Innate Immune Suppression by Sars-cov-2 Mrna Vaccinations: the Role of Gquadruplexes, Exosomes, and Micrornas

Journal Pre-proof

Stephanie Seneff, Greg Nigh, Anthony M. Kyriakopoulos, Peter A. McCullough

PII: S0278-6915(22)00206-X
DOI: https://doi.org/10.1016/j.fct.2022.113008
Reference: FCT 113008
To appear in: Food and Chemical Toxicology

Received Date: 9 February 2022
Revised Date: 3 April 2022
Accepted Date: 8 April 2022

Please cite this article as: Seneff, S., Nigh, G., Kyriakopoulos, A.M., McCullough, P.A., Innate immune
suppression by SARS-CoV-2 mRNA vaccinations: The role of G-quadruplexes, exosomes, and
MicroRNAs, Food and Chemical Toxicology (2022), doi: https://doi.org/10.1016/j.fct.2022.113008.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2022 Published by Elsevier Ltd.

Author Contributions: S.S., G.N and A.K. all contributed substantially to the writing of the
original draft. P.M. participated in the process of editorial revisions.
Journal Pre-proof
Journal Pre-proof
1
Innate Immune Suppression by SARS-CoV-2 mRNA
Vaccinations: The role of G-quadruplexes,
Exosomes, and MicroRNAs

Stephanie Seneff1*, Greg Nigh2, Anthony M. Kyriakopoulos3, and Peter A. McCullough4
1. Senior Research Scientist, Computer Science and Artificial Intelligence Laboratory, MIT,
Cambridge MA USA 02139. seneff@csail.mit.edu.
2. Naturopathic Oncologist, Immersion Health, Portland, OR 97214, USA.
drnigh@immersionhealthpdx.com
3. Director and Head of Research and Development, Nasco AD Biotechnology Laboratory,
Department of Research and Development, Sachtouri 11, 18536, Piraeus, Greece.
antkyriak@gmail.com.
4. Chief Medical Advisor, Truth for Health Foundation, Tucson, AZ USA.
peteramccullough@gmail.com.

* Correspondence: seneff@csail.mit.edu; Tel.: 1-617-901-0442.

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

Keywords: SARS-CoV-2 mRNA vaccines; Type I interferon response; exosomes; Gquadruplexes;
microRNAs; cancer

Highlights

• mRNA vaccines promote sustained synthesis of the SARS-CoV-2 spike protein
• The spike protein is neurotoxic, and it impairs DNA repair mechanisms

Journal Pre-proof

3
• Suppression of type I interferon responses results in impaired innate immunity
• The mRNA vaccines potentially cause increased risk to infectious diseases and cancer
• Codon optimization results in G-rich mRNA that has unpredictable complex effects
Introduction

Vaccination is an endeavor to utilize non-pathogenic material to mimic the immunological
response of a natural infection, thereby conferring immunity in the event of pathogen exposure.
This goal has been primarily pursued through the use of both whole organism and attenuated
virus vaccines. Use of fragments of virus or their protein products, referred to as “subunit
vaccines,” has been more technically challenging [1]. In any event, an implicit assumption behind
the deployment of any vaccination campaign is that the vaccine confers the effects of a ‘benign
infection,’ activating the immune system against future exposure, while avoiding the health
impacts of actual infection.
Much of the literature on this related to COVID-19 suggests that the immune response to mRNAbased
vaccination is similar to natural infection. A preprint study found “high immunogenicity
of BNT162b2 vaccine in comparison with natural infection.” The authors found there to be many
qualitative similarities though quantitative differences [2]. Jhaveri (2021) suggests that mRNA
vaccines do what infection with the virus does: “The protein is produced and presented in the
same way as natural infection” [3]. The U.S. Centers for Disease Control and Prevention (CDC)
makes the case based upon antibody titers generated by prior infection vs. vaccination, in
addition to production of memory B cells, to argue that the immune response to vaccination is
analogous to the response to natural infection [4]. It is this similarity in the humoral immune
response to vaccination vs natural infection, paired with both trial and observational data

Journal Pre-proof
4
demonstrating reduced risk of infection following vaccination, that stands as the justification for
the mass vaccination campaign.
Our paper summarizes the current literature on mRNA and its effects on the molecular biology
within human cells. We recognize that there is a wide range of opinions in this nascent phase of
mRNA technology. Given its widespread deployment ahead of basic work on so many of the
mechanisms we discuss here, we believe that our work is important for providing a broad
understanding of present and future reviews that relate to the burgeoning preclinical molecular
work being done in this area.
In this paper, we explore the scientific literature suggesting that vaccination with an mRNA
vaccine initiates a set of biological events that are not only different from that induced by infection
but are in several ways demonstrably counterproductive to both short- and long-term immune
competence and normal cellular function. These vaccinations have now been shown to
downregulate critical pathways related to cancer surveillance, infection control, and cellular
homeostasis. They introduce into the body highly modified genetic material. A preprint has
revealed a remarkable difference between the characteristics of the immune response to an
infection with SARS-CoV-2 as compared with the immune response to an mRNA vaccine against
COVID-19 [5]. Differential gene expression analysis of peripheral dendritic cells revealed a
dramatic upregulation of both type I and type II interferons (IFNs) in COVID-19 patients, but not
in vaccinees. One remarkable observation they made was that there was an expansion of
circulating hematopoietic stem and progenitor cells (HSPCs) in COVID-19 patients, but this
expansion was notably absent following vaccination. A striking expansion in circulating
Journal Pre-proof
5
plasmablasts observed in COVID-19 patients was also not seen in the vaccinees. All of these
observations are consistent with the idea that the anti-COVID-19 vaccines actively suppress type
I IFN signaling, as we will discuss below. In this paper we will be focusing extensively, though
not exclusively, on vaccination-induced type I IFN suppression and the myriad downstream
effects this has on the related signaling cascade.
Since long-term pre-clinical and Phase I safety trials were combined with Phase II trials, then
phase II and III trials were combined [6]; and since even those were terminated early and placebo
arms given the injections, we look to the pharmacosurveillance system and published reports for
safety signals. In doing so, we find that that evidence is not encouraging. The biological response
to mRNA vaccination as it is currently employed is demonstrably not similar to natural infection.
In this paper we will illustrate those differences, and we will describe the immunological and
pathological processes we expect are being initiated by mRNA vaccination. We will connect these
underlying physiological effects with both realized and yet-to-be-observed morbidities. We
anticipate that implementation of booster vaccinations on a wide scale will amplify all of these
problems.
The mRNA vaccines manufactured by Pfizer/BioNTech and Moderna have been viewed as an
essential aspect of our efforts to control the spread of COVID-19. Countries around the globe have
been aggressively promoting massive vaccination programs with the hope that such efforts might
finally curtail the ongoing pandemic and restore normalcy. Governments are reticent to consider
the possibility that these injections might cause harm in unexpected ways, and especially that
such harm might even surpass the benefits achieved in protection from severe disease. It is now
Journal Pre-proof
6
clear that the antibodies induced by the vaccines fade in as little as 3 to 10 weeks after the second
dose [7], such that people are being advised to seek booster shots at regular intervals [8]. It has
also become apparent that rapidly emerging variants such as the Delta and now the Omicron
strain are showing resistance to the antibodies induced by the vaccines, through mutations in the
spike protein [9]. Furthermore, it has become clear that the vaccines do not prevent transmission
of the disease, but can only be claimed to reduce symptom severity [10]. A study comparing
vaccination rates with COVID-19 infection rates across 68 countries and 2947 counties in the
United States in early September, 2021, found no correlation between the two, suggesting that
these vaccines do not protect from spread of the disease [11]. Regarding symptom severity, even
this aspect is beginning to be in doubt, as demonstrated by an outbreak in an Israeli hospital that
led to the death of five fully vaccinated hospital patients [12]. Similarly, Brosh-Nissimov et.al.
(2021) reported that 34/152 (22%) of fully vaccinated patients among 17 Israeli hospitals died of
COVID-19 [13].
The increasing evidence that the vaccines do little to control disease spread and that their
effectiveness wanes over time make it even more imperative to assess the degree to which the
vaccines might cause harm. That SARS-CoV-2 modified spike protein mRNA vaccinations have
biological impacts is without question. Here we attempt to distinguish those impacts from natural
infection, and establish a mechanistic framework linking those unique biological impacts to
pathologies now associated with vaccination. We recognize that the causal links between
biological effects initiated by mRNA vaccination and adverse outcomes have not been established
in the large majority of cases.
Journal Pre-proof
7
2. Interferons: An Overview with Attention to Cancer Surveillance
Discovered in 1957, interferon (IFN) earned its name with the recognition that cells challenged by
attenuated influenza A virus created a substance that “interfered with” a subsequent infection by
a live virus [14]. IFN is now understood to represent a very large family of immune-modulating
proteins, divided into three types, designated as type I, II, and III based upon the receptors each
IFN interacts with. Type I IFN includes both IFN-α and IFN-β, and this type is the most diverse,
being further divided into seventeen subtypes. IFN-α alone has thirteen subtypes currently
identified, and each of those is further divided into multiple categories [15]. Type I IFNs play a
powerful role in the immune response to multiple stressors. In fact, they have enjoyed clinical
therapeutic value as a treatment option for a variety of diseases and conditions, including viral
infections, solid tumors, myeloproliferative disorders, hematopoietic neoplasms and
autoimmune diseases such as multiple sclerosis [16].
As a group, IFNs play exceedingly complicated and pleiotropic roles that are coordinated and
regulated through the activity of the family of IFN regulatory factors, or IRFs [17]. IRF9 is most
directly involved in anti-viral as well as anti-tumor immunity and genetic regulation [18-20].
Closely related to this are plasmacytoid dendritic cells (pDCs), a rare type of immune cell that
circulate in the blood but migrate to peripheral lymphoid organs during a viral infection. They
respond to a viral infection by sharply upregulating production of type I IFNs. The IFN-α released
in the lymph nodes induces B cells to differentiate into plasmablasts. Subsequently, interleukin-
6 (Il-6) induces plasmablasts to evolve into antibody-secreting plasma cells [21]. Thus, IFNs play
a critical role in both controlling viral proliferation and inducing antibody production. Central to
Journal Pre-proof
8
both antiviral and anticancer immunity, IFN-α is produced by macrophages and lymphocytes
when either is challenged with viral or bacterial infection or encounters tumor cells [22]. Its role
as a potent antiviral therapy has been recognized in the treatment of hepatitis C complications
[23], Cytomegalovirus infection [24], chronic active ebola virus infection [25], inflammatory
bowel disease associated with herpes virus infection [26], and others.
Impaired type I IFN signaling is linked to many disease risks, most notably cancer, as type I IFN
signaling suppresses proliferation of both viruses and cancer cells by arresting the cell cycle, in
part through upregulation of p53, a tumor suppressor gene, and various cyclin-dependent kinase
inhibitors [27,28]. IFN-α also induces major histocompatibility (MHC) class 1 antigen
presentation by tumor cells, causing them to be more readily recognized by the cancer
surveillance system [29,30]. The range of anticancer effects initiated by IFN-α expression is
astounding and occurs through both direct and indirect mechanisms. Direct effects include cell
cycle arrest, induction of cell differentiation, initiation of apoptosis, activation of natural killer
and CD8+ T cells, and others [31].
The indirect anticancer effects are predominantly carried out through gene transcription
activation of the Janus kinase signal transducer and activator of transcription (JAK/STAT)
pathway. IFN-α binding on the cell surface initiates JAK, a tyrosine kinase, to phosphorylate
STAT1 and STAT2 [32]. Once phosphorylated, these STATs form a complex with IRF9, one of a
family of IRFs that play a wide range of roles in oncogene regulation and other cell functions [33].
It is this complex, named IFN-stimulated gene factor 3 (ISGF3), that translocates to the cell
nucleus to enhance the expression of at least 150 genes [31]. IRF9 has been suggested to be the
Journal Pre-proof
9
primary member of the IRF family of proteins responsible for activation of the IFN-α
antiproliferative effects, and that appears to be through its binding to the tumor necrosis factorrelated
apoptosis-inducing ligand (TRAIL) receptor 1 and 2 (TRAIL-R1/2) [34]. IRF7 is another
crucial member of the IRF family of proteins involved early in the response to a viral infection. It
is normally expressed in low amounts but is strongly induced by ISGF3. IRF7 also undergoes
serine phosphorylation and nuclear translocation to further activate the immune response. IRF7
has a very short half-life, so its gene-induction process is transient, perhaps to avoid
overexpression of IFNs [35].
Once TRAIL is bound by IRF9, it is then able to act as a ligand for Death Receptor 4 (DR4) or DR5,
initiating a cascade of events involving production of caspase 8 and caspase 3, and ultimately
triggering apoptosis [36]. Dysregulation of this pathway, through suppression of either IFN-α or
IRF9 and the resulting failure to bind TRAIL-R, has been associated with several hematologic
malignancies [37], and has been shown to increase the metastatic potential in animal models of
melanoma, colorectal cancer, and lymphoma [38].
IFN-α both initiates and orchestrates a wide range of cancer suppressing roles. Dunn et al. (2005)
showed that IFN-α plays an active role in cancer immunoediting, its locus of action being
hematopoietic cells that are “programmed” via IFN-α binding for tumor surveillance [39]. It is
via the exceedingly complex interactions between type I IFNs and IRF7 and IRF9 in particular
that a great deal of antiproliferative effects are carried out. This is evidenced by the large number
of studies showing increased tumor growth and/or metastases associated with a wide number of
cancer types.
Journal Pre-proof
10
For example, Bidwell et al. (2012) found that, among over 800 breast cancer patients, those with
high expression of IRF7-regulated genes had significantly fewer bone metastases, and they
propose assessment of these IRF7-related gene signatures as a way to predict those at greatest
risk [40]. Use of microRNA to target IRF7 expression has also been shown to enhance breast
cancer cell proliferation and invasion in vitro [41]. Zhao et al. (2017) found a similar role for IRF7
in relation to bone metastases in a mouse model of prostate cancer [42]. Regarding the anti-cancer
mechanism behind IRF7 expression, Solis et al. (2006) found that IRF7 induces transcription of
multiple genes and translation of their downstream protein products including TRAIL, IL-15,
ISG-56 and CD80, with the noted therapeutic implications [43].
IRF9, too, has a central role to play in cancer surveillance and prevention. Erb et al. (2013)
demonstrated that IRF9 is the mediator through which IL-6 augments the anti-proliferation
effects of IFN-α against prostate cancer cells [44]. Tian et al. (2018) found IRF9 to be a key negative
regulator of acute myeloid leukemia cell proliferation and evasion of apoptosis [45]. It does so, at
least in part, through acetylation of the master regulatory protein p53.
Both IFN-α and IRF9 are also apparently necessary for the cancer-preventative properties of a
fully functional BRCA2 gene. In a study presented as an abstract at the First AACR International
Conference on Frontiers in Basic Cancer Research, Mittal and Chaudhuri (2009) describe a set of
experiments which show for the first time that BRCA2 expression leads to increased IFN-α
production and augments the signal transduction pathway resulting in the complexing of IRF9,
STAT1 and STAT2 described previously [46]. Two years prior, Buckley et al. (2007) had
established that BRCA1 in combination with IFN-γ promotes type I IFNs and subsequent
Journal Pre-proof
11
production of IRF7, STAT1, and STAT2 [47]. Thus, the exceedingly important cancer regulatory
genes BRCA1 and BRCA2 rely on IRF7 and IRF9, respectively, to carry out their protective effects.
Rasmussen et al. (2021) reviewed compelling evidence that deficiencies of either IRF7 or IRF9 lead
to significantly greater risk of severe COVID-19 illness [48]. Importantly, they also note that
evidence suggests type I IFNs play a singularly important role in protective immunity against
COVID-19 illness, a role that is shared by multiple cytokines in most other viral illnesses
including influenza.
As will be discussed in more detail below, the SARS-CoV-2 spike protein modifies host cell
exosome production. Transfection of cells with the spike protein’s gene and subsequent SARSCoV-
2 spike glycoprotein production results in those cells generating exosomes containing
microRNAs that suppress IRF9 production while activating a range of pro-inflammatory gene
transcripts [49]. Since these vaccines are specifically designed to induce high and ongoing
production of SARS-CoV-2 spike glycoproteins, the implications are ominous. As described
above, inhibition of IRF9 will suppress TRAIL and all its regulatory and downstream apoptosisinducing
effects. IRF9 suppression via exosomal microRNA should also be expected to impair the
cancer-protective effects of BRCA2 gene activity, which depends on that molecule for its activity
as described above. BRCA2-associated cancers include breast, fallopian tube, and ovarian cancer
for women, prostate and breast cancer for men, acute myeloid leukemia in children, and others
[50].
Vaccination has also been demonstrated to suppress both IRF7 and STAT2 [51]. This can be
expected to interfere with the cancer-protective effects of BRCA1 as described above. Cancers
Journal Pre-proof
12
associated with impaired BRCA1 activity include breast, uterine, and ovarian cancer in women;
prostate and breast cancer in men; and a modest increase in pancreatic cancer for both men and
women [52].
Reduced BRCA1 expression is linked to both cancer and neurodegeneration. BRCA1 is a wellknown
breast cancer susceptibility gene. BRCA1 inhibits breast cancer cell proliferation through
activation of SIRT1 and subsequent suppression of the androgen receptor [53]. In a study
conducted by Suberbielle et al. (2015), reduced levels of BRCA1 were found in the brains of
Alzheimer’s patients [54]. Furthermore, experiments with knocking down neuronal BRCA1 in
the dentate gyrus of mice showed that DNA double-strand breaks were increased, along with
neuronal shrinkage and impairments in synaptic plasticity, learning and memory.
Analysis detailed in a recent case study on a patient diagnosed with a rare form of lymphoma
called angioimmunoblastic T cell lymphoma provided strong evidence for unexpected rapid
progression of lymphomatous lesions after administration of the BNT162b2 mRNA booster shot
[55]. Comparisons of detailed metrics for hypermetabolic lesions conducted immediately before
and 21 days after the vaccine booster revealed a five-fold increase after the vaccine, with the postbooster
test revealing a 2-fold higher activity level in the right armpit compared to the left one.
The vaccine had been injected on the right side. It is worth pointing out in this regard that
lymphoid malignancies have been associated with suppression of TRAIL R1 [56].
Given the universally recognized importance of optimally functioning BRCA1/2 for cancer
prevention and given the central role of the TRAIL signal transduction pathway for additional
cancer surveillance, the suppression of IRF7 and IRF9 through vaccination and subsequent SARSJournal
Pre-proof
13
CoV-2 spike glycoprotein production is extremely concerning for long-term cancer control in
injected populations.
3. Considerations in the Design of mRNA Vaccines
Over the last three decades, the mRNA technological platform aimed to develop effective and
safe nucleic acid therapeutic tools is said to have overcome serious obstacles on the coded product
instability, the overwhelming innate immunogenicity, and on the delivery methodologies [57].
One of the major success stories of mRNA use as a genetic vaccination tool is on the introduction
of robust immunity against cancer [58]. In addition, the potential of mRNAs to restore or replace
various types of proteins in cases of rare genetic metabolic disorders like Fabry disease has
offered great potential therapeutic alternatives where no other medication has proved to be
successful [59]. However, in the case of mRNA use as genetic vaccines against infectious diseases,
the preliminary safety investigations seemed to be premature for a world-wide use in the general
population [57,60].
Although there are essential epitopes on other SARS-CoV-2 proteins where an antibody response
could have provided essential immunogenicity, well known from SARS-CoV-1 [61], the primary
goal of the developers of the SARS-CoV-2 mRNA vaccines was to design a vaccine that could
induce a robust antibody response exclusively to the spike glycoprotein. Such antibodies,
specially IgA in the nasopharynx, should cause the invading viruses to be quickly cleared before
they could invade host cells, thus arresting the disease process early on. As stated succinctly by
Kaczmarek et. al. (2021) [62]:
Journal Pre-proof
14
“The rationale behind vaccination is to provide every vaccinated person with protection against
the SARS‐CoV‐2 virus. This protection is achieved by stimulating the immune system to produce
antibodies against the virus and to develop lymphocytes that will retain memory and the ability
to fight off the virus for a long time.” However, since vaccination is given parenterally, IgG is the
principal antibody class that is raised against the SARS-CoV-2 spike glycoprotein, not IgA [63].
Vaccines generally depend upon adjuvants such as aluminum and squalene to provoke immune
cells to migrate to the injection site immediately after vaccination. In the history of mRNA vaccine
development, it was initially hoped that the mRNA itself could serve as its own adjuvant. This is
because human cells recognize viral RNA as foreign, and this leads to upregulation of type I IFNs,
mediated via toll like receptors such as TLR3, TLR7 and TLR8 [64].
However, with time it became clear that there were problems with this approach, both because
the intense reaction could cause flu-like symptoms and because IFN-α could launch a cascade
response that would lead to the breakdown of the mRNA before it could produce adequate
amounts of SARS-CoV-2 spike glycoprotein to induce an immune response [65]. A breakthrough
came when it was discovered experimentally that the mRNA coding for the spike protein could
be modified in specific ways that would essentially fool the human cells into recognizing it as
harmless human RNA. A seminal paper by Karikó et al. (2005) demonstrated through a series of
in vitro experiments that a simple modification to the mRNA such that all uridines were replaced
with pseudouridine could dramatically reduce innate immune activation against exogenous
mRNA [64]. Andries et al. (2015) later discovered that 1-methylpseudouridine as a replacement
for uridine was even more effective than pseudouridine and could essentially abolish the TLR
Journal Pre-proof
15
response to the mRNA, preventing the activation of blood-derived dendritic cells [66]. This
modification is applied in both the mRNA vaccines on the market [67].
Rather prophetically, the extensive review by Forni G et al., 2021, has raised serious questions
about the development of innate immunity by the mRNA SARS-CoV-2 genetic vaccinations [68].
As the authors declared: “Due to the short development time and the novelty of the technologies
adopted, these vaccines will be deployed with several unresolved issues that only the passage of
time will permit to clarify.” Subsequently, the authors recommended including certain molecules
such as the long pentraxin PTX3 as representative humoral immunity markers to assess the early
activation of innate immune mechanisms and the underlying reactogenicity under the
BIOVACSAFE consortium protocols [68,69]. However, to the best of our knowledge these safety
protocols have not been included in the assessment of induced innate immunity by the SARSCoV-
2 mRNA genetic vaccines [70].
In this regard, in the case of SARS-CoV-2 BNT162b2 mRNA vaccine, unlike the immune response
induced by natural SARS-CoV-2 infection, where a robust interferon response is observed, those
vaccinated with BNT162b2 mRNA vaccines developed a robust adaptive immune response
which was restricted only to memory cells, i.e., an alternative route of immune response that
bypassed the IFN mediated pathways [70]. Furthermore, due to subsequent mutations in the
SARS-CoV-2 spike protein, there is a substantial loss of neutralising antibodies induced by the
BNT162b2 mRNA vaccine compared to those conferred by the SARS-CoV-2 mutants alone [71].
In that respect, as vaccine developers admit: “Vaccine RNA can be modified by incorporating 1-
methylpseudouridine, which dampens innate immune sensing and increases mRNA translation
Journal Pre-proof
16
in vivo.” [70,72]. Bearing in mind the multiple mutations that SARS-CoV-2 develops, as for
example in the Brazil outbreaks [73], an effective immune response that prevents the spread of
SARS-CoV2 mutants necessarily involves the development of a robust IFN-I response as a part
of the innate immune system. This response also requires the involvement of a functional NF-κB
response. Unfortunately, spike glycoprotein overexpression dismantles the NF-κB pathway
responses, and this molecular event can be augmented by spike-protein-coding mRNAs [74,75].
For successful mRNA vaccine design, the mRNA needs to be encapsulated in carefully
constructed particles that can protect the RNA from degradation by RNA depolymerases. The
mRNA vaccines are formulated as lipid nanoparticles containing cholesterol and phospholipids,
with the modified mRNA complexed with a highly modified polyethylene glycol (PEG) lipid
backbone to promote its early release from the endosome and to further protect it from
degradation [76]. The host cell’s existing biological machinery is co-opted to facilitate the natural
production of protein from the mRNA through endosomal uptake of a lipid particle [76]. A
synthetic cationic lipid is added as well, since it has been shown experimentally to work as an
adjuvant to draw immune cells to the injection site and to facilitate endosomal escape. De
Beuckelaer et al. (2016) observed that “condensing mRNA into cationic lipoplexes increases the
potency of the mRNA vaccine evoked T cell response by several orders of magnitude.” [65]
Another important modification is that they replaced the code for two adjacent amino acids in
the genome with codes for proline, which causes the spike glycoprotein to stay in a prefusion
stabilized form [77].
Journal Pre-proof
17
The SARS-CoV-2 spike glycoprotein mRNA is further “humanized” with the addition of a
guanine-methylated cap, 3’ and 5’ untranslated regions (UTRs) copied from those of human
proteins, and finally a long poly(A) tail to further stabilize the RNA [74]. In particular, researchers
have cleverly selected the 3’UTR taken from globins which are produced in large quantities by
erythrocytes, because it is very effective at protecting the mRNA from degradation and
maintaining sustained protein production [78]. This is to be expected, since erythrocytes have no
nucleus, so they are unable to replace the mRNAs once they are destroyed. Both the Moderna
and the Pfizer vaccines adopted a 3’UTR from globins, and the Pfizer vaccine also uses a slightly
modified globin 5’UTR [79]. De Beuckelaer et al. (2016) aptly summed up the consequences of
such modifications as follows: “Over the past years, technical improvements in the way IVT [in
vitro transcribed] mRNAs are prepared (5′ Cap modifications, optimized GC content, improved
polyA tails, stabilizing UTRs) have increased the stability of IVT mRNAs to such extent protein
expression can now be achieved for days after direct in vivo administration of the mRNA.” [65]
However, the optimized analogue cap formation of synthetic mRNAs inevitably forces the
recipient cells to undergo a cap-dependent prolonged translation, ignoring homeostatic demands
of cellular physiology [74]. The cap 2’ O methylation carried out by cap 2’ O methyltransferase
(CMTR1) serves as a motif that marks the mRNA as “self,” to prevent recognition by IFN-induced
RNA binding proteins [80]. Thus, the mRNA in the vaccines, equipped with the cap 2’ O
methylation motif, evades detection as a viral invasion. Furthermore, the overwhelming impetus
for cells to perform a single and artificial approach to translation according to the robust capping
and synthetic methylations of mRNAs in vaccines is fundamentally associated with disease
Journal Pre-proof
18
progression due to differential rather than normal signaling of pattern recognition receptors
(PRRs) [81].
The regulatory process controlling mRNA translation is extremely complex, and it is highly
disturbed in the context of mRNA vaccines [74,81]. Briefly, the idea is for mRNA vaccines to
achieve the intended goal (i.e., production of the modified spike protein) through a stealth
strategy that bypasses the natural immunological response to RNA-type viral infection. Injected
lipid nanoparticles containing mRNA are brought to the cell interior via endocytosis. The mRNA
escapes its lipid carrier and migrates to the ribosome, where it is abundantly translated into its
final protein product, following an optimized program for producing large quantities of a specific
protein over an extended period of time. These modified SARS-CoV-2 spike glycoproteins then
follow one of three primary pathways. Some are proteolytically degraded and fragments are
bound by MHC class I molecules for surface presentation to cytotoxic T-cells. A second pathway
has those same spike glycoprotein fragments bind MHC class II molecules, move to the cell
surface, and activate T-helper cells. A final pathway has soluble spike glycoproteins extruded
from the cell in exosomes, where they can be recognized by B-cell-activated spike-glycoproteinspecific
antibodies [82].
A recent early-release study has found that the mRNA in the COVID-19 vaccines is present in
germinal centers in secondary lymphoid tissue long after the vaccine is administered, and that it
continues to synthesize spike glycoprotein up to at least sixty days post-vaccination [83]. This
suggests that immune cells taking up the mRNA in the arm muscle migrate into the lymph system
to the lymph nodes, presumably in order to expose B-cells and T-cells to the toxic antigen. The
Journal Pre-proof
19
persistence of the mRNA in the lymph nodes and its sustained synthesis of SARS-CoV-2 spike
glycoprotein reflect the clever engineering involved in the mRNA technology, as described above.
In the end, it is through utilization of nanolipids and sophisticated mRNA technology that the
normal immune response to exogenous RNA is evaded in order to produce a strong antibody
response against an exogenous RNA virus.
4. GC enrichment and potential G4 (pG4) structures in vaccine mRNAs
Recently, members of our team investigated possible alterations in secondary structure of
mRNAs in SARS-CoV-2 vaccines due to codon optimization of synthetic mRNA transcripts [84].
This study has shown that there is a significant enrichment of GC content in mRNAs in vaccines
(53% in BNT162b2 and 61% in Moderna mRNA-1273) as compared to the native SARS-CoV-2
mRNA (36%). The enriched GC content of mRNAs is the result of codon optimization performed
during the development of the mRNAs used in SARS-CoV-2 vaccines, apparently without
determining the effect on secondary structures, particularly the Guanine quadruplex (G
quadruplex) formation [84].
Codon optimization describes the production of synthetic, codon-optimized polypeptides and
proteins used in biotechnology therapeutics (such as the synthetic mRNAs used for SARS-CoV-2
vaccination). The altered codon assignments within the mRNA template dramatically increase
the quantity of polypeptides and/or proteins produced [85]. Synonymous codon replacement also
results in a change in the multifunctional regulatory and structural roles of resulting proteins [86].
For this reason, codon optimization has been cautioned against due to its consequent changes
causing perturbation in the secondary conformation of protein products with potentially
Journal Pre-proof
20
devastating effects on their resulting immunogenicity, efficacy and function [87,88]. Notably,
various human diseases are the result of synonymous nucleotide polymorphisms [89].
In an experiment where GC-rich and GC-poor versions of mRNA transcripts for heat shock
protein 70 were configured in the context of identical promoters and UTR sequences, it was found
that GC-rich genes were expressed several-fold to over a hundred-fold more efficiently than their
GC-poor counterparts [90]. This is partly because all of the preferred mammalian codons have G
or C nucleotides in the third position. It is also well documented that AU-rich elements in the 3’
UTRs can destabilize mRNA [91]. What may be of particular concern is the fact that GC
enrichment content in vaccine mRNAs results in an enhanced ability for potential G-quadruplex
(pG4) formations in these structures, and this could cause onset of neurological disease [92].
Remarkably, the human prion protein (PrP) genetic sequence contains multiple G4 forming
motifs, and their presence may form the missing link in the initial conversion of PrP to the
misfolded form, PrPsc [93]. PrP binding to its own mRNA may be the seed that causes the protein
to misfold. This observation is particularly concerning in light of the fact that the SARS-CoV-2
spike glycoprotein has prion-like characteristics [94].
On the one hand, the GC content has a key role in the modulation of translation efficiency and
control of mRNA expression in mammals [95]. Especially during translation initiation, the GC
content operating as a cis-acting mRNA element orchestrates the 43S ribosomal pre-initiation
complex attachment and thereafter the assembly of the eukaryotic translation initiation factor 4EF
(eIF4F) complex. One representative example of this system in action is the regulation of α and β
globin mRNA expression through their 5’ untranslated regions (5’UTRs) [95].
Journal Pre-proof
21
On the other hand, the presence of pG4s in RNAs is implicated in cancer biology as key
determinants of the regulation of G4 RNA binding proteins such as helicase [96]. Generally, the
G-quadruplexes in RNAs have essential roles in a) the regulation of gene expression, b) the
localization of ribonuclear proteins, c) the mRNA localization and d) the regulation of protooncogene
expression [97].
Regarding SARS-CoV-2, relevant studies reveal overwhelming similarities between SARS-CoV-
2 pG4s, including in RNA coding for SARS-CoV-2 spike glycoprotein, and those sequenced in
the human transcriptome [98]. Thus, it can be inferred that synthetic mRNAs in vaccines carrying
more pG4 structures in their coding sequence for SARS-CoV-2 spike glycoprotein will amplify
and compound the potential post-transcriptional disorganization due to G4-enriched RNA
during natural SARS-CoV-2 infection. Moreover, the cellular nucleic acid binding protein
(CNBP), which is the main cellular protein that binds to the SARS-CoV-2 RNA genome in humaninfected
cells [99], binds to and promotes the unfolding of SARS-CoV-2 G4s formed by both
positive and negative sense template strands of the SARS-CoV-2 RNA genome. A similar
modulation of CNBP on vaccine mRNA G4s and promotion of G4 equilibrium towards unfolded
conformations create favorable conditions for miRNA binding, and this will have a direct impact
on miRNA-dependent regulation of gene expression [100].
The negative-sense RNAs are intermediate molecules produced by the replicase transcriptase
complex (RTC) formed by the nonstructural proteins of coronaviruses (including SARS-CoV-2)
to provide efficiency in replication and transcription [101,102]. This, however, introduces another
potentially serious complication associated with vaccination. Co-infection with other negative
Journal Pre-proof
22
sense RNA viruses such as hepatitis C [103] or infection by other coronaviruses contemporaneous
with vaccination periods would provide the necessary machinery of RTC to reproduce negative
sense intermediates from synthetic mRNAs and therefore amplify the presence of pG4s by
negative sense templates. This would result in further epitranscriptomic dysregulation [104].
Summarizing the topic to this point, the enrichment of GC content in vaccine mRNA will
inevitably lead to an increase in the pG4 content of the vaccines. This, in turn, will lead to
dysregulation of the G4-RNA-protein binding system and a wide range of potential diseaseassociated
cellular pathologies including suppression of innate immunity, neurodegeneration,
and malignant transformation [96].
Concerning the post translational dysregulation due to emergence of new G4 structures
introduced by vaccination, one other important issue related to miRNA regulation and pG4s
arises. In miRNA structures, hundreds of pG4 sequences are identified [105]. In their unfolded
conformation, as during binding to their respective targets in 3’ to 5’ sequences of mRNAs,
miRNAs switch off the translation of their respective target mRNA. Alternatively, when in the
presence of a G4 ligand, the translation of their target mRNAs is promoted [106]. Moreover, a
vast number of putative miRNA binding sites overlap with G4s in 3’ UTRs of mRNAs as there
are at least 521 specific miRNAs that are predicted to bind to at least one of these G4s. Overall,
44,294 G4-miRNA potential binding sites have been traced to possess putative overlapping G4s
in humans [100].
As described elsewhere, during the cellular translation of vaccine mRNAs, an increased assembly
of a number of RNA binding protein helicases, such as eIF4A bound to eIF4G, will occur [74]. The
Journal Pre-proof
23
presence of increased pG4s in synthetic mRNAs can potentially amplify binding of RNA binding
proteins and miRNAs. This form of molecular crowding of protein components (helicases) with
great affinity for G4 binding [100] will decrease the number of RNA binding proteins binding G4s
normally available for miRNA regulation. This loss of RNA binding proteins as well as miRNA
availability for regulation by binding to G4s can dramatically alter the translational regulation of
miRNAs present in cells and thereby disrupt essential regulation of oncogene expression. An
example is the p16-dependent regulation of the p53 tumor suppressor protein [100,107].
This process is exceedingly complicated yet tantamount to cellular homeostasis. So, again, it
merits summarizing. If pG4s accumulate, as would be expected with an increased amount of GC
content in the vaccine mRNA, this would have an effect of increasing potential G4 structures
available during translation events and this can affect miRNA post-transcriptional regulation.
This, in turn, would either favor greater expression of the oncogenes related to a range of cancers,
or drive cells towards apoptosis and cell death [108]. The case study described earlier in this paper
strongly supports the hypothesis that these injections induce accelerated lymphoma progression
in follicular B cells [55].
miRNA binding recognition patterns are imperfectly complementary to their target regions, and
for this reason they are referred to as “master regulators,” since one miRNA affects a plethora of
different targets [105]. The multitude of pG4s in the mRNA of the vaccine would predictably act
as decoys, distracting miRNAs from their normal function in regulating human protein
expression. The increase in G4 targets due to the vaccine would decrease the availability of
miRNAs to target human-expressed G4s for regulation of gene expression. This can result in
Journal Pre-proof
24
downregulation of miRNA expression which is implicated in cardiovascular pathology [109],
onset of neurodegeneration [110], and/or cancer progression [111].
In most respects within epitranscriptomic machinery, miRNAs are involved in translation
repression. One example, vital for cellular normal housekeeping, is that of Mouse double minute
2 homolog (MDM2), a physical negative regulatory protein of p53. P53 itself is considered the
master regulator of the cellular tumor suppression network of genes. P16 controls the expression
of many miRNAs, and, via miR-141 and mIR-146b-5p binding to MDM2 mRNA, it induces the
negative regulation of MDM2, thus enabling p53 ubiquitination and promotion of cell survival
upon DNA damage events [107]. Dysregulation of miRNAs that control MDM2 suppression of
p53 would predictably lead to an increased risk to a range of cancers [112].
5. Type I IFNs and COVID-19
Type I IFNs play an essential role in fighting viral infections, and deficiencies in type I IFN
signaling have been associated with poor outcomes from COVID-19 in multiple studies. These
cases are often associated with autoantibodies to type I IFNs. As reviewed below, type I IFNs
have been used with some success in treating severe COVID-19, particularly if administered very
early in the disease process. If, as argued above, the mRNA vaccines interfere with type I
signaling, this could lead to increased susceptibility to COVID-19 in the two weeks following the
first vaccine, before an antibody response has been initiated.
Cells infected with a virus detect the presence of virus replication through a number of pattern
recognition receptors (PRRs), which serve as sentinels sensing aberrant RNA structures that often
form during viral replication. These receptors respond by oligomerizing and subsequently
Journal Pre-proof
25
inducing type I IFNs, ultimately upregulating a large number of proteins involved in suppressing
viral proliferation [113].
A multi-author study by researchers in Paris, France, involving a cohort of 50 COVID-19 patients
with varying degrees of disease severity, revealed that patients with severe disease were
characterized by a highly impaired type I IFN response [114]. These patients had essentially no
IFN-β and low IFN-α production and activity. This was associated with a persistent blood viral
load and an exacerbated inflammatory response, characterized by high levels of tumor necrosis
factor α (TNF-α) and Il-6. The authors proposed type I IFN therapy as a potential treatment
option. A paper by several researchers in the United States also identified a unique and
inappropriate inflammatory response in severe COVID-19 patients, characterized by low levels
of both type I and type III IFNs along with elevated chemokines and elevated expression of Il-6
[115].
Type I IFNs have even been proposed as a treatment option for severe COVID-19. In a hamster
model, researchers exposed hamsters to SARS-CoV-2 and induced an inflammatory response in
the lungs and systemic inflammation in distal tissues. They found that intranasal administration
of recombinant IFN-α resulted in a reduced viral load and alleviation of symptoms [116]. A
retrospective cohort study of 446 COVID-19 patients determined that early administration of IFN-
α2b was associated with reduced in-hospital mortality. However, late IFN therapy increased
mortality and delayed recovery, revealing that early administration of interferon therapy is
essential for a favorable response [117].
Journal Pre-proof
26
A surprising number of people have neutralizing autoantibodies against type I IFNs, although
the underlying etiology of this phenomenon is not understood. A study using longitudinal
profiling of over 600,000 peripheral blood mononuclear cells and transcriptome sequencing from
54 patients with COVID-19 and 26 controls found a notable lack of type I IFN-stimulated gene
responses in myeloid cells from patients with critical disease [118]. Neutralizing autoantibodies
against type I IFNs were found in 19% of patients with critical disease, 6% of patients with severe
disease, and 0% of patients with moderate disease. Another study based in Madrid, Spain
revealed that 10% of patients with severe COVID-19 disease had autoimmune antibodies to type
I IFNs [119]. A multi-author study based in France found that COVID-19 mortality was
significantly more frequent in patients with neutralizing autoantibodies against type I interferon
than those without neutralizing antibodies (55% vs. 23%) [120]. Finally, Stertz and Hale (2021)
note that, whether due to autoantibodies or perhaps loss-of-function polymorphisms associated
with interferon system genes, deficiencies in interferon production are associated with as many
as 15% of all life-threatening COVID-19 cases [121].
6. Are the methylation strategies for cellular housekeeping generally omitted by vaccine
mRNAs?
Methylation of mRNAs has been evolutionarily devised to control translation of transcripts and
therefore expression of genes by a complex cascade of methylator (writers), de-methylator
(eraser) and reader proteins. Adenosine methylation is the most abundant epitranscriptomic
mRNA modification, and it occurs at multiple sites across the mRNA molecule [122]. A key
Journal Pre-proof
27
methylation of adenosine “N6-methyladenosine (m6A)” specifically in the 5′ UTR of mRNAs
regulates normal cell physiology, the inflammatory response and cancer progression. The role
and mechanisms of m6A in human disease is extensive, and it is excellently covered in other
comprehensive reviews [123,124]. Foremost among these, the SARS-CoV-2 molecular vaccination
induces cell stress conditions, as is described by the elevated NF-κB signaling after vaccination
[51,125].
Under conditions of cellular stress, which can be induced by a viral infection or disease states
such as cancer, m6A mediates mRNAs to undergo translation preferentially in a cap-independent
way [126]. As discussed previously, this is opposite to the impact of mRNA SARS-CoV-2
vaccination, which drives cells toward a cap-dependent translation. Furthermore, under
diversified conditions of cellular stress, there is an overwhelming induction of transcriptomewide
addition of m6A that causes an increased number of mRNAs to possess 5’UTRs enriched
with m6A [126].
Eukaryotic translation initiation factor 4E (eIF4E) is the initial mRNA cap-binding protein that
directs ribosomes to the cap structure of mRNAs, in order to initiate translation into protein. The
dependence on cap-dependent translation of vaccine mRNAs will consume a surplus of eIF4E
availability needed to translate an unnaturally high number of synthetic mRNAs. However, capindependent
translation takes place without requiring eIF4E to be bound to eIF4F. The
competition for ribosomes will shift towards the cap-independent translation of transcripts, since
the mRNAs undergoing cap-independent translation are equipped, apart from internal ribosome
Journal Pre-proof
28
entry sites (IRES), with special binding motifs that bind to factors that actively recruit mRNAs to
the ribosome cap-independent translational enhancers (CITEs) [127].
Furthermore, this also means that eIF4E, which is a powerful oncogene regulator and cell
proliferation modulator, will sustain its activities by this competition for an unnaturally
prolonged period of time, trying to counterbalance the competition between robustly-capped
mRNAs in vaccines and IRES-containing mRNAs [74,128]. This type of condition results in
dysregulation of co-transcriptional m6A mRNA modifications and seriously links to molecular
progressions of various cancers [129], as well as creating predisposing conditions for subsequent
viral infections [128].
We next consider the impact of mRNA-vaccination-derived SARS-CoV-2 spike glycoprotein on
the cellular IFN system via massive exosome production.
7. Exosomes and MicroRNAs
An important communication network among cells consists of extracellular vesicles (EVs) that
are constantly released by one cell and later taken up by another cell, which could be in a distant
organ. Small vesicles known as exosomes, formed inside endosomes, are similar in size to viruses,
and are released through exocytosis into the extracellular space to subsequently circulate
throughout the body [130]. Exosomes can deliver a diverse collection of biologically active
molecules, including mRNA, microRNAs, proteins, and lipids [131]. During a viral infection,
infected cells secrete large quantities of exosomes that act as a communication network among
the cells to orchestrate the response to the infection [132].
Journal Pre-proof
29
In a collaborative effort by a team of researchers from Arizona and Connecticut, it was found that
people who were vaccinated with the mRNA vaccines acquired circulating exosomes containing
the SARS-CoV-2 spike glycoprotein by day 14 following vaccination [133]. They also found that
there were no circulating antibodies to the spike glycoprotein fourteen days after the first vaccine.
After the second vaccine, however, the number of circulating spike-glycoprotein-containing
exosomes increased by up to a factor of 12. Furthermore, antibodies first appeared on day 14. The
exosomes presented spike glycoprotein on their surface, which, the authors argued, facilitated
antibody production. When mice were exposed to exosomes derived from vaccinated people,
they developed antibodies to the spike glycoprotein. Interestingly, following peak expression, the
number of circulating spike-glycoprotein-containing exosomes decreased over time, in step with
the decrease in the level of antibodies to the spike glycoprotein.
Exosomes exist as a part of the mRNA decay mechanism in close association under stress
conditions with stress granules (SGs) and P-bodies (PBs) [134,135]. Under conditions of vaccinemRNA-
induced translation, which could be called “excessive dependence on cap-dependent
translation,” there is an obvious resistance to promotion and assembly of the large decapping
complex [74], and therefore resistance against physiological mRNA decay processes [134]. This
would mean that the fate of particular synthetic mRNAs that otherwise would be determined by
the common cellular strategy for mRNA turnover involving messenger ribonucleinproteins
(mRNPs) is being omitted [136].
Furthermore, under conditions of over-reliance on cap-dependent translation by the synthetic
mRNAs in SARS-CoV-2 vaccines [74], many native mRNAs holding considerable IRES and
Journal Pre-proof
30
specific methylations (m6A) in their structure will favorably choose cap-independent translation,
which is strongly linked to mRNA decay quality control mechanisms [129]. In this sense,
considerable deadenylated mRNA products as well as products derived from mRNA metabolism
(decay) are directly linked to exosome cargoes [136].
An example of dependence on cap-dependent translation is described in T-cell acute
lymphoblastic leukaemia (T-ALL). Due to mechanistic target of rapamycin C (mTORC)-1 overfunctioning
in T-ALL, the cells are driven completely towards cap-dependent translation [137].
An analogous condition is described by Kyriakopoulos and McCullough (2021) [74].. Even in this
highly aggressive cancerous state, during inhibition of cap-dependent translation in T-ALL cells,
there is a rapid reversion to cap-independent translation [137]. Similarly, a picornavirus infection
[138] drives cells towards cap-independent translation due to inhibition of components of eIF4F
complex and pluralism of IRES in viral RNA.
In humans, there is an abundance of mostly asymptomatic picornavirus infections like the Safford
Virus with an over 90% seroprevalence in young children and adults [139]. In either case, whether
an apoptotic event due to a stress-like condition[140] or an mRNA-cap-driven-like carcinomatous
effect [141], the miRNA levels will be increased due to the increased epitranscriptomic
functioning and enhanced mRNA decay. Because of the high demand for gene expression, high
levels of certain miRNAs will be expected to be contained in exosomes via P bodies [142].
Also, under conditions of overwhelming production of SARS-CoV-2 spike glycoprotein due to
SARS-CoV-2 molecular vaccination, it would of course be expected that a significant proportion
Journal Pre-proof
31
of over-abundant intracellular spike glycoproteins would also be exported via exosome cargoes
[143].
Mishra and Banerjea [49] investigated the role of exosomes in the cellular response of SARS-CoV-
2 Spike-transfected cells. They wrote in the abstract:
“We propose that SARS-CoV-2 gene product, Spike, is able to modify the host exosomal
cargo, which gets transported to distant uninfected tissues and organs and can initiate a
catastrophic immune cascade within Central Nervous System (CNS).”
Their experiments involved growing human HEK293T cells in culture and exposing them to
SARS-CoV-2 spike gene plasmids, which induced synthesis of spike glycoprotein within the cells.
They found experimentally that these cells released abundant exosomes housing spike
glycoprotein along with specific microRNAs. They then harvested the exosomes and transferred
them to a cell culture of human microglia (the immune cells that are resident in the brain). They
showed that the microglia readily took up the exosomes and responded to the microRNAs by
initiating an acute inflammatory response. The role of microglia in causing neuroinflammation
in various viral diseases, such as Human Immunodeficiency Virus (HIV), Japanese Encephalitis
Virus (JEV), and Dengue, is well established. They proposed that long-distance cell-cell
communication via exosomes could be the mechanism by which neurological symptoms become
manifest in severe cases of COVID-19.
In further exploration, the authors identified two microRNAs that were present in high
concentrations in the exosomes: miR-148a and miR-590. They proposed a specific mechanism by
Journal Pre-proof
32
which these two microRNAs would specifically disrupt type I interferon signaling, through
suppression of two critical proteins that control the pathway: ubiquitin specific peptidase 33
(USP33) and IRF9. Phosphorylated STAT1 and STAT2 heterodimers require IRF9 in order to bind
IFN-stimulated response elements, and therefore IRF9 plays an essential role in the signaling
response. The authors showed experimentally that microglia exposed to the exosomes extracted
from the HEK293 culture had a 50% decrease in cellular expression of USP33 and a 60% decrease
in IRF9. They further found that miR-148a specifically blocks USP33 and miR-590 specifically
blocks IRF9. USP33 removes ubiquitin from IRF9, and in so doing it protects it from degradation.
Thus, the two microRNAs together conspire to interfere with IRF9, thus blocking receptor
response to type I interferons.
A study by de Gonzalo-Calvo et. al. (2021) looked at the microRNA profile in the blood of COVID-
19 patients and their quantitative variance based upon disease severity [144]. Multiple miRNAs
were found to be up- and down-regulated. Among these was miR-148a-3p, the guide strand
precursor to miR-148a. However, miR-148a itself was not among the microRNAs catalogued as
excessive or deficient in their study, nor was miR-590. It appears from these findings that miR148a
and miR-590 and their inflammatory effects are unique to vaccination-induced SARS-CoV-2 spike
glycoprotein production.
Tracer studies have shown that, following injection into the arm muscle, the mRNA in mRNA
vaccines is carried into the lymph system by immune cells and ultimately accumulates in the
spleen in high concentrations [145]. Other studies have shown that stressed immune cells in
germinal centers in the spleen release large quantities of exosomes that travel to the brain stem
Journal Pre-proof
33
nuclei along the vagus nerve (as reviewed in Seneff and Nigh (2021) [146]). The vagus nerve is
the 10th cranial nerve and it enters the brainstem near the larynx. The superior and recurrent
laryngeal nerves are branches of the vagus that innervate structures involved in swallowing and
speaking. Lesions in these nerves cause vocal cord paralysis associated with difficulty swallowing
(dysphagia) difficulty speaking (dysphonia) and/or shortness of breath (dyspnea) [147,148]. We
will return to these specific pathologies in our review of VAERS data below.
HEK293 cells were originally derived from cultures taken from the kidney of a human fetus
several decades ago and immortalized through infection with adenovirus DNA. While they were
extracted from the kidney, the cells show through their protein expression profile that they are
likely to be of neuronal origin [149]. This suggests that neurons in the vagus nerve would respond
similarly to the SARS-CoV-2 spike glycoprotein. Thus, the available evidence strongly suggests
that endogenously produced SARS-CoV-2 spike glycoprotein creates a different microRNA
profile than does natural infection with SARS-CoV-2, and those differences entail a potentially
wide range of deleterious effects.
A central point of our analysis below is the important distinction between the impact of
vaccination versus natural infection on type I IFN. While vaccination actively suppresses its
production, natural infection promotes type I IFN production very early in the disease cycle.
Those with preexisting conditions often exhibit impaired type I IFN signaling, which leads to
more severe, critical, and even fatal COVID-19. If the impairment induced by the vaccine is
maintained as antibody levels wane over time, this could lead to a situation where the vaccine
Journal Pre-proof
34
causes a more severe disease expression than would have been the case in the absence of the
vaccine.
Another expected consequence of suppressing type I IFN would be reactivation of preexisting,
chronic viral infections, as described in the next section.
8. Impaired DNA Repair and Adaptive Immunity
The immune system and the DNA repair system are the two primary systems that higher
organisms rely on for defense against diverse threats, and they share common elements. Loss of
function of key DNA repair proteins leads to defects in repair that inhibit the production of
functional B and T cells, resulting in immunodeficiency. Non-homologous end joining (NHEJ)
repair plays a critical role in lymphocyte-specific V(D)J recombination, which is essential for
producing the highly diverse repertoire of B-cell antibodies in response to antigen exposure [75].
Impaired DNA repair is also a direct pathway towards cancer.
A paper published by Liu et al. in 2021 monitored several parameters associated with immune
function in a cohort of patients by conducting single-cell mRNA sequencing of peripheral blood
mononuclear cells (PBMCs) harvested from the patients before and 28 days after the first injection
of a COVID-19 vaccine based on a weakened version of the virus [51]. While these vaccines are
different from the mRNA vaccines, they also work by injecting the contents of the vaccine into
the deltoid muscle, bypassing the mucosal and vascular barriers. The authors found consistent
alteration of gene expression following vaccination in many different immune cell types.
Observed increases in NF-κB signaling and reduced type I IFN responses were further confirmed
by biological assays. Consistent with other studies, they found that STAT2 and IRF7 were
Journal Pre-proof
35
significantly downregulated 28 days after vaccination, indicative of impaired type I IFN
responses. They wrote: “Together, these data suggested that after vaccination, at least by day 28,
other than generation of neutralizing antibodies, people’s immune systems, including those of
lymphocytes and monocytes, were perhaps in a more vulnerable state.” [51].
These authors also identified disturbing changes in gene expression that would imply impaired
ability to repair DNA. Up to 60% of the total transcriptional activity in growing cells involves the
transcription of ribosomal DNA (rDNA) to produce ribosomal RNA (rRNA). The enzyme that
transcribes ribosomal DNA into RNA is RNA polymerase I (Pol I). Pol I also monitors rDNA
integrity and influences cell survival [150]. During transcription, RNA polymerases (RNAPs)
actively scan DNA to find bulky lesions (double-strand breaks) and trigger their repair. In
growing eukaryotic cells, most transcription involves synthesis of ribosomal RNA by Pol I. Thus,
Pol I promotes survival following DNA damage [150]. Many of the downregulated genes
identified by Liu et al. (2021) were linked to the cell cycle, telomere maintenance, and both
promoter opening and transcription of POL I, indicative of impaired DNA repair processes [51]
One of the gene sets that were suppressed was due to “deposition of new CENPA [centromere
protein A] containing nucleosomes at the centromere.” Newly synthesized CENPA is deposited
in nucleosomes at the centromere during late telophase/early G1 phase of the cell cycle. This
points to arrest of the cell cycle in G1 phase as a characteristic feature of the response to the
inactivated SARS-CoV-2 vaccine. Arrest of pluripotent embryonic stem cells in the G1 phase
(prior to replication initiation) would result in impaired self-renewal and maintenance of
pluripotency [151].
Journal Pre-proof
36
Two checkpoint proteins crucially involved in DNA repair and adaptive immunity are BRCA1
and 53BP1, which facilitate both homologous recombination (HR) and NHEJ, the two primary
repair processes [152,153]. In an in vitro experiment on human cells, the SARS-CoV-2 full-length
spike glycoprotein was specifically shown to enter the nucleus and hinder the recruitment of
these two repair proteins to the site of a double-strand break [75]. The authors summarized their
findings by saying, “Mechanistically, we found that the spike protein localizes in the nucleus and
inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment
to the damage site.”
Another mechanism by which the mRNA vaccines could interfere with DNA repair is through
miR-148. This microRNA has been shown to downregulate HR in the G1 phase of the cell cycle
[154]. As was mentioned earlier in this paper, this was one of the two microRNAs found in
exosomes released by human cells following SARS-CoV-2 spike glycoprotein synthesis in the
experiments by Mishra and Banerjea (2021) [49].
9. Reactivation of Varicella-zoster
Type I IFN receptor signaling in CD8+ T cells is critical for the generation of effector and memory
cells in response to a viral infection [155]. CD8+ T cells can block reactivation of latent herpes
infection in sensory neurons [156]. If type I IFN signaling is impaired, as happens following
vaccination but not following natural infection with SARS-CoV-2, CD8+ T cells’ ability to keep
herpes in check would also be impaired. Might this be the mechanism at work in response to the
vaccines?
Journal Pre-proof
37
Shingles is an increasingly common condition caused by reactivation of latent herpes zoster
viruses (HZV), which also causes chicken pox in childhood. In a systematic review, Katsikas et
al., (2021) identified 91 cases of herpes zoster occurring an average of 5.8 days following mRNA
vaccination [157]. While acknowledging that causality is not yet confirmed, “Herpes zoster is
possibly a condition physicians and other healthcare professionals may expect to see in patients
receiving COVID-19 vaccines” [157]. In a letter to the editor published in September 20201, Fathy
et al. (2021) reported on 672 cases of skin reactions that were presumably vaccine-related,
including 40 cases of herpes zoster and/or herpes simplex reactivation [158]. These cases had been
reported to the American Academy of Dermatology and the International League of
Dermatologic Societies’ COVID-19 Dermatology Registry, established specifically to track
dermatological sequalae from the vaccines. There are multiple additional case reports of herpes
zoster reactivation following COVID-19 vaccination in the literature [159,160]. Lladó et al. (2021)
noted that 51 of 52 reports of reactivated herpes zoster infections happened following mRNA
vaccination [161]. Herpes zoster itself also interferes with IFN-α signaling in infected cells both
through interfering with STAT2 phosphorylation and through facilitating IRF9 degradation [162].
An additional case of viral reactivation is noteworthy as well. It involved an 82-year-old woman
who had acquired a hepatitis C viral (HCV) infection in 2007. A strong increase in HCV load
occurred a few days after vaccination with an mRNA Pfizer/BioNTech vaccine, along with an
appearance of jaundice. She died three weeks after vaccination from liver failure [163].
10. Immune Thrombocytopenia
Journal Pre-proof
38
Immune thrombocytopenia is an autoimmune disorder, where the immune system attacks
circulating platelets. Immune thrombocytopenic purpura (ITP) has been associated with several
vaccinations, including measles, mumps, rubella (MMR), hepatitis A, varicella, diphtheria,
tetanus, pertussis (DPT), oral polio and influenza [164]. While there is broad awareness that the
adenovirus DNA-based vaccines can cause vaccine-induced immune thrombotic
thrombocytopenia (VITT) [165], the mRNA vaccines are not without risk to VITT, as case studies
have been published documenting such occurrences, including life threatening and fatal cerebral
venous sinus thrombosis [166-169]. The mechanism is believed to involve VITT antibodies
binding to platelet factor 4 (PF4) and forming immune complexes that induce platelet activation.
Subsequent clotting cascades cause the formation of diffuse microclots in the brain, lungs, liver,
legs and elsewhere, associated with a dramatic drop in platelet count (Kelton et al., 2021). The
reaction to the vaccine has been described as being very similar to heparin-induced
thrombocytopenia (HIT), except that heparin administration is notably not involved [170].
It has been shown that the mRNA vaccines elicit primarily an immunoglobulin G (IgG) immune
response, with lesser amounts of IgA induced [63], and even less IgM production [171]. The
amount of IgG antibodies produced is comparable to the response seen in severe cases of COVID-
19. It is IgG antibodies in complex with heparin that induce HIT. One can hypothesize that IgG
complexed with the SARS-CoV-2 spike glycoprotein and PF4 is the complex that induces VITT in
response to mRNA vaccines. It has in fact been shown experimentally that the receptor binding
domain (RBD) of the spike protein binds to PF4 [172].
Journal Pre-proof
39
The underlying mechanism behind HIT has been well studied, including through the use of
humanized mouse models. Interestingly, human platelets, but not mouse platelets, express the
FcγRIIA receptor, which responds to PF4/heparin/IgG complexes through a tyrosine
phosphorylation cascade to induce platelet activation. Upon activation, platelets release granules
and generate procoagulant microparticles. They also take up calcium, activate protein kinase C,
clump together into microthrombi, and launch a cell death cascade via calpain activation. These
activated platelets release PF4 into the extracellular space, supporting a vicious cycle, as this
additional PF4 also binds to heparin and IgG antibody to further promote platelet activation.
Thus, FcγRIIA is central to the disease process [173].
Studies on mice engineered to express the human FcγRIIA receptor have shown that these
transgenic mice are far more susceptible to thrombocytopenia than their wild type counterparts
[174]. It has been proposed that platelets may serve an important role in the clearance of antibodyantigen
complexes by trapping the antigen in thrombi and/or carrying them into the spleen for
removal by immune cells. Platelets are obviously rapidly consumed in the process, which then
results in low platelet counts (thrombocytopenia).
Platelets normally circulate with an average lifespan of only five to nine days, so they are
constantly synthesized in the bone marrow and cleared in the spleen. Antibody-bound platelets,
subsequent to platelet activation via Fcγ receptors, migrate to the spleen where they are trapped
and removed through phagocytosis by macrophages [175]. Fully one third of the body’s total
platelets are found in the spleen. Since the mRNA vaccines are carried into the spleen by immune
cells initially attracted to the injection site in the arm muscle, there is tremendous opportunity for
Journal Pre-proof
40
the release of spike-glycoprotein-containing exosomes by dendritic cells in the spleen
synthesizing spike protein. One can speculate that platelet activation following the formation of
a P4F/IgG/spike protein complex in the spleen is part of the mechanism that attempts to clear the
toxic spike glycoprotein.
We mentioned earlier that one of the two microRNAs highly expressed in exosomes released by
human cells exposed to the SARS-CoV-2 spike glycoprotein was miR-148a. miR-148a has been
shown experimentally to suppress expression of a protein that plays a central role in regulating
FcγRIIA expression on platelets. This protein, called T-cell ubiquitin ligand-2 (TULA-2),
specifically inhibits activity of the platelet Fcγ receptor. miR-148a targets TULA-2 mRNA and
downregulates its expression. Thus, miR-148a, present in exosomes released by macrophages that
are compelled by the vaccine to synthesize SARS-CoV-2 spike glycoprotein, acts to increase the
risk of thrombocytopenia in response to immune complexes formed by spike glycoprotein
antigen and IgG antibodies produced against the spike glycoprotein.
11. PPAR-α, Sulfatide and Liver Disease
As we have already stated, an experiment by Mishra and Banerjea (2021) demonstrated that the
SARS-CoV-2 spike glycoprotein induces the release of exosomes containing microRNAs that
specifically interfere with IRF9 synthesis [49]. In this section we will show that one of the
consequences of suppression of IRF9 would be reduced synthesis of sulfatide in the liver,
mediated by the nuclear receptor peroxisome proliferator-activated receptor α (PPAR-α).
Journal Pre-proof
41
Sulfatides are major mammalian serum sphingoglycolipids which are synthesized and secreted
mainly from the liver [176]. They are the only sulfonated sphingolipids in the body. Sulfatides
are formed by a two-step process involving the conversion of ceramide to galactocerebroside and
its subsequent sulfation. Sulfatide is expressed on the surface of platelets, erythrocytes and
lymphocytes. Serum sulfatides exert both anti-coagulative and anti-platelet-activation functions.
The enzyme in the liver that synthesizes sulfatide, cerebroside sulfotransferase, has specifically
been found to be induced by activation of PPAR-α in mice [177]. Therefore, reduced expression
of PPAR-α leads to sulfatide deficiency.
PPAR-α ligands exhibit anti-inflammatory and anti-fibrotic effects, whereas PPAR-α deficiency
leads to hepatic steatosis, steatohepatitis, steatofibrosis, and liver cancer [178]. In 2019, an
experiment was conducted by a team of researchers in Japan on mice with a defective gene for
PPAR-α [176]. These mice, when fed a high cholesterol diet, were susceptible to excess
triglyceride accumulation and exacerbated inflammation and oxidative stress in the liver, along
with increased levels of coagulation factors. The mice also manifested with decreased levels of
sulfatides in both the liver and the serum. The authors hypothesized that cholesterol overload
exerts its toxic effects in part by enhancing thrombosis, following abnormal hepatic lipid
metabolism and oxidative stress. They showed that PPAR-α can attenuate these toxic effects
through transcriptional regulation of coagulation factors and upregulation of sulfatide synthesis,
in addition to its effects in ameliorating liver disease. They proposed that therapies such as
fibrates aimed at activating PPAR-α might prevent high-cholesterol-diet-induced cardiovascular
disease.
Journal Pre-proof
42
Tracer studies have shown that the mRNA from mRNA vaccines migrates preferentially to the
liver and spleen, reaching higher concentration there than in any other organs [145]. Thus, there
is potential for suppression of IRF9 in the liver by the vaccine. IRF9 is highly expressed in
hepatocytes, where it interacts with PPAR-α, activating PPAR-α target genes. A study on IRF9
knockout mice showed that these mice developed steatosis and hepatic insulin resistance when
exposed to a high-fat diet. In contrast, adenoviral-mediated hepatic IRF9 overexpression in obese
mice improved insulin sensitivity and ameliorated steatosis and inflammation [179].
Multiple case reports in the research literature describe liver damage following mRNA vaccines
[180-182]. A plausible factor leading to these outcomes is the suppression of PPAR-α through
downregulation of IRF9, and subsequently decreased sulfatide synthesis in the liver.
12. Guillain Barré Syndrome and Neurologic Injury Syndromes
GBS is an acute inflammatory demyelinating neuropathy associated with long-lasting morbidity
and a significant risk of mortality [183]. The disease involves an autoimmune attack on the nerves
associated with the release of pro-inflammatory cytokines.
GBS is often associated with autoantibodies to sulfatide and other sphingolipids [184]. Activated
T cells produce cytokines in response to antigen presentation by macrophages, and these
cytokines can induce autoantibody production through epitope spreading [185]. The antibodies,
in turn, induce complement activation, which causes demyelination and axonal damage, leading
to severe injury to peripheral neurons [186]. The SARS-CoV-2 spike glycoprotein has been shown
to bind to heparan sulfate, which is a sulfated amino-sugar complex resembling the sulfated
Journal Pre-proof
43
galactose in sulfatide [187]. Thus, it is conceivable that the spike glycoprotein also binds to
sulfatide, and this might trigger an immune reaction to the spike-glycoprotein-sulfatide complex.
As described in the previous section, impaired sulfatide synthesis in the liver due to suppression
of IRF9 will lead to systemic sulfatide deficiency over time. Sulfatide deficiency can have major
impact in the brain and nervous system. Twenty percent of the galactolipids found in the myelin
sheath are sulfatides. Sulfatide is a major component of the nervous system, found in especially
high concentrations in the myelin sheath in both the peripheral and the central nervous system.
Deficiencies in sulfatide can lead to muscle weakness, tremors, and ataxia [188], which are
common symptoms of GBS. Chronic neuroinflammation mediated by microglia and astrocytes in
the brain leads to dramatic losses of brain sulfatide, and brain deficiencies in sulfatide are a major
feature of Alzheimer’s disease [189]. Mice with a defect in the ability to synthesize sulfatide from
ceramide show an impaired ability to maintain the health of axons as they age. Over time, they
develop redundant, uncompacted and degenerating myelin sheaths as well as deteriorating
structure at the nodes of Ranvier in the axons, causing the loss of a functionally competent
axoglial junction [190].
Angiotensin II (Ang II), in addition to its profound effects on cardiovascular disease, also plays a
role in inflammation in the brain leading to neurodegenerative disease [191]. The SARS-CoV-2
spike glycoprotein contains a unique furin cleavage site not found in SARS-CoV, which allows
the extracellular enzyme furin to detach the S1 segment of the spike glycoprotein and release it
into circulation [192]. S1 has been shown to cross the blood-brain barrier in mice [193]. S1 contains
the receptor binding domain that binds to ACE2 receptors, disabling them. When ACE2 receptor
Journal Pre-proof
44
signaling is reduced, Ang II synthesis is increased. Neurons in the brain possess ACE2 receptors
that would be susceptible to disruption by S1 released from spike-glycoprotein-containing
exosomes or spike-glycoprotein-producing cells that had taken up the nanoparticles in the
vaccines. Ang II enhances TLR4-mediated signaling in microglia, inducing microglial activation
and increasing the production of reactive oxygen species leading to tissue damage, within the
paraventricular nucleus in the brain [194].
Elevated levels of Ang II is a causal factor in neurodegeneration of the optic nerve, causing optic
neuritis, which can result in severe irreversible visual loss [195]. Multiple case reports have
described cases of optic neuropathy appearing shortly after mRNA vaccination for COVID-19
[196,197]. Other debilitating neurological conditions are also appearing shortly after vaccination,
where a causal relationship is suspected. A case study based in Europe tracking neurological
symptoms following COVID-19 vaccination identified 21 cases developing within a median of 11
days post-vaccination. The cases had diverse diagnoses including cerebral venous sinus
thrombosis, nervous system demyelinating diseases, inflammatory peripheral neuropathies,
myositis, myasthenia, limbic encephalitis, and giant cell arteritis [198]. Khayat-Khoei et.al. (2021)
describe a case series of 7 patients, ages ranging from 24 to 64, presenting with demyelinating
disease within 21 days of a first or second mRNA vaccination [199]. Four had a prior history of
(controlled) MS, while three were previously healthy.
Hearing loss and tinnitus are also well-known side effects of COVID-19. A case study involved
a series of ten COVID-19 patients who suffered from audiovestibular symptoms such as hearing
loss, vestibular dysfunction and tinnitus [200]. The authors demonstrated that human inner ear
Journal Pre-proof
45
tissue expresses ACE2, furin and the transmembrane protease serine 2 (TMPRSS2), which
facilitates viral entry. They also showed that SARS-CoV-2 can infect specific human inner ear cell
types.
Another study evaluating the potential for the SARS-CoV-2 virus to infect the ear specifically
examined expression of the receptor ACE2 and the enzymes furin and TM-PRSS2 various types
of cells in the middle and inner ears of mice. They found that ACE2 and furin were “diffusely
present in the eustachian tube, middle ear spaces, and cochlea, suggesting that these tissues are
susceptible to SARS-CoV-2 infection.” [201]. Tinnitus is positively associated with hypertension,
which is induced by elevated levels of Ang II [202].
Headache is a very common adverse reaction to the COVID-19 mRNA vaccines, particularly for
people who are already susceptible to headaches. In a study based on a questionnaire involving
171 participants, the incidence of headaches was found to be 20.5% after the first vaccine, rising
to 45.6% after the second shot [203]. A case study described a 37-year-old woman suffering from
a debilitating migraine attack lasting for 11 days following the second Pfizer/BioNtech mRNA
vaccine [204].
Steroids are often used as adjunct therapy to treat migraine [205]. Dexamethasone and other
steroids stimulate PPAR-α receptors in the liver through the steroid receptor, thus offsetting the
effects of IRF9 suppression [206]. A theory for the origins of migraine involves altered processing
of sensory input in the brainstem, primarily trigeminal neurons [207]. The trigeminal nerve is in
close proximity to the vagus nerve in the brainstem, so spike-glycoprotein-carrying exosomes
could easily reach it along the vagal route. Magnetic resonance imaging has revealed that
Journal Pre-proof
46
structural changes in the trigeminal nerve reflecting aberrant microstructure and demyelination
are a characteristic feature of people who suffer from frequent migraine headaches [208]. A
potential factor linked to either SARS-CoV-2 infection or mRNA vaccination is an excessive level
of Ang II in the brainstem due to SARS-CoV-2 spike glycoprotein inhibition of ACE2 receptors.
ACE inhibitors and Ang II receptor antagonists have become popular drugs to treat migraine
headaches off-label [209,210]. Migraine headache could thus arise from both the spike
glycoprotein’s disruption of ACE2 receptors and the destruction of the myelin sheath covering
critical facial nerves through a microglial inflammatory response and loss of sulfatide. The source
of that spike glycoprotein could be either exogenous or endogenous.
13. Bell’s Palsy
Bell’s palsy is a common cranial neuropathy causing unilateral facial paralysis. Even in the Phase
III clinical trials, Bell’s palsy stood out, with seven cases appearing in the treatment arm as
compared to only one in the placebo group [211,212]. A case study reported in the literature
involved a 36-year-old man who developed weakness in the left arm one day after vaccination,
progressing to numbness and tingling in the arm and subsequent symptoms of Bell’s palsy over
the next few days. A common cause of Bell’s palsy is reactivation of herpes simplex virus infection
centered around the geniculate ganglion [213]. This, in turn, can be caused by disruption of type
I IFN signaling.
14. Myocarditis
There has been considerable media attention devoted to the fact that COVID-19 vaccines cause
myocarditis and pericarditis, with an increased risk in particular for men below the age of 50
Journal Pre-proof
47
[214,215]. The SARS-CoV-2 spike glycoprotein has been demonstrated to injure cardiac pericytes,
which support the capillaries and the cardiomyocytes [216]. Myocarditis is associated with
platelet activation, so this could be one factor at play in the response to the vaccines [217].
However, another factor could be related to exosomes released by macrophages that have taken
up the mRNA nanoparticles, and the specific microRNAs found in those exosomes.
A study involving patients suffering from severe COVID-19 disease looked specifically at the
expression of circulating microRNAs compared to patients suffering from influenza and to
healthy controls. One microRNA that was consistently upregulated in association with COVID-
19 was miR-155, and the authors suggested that it might be a predictor of chronic myocardial
damage and inflammation. By contrast, influenza infection was not associated with increased
miR-155 expression. They concluded: “Our study identified significantly altered levels of cardiacassociated
miRs [microRNAs] in COVID-19 patients indicating a strong association of COVID-19
with cardiovascular ailments and respective biomarkers” [218].
A study comparing 300 patients with cardiovascular disease to healthy controls showed a
statistically significant increase in circulating levels of miR-155 in the patients compared to
controls. Furthermore, those with more highly constricted arteries (according to a Gensini score)
had higher levels than those with lesser disease [219].
Importantly, exosomes play a role in inflammation in association with heart disease. During
myocardial infarction, miR-155 is sharply upregulated in macrophages in the heart muscle and
released into the extracellular milieu within exosomes. These exosomes are delivered to
Journal Pre-proof
48
fibroblasts, and miR-155 downregulates proteins in the fibroblasts that protect from inflammation
and promote fibroblast proliferation. The resulting impairment leads to cardiac rupture [220].
We have already discussed how the S1 segment of the SARS-CoV-2 spike glycoprotein can be
cleaved by furin and released into circulation. It binds to ACE2 receptors through its receptor
binding domain (RBD), and this inhibits their function. Because ACE2 degrades Ang II, disabling
ACE2 leads directly to overexpression of Ang II, further enhancing risk to cardiovascular disease.
AngII-induced vasoconstriction is an independent mechanism to induce permanent myocardial
injury even when coronary obstruction is not present. Repeated episodes of sudden constriction
of a cardiac artery due to Ang II can eventually lead to heart failure or sudden death [221]. Fatal
cases of COVID-19 vaccination have been described by Choi et al. and Verma et al. [222,223].
ACE2 suppression had already been seen in studies on the original SARS-CoV virus. An autopsy
study on patients succumbing to SARS-CoV revealed an important role for ACE2 inhibition in
promoting heart damage. SARS-CoV viral RNA was detected in 35% of 20 autopsied human heart
samples taken from patients who died. There was a marked increase in macrophage infiltration
associated with myocardial damage in the patients whose hearts were infected with SARS-CoV.
Importantly, the presence of SARS-CoV in the heart was associated with marked reduction in
ACE2 protein expression [224].
15. Considerations Regarding the Vaccine Adverse Event Reporting System (VAERS)
The Food and Drug Administration’s Vaccine Adverse Event Reporting System (VAERS) is an
imperfect but valuable resource for identifying potential adverse reactions to vaccines.
Established through collaboration between the CDC and FDA, VAERS is “a national early
Journal Pre-proof
49
warning system to detect possible safety problems in U.S.-licensed vaccines.” According to the
CDC it is “especially useful for detecting unusual or unexpected patterns of adverse event
reporting that might indicate a possible safety problem with a vaccine.”
(https://vaers.hhs.gov/about.html) Even the CDC recognizes that adverse events reported to
VAERS represent “only a small fraction of actual adverse events” [225]. A widely cited report
noted that fewer than 1% of all vaccine-related adverse events are reported to VAERS [226]. That
assertion, though, has no citation so the basis for the claim is unclear. Rose (2021) published a
much more sophisticated analysis of VAERS data to offer an estimate of underreporting by a
factor of 31 [227]. While it is impossible to determine underreporting with precision, the available
evidence is that underreporting very strongly characterizes the VAERS data. The information
presented below should be understood in that light.
In mining VAERS for ‘signals’ that might indicate adverse reactions (AEs) to mRNA vaccinations,
we acknowledge that no report to VAERS establishes a causal link with the vaccination. That said,
the possibility of a causal relationship is strengthened through both the causal pathways we have
described in this paper, and the strong temporal association between injections and reported AEs.
Nearly 60% of all mRNA-injection-related -AEs have happened within 48 hours of injection
(https://medalerts.org/vaersdb/findfield.php?TABLE=ON&GROUP1=ONS&EVENTS=ON&VA
X=COVID19&VAXTYPES=COVID-19&STATE=NOTFR).
Two important cautions regarding analysis of VAERS data should be noted. The first is that, in
addition to health care professionals submitting reports, VAERS is open for public submissions
as well. Members of the public may lack the skills necessary to evaluate a symptom appropriately
Journal Pre-proof
50
to determine if it merits a VAERS entry. A second caution is that public access also allows for the
possibility of anti-vaccination activists to populate VAERS with false reports to exaggerate the
appearance of AE risk.
An interim analysis of deaths cited previously found that health service employees were the
VAERS reporter in 67% of reports analyzed [210], suggesting a large portion of VAERS reports
are submitted by medical professionals and not the public. This finding also belies the notion that
anti-vaccination activists are filing an excessive number of egregious reports of vaccine injury.
All of the data reported in this section were obtained by querying the online resource,
http://wonder.cdc.gov/vaers.html. Over the 31-year history of VAERS, up to February 3, 2022,
there were a total of 10,321 deaths reported as a “symptom” in association with any vaccine, and
8,241 (80%) of those deaths were linked to COVID-19 vaccines. Importantly, only 14% of COVID-
19 VAERS-reported deaths as of June 2021 could have vaccination ruled out as a cause [228]. This
strongly suggests that these unprecedented vaccines exhibit unusual mechanisms of toxicity that
go well beyond what is seen with more traditional vaccines.
We decided that a reasonable way to characterize the significance of adverse events linked to
COVID-19 vaccines was to focus on events received in the year 2021, and to compare the counts
in the “SYMPTOM” field for the events associated with COVID-19 vaccines to the total counts for
that same symptom for all vaccines over that same year. In total, there were 737,689 events
reported in VAERS for COVID-19 vaccines in 2021, representing a shocking 93% of the total cases
reported for any vaccine that same year. While we recognize that some of the COVID-19 vaccines
are based on DNA vector technology rather than mRNA technology, this class (i.e., the Johnson
Journal Pre-proof
51
& Johnson vaccine) represents less than 9% of the COVID-19 reports, and its reaction profile is
surely much more similar to that of the mRNA vaccines than to that of all the other vaccines.
The total number of adverse event reports for COVID-19 injections is far greater than the
cumulative number of annual vaccine adverse event reports combined in all prior years, as shown
by Rose [227]. The influenza vaccine is a good one to compare against. Given that the protocol for
the mRNA vaccines requires two doses, and that many were persuaded to receive a booster shot
as well, it is clear that the sheer number of COVID-19 vaccines administered is large compared to
other vaccines. We can actually estimate what percent of the adverse reactions in 2021 would be
expected to be associated with COVID-19 vaccines if the likelihood of an adverse reaction were
similar to that of the influenza vaccine. The CDC tells us that 52% of the US population received
a flu shot in 2021. The USAFacts web site provides percentages of the US population that received
one, two or three doses of COVID-19 vaccines as a function of time (see:
https://usafacts.org/visualizations/covid-vaccine-tracker-states/). The numbers they report for
December 30, 2021 are 73% single dose, 62% fully vaccinated, and 21% boosted. This tallies up to
156% of the population as the total number of COVID-19 vaccines administered. This is exactly
three times as many COVID vaccines as flu shots.
From VAERS, one can easily obtain the total number of adverse reactions associated with COVID-
19 vaccines, the total number associated with flu vaccines, and the total number associated with
all vaccines, for the US-restricted VAERS data from 2021. These come out as: COVID-19: 737,587,
FLU: 9,124, and ALL: 792,935. First, we can observe that 93% of all the events reported were
linked to COVID-19 vaccines. If we remove the counts for COVID-19 and replace them with three
Journal Pre-proof
52
times the counts for flu (since COVID-19 vaccines were administered three times as often), we
find that COVID-19 should have accounted for 32.6% of all the events, which can be compared
with the actual result, which is 93%. We can also conclude that any event that shows up more
than 93% as often for COVID-19 vaccines as for all other vaccines is especially significant as a
potential toxic effect of these vaccines. Finally, we find that there are 27 times as many reports for
COVID-19 vaccines as would be expected if its adverse reactions were comparable to those from
the flu vaccine.
Symptom Inflamed Nerve(s) Covid-19
Vaccines
All
Vaccines
Percent
COVID-19
Anosmia olfactory nerve 3,657 3,677 99.5
Tinnitus vestibulo-cochlear nerve 13,275 13,522 98.2
Deafness cochlea 2,895 3,033 95.5
Bell’s Palsy/facial palsy facial nerve 5,881 6,129 96.0
Vertigo vestibular nerve 7,638 7,819 97.7
Migraine headache trigeminal nerve 8,872 9,059 97.9
Dysphonia glossopharyngeal nerve 1,692 1,751 96.6
Dysphagia several lower cranial nerves 4,711 4,835 97.4
Nausea vagus nerve 69,121 71,275 97.0
Vomiting vagus nerve 27,885 28,955 96.3
Dyspnea vagus nerve 39,551 40,387 97.9
Syncope vagus nerve 14,701 15,268 96.3
Journal Pre-proof
53
Bradycardia vagus nerve 673 699 96.3
TOTAL — 200,552 206,409 97.2
Table 1. Number of symptoms reported in VAERS, restricted to the US population, for
the year 2021, for various adverse effects that could be caused by inflammation in
associated major nerves, showing total counts for COVID-19 vaccines and for all vaccines.
15.1 VAERS Data Indicative of Nerve Damage and Vagus Nerve Involvement
Table 1 lists a number of symptoms in VAERS that can be associated with inflammation of or
damage to various major nerves of the body, particularly those in the head. Strikingly, COVID-
19 vaccines represented from 96 to 98% of the reports in the year 2021 related to each of these
debilitating conditions. There were nearly 100,000 cases of nausea or vomiting, which are
common symptoms of vagus nerve stimulation or damage [229]. 14,701 cases of syncope linked
to COVID-19 vaccines represented 96.3% of all cases of syncope, a well-established feature of
vagus nerve dysfunction [230]. There were 3,657 cases of anosmia (loss of smell), clearly
demonstrating that the SARS-CoV-2 spike glycoprotein from the injection in the arm was
reaching the olfactory nerve. Dyspnea (shortness of breath) is related to vagus nerve impairment
in the lungs, and there were 39,551 cases of dyspnea connected to COVID-19 vaccines in 2021.
Altogether, these events add up to a total of over 200,000 events, representing 97.2% of all the
entries related to any vaccine in 2021. This is also a substantial 27.2% of all the events listed for
2021 in association with COVID-19 vaccines.
15.2 VAERS data on the Heart and Liver
Journal Pre-proof
54
In this paper, we have identified both the heart and the liver as organs that can be expected to be
affected by the mRNA vaccines. The VAERS database shows a strong signal for both organs.
Table 2 shows the statistics for 2021 on major disorders of the heart, including myocarditis, arrest
(cardiac, cardiorespiratory and sinus arrest), arrhythmia (including supraventricular, nodal,
sinus, tachyarrhythmia and ventricular arrhythmia), myocardial infarction (including acute and
silent), and cardiac failure (including acute, chronic and congestive). Altogether, there were a
total of 8,090 COVID-19 events related to these heart conditions, representing nearly 98% of all
the events for all the vaccines for these symptoms in 2021.
Symptom Covid-19 Vaccines All Vaccines Percent COVID-19
Myocarditis 2,322 2,361 98.3
Arrest 1,319 1,371 96.2
Arrhythmia 1,069 1,087 98.3
Myocardial infarction 2,224 2,272 97.9
Cardiac failure 1,156 1,190 97.1
TOTAL 8,090 8,281 97.7
Table 2. Number of symptoms reported in VAERS, restricted to the US population, for
the year 2021, for various disorders of the heart, showing total counts for COVID-19
vaccines and for all vaccines.
Journal Pre-proof
55
It is difficult to find all of the symptoms associated with liver damage in VAERS, but we selected
a number that had high enough counts to be of interest and that clearly represent serious liver
problems. Altogether there were 731 events in these categories for COVID-19 vaccines, as shown
in Table 3, representing over 97% of all the cases connecting these conditions to any vaccine in
2021.
Symptom Covid-19
Vaccines
All Vaccines Percent COVID-
19
Liver disorder 83 87 95.4
[Drug-induced] liver injury 65 65 100
[Acute] hepatic failure 86 88 97.7
Hepatic cancer [metastatic] 12 12 100
Hepatic cirrhosis 67 69 97.1
Hepatic cyst 33 34 97.0
Liver function test increased 238 245 97.1
Liver function test abnormal 90 94 95.7
Hepatic function abnormal 34 34 100
Haemangioma of liver 10 10 100
Journal Pre-proof
56
Liver abscess 7 7 100
Liver transplant 6 6 100
TOTAL 731 751 97.3
Table 3. Number of symptoms reported in VAERS, restricted to the US population, for
the year 2021, for various indicators of liver disease, showing total counts for COVID-19
vaccines and for all vaccines.
15.3 VAERS Data Related to Thrombosis
There were 78 unique symptoms in VAERS involving thrombosis, specifying different arteries
and veins. Table 4 shows nine symptoms with the highest counts, totaling 7,356 events. We
investigated the time interval for the three dominant ones (thrombosis, deep vein thrombosis and
pulmonary thrombosis), and found that these all have a sharp peak in the 15-30-day range for
onset interval (time after vaccination). This coincides with a sharp peak in pulmonary embolism,
a life-threatening condition, also in the 15-30-day time interval. Overall, for these nine thrombotic
symptoms, a random sampling from the year 2021 would yield a COVID vaccine as opposed to
any other vaccine 98.7% of the time. Pulmonary embolism, a life-threatening condition that can
be caused by a blood clot that travels to the lungs, has a slightly higher probability of 98.8%, with
3,100 cases listed for COVID-19.
Journal Pre-proof
57
Symptom Covid-19
Vaccines
All Vaccines Percent COVID-19
Thrombosis 3,899 3,951 98.7
Deep vein thrombosis 2,275 2,297 99.0
Pulmonary thrombosis 631 646 97.7
Cerebral thrombosis 211 215 98.1
Portal vein thrombosis 89 90 98.9
Superficial vein thrombosis 81 81 100
Peripheral artery thrombosis 74 74 100
Mesenteric vein thrombosis 55 56 98.2
Venous thrombosis 41 41 100
TOTAL 7,356 7,451 98.7
Pulmonary embolism 3,100 3,137 98.8
Table 4. Number of symptoms reported in VAERS, restricted to the US population, for
the year 2021, for various specific types of thrombosis, showing total counts for COVID-
19 vaccines and for all vaccines. Pulmonary embolism, a highly related symptom, is also
shown.
15.4 VAERS data related to Neurodegenerative Disease
Journal Pre-proof
58
Table 5 lists results for several conditions that are linked to neurodegenerative disease. Decreased
mobility can be caused by Parkinson’s disease, and there were a striking 8,975 cases listed for
2021 and COVID-19 vaccines. Alzheimer’s and Parkinson’s are diseases that normally take
decades to develop, and ordinarily one would assume that a vaccine has nothing to do with it.
While the numbers are small, most of the cases in VAERS were linked to COVID-19 vaccines.
Anosmia, also included in the table on the vagus nerve, is especially interesting, because it is a
well-known early sign of Parkinson’s disease, and it is also a well-identified feature of SARSCoV-
2 infection. 99.5% of the cases with anosmia as a symptom were linked to COVID-19
vaccines. Overall, the symptoms in this table were linked to COVID-19 vaccines nearly 95% of
the time.
Symptom Covid-19
Vaccines
All Vaccines Percent COVID-19
Alzheimer’s dementia 37 39 94.9
Parkinsonian symptoms 83 89 93.3
Memory impairment 1,681 1,720 97.7
Anosmia 3,657 3,677 99.5
Mobility decreased 8,975 9,743 92.1
Cognitive disorder 779 815 92.1
Journal Pre-proof
59
TOTAL 15,212 16,083 94.6
Table 5. Number of symptoms reported in VAERS, restricted to the US population, for
the year 2021, for various disorders linked to neurodegenerative disease, showing total
counts for COVID-19 vaccines and for all vaccines.
15.5 VAERS Signal for Cancer
Cancer is a disease generally understood to take months or, more commonly, years to progress
from an initial malignant transformation in a cell to development of a clinically recognized
condition. Since VAERS reports of adverse events are happening primarily within the first month
or even the first few days after vaccination [227], it seems likely that the acceleration of cancer
progression following vaccines would be a difficult signal to recognize. Furthermore, most people
do not expect cancer to be an adverse event that could be caused by a vaccine, and hence they fail to
enter a report when cancer develops shortly after vaccination. However, as we have outlined in our
paper, if the mRNA vaccinations are leading to widespread dysregulation of oncogene controls, cell
cycle regulation, and apoptosis, then VAERS reports should reflect an increase in reports of cancer,
relative to the other vaccines, even if the numbers are small. The experiment demonstrating
impairment of DNA repair mechanisms by SARS-CoV-2 spike protein in an in vitro study provides
compelling evidence that the vaccines could accelerate the rate of DNA mutations, increasing cancer
risk [75].
For our analysis of evidence of increased cancer risk in VAERS, we focused on two somewhat distinct
approaches. One, represented by the results in Table 6, was to gather the counts for any terms that
Journal Pre-proof
60
contained keywords clearly linked to cancer, namely, “cancer,” “lymphoma,” “leukaemia,”
“metastasis,” “carcinoma,” and “neoplasm.” Overall, we found 1,474 entries linking these terms to
COVID-19 vaccines, representing 96% of all the entries for any of these terms for any vaccine in that
year.
The complementary approach was to find terms involving cancer in specific organs, namely, breasts,
prostate, bladder, colon, brain, lungs, pancreas and ovaries, as shown in Table 7. Although all the
numbers are small, the highest by far was for breast cancer (246 cases), with nearly four times as many
hits as for lung cancer, the second most common type. All of the cases for pancreatic, ovarian and
bladder cancer were linked to COVID-19 vaccines, with zero cases for any other vaccine. Altogether,
we tabulated 534 cases of cancer of specific organs linked to COVID-19 vaccines, representing 97.3%
of all the cases for any vaccine in 2021.
Symptom Counts COVID-19
vaccines
Counts All
Vaccines
Percent
COVID-19
Cancer 396 403 98.3
Lymphoma 144 153 94.1
Leukaemia 155 161 96.3
Metastatic/metastasis 175 179 97.8
Carcinoma 176 187 94.1
Neoplasm 428 452 94.7
Journal Pre-proof
61
TOTAL 1,474 1,535 96.0
Table 6. Number of symptoms reported in VAERS, restricted to the US population, for the year 2021,
for various cancer-related terms, showing total counts for COVID-19 vaccines and for all vaccines.
Symptom Counts COVID-19
vaccines
Counts All Vaccines Percent COVID-19
Breast cancer 246 254 96.8
Prostate cancer 50 52 96.2
Bladder cancer 30 30 100
Colon cancer 40 41 97.6
Brain neoplasm 53 55 96.4
Lung cancer 64 66 97.0
Pancreatic cancer 24 24 100
Ovarian cancer 27 27 100
Total 534 549 97.3
Journal Pre-proof
62
Table 7. Number of symptoms reported in VAERS, restricted to the US population, for the
year 2021, for cancer of specific organs, showing total counts for COVID-19 vaccines and for
all vaccines.
16. Conclusions
There has been an unwavering message about the safety and efficacy of mRNA vaccinations
against SARS-CoV-2 from the public health apparatus in the US and around the globe. The
efficacy is increasingly in doubt, as shown in a recent letter to the Lancet Regional Health by
Günter Kampf [231]. Kampf provided data showing that the vaccinated are now as likely as the
unvaccinated to spread disease. He concluded: “It appears to be grossly negligent to ignore the
vaccinated population as a possible and relevant source of transmission when deciding about
public health control measures.” Moreover, the inadequacy of phase I, II, and III trials to evaluate
mid-term and long-term side effects from mRNA genetic vaccines may have been misleading on
their suppressive impact on the innate immunity of the vaccinees.
In this paper, we call attention to three very important aspects of the safety profile of these
vaccinations. First is the extensively documented subversion of innate immunity, primarily via
suppression of IFN-α and its associated signaling cascade. This suppression will have a wide
range of consequences, not the least of which include the reactivation of latent viral infections
and the reduced ability to effectively combat future infections. Second is the dysregulation of the
system for both preventing and detecting genetically driven malignant transformation within
cells and the consequent potential for vaccination to promote those transformations. Third,
mRNA vaccination potentially disrupts intracellular communication carried out by exosomes,
Journal Pre-proof
63
and induces cells taking up spike glycoprotein mRNA to produce high levels of spikeglycoprotein-
carrying exosomes, with potentially serious inflammatory consequences. Should
any of these potentials be fully realized, the impact on billions of people around the world could
be enormous and could contribute to both the short-term and long-term disease burden our
health care system faces.
Given the current rapidly expanding awareness of the multiple roles of G4s in regulation of
mRNA translation and clearance through stress granules, the increase in pG4s due to enrichment
of GC content as a consequence of codon optimization has unknown but likely far-reaching
consequences. Specific analytical evaluation of the safety of these constructs in vaccines is
urgently needed, including mass spectrometry for identification of cryptic expression and
immunoprecipitation studies to evaluate the potential for disturbance of or interference with the
essential activities of RNA and DNA binding proteins.
It is essential that further studies be conducted to determine the extent of the potential
pathological consequences outlined in this paper. It is not practical for these vaccinations to be
considered part of a public health campaign without a detailed analysis of the human impact of
the potential collateral damage. VAERS and other monitoring systems should be optimized to
detect signals related to the health consequences of mRNA vaccination we have outlined. We
believe the upgraded VAERS monitoring system described in the Harvard Pilgrim Health Care,
Inc. study, but unfortunately not supported by the CDC, would be a valuable start in this regard
[226].
Journal Pre-proof
64
In the end, billions of lives are potentially at risk, given the large number of individuals injected
with the SARS-CoV-2 mRNA vaccines and the broad range of adverse outcomes we have
described. We call on the public health institutions to demonstrate, with evidence, why the issues
discussed in this paper are not relevant to public health, or to acknowledge that they are and to
act accordingly. Furthermore, we encourage all individuals to make their own health care
decisions with this information as a contributing factor in those decisions.
Author Contributions: S.S., G.N and A.K. all contributed substantially to the writing of the
original draft. P.M. participated in the process of editorial revisions.
Funding: This research was funded in part by Quanta Computers, Inc., Taipei, Taiwan, under the
auspices of the Qmulus project.
Competing Interests: The authors declare no competing interests.
References
1. Bhurani, V.; Mohankrishnan, A.; Morrot, A.; Dalai, S. K.. Developing effective vaccines: cues from
natural infection. Int Rev Immunol 2018, 37(5), 249-265. doi: 10.1080/08830185.2018.1471479.
2. Psichogiou, M.; Karabinis, A.; Poulakou, G.; Antoniadou, A.; Kotanidou, A.; Degiannis, D.;
Pavlopoulou, I.D.; Chaidaroglou, A.; Roussos, S.; Mastrogianni E.; et al. Comparative
immunogenicity of BNT162b2 mRNA vaccine with natural COVID-19 infection. Vaccines (Basel)
2021, 9(9), 1017. doi: 10.3390/vaccines9091017.
3. Jhaveri, R. The COVID-19 mRNA vaccines and the pandemic: Do they represent the beginning of
the end or the end of the beginning? Clin Ther 2021, 43(3), 549-556. doi:
10.1016/j.clinthera.2021.01.014
Journal Pre-proof
65
4. Centers for Disease Control and Prevention. 2021. Coronavirus Disease 2019 (COVID-19). [online]
Available at: <https://www.cdc.gov/coronavirus/2019-ncov/science/science-briefs/vaccineinduced-
immunity.html#anchor_1635540449320 [Accessed 28 November 2021].
5. Ivanova, E.N.; Devlin, J.C.; Buus, T.B.; Koide, A.; Cornelius, A.; Samanovic, M.I.; Herrera, A.;
Zhang, C.; Desvignes, L.; Odum, N.; Ulrich, R.; Mulligan, M.J.; Koide, S.; Ruggles, K.V.; Herati,
R.S.; Koralov, S.B. Discrete immune response signature to SARS-CoV-2 mRNA vaccination
versus infection. medRxiv preprint April 21, 2021. doi:
https://doi.org/10.1101/2021.04.20.21255677.
6. Kwok, H.F. Review of COVID-19 vaccine clinical trials — A puzzle with missing pieces. Int J Biol
Sci 2021, 7(6), 1461.
7. Shrotri, M.; Navaratnam, A.M.; Nguyen, V.; Byrne, T.; Geismar, C.; Fragaszy, E.; Beale, S.; Fong,
W.L.E.; Patel, P.; Kovar, J.; et al. Spike-antibody waning after second dose of BNT162b2 or
ChAdOx1. The Lancet 2021, 398(10298), 385-387.
8. Centers for Disease Control and Prevention. 2021. COVID-19 Booster Shot. [online] Available at:
https://www.cdc.gov/coronavirus/2019-ncov/vaccines/booster-shot.html [Accessed 28
November 2021].
9. Yahi, N.; Chahinian, H.; Fantini, J. Infection-enhancing anti-SARS-CoV-2 antibodies recognize
both the original Wuhan/D614G strain and Delta variants. A potential risk for mass vaccination?
J Infect 2021, 83(5), 607-635. doi: 10.1016/j.jinf.2021.08.010.
10. Kampf, G. The epidemiological relevance of the COVID-19-vaccinated population is increasing.
Lancet Reg Health – Europe 2021, 11, 100272. Doi: 10.1016/j.lanepe.2021.100272.
Journal Pre-proof
66
11. Subramanian, S.V.; Kumar, A. Increases in COVID-19 are unrelated to levels of vaccination across
68 countries and 2947 counties in the United States. Eur J Epidemiol 2021, 1-4. doi: 10.1007/s10654-
021-00808-7.
12. Shitrit, P.; Zuckerman, N.S.; Mor, O.; Gottesman, B.-S.; Chowers, M. Nosocomial outbreak caused
by the SARS-CoV-2 Delta variant in a highly vaccinated population, Israel, July 2021. Euro Surveill
2021, 26(39), 2100822. doi: 10.2807/1560-7917.ES.2021.26.39.2100822.
13. Brosh-Nissimov, T.; Orenbuch-Harroch, E.; Chowers, M.; Elbaz, M.; Nesher, L.; Stein, M.; Maor,
Y.; Cohen, R.; Hussein, K.; Weinberger, M.; et al. BNT162b2 vaccine breakthrough: clinical
characteristics of 152 fully vaccinated hospitalized COVID-19 patients in Israel. Clin Microbiol
Infect 2021, 27(11), 1652-1657. doi: 10.1016/j.cmi.2021.06.036.
14. Lindenmann, J. From interference to interferon: a brief historical introduction. Philos Trans R Soc
Lond B, Biol Sci 1982, 299(1094), 3-6.
15. Wang, H.; Hu, H.; Zhang, K. Overview of interferon: characteristics, signaling and anti-cancer
effect. Arch Biotechnol Biomed 2017, 1, 1-16.
16. Passegu, E.; Ernst, P.A. IFN-alpha wakes up sleeping hematopoietic stem cells. Nat Med 2009,
15(6), 612613. doi: 10.1038/nm0609-612.
17. Kaur, A.; Fang, C. M. (2020). An overview of the human immune system and the role of interferon
regulatory factors (IRFs). Prog Microbes Mol Biol 2020, 3(1). doi: 10.36877/pmmb.a0000129.
18. Alsamman, K.; El-Masry, O.S. (2018). Interferon regulatory factor 1 inactivation in human cancer.
Biosci Reports 2018, 38(3), BSR20171672. doi: 10.1042/BSR20171672.
19. Huang, F.T.; Sun, J.; Zhang, L.; He, X.; Zhu, Y.H.; Dong, H.J.; Wang, H.-Y.; Zhu, L.; Zou, Huang,
J.-W.; et al. Role of SIRT1 in hematologic malignancies. J Zhejiang Univ-Sci B 2019, 20(5), 391-398.
doi: 10.1631/jzus.B1900148.
Journal Pre-proof
67
20. Zitvogel, L.; Galluzzi, L.; Kepp, O.; Smyth, M.J.; Kroemer, G. Type I interferons in anticancer
immunity. Nat Rev Immunol 2015, 15(7), 405-414. doi: 10.1038/nri3845.
21. Jego, G.A.; Palucka, K.; Blanck, J.-P.; Chalouni, C.; Pascual, V.; Banchereau, J. Plasmacytoid
dendritic cells induce plasma cell differentiation through type I interferon and interleukin 6.
Immunity 2003, 19, 225234. doi: 10.1016/s1074-7613(03)00208-5.
22. De Andrea, M.; Ravera, R.; Gioia, D.; Gariglio, M.; Landolfo, S. The interferon system: an
overview. Eur J Paedia Neurol 2002, 6, A41-A46. doi: 10.1053/ejpn.2002.0573
23. Feng, B.; Eknoyan, G.; Guo, Z.S.; Jadoul, M.; Rao, H.Y.; Zhang, W.; Wei, L. Effect of interferonalpha-
based antiviral therapy on hepatitis C virus-associated glomerulonephritis: a metaanalysis.
Nephrol Dial Transplant 2012, 27(2), 640-646.
24. Delannoy, A.S.; Hober, D.; Bouzidi, A.; Wattre, P. Role of interferon alpha (IFN‐α) and interferon
gamma (IFN‐γ) in the control of the infection of monocyte‐like cells with Human
Cytomegalovirus (HCMV). Microbiol Immunol 1999, 43(12), 1087-1096.
25. Sakai, Y.; Ohga, S., Tonegawa, Y.; Takada, H., Nakao, F.; Nakayama, H.; Aoki, T.; Yamamori, S.;
Hara, T. Interferon-alpha therapy for chronic active Epstein-Barr virus infection: potential effect
on the development of T- lymphoproliferative disease. J Pediatr Hematol Oncol 1998, 20(4), 342-
346.
26. Ruther, U.; Nunnensiek, C.; Muller, H.A.; Bader, H.; May, U.; Jipp, P. Interferon alpha (IFN alpha
2a) therapy for herpes virus-associated inflammatory bowel disease (ulcerative colitis and
Crohn’s disease). Hepato-gastroenterology 1998, 45(21), 691-699. Doi: 10.1111/j.1348-
0421.1999.tb03365.x.
Journal Pre-proof
68
27. Musella, M.; Manic, G.; de Maria, R.; Vitale, I.; Sistigue, A. Type-I-interferons in infection and
cancer: Unanticipated dynamics with therapeutic implications. Oncoimmunology 2017, 6(5),
e1314424. doi: 10.1080/2162402X.2017.1314424.
28. Matsuoka, M.; Tani, K.; Asano, S. Interferon-alpha-induced G1 phase arrest through upregulated
expression of CDK inhibitors, p19Ink4D and p21Cip1 in mouse macrophages. Oncogene 1998, 16,
2075-86. doi: 10.1038/sj.onc.1201745.
29. Heise, R.; Amann, P.M.; Ensslen, S.; Marquardt, Y.; Czaja, K.; Joussen, S.; Beer, D.; Abele, R.;
Plewnia, G.; Tampé, R.; et al. Interferon alpha signaling and its relevance for the upregulatory
effect of transporter proteins associated with antigen processing (TAP) in patients with malignant
melanoma. PLoS One 2016, 11(1), e0146325. doi: 10.1371/journal.pone.0146325.
30. Sundstedt, A.; Celander, M.; Hedlund, G. (2008). Combining tumor-targeted superantigens with
interferon-alpha results in synergistic anti-tumor effects. Int Immunopharmacol 2008, 8(3), 442- 452.
doi: 10.1016/j.intimp.2007.11.006.
31. Schneider, W.M.; Chevillotte, M.D.; Rice, C.M. Interferon-stimulated genes: a complex web of
host defenses. Anni Rev Immunol 2014, 32, 513-545.
32. Asmana Ningrum, R. Human interferon α-2b: a therapeutic protein for cancer treatment.
Scientifica (Cairo) 2014, 2014, 970315. doi: 10.1155/2014/970315.
33. Takaoka, A.; Tamura, T.; Taniguchi, T. Interferon regulatory factor family of transcription factors
and regulation of oncogenesis. Cancer Science 2008, 99(3), 467-478. doi: 10.1111/j.1349-
7006.2007.00720.
34. Tsuno, T.; Mejido, J.; Zhao, T.; Morrow, A.; Zoon, K.C. IRF9 is a key factor for eliciting the
antiproliferative activity of IFN-α. J Immunother 2009, 32(8), 803. doi:
10.1097/CJI.0b013e3181ad4092.
Journal Pre-proof
69
35. Honda, K.; Takaoka, A.; Taniguchi, T. Type I interferon [corrected] gene induction by the
interferon regulatory factor family of transcription factors. Immunity 2006, 25(3), 349-360. doi:
10.1016/j.immuni.2006.08.009.
36. Sayers, T.J. Targeting the extrinsic apoptosis signaling pathway for cancer therapy. Cancer
Immunol Immunother 2011, 60(8), 1173-1180. doi: 10.1007/s00262-011-1008-4.
37. Testa, U. TRAIL/TRAIL‐R in hematologic malignancies. J Cell Biochem 2010, 110(1), 21-34. doi:
10.1002/jcb.22549
38. Finnberg, N.K.; El-Deiry, W.S. TRAIL death receptors as tumor suppressors and drug targets. Cell
Cycle 2008, 7(11), 1525-1528. doi: 10.4161/cc.7.11.5975
39. Dunn, G.P.; Bruce, A.T.; Sheehan, K.C.F.; Shankaran, V.; Uppaluri, R.; Bui, J.D.; Diamond, M.S.;
Koebel, C.M.; Arthur, C.; White, J.M. et al. A critical function for type I interferons in cancer
immunoediting. Nat Immunol 2005, 6(7), 722-9. doi: 10.1038/ni1213.
40. Bidwell, B.N.; Slaney, C.Y.; Withana, N.P.; Forster, S.; Cao, Y.; Loi, S.; Andrews, D.; Mikeska, T.;
Mangan, N.E.; Samarajiwa, S.A.; et al. Silencing of Irf7 pathways in breast cancer cells promotes
bone metastasis through immune escape. Nature Med 2012, 18(8), 1224-1231. doi: 10.1038/nm.2830.
41. Li, Y.; Huang, R.; Wang, L.; Hao, J.; Zhang, Q.,; Ling, R.; Yun, J. micro RNA‐762 promotes breast
cancer cell proliferation and invasion by targeting IRF7 expression. Cell Prolif 2015, 48(6), 643-649.
doi: 10.1111/cpr.12223.
42. Zhao, Y.; Chen, W.; Zhu, W.; Meng, H.; Chen, J.; Zhang, J. Overexpression of interferon regulatory
factor 7 (IRF7) reduces bone metastasis of prostate cancer cells in mice. Oncol Res 2017, 25(4), 511.
doi: 10.3727/096504016X14756226781802.
Journal Pre-proof
70
43. Solis, M.; Goubau, D.; Romieu-Mourez, R.; Genin, P.; Civas, A.; Hiscott, J. Distinct functions of
IRF-3 and IRF-7 in IFN-alpha gene regulation and control of anti-tumor activity in primary
macrophages. Biochem Pharmacol 2006, 72(11), 1469-1476. doi: 10.1016/j.bcp.2006.06.002.
44. Erb, H.H.; Langlechner, R.V.; Moser, P.L; Handle, F.; Casneuf, T.; Verstraeten, K.; Schlick, B.;
Schäfer, G.; Hall, B.; Sasser, K.; Culig, Z.; Santer, F.R.; et al. IL6 sensitizes prostate cancer to the
antiproliferative effect of IFNα2 through IRF9. Endocrine-related Cancer 2013, 20(5), 677. doi:
10.1530/ERC-13-0222.
45. Tian, W.-L.; Guo, R.; Wang, F.; Jiang, Z.-X.; Tang, P.; Huang, Y.-M.; Sun, L. The IRF9-SIRT1-P53
axis is involved in the growth of human acute myeloid leukemia. Exper Cell Res 2018, 365, 185-
193. doi: 10.1016/j.yexcr.2018.02.036.
46. Mittal, M.K.; Chaudhuri, G. Abstracts: First AACR International Conference on Frontiers in Basic
Cancer Research–Oct 8–11, 2009. Boston, MA. 2009. doi: 10.1158/0008-5472.FBCR09-A16.
https://cancerres.aacrjournals.org/content/69/23_Supplement/A16.short
47. Buckley, N.E.; Hosey, A.M.; Gorski, J.J.; Purcell, J.W.; Mulligan, J.M.; Harkin, D.P.; Mullan, P.B.
BRCA1 regulates IFN-γ signaling through a mechanism involving the type I IFNs. Mol Cancer Res
2007, 5(3), 261-270. doi: 10.1158/1541-7786.MCR-06-0250.
48. Rasmussen, S.A.; Abul-Husn, N.S.; Casanova, J.L; Daly, M.J.; Rehm, H.L; Murray, M.F. The
intersection of genetics and COVID-19 in 2021: preview of the 2021 Rodney Howell Symposium.
Genetics in Medicine 2021, 23(6), 1001-1003. doi: 10.1038/s41436-021-01113-0.
49. Mishra, R.; Banerjea, A.C. SARS-CoV-2 Spike targets USP33-IRF9 axis via exosomal miR-148a to
activate human microglia. Front Immunol 2021, 12, 656700. doi: 10.3389/fimmu.2021.656700.
50. National Cancer Institute. 2021. BRCA Gene Mutations: Cancer Risk and Genetic Testing Fact
Sheet. [online] Available at: https://www.cancer.gov/about-cancer/causes-
Journal Pre-proof
71
prevention/genetics/brca-fact-sheet#what-other-cancers-are-linked-to-harmful-variants-inbrca1-
and-brca2. [Accessed 27 November 2021].
51. Liu, J.; Wang, J.; Xu, J.; Xia, H.; Wang, Y.; Zhang, C.; Chen, W.; Zhang, H.; Liu, Q.; Zhu, R.; et al.
Comprehensive investigations revealed consistent pathophysiological alterations after
vaccination with COVID-19 vaccines. Cell Discov 2021, 7(1), 99. doi: 10.1038/s41421-021-00329-3.
52. Cancer risk and BRCA1 gene mutations. 2021. Available at:
https://www.facingourrisk.org/info/hereditary-cancer-and-genetic-testing/hereditary-cancergenes-
and-risk/genes-by-name/brca1/cancer-risk [Accessed 27 November 2021].
53. Zhang, W.; Luo, J.; Yang, F.; Wang, Y.; Yin, Y.; Strom, A.; Gustafsson, J.Å.; Guan, X. BRCA1
inhibits AR-mediated proliferation of breast cancer cells through the activation of SIRT1. Sci
Reports 2016, 6, 22034. doi: 10.1038/srep22034.
54. Suberbielle, E.; Djukic, B.; Evans, M.; Kim, D.H.; Taneja, P.; Wang, X.; Finucane, M.; Knox, J.; Ho,
K.; Devidze, N.; et al. DNA repair factor BRCA1 depletion occurs in Alzheimer brains and impairs
cognitive function in mice. Nat Comm 2015, 6, 8897. doi: 10.1038/ncomms9897.
55. Goldman, S.; Bron, D.; Tousseyn, T.; Vierasu, I.; Dewispelaere, L.; Heimann, P.; Cogan, E.;
Goldman, M. Rapid progression of angioimmunoblastic T cell lymphoma following BNT162b2
mRNA vaccine booster shot: A case report. Front Med 2021, 8, 798095. doi:
10.3389/fmed.2021.798095.
56. MacFarlane, M.; Kohlhaas, S.L.; Sutcliffe, M.J.; Dyer, M.J.; Cohen, G.M. TRAIL receptor-selective
mutants signal to apoptosis via TRAIL-R1 in primary lymphoid malignancies. Cancer Res 2005,
65(24), 11265-11270. doi: 10.1158/0008-5472.CAN-05-2801.
57. Pardi, N.; Hogan, M.J.; Porter, F.W.; Weissman, D. mRNA vaccines – a new era in vaccinology.
Nat Rev Drug Discov 2018, 17(4), 261-279. doi: 10.1038/nrd.2017.243.
Journal Pre-proof
72
58. Van Lint, S.; Renmans, D.; Broos, K.; Dewitte, H.; Lentacker, I.; Heirman, C.; Breckpot, K.;
Thielemans, K. The ReNAissanCe of mRNA-based cancer therapy. Expert Rev Vaccines 2015, 14(2),
235-51. doi: 10.1586/14760584.2015.957685.
59. Martini, P.G.V.; Guey, L.T. A new era for rare genetic diseases: Messenger RNA therapy. Hum
Gene Ther 2019, 30(10), 1180-1189. doi: 10.1089/hum.2019.090.
60. Doulberis, M.; Papaefthymiou,A.; Kotronis, G.; Gialamprinou, D.; Soteriades, E.S.;
Kyriakopoulos, A.; et al. Does COVID-19 vaccination warrant the classical principle “ofelein i mi
vlaptin”?. Medicina (Kaunas). 2021, 57(3), 253. doi:10.3390/medicina57030253.
61. Gordon, D.E.; Hiatt, J. Bouhaddou, M.; Rezelj, V.V.; Ulferts, S.; Braberg, H.; et al. Comparative
host-coronavirus protein interaction networks reveal pan-viral disease mechanisms. Science 2020,
370(6521), eabe9403. doi: 10.1126/science.abe9403.
62. Kaczmarek, R.; El Ekiaby, M.; Hart, D. P.; Hermans, C.; Makris, M.; Noone, D.; O’Mahony, B.;
Page, D.; Peyvandi, F.; Pipe, S.W.; et al. Vaccination against COVID‐19: Rationale, modalities and
precautions for patients with haemophilia and other inherited bleeding disorders. Haemophilia
2021, 27(4), 515-518. doi: 10.1111/hae.14271.
63. Wisnewski, A.V.; Campillo Luna, J.; Redlich, C.A. Human IgG and IgA responses to COVID-19
mRNA vaccines. PLoS ONE 2021, 16(6), e0249499. doi: 10.1371/journal.pone.0249499.
64. Karikó, K.; Buckstein, M.; Ni, H.; Weissman, D. Suppression of RNA recognition by toll-like
receptors: The impact of nucleoside modification and the evolutionary origin of RNA. Immunity
2005, 23, 165175. doi: 10.1016/j.immuni.2005.06.008.
65. de Beuckelaer, A.; Pollard, C.; Van Lint, S.; Roose, K.; Van Hoecke,L.V.; Naessens, T.;
Udhayakumar, V.K.; Smet, M.; Sanders, N.; Lienenklaus, S.; et al. Type I interferons interfere with
Journal Pre-proof
73
the capacity of mRNA lipoplex vaccines to elicit cytolytic T cell responses. Mol Ther 2016, 24(11),
2012-2020. doi: 10.1038/mt.2016.161.
66. Andries, O.; Mc Cafferty, S.; De Smedt, S.C.; Weiss, R.; Sanders, N.N.; Kitada, T. N1-
methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by
providing enhanced protein expression and reduced immunogenicity in mammalian cell lines
and mice. J Control Release 2015, 217, 337-344. doi: 10.1016/j.jconrel.2015.08.051.
67. Park, J.W.; Lagniton, P.; Liu, Y.; Xu, R.H. (2021). mRNA vaccines for COVID-19: what, why and
how. Int J Biol Sci 2021, 17(6), 1446–1460. doi: 10.7150/ijbs.59233.
68. Forni, G.; Mantovani, A.; COVID-19 Commission of Accademia Nazionale dei Lincei, Rome.
COVID-19 vaccines: where we stand and challenges ahead. Cell Death Differ 2021, 28(2), 626-639.
doi: 10.1038/s41418-020-00720-9.
69. Weiner, J.; Lewis, D.; Maertzdorf, J.; Mollenkopf, H.; Bodinham, C.; Pizzoferro, K.; Linley, C.;
Greenwood, A.; Mantovani, A.; Bottazzi, B.; Denoel, P.; Leroux-Roels, G.; Kester, K.; Jónsdóttir,
I.; van den Berg, R.A.; Kaufmann, S.; Del Giudice, G. Characterization of potential biomarkers of
reactogenicity of licensed antiviral vaccines: randomized controlled clinical trials conducted by
the BIOVACSAFE consortium. Scientific Reports 2019, 9(1), 20362. Doi: 10.1038/s41598-019-56994-
8.
70. Mulligan, M.J.; Lyke, K.E.; Kitchin, N.; Absalon, J.; Gurtman, A.; Lockhart, S.; Neuzil, K.; Raabe,
V.; Bailey, R.; Swanson, K.A.; Li, P.; Koury, K.; Kalina, W.; Cooper, D.; Fontes-Garfias, C.; Shi, P.-
Y.; Türeci, Ö.: Tompkins, K.R.; Walsh, E.E.; Frenck, R.; Falsey, A.R.; Dormitzer, P.R.; Gruber, W.C.;
Şahin U.; Jansen K.U. Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults. Nature
2020, 586(7830), 589-593. doi: 10.1038/s41586-020-2639-4.
Journal Pre-proof
74
71. Collier, D.A.; De Marco, A.; Ferreira, I.A.T.M.; Meng, B.; Datir, R.P.; Walls, A.C.; et al. Sensitivity
of SARS-CoV-2 B.1.1.7 to mRNA vaccine-elicited antibodies. Nature 2021, 593, 136-141. Doi:
10.1038/s41586-021-03412-7.
72. Katalin Karikó, K.; Muramatsu, H.; Welsh, F.A.; Ludwig, J.; Kato, H.; Akira, S.; Weissman, D.;
Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with
increased translational capacity and biological stability. Mol Ther 2008, 16, 1833-1840. Doi:
10.1038/mt.2008.200.
73. Timmers, L.F.S.M.; Peixoto, J.V.; Ducati, R.G.; Bachega, J.F.R.; de Mattos Pereira, L.; Caceres, R.A.;
Majolo, F.; da Silva, G.L.; Anton, D.B.; Dellagostin, O.A.; Henriques J.A.P.; Xavier L.L.; Goettert,
M.I.; Laufer, S. SARS-CoV-2 mutations in Brazil: from genomics to putative clinicalconditions.
Sci Rep 2021, 11, 11998. Doi: 10.1038/s41598-021-91585-6.
74. Kyriakopoulos, A.M.; McCullough, P.A. Synthetic mRNAs; Their Analogue Caps and
Contribution to Disease. Diseases 2021, 9(3), 57. doi: 10.3390/diseases9030057.
75. Jiang, H.; Mei, Y.-F. SARS-CoV-2 spike impairs DNA damage repair and inhibits V(D)J
recombination in vitro. Viruses 2021, 13, 2056. doi: 10.3390/v13102056.
76. Hou, X.; Zaks, T.; Langer, R.; Dong, Y. Lipid nanoparticles for mRNA delivery. Nat Rev Mater
2021, 6, 1078-1094. doi: 10.1038/s41578-021-00358-0.
77. Wrapp, D.; Wang, N.; Corbett, K.S.; Goldsmith, J.A.; Hsieh, C.L.; Abiona, O.; Graham, B.S.;
McLellan, J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science
2020, 367(6483), 1260-1263. doi: 10.1126/science.abb2507.
78. Orlandini von Niessen, A.G.; Poleganov, M.A.; Rechner, C.; Plaschke, A.; Kranz, L.M.; Fesser, S.;
Diken, M.; Löwer, M.; Vallazza, B.; Beissert, T.; et al. Improving mRNA-based therapeutic gene
Journal Pre-proof
75
delivery by expression-augmenting 3’ UTRs identified by cellular library screening. Mol Ther
2019, 27(4), 824-836. doi: 10.1016/j.ymthe.2018.12.011.
79. Xia, X. Detailed dissection and critical evaluation of the Pfizer/BioNTech and Moderna mRNA
vaccines. Vaccines 2021, 9, 734. doi: 10.3390/vaccines9070734.
80. Williams, G.D.; Gokhale, N.S.; Snider, D.L.; Horner, S.M. The mRNA cap 2′-O-methyltransferase
CMTR1 regulates the expression of certain interferon-stimulated genes. mSphere 2020, 5(3),
e00202-20. doi: 10.1128/mSphere.00202-20.
81. Leung, D.W.; Amarasinghe, G.K. When your cap matters: structural insights into self vs non-self
recognition of 5′ RNA by immunomodulatory host proteins. Curr Opin Struct Biol 2016, 36, 133-
141. doi: 10.1016/j.sbi.2016.02.001.
82. Chaudhary, N.; Weissman, D.; Whitehead, K.A. mRNA vaccines for infectious diseases:
principles, delivery and clinical translation. Nat Rev Drug Discov 2021, 20, 817–838. doi:
10.1038/s41573-021-00283-5.
83. Röltgen, K.; Nielsen, S.C.A.; Silva, O.; Younes, S.F.; Zaslavsky, M.; Costales, C.; Yang, F.; Wirz,
O.F.; Solis, D.; Hoh, R.A. Immune imprinting, breadth of variant recognition and germinal center
response in human SARS-CoV-2 infection and vaccination. Cell 2022 Jan 25; S0092-8674(22)00076-
9. [Epub ahead of print]. doi: 10.1016/j.cell.2022.01.018.
84. McKernan, K.; Kyriakopoulos, A.M.; McCullough, P.A. Differences in vaccine and SARS-CoV-2
replication derived mRNA: Implications for cell biology and future disease. OSF Preprints
November 26, 2021. doi: 10.31219/osf.io/bcsa6.
85. Mauro, V.P.; Chappell, S.A. A critical analysis of codon optimization in human therapeutics.
Trends Mol Med 2014, 20(11), 604-13. doi: 10.1016/j.molmed.2014.09.003.
Journal Pre-proof
76
86. Shabalina, S.A.; Spiridonov, N.A.; Kashina, A. Sounds of silence: synonymous nucleotides as a
key to biological regulation and complexity. Nucleic Acids Res 2013, 41(4), 2073-94. doi:
10.1093/nar/gks1205.
87. Zhou, M.; Guo, J.; Cha, J.; Chae, M.; Chen, S.; Barral, J.M.; Sachs, M.S.; Liu, Y. Non-optimal codon
usage affects expression, structure and function of clock protein FRQ. Nature 2013, 495(7439), 111-
5. doi: 10.1038/nature11833.
88. Agashe, D.; Martinez-Gomez, N.C.; Drummond, D.A.;Marx, C.J. Good codons, bad transcript:
large reductions in gene expression and fitness arising from synonymous mutations in a key
enzyme. Mol Biol Evol 2013, 30, 549-560. doi: 10.1093/molbev/mss273
89. McCarthy, C.; Carrea, A.; Diambra, L.Bicodon bias can determine the role of synonymous SNPs
in human diseases. BMC Genomics 2017, 18(1), 227. doi: 10.1186/s12864-017-3609-6.
90. Kudla, G.; Lipinski, L.; Caffin, F.; Helwak, A.; Zylicz, M. High guanine and cytosine content
increases mRNA levels in mammalian cells. PLoS Biol 2006, 4(6), e180. doi:
10.1371/journal.pbio.0040180.
91. Otsuka, H.; Fukao, A.; Funakami, Y.; Duncan, K.E.; Fujiwara, T. Emerging evidence of
translational control by AU-rich element-binding proteins. Front. Genet 2019, 10, 332. doi:
10.3389/fgene.2019.00332.g.
92. Wang, E.; Thombre, R.; Shah, Y.; Latanich, R.; Wang, J. G-Quadruplexes as pathogenic drivers in
neurodegenerative disorders. Nucleic Acids Research 2021, 49(9), 4816-4830. doi:
10.1093/nar/gkab164.
93. Olsthoorn, R.C. G-quadruplexes within prion mRNA: the missing link in prion disease? Nucleic
Acids Res 2014, 42, 9327-9333. doi: 10.1093/nar/gku559.
Journal Pre-proof
77
94. Tetz, G.; Tetz, V. Prion-like domains in spike protein of SARS-CoV-2 differ across its variants
and enable changes in affinity to ACE2. Microorganisms 2022, 10, 280. doi:
10.3390/microorganisms10020280.
95. Babendure, J.R.; Babendure, J.L.; Ding, J.H.; Tsien, R.Y. Control of mammalian translation by
mRNA structure near caps. RNA 2006, 12(5), 851-861. doi:10.1261/rna.2309906
96. Herdy, B.; Mayer, C.; Varshney, D.; Marsico, G.; Murat, P.; Taylor, C.; D’Santos, C.; Tannahill, D.;
Balasubramanian, S. Analysis of NRAS RNA G-quadruplex binding proteins reveals DDX3X as
a novel interactor of cellular G-quadruplex containing transcripts. Nucleic Acids Res 2018, 46(21),
11592-11604. doi: 10.1093/nar/gky861.
97. Fay, M.M.; Lyons, S.M.; Ivanov, P. RNA G-quadruplexes in biology: principles and molecular
mechanisms. J Mol Biol 2017, 429(14), 2127–2147. doi: 10.1016/j.jmb.2017.05.017.
98. Zhang, R.; Xiao, K.; Gu, Y.; Liu, H.; Sun, X. Whole genome identification of potential Gquadruplexes
and analysis of the G-quadruplex binding domain for SARS-CoV-2. Front Genet
2020, 11, 587829. doi: 10.3389/fgene.2020.587829.
99. Schmidt, N.; Lareau, C.A.; Keshishian, H.; Ganskih, S.; Schneider, C.; Hennig, T.; Melanson, R.;
Werner, S.; Wei, Y.; Zimmer, M.; et al. The SARS-CoV-2 RNA-protein interactome in infected
human cells. Nat Microbiol 2021, 6(3), 339-353. doi: 10.1038/s41564-020-00846-z.
100. Rouleau, S.; Glouzon, J.S.; Brumwell, A.; Bisaillon, M.; Perreault, J.P. 3′ UTR G-quadruplexes
regulate miRNA binding. RNA, 2017, 23(8), 1172-1179. doi:10.1261/rna.060962.117.
101. Bezzi, G.; Piga, E.J.; Binolfi, A.; Armas, P. CNBP binds and unfolds in vitro G-quadruplexes
formed in the SARS-CoV-2 positive and negative genome strands. Int J Mol Sci 2021, 22(5), 2614.
doi: 10.3390/ijms22052614.
Journal Pre-proof
78
102. Sola, I.; Almazán, F.; Zúñiga, S.; Enjuanes, L. Continuous and discontinuous RNA synthesis in
coronaviruses. Annu Rev Virol 2015, 2(1), 265-88. doi: 10.1146/annurev-virology-100114-055218.
103. Jaubert, C.; Bedrat, A.; Bartolucci, L.; Di Primo, C.; Ventura, M.; Mergny, J.-L.; Amrane, S.;
Andreola, M.-L RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus
negative RNA strand. Sci Rep 2018, 8, 8120. https://doi.org/10.1038/s41598-018-26582-3.
104. Spiegel, J.; Adhikari, S.;Balasubramanian, S. The structure and function of DNA G-quadruplexes.
Trends Chem 2020, 2(2), 123-136. doi: 10.1016/j.trechm.2019.07.002.
105. Rouleau, S.G.; Garant, J.-M.; Balduc, F.; Bisaillon, M.; Perreault, J.-P. G-Quadruplexes influence
pri-microRNA processing. RNA Biology 2018, 15(2), 198-206. doi: 10.1080/15476286.2017.1405211.
106. Chan, K.L.; Peng, B.; Umar, M.I.; Chan, C.Y.; Sahakyan, A.B.; Le, M.T.N.; Kwok, C.K. Structural
analysis reveals the formation and role of RNA G-quadruplex structures in human mature
microRNAs. Chem Commun (Camb) 2018, 54(77), 10878-10881. doi: 10.1039/c8cc04635b.
107. Al-Khalaf, H.H.; Aboussekhra, A. p16 controls p53 protein expression through miR-dependent
destabilization of MDM2. Mol Cancer Res 2018, 16(8), 1299-1308. doi: 10.1158/1541-7786.MCR-18-
0017.
108. Weldon, C.; Dacanay, J.G.; Gokhale, V.; Boddupally, P.V.L.; Behm-Ansmant, I.; Burley, G.A.;
Branlant, C.; Hurley, L.M.; Dominguez, C.; Eperon, I.C. Specific G-quadruplex ligands modulate
the alternative splicing of Bcl-X. Nucleic Acids Res 2018, 46(2), 886-896. doi: 10.1093/nar/gkx1122.
109. Small, E.M.; Olson, E.N. Pervasive roles of microRNAs in cardiovascular biology. Nature 2011,
469(7330), 336-342. doi:10.1038/nature09783.
110. Abe, M.; Bonini, N.M. MicroRNAs and neurodegeneration: role and impact. Trends Cell Biol 2013,
23(1), 30-6. doi: 10.1016/j.tcb.2012.08.013.
Journal Pre-proof
79
111. Farazi, T.A.; Hoell, J.I.; Morozov, P.; Tuschl, T. MicroRNAs in human cancer. Adv Exp Med Biol
2013, 774, 1-20. doi: 10.1007/978-94-007-5590-1_1.
112. Ozaki, T.; Nakagawara, A. Role of p53 in Cell Death and Human Cancers. Cancers (Basel) 2011,
3(1), 994-1013. doi:10.3390/cancers3010994.
113. Janeway, C.A., Jr.; Medzhitov, R. Innate immune recognition. Annu Rev Immunol 2002, 20, 197-
216. doi: 10.1146/annurev.immunol.20.083001.084359.
114. Hadjadj, J.; Yatim, N.; Barnabei, L.; Corneau, A.; Boussier, J.; Smith, N.; Péré, H.; Charbit, B.;
Bondet, V.; Chenevier-Gobeaux, C.; et al. Impaired type I interferon activity and inflammatory
responses in severe COVID-19 patients. Science 2020, 369(6504), 718-724. doi:
10.1016/j.cell.2020.04.026.
115. Blanco-Melo, D.; Nilsson-Payant, B.E.; Liu, W.C.; Uhl, S.; Hoagland, D.; Møller, R.; Jordan, T.X.;
Oishi, K.; Panis, M.; Sachs, D.; et al. Imbalanced host response to SARS-CoV-2 drives development
of COVID-19. Cell. 2020, 181(5), 1036-1045 e9.
116. Hoagland, D.A.; Møller, R.; Uhl, S.A.; Oishi, K.; Frere, J.; Golynker, T.; Horiuchi, S.; Panis, M.;
Blanco-Melo, D.; Sachs, D.; et al. Leveraging the antiviral type I interferon system as a first line
of defense against SARS-CoV-2 pathogenicity. Immunity 2021, 54, 557570. doi:
10.1016/j.immuni.2021.01.017.
117. Wang, N.; Zhan, Y.; Zhu, L.; Hou, Z.; Liu, F.; Song, P.; Qiu, F.; Wang, X.; Zou, X.; Wan, D.; et al.
Retrospective multicenter cohort study shows early interferon therapy is associated with
favorable clinical responses in COVID-19 patients. Cell Host Microbe 2020, 28(3), 455-464.e2. doi:
10.1016/j.chom.2020.07.005.
118. van der Wijst, M.G.P.; Vazquez, S.E.; Hartoularos, G.C.; Bastard, P.; Grant, T.; Bueno, R>; Lee,
D.S.; Greenland, J.R.; Sun, Y.; Perez, R.; et al. Type I interferon autoantibodies are associated with
Journal Pre-proof
80
systemic immune alterations in patients with COVID-19. Sci Transl Med 2021, 13(612), eabh2624.
doi: 10.1126/scitranslmed.abh2624.
119. Troya, J.; Bastard, P.; Planas-Serra, L.; Ryan, P.; Ruiz, M.; de Carranza, M.; Torres, J.; Martnez, A.;
Abel, L.; Casanova, J.-L.; Pujol, A. Neutralizing autoantibodies to type I IFNs in >10% of patients
with severe COVID-19 pneumonia hospitalized in Madrid, Spain. J Clin Immunol 2021, 41, 914922.
doi: 10.1007/s10875-021-01036-0.
120. Chauvineau‐Grenier, A.; Bastard, P.; Servajeank, A.; Gervais, A.; Rosain, J.; Jouanguy, E.; Cobat,
A.; Casanova, J.-L.; Rossi, B. Autoantibodies neutralizing type I interferons in 20% of COVID‐19
deaths in a French hospital. J Clin Immunol January 27, 2022 [Epub ahead of print]. doi:
10.1007/s10875-021-01203-3.
121. Stertz, S.; Hale, B.G. Interferon system deficiencies exacerbating severe pandemic virus infections.
Trends Microbiol 2021, 29(11), 973-982. doi: 10.1016/j.tim.2021.03.001.
122. Zaccara, S.; Ries, R.J.; Jaffrey S.R. Reading, writing and erasing mRNA methylation. Nature
Reviews 2019, 20, 608-624. Doi: 10.1038/s41580-019-0168-5.
123. Yang, C.; Hu, Y.; Zhou, B.; Bao, Y.; Li, Z.; Gong, C.; Yang, H.; Wang, S.; Xiao, Y. The role of m6A
modification in physiology and disease. Cell Death Dis 2020, 11, 960.
https://doi.org/10.1038/s41419-020-03143-z
124. Knuckles, P.; Bühler, M. Adenosine methylation as a molecular imprint defining the fate of RNA.
FEBS Lett 2018, 592(17), 2845-2859. doi:10.1002/1873-3468.13107.
125. Koo, J.W.; Russo, S.J.; Ferguson, D.; Nestler, E.J.; Duman, R.S. Nuclear factor-kappaB is a critical
mediator of stress-impaired neurogenesis and depressive behavior. Proc Natl Acad Sci U S A 2010,
107(6), 2669-2674. doi:10.1073/pnas.0910658107.
Journal Pre-proof
81
126. Meyer, K.D.; Patil, D.P.; Zhou, J.; Zinoviev, A.; Skabkin, M.A.; Elemento, O.; Pestova, T.V.; Qian,
S.-B.; Jaffrey, S.R. 5′ UTR m(6)A promotes cap-independent translation. Cell 2015, 163(4), 999-1010.
doi: 10.1016/j.cell.2015.10.012.
127. Shatsky, I.N.; Terenin, I.M.; Smirnova, V.V.; Andreev, D.E.. Cap-independent translation: What’s
in a name? Trends Biochem Sci 2018, 43(11), 882-895. doi: 10.1016/j.tibs.2018.04.011.
128. Svitkin, U.V.; Herdy, B.; Costa-Mattioli, M.; Gingras, A.-C.; Raught, B.; Sonenberg, N. Eukaryotic
translation initiation factor 4E availability controls the switch between cap-dependent and
internal ribosomal entry site-mediated translation. Mol Cell Biol 2005, 25(23), 10556-65. doi:
10.1128/MCB.25.23.10556-10565.2005.
129. Han, S.H.; Choe, J. Diverse molecular functions of m6A mRNA modification in cancer. Exp Mol
Med 2020, 52(5), 738-749. doi:10.1038/s12276-020-0432-y.
130. Yoshikawa, F.S.; Teixeira, F.M.; Sato, M.N.; Oliveira, L.M.Delivery of microRNAs by extracellular
vesicles in viral infections: Could the news be packaged? Cells 2019, 8((6), 611. doi:
10.3390/cells8060611.
131. Ratajczak, M.Z.; Ratajczak, J. Horizontal transfer of RNA and proteins between cells by
extracellular microvesicles: 14 years later. Clin Trans Med 2016, 5, 7. doi: 10.1186/s40169-016-0087-
4.
132. Chahar, H.S.; Bao, X.; Casola, A. Exosomes and their role in the life cycle and pathogenesis of
RNA viruses. Viruses 2015, 7, 3204-3225; doi: 10.3390/v7062770.
133. Bansal, S.; Perincheri, S.; Fleming, T.; Poulson, C.; Tiffany, B.; Bremner, R.M.; Mohanakumar, T..
Cutting edge: circulating exosomes with COVID spike protein are induced by BNT162b2
(PfizerBioN-Tech) vaccination prior to development of antibodies: A novel mechanism for
Journal Pre-proof
82
immune activation by mRNA vaccines. J Immunol 2021, 207(10), 2405-2410. doi:
10.4049/jimmunol.2100637.
134. Decker, C.J.; Parker, R. P-bodies and stress granules: possible roles in the control of translation
and mRNA degradation. Cold Spring Harb Perspect Biol 2012, 4(9), a012286.
doi:10.1101/cshperspect.a012286.
135. Kothandan, V.K.; Kothandan, S.; Kim, D.H.; Byun, Y.; Lee, Y.-K.; Park, I.-K.; Hwang, S.R.
Crosstalk between stress granules, exosomes, tumour antigens, and immune cells: Significance
for cancer immunity. Vaccines 2020, 8(2), 172, doi:10.3390/vaccines8020172.
136. Borbolis, F.; Syntichaki, P. Cytoplasmic mRNA turnover and ageing. Mech Ageing Dev 2015, 152,
32-42. doi:10.1016/j.mad.2015.09.006.
137. Girardi, T.; De Keersmaecker, K. T-ALL: ALL a matter of translation?. Haematologica 2015, 100(3),
293-295. doi: 10.3324/haematol.2014.118562.
138. Jang, S.K.; Pestova, T.V.; Hellen, C.U.T.; Witherell, G.W.; Wimmer, E. Cap-independent
translation of picornavirus RNAs: structure and function of the internal ribosomal entry site.
Enzyme 1990, 44, 292-309. doi: 10.1159/000468766.
139. Zoll, J.; Erkens Hulshof, S.; Lanke, K.; Verduyn Lunel. F.; Melchers, W.J.; Schoondermark-van de
Ven, E.; Roivainen, M.; Galama, J.M.; van Kuppeveld, F.J. Saffold virus, a human Theiler’s-like
cardiovirus, is ubiquitous and causes infection early in life. PLoS Pathog 2009, 5(5), e1000416. doi:
10.1371/journal.ppat.1000416.
140. Rusk, N. When microRNAs activate translation. Nat Methods 2008, 5, 122–123. doi:
10.1038/nmeth0208-122a.
Journal Pre-proof
83
141. De Paolis, V.; Lorefice, E.; Orecchini, E.; Carissimi, C.; Laudadio, I.; Fulci, V.. Epitranscriptomics:
A new layer of microRNA regulation in cancer. Cancers (Basel). 2021, 13(13), 3372.
doi:10.3390/cancers13133372.
142. Yu, X.; Odenthal, M.; Fries, J.W.U. Exosomes as miRNA carriers: formation–function–future. Int
J Mol Sci 2016, 17, 2028. doi: 10.3390/ijms17122028.
143. Wei, H.; Chen, Q.; Lin, L.; Sha, C.; Li, T.; Liu, Y.; Yin, X.; Xu, Y.; Chen, L.; Gao, W.; Li, Y.; Zhu, X..
Regulation of exosome production and cargo sorting. Int J Biol Sci 2021, 17(1), 163–177. doi:
10.7150/ijbs.53671.
144. de Gonzalo-Calvo, D.; Benítez, I.D.; Pinilla, L.; Carratalá, A.; Moncusí-Moix, A.; Gort-Paniello, C.;
Molinero, M.; González, J.; Torres, G.; Bernal, M.; et al. Circulating microRNA profiles predict the
severity of COVID-19 in hospitalized patients. Transl Res 2021, 236, 147-159. doi:
10.1016/j.trsl.2021.05.004.
145. Bahl, K.; Senn, J.J.; Yuzhakov, O.; Bulychev, A.; Brito, L.A.; Hassett, K.J.; Laska M.E.; Smith, M.;
Almarsson, Ö.; Thompson, J.; et al. Preclinical and clinical demonstration of immunogenicity by
mRNA vaccines against H10N8 and H7N9 influenza viruses. Molecular Therapy 2017, 25(6), 1316-
1327. doi: 10.1016/j.ymthe.2017.03.035.
146. Seneff, S.; Nigh, G. Worse Than the Disease? Reviewing Some Possible Unintended Consequences
of the mRNA Vaccines Against COVID-19. IJVTPR 2021, 2(1), 38-79.
147. Gould, F.D.H.; Lammers, A.R.; Mayer, C.J.; German, R.Z. Specific vagus nerve lesion have
distinctive physiologic mechanisms of dysphagia. Front Neurol 2019, 10, 1301. doi:
10.3389/fneur.2019.01301.
148. Erman, A.B.; Kejner, A.E.; Norman, B.S.; Hogikyan, D.; Feldman, E.L. Disorders of cranial nerves
IX and X. Semin Neurol 2009, 29(1), 8592. doi: 10.1055/s-0028-1124027.
Journal Pre-proof
84
149. Shaw, G.; Morse. S.; Ararat, M.; Graham, F.L. Preferential transformation of human neuronal cells
by human adenoviruses and the origin of HEK 293 cells. FASEB J 2002, 16(8), 869-71. doi:
10.1096/fj.01-0995fje.
150. Kakarougkas, A.; Ismail, A.; Klement, K.; Goodarzi, A.A.; Conrad, S.; Freire, R.; Shibata, A.;
Lobrich, M.; Jeggo, P.A. Opposing roles for 53BP1 during homologous recombination. Nucleic
Acids Res 2013, 41(21), 9719-31. doi: 10.1093/nar/gkt729.
151. Choi, H.S.; Lee, H.M.; Jang, Y.-J.; Kim, C.-H.; Ryua, C.J. Heterogeneous nuclear ribonucleoprotein
A2/B1 regulates the self-renewal and pluripotency of human embryonic stem cells via the control
of the G1/S transition. Stem Cells 2013, 31, 2647-2658. doi: 10.1002/stem.1366.
152. Zhang, J.; Powell, S.N. The role of the BRCA1 tumor suppressor in DNA double-strand break
repair. Mol Cancer Res 2005, 3(10), 531-9. doi: 10.1158/1541-7786.MCR-05-0192.
153. Panier, S.; Boulton, S.J. Double-strand break repair: 53BP1 comes into focus. Nature Reviews 2014,
15, 9. doi: Ihttps://doi.org/10.1038/nrm3719.
154. Choi, Y.E.; Pan, Y.; Park, E.; Konstantinopoulos, P.; De, S.; D’Andrea, A.; Chowdhury, D.
MicroRNAs downregulate homologous recombination in the G1 phase of cycling cells to
maintain genomic stability. eLife 2014, 3, e02445. doi: 10.7554/eLife.02445.
155. Kolumam, G.A.; Thomas, S.; Thompson, L.J.; Sprent, J.; Murali-Krishna, K. Type I interferons act
directly on CD8 T cells to allow clonal expansion and memory formation in response to viral
infection. J Exp Med 2005, 202(5), 637650. doi: 10.1084/jem.20050821.
156. Liu, T.; Khanna, K.M.; Chen, X.; Fink, D.J.; Hendricks, R.L.. CD8(+) T cells can block herpes
simplex virus type 1 (HSV-1) reactivation from latency in sensory neurons. J Exp Med 2000, 191(9),
1459-66. doi: 10.1084/jem.191.9.1459.
Journal Pre-proof
85
157. Katsikas Triantafyllidis, K.; Giannos, P.; Mian, I. T.; Kyrtsonis, G.; Kechagias, K.S.). Varicella
zoster virus reactivation following COVID-19 vaccination: a systematic review of case reports.
Vaccines 2021, 9(9), 1013. doi: 10.3390/vaccines9091013.
158. Fathy, R.A.; McMahon, D.E.; Lee, C.; Chamberlin, G.C.; Rosenbach, M.; Lipoff, J.B.; Tyagi, A.;
Desai, S.R.; French, L.E.; Lim. H.W.; et al. Varicella-zoster and herpes simplex virus reactivation
post-COVID-19 vaccination: a review of 40 cases in an International Dermatology Registry.
JEADV 2022, 36(1), e6-e9. doi: 10.1111/jdv.17646.
159. Psichogiou, M.; Samarkos, M.; Mikos, N.; Hatzakis, A. Reactivation of Varicella zoster virus after
vaccination for SARS-CoV-2. Vaccines 2021, 9, 572. doi: 10.3390/vaccines9060572.
160. Iwanaga, J.; Fukuoka, H.; Fukuoka, N.; Yutori, H.; Ibaragi, S.; Tubbs, R.S.A narrative review and
clinical anatomy of Herpes zoster infection following COVID‐19 vaccination. Clin Anat 2021,
35(1), 45-51. doi: 10.1002/ca.23790.
161. Lladó, I.; Fernández-Bernáldez, A.; Rodríguez-Jiménez, P. Varicella zoster virus reactivation and
mRNA vaccines as a trigger. JAAD Case Reports 2021, 15, 62-63. doi: 10.1016/j.jdcr.2021.07.011.
162. Verweij, M.C.; Wellish, M.; Whitmer, T.; Malouli, D.; Lapel, M.; Jonjić, S.; Haas, J.G.; DeFilippis,
V.R.; Mahalingam, R.; Früh, K. Varicella viruses inhibit interferon-stimulated JAK-STAT
signaling through multiple mechanisms. PLoS Pathog 2015, 11(5), e1004901. doi:
10.1371/journal.ppat.1004901.
163. Lensen, R.; Netea, M.G.; Rosendaal, F.R. Hepatitis C virus reactivation following COVID-19
vaccination — A case report. Int Med Case Rep J 2021, 14, 573-575. doi: 10.2147/IMCRJ.S328482.
164. Perricone, C.; Ceccarelli, F.; Nesher, G.; Borella, E.; Odeh, Q.; Conti, F.; Shoenfeld, Y.; Valesini, G.
Immune thrombocytopenic purpura (ITP) associated with vaccinations: a review of reported
cases. Immunol Res 2014, 60, 226-35. 10.1007/s12026-014-8597-x
Journal Pre-proof
86
165. Kelton, J.G.; Arnold, D.M.; Nazy, I. Lessons from vaccine-induced immune thrombotic
thrombocytopenia. Nat Rev Immunol 2021, 21(12), 753-755. doi: 10.1038/s41577-021-00642-8.
166. Lee, E.-J.; Cines, D.B.; Gernsheimer, T.; Kessler, C.; Michel, M.; Tarantino, M.D.; Semple, J.W.;
Arnold, D.M.; Godeau, B.; Lambert, M.P.; Bussel, J.B. Thrombocytopenia following Pfizer and
Moderna SARS-CoV-2 vaccination. Am J Hematol 2021, 96(5), 534-537. https://doi.org/10.1002/a
jh.26132.
167. Akiyama, H.; Kakiuchi, S.; Rikitake, J.; Matsuba, H.; Sekinada, D.; Kozuki, Y.; Iwata, N.. Immune
thrombocytopenia associated with Pfizer-BioNTech’s BNT162b2 mRNA COVID-19 vaccine.
IDCases 2021, 25, e01245. doi: 10.1016/j.idcr.2021.e01245.
168. Atoui, A.; Jarrah, K.; Al Mahmasani, L.; Bou‐Fakhredin, R.; Taher, A.T. Deep venous thrombosis
and pulmonary embolism after COVID‐19 mRNA vaccination. Ann Hematol 2022,: 1–3. [Epub
ahead of print] Doi: : 10.1007/s00277-021-04743-1.
169. Zakaria, Z.; Sapiai, N.A.; Izaini Ghani, A.R. Cerebral venous sinus thrombosis 2 weeks after the
first dose of mRNA SARS‐CoV‐2 vaccine. Acta Neurochir (Wien) 2021, 163(8), 2359-2362. doi:
10.1007/s00701-021-04860-w.
170. Cines, D.B.; Bussel, J.B. SARS-CoV-2 vaccine-induced immune thrombotic thrombocytopenia. N
Engl J Med 2021, 384, 2254-2256. doi: 10.1056/NEJMe2106315.
171. Danese, E.; Montagnana, M.; Salvagno, G.L.; Peserico, D.; Pighi, L.; De Nitto, S.; Henry B.M.;
Porru, S.; Lippi, G. Comprehensive assessment of humoral response after Pfizer BNT162b2
mRNA Covid-19 vaccination: a three-case series. Clin Chem Lab Med 2021, 59(9), 1585-1591. doi:
10.1515/cclm-2021-0339.
Journal Pre-proof
87
172. Passariello, M.; Vetrei, C.; Amato, F.; De Lorenzo, C. Interactions of Spike-RBD of SARS-CoV-2
and Platelet Factor 4: New Insights in the Etiopathogenesis of Thrombosis. Int J Mol Sci 2021, 22,
8562. doi: 10.3390/ijms22168562.
173. Nevzorova, T.A.; Mordakhanova, E.R.; Daminova, A.G.; Ponomareva, A.A.; Andrianova, I.A.;
Minh, G.L.; Rauova, L.; Litvinov, R.L.; Weisel, J.W. Platelet factor 4-containing immune
complexes induce platelet activation followed by calpain-dependent platelet death. Cell Death
Discov 2019, 5, 106. doi: 10.1038/s41420-019-0188-0.
174. McKenzie, S.E.; Taylor, S.M.; Malladi, P.; Yuhan, H.; Cassel, D.L.; Chien, P.; Schwartz, E.;
Schreiber, A.D.; Surrey, S.; Reilly, M.P. The role of the human Fc receptor FcRIIA in the immune
clearance of platelets: A transgenic mouse model. J Immunol 1999, 162, 4311-4318.
http://www.jimmunol.org/content/162/7/4311.
175. Crow, A.R.; Lazarus, A.H. Role of Fcgamma receptors in the pathogenesis and treatment of
idiopathic thrombocytopenic purpura. J Pediatr Hematol Oncol 2003, 25(Suppl 1), S14S18. doi:
10.1097/00043426-200312001-00004.
176. Lu, Y.; Harada, M.; Kamijo, Y.; Nakajima, T.; Tanaka, N.; Sugiyama, E.; Kyogashima, M.;
Gonzalez, F.J.; Aoyama, T. Peroxisome proliferator-activated receptor attenuates high-cholesterol
diet-induced toxicity and pro-thrombotic effects in mice. Arch Toxicol 2019, 93(1), 149161. doi:
10.1007/s00204-018-2335-4.
177. Kimura, T.; Nakajima, T.; Kamijo, Y.; Tanaka, N.; Wang, L.; Hara, A.; Sugiyama, E.; Tanaka, E.;
Gonzalez, F.J.; Aoyama, T. Hepatic cerebroside sulfotransferase is induced by PPAR activation in
mice. PPAR Research 2012, 2012, 174932. doi: 10.1155/2012/174932
178. Wang, Y.; Nakajima, T.; Gonzalez, F.J.; Tanaka, N. PPARs as metabolic regulators in the liver:
Lessons from liver-specific PPAR-null mice. Int J Mol Sci 2020, 21, 2061. doi: 10.3390/ijms21062061.
Journal Pre-proof
88
179. Wang, X.-A.; Zhang, R.; Jiang, D.; Deng, W.; Zhang, S.; Deng, S.; Zhong, J.; Wang, T.; Zhu, L.-H.;
Yang, L.; et al. Interferon regulatory factor 9 protects against hepatic insulin resistance and
steatosis in male mice. Hepatology 2013, 58(2), 603-16. doi: 10.1002/hep.26368.
180. Zin Tun, G.S.; Gleeson, D.; Al-Joudeh, A.; Dube, A. Immune-mediated hepatitis with the Moderna
vaccine, no longer a coincidence but confirmed. J Hepatol 2021, Oct 5. [Epub ahead of print]. doi:
10.1016/j.jhep.2021.09.031
181. Dumortiera, J. Liver injury after mRNA-based SARS-CoV-2 vaccination in a liver transplant
recipient. Clin Res Hepatol Gastroenterol 2022, 46, 101743. doi: 10.1016/j.clinre.2021.101743.
182. Mann, R.; Sekhon, S.; Sekhon, S. Drug-induced liver injury after COVID-19 vaccine. Cureus 2021,
13(7), e16491. doi: 10.7759/cureus.16491.
183. Créange, A. A role for interferon-beta in Guillain-Barré Syndrome? BioDrugs 2000, 14(1), 1-11. doi:
10.2165/00063030-200014010-00001.
184. Ilyas, A.A.; Mithen, F.A.; Dalakas, M.C.; Wargo, M.; Chen, Z.W.; Bielory, L.; Cook, S.D.
Antibodies to sulfated glycolipids in Guillain-Barr syndrome. J Neurol Sci 1991, 105(1), 108-17.
doi: 10.1016/0022-510x(91)90126-r.
185. Vanderlugt, C.L.; Miller, S.D. Epitope spreading in immune-mediated diseases: Implications for
immunotherapy. Nat Rev Immunol 2002, 2, 85-95. doi: 10.1038/nri724.
186. Kuwahara, M.; Kusunoki, S. Mechanism and spectrum of anti-glycolipid antibody-mediated
chronic inflammatory demyelinating polyneuropathy. Clin Exper Neuroimmunol 2018, 9(1), 65-74.
doi: 10.1111/cen3.12452.
187. Kalra, R.S.; Kandimalla, R. Engaging the spikes: heparan sulfate facilitates SARS-CoV-2 spike
protein binding to ACE2 and potentiates viral infection. Signal Transduct Target Ther 2021, 6, 39.
doi: 10.1038/s41392-021-00470-1.
Journal Pre-proof
89
188. Honke, K. Biosynthesis and biological function of sulfoglycolipids. Proc Jpn Acad Ser B Phys Biol
Sci 2013, 89(4), 129138. doi: 10.2183/pjab.89.129.
189. Qiu, S.; Palavicini, J.P.; Wang, J.; Gonzalez, N.S.; He, S.; Dustin, E.; Zou, C.; Ding, L.; Bhattacharjee,
A.; Van Skike, C.E.; et al. Adult-onset CNS myelin sulfatide deficiency is sufficient to cause
Alzheimer’s disease-like neuroinflammation and cognitive impairment. Mol Neurodegen 2021, 16,
64. doi: 10.1186/s13024-021-00488-7.
190. Marcus, J.; Honigbaum, S.; Shroff, S.; Honke, K.; Rosenbluth, J.; Dupree, J.L. Sulfatide is essential
for the maintenance of CNS myelin and axon structure. Glia 2006, 53(4), 372-81. doi:
10.1002/glia.20292.
191. Lanz. T.V.; Ding, Z.; Ho, P.P.; Luo, J.; Agrawal, A.N.; Srinagesh, H.; Axtell, R.; Zhang, H.; Platten,
M.; Wyss-Coray, T.; Steinman, L. Angiotensin II sustains brain inflammation in mice via TGFbeta.
J Clin Invest 2010, 120(8), 2782-94. doi: 10.1172/JCI41709.
192. Letarov, A.V.; Babenko, V.V.; Kulikov, E.E.; Free SARS-CoV-2 spike protein S1 particles may play
a role in the pathogenesis of COVID-19 infection. Biochemistry (Moscow) 2021, 86(3), 257-261. doi:
10.1134/S0006297921030032.
193. Rhea, E.M.; Logsdon, A.F.; Hanse, K.M.; Williams, L.M.; Reed, M.J.; Baumann, K.K.; Holden, S.J.;
Raber, J.; Banks, W.A.; Erickson, M.A. The S1 protein of SARS-CoV-2 crosses the blood-brain
barrier in mice. Nature Neurosci 2021, 24, 368-378. doi: 10.1038/s41593-020-00771-8.
194. Rodriguez-Perez, A.I.; Borrajo, A.; Rodriguez-Pallares, J.; Guerra, M.J.; Labandeira-Garcia, J.L.
Interaction between NADPH-oxidase and Rho-kinase in angiotensin II-induced microglial
activation. Glia 2015, 63, 466e482. doi: 10.1002/glia.22765.
Journal Pre-proof
90
195. Guo, X.; Namekata, K.; Kimura, A.; Harada, C.; Harada, T. The renin-angiotensin system
regulates neurodegeneration in a mouse model of optic neuritis. Am J Pathol 2017, 187(12), 2876-
2885. doi: 10.1016/j.ajpath.2017.08.012.
196. Maleki, A. COVID-19 recombinant mRNA vaccines and serious ocular inflammatory side effects:
Real or coincidence? J Ophthalmic Vis Res 2021, 16(3), 490501. doi: 10.18502/jovr.v16i3.9443.
197. Barone, V.; Camilli, F.; Crisci, M.; Scandellari, C.; Barboni, P.; Lugaresia, A.. Inflammatory optic
neuropathy following SARS-CoV-2 mRNA vaccine: Description of two cases. J Neurol Sci 2021,
429, 118186. doi: 10.1016/j.jns.2021.118186
198. Kaulen, L.D.; Doubrovinskaia, S.; Mooshage, C.; Jordan, B.; Purrucker, J.; Haubner, C.; Seliger, C.;
Lorenz, H.-M.; Nagel, S.; Wildemann, B.; Bendszus, M.; Wick, W.; Schnenberger, S. Neurological
autoimmune diseases following vaccinations against SARS-CoV-2: a case series. Eur J Neurol 2021,
00, 1-9. [Epub ahead of print] doi: 10.1111/ene.15147.
199. Khayat-Khoei, M.; Bhattacharyya, S.; Katz, J.; Harrison, D.; Tauhid, S.; Bruso, P.; Houtchens, M.K.;
Edwards, K.R.; Bakshi, R.). COVID-19 mRNA vaccination leading to CNS inflammation: a case
series. J Neurol 2021 Sep 4, 1-14. [Epub ahead of print] doi: 10.1007/s00415-021-10780-7.
200. Jeong, M.; Ocwieja, K.E.; Han, D.; Wackym, P.A.; Zhang, Y.; Brown, A.; Moncada, C.; Vambutas,
A.; Kanne, T.; Crain, R.; et al. Direct SARS-CoV-2 infection of the human inner ear may underlie
COVID-19-associated audiovestibular dysfunction. Comm Med 2021, 1, 44. doi: 10.1038/s43856-
021-00044-w.
201. Uranaka, T.; Kashio, A.; Ueha, R.; Sato, T.; Bing, H.; Ying, G.; Kinoshita, M.; Kondo, K.; Yamasoba,
T. Expression of ACE2, TMPRSS2, and furin in mouse ear tissue, and the implications for SARSCoV-
2 infection. Laryngoscope 2021, 131(6), E2013-E2017. doi: 10.1002/lary.29324.
Journal Pre-proof
91
202. Rodrigues Figueiredo, R.; Aparecida Azevedo, A.; De Oliveira Penido, N. Positive association
between tinnitus and arterial hypertension. Front Neurol 2016, 7, 171. doi:
10.3389/fneur.2016.00171
203. Sekiguchi, K.; Watanabe, N.; Miyazaki, N.; Ishizuchi, K.; Iba, C.; Tagashira, Y.; Uno, S.; Shibata,
M.; Hasegawa, N.; Takemura, R.; et al. Incidence of headache after COVID-19 vaccination in
patients with history of headache: A cross-sectional study. Cephalalgia 2021, 3331024211038654.
[Epub ahead of print.] doi: 10.1177/03331024211038654.
204. Consoli, S.; Dono, F.; Evangelista, G.; D’Apolito, M.; Travaglini, D.; Onofrj, M.; Bonanni, L. Status
migrainosus: A potential adverse reaction to Comirnaty (BNT162b2, BioNtech/Pfizer) COVID-19
vaccinea case report. Neurol Sci 2021 Nov 22, 1-4. [Epub ahead of print] doi: .10.1007/s10072-021-
05741-x.
205. Huang, Y.; Cai, X.; Song, X.; Tang, H.; Huang, Y.; Xie, S.; Hu, Y. Steroids for preventing recurrence
of acute severe migraine headaches: a meta-analysis. Eur J Neurol. 2013, 20(8), 1184-1190. doi:
10.1111/ene.12155.
206. Lemberger, T.; Staels, B.; Saladin, R.; Desvergne, B.; Auwerx, J.; Wahli, W. Regulation of the
peroxisome proliferator-activated receptor alpha gene by glucocorticoids. J Biol Chem 1994,
269(40), 24527-30. doi: 10.1093/toxsci/kfn260.
207. Dodick, D.; Silberstein, S. Central sensitization theory of migraine: clinical implications. Headache
2006, 46(suppl 4), S18291. doi: 10.1111/j.1526-4610.2006.00602.x.
208. Mungoven, T.J.; Meylakh, N.; Marciszewski, K.K.; Macefield, V.G.; Macey, P.M.; Henderson, L.A.
Microstructural changes in the trigeminal nerve of patients with episodic migraine assessed using
magnetic resonance imaging. J Headache Pain 2020, 21, 59. doi: 10.1186/s10194-020-01126-1.
Journal Pre-proof
92
209. Tronvik, E.; Stovner, L.J.; Helde, G.; Sand, T.; Bovim, G. Prophylactic treatment of migraine with
an angiotensin II receptor-blocker: A randomized controlled trial. JAMA 2003, 289(1), 65-69.
doi:10.1001/jama.289.1.65.
210. Nandha, R.; Singh, H. Renin angiotensin system: A novel target for migraine prophylaxis. Indian
J Pharmacol 2012, 44(2), 157160. doi: 10.4103/0253-7613.93840.
211. FDA. Vaccines and related biological products advisory committee December 10, 2020 meeting
announcement; 2021. https://www.fda.gov/advisory-committees/advisory-committeecalendar/
vaccines-and-related-biological-products-advisory-committee-december-10-2020-
meeting- announcement. [Accessed March 29, 2021].
212. FDA. Vaccines and related biological products advisory committee December 17, 2020 meeting
announcement; 2021. https://www.fda.gov/advisory-committees/advisory-committeecalendar/
vaccines-and-related-biological-products-advisory-committee-december-17-2020-
meeting-announcement. [Accessed March 29, 2021].
213. Eviston, T.; Croxson, G.R.; Kennedy, P.G.E.; Hadlock, T.; Krishnan, A.V. Bell’s palsy: aetiology,
clinical features and multidisciplinary care. J Neurol Neurosurg Psychiatry 2015, 86, 13561361. doi:
10.1136/jnnp-2014-309563.
214. Simone, A.; Herald, J.; Chen, A. Acute myocarditis following COVID-19 mRNA vaccination in
adults aged 18 years or older. JAMA Intern Med 2021, 181(12), 1668-1670. doi:
10.1001/jamainternmed.2021.5511.
215. Jain, S.S.; Steele, J.M.; Fonseca, B.; Huang, S.; Shah, S.; Maskatia, S.A.; Buddhe, S.; Misra, N.;
Ramachandran, P.; Gaur, L.; et al. COVID-19 vaccination–associated myocarditis in adolescents.
Pediatrics 2021, 148(5), e2021053427. doi: 10.1542/peds.2021-053427.
Journal Pre-proof
93
216. Avolio, E.; Gamez, M.; Gupta, K.; Foster, R.; Berger, I.; Caputo, M.; Davidson, A.; Hill, B.;
Madeddu, P. The SARS-CoV-2 spike protein disrupts the cooperative function of human cardiac
pericytes – endothelial cells through CD147 receptor-mediated signalling: a potential noninfective
mechanism of COVID-19 microvascular disease. bioRxiv preprint. December 21, 2020.
doi: 10.1101/2020.12.21.423721.
217. Weikert. U.; Kühl, U.; Schultheiss, H.-P.; Rauch, U. Platelet activation is increased in patients with
cardiomyopathy: myocardial inflammation and platelet reactivity. Platelets 2002, 13(8), 487-91.
doi: 10.1080/0953710021000057857.
218. Garg, A.; Seeliger, B.; Derda, A.A.; Xiao, K.; Gietz, A.; Scherf, K.; Sonnenschein, K.; Pink, I.;
Hoeper, M.M.; Welte, T.; et al. Circulating cardiovascular microRNAs in critically ill COVID-19
patients. Eur J Heart Fail. 2021, 23(3), 468-475. doi: 10.1002/ejhf.2096.
219. Qiu, X.-K., Ma, J. Alteration in microRNA-155 level correspond to severity of coronary heart
disease. Scand J Clin Lab Invest 2018, 78(3), 219-223. doi: 10.1080/00365513.2018.1435904.
220. Wang, C.; Zhang, C.; Liu, L.; A, X.; Chen, B.; Li, Y.; Du, J. Macrophage-derived mir-155-containing
exosomes suppress fibroblast proliferation and promote fibroblast inflammation during cardiac
injury. Mol Ther 2017, 25(1), 192-204. doi: 10.1016/j.ymthe.2016.09.001.
221. Gavras, I.; Gavras, H. Angiotensin II as a cardiovascular risk factor. J Hum Hypertens 2002,
16(Suppl 2), S2-6. doi: 10.1038/sj.jhh.1001392.
222. Choi, S.; Lee, S.; Seo, J.-W.; Kim, M.-J.;Jeon, Y.H.; Park, J.H.; Lee, J.K.; Yeo, N.S. Myocarditisinduced
Sudden Death after BNT162b2 mRNA COVID-19 Vaccination in Korea: Case Report
Focusing on Histopathological Findings. J Korean Med Sci 2021, 36(40), e286. doi:
10.3346/jkms.2021.36.e286.
Journal Pre-proof
94
223. Verma, A.K.; Lavine, K.J.;. Lin, C.-Y. Myocarditis after Covid-19 mRNA Vaccination. NEJM 2021,
385, 1332-1334. doi: 10.1056/NEJMc2109975.
224. Oudit, G.Y.; Kassiri, Z.; Jiang, C.; Liu, P.P.; Poutanen, S.M.; Penninger, J.M.; Butany, J. SARS
coronavirus modulation of myocardial ACE2 expression and inflammation in patients with
SARS. Eur J Clin Invest 2009, 39(7), 618625. doi: 10.1111/j.1365-2362.2009.02153.
225. Vaers Home. VAERS. (n.d.). Retrieved December 5, 2021, from
https://vaers.hhs.gov/data/dataguide.html.
226. Lazarus, R.; Klompas, M.; Bernstein, S. Electronic Support for Public Health–Vaccine Adverse
Event Reporting System (ESP: VAERS). Grant. Final Report, Grant ID: R18 HS, 17045. 2010.
227. Rose, J. Critical appraisal of VAERS pharmacovigilance: is the U.S. vaccine adverse events
reporting system (VAERS) a Functioning pharmacovigilance system? Science, Public Health Policy,
and the Law 2021, 3, 100-129.
228. McLachlan, S.; Osman, M.; Dube, K.; Chiketero, P.; Choi, Y.; Fenton, N. Analysis of COVID-19
vaccine death reports from the Vaccine Adverse Events Reporting System (VAERS) Database.
Preprint. 2021. doi: 10.13140/RG.2.2.26987.26402.
229. Babic, T.; Browning, K.N. The role of vagal neurocircuits in the regulation of nausea and
vomiting. Eur J Pharmacol. 2014, 722, 38-47. doi: 10.1016/j.ejphar.2013.08.047.
230. Fenton, A.M.; Hammill, S.C.; Rea, R.F.; Low, P.A.; Shen, W.-K. Vasovagal syncope. Annals Intern
Med 2000, 133(9), 714-725. doi: 10.7326/0003-4819-133-9-200011070-00014.
231. Kampf, G. The epidemiological relevance of the COVID-19-vaccinated population is increasing.
The Lancet Regional Health – Europe 2021, 11, 100272. doi: 10.1016/j.lanepe.2021.100272.
Journal Pre-proof
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐ The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: