Note that many of these reference implications for SARS-CoV-2 infection, but the same conclusion applies to injection: the mechanistic studies do not use the virus, they use virus-like-particles (VLPs) with spike protein on the surface. Thus, the findings and conclusions of such studies apply equally well to vaccines that induce spike protein.
VERY IMPORTANT (Study showing variants were a deliberate laboratory evolution) – Unnatural evolutionary processes of SARS-CoV-2 variants and possibility of deliberate natural selection https://zenodo.org/record/8248320
Authors found:
In this study, we aimed to clarify the evolutionary processes leading to the formation of SARS-CoV-2 Omicron variants, focusing on Omicron variants with many amino acid mutations in the spike protein among SARS-CoV-2 isolates. To determine the order in which the mutations leading to the formation of the SARS-CoV-2 Omicron variants, we compared the sequences of 129 Omicron BA.1-related isolates, 141 BA.1.1-related isolates, and 122 BA.2-related isolates, and tried to dissolve the evolutionary processes of the SARS-CoV-2 Omicron variants, including the order of mutations leading to the formation of the SARS-CoV-2 Omicron variants and the occurrence of homologous recombination. As a result, we concluded that the formations of a part of Omicron isolates BA.1, BA.1.1, and BA.2 were not the products of genome evolution as is commonly observed in nature…
In the genetic variation in the S protein in these variants, most of the mutations were non-synonymous (Fig. 1). There were no synonymous mutations in the Alpha, Beta, Gamma, Delta, or Mu variants, but only one each in the Lambda and Omicron variants. Among these variants, the Omicron variant (BA.1 lineage), which shows the greatest accumulation of mutations in the S protein, is primarily non-synonymous in the S protein and has only one synonymous mutation at c25000u. The synonymous/non-synonymous ratio is abnormal, given how human coronaviruses have mutated.
HOW THE COVID-19 mRNA VACCINE IS SUPPOSED TO WORK
Where this video is incorrect is the claim that the LNP’s target ‘dendritic cells’ when in reality there is no applied targeting at all and can transfect ANY cell in the body through the positive to negative ion exchange.
SEE – Distinguishing features of current COVID-19 vaccines: knowns and unknowns of antigen presentation and modes of action https://www.nature.com/articles/s41541-021-00369-6 Detailed that mRNA vaccines are a full length spike with only the changes to the S2. The S1 is identical to the virus.
The two mRNA vaccines in current widespread application (BioNTech-Pfizer and Moderna) (Table 1) are technologically very similar. They contain codon-optimized sequences for efficient expression of the full-length S protein and use the authentic signal sequence for its biosynthesis44,45,46,47 (Fig. 1b). Both constructs include the two stabilizing mutations in S2 (K986P and V987P) that were shown to prevent the conformational change of the pre-fusion into the post-fusion structure of S (section “Introduction” and Fig. 2c)20,21.
VERY IMPORTANT (Morbidities and pathologies based on basic biochemistry of coronaviruses) – Back to the Basics of SARS-CoV-2 Biochemistry: Microvascular Occlusive Glycan Bindings Govern Its Morbidities and Inform Therapeutic Responses https://www.mdpi.com/1999-4915/16/4/647
Consistent with the biochemistry of coronaviruses as well established over decades, SARS-CoV-2 makes its initial attachment to host cells through the binding of its spike protein (SP) to sialylated glycans (containing the monosaccharide sialic acid) on the cell surface. The virus can then slide over and enter via ACE2. SARS-CoV-2 SP attaches particularly tightly to the trillions of red blood cells (RBCs), platelets and endothelial cells in the human body, each cell very densely coated with sialic acid surface molecules but having no ACE2 or minimal ACE2. These interlaced attachments trigger the blood cell aggregation, microvascular occlusion and vascular damage that underlie the hypoxia, blood clotting and related morbidities of severe COVID-19. Notably, the two human betacoronaviruses that express a sialic acid-cleaving enzyme are benign, while the other three—SARS, SARS-CoV-2 and MERS—are virulent. RBC aggregation experimentally induced in several animal species using an injected polysaccharide caused most of the same morbidities of severe COVID-19. This glycan biochemistry is key to disentangling controversies that have arisen over the efficacy of certain generic COVID-19 treatment agents and the safety of SP-based COVID-19 vaccines. More broadly, disregard for the active physiological role of RBCs yields unreliable or erroneous reporting of pharmacokinetic parameters as routinely obtained for most drugs and other bioactive agents using detection in plasma, with whole-blood levels being up to 30-fold higher. Appreciation of the active role of RBCs can elucidate the microvascular underpinnings of other health conditions, including cardiovascular disease, and therapeutic opportunities to address them.
VERY IMPORTANT (Strategies to combat toxicity of LNP modRNA) – Strategies to reduce the risks of mRNA drug and vaccine toxicity https://pubmed.ncbi.nlm.nih.gov/38263456/
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.
Liver and spleen toxicity
Owing to pronounced hepatic and splenic biodistribution of LNP–mRNA, microscopic observations and histopathology of the liver and spleen are standard practice during preclinical development.
Acute drug-induced liver injury is routinely evaluated by measuring blood plasma levels of alanine aminotransferase (ALT), aspartate ami-notransferase (AST) and alkaline phosphatase (APT)98,113. Expectedly, most publicly available studies promoting LNP–mRNA therapeutic applications report only minor pathological findings. In one such study, modified mRNA that encoded human methylmalonyl-CoA mutase (hMUT) was synthesized to treat a hypomorphic mouse model of meth-ylmalonic acidaemia/aciduria (MMA, an ultra-rare metabolic disorder). At the highest intravenously administered dose of LNP–mRNA formulation, there were no clinical chemistry findings, but 80% of mice
presented with a mild decrease in lymphocytes next to the central splenic arteries 113. This effect was attributed to the LNPs and not the hMUT mRNA or its expression, as it was also observed in LNPs carry-
ing mRNA for enhanced green fluorescent protein (EGFP). In another study, modified mRNA encoding human arginase was used in a mouse model of arginase deficiency114. Although there were no biochemical or histopathological findings, electron microscopy on liver sections from the group that received a single control formulation of firefly luciferase mRNA presented with sub-micrometre-sized lipid droplets. Finally, a single intramuscular injection of LNP–mRNA formulation encoding the influenza haemagglutinin H3 antigen showed increased AST, ALT and C-reactive protein levels in rabbits 115
. Liver histopathological findings included focal subscapular vacuolation, inflammatory cell and erythrocyte infiltration, and increased cellularity (lymphocyte expansion) was observed in the germinal centres of the spleen. In summary, hepatic or splenic pathogenicity can arise after intravenous as
well as intramuscular administration of LNP–mRNA, and hepatocyte uptake of the lipid component seems capable of disturbing fatty acid and lipid management.
VERY IMPORTANT (Narrative regarding pathogenicity of the spike) – Understanding the Pharmacology of COVID-19 mRNA Vaccines: Playing Dice with the Spike? https://pubmed.ncbi.nlm.nih.gov/36142792/
Coronavirus disease-19 (COVID-19) mRNA vaccines are the mainstays of mass vaccination campaigns in most Western countries. However, the emergency conditions in which their development took place made it impossible to fully characterize their effects and mechanism of action. Here, we summarize and discuss available evidence indicating that COVID-19 mRNA vaccines better reflect pharmaceutical drugs than conventional vaccines, as they do not contain antigens but an active SARS-CoV-2 S protein mRNA, representing at the same time an active principle and a prodrug, which upon intracellular translation results in the endogenous production of the SARS-CoV-2 S protein. Both vaccine-derived SARS-CoV-2 S protein mRNA and the resulting S protein exhibit a complex pharmacology and undergo systemic disposition. Defining COVID-19 mRNA vaccines as pharmaceutical drugs has straightforward implications for their pharmacodynamic, pharmacokinetic, clinical and post-marketing safety assessment. Only an accurate characterization of COVID-19 mRNA vaccines as pharmaceutical drugs will guarantee a safe, rational and individualized use of these products.
Adverse Effects Following COVID-19 Vaccination: Too Much S Protein, for Too Long and/or in the WRONG Place?
A comprehensive review of the literature recently discussed the role of COVID-19-mRNA-vaccine-induced S protein in adverse effects following vaccination [14], and we showed that production of S protein induced by COVID-19 mRNA vaccines may well compare to the estimated production during SARS-CoV-2 infection [15]. The present opinion paper identifies, develops and discusses the implications of the role of the S protein in adverse effects following vaccination and indicates the most appropriate pharmacological approaches for a better characterization of these vaccines, with the aim of providing guidance towards their rational and individualized use. Indeed, based on these premises, a major explanation of adverse effects following COVID-19 vaccination could well be that mRNA vaccines induce in selected individuals excessive production of S protein, for too long and/or in inappropriate tissues and organs, and this occurrence is at present unpredictable, since systemic biodistribution and disposition of the COVID-19 mRNA vaccine has so far never been considered an issue, and as a consequence it has never been studied as it would have actually deserved. Remarkably, the inadequate understanding of how to target specific organs and cells for protein expression is well-acknowledged as one of the major limitations of mRNA gene therapy [16]; however, for mRNA vaccines, it has been so far ignored.
Pharmacology of the SARS-CoV-2 S Protein mRNA
The SARS-CoV-2 S protein mRNA contained in COVID-19 mRNA vaccines has a complex pharmacology. The topic has recently been the subject of an excellent review that focused on the possibility that mRNA vaccines eventually alter the genomes of human cells through retroposition and integration [21]. The author concludes by asking for experiments that specifically address the issue of genome integration safety [21]. Indeed, integration of DNA copies of SARS-CoV-2 sequences into the genome of infected human cells has been recently described, and chimeric transcripts were detected in patient-derived tissues [22], a finding which raised significant controversy and debate [23,24,25]. Nonetheless, recently, a case report was published showing the persistence of residual SARS-CoV-2 RNA and antigens in the appendix, skin and breast tissues of two patients who exhibited long COVID-19 symptoms 163 and 426 days after symptom onset [26]. Remarkably, intracellular reverse transcription of the BioNTech–Pfizer COVID-19 mRNA vaccine has been shown in vitro in the human liver cell line Huh7 [27], although its relevance still awaits to be assessed in in vivo models [28]. Nevertheless, the issue deserves careful consideration in view, for example, of the presence of vaccine mRNA and vaccine-induced S protein in axillary lymph nodes up to 60 days after both mRNA-1273 or BNT162b2 COVID-19 vaccines [9] as well as the occurrence of the S protein in circulating monocytes of vaccinated subjects with PASC-like symptoms many months after vaccination [11].
The SARS-CoV-2 S Protein Alone Directly Affects Human Cells
The SARS-CoV-2 S protein exerts direct effects on human cells even in the absence of other viral components [48]. For example, treatment of human pulmonary artery smooth muscle cells or endothelial cells with 10 ng/mL (0.13 nM) SARS-CoV-2 S protein S1 subunit activates cell growth signaling, an effect which is consistent with the pulmonary vascular wall thickening of patients who died of COVID-19 [49]. Another study showed that transient transfection of the human lung alveolar epithelial cell line A549 or of the human liver epithelial cell line Huh7.5 with SARS-CoV-2 S protein results in increased activation of proinflammatory NF-κB and AP-1 transcription factors, and of p38 and ERK mitogen-activated protein kinases, finally resulting in increased release of interleukin (IL)-6, through downregulation of ACE2 protein expression and subsequently activation of angiotensin II type 1 receptors [50]. Cell lines transiently transfected with the SARS-CoV-2 S protein may well reflect the consequences of vaccine-induced endogenous production of the S protein, while in vitro experiments with the SARS-CoV-2 S protein may predict the effects of the free-floating protein in plasma and extracellular fluids.
In summary, both the SARS-CoV-2 S protein mRNA and the S protein itself exhibit a complex pharmacological profile with potential toxicological issues. None of these issues, however, were taken into consideration in the studies that led to the marketing authorization [1,2], precisely because, first of all, from a regulatory point of view, these products were treated as conventional vaccines.
VERY IMPORTANT (Strategies to deactivate the action of the modRNA shots) – Hulscher, McCullough and Marotta – Strategic deactivation of mRNA COVID-19 vaccines: New applications for siRNA therapy and RIBOTACs https://onlinelibrary.wiley.com/doi/10.1002/jgm.3733
We highlight emerging concerns regarding the wide systemic biodistribution of these mRNA vaccines leading to prolonged inflammatory responses and other safety concerns. The regulatory framework guiding the biodistribution studies is pivotal in assessing the safety profiles of new mRNA formulations in use today. The stability of mRNA vaccines, their pervasive distribution, and the longevity of the encapsulated mRNA along with unlimited production of the damaging and potentially lethal spike (S) protein call for strategies to mitigate potential adverse effects. Here, we explore the potential of small interfering RNA (siRNA) and ribonuclease targeting chimeras (RIBOTACs) as promising solutions to target, inactivate, and degrade residual and persistent vaccine mRNA, thereby potentially preventing uncontrolled S protein production and reducing toxicity. The targeted nature of siRNA and RIBOTACs allows for precise intervention, offering a path to prevent and mitigate adverse events of mRNA-based therapies. This review calls for further research into siRNA and RIBOTAC applications as antidotes and detoxication products for mRNA vaccine technology.
VERY IMPORTANT (Pfizer study showing 1.4 trillion LNP per shot) – New insights into the structure of Comirnaty Covid-19 vaccine: A theory on soft nanoparticles with mRNA-lipid supercoils stabilized by hydrogen bonds https://www.biorxiv.org/content/10.1101/2022.12.02.518611v1.full.pdf
IMPORTANT (Batches tested by Dutch scientists show differing adverse events – Schmeling et al) – Batch-dependent safety of the BNT162b2 mRNA COVID-19 vaccine https://onlinelibrary.wiley.com/doi/10.1111/eci.13998
The total number of SAEs associated with each batch was divided by the number of doses in the batch to obtain the rate of SAEs per 1000 doses. Since the observed relationship between the numbers of SAEs and BNT162b2 vaccine doses was highly heterogeneous, conventional regression statistics were not considered to be applicable. Therefore, heterogeneity in the relationship between the numbers of SAEs and doses per vaccine batch was assessed by log-transformation followed by non-hierarchical cluster analysis and general linear model (GLM) test for differences in SAE rates between batches. Reporting of the study conforms to broad EQUATOR guidelines.3
The observed variation in SAE rates and seriousness between BTN162b2 vaccine batches in this nationwide study was contrary to the expected homogenous rate and distribution of SAEs between batches. In Denmark and other EU/EEA countries, vaccine quality is monitored according to Official Control Authority Batch Release (OCABR) guidelines and to our knowledge, potential differences in BNT162b2 vaccine batch clinical safety or effectiveness have not been reported previously, for example in pre-authorization trials and subsequent population-based studies.4, 5 Such effects may be easier to detect in small countries like Denmark where BNT162b2 vaccines during the study period were generally provided in several smaller batches. Also, regulatory monitoring and scientific interest in COVID-19 vaccine safety have primarily focused on serious adverse events, for example myocarditis.6 In any case, identification of such effects evidently requires that observed adverse events are linked with the respective individual batch labels and sizes (dose numbers). Previously, variation in the production (culture growth) of the Bacille Calmette-Guérin vaccine has been shown to influence important immunological effects of this vaccine,7 and two cases of myocarditis have been reported in two young males after receiving mRNA-1273 COVID-19 vaccine (Moderna) from the same vaccine batch on the same day.8 Indeed, variations (batch-to-batch, vial-to-vial and even dose-to-dose) in vaccines may occur as a result of variabilities and practice breaches in, for example vaccine manufacturing, storage, transportation, clinical handling and control aspects, and in 2021, three lots of the mRNA1273 vaccine totalling more than 1.6 million doses were recalled in Japan after 39 vials of the vaccine were found to contain foreign materials.9 Leaked and contested data have also suggested that some early commercial batches of the BNT162b2 vaccine contained lower than expected levels of intact mRNA.10
VERY IMPORTANT (Literature review and fully referenced, spike from vaccine or virus dangerous) – ‘Spikeopathy’: COVID-19 Spike Protein Is Pathogenic, from Both Virus and Vaccine mRNA https://www.mdpi.com/2227-9059/11/8/2287 Published 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.
VERY IMPORTANT (Truncated mRNA in vax analysis of off-target antigen, so broken mRNA can’t be translated and present no danger) – 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
IMORTANT (Pseudouridine shifts the reading of the modRNA by ribosomes and can cause a misreading of the protein and hence create an immune response to something not representative of the antigen – 1 in 3 people who took Pfizer exhibited this effect according to the BBC) – 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).
Follow up to the above – 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.
IMPORTANT (Frame shifting evidenced) – 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.
IMPORTANT (Fibril formation from spike) – SARS-CoV-2 Spike amyloid fibrils specifically and selectively accelerates amyloid fibril formation of human prion protein and the amyloid β peptide https://www.biorxiv.org/content/10.1101/2023.09.01.555834v1
An increasing number of reports suggest an association between COVID-19 infection and initiation or acceleration of neurodegenerative diseases including Alzheimer’s disease (AD) and Creutzfeldt-Jakob disease (CJD). Both these diseases and several other neurodegenerative diseases are caused by conversion of human proteins into a misfolded, aggregated amyloid fibril state. The fibril formation process is self-perpetuating by seeded conversion from preformed fibril seeds. We recently described a plausible mechanism for amyloid fibril formation of SARS-CoV-2 spike protein. Spike-protein formed amyloid fibrils upon cleavage by neutrophil elastase, abundant in the inflammatory response to COVID-19 infection. We here provide evidence of significant Spike-amyloid fibril seeded acceleration of amyloid formation of CJD associated human prion protein (HuPrP) using an in vitro conversion assay. By seeding the HuPrP conversion assay with other in vitro generated disease associated amyloid fibrils we demonstrate that this is not a general effect but a specific feature of spike-amyloid fibrils. We also showed that the amyloid fibril formation of AD associated Aβ1-42 was accelerated by Spike-amyloid fibril seeds. Of seven different 20-amino acid long peptides, Spike532 (532NLVKNKCVNFNFNGLTGTGV551) was most efficient in seeding HuPrP and Spike601 (601GTNTSNQVAVLYQDVNCTEV620) was most effective in seeding Aβ1-42, suggesting substrate dependent selectivity of the cross-seeding activity. Albeit purely in vitro, our data suggest that cross-seeding by Spike-amyloid fibrils can be implicated in the increasing number of reports of CJD, AD, and possibly other neurodegenerative diseases in the wake of COVID-19.
VERY IMPORTANT (LNP technology resulting in myocarditis, not the spike, so all mRNA tech against any antigen will produce more cases of vaccine-induced myocarditis) – Cytokinopathy with aberrant cytotoxic lymphocytes and profibrotic myeloid response in SARS-CoV-2 mRNA vaccine–associated myocarditis https://www.science.org/doi/10.1126/sciimmunol.adh3455
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.
IMPORTANT (LNP adjuvants causing cardiac issues) – Adjuvants in COVID-19 vaccines: innocent bystanders or culpable abettors for stirring up COVID-heart syndrome https://journals.sagepub.com/doi/10.1177/25151355241228439
COVID-19 infection is a multi-system clinical disorder that was associated with increased morbidity and mortality. Even though antiviral therapies such as Remdesvir offered modest efficacy in reducing the mortality and morbidity, they were not efficacious in reducing the risk of future infections. So, FDA approved COVID-19 vaccines which are widely administered in the general population worldwide. These COVID-19 vaccines offered a safety net against future infections and re-infections. Most of these vaccines contain inactivated virus or spike protein mRNA that are primarily responsible for inducing innate and adaptive immunity. These vaccines were also formulated to contain supplementary adjuvants that are beneficial in boosting the immune response. During the pandemic, clinicians all over the world witnessed an uprise in the incidence and prevalence of cardiovascular diseases (COVID-Heart Syndrome) in patients with and without cardiovascular risk factors. Clinical researchers were not certain about the underlying reason for the upsurge of cardiovascular disorders with some blaming them on COVID-19 infections while others blaming them on COVID-19 vaccines. Based on the literature review, we hypothesize that adjuvants included in the COVID-19 vaccines are the real culprits for causation of cardiovascular disorders. Operation of various pathological signaling events under the influence of these adjuvants including autoimmunity, bystander effect, direct toxicity, anti-phospholipid syndrome (APS), anaphylaxis, hypersensitivity, genetic susceptibility, epitope spreading, and anti-idiotypic antibodies were partially responsible for stirring up the onset of cardiovascular disorders. With these mechanisms in place, a minor contribution from COVID-19 virus itself cannot be ruled out. With that being said, we strongly advocate for careful selection of vaccine adjuvants included in COVID-19 vaccines so that future adverse cardiac disorders can be averted.
IMPORTANT HISTORICAL LNP (Oxidative stress caused by nanocarriers) – Nanoparticles Induced Oxidative Damage in Reproductive System and Role of Antioxidants on the Induced Toxicity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10059981/
Impact on the Male Reproductive System
Both oxidative stress and inflammation are believed to be sensitive to the male reproductive system; where nanoparticles’ exposure readily increases oxidative stress and causes cell death and poor spermatogenesis, oxidative stress is reported to be the primary cause of 30–80% of infertility problems in male [39,40]. Studies suggest that nanoparticles react to distinct germ cells and damage cells differently. The changes of the cytoskeleton on the entry of nanoparticles can affect the production of sperm flagella and their ability to migrate, as well as the formation of Sertoli cell tight junctions and the tight junctions between them; all these changes can affect spermatogenesis [41]. Liu et al. [42] have proposed that ZnO nanoparticles caused down-regulation of tight junction proteins in Sertoli cells resulting in BTB dysfunction and no changes in cytoskeleton dynamics were identified [42]. The various nanoparticles involved in the toxicity of the male reproductive system are tabulated in Table 1.
The buildup of nanoparticles destroys germ cells, Leydig cells, and Sertoli cells, impairing the motility, shape, quality, and quantity of the sperm and limiting the number of mature oocytes or preventing the growth of primary and secondary follicles. Further, nanoparticles can alter the number of hormones released, altering sexual behavior [34]. In a study, Mathias et al. [43] found there were no differences in any of the sexual activities in silver nanoparticles on the following sexual behaviors like number of mounts, intromissions, ejaculatory intervals, attempted mounts, and ejaculations, but the exposure to nanoparticles was reducing sperm quality. Nanoparticles such as silica nanoparticles were found to increase the levels of micronucleus frequencies, malondialdehyde levels, and lower activity of catalase and glutathione content in testicular tissues at a higher concentration treated group, pointing to mechanisms of DNA damage and oxidative stress. Significant testicular histological changes were also seen in this group along with inflammation, testicular apoptosis, and oxidative stress by enhancing the gene expression corresponding to the pro-inflammatory activity, apoptotic activity, and oxidative stress caspase 3 and iNOS [44].
Spermatogenesis and Toxicity
According to some of the research, nanoparticles are found to have the ability to quickly pass the BTB, after building up in the testis and having a negative impact on spermatogenesis. ROS generation in the seminiferous tubule, the place where spermatogenesis takes place, can cause DNA damage to spermatogenic cells [59]. While the testicular tissues are exposed to nanoparticles, seminiferous tubules undergo histological changes, which damage the testicles and diminish sperm production [40] (Figure 3).
IMPORTANT HISTORICAL LNP (Nanocarriers toxicity) – Nanoparticles-induced potential toxicity on human health: Applications, toxicity mechanisms, and evaluation models https://pubmed.ncbi.nlm.nih.gov/37457660/
Nanoparticles (NPs) have become one of the most popular objects of scientific study during the past decades. However, despite wealth of study reports, still there is a gap, particularly in health toxicology studies, underlying mechanisms, and related evaluation models to deeply understanding the NPs risk effects. In this review, we first present a comprehensive landscape of the applications of NPs on health, especially addressing the role of NPs in medical diagnosis, therapy. Then, the toxicity of NPs on health systems is introduced. We describe in detail the effects of NPs on various systems, including respiratory, nervous, endocrine, immune, and reproductive systems, and the carcinogenicity of NPs. Furthermore, we unravels the underlying mechanisms of NPs including ROS accumulation, mitochondrial damage, inflammatory reaction, apoptosis, DNA damage, cell cycle, and epigenetic regulation. In addition, the classical study models such as cell lines and mice and the emerging models such as 3D organoids used for evaluating the toxicity or scientific study are both introduced. Overall, this review presents a critical summary and evaluation of the state of understanding of NPs, giving readers more better understanding of the NPs toxicology to remedy key gaps in knowledge and techniques.
IMPORTANT (Spike alone causes body to produce more IL interleukin) – SARS-CoV-2 spike protein induces the cytokine release syndrome by stimulating T cells to produce more IL-2 https://www.biorxiv.org/content/10.1101/2023.11.01.565098v1?publication=on
Cytokine release syndrome (CRS) is one of the leading causes of mortality in COVID-19 patients caused by the SARS-CoV-2 coronavirus. However, the mechanism of CRS induced by SARS-CoV-2 is vague. This study shows that dendritic cells loaded with spike protein of SARS-CoV-2 stimulate T cells to release much more IL-2, which subsequently cooperates with spike protein to facilitate peripheral blood mononuclear cells to release IL-1β, IL-6, and IL-8. These effects are achieved via IL-2 stimulation of NK cells to release TNF-α and IFN-γ, as well as T cells to release IFN-γ. Mechanistically, IFN-γ and TNF-α enhance the transcription of CD40, and the interaction of CD40 and its ligand stabilizes the membrane expression of TLR4 which serves as a receptor of spike protein on the surface of monocytes. As a result, there is a constant interaction between spike protein and TLR4, leading to continuous activation of NF-κB. Furthermore, TNF-α also activates NF-κB signaling in monocytes, which further cooperates with IFN-γ and spike protein to modulate NF-κB-dependent transcription of CRS-related inflammatory cytokines.
IMPORTANT (LNP mechanism possibly causative of low level cytokines resulting in inflammatory adverse events) – mRNA-LNP COVID-19 vaccine lipids induce low level complement activation and production of proinflammatory cytokines: Mechanisms, effects of complement inhibitors, and relevance to adverse reactions https://www.biorxiv.org/content/10.1101/2024.01.12.575122v1
In serum, Comirnaty and the control PEGylated liposome (Doxebo) caused different rises of C split products, C5a, sC5b-9, Bb and C4d, indicating stimulation of the classical pathway of C activation mainly by the liposomes, while a stronger stimulation of the alternative pathway was equal with the vaccine and the liposomes. Spikevax had similar C activation as Comirnaty, but viral or synthetic mRNAs had no such effect. In autologous serum-supplemented peripheral blood mononuclear cell (PBMC) cultures, Comirnaty caused increases in the levels of sC5b-9 and proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1β < TNF-α < IL-6 < IL-8, whereas heatinactivation of serum prevented the rises of IL-1α, IL-1β, and TNF-α. Clinical C inhibitors, Soliris and Berinert, suppressed vaccine-induced C activation in serum but did not affect cytokine production when applied individually. These findings suggest that the PEGylated lipid coating of mRNA-LNP nanoparticles can trigger C activation mainly via the alternative pathway, which may be causally related to the induction of some, but not all inflammatory cytokines. While innate immune stimulation is essential for the vaccine’s efficacy, concurrent production of C- and PBMC-derived inflammatory mediators may contribute to some of the AEs
IMPORTANT (mRNA and LNP details) German Translation – mRNA vaccination: Incomprehensible secrecy continues https://www.infosperber.ch/gesundheit/mrna-impfung-unbegreifliche-geheimnistuerei-geht-weiter/
IMPORTANT (Role of APC and biodistribution) – Role of the antigen presentation process in the immunization mechanism of the genetic vaccines against COVID‐19 and the need for biodistribution evaluations https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9135042/
Authors conclude: In conclusion, it is essential to underline that every human cell that intakes the LNPs and translates the viral protein (in case of the mRNA vaccines), or that gets infected by the adenovirus and expresses and translates the viral protein (in case of the adenovirus‐based vaccines), is inevitably recognized as a threat by the immune system and killed (Figure 1). There are no exceptions to this mechanism. The severity of the resulting damage and the consequences for health depend on the quantity of the cells involved, on the type of tissue and on the strength of the following autoimmune reaction. For instance, if the mRNA contained in the LNPs would get internalized by cardiac myocytes, and such cells would produce the spike protein, the resulting inflammation would likely lead to the necrosis of the myocardium, with an extent proportional to the number of involved cells. Therefore, it is fundamental to perform pharmacokinetic evaluations in humans, in order to determine the exact biodistribution of the vaccines against COVID‐19, and thus to identify the possible tissues at threat.
See the below research into the spike protein and the use of mRNA lipid nanoparticles as a delivery system:
IMPORTANT HISTORICAL 2021 STUDY (LNP crossing the BBB) Published 2012- Chapter twelve – Lipid Nanoparticles for Drug Targeting to the Brain https://www.sciencedirect.com/science/article/abs/pii/B9780123918604000124
IMPORTANT HISTORIC 2006 STUDY (Cationic lipids in the LNPs are toxic) – Toxicity of cationic lipids and cationic polymers in gene delivery https://www.sciencedirect.com/science/article/abs/pii/S0168365906002045?via%3Dihub PDF Download https://sci-hub.se/downloads/2019-01-07/0b/lv2006.pdf
Authors stated:
2.1. Lipid structure vs. toxicity
Cationic lipids used for gene therapy are composed of three basic domains: a positive charged headgroup, a hydrophobic chain, and a linker which joins the polar and non-polar regions [18]. Fig. 1 displays the three basic domains of 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP). The polar and hydrophobic domains of cationic lipids may have dramatic effects on both transfection and toxicity levels.
IMPORTANT HISTORIC 2012 STUDY – Accumulation of nanocarriers in the ovary: A neglected toxicity risk? https://www.sciencedirect.com/science/article/abs/pii/S0168365912000892?via%3Dihub
Authors found: 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.
IMPORTANT HISTORICAL mRNA RABIES VACCINE (Begun in 2013, published in 2017) – Safety and immunogenicity of a mRNA rabies vaccine in healthy adults: an open-label, non-randomised, prospective, first-in-human phase 1 clinical trial
ClinicalTrials.gov link – https://clinicaltrials.gov/ct2/show/NCT02241135?id=NCT02241135&draw=2&rank=1
Authors found:
Findings
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.
IMPORTANT HISTORIC 2018 STUDY – Potential adverse effects of nanoparticles on the reproductive system https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294055/
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.
VERY IMPORTANT HISTORIC 2017 STUDY (Biodistribution study of mRNA vaccines against influenza) – Preclinical and Clinical Demonstration of Immunogenicity by mRNA Vaccines against H10N8 and H7N9 Influenza Viruses https://www.sciencedirect.com/science/article/pii/S1525001617301569
Authors found:
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 (Cmax) of the injection site muscle was 5,680 ng/mL, and the level declined with an estimated t1/2 of 18.8 hr (Table 1). Proximal lymph nodes had the second highest concentration at 2,120 ng/mL (tmax of 8 hr with a relatively long t1/2 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 Cmax of 86.9 ng/mL (area under the curve [AUC]0–264 of 2,270 ng.hr/mL) and 47.2 ng/mL (AUC0–264 of 276 ng.hr/mL), respectively. In the remaining tissues and plasma, H10 mRNA was found at 100- to 1,000-fold lower levels.
VERY IMPORTANT (Biodistribution of LNP based on particle size) – 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.
VERY IMPORTANT (Showing how LNP’s can be used to deliver genes into nucleus) – mRNA-loaded lipid nanoparticles reprogram cells and edit genes https://cen.acs.org/pharmaceuticals/gene-therapy/mRNA-loaded-lipid-nanoparticles-reprogram-cells/101/web/2023/08
A team of researchers have used lipid nanoparticles loaded with mRNA—the starring technology in some COVID-19 vaccines—to noninvasively and selectively trigger cell death in living mice’s blood stem cells. And in a second experiment, they used the nanoparticle system to remove a sickle cell–producing gene in human cells (Science 2023; DOI: 10.1126/science.ade6967).
LINKED TO ABOVE – High spontaneous integration rates of end-modified linear DNAs upon mammalian cell transfection https://www.nature.com/articles/s41598-023-33862-0
ALSO LINKED TO ABOVE – Cytoplasmic transport and nuclear import of plasmid DNA https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705778/
VERY IMPORTANT (DNA Plasmid SV40 Promotor contamination in vials) – 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/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.
VERY IMPORTANT (Related to above) – SV40 As an Effective Gene Transfer Vector in Vivo* https://www.jbc.org/article/S0021-9258(18)40066-X/fulltext
SV40 was used to transduce gene expression in vitro and in vivo. Using cloned SV40 genome, we replaced large T antigen gene (Tag) with a polylinker, and inserted firefly luciferase, controlled by SV40 early promoter. Transfection into Tag-expressing cells yielded Tag-deficient virus, SVluc. SVluc was Tag-deficient and therefore replication-deficient in cells that did not supply Tag. SVluc transduced functional luciferase expression in vitro. BALB/c mice were inoculated with SVluc, and their tissues were assayed 3-21 days post-inoculation (dpi) for luciferase protein production and enzyme activity. Luciferase protein was detected by immunohistochemistry throughout the experiment, from 3 to 21 dpi. There was no inflammatory reaction against SVluc-infected cells at any time, in any tissue studied. Luciferase activity was first detected by luminometry 14 dpi, and remained level through day 21. Thus, replication-deficient recombinant SV40 can mediate gene transfer in vitro and in vivo.
VERY IMPORTANT (Related to above) – 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.
VERY IMPORTANT (Related to above – 7% of plasmid DNA enters the nucleus) – 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.
VERY IMPORTANT (P53 elevates and inhibits SV40 and S protein binds to P53) – p53 elevation in human cells halt SV40 infection by inhibiting T-ag expression https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288138/
We found that p53 is activated early after SV40 infection and defends the host against the infection. Using live cell imaging and single cell analyses we found that p53 dynamics are variable among individual cells, with only a subset of cells activating p53 immediately after SV40 infection. This cell-to-cell variabilty had clear consequences on the outcome of the infection. None of the cells with elevated p53 at the beginning of the infection proceeded to express T-ag, suggesting a p53-dependent decision between abortive and productive infection. In addition, we show that artificial elevation of p53 levels prior to the infection reduces infection efficiency, supporting a role for p53 in defending against SV40. We further found that the p53-mediated host defense mechanism against SV40 is not facilitated by apoptosis nor via interferon-stimulated genes. Instead p53 binds to the viral DNA at the T-ag promoter region, prevents its transcriptional activation by Sp1, and halts the progress of the infection.
VERY IMPORTANT (P53 inhibited – Cancer suppression pathway) – 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.
RELATED TO ABOVE – Tumor Suppressor p53: Biology, Signaling Pathways, and Therapeutic Targeting https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8730328/
TP53 is the most commonly mutated gene in human cancer with over 100,000 literature citations in PubMed. This is a heavily studied pathway in cancer biology and oncology with a history that dates back to 1979 when p53 was discovered. The p53 pathway is a complex cellular stress response network with multiple diverse inputs and downstream outputs relevant to its role as a tumor suppressor pathway. While inroads have been made in understanding the biology and signaling in the p53 pathway, the p53 family, transcriptional readouts, and effects of an array of mutants, the pathway remains challenging in the realm of clinical translation. While the role of mutant p53 as a prognostic factor is recognized, the therapeutic modulation of its wild-type or mutant activities remain a work-in-progress. This review covers current knowledge about the biology, signaling mechanisms in the p53 pathway and summarizes advances in therapeutic development.
A.Tumor Suppressor TP53: “Guardian of the Genome”
TP53 is a gene that encodes for the p53 tumor suppressor protein, commonly referred to as the “Guardian of the Genome” [1]. Its main biological function appears to involve the protection of the DNA integrity of the cell. TP53 plays additional roles in development, aging and cell differentiation [2]. For example, p53-nullizygous genetic models exhibit phenotypes related to aging, pluripotency and development characterized by early aging onset, induction of cell pluripotency and inability of embryos undergo gastrulation, respectively [3–5]. The p53 protein is a transcription factor that controls the output of many biological processes according to the type of cellular stress signal input. Stress signals known to activate p53 include oncogene activation, DNA damage, and replication stress [6]. In response to these stresses, p53 undergoes post-translational modifications, promotes the transcription of genes involved in specific cell responses according to the stress type thereby controlling the cell’s fate [7]. Vastly studied biological processes where p53 has been shown to play a role are cell cycle arrest, senescence, DNA repair, and apoptosis (Figure 1). Over the last few years, researchers have elucidated many other pathways that p53 participates in such as autophagy, cell metabolism, ferroptosis, and pathways that involve the generation of reactive oxygen species. In some of these pathways, p53 does not execute signals directly as a transcription factor, but rather from its interaction with other proteins. For example, in apoptosis, activation of this pathway can occur via the interaction of p53 with anti-apoptotic proteins located in the mitochondria.
VERY IMPORTANT (EMA DNA Plasmid genome integration) – Guideline on plasmid DNA vaccines for veterinary use https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-plasmid-dna-vaccines-veterinary-use_en.pdf
The plasmid DNA which is internalised by the cells of the vaccinated animal may integrate into the
chromosomes of the vaccinated animal and disrupt the normal replicative state of that cell, causing
uncontrolled cell division and oncogenesis:
After the injection of DNA into an animal, a small proportion of the DNA molecules enters cells. The
probability of any DNA molecule integrating into the chromosome is low and given that
oncogenesis is a multi-factorial event, the risk of insertional mutagenesis is exceedingly low.
Integration studies, where relevant, should be undertaken with the finished product and the
percentage of supercoiled plasmid used should be stated. So far, the integration of plasmid DNA
into chromosomal DNA of a vaccinated animal has not been observed (EFSA, EFSA Journal 2017).
However, integration (e.g. into the muscle cells surrounding the vaccination site or into germ line
cells in the gonads) cannot be discounted1.
The current testing methods are not sufficiently sensitive to routinely detect actual integration that
may be orders of magnitude below the limits of detection of the methods. Therefore, each product
should be assessed on a case-by-case basis, taking into consideration the specific limits of
detection, the route of administration, the target tissue, the amount of plasmid administered, and
the age of the vaccinated animal. The information should be compiled in a risk assessment.
VERY IMPORTANT (Lipid nanoparticles linked to adverse events causation) – mRNA-LNP COVID-19 Vaccine Lipids Induce Complement Activation and Production of Proinflammatory Cytokines: Mechanisms, Effects of Complement Inhibitors, and Relevance to Adverse Reactions https://www.mdpi.com/1422-0067/25/7/3595
Incubation of Pfizer-BioNTech’s Comirnaty and Moderna’s Spikevax with 75% human serum led to significant increases in C5a, sC5b-9, and Bb but not C4d, indicating C activation mainly via the alternative pathway. Control PEGylated liposomes (Doxebo) also induced C activation, but, on a weight basis, it was ~5 times less effective than that of Comirnaty. Viral or synthetic naked mRNAs had no C-activating effects. In peripheral blood mononuclear cell (PBMC) cultures supplemented with 20% autologous serum, besides C activation, Comirnaty induced the secretion of proinflammatory cytokines in the following order: IL-1α < IFN-γ < IL-1β < TNF-α < IL-6 < IL-8. Heat-inactivation of C in serum prevented a rise in IL-1α, IL-1β, and TNF-α, suggesting C-dependence of these cytokines’ induction, although the C5 blocker Soliris and C1 inhibitor Berinert, which effectively inhibited C activation in both systems, did not suppress the release of any cytokines. These findings suggest that the inflammatory AEs of mRNA-LNP vaccines are due, at least in part, to stimulation of both arms of the innate immune system, whereupon C activation may be causally involved in the induction of some, but not all, inflammatory cytokines.
VERY IMPORTANT (Biodistribution study from Pfizer (Wistar Rat) showing accumulation in distant organs) – PHARMACOKINETICS: ORGAN DISTRIBUTION CONTINUED – Page 7 https://phmpt.org/wp-content/uploads/2022/03/125742_S1_M2_26_pharmkin-tabulated-summary.pdf Biodistribution – https://phmpt.org/wp-content/uploads/2022/03/125742_S1_M4_4223_185350.pdf
VERY IMPORTANT (Further information on Pfizer vaccine on Wistar-Han rat IM injection regarding fertility) – A Combined Fertility and Developmental Study (Including Teratogenicity and Postnatal Investigations) of BNT162b1, BNT162b2 and BNT162b3 by Intramuscular Administration in the Wistar Rat https://pdata0916.s3.us-east-2.amazonaws.com/pdocs/110122/125742_S1_M4_20256434.pdf
Abstract & Conclusion
The objective of the study was to assess potential effects of the vaccine candidates and the immune response, on fertility and pre and postnatal development in the female Wistar rat.
The vaccines were administered intramuscularly to Female Wistar rates 21 and 14 days before mating and then on gestation day 9 and 20 (4 doses in total). The control group had a saline solution on the same days and under the same conditions. Each group had 44 females, 22 which were assigned to the caesarean subgroup and 22 to the littering subgroup.
After mating with unvaccinated males (so we have no idea if there are any fertility issues with males), 22 rats underwent caesarean section on day 21, whilst the rest were allowed to litter and offspring observed up to weaning on postnatal day 21.
Main report
Unscheduled deaths
Although the report states that there were no deaths throughout the study related to any of the 3 vaccine candidates, later in the text it says there were three unscheduled deaths in the female rates.
A moribund female showing signs of parturition difficulties and 2 females with total litter death were euthanized by carbon dioxide inhalation and exsanguination.
Mortality
Further down the report it contradicts itself again and says there was no unscheduled death related to any of the 3 vaccine candidates.
However it notes that one female from the BNT162b3 group had parturition difficulties and was euthanised for ethical reasons.
The female delivered 8 pups. On LD0, distended/blue abdomen was noted. On LD1, hunched posture, pale, marked piloerection, bleeding at the vulva/red vaginal discharge, distended/purple abdomen were noted and 4 of the delivered pups were missing/dead/cannibalized. At necropsy, the female had 13 implantations, 3 fetuses were retained in the uterus. No macroscopic findings were noted.
The authors say that such cases of parturition difficulties are present in historical control data (a study in 2016) for this strain of rat so these finding were considered to be incidental.
One female in the BNT162b1 group incurred total litter death of 15 pups (9 stillborn, 3 cannibalised, 1 dead and 2 missing pups). Another female from the BNT162b3 group delivered 8 stillborn pups.
Again, the authors say total litter death at or shortly after birth is present in historical data (two studies out of 18 between 2015 and 2019) so they conclude the deaths were incidental and not related to the vaccines.
Clinical Observations
Apparently there were no adverse clinical signs because again they were considered incidental.
Other clinical signs such as abnormal vocalization, chromodacryorrhea, desquamation, erythema, localized hairloss, malocclusion, long or missing teeth, red vaginal discharge, red stained fur, scab(s), sore(s) noted sporadically across the groups were considered to be incidental, related to the method of dose administration or to the pregnancy status of the females.
Body Weight
Mean body weight gain was lower in the BNT162b1 and BNT162b3 groups (26 g and 30 g), compared with the control group (33 g) throughout the lactation phase. This was not considered vaccine-related, but due to an atypical high value in the control group compared with the historical control data range (from 10.9 g to 32.6 g).
Food Consumption
Reduced mean food consumption was noted after the first 3 dose administrations (M-21, M-14 and GD9) in the BNT162b1, BNT162b2 and BNT162b3 groups compared with the control group (up to -14, -16 and -17% on GD9, respectively). Complete recovery was noted between each of the dose administrations such that mean food consumption was comparable with the control group during the premating and gestation periods, therefore none of the transient differences from control were considered adverse.
Pre-Implantation Data
The mean percentage pre-implantation loss was higher in the BNT162b2 and BNT162b3 groups (9.77% and 7.96%, respectively) compared with the control group (4.09%). However, the differences were not biologically meaningful and the values remained within the historical control data range (5.1% to 11.5%) for pivotal studies, so the difference was considered to be incidental.
External Observations
From the BNT162b1 group, Fetus 17 (F35) had exencephaly, open eye and spina bifida in the cervical region. Exencephaly and open eye are part of the background data for this strain of rat. These malformations noted for a single fetus were therefore considered incidental in view of the isolated incidence.
In the BNT162b2 group, there was Fetus 14 (F58) had gastroschisis and Fetus 14 (F64) had a small mouth and agnathia. These malformations are part of the background data for this strain of rat (CRL:WI(Han)) and were considered incidental in view of their isolated and sporadic nature
Visceral Observations
Again, the report states there were no effects on fetal soft tissue morphology due to the vaccines.
However, from the BNT162b1 group, one fetus has narrowed ductus arteriosus and another had a retinal fold. One fetus in the BNT162b2 group has a right-sided aortic arch and one from the BNT162b3 group has a ventricular septum defect. These were, again, all considered incidental, as were less severe soft tissue anomalies.
Skeletal Observations
No effects on fetal skeletal morphology were reported but in the BNT162b1 group a fetus had acrania and multiple abnormalities of thoracic and cervical vertebrae. In the BNT162b2 group a fetus had short and fused mandibles. All of these, including other less severe anomalies, such as supernumerary lumbar ribs, 7 lumbar vertebrae or incomplete ossification of thoracic centrum, were considered incidental.
Pre-Birth Loss
No effect on pre-birth loss reported. However, in the BNT162b1 group pre-birth loss was 12.2% and in the BNT162b3 group it was 13.8%. This compared with the control group at 6.8% but was within historical ranges so considered incidental.
Consequently, the mean number of pups delivered was marginally lower in the BNT162b1 and BNT162b3 groups (11.9 and 11.4, respectively) compared with the control group (13.3). However, the values remained consistent with the historical control data range (from 9.9 to 11.8) for pivotal studies.
Pup Viability and Litter Sizes
No effects were observed which were related to the vaccines. However, in the BNT162b1 group the live birth rate was 93.2% and in BNT162b3 it was 94.7%, compared with 98% in the control group.
Consequently, the mean live litter size was marginally lower in the BNT162b1 and BNT162b3 groups (11.0 and 11.3, respectively) compared with concurrent control group (13.0). However, the values were consistent with the background data for this strain of rat (from 9.8 to 11.6)
Necropsy finding of adult females
Abnormalities of the liver (diaphragmatic hernia, mottled surface, abnormal shape or adherent mass) were occasionally noted for isolated females across all groups (including controls) and were considered incidental.
It seems all of the adverse events above were considered incidental by the authors but I guess they are in a much better position to analyse the data. Furthermore, many of the adverse events related to the other two vaccine candidates, it was BNT162b2 which was eventually selected for emergency use authorisation.
However, with so many adverse events only happening in the vaccine groups I would want to study the effects on more than 44 rats before advising every pregnant woman in the world to get injected.
VERY IMPORTANT (Biodistribution of spike) – 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
Authors found:
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has been associated mainly with a range of neurological symptoms, including brain fog and brain tissue loss, raising concerns about the virus’s acute and potential chronic impact on the central nervous system. 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.
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has been associated mainly with a range of neurological symptoms, including brain fog and brain tissue loss, raising concerns about the virus’s acute and potential chronic impact on the central nervous system. 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.
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has been associated mainly with a range of neurological symptoms, including brain fog and brain tissue loss, raising concerns about the virus’s acute and potential chronic impact on the central nervous system. 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.
VERY IMPORTANT (S protein causing neurological development issues) – Effects of spike protein and toxin-like peptides found in COVID-19 patients on human 3D neuronal/glial model undergoing differentiation: Possible implications for SARS-CoV-2 impact on brain development https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9068247/
Apart from the possible direct involvement of SARS-CoV-2 or its specific viral components in the occurrence of neurological and neurodevelopmental manifestations, we recently reported the presence of toxin-like peptides in plasma, urine and fecal samples specifically from COVID-19 patients. In this study, we investigated the possible neurotoxic effects elicited upon 72-hour exposure to human relevant levels of recombinant spike protein, toxin-like peptides found in COVID-19 patients, as well as a combination of both in 3D human iPSC-derived neural stem cells differentiated for either 2 weeks (short-term) or 8 weeks (long-term, 2 weeks in suspension + 6 weeks on MEA) towards neurons/glia. Whole transcriptome and qPCR analysis revealed that spike protein and toxin-like peptides at non-cytotoxic concentrations differentially perturb the expression of SPHK1, ELN, GASK1B, HEY1, UTS2, ACE2 and some neuronal-, glia- and NSC-related genes critical during brain development. Additionally, exposure to spike protein caused a decrease of spontaneous electrical activity after two days in long-term differentiated cultures.
IMPORTANT (Time to express spike following transfection) – Correlation of mRNA delivery timing and protein expression in lipid-based transfection https://academic.oup.com/ib/article/11/9/362/5673747?login=false
To investigate whether nanocarriers are taken up, we prepared lipoplexes containing mRNA with a Cy5-labeled fraction to visualize the lipoplexes during the time-lapse measurement. The fluorescence kinetics of the Cy5-labeled mRNAs show the adsorption onto the cell surfaces (see Supplementary Fig. 2B). As a reference, we observed on areas with no cells that the fluorescence time courses of nanocarriers increase during the incubation period and abruptly decrease when the chamber is flushed with fresh carrier-free medium indicating that some partially adherent nanocarriers are rinsed away. Interestingly, in contrast, the fluorescence of nanocarriers adsorbed to cells not only does not decrease but also increases within the next 1 hour after flushing. We interpret this time course such that firstly, nanocarriers are strongly adsorbed and are not rinsed away and secondly, the fluorescence of the Cy5-labeled mRNAs is likely to increase during the time of uptake due to unpacking of the lipid-based carriers and thus dequenching the Cy5 fluorescence.
The average width of the onset time distribution measured as full width half maximum (FWHM) of the estimated distribution is about 2–3 hours. The expression rate is described by a log-normal distribution (see Supplementary Fig. 4B). The mRNA degradation rates δ also exhibit a distribution, an observation that will not be further discussed since it is outside the scope of this report (see Supplementary Fig. 3).
IMPORTANT (1% to 2% of lipid nanoparticle (LNP)-mediated nucleic acid delivery leads to successful cell transfection) – A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery https://www.nature.com/articles/s42003-021-01728-8.pdf
VERY IMPORTANT (SV40 Promotor contamination and possible link to cancer) – Association Between Simian Virus 40 and Human Tumors https://pubmed.ncbi.nlm.nih.gov/31403031/ News article https://expose-news.com/2023/07/14/cancer-agent-pfizer-vaccine/
IMPORTANT (Spike interfers with calcium pathways) – COVID-19: the CaMKII-like system of S protein drives membrane fusion and induces syncytial multinucleated giant cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8374125/
IMPORTANT (N1-methyl pseudouridine cause of cancer) – Review: N1-methyl-pseudouridine (m1Ψ): Friend or foe of cancer? https://pubmed.ncbi.nlm.nih.gov/38583833/
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.
VERY IMPORTANT (Australian TGA Pfizer assessment) – Nonclinical Evaluation Report; BNT162b2 [mRNA] COVID-19 vaccine (COMIRNATY) https://www.tga.gov.au/sites/default/files/foi-2389-06.pdf
Genotoxicity
No genotoxicity studies were conducted for the vaccine. This is in line with relevant guidelines for
vaccines. There were also no genotoxicity studies with the novel excipients.Carcinogenicity
Carcinogenicity studies were not conducted. This is acceptable based on its duration of use. The
novel lipid excipients are not expected to be carcinogenic based on the low exposure, duration of
exposure, absence of structure alerts for mutagenicityImmunotoxicty
No dedicated immunotoxicity study was conducted. An in vitro study on stimulation of cytokine
release in human PBMC cells provided inconclusive results. As expected, immune-stimulatory effects
were observed in pharmacology and repeat dose toxicity studies. No vaccine-related systemic
intolerance or mortality was observed in the studies.Paediatric use
BNT162b2 is not proposed for paediatric use and no specific studies in juvenile animals were
submitted.DISTRIBUTION
The distribution of LNP-BNT162b2 (V9) mRNA or expressed S protein was not studied.
VERY IMPORTANT (Biodistribution) – The S1 protein of SARS-CoV-2 crosses the blood–brain barrier in mice https://www.nature.com/articles/s41593-020-00771-8
VERY IMPORTANT (Patent: Moderna Lipid Nanoparticle containing hydrogel delivery that may be causing fibrous blood clotting) – Modified polynucleotides for the production of secreted proteins https://pubchem.ncbi.nlm.nih.gov/patent/US-10703789-B2
Abstract: A pharmaceutical composition which has a plurality of lipid nanoparticles that has a mean particle size of between 80 nm and 160 nm and contains a modified mRNA encoding a polypeptide. The lipid nanoparticles include a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid. The mRNA contains a 5′-cap, 5′-UTR, N1-methyl-pseudouridine, a 3′-UTR, and a poly-A region with at least 100 nucleotides.
VERY IMPORTANT (Pseudouridine increasing protein production in mRNA spike production) – 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).
IMPORTANT (mRNA protein replication in vaccine more than virus comparison) – Differences in Vaccine and SARS-CoV-2 Replication Derived mRNA: Implications for Cell Biology and Future Disease https://osf.io/bcsa6/
In this study, we performed a RNAfold analysis to investigate alterations in secondary structures of mRNAs in SARS-CoV-2 vaccines due to codon optimization. We show a significant increase in the GC content of mRNAs in vaccines as compared to native SARS-CoV-2 RNA sequences encoding the spike protein. As the GC enrichment leads to more G-quadruplex structure formations, these may contribute to potential pathological processes initiated by SARS-CoV-2 molecular vaccination.
VERY IMPORTANT (Pseudouridine in mRNA) – Modifications in an Emergency: The Role of N1-Methylpseudouridine in COVID-19 Vaccines https://pubs.acs.org/doi/10.1021/acscentsci.1c00197?ref=pdf#
As in the case of 5′-UTRs, it was found that m1Ψ stabilizes structure. Further studies provided support for a model in which secondary structure in the coding sequence, which can be enforced by m1Ψ, may increase mRNA functional half-life independent of codon optimality.
VERY IMPORTANT (Pseudouridine in mRNA increases output) – Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability https://www.cell.com/molecular-therapy-family/molecular-therapy/fulltext/S1525-0016(16)32681-8
We found that mRNAs containing pseudouridines have a higher translational capacity than unmodified mRNAs when tested in mammalian cells and lysates or administered intravenously into mice at 0.015–0.15 mg/kg doses. The delivered mRNA and the encoded protein could be detected in the spleen at 1, 4, and 24 hours after the injection, where both products were at significantly higher levels when pseudouridine-containing mRNA was administered. Even at higher doses, only the unmodified mRNA was immunogenic, inducing high serum levels of interferon-α (IFN-α). These findings indicate that nucleoside modification is an effective approach to enhance stability and translational capacity of mRNA while diminishing its immunogenicity in vivo. Improved properties conferred by pseudouridine make such mRNA a promising tool for both gene replacement and vaccination.
IMPORTANT (Mechanism that switched off the receptors to allow modRNA to escape neutralisation) – 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.
IMPORTANT (Pseudouridine and LNP stability / degredation) – Research Advances on the Stability of mRNA Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10051489/
VERY IMPORTANT (Spike protein directly responsible for disrupting cardiac mechanism) – SARS-CoV-2 direct cardiac damage through spike-mediated cardiomyocyte fusion https://europepmc.org/article/ppr/ppr232448
IMPORTANT (Circulating spike cause of myocarditis) – Circulating Spike Protein Detected in Post–COVID-19 mRNA Vaccine Myocarditis https://www.ahajournals.org/doi/suppl/10.1161/circulationaha.122.061025
IMPORTANT (Adverse effects of S Protein) – Adverse effects of COVID-19 mRNA vaccines: the spike hypothesis https://www.sciencedirect.com/science/article/pii/S1471491422001034
IMPORTANT (Comparison of vaccine induced spike (0.9 billion) to viral (72 billion) but doesn’t consider circulation of mRNA or spike post injection – see biodistribution) – The spike hypothesis in vaccine-induced adverse effects: questions and answers https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9494717/
IMPORTANT (Spike causes inflammation on its own, other proteins of the virus do not) – SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway https://elifesciences.org/articles/68563
We observed that spike (S) protein potently induced inflammatory cytokines and chemokines, including IL-6, IL-1β, TNFα, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and nucleocapsid (N) proteins. When stimulated with extracellular S protein, human and mouse lung epithelial cells also produced inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly were non-inflammatory, but elicited an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-κB pathway in a MyD88-dependent manner. ‘
Further, such an activation of the NF-κB pathway was abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein-induced IL-6, TNF-α, and IL-1β in wild-type, but not Tlr2-deficient mice. Notably, upon recognition of S protein, TLR2 dimerizes with TLR1 or TLR6 to activate the NF-κB pathway. Taken together, these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.’
VERY IMPORTANT (Spike protein causes endoplasmic reticulum stress – Published 2007) – The coronavirus spike protein induces endoplasmic reticulum stress and upregulation of intracellular chemokine mRNA concentrations https://pubmed.ncbi.nlm.nih.gov/17670839/
VERY IMPORTANT (Spike protein and endothelial disease) – The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB signaling https://www.jbc.org/article/S0021-9258(22)00135-1/fulltext
RELATED TO ABOVE – IMPORTANT (Body unable to clear spike protein or it is constantly being produced) – Liquid biomarkers of macrophage dysregulation and circulating spike protein illustrate the biological heterogeneity in patients with post-acute sequelae of COVID-19 https://onlinelibrary.wiley.com/doi/10.1002/jmv.28364
IMPORTANT (Spike protein method of action) – The S Protein of SARS-CoV-2 Injures Cardiomyocytes Indirectly through the Release of Cytokines Instead of Direct Action https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8593489/
IMPORTANT (Non invasive study showing spike reducing neuron activity) – SARS-CoV-2 Spike Protein Reduces Burst Activities in Neurons Measured by Micro-Electrode Arrays https://www.biorxiv.org/content/10.1101/2023.04.24.538161v1 Published https://journals.lww.com/annals-of-medicine-and-surgery/fulltext/2023/07000/sars_cov_2_spike_protein_reduces_burst_activities.33.aspx
IMPORTANT – (S1 causes lung injury on its own proving cytotoxicity) Published June 22 – The SARS-CoV-2 spike protein subunit S1 induces COVID-19-like acute lung injury in Κ18-hACE2 transgenic mice and barrier dysfunction in human endothelial cells https://pubmed.ncbi.nlm.nih.gov/34156871/
Authors found:
[W]e intratracheally instilled the S1 subunit of SARS-CoV-2 spike protein (S1SP) in K18-hACE2 transgenic mice that overexpress human ACE2 and examined signs of COVID-19-associated lung injury 72 h later.
K18-hACE2 mice instilled with S1SP exhibited a decline in body weight, dramatically increased white blood cells and protein concentrations in bronchoalveolar lavage fluid (BALF), upregulation of multiple inflammatory cytokines in BALF and serum, histological evidence of lung injury, and activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways in the lung.
IMPORTANT (Virus mediates myocardial inflammation and ACE2 reduction) Historical 2009 study – SARS-coronavirus modulation of myocardial ACE2 expression and inflammation in patients with SARS https://pubmed.ncbi.nlm.nih.gov/19453650/
We hypothesized that the interaction between SARS‐CoV and ACE2 in the heart could contribute to SARS‐mediated myocardial inflammation and damage. We showed that pulmonary infection with the SARS‐CoV in mice leads to myocardial SARS‐CoV in an ACE2‐dependent manner coupled with down‐regulation of the myocardial Ace2 mRNA and loss of ACE2 protein. In patients who died from SARS, presence of SARS‐CoV in the heart was associated with greater macrophage infiltration and myocardial damage in association with decreased myocardial ACE2 protein expression. Our results demonstrate that ACE2 plays a key role in mediating SARS‐CoV infection in the heart, which in combination with down‐regulation of the myocardial ACE2 could be the underlying pathophysiological mechanism of SARS‐associated heart disease.
IMPORTANT (Spike protein endothelial cells damage) – The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB signaling https://www.jbc.org/article/S0021-9258(22)00135-1/fulltext
‘Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries.
We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19.’
IMPORTANT (S1 sub unit issues) – The SARS-CoV-2 spike protein subunit S1 induces COVID-19-like acute lung injury in Κ18-hACE2 transgenic mice and barrier dysfunction in human endothelial cells https://pubmed.ncbi.nlm.nih.gov/34156871/
The S1 subunit of the SARS-CoV-2 spike protein (S1SP) causes acute lung injury and the activation of the STAT3 and NF-κB inflammatory pathways 72 h after exposure. A: H&E staining of lung sections demonstrates septal thickening, neutrophil infiltration, and edema in S1SP-instilled K18-hACE2 mice, minimal edema and leucocyte infiltration in S1SP-instilled WT, whereas SP-instilled K18-hACE2 display minimal changes compared with controls. B: the Lung Injury Score quantifies maximal injury in S1SP-instilled K18-hACE2 mice, a milder pathology in S1SP-instilled WT and no significant changes in SP- or saline-instilled K18-hACE2 mice. C: Western blot analysis of lung homogenates revealed increased phosphorylation of I-kappaB alpha (IκBα) and STAT3. Bands were normalized to β-actin and are shown as fold of control. (means ± SE; n = 5 mice/group; *P < 0.05; **P < 0.01; ***P < 0.001 with one-way ANOVA and Tukey’s). H&E, hematoxylin-eosin; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; VEH, vehicle; SP, spike protein; S1SP, subunit 1 of spike protein; WT, wild type.
IMPORTANT (S2 sub unit tumour suppression) – S2 Subunit of SARS-nCoV-2 Interacts with Tumor Suppressor Protein p53 and BRCA: an In Silico Study https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324311/
IMPORTANT (Role of Omicron spike in pathogenicity) – Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1 Omicron https://www.biorxiv.org/content/10.1101/2022.10.13.512134v1
IMPORTANT (LNP stability) – Achieving long-term stability of lipid nanoparticles: examining the effect of pH, temperature, and lyophilization https://pubmed.ncbi.nlm.nih.gov/28115848/
IMPORTANT (LNP can cause clotting risk) – Physicochemical Targeting of Lipid Nanoparticles to the Lungs Induces Clotting: Mechanisms and Solutions https://www.biorxiv.org/content/10.1101/2023.07.21.550080v1.full
IMPORTANT (Myocarditis risk) – Potential implications of lipid nanoparticles in the pathogenesis of myocarditis associated with the use of mRNA vaccines against SARS-CoV-2 https://www.sciencedirect.com/science/article/pii/S2589936821000839
VERY IMPORTANT (mRNA in breastmilk) – Detection of Messenger RNA COVID-19 Vaccines in Human Breast Milk https://jamanetwork.com/journals/jamapediatrics/article-abstract/2796427
VERY IMPORTANT (Biodistribution of mRNA in breast milk) – Biodistribution of mRNA COVID-19 vaccines in human breast milk https://www.thelancet.com/journals/ebiom/article/PIIS2352-3964(23)00366-3/fulltext
Furin Cleavage Site studies:
IMPORTANT (Furin Cleavage Site on spike) – The genetic structure of SARS-CoV-2 does not rule out a laboratory origin https://onlinelibrary.wiley.com/doi/full/10.1002/bies.202000240
IMPORTANT (Viral analysis leads to interesting conclusions) – 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-vaccine-for-covid19-sarscov2-developed-from-analysis-of-its-general-method-of-action-for-infectivity/DBBC0FA6E3763B0067CAAD8F3363E527
IMPORTANT (Furin Cleavage Site on spike) – In silico comparison of SARS-CoV-2 spike protein-ACE2 binding affinities across species and implications for virus origin https://www.nature.com/articles/s41598-021-92388-5
IMPORTANT (Furin Cleavage Site on spike) – Call for an independent inquiry into the origin of the SARS-CoV-2 virus https://www.pnas.org/doi/10.1073/pnas.2202769119
IMPORTANT (Furin Cleavage Site on Spike) – The Emergence of the Spike Furin Cleavage Site in SARS-CoV-2 https://academic.oup.com/mbe/article/39/1/msab327/6426085
Authors found:
How Does the SARS-CoV-2 Spike Compare with Those of Other SARSr-CoVs?
Several novel SARS-CoV-2-like genomes from horseshoe bats in Japan (Rc-o319), Cambodia (RshSTT182 and RshSTT200), Thailand (RacCS203, 224, 253, 263, and 271), Laos (BANAL-52, -103, -116, -236, and -247), and China (RpYN03-09) have been published (Murakami et al. 2020; Delaune et al. 2021; Temmam et al. 2021; Wacharapluesadee et al. 2021; Zhou et al. 2021). However, to this day, no known sarbecovirus except SARS-CoV-2 has an FCS insertion at the S1/S2 junction. To understand how unique the S1/S2 FCS in SARS-CoV-2 is, we re-aligned the S1/S2 regions of known SARS-CoV-2-like CoVs. We found that the alignment is sensitive to lineage sampling by visually comparing a codon sequence alignment of all known spikes (fig. 1; supplementary fig. S1, Supplementary Material online) to alignments of a few subsets of the spikes (fig. 2). The full alignment appears to feature numerous insertions at different positions in the S1/S2 region across the range of spike sequences examined (fig. 1). Thus far, the PRRA motif is unique in its position, whereas spike sequences more distantly related to that of SARS-CoV-2 present other commonly featured insertions at other positions. For instance, despite being collected from different locations RmYN02 (Yunnan, China), RacCS203 (Thailand), BANAL-20-116 and BANAL-20-247 (Laos), the NSPXARVG motif appears to be conserved across these four more closely related spikes. It is currently difficult to know exactly how unique the SARS-CoV-2 PRRA motif is among SARS-CoV-2-like spike sequences because these viral lineages are relatively under-sampled.
[..] As more bat CoVs are sampled, it is possible that another SARSr-CoV will be discovered with an S1/S2 FCS insertion. FCSs have evolved naturally in other non-sarbecovirus families of betacoronaviruses (Wu and Zhao 2020). Therefore, an S1/S2 FCS emerging in a sarbecovirus is consistent with natural evolution. Even so, the knowledge that scientists had a workflow for identifying novel cleavage sites in diverse SARSr-CoVs and experimentally characterizing these cleavage sites in SARSr-CoVs—likely in a manner that makes the resulting recombinant SARSr-CoV practically indistinguishable from a rare SARSr-CoV with a naturally emerging FCS—makes it challenging to rule out an artificial origin of the SARS-CoV-2 S1/S2 FCS (Daszak 2018; Lerner and Hibbett 2021).
IMPORTANT (Furin cleavage site) – MSH3 Homology and Potential Recombination Link to SARS-CoV-2 Furin Cleavage Site https://www.frontiersin.org/articles/10.3389/fviro.2022.834808/full
IMPORTANT (Furin Cleavage Site on spike) – The Polybasic Cleavage Site in SARS-CoV-2 Spike Modulates Viral Sensitivity to Type I Interferon and IFITM2 https://pubmed.ncbi.nlm.nih.gov/33563656/
Biodistribution studies showing the length of time antigen and mRNA stay in body:
VERY IMPORTANT (Need for biodistribution) – Role of the antigen presentation process in the immunization mechanism of the genetic vaccines against COVID-19 and the need for biodistribution evaluations https://pubmed.ncbi.nlm.nih.gov/35298029/
IMPORTANT (Biodistribution – observed 1 day later) – 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
IMPORTANT (BIODISTRIBUTION) – Vaccine mRNA Can Be Detected in Blood at 15 Days Post-Vaccination https://www.mdpi.com/2227-9059/10/7/1538
IMPORTANT (Biodistribution – mRNA seen 14 to 28 days later in blood) – Blood Distribution of SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine in Humans https://www.medrxiv.org/content/10.1101/2024.07.25.24311039v1
We found that both vaccine mRNA and ionizable lipids can be detected from plasma at 4 hours following the bivalent Moderna SPIKEVAX booster, peak at around day 1 and showed a subsequent log-linear decay profile. We further showed a slow degradation of intact vaccine mRNA in blood. The similar kinetics of intact mRNA and the ionizable lipid in blood and the slow degradation of the mRNA suggests mRNA lipid nanoparticles remain intact and travel from injection sites or lymph nodes into blood stream within 4 hours post-vaccination. The rapid dissemination of mRNA lipid nanoparticles in blood found in our study is consistent with the recent findings on the detection of mRNA in breast milk at 3–45 hours post-vaccination.3 We detected low levels of mRNA in plasma 14-28 days after vaccination. This is consistent with recent cross-sectional and autopsy studies.2,5 Taken together, our results suggest vaccine mRNA lipid nanoparticles recirculate for up to 1-month post-vaccination.
We initially hypothesised that the decay rate of mRNA (lipid nanoparticles) would be influenced by the levels of anti-PEG antibodies, as many animal studies have demonstrated the phenomenon of accelerated blood clearance.19 However, we did not observe such a correlation (Figure 4b). The relatively low levels of anti-PEG antibodies in the blood and relatively uniform levels of mRNA decay across the subjects suggests mRNA decay may be more of an intrinsic feature of humans and less susceptible to external factors such as PEG antibodies. We speculate that humans with much higher levels of PEG antibodies, such as those receiving PEGylated therapeutics intravenously, may clear lipid nanoparticle mRNA vaccines more quickly.
We did however observe that the peak amounts of mRNA and ionizeable lipid (lipid nanoparticles) detected in the blood had a positive correlation with the subsequent expansion of anti-PEG IgG and IgM (Figure 4c,d). We also observed a negative correlation between the level of in vitro monocyte phagocytosis of lipid nanoparticles and anti-PEG IgG expansion (Figure 6c). These findings suggest the amounts of mRNA lipid nanoparticles that remain the blood (and free of phagocytosis) may influence the PEG antibody immunogenicity in humans. This phenomenon only affected PEG immunogenicity, as the mRNA levels in the blood were not significantly correlated with the expansion of spike-binding IgG or neutralizing antibodies (Figure 5d). This is consistent with PEG being expressed on the surface of the lipid nanoparticle, whereas the spike protein, the target of neutralization and spike binding IgG, is only expressed following mRNA transfection of cells.
IMPORTANT (Biodistribution – 28 days later) – 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
IMPORTANT (Biodistribution in lymph 30 days and in cardiac system) – 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.
IMPORTANT (Seen in Lymph nodes 2 months ) – 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?rss=yes#relatedArticles
IMPORTANT (Exosomes and spike circulating 4 months post vaccination) Published Nov 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
LINKED TO ABOVE (Exosomes now circulating with S1 spike can traverse the blood brain barrier) Published 2020 – Transport of Extracellular Vesicles across the Blood-Brain Barrier: Brain Pharmacokinetics and Effects of Inflammation https://pubmed.ncbi.nlm.nih.gov/32575812/
IMPORTANT (Spike seen 187 days after vaccination) – 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.
IMPORTANT (Spike seen 15 months post infection) – 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
IMORTANT (Circulating exosomes with spike in severe Covid-19) – Persistent circulation of soluble and extracellular vesicle-linked Spike protein in individuals with postacute sequelae of COVID-19 https://onlinelibrary.wiley.com/doi/10.1002/jmv.28568
We investigated the levels of both Spike protein (Spike) and viral RNA circulating in patients hospitalized with acute COVID-19 and in patients with and without PASC. We found that Spike and viral RNA were more likely to be present in patients with PASC. Among these patients, 30% were positive for both Spike and viral RNA; whereas, none of the individuals without PASC were positive for both. The levels of Spike and/or viral RNA in the PASC+ve patients were found to be increased or remained the same as in the acute phase; whereas, in the PASC−ve group, these viral components decreased or were totally absent. Additionally, this is the first report to show that part of the circulating Spike is linked to extracellular vesicles without any presence of viral RNA in these vesicles. In conclusion, our findings suggest that Spike and/or viral RNA fragments persist in the recovered COVID-19 patients with PASC up to 1 year or longer after acute SARS-CoV-2 infection.
IMPORTANT (Biodistribution) – A comparative biodistribution study of polymeric and lipid-based nanoparticles https://link.springer.com/article/10.1007/s13346-022-01157-y
IMPORTANT (Biodistribution to Brain and Heart) – A Case Report: Multifocal Necrotizing Encephalitis and Myocarditis after BNT162b2 mRNA Vaccination against COVID-19 https://www.mdpi.com/2076-393X/10/10/1651
IMPORTANT (Biodistribution – Liver and spleen destinations for mRNA delivery) – Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453548/
Biodegradability and Targeting Issues with Non-viral Vectors
Systemically delivered LNPs carrying mRNA face multiple barriers to delivery (Figure 1). The mononuclear phagocytic system (MPS), especially in the liver and spleen, is a frequent destination for injected nanoparticles, owing to its native role in policing the body for nano-sized infectious agents.86 The kidney filters off naked mRNA or any nanoparticle with hydrodynamic diameter less than 5.5 nm.87 Most of the LNPs are about 100 nm in diameter and large enough to prevent them from escaping the MPS and reaching other organs of interest.88 The liver, which forms a major part of the MPS, has a fenestrated vasculature and contains phagocytic cells such as Kupffer cells, which retain cationic LNPs. Furthermore, large cationic LNPs fail to extravasate from the capillaries found in the lungs and cannot be filtered from the bloodstream by the kidney.86 This can lead to the accumulation of delivery materials in the liver, lungs, or other organs.89 Therefore, the clearance and biodegradability of the delivery system components is one consideration when developing mRNA delivery materials.
IMPORTANT (mRNA INTEGRITY) – The EMA covid-19 data leak, and what it tells us about mRNA instability https://www.bmj.com/content/372/bmj.n627
IMPORTANT (mRNA INTEGRITY) – Addressing the Cold Reality of mRNA Vaccine Stability https://pubmed.ncbi.nlm.nih.gov/33321139/
IMPORTANT (mRNA vaccine manufacturing process) – mRNA vaccines manufacturing: Challenges and bottlenecks https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987532/
IMPORTANT (mRNA manufacturing integrity and purity) – Failure to Scale: Covid-19 Injection Vials Must be Independently Tested for Conformity to Label https://www.trialsitenews.com/a/failure-to-scale-covid-19-injection-vials-must-be-independently-tested-for-conformity-to-label.-9a77eba4
IMPORTANT (Good manufacturing process and mRNA integrity – Sasha Latypova) – Broken Bioweapon – Lack of mRNA Integrity in Pfizer Batches: All Regulators Knew This When they “Pretend-Approved” the Shots https://sashalatypova.substack.com/p/broken-bioweapon?publication_id=870364&post_id=96421076&isFreemail=true
IMPORTANT (Vaccine development) – SARS-CoV-2 Vaccines Based on the Spike Glycoprotein and Implications of New Viral Variants https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8311925/
IMPORTANT – 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
IMPORTANT (Skin lesions showing spike protein after 3 months) – Persistent varicella zoster virus infection following mRNA COVID-19 vaccination was associated with the presence of encoded spike protein in the lesion https://onlinelibrary.wiley.com/doi/10.1002/cia2.12278
IMPORTANT – SARS-CoV-2 Spike Protein in the Pathogenesis of Prion-like Diseases https://www.authorea.com/users/455597/articles/582067-sars-cov-2-spike-protein-in-the-pathogenesis-of-prion-like-diseases
IMPORTANT (Spike in the nucleus) – Nuclear translocation of spike mRNA and protein is a novel pathogenic feature of SARS-CoV-2 https://www.biorxiv.org/content/10.1101/2022.09.27.509633v1
SARS-CoV-2 and vaccine DNA research:
IMPORTANT (mRNA reverse transcription into nucleus) – Intracellular Reverse Transcription of Pfizer BioNTech COVID-19 mRNA Vaccine BNT162b2 In Vitro in Human Li ver Cell Line https://www.mdpi.com/1467-3045/44/3/73/htm
IMPORTANT (Virus reverse transcribes into DNA) – SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome https://www.biorxiv.org/content/10.1101/2020.12.12.422516v1
SARS–CoV–2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC8538446/
IMPORTANT (LNP optimisation for injection) – Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383180/
IMPORTANT (Long Covid) – Persistent Presence of Spike protein and Viral RNA in the Circulation of Individuals with Post-Acute Sequelae of COVID-19 https://www.medrxiv.org/content/10.1101/2022.08.07.22278520v1
IMPORTANT (Long Covid) – Persistent circulating SARS-CoV-2 spike is associated with post-acute COVID-19 sequelae https://www.medrxiv.org/content/10.1101/2022.06.14.22276401v1
IMPORTANT (Long Covid) – SARS-CoV-2 S1 Protein Persistence in SARS-CoV-2 Negative Post-Vaccination Individuals with Long COVID/ PASC-Like Symptoms https://www.researchsquare.com/article/rs-1844677/v1
IMPORTANT (Long Covid – 15% of modRNA recipients expressed PASC, of which 94% took Pfizer or Moderna) – Persistent Circulating Severe Acute Respiratory Syndrome Coronavirus 2 Spike Is Associated With Post-acute Coronavirus Disease 2019 Sequelae https://pubmed.ncbi.nlm.nih.gov/36052466/
IMPORTANT (Microclotting) – A central role for amyloid fibrin microclots in long COVID/PASC: origins and therapeutic implications https://pubmed.ncbi.nlm.nih.gov/35195253/
IMPORTANT (Amyloid proteins in the spike) – Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes https://www.nature.com/articles/s41467-023-36234-4
An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions (APRs) in these proteomes and revealed a strong aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone SARS-CoV and SARS-CoV-2 proteins and protein regions, including the signal sequence peptide and fusion peptides 1 and 2 of the spike protein, a peptide from the NSP6 protein, and the ORF10 and NSP11 proteins. Our results show that these peptides and proteins can form amyloid aggregates.
IMPORTANT (mRNA in breast milk) – Neutralizing Activity and SARS-CoV-2 Vaccine mRNA Persistence in Serum and Breastmilk After BNT162b2 Vaccination in Lactating Women https://ncbi.nlm.nih.gov/pmc/articles/PMC8787073/
IMPORTANT – Coronavirus spike protein activated natural immune response, damaged heart muscle cells, study finds https://www.sciencedaily.com/releases/2022/07/220725105801.htm
IMPORTANT (Circulating spike in PASC patients) – Persistent Circulating Severe Acute Respiratory Syndrome Coronavirus 2 Spike Is Associated With Post-acute Coronavirus Disease 2019 Sequelae https://academic.oup.com/cid/advance-article/doi/10.1093/cid/ciac722/6686531?login=false
IMPORTANT – SARS-CoV-2 Spike Protein Suppresses ACE2 and Type I Interferon Expression in Primary Cells From Macaque Lung Bronchoalveolar Lavage https://www.frontiersin.org/articles/10.3389/fimmu.2021.658428/full
IMPORTANT – The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease https://portlandpress.com/clinsci/article/135/24/2667/230273/The-SARS-CoV-2-Spike-protein-disrupts-human
IMPORTANT – The mRNA-LNP platform’s lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory https://www.jimmunol.org/content/206/1_Supplement/30.01
IMPORTANT – SARS-CoV-2 spike S1 subunit induces hypercoagulability https://www.news-medical.net/news/20210310/SARS-CoV-2-spike-S1-subunit-induces-hypercoagulability.aspx
IMPORTANT– Free SARS-CoV-2 Spike Protein S1 Particles May Play a Role in the Pathogenesis of COVID-19 Infection https://pubmed.ncbi.nlm.nih.gov/33838638/
IMPORTANT – SARS-CoV-2 spike protein alone may cause lung damage https://medicalxpress.com/news/2021-04-sars-cov-spike-protein-lung.html
IMPORTANT (AUTOPSY CASE STUDY) – High viral loads: what drives fatal cases of COVID-19 in vaccinees? – an autopsy study https://pubmed.ncbi.nlm.nih.gov/35365771/
IMPORTANT– Polish Preprint: mRNA COVID Vaccine Caused In Vitro Brain Cell Changes “Similar to Those in Cancer Cells” https://trialsitenews.com/polish-preprint-mrna-covid-vaccine-caused-changes-to-brain-cells-in-vitro-similar-to-those-in-cancer-cells/?utm_source=Contextly&utm_medium=ChannelEmail&utm_campaign=COVID-19&utm_content=Notification
IMPORTANT – Vascular dysregulation following SARS-CoV-2 infection involves integrin signalling through a VE-Cadherin mediated pathway https://www.biorxiv.org/content/10.1101/2022.03.15.484274v1
IMPORTANT LN 2 MONTHS – 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?rss=yes#relatedArticles
IMPORTANT– In vitro Characterization of SARS-CoV-2 Protein Translated from the Moderna mRNA-1273 Vaccine https://www.medrxiv.org/content/10.1101/2022.03.01.22271618v1
IMPORTANT – Comprehensive investigations revealed consistent pathophysiological alterations after vaccination with COVID-19 vaccines https://pubmed.ncbi.nlm.nih.gov/34697287/
IMPORTANT – The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype https://www.biorxiv.org/content/10.1101/2022.04.28.489537v1
Polyethylene Glycol (PEG) research
Research pertaining to Polyethylene Glycol (PEG) immune responses to help the lipid nanoparticles (LNP) evade the body immune system:
IMPORTANT (Anti-PEG Abs) – Anti-PEG Antibodies Boosted in Humans by SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine https://pubs.acs.org/doi/10.1021/acsnano.2c04543
We studied plasma from 130 adults receiving either the BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) mRNA vaccines or no SARS-CoV-2 vaccine for PEG-specific antibodies. Anti-PEG IgG was commonly detected prior to vaccination and was significantly boosted a mean of 13.1-fold (range 1.0–70.9) following mRNA-1273 vaccination and a mean of 1.78-fold (range 0.68–16.6) following BNT162b2 vaccination. Anti-PEG IgM increased 68.5-fold (range 0.9–377.1) and 2.64-fold (0.76–12.84) following mRNA-1273 and BNT162b2 vaccination, respectively. The rise in PEG-specific antibodies following mRNA-1273 vaccination was associated with a significant increase in the association of clinically relevant PEGylated LNPs with blood phagocytes ex vivo. PEG antibodies did not impact the SARS-CoV-2 specific neutralizing antibody response to vaccination. However, the elevated levels of vaccine-induced anti-PEG antibodies correlated with increased systemic reactogenicity following two doses of vaccination. We conclude that PEG-specific antibodies can be boosted by LNP mRNA vaccination and that the rise in PEG-specific antibodies is associated with systemic reactogenicity and an increase of PEG particle–leukocyte association in human blood. The longer-term clinical impact of the increase in PEG-specific antibodies induced by lipid nanoparticle mRNA vaccines should be monitored.
IMPORTANT (PEG antibodies elevated accumulation of modRNA in liver and spleenand was fatal in higher doses) – Effects of PEG antibodies on in vivo performance of LNP-mRNA vaccines https://pubmed.ncbi.nlm.nih.gov/38081560/
By illustrating the distribution characteristics of PEG antibodies in human, the present study focused on the influence of PEG antibodies on the safety and efficacy of LNP-mRNA vaccine against COVID-19 in animal models. It was found that PEG antibodies led to shortened blood circulation duration, elevated accumulation and mRNA expression in liver and spleen, enhanced expression in macrophage and dendritic cells, while without affecting the production of anti-Spike protein antibodies of COVID-19 LNP vaccine. Noteworthily, PEG antibodies binding on the LNP vaccine increased probability of complement activation in animal as well as in human serum and led to lethal side effect in large dosage via intravenous injection of mice. Our data suggested that PEG antibodies in human was a risky factor of LNP-based vaccines for biosafety concerns but not efficacy.
Time course of adverse reactions following BNT162b2 vaccination in healthy and allergic disease individuals aged 5–11 years and comparison with individuals aged 12–15 years: an observational and historical cohort study https://link.springer.com/article/10.1007/s00431-022-04643-0
Role of anti-polyethylene glycol (PEG) antibodies in the allergic reactions and immunogenicity of PEG-containing Covid-19 vaccines https://www.medrxiv.org/content/10.1101/2022.10.03.22280227v1
Anti-PEG Antibodies Boosted in Humans by SARS-CoV-2 Lipid Nanoparticle mRNA Vaccine https://www.medrxiv.org/content/10.1101/2022.01.08.22268953v2
Effect of PEG density on draining Lymph nodes https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3849130/
IMPORTANT (PEG anaphylactic shock) – The COVID-19 mRNA vaccine Comirnaty induces anaphylactic shock in an anti-PEG hyperimmune large animal model: Role of complement activation in cardiovascular, hematological and inflammatory mediator changes https://www.biorxiv.org/content/10.1101/2023.05.19.541479v1
IMPORTANT (Structure of vaccine; plus numbers of LNP) – Differences in Vaccine and SARS-CoV-2 Replication Derived mRNA: Implications for Cell Biology and Future Disease https://osf.io/bcsa6/
What happens to those billions of Lipid NanoParticles you’ve become host to? https://covidmythbuster.substack.com/p/what-happens-to-those-billions-of?token=eyJ1c2VyX2lkIjo1ODU2MjY2NywicG9zdF9pZCI6NDgzOTE3MzksIl8iOiJOTE8yKyIsImlhdCI6MTY1MDQ0MjgxMCwiZXhwIjoxNjUwNDQ2NDEwLCJpc3MiOiJwdWItNjY1MjQwIiwic3ViIjoicG9zdC1yZWFjdGlvbiJ9.DnkOJDGbZD9JxXLBOWPF_1JhGux96mpU3LUGqNhsERk
IMPORTANT (Removing the spike from your system) – Rapid Degradation of SARS-CoV-2 Spike S Protein by A Specific Serine Protease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8954242/
IMPORTANT (mRNA adjuvants) – Making innate sense of mRNA vaccine adjuvanticity https://www.nature.com/articles/s41590-022-01168-4
IMPORTANT (mRNA LNP transfection pathways) – Intracellular Routing and Recognition of Lipid-Based mRNA Nanoparticles https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8308975/
SARS-COV-2 Vaccines and Neurodegenerative Disease https://www.theepochtimes.com/sars-cov-2-vaccines-and-neurodegenerative-disease_4207235.html?est=od2CNcQs0MnayM4t26ddQxcXH3jdIIeAQP3CWzyAA043jJWySn8eACp41fBCyqNrGfZiVsGw
Worse Than the Disease? Reviewing Some Possible Unintended Consequences of the mRNA Vaccines Against COVID-19 https://ijvtpr.com/index.php/IJVTPR/article/view/23
The SARS-CoV-2 spike protein subunit S1 induces COVID-19-like acute lung injury in Κ18-hACE2 transgenic mice and barrier dysfunction in human endothelial cells https://pubmed.ncbi.nlm.nih.gov/34156871/
The S1 protein of SARS-CoV-2 crosses the blood–brain barrier in mice https://www.nature.com/articles/s41593-020-00771-8
SARS CoV-2 related microvascular damage and symptoms during and after COVID-19: Consequences of capillary transit-time changes, tissue hypoxia and inflammation https://physoc.onlinelibrary.wiley.com/doi/10.14814/phy2.14726
The BNT162b2 mRNA vaccine against SARS-CoV-2 reprograms both adaptive and innate immune responses https://www.medrxiv.org/content/10.1101/2021.05.03.21256520v1
The SARS-CoV-2 mRNA vaccine breakthrough infection phenotype includes significant symptoms, live virus shedding, and viral genetic diversity https://pubmed.ncbi.nlm.nih.gov/34117878/
mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032477/
What leads to more spike protein in the body: the vaccine or virus? Part 2 https://joomi.substack.com/p/what-leads-to-more-spike-protein
Concerns of Lipid Nanoparticle Carrying mRNA Vaccine into the Brain: What to Make of It https://medium.com/microbial-instincts/concerns-of-lipid-nanoparticle-carrying-mrna-vaccine-into-the-brain-what-to-make-of-it-42b1a98dae27
Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis https://pubmed.ncbi.nlm.nih.gov/15141377/
The tangled history of mRNA vaccines https://www.nature.com/articles/d41586-021-02483-w Non-paywall version: https://web.archive.org/web/20230215062722/https://www.nature.com/articles/d41586-021-02483-w
Nanomaterial Delivery Systems for mRNA Vaccines https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836001/
Lipid nanoparticles for mRNA delivery https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8353930/
mRNA vaccine delivery using lipid nanoparticles https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439223/
IMPORTANT (Differences between the types of vaccine mechanisms) – Understanding messenger RNA and other SARS-CoV-2 vaccines https://www.mdedge.com/hematology-oncology/article/233491/coronavirus-updates/understanding-messenger-rna-and-other-sars Non paywall https://web.archive.org/web/20201217133059/https://www.mdedge.com/hematology-oncology/article/233491/coronavirus-updates/understanding-messenger-rna-and-other-sars?sso=true
IMPORTANT (Adenovirus vector Covid vaccine operation) – aCOVID-19 Vaccines Based on Adenovirus Vectors https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955943/
IMPORTANT (Biodistribution potential to BBB) – Potential of Nanocarrier-Based Drug Delivery Systems for Brain Targeting: A Current Review of Literature https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8593899/
SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8091897/
IMPORTANT (S1 subunit on its own can cause hypertension) – SARS-CoV-2 Spike Protein Elicits Cell Signaling in Human Host Cells https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827936/
It was found that the treatment of cultured primary human pulmonary artery smooth muscle cells (SMCs) or human pulmonary artery endothelial cells with the recombinant SARS-CoV-2 spike protein S1 subunit is sufficient to promote cell signaling without the rest of the viral components.
PAH (Pulmonary Arterial Hypertension) is a serious disease without a cure that can affect males and females of any age including children. The increased pulmonary vascular resistance in PAH results in right heart failure and subsequently death.
Patients diagnosed with PAH only live for 2–3 years from the time of diagnosis on average if untreated. Even with currently available therapies, only 60–70% of PAH patients survive for three years. PAH is hard to detect because its symptoms (e.g., shortness of breath, fatigue, and dizziness) are similar to those of other common non-life threatening conditions, and the official diagnosis for PAH must be made through invasive right heart catheterization.
Self-assembled mRNA vaccines (LNP) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837307/
From sewer to saviour — targeting the lymphatic system to promote drug exposure and activity https://www.nature.com/articles/nrd4608
The novel coronavirus’ spike protein plays additional key role in illness https://web.archive.org/web/20210501153712/https://www.salk.edu/news-release/the-novel-coronavirus-spike-protein-plays-additional-key-role-in-illness/
Liposomal Formulations for Nucleic Acid Delivery https://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.486.368&rep=rep1&type=pdf
The mRNA-LNP platform’s lipid nanoparticle component used in preclinical vaccine
studies is highly inflammatory https://www.scienceopen.com/document_file/17b34a53-ff8b-4c2c-a2c8-8a57897a3bac/PubMedCentral/17b34a53-ff8b-4c2c-a2c8-8a57897a3bac.pdf
Nanomaterial Delivery Systems for mRNA Vaccines https://mdpi-res.com/d_attachment/vaccines/vaccines-09-00065/article_deploy/vaccines-09-00065-v2.pdf#page25
Encapsulation state of messenger RNA inside lipid nanoparticles https://www.cell.com/biophysj/fulltext/S0006-3495(21)00241-1
Incredible images reveal how cells exposed to AstraZeneca’s vaccine produce the key spike protein
https://www.dailymail.co.uk/sciencetech/article-9444607/Incredible-images-reveal-cells-exposed-AstraZenecas-vaccine-produce-key-spike-protein.html
Impact of lipid nanoparticle size on mRNA vaccine immunogenicity
https://www.sciencedirect.com/science/article/pii/S0168365921002376?via%3Dihub
Horrifying study reveals mRNA vaccine nanoparticles are circulated throughout the entire body: Brain, heart, liver, ovaries, testes and more https://stateofthenation.co/?p=68063
Dangerous New Revelations —-Leaky Blood Vessels: An Unknown Danger of COVID-19 Vaccination
https://peckford42.wordpress.com/2021/08/05/dangerous-new-revelations-leaky-blood-vessels-an-unknown-danger-of-covid-19-vaccination/
mRNA vaccines in blood vessels https://dundasvalley.files.wordpress.com/2021/08/pfizer-pharmacokinetics-and-toxicity.pdf
mRNA vaccine delivery using lipid nanoparticles https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439223/
Concerns of Lipid Nanoparticle Carrying mRNA Vaccine into the Brain: What to Make of It?
https://medium.com/microbial-instincts/concerns-of-lipid-nanoparticle-carrying-mrna-vaccine-into-the-brain-what-to-make-of-it-42b1a98dae27
Be aware of SARS-CoV-2 spike protein: There is more than meets the eye https://pubmed.ncbi.nlm.nih.gov/34100279/
Delta spike P681R mutation enhances SARS-CoV-2 fitness over Alpha variant https://pubmed.ncbi.nlm.nih.gov/34462752/
Covid-19 vaccine & Microvascular Injury https://www.talkingaboutthescience.com/whelan2020/
SARS-CoV-2 spike protein induces abnormal inflammatory blood clots neutralized by fibrin immunotherapy
https://www.biorxiv.org/content/10.1101/2021.10.12.464152v1
Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347890/?report=reader
Canceling the Spike Protein – Striking Visual Evidence http://www.orthomolecular.org/resources/omns/v17n24.shtml
Endothelial Cells https://pubmed.ncbi.nlm.nih.gov/28667554/
Distinguishing features of current COVID-19 vaccines: knowns and unknowns of antigen presentation and modes of action https://www.nature.com/articles/s41541-021-00369-6
Cutting Edge: Circulating Exosomes with COVID Spike Protein Are Induced by BNT162b2 (Pfizer–BioNTech) Vaccination prior to Development of Antibodies https://www.jimmunol.org/content/early/2021/10/11/jimmunol.2100637
Frontiers | Circulating Exosomes Are Strongly Involved in SARS-CoV-2 Infection https://www.frontiersin.org/articles/10.3389/fmolb.2021.632290/full
SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2 https://www.cell.com/immunity/fulltext/S1074-7613%2821%2900212-0
The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human blood–brain barrier https://www.sciencedirect.com/science/article/pii/S096999612030406X
SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: Implications for microclot formation in COVID-19 https://www.medrxiv.org/content/10.1101/2021.03.05.21252960v1
The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in vitro models of the human blood–brain barrier https://www.semanticscholar.org/paper/The-SARS-CoV-2-spike-protein-alters-barrier-in-2D-Buzhdygan-DeOre/fb6a7497c9dab20b8891d82d7a4d4547f1159e8a