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The Matryoshka code of COVID-19 mRNA vaccines: overlapping viral sequences?

Authors:
  • Patton State Hospital
  • Progenabiome
Preprints and early-stage research may not have been peer reviewed yet.

Abstract

We read with great interest the paper by Beaudoin CA et al. “Are There Hidden Genes in DNA/RNA Vaccines?”, reporting overlapping sequences between the SARS-CoV-2 spike (S) glycoprotein and two viral genes (1). If translated, the undesired proteins may cause rare, untoward effects, including those recorded in Vaccine Adverse Event Reporting System (VAERS).
The Matryoshka code of COVID-19 mRNA vaccines: overlapping viral
sequences?
Adonis Sfera1, 2*, Sabine Hazan1, Dan O. Sfera1, Zisis Kozlakidis3
1Patton State Hospital, United States, 2University of California,
Riverside, United States, 3International Agency For Research On Cancer
(IARC), France
Submitted to Journal:
Frontiers in Immunology
Specialty Section:
Viral Immunology
Article type:
General Commentary Article
Manuscript ID:
1035520
Received on:
02 Sep 2022
Journal website link:
www.frontiersin.org
We read with great interest the paper by Beaudoin CA et al. “Are There
Hidden Genes in DNA/RNA Vaccines?”, reporting overlapping
sequences between the SARS-CoV-2 spike (S) glycoprotein and two viral
genes (1). If translated, the undesired proteins may cause rare,
untoward effects, including those recorded in Vaccine Adverse Event
Reporting System (VAERS).
These findings are in line with our own research and that of others
however, aside from overlapping genes (OLGs), the S protein also
contains overlapping molecular structures and signals (heptad repeats,
simple sequence repeats, calcium calmodulin kinase II, and prion-like
domains) that can lead to VAERS-recorded pathology (2-6).
Overlapping genetic and structural information are important to
viruses, as they maximize the number of translated proteins derived
from the same genetic information (7). This compact arrangement also
allows for the emergence of mutations without major genetic
restructuring (8). Furthermore, there is evidence that such structures
also regulate gene expression in many viruses (9), including
coronaviruses (10-11)(Fig. 1).
Taking the above viral-derived complication into account, messenger
RNA (mRNA) vaccines encode the full-length S protein that when
expressed on the surface of cells, prompts the generation of
neutralizing antibodies (12). Thus, both OLGs and molecular systems
may be translated too, contributing to vaccine complications and
potential adverse effects.
Fig. 1 The overlapping molecular structures and signals in the S protein of SARS-CoV-2 virus.
Glycosylation is a viral strategy for successfully exploiting host translational machinery.
Vaccination with SARS-CoV-2 S protein lacking glycan shields elicits enhanced responses
therefore, glycosylation may have been altered in mRNA vaccines. RBD (receptor binding
domain), CaMKII (calcium calmodulin kinase II), SSR(simple sequence repeats).
Messenger RNA vaccines, known modifications
To elicit the generation of neutralizing antibodies, exogenously
administered mRNA must be heavily engineered to avoid hydrolysis by
the extracellular RNAases and detection by cytosolic immune sensors
(13-14). Placing the nucleic acid backbone into lipid nanoparticles
(LNPs), hides it from RNAases, while codon optimization, replacing
uridine with N1-methylpseudouridine (m1Ψ), renders the vaccine
undetectable to sensors (15-16). Other adjustments were made in the
untranslated regions (UTRs) and polyadenylated (polyA) tail to protect
and stabilize the vaccine (14)(16-17). Another known change, addition
of two proline residues, maintain the S antigen in prefusion
conformation to augment immune system exposure (18). Moreover,
aside from m1Ψ, codon optimization includes increased the CG content
and possibly G-quadruplex structures to enhance translation (6).
Potential unknown changes
Aside from the reported changes, the mRNA encoded S antigen may
have been engineered further to increase efficacy and translation.
Sense codon reassignment?
Pfizer/BioNTech has published the mRNA vaccine sequences, allowing
scientists and clinicians to compare codons with the wild type S protein.
However, the translated peptides remain proprietary therefore, at this
time, it is not possible to rule out sense codon reassignment or
introduction of unnatural proteins (19). This is important as genetic
code expansion and incorporation of immunogenic noncanonical amino
acids, patented in 2018 (WO2019193416A1), were evaluated for
utilization in genetic vaccines (20). Some unnatural amino acids,
especially homoarginine, was associated with heart disease and sudden
death therefore, these artificial building blocks may in rare occasions
directly contribute to VAERS-recorded events (21-22).
Sugar coating or not?
It is unknown at this time whether the S protein glycans were altered to
increase the efficacy of the mRNA therapeutics. However, vaccine-
elicited neutralizing antibodies exhibit a distinct glycosylation pattern
than post-infection antibodies, indicating possible manipulation (23-
25). This is significant as glycosylation plays a major role in
cardiovascular and endothelial homeostasis, providing a potential link
to VAERS-recorded events (26-27)(Fig. 1).
The S antigen molecular systems
Several biomolecular systems are present in the S protein of SARS-CoV-
2 that when translated may trigger secondary pathways linked to
vaccine adverse effects. These systems include simple sequence
repeats, heptad repeats, calcium calmodulin kinase II, and prion-like
domains (3-6). Translation of these molecular structures may lead to
new viral variants, pathological cell-cell fusion, and defective
proteostasis. These potential links, derived from the viral legacy of
overlapping genetic and structural information will be briefly presented
below:
-Simple sequence repeats (SSRs)
Also called microsatellites, SSRs are present in the genomes of many
viruses, including SARS-CoV-2, accounting for a number of the new,
emerging variants (28-29). Trinucleotide and hexanucleotide repeats
are the most common SSRs that, aside from their role in viral genomes,
contribute to skeletal muscle pathology and neurodegeneration,
possibly explaining vaccine-induced neuropsychiatric and
neuromuscular symptoms (30-31). Interestingly, SSRs are influenced by
the CG content of nucleic acids which is elevated in COVID-19 mRNA
vaccines, linking these therapeutics to the emergence of new variants
(32-33).
The DNA mismatch repair factor, MSH3, previously associated with
trinucleotide repeats, was also found to function as a sensor for G-
quadruplexes therefore, opposing codon optimization (34-35). This is
interesting as a novel study found a proprietary, Moderna-owned,
reverse MSH3 sequence that matches the SARS-CoV-2 furin cleavage
site, suggesting an OLG (36). Indeed, to protect the optimized CG
content and G-quadruplexes, MSH3 may need to be attenuated or
inhibited, explaining the reason this reverse sequence could have been
patented (US-9587003-B2).
-Heptad repeats
There are two heptad repeats in the S protein of SARS-CoV-2 that
assemble into a six-helix bundle to execute membrane fusion (37).
Translation of these structures likely accounts for vaccine-induced
pathological cell-cell fusion, that could result in rare post-vaccination
events, such as giant cell myocarditis (38-39).
-Calcium calmodulin kinase II
Cell-cell fusion can also be promoted by calcium calmodulin kinase II
(CaMKII), a system detected in the S antigen of the SARS-CoV-2 virus
(4). CaMKII may promote post-vaccination pathological syncytia,
probably accounting for VAERS-reported multinucleated giant cells
thyroiditis or myocarditis (4)(40).
-Prion-like domains
The receptor-binding domain (RBD) of the SARS-CoV-2 virus contains a
prion motif that could be translated, leading to pathology (5). Indeed,
post-vaccination Creutzfeldt-Jakob disease (CJD) was reported by two
separate studies, indicating that the prion motif may get translated (41-
42).
In summary
OLG and overlapping molecular structures are common occurrence in
viruses and contribute to a number of biological processes. However,
such overlapping information may also be translated with the vaccine
mRNA, thus inadvertently increasing the odds of pathology. An mRNA
vaccine expressing only the RBD may lower the susceptibility for
adverse effects.
Disclaimer:
Where authors are identified as personnel of the International Agency
for Research on Cancer/WHO, the authors alone are responsible for the
views expressed in this article and they do not necessarily represent the
decisions, policy or views of the International Agency for Research on
Cancer/WHO.
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The CVnCoV (CureVac) mRNA vaccine for SARS-CoV-2 has recently been evaluated in a phase 2b/3 efficacy trial in humans¹. CV2CoV is a second-generation mRNA vaccine with non-modified nucleosides but optimized non-coding regions and enhanced antigen expression. Here we report a head-to-head study of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in nonhuman primates. We immunized 18 cynomolgus macaques with two doses of 12 ug of lipid nanoparticle formulated CVnCoV, CV2CoV, or sham (N=6/group). CV2CoV induced substantially higher binding and neutralizing antibodies, memory B cell responses, and T cell responses as compared with CVnCoV. CV2CoV also induced more potent neutralizing antibody responses against SARS-CoV-2 variants, including the delta variant. Moreover, CV2CoV proved comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. While CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tract. Binding and neutralizing antibody titers correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in nonhuman primates.
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With the success of COVID-19 vaccines, newly created mRNA vaccines against other infectious diseases are beginning to emerge. Here, we review the structural elements required for designing mRNA vaccine constructs for effective in vitro synthetic transcription reactions. The unprecedently speedy development of mRNA vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was enabled with previous innovations in nucleoside modifications during in vitro transcription and lipid nanoparticle delivery materials of mRNA. Recent updates are briefly described in the status of mRNA vaccines against SARS-CoV-2, influenza virus, and other viral pathogens. Unique features of mRNA vaccine platforms and future perspectives are discussed.