple variants increased substantially following XBB infection, suggesting that the monovalent XBB.1.5 booster will likely increase
these NAb responses. It will also be important to monitor for potential further evolution or recombination of BA.2.86.

Kobbe et al; 2024; “Delayed Induction of Noninflammatory SARS-CoV-2 Spike-Specific IgG4 Antibodies Detected 1 Year After BNT162b2 Vaccination in Children” (https://pubmed.ncbi.nlm.nih.gov/39078156/)

Humoral immune responses after BNT162b2 vaccination are predominantly composed of immunoglobulin (Ig) G1 and IgG3 subclass antibodies. As previously described in adults, S1-specific and receptor-binding domain-specific IgG4 levels increase significantly 1 year after the second BNT162b2 vaccination in children 5-11 years of age.

Sheehan et al; 2023; “Dynamics of Serum-Neutralizing Antibody Responses in Vaccinees through Multiple Doses of the BNT162b2 Vaccine” (https://www.mdpi.com/2076-393X/11/11/1720)

To enhance the durability of post-vaccination immunity and combat SARS-CoV-2 variants, boosters have been administered to two-dose vaccinees. However, long-term humoral responses following booster vaccination are not well characterized. A 16-member cohort of healthy SARS-CoV-2 naïve participants were enrolled in this study during a three-dose BNT162b2 vaccine series. Serum samples were collected from vaccinees over 420 days and screened for antigen (Ag)-specific antibody titers, IgG subclass distribution, and neutralizing antibody (nAb) responses. Vaccine boosting restored peak Ag-specific titers with sustained α-RBD IgG and IgA antibody responses when measured at six months post-boost. RBD- and spike-specific IgG4 antibody levels were markedly elevated in three-dose but not two-dose immune sera. Although strong neutralization responses were detected in two- and three-dose vaccine sera, these rapidly decayed to pre-immune levels by four and six months, respectively. While boosters enhanced serum IgG Ab reactivity and nAb responses against variant strains, all variants tested showed resistance to two- and three-dose immune sera. Our data reflect the poor durability of vaccine-induced nAb responses which are a strong predictor of protection from symptomatic SARS-CoV-2 infection. The induction of IgG4-switched humoral responses may permit extended viral persistence via the downregulation of Fc-mediated effector functions.

Nkinda et al; 2024; “Evaluation of cross-neutralizing immunity following COVID-19 primary series vaccination during the Omicron surge in Tanzania” (https://pubmed.ncbi.nlm.nih.gov/39056238/)

[W]e tested cross-neutralizing capacity of antibodies elicited by infection, vaccination, or both against SARS-CoV-2 Omicron subvariants BA.1, and the newer subvariants BQ.1.1 and XBB.1.5. that were unexperienced by this population. Participants who were either SARS-CoV-2 infected-only (n = 28), infected vaccinated (n = 22), or vaccinated-only (n = 73) were recruited from Dar-es-Salaam, Tanzania, between April and December 2022. Plasma 50% neutralization titers (NT₅₀) against SARS-CoV-2 wild-type strain and Omicron subvariants were quantified by a lentiviral-based pseudo-virus assay. Percentage of participants with neutralizing activity against WT and BA.1 was high (>85%) but was reduced against BQ.1.1 (64%-77%) and XBB.1.5 (35%-68%) subvariants. The low median cross-neutralization titer was slightly higher in the infected vaccinated group compared to vaccine-only group against BQ.1.1 (NT₅₀ 148 vs. 85, p = 0.032) and XBB.1.5 (NT₅₀ 85 vs. 37 p = 0.022) subvariants. In contrast, vaccine-boost among the infected vaccinated did not result to increased cross-neutralization compared to infected-only participants (BQ.1.1 [NT₅₀ of 148 vs. 100, p = 0.501] and XBB.1.5 [NT₅₀ 86 vs. 45, p = 0.474]). We report severely attenuated neutralization titers against BQ.1.1 and XBB.1.5 subvariants among vaccinated participants, which marginally improved in the infected vaccinated participants.

Saiag et al; 2024; “Antibody Response After a Fifth Dose (Third Booster) of BNT162b2 mRNA COVID-19 Vaccine in Healthcare Workers” (https://pubmed.ncbi.nlm.nih.gov/39518677/)

Although a fourth dose of SARS-CoV-2 vaccine was shown to be effective, the immunogenicity of a fifth dose in immunocompetent individuals had not been well described. This was a prospective observational cohort study of previously vaccinated healthcare workers at a single tertiary hospital in Israel. Individuals were administered up to three booster doses of the BNT162b2 mRNA vaccine (i.e., up to five overall doses), during the period between July 2021 and January 2023. Immunogenicity was assessed using the SARS-CoV-2 IgG (sCOVG) semi-quantitative assay, performed at several time points. The cohort consisted of 162 individuals (median age 69 years, 62% female). Of these, 104 (64%) received four doses and 58 (36%) received five doses. Anti-SARS-CoV-2 antibody levels increased in all cases, regardless of the baseline levels. The fold-change increase in the mean sCOVG index was 29.2 (SD 2.6) after the third vaccine, 3.8 (SD 2.4) after the fourth vaccine, and 3.6 (SD 3.0) after the fifth vaccine. A waning effect over time was seen in 78% and 43% of participants for the third and fourth doses, respectively. Adverse events following the fifth dose were limited and mild. Similar to previous booster vaccines, a fifth dose of BNT162b2 is immunogenic and safe in healthy individuals, although the clinical implications remain unclear.

Chen et al; 2024; “Recall of cross-reactive memory B cells enhances antibody durability and breadth against SARS-CoV-2 variants” (https://journals.aai.org/jimmunol/article/212/1_Supplement/0141_4393/267367/Recall-of-cross-reactive-memory-B-cells-enhances)

Long-lasting antibody production with broad recognition breadth is crucial to combat with many pathogens. After infection or vaccination, B cell memory is preserved via two key elements: memory B cells, which can be efficiently recalled during subsequent encounters, and long-lived plasma cells, which continue to produce antibodies over the long term. It’s critical to understand antibody durability and how memory can identify different viral variants following iterative exposures. We examined the recognition of SARS-CoV-2 variants, the dynamics of memory B cells, and the secretion of antibodies over time after recurrent infections and vaccinations.

Within unvaccinated individuals who recovered from COVID, enhanced antibody stability over time was observed within a subgroup of individuals who recovered more quickly from COVID, had greater percentage of spike specific memory circulating T follicular helper (cTfh), and harbored significantly more memory B cells cross-reactive to endemic coronaviruses early after infection. These cross-reactive clones map to the conserved S2 region of SARS-CoV-2 spike with higher somatic hypermutation levels and affinity. Following SARS-CoV-2 inactivated vaccination, frequency of cross-reactive memory B cells after BF.7 infection links to antibody longevity and neutralization breadth to SARS-CoV-2 variants.

We conclude that memory B cell recall shapes functional cross-variant antibody repertoire composition and longevity.

Samanovic et al; 2021; “Robust immune responses after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experienced individuals” (Robust immune responses after one dose of BNT162b2 mRNA vaccine dose in SARS-CoV-2 experienced individuals – PubMed)

The use of COVID-19 vaccines will play the major role in helping to end the pandemic that has killed millions worldwide. COVID-19 vaccines have resulted in robust humoral responses and protective efficacy in human trials, but efficacy trials excluded individuals with a prior diagnosis of COVID-19. As a result, little is known about how immune responses induced by mRNA vaccines differ in individuals who recovered from COVID-19. Here, we evaluated longitudinal immune responses to two-dose BNT162b2 mRNA vaccination in 15 adults who recovered from COVID-19, compared to 21 adults who did not have prior COVID-19 diagnosis. Consistent with prior studies of mRNA vaccines, we observed robust cytotoxic CD8⁺ T cell responses in both cohorts following the second dose. Furthermore, SARS-CoV-2-naive individuals had progressive increases in humoral and antigen-specific antibody-secreting cell (ASC) responses following each dose of vaccine, whereas SARS-CoV-2-experienced individuals demonstrated strong humoral and antigen-specific ASC responses to the first dose but muted responses to the second dose of the vaccine at the time points studied. Together, these data highlight the relevance of immunological history for understanding vaccine immune responses and may have significant implications for personalizing mRNA vaccination regimens used to prevent COVID-19, including booster shots.

Turner et al; 2021; “SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans” (SARS-CoV-2 infection induces long-lived bone marrow plasma cells in humans | Nature)

Long-lived bone marrow plasma cells (BMPCs) are a persistent and essential source of protective antibodies^{1,2,3,4,5,6,7}. Individuals who have recovered from COVID-19 have a substantially lower risk of reinfection with SARS-CoV-2^8,9,10. Nonetheless, it has been reported that levels of anti-SARS-CoV-2 serum antibodies decrease rapidly in the first few months after infection, raising concerns that long-lived BMPCs may not be generated and humoral immunity against SARS-CoV-2 may be short-lived^11,12,13. Here we show that in convalescent individuals who had experienced mild SARS-CoV-2 infections (n = 77), levels of serum anti-SARS-CoV-2 spike protein (S) antibodies declined rapidly in the first 4 months after infection and then more gradually over the following 7 months, remaining detectable at least 11 months after infection. Anti-S antibody titres correlated with the frequency of S-specific plasma cells in bone marrow aspirates from 18 individuals who had recovered from COVID-19 at 7 to 8 months after infection. S-specific BMPCs were not detected in aspirates from 11 healthy individuals with no history of SARS-CoV-2 infection. We show that S-binding BMPCs are quiescent, which suggests that they are part of a stable compartment. Consistently, circulating resting memory B cells directed against SARS-CoV-2 S were detected in the convalescent individuals. Overall, our results indicate that mild infection with SARS-CoV-2 induces robust antigen-specific, long-lived humoral immune memory in humans.

9g. Misc immune system studies

Rubio-Casillas et al; 2023; “Do vaccines increase or decrease susceptibility to diseases other than those they protect against?” (https://pubmed.ncbi.nlm.nih.gov/38158298/)

Contrary to the long-held belief that the effects of vaccines are specific for the disease they were created; compelling evidence has demonstrated that vaccines can exert positive or deleterious non-specific effects (NSEs). In this review, we compiled research reports from the last 40 years, which were found based on the PubMed search for the epidemiological and immunological studies on the non-specific effects (NSEs) of the most common human vaccines. Analysis of information showed that live vaccines induce positive NSEs, whereas non-live vaccines induce several negative NSEs, including increased female mortality associated with enhanced susceptibility to other infectious diseases, especially in developing countries. These negative NSEs are determined by the vaccination sequence, the antigen concentration in vaccines, the type of vaccine used (live vs. non-live), and also by repeated vaccination. We do not recommend stopping using non-live vaccines, as they have demonstrated to protect against their target disease, so the suggestion is that their detrimental NSEs can be minimized simply by changing the current vaccination sequence. High IgG4 antibody levels generated in response to repeated inoculation with mRNA COVID-19 vaccines could be associated with a higher mortality rate from unrelated diseases and infections by suppressing the immune system. Since most COVID-19 vaccinated countries are reporting high percentages of excess mortality not directly attributable to deaths from such disease, the NSEs of mRNA vaccines on overall mortality should be studied in depth.

Adhikari et al; 2024; “Brief research report: impact of vaccination on antibody responses and mortality from severe COVID-19” (https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1325243/full)

Results: While mortality rates were 36% (n=25) and 27% (n=15) for non-COVID-19 NVax and Vax patients, respectively, in COVID-19 patients mortality rates were 37% (NVax, n=89) and 70% (Vax, n=23). Among COVID-19 patients, mortality rate was significantly higher among Vax vs. NVax patients (p=0.002). The Charlson’s Comorbidity Index score (CCI) was also significantly higher among Vax vs. NVax COVID-19 patients. However, the mortality risk remained significantly higher (p=0.02) when we compared COVID-19 Vax vs. NVax patients with similar CCI score, suggesting that additional factors may increase risk of mortality. Higher levels of SARS-CoV-2 Abs were noted among survivors, suggestive of their protective role. We observed a trend for increased total IgG4 Ab, which promotes immune tolerance, in the Vax vs. NVax patients in week 3.

Conclusion: Although our cohort size is small, our results suggest that vaccination status of hospital-admitted COVID-19 patients may not be instructive in determining mortality risk. This may reflect that within the general population, those individuals at highest risk for COVID-19 mortality/immune failure are likely to be vaccinated. Importantly, the value of vaccination may be in preventing hospitalization as opposed to stratifying outcome among hospitalized patients, although our data do not address this possibility. Additional research to identify factors predictive of aberrant immunogenic responses to vaccination is warranted.

Sherriff et al; 2024; “A Study on the Self-Reported Physician-Diagnosed Cardiac Complications Post mRNA Vaccination in Saudi Arabia” (https://assets.cureus.com/uploads/original_article/pdf/216342/20240210-27678-1rqn0hz.pdf)

“The advent of mRNA-based vaccines has been a pivotal milestone in the global response to the pandemic, prompting widespread vaccination campaigns, including in Saudi Arabia. This study investigates self-reported physician-diagnosed cardiac complications post mRNA vaccination in Saudi Arabia, recognizing the need to monitor these rare events. The research aimed to study the self-reported physician-diagnosed incidence, nature, and associated factors of cardiac complications in this distinctive demographic group of post-mRNA vaccinations. Due to the scarcity of specific vaccine safety data, the study sought to provide data supporting public awareness and recommendations for global public health practices. Emphasizing ethical considerations, the study focuses on local factors, contributing valuable insights into the safety profile of mRNA vaccines, and aiding informed decision-making for public health strategies in Saudi Arabia and globally.

Results
Significant associations were found among demographic variables, vaccination behaviors, health diagnoses, and perceptions of self-reported physician-diagnosed cardiac complications post mRNA vaccination in Saudi Arabia. Key findings included a high mRNA vaccine uptake with a frequency of 747 (92.79%) and a mere frequency of 218 (27.11%) reporting cardiac complications post vaccination. The study highlighted diverse influences on vaccine decisions, with a frequency of 384 (47.76%) expressing neutral confidence in vaccine safety. The study contributes to the global understanding of mRNA vaccine safety, emphasizing the unique Saudi demographic context. Methodological rigor, ethical considerations, and acknowledgment of limitations enhance credibility. Collaborative efforts and tailored recommendations for public health policies and communication strategies are underscored.

Conclusion
This study on self-reported physician-diagnosed cardiac complications post mRNA vaccination in Saudi
Arabia is a crucial addition to global vaccine safety research. Providing insights shaped by local factors, the study aids in informed decision-making for public health strategies in Saudi Arabia and globally. It addresses the scarcity of specific vaccine safety data, fostering a nuanced understanding of mRNA vaccine-related cardiac complications worldwide.

Thuluvath et al; 2021; “Analysis of antibody responses after COVID-19 vaccination in liver
transplant recipients and those with chronic liver diseases” (https://www.journal-of-hepatology.eu/article/S0168-8278(21)02002-X/pdf)

Results: Of the 233 patients enrolled so far, 62 were LT recipients, 79 had cirrhosis (10 decompensated) and 92 had CLD without cirrhosis. Antibody titers were defined as undetectable (<0.40 U/ml), suboptimal (0.40–250 U/ml) and adequate (>250 U/ml). Of the 62 patients who had LT, antibody levels were un-
detectable in 11 patients and suboptimal (median titer 17.6, range 0.47–212 U/ml) in 27 patients. Among 79 patients with cirrhosis, 3 had undetectable antibody levels and 15 had suboptimal (median titer 41.3, range 0.49–221 U/L) antibody responses. Of the 92 patients without cirrhosis, 4 had undetectable
antibody levels and 19 had suboptimal (median titer 95.5, range 4.9–234 U/L) antibody responses. Liver transplantation, use of 2 or more immunosuppression medications and vaccination with a single dose of the Johnson & Johnson vaccine were associated with poor immune response on multivariable analysis. No patient had any serious adverse events.
Conclusions: Poor antibody responses after SARS-CoV-2 vaccination were seen in 61% of LT recipients and 24% of those with CLD.

10. Miscellaneous Research Studies

Hulscher et al; 2025; “Review of Calls for Market Removal of COVID-19 Vaccines Intensify: Risks Far Outweigh Theoretical Benefits” (https://publichealthpolicyjournal.com/review-of-calls-for-market-removal-of-covid-19-vaccines-intensify-risks-far-outweigh-theoretical-benefits/)

COVID-19 vaccination campaigns around the globe have failed to meet fundamental standards of safety and efficacy, leading to mounting evidence of significant harm. More than 81,000 physicians, scientists, researchers, and concerned citizens, 240 elected government officials, 17 professional public health and physician organizations, 2 State Republican Parties, 17 Republican Party County Committees, and 6 scientific studies from across the world have called for the market withdrawal of COVID-19 vaccines. As of September 6, 2024, the CDC has documented 19,028 deaths in the United States reported to the Vaccine Adverse Event Reporting System (VAERS) by healthcare professionals or pharmaceutical companies who believe the product is related to the death. The total number of COVID-19 vaccine deaths reported to VAERS (37,544 among all participating countries) have far exceeded the recall limits of past vaccine withdrawals by up to 375,340%. The criteria for an FDA Class I recall, which applies to products with a reasonable probability of causing serious adverse health consequences or death, have been far exceeded. Excess mortality, negative efficacy, widespread DNA contamination, and a lack of demonstrated reduction in transmission, hospitalization, or mortality have undermined the rationale for continued administration. These unified requests for regulatory action underscore substantial shortcomings in data safety monitoring and risk mitigation. Immediate removal of COVID-19 vaccines from the market is essential to prevent further loss of life and ensure next steps are taken for accountability of the harm incurred.

Mordue et al; 2024; “Medical Ethics and Informed Consent to Treatment: Past, Present and Future” (https://pmc.ncbi.nlm.nih.gov/articles/PMC11627192/)

It has been asserted that there was an erosion of medical ethics during the Covid-19 pandemic and a departure from the principle of obtaining fully informed consent from patients before treatment. In light of these assertions, this article reviews the historical development of medical ethics and the approach to obtaining informed consent and critiques the consent practices before and during the pandemic. It then describes a new tool for displaying key statistics on the benefits and risks of interventions to help explain them to patients and suggests a more rigorous process for seeking fully informed consent in the future.

From January 2021, (i) there was a rapid rollout of the novel mRNA and DNA vaccine technology under emergency authorisation and with no long-term safety data to almost the whole population, most notably children and pregnant women, regardless of their individual risk from Covid-19 or their immune status with respect to SARS-CoV-2 (compromises the principles of truth-telling/full disclosure, beneficence and non-maleficence) [13]. A recent paper performed a risk-benefit assessment and ethical analysis and concluded that ‘booster mandates in young adults are expected to cause a net harm’ [14]. Next, (ii) vaccine benefits were exaggerated, going beyond the reduction of symptomatic cases observed in the trials [15], with many in positions of authority claiming that mass vaccination would stop Covid-19 transmission through vaccine-induced herd immunity [16] and some stating that vaccination was the only route to avoiding ongoing lockdowns and masking (compromises truth-telling/disclosure and voluntary consent); (iii) governments and health officials heavily marketed the Covid-19 vaccines to the public using terms such as ‘safe and effective’ and ‘rigorously evaluated by MHRA’ (UK Medicines and Healthcare products Regulatory Agency) making an individual objective risk versus benefit analysis in order to give valid informed consent more difficult (compromises truth-telling/disclosure and voluntary consent); (iv) the requirement to disclose alternatives to the Covid-19 vaccines was not followed: information concerning potentially effective drugs for early treatment was not shared with the public [17], leaving those at significant risk with no apparent choice (compromises truth-telling, beneficence and non-maleficence); (v) in many countries, Covid-19 vaccines were mandated for certain groups and in order to access certain basic human freedoms (e.g. work, travel, shopping and leisure), a violation of the principle that consent must be entirely voluntary and without coercion, penalty or restriction (compromises autonomy and voluntary consent).

Next, (vi) there was a failure of regulators and governments around the world to interrogate or respond to safety signals in vaccine safety surveillance systems over the last three years (compromises truth-telling and non-maleficence) [18], and furthermore, (vii) when concerned professionals and vaccine-injured patients did try to inform people of the risks, they were smeared and silenced (compromises truth-telling/disclosure) [19]. Finally, the actions taken by governments to indemnify the Covid-19 vaccine manufacturers against liability for harms suffered, and the often very limited and inadequate government compensation schemes, have left patients holding the full risk.

It is hard not to conclude that the medical profession has stood by, largely silent, as under emergency laws in the United Kingdom and around the world, one by one, fundamental ethical principles have been abandoned and previously hard ethical red lines have been crossed. In Europe, this occurred despite the Parliamentary Assembly of the Council of Europe passing Resolution number 2361 on 27 January 2021, which states the following [20]: 1.1 Paragraph 7.3.1 – ‘ensure that citizens are informed that (the Covid-19) vaccination is NOT mandatory and that no one is politically, socially, or otherwise pressured to get themselves vaccinated, if they do not wish to do so themselves’; 1.2 Paragraph 7.3.2 – ‘ensure that no one is discriminated against for not having been vaccinated, due to possible health risks or not wanting to be vaccinated’.

Bolormaa et al; 2024; “Methodology of comparative studies on the relative effectiveness of COVID-19 vaccines: a systematic review” (https://pubmed.ncbi.nlm.nih.gov/39511961/)

Methods: A systematic search was conducted on June 13, 2024, to identify comparative studies evaluating the effectiveness of mRNA versus non-mRNA and monovalent versus bivalent COVID-19 vaccines. We screened titles, abstracts, and full texts, collecting data on publication year, country, sample size, study population composition, study design, VE estimates, outcomes, and covariates. Studies that reported relative VE (rVE) were analyzed separately from those that did not.

Results: We identified 25 articles comparing rVE between mRNA and non-mRNA COVID-19 vaccines, as well as between monovalent and bivalent formulations. Among the studies assessing VE by vaccine type, 126 did not provide rVE estimates. Comparative VE studies frequently employed retrospective cohort designs. Among the definitions of rVE used, the most common were hazard ratio and absolute VE, calculated as (1-odds ratio)×100. Studies were most frequently conducted in the United Kingdom and the United States, and the most common outcome was infection. Most targeted the general population and assessed the VE of mRNA vaccines using the AstraZeneca vaccine as a reference. A small proportion, 7.3% (n=11), did not adjust for any variables. Only 3 studies (2.0%) adjusted for all core confounding variables recommended by the World Health Organization.

Conclusion: Few comparative studies of COVID-19 vaccines have incorporated rVE methodologies. Reporting rVE and employing a consistent set of covariates can broaden our understanding of COVID-19 vaccines.

Eales et al; 2023; “Dynamics of SARS-CoV-2 infection hospitalisation and infection fatality ratios over 23 months in England” (https://pmc.ncbi.nlm.nih.gov/articles/PMC10212114/)

During 2020, we estimated the IFR to be 0.67% and the IHR to be 2.6%. By late 2021/early 2022, the IFR and IHR had both decreased to 0.097% and 0.76%, respectively. The average case ascertainment rate over the entire duration of the study was estimated to be 36.1%, but there was some significant variation in continuous estimates of the case ascertainment rate. Continuous estimates of the IFR and IHR of the virus were observed to increase during the periods of Alpha and Delta’s emergence. During periods of vaccination rollout, and the emergence of the Omicron variant, the IFR and IHR decreased. During 2020, we estimated a time-lag of 19 days between hospitalisation and swab positivity, and 26 days between deaths and swab positivity. By late 2021/early 2022, these time-lags had decreased to 7 days for hospitalisations and 18 days for deaths. Even though many populations have high levels of immunity to SARS-CoV-2 from vaccination and natural infection, waning of immunity and variant emergence will continue to be an upwards pressure on the IHR and IFR.

Liu et al; 2022; “Reduction in the infection fatality rate of Omicron variant compared with previous variants in South Africa” (https://pmc.ncbi.nlm.nih.gov/articles/PMC9022446/)

Results

We found that the high relative transmissibility of the Omicron variant was mainly due to its immune evasion ability, whereas its infection fatality rate substantially decreased by approximately 78.7% (95% confidence interval: 66.9%, 85.0%) with respect to previous variants.

Conclusion

On the basis of data from South Africa and mathematical modeling, we found that the Omicron variant is highly transmissible but with significantly lower infection fatality rates than those of previous variants of SARS-CoV-2.

Girma & Paton; 2024; “Using double-debiased machine learning to estimate the impact of Covid-19 vaccination on mortality and staff absences in elderly care homes.” (https://www.sciencedirect.com/science/article/pii/S0014292124002113)

Machine learning approaches provide an alternative to traditional fixed effects estimators in causal inference. In particular, double-debiased machine learning (DDML) can control for confounders without making subjective judgements about appropriate functional forms. In this paper, we use DDML to examine the impact of differential Covid-19 vaccination rates on care home mortality and other outcomes. Our approach accommodates fixed effects to account for unobserved heterogeneity. In contrast to standard fixed effects estimates, the DDML results provide some evidence that higher vaccination take-up amongst residents, but not staff, reduced Covid mortality in elderly care homes. However, this effect was relatively small, is not robust to alternative measures of mortality and was restricted to the initial vaccination roll-out period.

Even using DDML, we are unable to identify strong evidence that vaccination rates amongst care home staff reduced mortality or that resident vaccination reduced mortality during booster roll out period (from September 2021). Indeed, in the later period, we find some evidence that higher vaccination rates are associated with higher Covid mortality.

The lack of evidence that vaccination of care home staff has any causal effect in reducing mortality amongst residents is unsurprising in the light of research noted above suggesting vaccination has modest impacts on contagiousness and, hence transmission. The limited effect of resident vaccination on mortality requires more explanation given evidence from some randomised controlled trials (RCTs) that, other thing equal, Covid vaccination lowers the risk of serious illness and death. However, there are good reasons why such trial outcomes may not be replicated in real world data.

First, is the well-known difficult of controlling for healthy vaccinee effects even within RCTs and which may lead to effectiveness being overstated. Second, RCTs typically compare mortality rates amongst those vaccinated with those unvaccinated and who have not previously had Covid. Even at the start of the vaccine rollout in the UK, many residents and staff in care homes had previously recovered from a Covid infection. Previous infection is known to reduce very significantly the chance both of subsequent infection and of mortality given subsequent infection. As a result, the potential for any beneficial mortality impact of vaccination amongst this group will be correspondingly smaller. Further, if the previously infected group are less likely than others to be vaccinated, this would provide another mechanism that pushes up population-level mortality rates amongst the vaccinated relative to unvaccinated cohorts.

Apart from the novel application of DDML techniques, a key strength of our analysis is the construction of a measure of ‘vaccination stock’ that takes account both of waning vaccination effectiveness over time as well as the impact of second and booster vaccination doses.

The limited effectiveness of vaccination on Covid mortality is consistent with research (for example, Fabiani et al. (2020), Simonsen et al., 2007 and Verhees et al., 2019) on the impact of influenza vaccination and which has similarly found it difficult to establish a clear causal impact on mortality rates.

Qasmieh et al; 2024; “Magnitude of Potential Biases in COVID-19 Vaccine Effectiveness Studies due to Differential Healthcare seeking following Home Testing: Implications for Test Negative Design Studies” (https://www.medrxiv.org/content/10.1101/2024.12.30.24319700v1)

The test-negative design (TND) is widely used to estimate COVID-19 vaccine effectiveness (VE). Biased estimates of VE may result from effects of at-home SARS-CoV-2 rapid diagnostic test (RDT) results on decisions to seek healthcare. To investigate magnitude of potential bias, we constructed decision trees with input probabilities obtained from longitudinal surveys of U.S. adults between March 2022 – October 2023. Prevalence of at-home RDT use and healthcare seeking following a positive or negative RDT result was estimated by participant vaccination status and socio-demographic characteristics. At true VE values ranging from 5% to 95%, we defined bias as the difference between the observed and true VE. Among 1,918 symptomatic adults, prevalence of at-home RDT use was higher among vaccinated (37%) versus unvaccinated (22%) participants. At-home RDT use was associated with seeking care, and participants reporting positive RDT were more likely than those reporting negative RDT to have sought care when ill. In primary analyses, we observed downward bias in VE estimates that increased in magnitude when true VE was low. Variations in proportions of vaccination, at-home RDT use and healthcare seeking by socio-demographic characteristics may impact VE estimates.

Nakatani et al; 2024; “Behavioral and Health Outcomes of mRNA COVID-19 Vaccination: A Case-Control Study in Japanese Small and Medium-Sized Enterprises” (https://www.cureus.com/articles/313843-behavioral-and-health-outcomes-of-mrna-covid-19-vaccination-a-case-control-study-in-japanese-small-and-medium-sized-enterprises)

The study found a higher reported incidence of COVID-19 infection among vaccinated individuals, with the risk increasing alongside the number of vaccine doses received. Adjusted odds ratios (ORs) indicated that vaccinated individuals were 1.85 times more likely to contract COVID-19 compared to their unvaccinated counterparts (95% CI: 1.33-2.57, p < 0.001). The odds rose with additional doses: one to two doses (OR: 1.63, 95% CI: 1.08-2.46, p = 0.020), three to four doses (OR: 2.04, 95% CI: 1.35-3.08, p = 0.001), and five to seven doses (OR: 2.21, 95% CI: 1.07-4.56, p = 0.033). Behavioral analysis further revealed that reduced frequency of bathing and exercising significantly correlated with higher infection rates (p < 0.05). These paradoxical findings suggest that factors such as immune response mechanisms; including antibody-dependent enhancement (ADE) and original antigenic sin; as well as behavioral changes and exposure risk, may play a role in the observed outcomes.

Anderson et al; 2020; “Challenges in creating herd immunity to SARS-CoV-2 infection by mass vaccination” (https://pmc.ncbi.nlm.nih.gov/articles/PMC7836302/?report=reader)

For a vaccine with 100% efficacy that gives life-long protection, the level of herd immunity as a proportion of the population, pc, required to block transmission is [1 – 1 / R0], where R0 is the basic reproduction number.16 Given an R0 value before lockdowns in most countries of between 2·5 to 3·5, we estimate the herd immunity required is about 60–72%. If the proportional vaccine efficacy, ε, is considered, the simple expression for pcbecomes [1 – 1 / R0] / ε. If we assume ε is 0·8 (80%), then the herd immunity required becomes 75–90% for the defined range of R0 values. For lower efficacies, the entire population would have to be immunised. 

Dun-Dery et al; 2025; “No Association between SARS-CoV-2 Infection and Quality of Life 6- and 12-Months After Infection” (https://www.academicpedsjnl.net/article/S1876-2859(24)00273-0/fulltext)

Results

Among SARS-CoV-2 positive and negative participants eligible for long-term follow-up, 74.8% (505/675) and 71.8% (1106/1541) at 6- and 59.0% (727/1233) and 68.1% (2520/3699) at 12-months, completed follow-up, respectively. Mean ± SD PedsQL scores did not differ between positive and negative groups; difference: −0.86 (95% CI: −2.33, 0.61) at 6- and −0.48 (95% CI: −1.6, 0.64) at 12-months, respectively. SARS-CoV-2 test-positivity was associated with higher social subscale scores. Although in bivariate analysis, overall health status at 6-months was higher among SARS-CoV-2 cases [difference: 2.16 (95% CI: 0.80, 3.53)], after adjustment for co-variates, SARS-CoV-2 infection was not independently associated with total PedsQL or overall health status at either time point. Parental perception of recovery did not differ based on SARS-CoV-2 test-status at either time point.

Conclusions

SARS-CoV-2 infection was not associated with QoL, overall health status, or parental perception of recovery 6- and 12-months following infection.

Igyato et al; 2022; ““Don’t Look Up” Your Science—Herd Immunity or Herd Mentality?” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9316410/)

How Not to Vaccinate Ourselves out of a Pandemic

Scientists have been working on an effective universal influenza (RNA virus) vaccine for decades. We still do not have one, and we have not developed herd immunity from natural infections. As possible reasons for failure, we could mention that: 1. RNA viruses frequently mutate to generate new variants to evade pre-existing immunity; 2. It is hard to make the immune system to react to conserved viral elements; and 3. For ill-defined reasons many people do not develop long-lasting immune memory after infection or vaccination [24,25,26]. SARS-CoV-2 is also an RNA virus, and therefore prone to mutations, though, it was initially thought to mutate at a slightly lower rate than other RNA viruses [27]. Thus, through mutations, it is expected to evade pre-existing immunity sooner or later, especially if we focus the vaccine on a single antigenic target such as the spike protein that is critical for the virus to propagate. The so-called common human coronaviruses that contribute to 15–30% of cases of common colds in humans, also belong to this virus family, and can sometimes cause life-threatening lower respiratory tract infections in infants, elderly people, or immunocompromised patients [28]. However, mainly for the above-mentioned reasons, no serious attempts have been made by scientists to generate a vaccine targeting these virus strains. Therefore, from a scientific perspective, thinking that we can make an effective vaccine against SARS-CoV-2 by focusing on a constantly changing protein in a year or so to end the pandemic lacked a solid scientific rationale. Nevertheless, if we still wanted to fight this pandemic with a vaccine, then the “old school” vaccine platform, relying on whole killed/live attenuated virus, that helped us to control or eradicate other viruses, such as smallpox, polio, yellow fever, rabies, etc., should have been given priority [29]. The approach of a killed or live-attenuated SARS-CoV-2 vaccine formulation delivered intranasally would have been the most supported approach by a strong scientific rationale. From an immunological perspective, this vaccine formulation would have contained all the viral proteins and molecular determinants that the immune system could react to, providing the most comprehensive possible protection, even from subsequent variants [30]. Furthermore, a vaccine delivered intranasally, unlike the present ones injected intramuscularly, would have likely generated protective immunity at the mucosal sites (airways) where we are exposed to the virus, and where we need it the most [31,32,33,34]. While the generation and optimization of an intranasal vaccine could take longer than the mRNA-LNP (mRNA combined with lipid nanoparticles) vaccine and possible adverse reactions from disease-causing potential apply for a live-attenuated vaccine [35], the fact that these vaccines could have been made almost anywhere, would have increased the likelihood of fast, even, and fair worldwide distribution at a more affordable price.

All That Glitters Is Not Gold

Selecting, supporting, and promoting the mRNA-LNP platform, which has never been used in humans before and for which we lack long-term safety data over well-established killed/attenuated vaccine platforms to fight COVID, lacked solid scientific rationale. In this section, we will take a closer look at the clinical trial data with the mRNA-LNP platform, briefly discuss how this platform works and present some of its drawbacks.

The mRNA-LNP-based vaccine clinical trials compared the number of COVID cases in healthy individuals divided into vaccinated vs. control (placebo) groups. The placebo group received pure saline, and no control group injected with empty LNPs was used. A case was defined as an individual who experienced symptoms and had a positive test for SARS-CoV-2 infection [39]. The incidence of severe disease and death, as endpoints, were not considered [39,40,41]. These trials reported that the mRNA-LNP vaccines are around 95% effective in relative risk reduction (RRR) [42]. However, the companies failed to report the absolute risk reduction (ARR) and did not make the complete clinical trial data publicly available [36,37,43]. While the RRRs give a percentage reduction in one group (vaccinated) compared to another (placebo), the ARRs show the actual difference in risk between one group and another. The RRR for the mRNA-LNP vaccines was ~95%, while the ARR was later estimated to be ~1%. The authorities would probably have had a hard time convincing people to take the vaccine if they had stated that the shot reduces your risk of becoming infected by only ~1% or that you need to give ~100 people shots to prevent one infection. “Omitting ARR findings in public health and clinical reports of vaccine efficacy is an example of outcome reporting bias, which ignores unfavorable outcomes and misleads the public’s impression and scientific understanding of a treatment’s efficacy and benefits. Furthermore, the ethical and legal obligation of informed consent requires that patients are educated about the risks and benefits of a healthcare procedure or intervention [44]”.

The novelty of the mRNA-LNP platform is that the body makes proteins using the mRNA as a template. Thus, vaccine companies do not have to produce the proteins upfront. The component of this platform that supports potent immune responses is the lipid nanoparticles’ (LNP) ionizable lipid (a synthetic molecule with a long life span; [45]), which is shown to be highly inflammatory [46,47,48] and the likely driver of many of the documented side effects of this platform. If you take these LNPs and mix them with proteins, you obtain similar immune responses to the mRNA-LNP [49]. Thus, the critical component here is the LNP with ill-defined biological effects but strong adjuvant and inflammatory properties, which likely also contribute to the immune system’s reprogramming [50,51]. While the positive aspect of this platform is widely publicized, the authorities seem to not be concerned by the severe side effects and thousands of deaths reported so far for this platform in the Vaccine Adverse Event Reporting System (VAERS) database [52]. This might be because it is hard to establish direct causation in most cases, which becomes almost impossible with time. The VAERS is known to underreport certain cases [53]. Nevertheless, the number of already reported death cases surpasses the ones reported for all the other vaccines pooled together. This should raise a red flag at the regulatory agencies and trigger some sort of investigation, especially since more and more peer-reviewed and preprint case reports document the existence of the short- and long-term side effects of these vaccines [54]. These include, but are not limited to, fatal and non-fatal cases affecting the cardiovascular and nervous systems. Autoimmune cases targeting different organs, hepatitis, virus reactivation, multisystem inflammatory syndrome cases, etc., were also reported [54,55]. Whether the documented side effects are linked to the highly inflammatory properties of the LNPs, the autoimmune reactions targeting the spike protein-expressing cells [47], the pathogenicity of the spike protein coded by the vaccines, or the combination of these and other factors, remains to be determined. The spike protein coded in these vaccines is stabilized in a pre-fusion form and contains a membrane anchor sequence. These modifications might make it less pathogenic than the wild-type viral spike protein [56,57], but since the vaccine components can directly or indirectly reach almost any organ in the body [46,58,59,60,61], people exposed to multiple shots might be at higher risk of developing the presented side effects. A recent study revealed another potential, unexpected problem with the mRNA-LNP platform. This study showed that the vaccine mRNA could be reverse transcribed into DNA in a human hepatic cell line in vitro [62]. It remains to be determined whether this can be observed in vivo and at physiologically relevant levels [63]. However, if this phenomenon exists combined with genomic insertion, it might bear serious health concerns, especially if reproductive cells are affected.

SARS-CoV-2 Vaccines and Herd Immunity—A Fictional Romance

After the partial clinical trial data became public, the mRNA-LNP vaccines were marketed by government officials and experts, who suggested that if you take the vaccine, you will not get infected; therefore, you will protect yourself and the people around you. This narrative, however, was formulated on the pretext that the vaccines provided “sterilizing” immunity from SARS-CoV-2. Nevertheless, experts suggested that we can reach herd immunity if ~60% of the world population become immunized. After the “breakthrough” infections started to become apparent and later proved that vaccines do not stop people from getting infected, or spreading the virus, and that vaccinated individuals can contain similar levels of viral loads as unvaccinated people [65], the narrative changed, stating that at least you are less likely to get seriously ill and die. Experts responded to these data by bumping up their estimates on the percent of the population that must be vaccinated to reach herd immunity. To achieve the very high numbers (80–90%), some experts suggested that teenagers and, later, children must be included in the pool and get vaccinated. The idea behind herd immunity is that if enough people are immune, then the pathogen will not find enough susceptible hosts through which to replicate and spread and will eventually die out. Thus, one might wonder, if the vaccines do not provide sterilizing immunity, and people can still become infected and spread the virus, is it even theoretically possible to reach herd immunity? Data from countries with high vaccination rates prove that these vaccines, even combined with endless boosters, will not achieve herd immunity nor protect us from new variants and waves. The uncontrolled spread of Omicron through the US/world further highlights the subpar performance of the vaccines in preventing viral infections and spread. More than 70% of the first Omicron cases reported by the CDC were fully vaccinated [66]. The boosters based on the spike protein from the original virus variant, as expected and indicated, are becoming less and less effective with the new variants but might still provide some benefits in preventing severe illness and death. These benefits are probably provided by vaccine-induced antibodies circulating in the bloodstream and preventing systemic viral spread. However, the same antibodies might also contribute to an increased risk of antibody-mediated enhancement upon exposure to the virus [67,68]. While some research supports the generation of memory responses [69,70], the overwhelming majority shows a waning immunity, leading manufacturers and regulatory agencies to recommend frequent booster shots [71]. The need for continuous boosters targeting earlier variants also argues that the vaccines do not induce long-lasting memory responses or that natural infections cannot reactivate them for unknown reasons.

Layman’s Summary

The virus fatality rate did not support lockdowns, blanket restrictive measures, and non-selective mass vaccinations. There was no solid scientific rationale to adopt the untested mRNA-LNP platform over other well-established vaccine formulations to fight COVID. Scientists, experts, government officials, the media, and scientific journals, all contributed to suppressing alternative ideas on how to manage the pandemic. Some of these groups are still promoting the scientifically debunked idea that the present vaccines protect you from catching COVID and becoming sick [116], and that this is a pandemic of the unvaccinated [117]. Unfortunately, the virus is likely here to stay in one form or another, and we must learn to live with it. How effective the lockdowns and other restrictive measures were in reducing COVID deaths are still a matter of debate [118,119,120,121], but we likely lost, or we will lose a lot more people, from the direct and indirect effects of these measures. The excess of global deaths is often exclusively attributed to COVID [122]. However, these reports fail to factor in the direct and indirect effects of COVID-related restrictions. Many people died because they did not have access to healthcare during this period (they were afraid of going to the hospitals, or the hospitals did not accept them). Furthermore, a significant increase in suicide rates, drug and alcohol abuse, domestic violence, obesity, economic hardships, vaccination-related events, etc., likely contributed to excess deaths. If the vaccines do not prevent infections and spread, it is time to stop coercing people to get vaccinated, vilifying unvaccinated people, and giving extra perks to the vaccinated. It is neither feasible nor economically sustainable to “vaccinate” everyone every few months or perform continuous testing, and more importantly, there is no scientific rationale for doing so. We must focus our resources on the vulnerable population and provide them with efficient treatment options and possible preventative steps in the form of lifestyle changes, supplements, and effective, long-lasting, safe, and affordable vaccines.

Liester et al; 2025; “A Narrative Review of the COVID-19 Infodemic and Censorship in
Healthcare” (https://scholarworks.sjsu.edu/cgi/viewcontent.cgi?article=1087&context=secrecyandsociety)

Ideological and financial motivations have undermined science for decades. In this narrative review, we explore how organizations and governments used misinformation, disinformation, censorship, and secrecy to manage the COVID-19 pandemic. Various rationales for employing censorship and secrecy during the COVID-19 pandemic are examined including how organizations and governments create confusion about the risks associated with their products and blame avoidance to shift responsibility and to avoid accountability for their actions. Methods of censorship employed during the COVID-19 pandemic are reviewed, examples are provided, and the consequences of these actions are reviewed. Information included in this review was obtained from scientific papers, government documents, mass media articles, books, and personal accounts of physicians and scientists. We examine how the use of censorship and secrecy created a challenge for scientists, physicians, politicians, and the general public in trying to understand COVID-related topics. Finally, strategies for managing censorship and secrecy during a pandemic are presented.

Yamamoto; 2025; “Need for validation of vaccination programs.” (https://link.springer.com/article/10.1007/s44337-025-00274-0)

Reevaluation of vaccination programs, including live-attenuated vaccines, is crucial. Recently, three cases of children who died a day after routine vaccination were reported in Japan. Despite detailed information, including autopsy findings, experts concluded that a causal relationship with vaccination could not be evaluated. This commentary highlights the challenges with mRNA vaccines and further discusses the need to reassess the efficacy and safety of vaccines that have already been approved.

Conclusion

In the post-mRNA vaccination era, marked by an increase in shingles cases, it is essential to re-evaluate the risks and benefits of currently approved vaccines.

Thung-Hong Lin et al; 2022; “Government-sponsored disinformation and the severity of respiratory infection epidemics including COVID-19: A global analysis, 2001–2020” (https://www.sciencedirect.com/science/article/pii/S0277953622000478)

Highlights

• Political disinformation was associated with the respiratory infections’ incidence.
• The disinformation was positively associated with the incidence of COVID-19.
• Internet censorship led to underreport the incidence of respiratory infections.
• Governments must stop sponsoring disinformation to avoid blame or gain advantage.

Internet misinformation and government-sponsored disinformation campaigns have been criticized for their presumed/hypothesized role in worsening the coronavirus disease 2019 (COVID-19) pandemic. We hypothesize that these government-sponsored disinformation campaigns have been positively associated with infectious disease epidemics, including COVID-19, over the last two decades. By integrating global surveys from the Digital Society Project, Global Burden of Disease, and other data sources across 149 countries for the period 2001–2019, we examined the association between government-sponsored disinformation and the spread of respiratory infections before the COVID-19 outbreak. Then, building on those results, we applied a negative binomial regression model to estimate the associations between government-sponsored disinformation and the confirmed cases and deaths related to COVID-19 during the first 300 days of the outbreak in each country and before vaccination began. After controlling for climatic, public health, socioeconomic, and political factors, we found that government-sponsored disinformation was significantly associated with the incidence and prevalence percentages of respiratory infections in susceptible populations during the period 2001–2019. The results also show that disinformation is significantly associated with the incidence rate ratio (IRR) of cases of COVID-19. The findings imply that governments may contain the damage associated with pandemics by ending their sponsorship of disinformation campaigns.

Alessandria et al; 2024; “A Critical Analysis of All-Cause Deaths during COVID-19 Vaccination in an Italian Province” (https://www.mdpi.com/2076-2607/12/7/1343)

We used data from an Italian study on COVID-19 vaccine effectiveness, with a large cohort, long follow-up, and adjustment for confounding factors, affected by ITB, with the aim to verify the real impact of the vaccination campaign by comparing the risk of all-cause death between the vaccinated population and the unvaccinated population. We aligned all subjects on a single index date and considered the “all-cause deaths” outcome to compare the survival distributions of the unvaccinated group versus various vaccination statuses. The all-cause-death hazard ratios in univariate analysis for vaccinated people with 1, 2, and 3/4 doses versus unvaccinated people were 0.88, 1.23, and 1.21, respectively. The multivariate values were 2.40, 1.98, and 0.99. Possible explanations of this trend of the hazard ratios as vaccinations increase could be a harvesting effect; a calendar-time bias, accounting for seasonality and pandemic waves; a case-counting window bias; a healthy-vaccinee bias; or some combination of these factors. With 2 and even with 3/4 doses, the calculated Restricted Mean Survival Time and Restricted Mean Time Lost have shown a small but significant downside for the vaccinated populations.

Anderson et al; 2020; “The Effect of Influenza Vaccination for the Elderly on Hospitalization and Mortality: An Observational Study With a Regression Discontinuity Design“

Results

The data included 170 million episodes of care and 7.6 million deaths. Turning 65 was associated with a statistically and clinically significant increase in rate of seasonal influenza vaccination. However, no evidence indicated that vaccination reduced hospitalizations or mortality among elderly persons. The estimates were precise enough to rule out results from many previous studies.

Limitation

The study relied on observational data, and its focus was limited to individuals near age 65 years.

Conclusion

Current vaccination strategies prioritizing elderly persons may be less effective than believed at reducing serious morbidity and mortality in this population, which suggests that supplementary strategies may be necessary.”

Hoang TNA et al; 2023; “Assessing the robustness of COVID-19 vaccine efficacy trials: systematic review and meta-analysis, January 2023” (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10236928/)

Based on the RoB2 scale, we assessed two studies to be at low risk of bias and 13 at unclear risk of bias, while most studies (25/40) were classified as at high risk of bias (Figure 5).

10a. Misc statistical studies

Rodrigues et al; 2024; “Evaluation of post-COVID mortality risk in cases classified as severe acute respiratory syndrome in Brazil: a longitudinal study for medium and long term” (https://www.frontiersin.org/journals/medicine/articles/10.3389/fmed.2024.1495428/full)

Results: In the medium and long-term periods studied, 5,157 deaths were recorded out of 15,147 reported SARS/COVID-19 cases. Of these deaths, 91.5% (N = 4,720) occurred within the first year, while 8,5% (N = 437) after the first year. People without formal education, the older adult, had higher percentages of deaths in both periods. In the medium-term post-COVID period, the risk of death was reduced by 8% for those who had been vaccinated while in the long-term post-COVID period, the risk of death almost doubled for those who had been vaccinated. While in the medium term, there was a reduction in mortality risk for those who took two or three doses, in the long term the risk of death was greater for those who took one or two doses.

Conclusion: The protective effect of COVID-19 immunization was observed up to one year after the first symptoms. After one year, the effect was reversed, showing an increased risk of death for those vaccinated. These results highlight the need for further research to elucidate the factors that contribute to these findings.

Anderson et al; 2020; “The Effect of Influenza Vaccination for the Elderly on Hospitalization and Mortality: An Observational Study With a Regression Discontinuity Design” (https://pubmed.ncbi.nlm.nih.gov/32120383/)

Objective: To determine the effectiveness of the influenza vaccine in reducing hospitalizations and mortality among elderly persons by using an observational research design that reduces the possibility of bias and confounding.

Design: A regression discontinuity design was applied to the sharp change in vaccination rate at age 65 years that resulted from an age-based vaccination policy in the United Kingdom. In this design, comparisons were limited to individuals who were near the age-65 threshold and were thus plausibly similar along most dimensions except vaccination rate.

Setting: England and Wales. Participants: Adults aged 55 to 75 years residing in the study area during 2000 to 2014. Measurements: Hospitalization and mortality rates by month of age.

Results: The data included 170 million episodes of care and 7.6 million deaths. Turning 65 was associated with a statistically and clinically significant increase in rate of seasonal influenza vaccination. However, no evidence indicated that vaccination reduced hospitalizations or mortality among elderly persons. The estimates were precise enough to rule out results from many previous studies.

Limitation: The study relied on observational data, and its focus was limited to individuals near age 65 years.

Conclusion: Current vaccination strategies prioritizing elderly persons may be less effective than believed at reducing serious morbidity and mortality in this population, which suggests that supplementary strategies may be necessary.

Garner J.,; 2022; “Health versus Disorder, Disease, and Death: Unvaccinated Persons Are Incommensurably Healthier than Vaccinated” (https://ijvtpr.com/index.php/IJVTPR/article/view/40)

Results from the 2019/2020 nationwide Control Group Survey of Unvaccinated Americans (CGS) show that those refusing vaccines are thriving while those accepting them are being injured and met with a multiplicity of grave injuries as well as sudden unexpected death. This survey quantified the long-term health risks of total vaccine avoidance against the health outcomes observed in the 99.74% vaccine-exposed American population. Based upon the sample sizes for the controls vs. the exposed population, the p-values and odds ratios evidence the astronomical odds against the innocence of vaccines as the actual cause of well over 90% of the disabling and life-threatening chronic conditions suffered by Americans. The true “controls” (calculated to represent 0.26% of the population in 2020) have established the baseline disease risk incurred by those without exposure to vaccination. The null hypothesis, that no significant difference would be found between vaccinated vs. unvaccinated persons in heart disease, diabetes, digestive disorders, eczema, asthma, allergies, developmental disabilities, birth defects, epilepsy, autism, ADHD, cancers, and arthritis, is rejected with overwhelming statistical confidence and power in every single contrast. Because 99.74% of the U.S. population is vaccine-exposed, published national disease rates invariably reflect the frequency of observed negative outcomes arising from exposure to vaccines. The Control Group comparison graphs lead to the inescapable conclusion, and near mathematical certainty, that vaccine exposure is the actual cause of the observed disparity in health outcomes between vaccinated and unvaccinated populations. Vaccines are NOT moving the population toward better health, as suggested by the World Health Organization and the US Department of Health & Human Services, but rather toward epidemic levels of lifelong debilitating chronic disorders.

Crommelynck et al; 2024; “The enhanced national pharmacovigilance system implemented for COVID-19 vaccines in France: A 2-year experience report” (https://www.sciencedirect.com/science/article/pii/S0040595724001999)

Here, we review the significant outcomes from a 2-year collaboration experience between the French National Agency for Medicines and Health Products Safety, the 30 Regional Pharmacovigilance Centres, disease-related experts and the pharmacovigilance and risk assessment committee at the European medicine agency. In France, until January 2023, over 155 million doses of COVID-19 vaccines were administrated, and 190,000 adverse events following immunizations (25% classified as serious) were analysed. Altogether 53 potential safety signals were reported to the Pharmacovigilance and Risk Assessment Committee at the European Medicine Agency by the French National Agency for Medicines and Health Products Safety: 13 were confirmed, 24 are still under investigation and 16 were not confirmed.

Henderson et al; 2024; “Reproducibility of COVID-era infectious disease models” (https://www.sciencedirect.com/science/article/pii/S1755436524000045?via%3Dihub)

Highlights

• 88 out of 100 randomly sampled studies Infectious disease models were not computationally reproducible.
• 67 out of 100 top cited Infectious disease models were not computationally reproducible.
• Journals mandating data release are significantly associated with code release.

Infectious disease modelling has been prominent throughout the COVID-19 pandemic, helping to understand the virus’ transmission dynamics and inform response policies. Given their potential importance and translational impact, we evaluated the computational reproducibility of infectious disease modelling articles from the COVID era. We found that four out of 100 randomly sampled studies released between January 2020 and August 2022 could be completely computationally reproduced using the resources provided (e.g., code, data, instructions) whilst a further eight were partially reproducible. For the 100 most highly cited articles from the same period we found that 11 were completely reproducible with a further 22 partially reproducible. Reflecting on our experience, we discuss common issues affecting computational reproducibility and how these might be addressed.

Makovec et al; 2023; “Analysis of COVID-19 Vaccination Effectiveness” (https://www.walshmedicalmedia.com/open-access/analysis-of-covid19-vaccination-effectiveness-120520.html)

Global research on the COVID-19 vaccination effectiveness is using methods that are misleading the scientific community and public opinion. There is a basic standard in medicine: To measure the effectiveness of an experimental medicine, we need to have two groups. The group that will take medicine and the group that will not take medicine. We follow the health status of both groups for a few months, and will get objective results. This is the only proper methodology to verify the effectiveness of a new medicine. Articles cited from 1-5 did not use the basic standard. They develop different kinds of methodologies that all have no statistical significance. On the basis of their methodologies, they conclude that COVID-19 vaccines have a positive effect on public health. By comparing graphs of the intensity of vaccination and the rate of mortality, we see that after the period of intense vaccination follows the period of higher excess mortality. Basic statistical data are confirming that COVID-19 vaccines increased the mortality rate.

Donzelli et al; 2021; “Comparison of hospitalizations and deaths from COVID-19 2021 versus 2020 in Italy: surprises and implications” (https://f1000research.com/articles/10-964)

Data from the Istituto Superiore di Sanità (ISS) emphasized by the media indicate that COVID-19 vaccination reduces related infections, hospitalizations and deaths.
However, a comparison showed significantly more hospitalizations and intensive care unit accesses in the corresponding months and days in 2021 versus 2020 and no significant differences in deaths.
The combination of non-alternative hypotheses may help explain the discrepancy between the results in the entire population and the vaccination’s success claimed by the ISS in reducing infections, serious cases and deaths:

• a bias: counting as unvaccinated also “those vaccinated with 1 dose in the two weeks following the inoculation”, and as incompletely vaccinated also “those vaccinated with 2 doses within two weeks of the 2nd inoculation”.
• a systematic error: counting as unvaccinated also “vaccinated with 1 dose in the two weeks following the inoculation”, and as incompletely vaccinated also “vaccinated with 2 doses within two weeks of the 2nd inoculation”.

Many reports show an increase in COVID-19 cases in these time-windows, and related data should be separated

• levels of protective effectiveness in vaccinated people, often considered stable, actually show signs of progressive reduction over time, which could contribute to reducing the overall population result
• unvaccinated people show more severe disease than in 2020, supporting also in humans the theory of imperfect vaccines, which offer less resistance to the entry of germs than the resistance later encountered inside the human body. This favors the selection of more resistant and virulent mutants, that can be spread by vaccinated people. This damages first the unvaccinated people, but ultimately the whole community.

An open scientific debate is needed to discuss these hypotheses, following the available evidence (as well as to discuss the inconsistent theory of unvaccinated young people as reservoirs of viruses/mutants), to assess the long-term and community impact of different vaccination strategies.