The studies listed below are peer reviewed papers on the topic of issues facing mRNA treatments. It is interesting to note that few, if any, of these issue were resolved prior to the release of the Covid-19 shots. Please check this page from time to time as new research is added as it is published.
How to use this page
Papers are listed by date, the most recent appearing at the top of the page. Click to expand for a full text link, author details, correspondence and abstract. Where a paper is published in multiple journals, the link provided is to a full text version. If we have missed important validated research, please log in and use the comment box below to send us a link. Acceptance of submitted links is at the discretion of the editors.
Please note that on occasion, a retracted study will still be listed. This is an editorial decision, based on the reasons for the papers retraction. Clear attempts to discredit research with a view to censorship does not warrant retraction.These papers are highlighted in red and where possible PDF versions exist on our servers.
Hypothesis: HPV E6 and COVID spike proteins cooperate in targeting tumor suppression by p53
Human Papilloma Virus (HPV) is a causative agent in several cancers including cervical cancer, head and neck cancer, anal cancer, penile, vulvar and vaginal cancers. HPV through its virus-encoded protein E6 and the cellular E6-Associated Protein (E6-AP) target the tumor suppressor p53 protein for degradation thereby contributing to cancer development after HPV infection. As viruses cause cancer, the author previously hypothesized that SARS-COV-2 virus may be associated with cancer. More recent insights on the present hypothesis have come from studies suggesting (1) Spike protein of SARS-COV-2 may suppress p53 function, (2) cancer has been associated with mRNA vaccines that produce Spike, and (3) a case mentioned by Dr. Patrick Soon Shiong of a patient who survived HPV-associated head and neck cancer, but the tumor recurred after COVID mRNA vaccination including with liver metastases. Thus, the present hypothesis is that virally encoded proteins such as HPV-E6 or SARS-COV-2 Spike may cooperate in suppressing host defenses including tumor suppressor mechanisms involving p53. The hypothesis can be further explored through epidemiologic and laboratory studies.
COVID-19 vaccine adverse events: Evaluating the pathophysiology with an emphasis on sulfur metabolism and endotheliopathy
In this narrative review, we assess the pathophysiology of severe adverse events that presented after vaccination with DNA and mRNA vaccines against COVID-19. The focus is on the perspective of an undersulfated and degraded glycocalyx, considering its impact on immunomodulation, inflammatory responses, coagulation and oxidative stress. The paper explores various factors that lead to glutathione and inorganic sulfate depletion and their subsequent effect on glycocalyx sulfation and other metabolites, including hormones. Components of COVID-19 vaccines, such as DNA and mRNA material, spike protein antigen and lipid nanoparticles, are involved in possible cytotoxic effects. The common thread connecting these adverse events is endotheliopathy or glycocalyx degradation, caused by depleted glutathione and inorganic sulfate levels, shear stress from circulating nanoparticles, aggregation and formation of protein coronas; leading to imbalanced immune responses and chronic release of pro-inflammatory cytokines, ultimately resulting in oxidative stress and systemic inflammatory response syndrome. By understanding the underlying pathophysiology of severe adverse events, better treatment options can be explored
Lipid Nanoparticle-Associated Inflammation is Triggered by Sensing of Endosomal Damage: Engineering Endosomal Escape Without Side Effects
Serena Omo-Lamai, Yufei Wang, Manthan N. Patel, Eno-Obong Essien, Mengwen Shen, Aparajeeta Majumdar, Carolann Espy, Jichuan Wu, Breana Channer, Michael Tobin, Shruthi Murali, Tyler E. Papp, Rhea Maheshwari, Liuqian Wang, Liam S. Chase, Marco E. Zamora, Mariah L. Arral, Oscar A. Marcos-Contreras, Jacob W. Myerson, Christopher A. Hunter, Andrew Tsourkas, Vladimir Muzykantov, Igor Brodsky, Sunny Shin, Kathryn A. Whitehead, Peter Gaskill, Dennis Discher, Hamideh Parhiz, Jacob S. Brenner
Lipid nanoparticles (LNPs) have emerged as the dominant platform for RNA delivery, based on their success in the COVID-19 vaccines and late-stage clinical studies in other indications. However, we and others have shown that LNPs induce severe inflammation, and massively aggravate pre-existing inflammation. Here, using structure-function screening of lipids and analyses of signaling pathways, we elucidate the mechanisms of LNP-associated inflammation and demonstrate solutions. We show that LNPs’ hallmark feature, endosomal escape, which is necessary for RNA expression, also directly triggers inflammation by causing endosomal membrane damage. Large, irreparable, endosomal holes are recognized by cytosolic proteins called galectins, which bind to sugars on the inner endosomal membrane and then regulate downstream inflammation. We find that inhibition of galectins abrogates LNP-associated inflammation, both in vitro and in vivo. We show that rapidly biodegradable ionizable lipids can preferentially create endosomal holes that are smaller in size and reparable by the endosomal sorting complex required for transport (ESCRT) pathway. Ionizable lipids producing such ESCRT-recruiting endosomal holes can produce high expression from cargo mRNA with minimal inflammation. Finally, we show that both routes to non-inflammatory LNPs, either galectin inhibition or ESCRT-recruiting ionizable lipids, are compatible with therapeutic mRNAs that ameliorate inflammation in disease models. LNPs without galectin inhibition or biodegradable ionizable lipids lead to severe exacerbation of inflammation in these models. In summary, endosomal escape induces endosomal membrane damage that can lead to inflammation. However, the inflammation can be controlled by inhibiting galectins (large hole detectors) or by using biodegradable lipids, which create smaller holes that are reparable by the ESCRT pathway. These strategies should lead to generally safer LNPs that can be used to treat inflammatory diseases.
Adjuvant Activity and Toxicological Risks of Lipid Nanoparticles Contained in the COVID‑19 “mRNA Vaccines”
The LNPs reportedly used as the platform by Pfizer/BioNTech for its SARS-CoV-2 “mRNA vaccines” allegedly consist of a mixture of phospholipids, cholesterol, PEGylated lipids, and an ionizable cationic lipid. This study reviews some of the main toxicological risks and immunostimulatory properties of such nanomaterials, with particular attention to the ionizable LNPs and their adjuvant properties, inflammatory responses, stimulation of immune cells, and formation of ROS inside transfected cells. The decision not to carry out safety pharmacology, carcinogenicity, and genotoxicity tests on these nanomaterials appears unjustifiable and in contradiction with the international policies provided for novel adjuvants. Important gaps are highlighted on the activities by the relevant regulatory bodies, related to the scientific evaluation, risk management, and pharmacovigilance for new medicinal products in the EU. Given the findings discussed here, it is strongly urged that the mRNA-LNP-based “vaccines” and their boosters should be removed from the worldwide market because of unacceptable and potentially fatal safety risks.
N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting
Thomas E. Mulroney, Tuija Pöyry, Juan Carlos Yam-Puc, Maria Rust, Robert F. Harvey, Lajos Kalmar, Emily Horner, Lucy Booth, Alexander P. Ferreira, Mark Stoneley, Ritwick Sawarkar, Alexander J. Mentzer, Kathryn S. Lilley, C. Mark Smales, Tobias von der Haar, Lance Turtle, Susanna Dunachie, Paul Klenerman, James E. D. Thaventhiran & Anne E. Willis
In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1,2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3,4,5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization.
The Novelty of mRNA Viral Vaccines and Potential Harms: A Scoping Review
Pharmacovigilance databases are showing evidence of injury in the context of the modified COVID-19 mRNA products. According to recent publications, adverse event reports linked to the mRNA COVID-19 injections largely point to the spike protein as an aetiological agent of adverse events, but we propose that the platform itself may be culpable. To assess the safety of current and future mRNA vaccines, further analysis is needed on the risks due to the platform itself, and not specifically the expressed antigen. If harm can be exclusively and conclusively attributed to the spike protein, then it is possible that future mRNA vaccines expressing other antigens will be safe. If harms are attributable to the platform itself, then regardless of the toxicity, or lack thereof, of the antigen to be expressed, the platform may be inherently unsafe, pending modification. In this work, we examine previous studies of RNA-based delivery by a lipid nanoparticle (LNP) and break down the possible aetiological elements of harm.
Apparent Cytotoxicity and Intrinsic Cytotoxicity of Lipid Nanomaterials Contained in a COVID-19 mRNA Vaccine
The medicinal preparation called Comirnaty by Pfizer-BioNTech is an aqueous dispersion of lipid nanomaterials, intended to constitute, after thawing and dilution, the finished product for intramuscular injection. In the present study, we examine some evident chemical-physical criticalities of the preparation, particularly regarding the apparent and the intrinsic pKa (acid dissociation constant) of its main excipient, the ionizable cationic lipid ALC-0315. The very high value of its intrinsic pKa causes, after internalization and endosomal escape of LNPs, a sudden increase of its cationic charge concentration and consequently the formation of pro-inflammatory cytokines and ROS (reactive oxygen species), that can disrupt the mitochondrial membrane and release its content, cause RNA mistranslation, polymerization of proteins and DNA, DNA mutations, destruction of the nuclear membrane and consequent release of its content. Additionally, the apparently low pKa value (6.09) of ALC-0315 associated with other lipids in the LNP, is not suitable for intramuscular application. Its value is too low to enable a proper transfection of host cells, despite what is stated by EMA (European Medicines Agency) in its Assessment report dated 19 February 2021, in flagrant contradiction with the same bibliographic source therein cited. Furthermore, the exceptional penetrability, mobility, chemical reactivity and systemic accumulation of uncontrollable cationic lipid nanoparticles, with high cytotoxicity levels, shed in unpredictable biological locations, even far from the site of inoculation, are all factors that can lead to an unprecedented medical disaster. Meanwhile, further immediate studies and verifications are recommended, taking into consideration, in accordance with the precautionary principle, the immediate suspension of vaccinations with the COVID-19 mRNA- LNP-based vaccines.
Autoimmune inflammatory reactions triggered by the COVID-19 genetic vaccines in terminally differentiated tissues
Panagis Polykretis, Alberto Donzelli, Janci C Lindsay, David Wiseman, Anthony M Kyriakopoulos, Michael Mörz, Paolo Bellavite, Masanori Fukushima, Stephanie Seneff, Peter A McCullough
As a result of the spread of SARS-CoV-2, a global pandemic was declared. Indiscriminate COVID-19 vaccination has been extended to include age groups and naturally immune people with minimal danger of suffering serious complications due to COVID-19. Solid immuno-histopathological evidence demonstrates that the COVID-19 genetic vaccines can display a wide distribution within the body, affecting tissues that are terminally differentiated and far away from the injection site. These include the heart and brain, which may incur in situ production of spike protein eliciting a strong autoimmunological inflammatory response. Due to the fact that every human cell which synthesises non-self antigens, inevitably becomes the target of the immune system, and since the human body is not a strictly compartmentalised system, accurate pharmacokinetic and pharmacodynamic studies are needed in order to determine precisely which tissues can be harmed. Therefore, our article aims to draw the attention of the scientific and regulatory communities to the critical need for biodistribution studies for the genetic vaccines against COVID-19, as well as for rational harm-benefit assessments by age group.
Chemical-physical criticality and toxicological potential of lipid nanomaterials contained in a COVID-19 mRNA vaccine
The medicinal preparation called Comirnaty by Pfizer-BioNTech is an aqueous dispersion of lipid nanomaterials, intended to constitute, after thawing and dilution, the finished product for intramuscular injection. In the present study, we examine some evident chemical-physical criticalities of the preparation, regarding the manifest instability of its qualitative-quantitative composition, as well as its consequent toxicological potential, in this case related to the possible formation of ROS (reactive oxygen species), after intramuscular inoculation, in different biological sites, such as, potentially, kidneys, liver, heart, brain, etc., causing dysfunctions and alterations thereof. Of particular concern is the presence in the formulation of the two functional excipients, ALC-0315 and ALC-0159, never used before in a medicinal product, nor registered in the European Pharmacopoeia, nor in the European C&L inventory. The current Safety Data Sheets of the manufacturer are omissive and non-compliant, especially with regard to the provisions of current European regulations on the registration, evaluation, authorization and restriction of nanomaterials. The presence of electrolytes in the preparation and the subsequent dilution phase after thawing and before inoculation raise well-founded concerns about the precarious stability of the resulting suspension and the Polydispersity index of the nanomaterials contained in it, factors that can be hypothesized as the root causes of numerous post-vaccination adverse effects recorded at statistical-epidemiological level. Further immediate studies and verifications are recommended, taking into consideration, if necessary and for purely precautionary purposes, the immediate suspension of vaccinations with the Pfizer-BioNTech Comirnaty preparation.
Skewed fate and hematopoiesis of CD34+ HSPCs in umbilical cord blood amid the COVID-19 pandemic
Benjamin K. Estep, Charles J. Kuhlmann, Satoru Osuka, Gajendra W. Suryavanshi, Yoshiko Nagaoka-Kamata, Ciearria N. Samuel, Madison T. Blucas, Chloe E. Jepson, Paul A. Goepfert, Masakazu Kamata
Umbilical cord blood (UCB) is an irreplaceable source for hematopoietic stem progenitor cells (HSPCs). However, the effects of SARS-CoV-2 infection and COVID-19 vaccination on UCB phenotype, specifically the HSPCs therein, are currently unknown. We thus evaluated any effects of SARS-CoV-2 infection and/or COVID-19 vaccination from the mother on the fate and functionalities of HSPCs in the UCB. The numbers and frequencies of HSPCs in the UCB decreased significantly in donors with previous SARS-CoV-2 infection and more so with COVID-19 vaccination via the induction of apoptosis, likely mediated by IFN-γ-dependent pathways. Two independent hematopoiesis assays, a colony forming unit assay and a mouse humanization assay, revealed skewed hematopoiesis of HSPCs obtained from donors delivered from mothers with SARS-CoV-2 infection history. These results indicate that SARS-CoV-2 infection and COVID-19 vaccination impair the functionalities and survivability of HSPCs in the UCB, which would make unprecedented concerns on the future of HSPC-based therapies.
Added to pre-existing inflammation, mRNA-lipid nanoparticles induce inflammation exacerbation (IE)
PUBLICATION: Elsevier, Journal of Controlled Release
AUTHORS
Hamideh Parhiz, Jacob S. Brenner, Priyal N. Patel, Tyler E. Papp, Hamna Shahnawaz, Qin Li , Ruiqi Shi, Marco E. Zamora, Amir Yadegari, Oscar A. Marcos-Contreras, Ambika Natesan, Norbert Pardi, Vladimir V. Shuvaev, Raisa Kiseleva, Jacob W. Myerson, Thomas Uhler, Rachel S. Riley, Xuexiang Han, Michael J. Mitchell, Kieu Lam, Vladimir R. Muzykantov
Current nucleoside-modified RNA lipid nanoparticle (modmRNA-LNP) technology has successfully paved the way for the highest clinical efficacy data from next-generation vaccinations against SARS-CoV-2 during the COVID-19 pandemic. However, such modmRNA-LNP technology has not been characterized in common pre-existing inflammatory or immune-challenged conditions, raising the risk of adverse clinical effects when administering modmRNA-LNPs in such cases. Herein, we induce an acute-inflammation model in mice with lipopolysaccharide (LPS) intratracheally (IT), 1 mg kg−1, or intravenously (IV), 2 mg kg−1, and then IV administer modmRNA-LNP, 0.32 mg kg−1, after 4 h, and screen for inflammatory markers, such as pro-inflammatory cytokines. ModmRNA-LNP at this dose caused no significant elevation of cytokine levels in naive mice. In contrast, shortly after LPS immune stimulation, modmRNA-LNP enhanced inflammatory cytokine responses, Interleukin-6 (IL-6) in serum and Macrophage Inflammatory Protein 2 (MIP-2) in liver significantly. Our report identifies this phenomenon as inflammation exacerbation (IE), which was proven to be specific to the LNP, acting independent of mRNA cargo, and was demonstrated to be time- and dose-dependent. Macrophage depletion as well as TLR3 −/− and TLR4−/− knockout mouse studies revealed macrophages were the immune cells involved or responsible for IE. Finally, we show that pretreatment with anti-inflammatory drugs, such as corticosteroids, can partially alleviate IE response in mice. Our findings characterize the importance of LNP-mediated IE phenomena in gram negative bacterial inflammation, however, the generalizability of modmRNA-LNP in other forms of chronic or acute inflammatory and immune contexts needs to be addressed.
The mRNA-LNP platform’s lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory
Vaccines based on mRNA-containing lipid nanoparticles (LNPs) are a promising new platform used by two leading vaccines against COVID-19. Clinical trials and ongoing vaccinations present with varying degrees of protection levels and side effects. However, the drivers of the reported side effects remain poorly defined. Here we present evidence that Acuitas’ LNPs used in preclinical nucleoside-modified mRNA vaccine studies are highly inflammatory in mice. Intradermal and intramuscular injection of these LNPs led to rapid and robust inflammatory responses, characterized by massive neutrophil infiltration, activation of diverse inflammatory pathways, and production of various inflammatory cytokines and chemokines. The same dose of LNP delivered intranasally led to similar inflammatory responses in the lung and resulted in a high mortality rate, with mechanism unresolved. Thus, the mRNA-LNP platforms’ potency in supporting the induction of adaptive immune responses and the observed side effects may stem from the LNPs’ highly inflammatory nature.
Mechanical insights into ribosomal progression overcoming RNA G-quadruplex from periodical translation suppression in cells
G-quadruplexes formed on DNA and RNA can be roadblocks to movement of polymerases and ribosome on template nucleotides. Although folding and unfolding processes of the G-quadruplexes are deliberately studied in vitro, how the mechanical and physical properties of the G-quadruplexes affect intracellular biological systems is still unclear. In this study, mRNAs with G-quadruplex forming sequences located either in the 5′ untranslated region (UTR) or in the open reading frame (ORF) were constructed to evaluate positional effects of the G-quadruplex on translation suppression in cells. Periodic fluctuation of translation suppression was observed at every three nucleotides within the ORF but not within the 5′ UTR. The results suggested that difference in motion of ribosome at the 5′ UTR and the ORF determined the ability of the G-quadruplex structure to act as a roadblock to translation in cells and provided mechanical insights into ribosomal progression to overcome the roadblock.
If you'd like to leave a link to papers we've missed that you feel contribute to the topic above, please provide a link to the paper in the comments below. You must be logged in to comment.
Articles, resources and investigative pieces commissioned by the Future of Health and features authored/collated by our staff are published under the banner of our Editorial team. Views expressed in these articles do not necessarily reflect the views of all our editorial staff. Get in touch at [email protected]
