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.