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 [115]. Exosomes
can deliver a diverse collection of biologically active molecules,
including mRNA, microRNAs, proteins, and lipids [116]. 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 [117].
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 spike protein by
day 14 following vaccination [118]. They also found that there were
no circulating antibodies to the spike protein fourteen days after the
first vaccine. After the second vaccine, however, the number of
circulating spike-containing exosomes increased by up to a factor of 12.
Furthermore, antibodies to spike first appeared on day 14. The exosomes
presented spike protein 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
protein. Interestingly, following peak expression, the number of
circulating spike-containing exosomes decreased over time, in step with
the decrease in the level of antibodies to the spike protein.
Exosomes exist as a part of the mRNA decay mechanism in close
association under stress conditions with stress granules (SGs) and
P-bodies (PBs) [119,120]. Under conditions of vaccine-mRNA-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 [65], and therefore
resistance against physiological mRNA decay processes [119]. 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
[121].
Furthermore, under conditions of over-reliance on cap-dependent
translation by the synthetic mRNAs in SARS-CoV-2 vaccines [65], many
native mRNAs holding considerable IRES and specific methylations (m6A)
in their structure will favorably choose cap-independent translation,
which is strongly linked to mRNA decay quality control mechanisms
[114]. In this sense, considerable deadenylated mRNA products as
well as products derived from mRNA metabolism (decay) are directly
linked to exosome cargoes [121].
A fine 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 over-functioning in T-ALL, the cells are
driven completely towards cap-dependent translation [122]. An
analogous condition is described by Kyriakopoulos and McCullough (2021)
[65]. 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 [122]. Similarly, a
picornavirus infection [123] 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 [124]. In either case, whether an
apoptotic event due to a stress-like condition[125] or an
mRNA-cap-driven-like carcinomatous effect [126], 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 [127].
Also, under conditions of overwhelming production of spike protein due
to SARS-CoV-2 molecular vaccination, it would of course be expected that
a significant proportion of over-abundant intra-cellular spike proteins
would also be exported via exosome cargoes [128].
A seminal paper by a research team in India investigated the role of
exosomes in the cellular response to internally synthesized SARS-CoV-2
spike protein [50]. 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 protein within the cells. They found experimentally that these
cells released abundant exosomes housing spike protein 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 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 [129]. 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 spike
protein 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
[130]. Other studies have shown that stressed immune cells in the
spleen release large quantities of exosomes that travel to the brain
stem nuclei along the vagus nerve (as reviewed in Seneff and Nigh (2021)
[81]). 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) [131,132].
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 [133]. This suggests that neurons in the
vagus nerve would respond similarly to the spike protein. Thus, the
available evidence strongly suggests that endogenously produced spike
protein 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 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.