Case for Immediate Suspension of All Covid Shots: Peer-reviewed Study

Authors: Veronika Kyrylenko New American Sptember 27, 2022

Dr. Malhotra’s two-part peer-reviewed article titled “Curing the pandemic of misinformation on COVID-19 mRNA vaccines through real evidence-based medicine” (available here and here) was presented during a press conference organized by the World Council for Health on Tuesday, and was streamed live by The New American.

Corruption of Medicine

Coming from the crème de la crème of the British medical establishment, Dr. Malhotra, vaccinated himself, first examined the “root cause” of the vaccines’ failure and the overall decline of public health. That, he believes, is a capture of the healthcare authorities and a part of the medical profession, including major medical journals, by large pharmaceutical companies. The profound corruption undermines the principles of ethical evidence-based medical practice and informed consent, argued Malhotra.

Citing a 2021 research paper by Dr. John Ioannidis, “How to Survive the Disinformation Mess,” Dr. Malhotra pointed out that “most published research is not reliable, offers no benefit to patients, or is not useful for decision makers.” One would assume that his or her doctor, who swore the Hippocratic Oath, would certainly recognize the issue and warn about it — but no, said the doctor, “most healthcare professionals ARE NOT AWARE of this problem,” since they often lack the necessary skills to properly evaluate such research papers. (Emphasis in original.) Most disturbingly, the people at the very top of the clinical and academic leadership are “ignorant” of this fact.

Not surprisingly, the doctor continued, “the greater the financial and other prejudices in a scientific field, the less likely the research findings are to be true.”

One should not expect pharmaceutical corporations to care about individuals’ personal well-being and health. “Drug companies have a legal obligation to produce a profit for their shareholders. They do not have a legal obligation to give you the best treatment, even though most of us would like that to be the case,” Dr. Malhotra said, proceeding to walk the audience through the scandalous history of the drug industry, marred by scientific fraud and corporate crimes.

Later in his speech, he touched on how the enormous amount of power transforms the psyches of corporate executives, making them practically inhumane.

Worse Than the Disease

Dr. Malhotra then presented overwhelming clinical evidence that vaccines cause more harm than Covid. In other words, he proved that the cure is actually worse than the disease.

As the number of Brits suffering heart attacks was increasing during the year 2021, with one such victim being Dr. Malhotra’s father, the doctor started urging his colleagues to closely look at the evidence of heart issues caused or aggravated by the shots.

The doctor cited his discovery of the November 8, 2021 article entitled “Mrna COVID Vaccines Dramatically Increase Endothelial Inflammatory Markers and ACS Risk as Measured by the PULS Cardiac Test: a Warning” by cardiac surgeon Stephen Gundry. The study concluded that mRNA vaccinations “dramatically increase inflammation on the endothelium,” which is a single layer of cells that lines the interior surface of blood and lymphatic vessels. That “may account for the observations of increased thrombosis, cardiomyopathy, and other vascular events following vaccination,” per the study’s abstract.

Dr. Malhotra explained then that what shots did was aggravate the underlying heart disease, increasing the risk of heart attack from 11 percent in five years to 25 percent, which the doctor called a “huge increase.”

Do the benefits of getting a shot outweigh the risk?

Not at all, maintained the doctor.

First, vaccines do not prevent infection; this has been admitted at the highest levels of healthcare agencies in the West. For example, the U.S. Food and Drug Administration published back in May 2021 that “Antibody Testing Is Not Currently Recommended to Assess Immunity After COVID-19 Vaccination,” meaning that the antibodies that a vaccinated person produces are unreliable protection! Yet, all clinical studies done by the vaccine makers quote “antibody levels” as proof that their shots work. That includes the latest trials of the booster that presumably targets the Omicron strain, which measured antibodies in eight mice. Malhotra called it a “miscarriage of medical science.”

Furthermore, real-world data shows that shots do not reduce one’s chances of landing in the hospital, since to prevent one single Covid death, thousands, if not tens and even hundreds of thousands, of people must receive a vaccine. And many of those who are vaccinated will have serious adverse reactions. Per Malhotra’s paper, “the MHRA [the Medicines and Healthcare Products Regulatory Agency of the U.K.] figures show around 1 in 120 suffering a likely adverse event that is beyond mild.”

Shouldn’t healthcare authorities and regulators be vigilant about such life-and-death issues? Isn’t that their primary duty?

In theory, yes. But in practice, they are also captured by Big Pharma, and in many countries around the world, most of the funding for regulatory agencies comes from the pharmaceutical industry. In particular, the FDA receives as much as 65 percent of its funding from the very companies it regulates, notes Malhotra.  

The analysis of real-world data on the vaccines allowed the doctor to come to the key following conclusions:

1. The shots offer no protection from Covid infection.

2. The shots did not reduce Covid mortality or all-cause mortality in the original randomized control studies.

3. As suggested by those studies, one in 800 mRNA vaccine recipients would be seriously harmed by the shot. That’s greater than the likelihood of being hospitalized with Covid.

4. To prevent one Omicron-related death in those over 80 years of age, 7,300 people in that age group must be vaccinated. For those under 50, the ratio is one to 10,000.

5. The shots are linked to an unprecedented number of reported adverse reactions.

6. According to real-world data, the rate of harm requiring hospitalization is close to one in 1,000 within a couple of months of the mRNA jab, which is likely a “significant underestimate.”

Dr. Malhotra’s observations were supported by Drs. Tess Lawrie and Ryan Cole.

Dr. Cole, participating virtually, supports Dr. Malhotra’s calls for immediate and complete suspension of Covid shots. Cole explained how the shots code the most toxic part of the virus — the spike protein — into body cells. That protein circulates in our bodies for weeks, and then is produced by our cells for at least 60 days.

What happens then? According to Dr. Cole, some of the spike-induced harms includes the following:

  • Viral reactivation;
  • DNA mismatch repair inhibition;
  • Neurologic damage;
  • Mitochondrial damage;
  • Heart damage;
  • Immune inhibition (original antigenic sin);
  • Antibody-dependent cellular cytotoxicity;
  • Decreased sperm counts and motility,
  • Hormonal dysregulation, etc.

The spike protein is to blame for increased rates of cancer, cardiac arrests, and blood clotting, argued the doctor. It is the Covid shots that are causing excess mortality in highly vaccinated countries.

Dr. Lawrie, a co-founder of the WCH, referenced the WCH report on the abundant pharmacovigilance data on WHO VigiAccess, CDC VAERS, EudraVigilance, and the U.K. Yellow Card Scheme to suspend the use of experimental Covid gene therapeutics. 

After the press conference, Dr. Lawrie said to The New American,

Doctors can no longer ignore the fact that they absolutely must join the conversation on vaccine injury linked with these Covid vaccines — a novel technology that has been widely rolled out and never proven safe in animals let alone healthy men, women, and children. They have been deceived and incentivized to turn a blind eye. Now is the time to speak up, and not a minute too soon.

Drs. Malhotra, Cole, and Lawrie are not alone in their calls. Earlier this month, more than 400 scientists and medical professionals from more than 34 countries signed the “Declaration of International Medical Crisis Due to the Diseases and Deaths Co-related to the ‘COVID-19 Vaccines.’” 

Speaking with The New American on the matter, one of the signatories of the Declaration, Dr. Naseeba Kathrada of the WCH, said that practicing physicians around the world are seeing an “unheard of” number of diseases and deaths in healthy people linked to the shots.

COVID-19 “Long-Haulers:” The Emergence of Auditory/Vestibular Problems After Medical Intervention

Authors: Robert M. DiSogra Audiology Today American Academy of Audiology

Johns Hopkins University’s Center for Systems Science and Engineering (CSSE) in the United States reported over seven million documented cases of COVID-19 and over 212,000 deaths since the virus was first identified in this country in January 2020 (2020).

Early in the pandemic, the medical profession, the Centers for Disease Control and Prevention (CDC), the National Institute of Health (NIH), and both federal and state governments worked 24/7 to develop testing protocols and intervention strategies (pharmacological management and vaccines).

Until a scientifically proven intervention strategy is identified along with a vaccine, the public continues to be advised by the CDC to wear face masks, socially distance from each other, wash their hands regularly, and avoid crowds/indoor events. This major change in our lifestyle/behavior and the associated economic impact is still with us today.

As a novel virus, no assumptions can be made about treatment or management strategies or prediction of late onset of new symptoms. Within a few months after the pandemic was declared, a variety of pharmacological interventions were proposed by the federal government—all without scientific evidence. The most popular unproven intervention strategy in the United States was the combined use of two known ototoxic drugs: hydroxychloroquine and azithromycin (Bortoli and Santiago, 2007; FDA, 2017; Prayuenyong. et al, 2020).

DiSogra (2020a) provides a detailed review of this strategy from an audiologist’s perspective. In Europe, hydroxychloroquine and chloroquine were prescribed for almost 12 percent of COVID-19 patients (Lechien et al, 2020).

Researchers attempted to determine if other FDA-approved drugs could be repurposed as an intervention strategy. A summary of several FDA-approved drugs that were being repurposed for COVID-19 patients appears in DiSogra (2020b).

Vaccines for COVID-19 are still undergoing clinical trials. The U.S. National Library of Medicine’s Clinical Trials website is monitoring over 80 COVID-19 vaccine-related clinical trials (in various phases of development) worldwide as of September 24, 2020.

COVID-19 Recovery

A self-organized group of COVID-19 “long-haul” patients, who are researchers in relevant fields (e.g., participatory design, neuroscience, public policy, data collection and analysis, human-centered design, health activism) and have intimate knowledge of COVID-19, have been working on patient-led research around the COVID experience and prolonged recoveries (Assaf et al, 2020).

To capture and share the experiences of patients suffering from prolonged or long-haul COVID-19 symptoms, survivor/researchers used a data-driven participatory-type survey and patient-centric analysis. With 640 survey respondents, many participants experienced fluctuations in the type (70 percent reporting) and intensity (89 percent reporting) of symptoms over the course of being symptomatic.

For approximately 10 percent who had recovered, the average length of time of being symptomatic was 27 days. Unrecovered respondents experienced symptoms for an average of 40 days, with a large proportion experiencing symptoms for five to seven weeks. The chance of full recovery by day 50 was smaller than 20 percent.

Most common auditory/vestibular symptoms were earaches and vertigo lasting up to eight weeks after the diagnosis. Sixty percent of the respondents reported balance issues that peaked by second week and subsided over the next four weeks. Earaches (~32 percent) and vertigo/motion sickness (~25 percent) persisted over six weeks. One patient reported hearing loss that recovered after three weeks. Subjects listed tinnitus as the second highest complaint on a write-in list of symptoms.

All patients experienced a full recovery after 90 days except for patients with pre-existing asthma. The majority of survey respondents were not hospitalized; however, a large number of participants (37.5 percent) had visited the emergency rooms or urgent care but were not admitted for further testing or overnight observation.

Auditory Symptoms After COVID-19 Treatment

For this manuscript, “auditory symptoms” is defined as hearing loss (any degree/type), earache, subjective tinnitus, or vertigo/balance problems.

Sensorineural Hearing Loss

Almufarrij et al (2020) conducted a rapid systematic review investigated audio-vestibular symptoms associated with coronavirus. They found five case reports and two cross-sectional studies that met the inclusion criteria (N=2300). No records of audio-vestibular symptoms were reported with the earlier types of coronavirus (i.e., severe-acute respiratory syndrome [SARS] and Middle East respiratory syndrome [MERS]).

Reports of hearing loss, tinnitus, and vertigo were rarely reported in individuals who tested positive for the SARS-CoV-2. They opined that reports of audio-vestibular symptoms in confirmed COVID-19 cases are few “with mostly minor symptoms, and the studies are of poor quality.”

Munro et al (2020) concluded that it was unclear which cases of hearing loss [and tinnitus] can be directly attributed to SARS-CoV-2 or perhaps related to the many possible causes of hearing loss associated with critical care including ototoxic mediations (Ciorba et al. 2020), local, or systematic infections, vascular disorders and auto-immune disease.

Elbiol (2020) reported only one case (N=121) of sudden hearing loss (0.6 percent). A case report of sudden hearing loss that occurred one week after hospitalization was also published by Koumpa et al (2020).

Conductive Hearing Loss

Fiden (2020) reported one COVID-19 patient with a unilateral otitis media. The conductive hearing loss was mild to moderate.

Tinnitus

Tinnitus was reported in four studies in 2020 (N = 8 patients; Cui et al, Fidan, Lechien et al, and Sun et al). The characteristics of the tinnitus and the impact on the individual were not reported.

Munro, et al (2020) followed 121 COVID-19 patients eight weeks after discharge. Sixteen (13.2 percent) patients reported a change in hearing and/or tinnitus after diagnosis of COVID-19. However, there was no pattern for the duration of the recovery.

Some patients showed no changes in tinnitus while one patient reported no tinnitus after eight weeks. There was self-reported tinnitus in eight cases with three reporting a pre-existing hearing loss. Another patient reported that their tinnitus resolved. Elibol (2020) noted that tinnitus is rarely seen in COVID-19 patients.

Liang et al (2020) attempted to identify and describe neurosensory dysfunctions (including tinnitus) of COVID-19 patients. A total of 86 patients were screened but only three (3.5 percent) were identified as having tinnitus. The average interval from onset of tinnitus was one day; while the average interval from onset of tinnitus to admission was 6 ± 5.29 days; the average duration of tinnitus was 5 ± 0 days. Finally, a non-organic component of the tinnitus (i.e., anxiety) cannot be ruled out (Xia et al, 2020). Although the current studies indicate a low incidence of tinnitus in these patients, development of tinnitus management protocol may be beneficial.

Vertigo

The Munro study (2020) identified one patient with hearing loss that also reported vertigo, which the authors concluded may have been vestibular in origin. TABLE 1 summarizes the earliest case reports and cross-sectional study designs that identified auditory/vestibular problems.

AUTHORTYPE OF STUDYNAUDITORY/VESTIBULAR SYMPTOMS
Asaaf et al, 2020Survey640Earaches (32 %)
Vertigo (60%)
Hearing Loss (0.15%)
Ciu et al, 2020Case Report20Tinnitus (N=1)
Otitis media (N=1)
Fiden, 2020Case Report1Tinnitus
Otitis media
Han et al, 2019Case Report1Vertigo
Lechien et al, 2019Cross Sectional1420Ear pain (N=358 or 25%)
Rotary vertigo (N=6 or 0.4%)
Tinnitus (N=5 or 0.3%)
Mustafa, 2020Cross Sectional20Sensorineural HL
Sriwijitalai and Wiwanitkit, 2020Case Report82Sensorineural HL (N=1 or 1.2%)
Sun et al, 2020Case Report1Sensorineural HL

TABLE 1. Summary of published case reports and cross-sectional research that identified some type of auditory/vestibular problems (adapted from Almufarrij et al, 2020).

The Mustafa study (2020) compared two groups of patients (asymptomatic SARS-CoV-2 vs. control), and the results found that the asymptomatic SARS-CoV-2 group had significantly poorer hearing thresholds at 4-8 kHz and lower amplitude transient evoked otoacoustic emissions (Mustafa, 2020). Almufarrij et al (2020) concluded that high-quality studies are required in different age groups to investigate the acute effects of coronavirus. These studies include temporary effects from medications as well as studies on long-term risks on the audio-vestibular system.

Some Intervention Strategies

Aside for re-purposed pharmaceuticals, dietary supplements are proposed as a treatment option (DiSogra, 2020c). In the Aasaf study (2020), Tylenol® (followed by an inhaler) were the top medications taken by respondents in their survey to treat symptoms. Supplements, such as vitamin C, vitamin D, zinc and electrolytes, were taken by many of the respondents over several weeks. Hot liquids were also very popular with the respondents.

Other popular entries for medications, supplements and treatments reported by participants included Mucinex®, prednisone, steroids, ginger, magnesium, steam, probiotics, oregano oil/supplements, Flonase®, and other nasal sprays.

The majority of respondents never consumed any of the following substances: smoke/vape nicotine, edible or liquid cannabis, smoke/vape recreational cannabis, consume or smoke cannabidiol-only products or consume recreational drugs. Many of the respondents said they occasionally or frequently consumed alcoholic beverages.

Conclusion

The auditory-vestibular side effects of any illness, or from a pharmaceutical, nutraceutical, noise, or trauma, as well as any psychogenic component, will always be a concern for audiologists. With COVID-19, it is still too early to predict auditory-vestibular side effects, although several studies have attempted to do so or at least guide us in our short and long-term management.

If these “long-hauler” patients can be followed more closely, a body of knowledge should emerge that will help audiologists better manage COVID-19 survivors when their auditory/vestibular symptoms result in a referral for testing. It would appear that conductive and sensorineural hearing loss, tinnitus (including its non-organic origin) and vertigo can be expected but with no predictable pattern.

Protocols for management will need to be developed; however, in the interim, an ototoxic drug monitoring protocol can serve as a reference (American Academy of Audiology, 2009). The duration of these symptoms (after the diagnosis) can last from one day to eight weeks but, again, it is still too early in the life of this pandemic to state definitively if these symptoms are temporary or permanent. Although no formal protocols have been developed, audiologists must keep in mind that the more severe, life-threatening side effects of COVID-19 will continue to get researcher’s attention.

This article is a part of the September/October 2020 Audiology Today issue.

References

Almufarrij I, Uus K, Munro KJ. (2020) Does coronavirus affect the audio-vestibular system? A rapid systematic review. Int J Audiol 59(7):487–491. 

American Academy of Audiology. (2009) Position statement and clinical practice guidelines – ototoxicity monitoring. Accessed at https://audiology-web.s3.amazonaws.com/migrated/OtoMonGuidelines.pdf_539974c40999c1.58842217.pdf.

Assaf G, Davis, H, McCorkell L, Wei H, Brooke O, Akrami A, Low R, Mercier J, Adetutu A. (2020) Report: What does COVID-19 recovery actually look like? Patient Led Res.  Accessed at https://patientresearchcovid19.com/research/report-1/#Recovery_Timecourse. Accessed online September 26, 2020.

Bortoli R, Santiago M. (2007) Chloroquine ototoxicity. Clin Rheumatol 26 (11):1809–1810 

Ciorba A, Corazzi V, Skarzynski PH, Skarzynska MB, Bianchini C, Pelucchi S, Hatzopoulos S. (2020) Don’t forget ototoxicity during the SARS-CoV-2 (Covid-19) pandemic. Int J Immunopath Pharmacol 34:1–3. 

Cui C, Yao Q, Zhang D, Zhao Y, Zhang K, Nisenbaum E, Liu X, Cao P, Zhao K, Huang X, Leng D,Liu C, Li N, Luo Y, Chen B, Casiano R,Weed D, Sargi Z, Telischi F, Lu H, Denneny III JC, Shu Y,  Liu X. (2020) Approaching otolaryngology patients during the COVID-19 pandemic. Otolaryngol Head Neck Surg 163(1):121–131

DiSogra RM. (2020a) Audiological management of COVID-19 survivors treated with hydroxychloroquine and azithromycin. Accessed September 24, 2020, at www.audiology.org/audiology-today-mayjune-2020/online-feature-audiological-management-covid-19-survivors-treated

DiSogra RM. (2020b) Ototoxicity of FDA-approved drugs being re-purposed for COVID-19 treatment. Accessed on September 24, 2020, at www.audiology.org/audiology-today-mayjune-2020/online-feature-ototoxicity-fda-approved-drugs-being-re-purposed-covid.

DiSogra RM. (2020c) Dietary supplements used for COVID-19 treatment. Accessed on September 24, 2020, at www.audiology.org/audiology-today-mayjune-2020/online-feature-dietary-supplements-used-covid-19-treatment. Accessed online 9/24/2020

Elibol E. (2020) Otolaryngological symptoms in COVID-19. Eur Arch Otorhinolaryngol 1:1–4. Accessed online ahead of print September 24, 2020.

Fidan, V. (2020) New type of corona virus induced acute otitis media in adult. Amer J Otolaryngol 41:3 Article in Press. Accessed online September 24, 2020.

Food and Drug Administration. Aralen® chloroquine phosphate USP. 2017. www.accessdata.fda.gov/drugsatfda_docs/label/2017/006002s044lbl.pdf. Accessed online September 22, 2020.

Han W, Quan B, Guo Y, Zhang J, Lu Y, Feng G, Wu Q, Fang F, Cheng L, Jiao N, Li X, Chen Q. (2019) The course of clinical diagnosis and treatment of a case infected with coronavirus disease. J Med Virol 2(5):461–463

Johns Hopkins University Coronavirus Resource Center. https://coronavirus.jhu.edu/map.html. Accessed 9/24/2020

Koumpa FS, Forde CT, Manjaly JG. (2020) Sudden irreversible hearing loss post COVID-19. BMJ Case Reports 13(11):e238419.

Lechien JR, Chiesa-Estomba CM, Place S, Van Laethem Y, Cabaraux P, Mat Q, Saussez S. (2019) Clinical and epidemiological characteristics of 1,420 European patients with mild-to-moderate coronavirus disease. J Intern Med 288:3, 1–10

Liang Y, Xu J, Chu M, Mai J, Lai N, Tang W, Yang T, Zhang S, Guan C, Zhong F, Yang L, Liao G. (2020) Neurosensory dysfunction: A diagnostic marker of early COVID-19. Int J Infect Dis. 98:347–352.

Mustafa MWM. (2020) Audiological profile of asymptomatic Covid-19 PCR-positive cases. AmerJ Otolaryngol 41:3.

National Library of Medicine. Clinical trials website (www.clinicaltrials.gov). Accessed September 24, 2020.

Prayuenyong P, Kasbekar AV, Baguley DM. Clinical Implications of chloroquine and hydroxychloroquine ototoxicity for COVID-19 treatment: a mini-review.” Frontiers Public Health 8 (252): 1–8

Sriwijitalai W, Wiwanitkit V. (2020) Hearing loss and COVID-19: a note. Amer J Otolaryngol. Letter to the Editor.

Sun R, Liu H, Wang X. (2020) Mediastinal emphysema, giant bulla, and pneumothorax developed during the course of COVID-19 pneumonia.” Kor J Radiol 21(5):541–544

World Health Organization. Timeline of WHO’s response to COVID-19. www.who.int/news-room/detail/29-06-2020-covidtimeline. Accessed September 24, 2020.

Xia L, Wang J, Chuan D, Fan J, Chen Z. COVID 19 associated anxiety enhances tinnitus. www.medrxiv.org/content. Accessed September 24, 2020.

European Medicines Agency Recommends Adding “Heavy Menstrual Bleeding” to Pfizer and Moderna’s COVID Shots Product Information as a Side Effect

By Jim Hoft Published October 28, 2022  TGP

On Friday, the European Medicines Agency (EMA) recommended adding “heavy menstrual bleeding” to the list of side effects caused by Pfizer and Moderna’s mRNA shots.

In April 2021, The Gateway Pundit first reported on the tens of thousands of women who complained about irregular menstruation after taking the COVID vaccines.

Women who received the Covid vaccine reported to have spotting in between their cycles, shortened cycles, and lengthened cycles.

However, anyone who spoke about a link between Covid vaccines and menstrual problems/fertility issues was labeled a “conspiracy theorist.”

Earlier this year, the EMA’s risk assessment committee announced that it would review reports of menstruation irregularities after thousands of women reported changes to their monthly cycle after getting the COVID vaccine.

“After reviewing the available evidence, the PRAC decided to request an in-depth evaluation of all available data, including reports from spontaneous reporting systems, clinical trials, and the published literature,” according to the news release.

“At this stage, it is not yet clear whether there is a causal link between the COVID-19 vaccines and the reports of heavy periods or amenorrhea. There is also no evidence to suggest that COVID-19 vaccines affect fertility,” the agency said.

Now, the regulatory body recommended including “heavy menstrual bleeding” as an adverse effect on the list of product warnings and precautions.

Read the EMA statement:

The PRAC has recommended that heavy menstrual bleeding should be added to the product information as a side effect of unknown frequency of the mRNA COVID-19 vaccines Comirnaty and Spikevax.

Heavy menstrual bleeding (heavy periods) may be defined as bleeding characterised by an increased volume and/or duration which interferes with the person’s physical, social, emotional and material quality of life. Cases of heavy menstrual bleeding have been reported after the first, second and booster doses of Comirnaty and Spikevax.

The PRAC finalised the assessment of this safety signal after reviewing the available data, including cases reported during clinical trials, cases spontaneously reported in Eudravigilance and findings from the medical literature.

After reviewing the data, the Committee concluded that there is at least a reasonable possibility that the occurrence of heavy menstrual bleeding is causally associated with these vaccines and therefore recommended the update of the product information.

The available data reviewed involved mostly cases which appeared to be non-serious and temporary in nature.

Menstrual disorders in general are quite common and they can occur for a wide range of reasons. This includes some underlying medical conditions. Any person who experiences postmenopausal bleeding or is concerned about a change in menstruation should consult their doctor.

There is no evidence to suggest the menstrual disorders experienced by some people have any impact on reproduction and fertility. Available data provides reassurance about the use of mRNA COVID-19 vaccines before and during pregnancy. A review carried out by EMA’s Emergency Task Force showed that mRNA COVID-19 vaccines do not cause pregnancy complications for expectant mothers and their babies, and they are as effective at reducing the risk of hospitalisation and deaths in pregnant people as they are in non-pregnant people.

The Committee reiterates that the totality of data available confirms that the benefits of these vaccines greatly outweigh the risks.

Healthcare professionals and patients are encouraged to continue to report cases of heavy menstrual bleeding to their national authorities.

The PRAC will continue to monitor for cases of this condition and will communicate further if new recommendations are warranted.

Adverse effects of COVID-19 mRNA vaccines: the spike hypothesis

Published:April 20, 2022DOI:https://doi.org/10.1016/j.molmed.2022.04.0 Authors: oannis P. Trougakos Evangelos Terpos Harry Alexopoulos Efstathios Kastritis Evangelos Andreakos Meletios A. Dimopoulos

Highlights

  • Coronavirus disease 2019 (COVID-19) mRNA vaccines induce robust immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), yet their cellular/molecular mode of action and the etiology of the induced adverse events (AEs) remain elusive.
  • Lipid nanoparticles (LNPs) probably have a broad distribution in human tissues/organs; they may also (along with the packaged mRNA) exert a proinflammatory action.
  • COVID-19 mRNA vaccines encode a transmembrane SARS-CoV-2 spike (S) protein; however, shedding of the antigen and/or related peptide fragments into the circulation may occur.
  • Binding of circulating S protein to angiotensin-converting enzyme 2 (ACE2) (that is critical for the renin–angiotensin system balance) or to other targets, along with the possibility of molecular mimicry with human proteins, may contribute to the vaccination-related AEs.
  • The benefit–risk profile remains in favor of COVID-19 vaccination, yet prospective pharmacovigilance and long-term monitoring of vaccinated recipients should be a public health priority.

Vaccination is a major tool for mitigating the coronavirus disease 2019 (COVID-19) pandemic, and mRNA vaccines are central to the ongoing vaccination campaign that is undoubtedly saving thousands of lives. However, adverse effects (AEs) following vaccination have been noted which may relate to a proinflammatory action of the lipid nanoparticles used or the delivered mRNA (i.e., the vaccine formulation), as well as to the unique nature, expression pattern, binding profile, and proinflammatory effects of the produced antigens – spike (S) protein and/or its subunits/peptide fragments – in human tissues or organs. Current knowledge on this topic originates mostly from cell-based assays or from model organisms; further research on the cellular/molecular basis of the mRNA vaccine-induced AEs will therefore promise safety, maintain trust, and direct health policies.

Fighting the COVID-19 pandemic with SARS-CoV-2 S protein-encoding mRNA vaccines

COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Box 1) and has resulted in millions of deaths worldwide. Nevertheless, for the majority of SARS-CoV-2-infected individuals, COVID-19 will remain asymptomatic or only mildly symptomatic [12.]. Although SARS-CoV-2 may also circulate in the gastrointestinal tract[3.

], being a respiratory virus, the virus itself or its related antigens will not, in most cases, impact tissues and organs other than the respiratory system (RS) (Box 1) [4.56.

]. In patients with severe disease, infection of airway and lung tissues may cause pneumonia and excessive inflammation which can lead to acute respiratory distress syndrome (ARDS) (see Glossary) (Box 1) [.8. 9.0.. ARDS may then lead to organ damage beyond the RS because of micro-/macro-thromboembolism, hyperinflammation, aberrant complement activation, or extended viremia 78.9.10.11.12.

. This may be due to the broad expression of its receptor angiotensin-converting enzyme 2 (ACE2) in several cell types and tissues [14.15.16.] which results in an expanding tropism of SARS-CoV-2 for various critical organs (heart, pancreas, kidneys, etc.). If systemic collapse and death are avoided, the postulated direct virus ‘attack’ – or indirect effects due to cytokine storm [13.] or imbalance of the renin–angiotensin system (RA13.

] – causing multiorgan damage, possibly foster systemic defects which cause a chronic condition (referred to as long COVID-19) which is independently associated with the severity of the initial illness17.].Box 1

Following an unprecedented effort of biomedical research and mobilization of resources, two mRNA vaccines – namely BNT162b2 (ComirnatyTM) from Pfizer-BioNTech and the mRNA-1273 of Moderna (encoded antigen: SARS-CoV-2 S protein of the Wuhan-Hu-1 strain) [18.1920.] – were the first to receive FDA emergency use authorization. In mRNA vaccines, which are characterized by relatively rapid prototyping and manufacturing on a large scale, the S protein-encoding mRNA is delivered via lipid nanoparticles (LNPs) to human cells that produce the mature viral protein or related antigens (Figure 1, Key figure), which can exhibit a rather wide tissue/organ distribution (discussed later) [202122.]. In addition to the plausible proinflammatory role of LNPs (evidenced also from reported immediate allergic reactions) [23.4.] and of packaged mRNA – which has nonetheless been engineered by a replacement of uridine with pseudo uridine [20.25.6.

] so as not to trigger innate immunity through pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) receptors – we surmise that vaccination-mediated adverse effects (AEs) can be attributed to the unique characteristics of the S protein itself (antigen) either due to molecular mimicry with human proteins or as an ACE2 ligand.

Figure 1
Figure 1Key figure. Antigen expression–localization following cell transfection with spike (S) protein mRNA-containing lipid nanoparticles (LNPs) used in anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines.Show full captionView Large ImageDownload Hi-res imageDownload (PPT)

As delivered mRNAs can theoretically trigger the production of distinct antigens that can distribute systemically [

20.

], they are radically different from conventional platforms (i.e., inactivated whole-virus vaccines or even protein-subunit nanoparticle vaccines) (Box 2) where the produced antigen and its distribution are more predictable. As all COVID-19 vaccines rely on the S protein of the original Wuhan-Hu-1 strain [19.20.

], the differences across different vaccination platforms thus far reported (Box 2) may relate to the various vectors and formulations and/or the S protein constructs employed. Box 2

Anti-SARS-CoV-2 mRNA vaccines and their reported adverse effects

Both the BNT162b2 and mRNA-1273 vaccines are administered intramuscularly and mobilize robust and likely durable innate, humoral, and cellular adaptive immune responses [27.28230.

]. Existing data on the available mRNA vaccines are mostly limited to serological analyses. Nonetheless, beyond the assessment of immune responses, the understanding of the safety profile of these vaccines is critical to ensure safety, maintain trust, and inform policy. Reportedly, mRNA vaccines are in general well tolerated, with very low frequencies of associated severe postimmunization AEs. Although rare, AEs include serious clinical manifestations such as acute myocardial infarction, Bell’s palsycerebral venous sinus thrombosisGuillain–Barré syndrome, myocarditis/pericarditis (mostly in younger ages), pulmonary embolism, stroke, thrombosis with thrombocytopenia syndrome, lymphadenopathy, appendicitis, herpes zoster reactivation, neurological complications, and autoimmunity (e.g., autoimmune hepatitis and autoimmune peripheral neuropathies [31.32.33.34.

]) (see Clinician’s corner). Apart from AEs documented in clinical trials, most of the syndromes or isolated manifestations have been reported in multicenter or even nationwide retrospective observational studies and case series. Although correlation does not necessarily mean causation, active monitoring and awareness regarding reported postvaccination AEs are essential. Importantly, these associated AEs are significantly less frequent than analogous or additional serious AEs induced after severe COVID-19 [31.3234.

]. Some vaccine-induced AEs (e.g., myocardial infarction, Guillain–Barré syndrome) were found to increase with age, while others (e.g., myocarditis, anaphylaxis, appendicitis) were more common in younger people [35.36.

]. Although myocarditis cases are rather rare, in a study of US military personnel the number was higher than expected among males after a second vaccine dose 37.

]; similarly, the rate of postvaccination cardiac AEs was higher in young boys following the second dose [38.9.. Finally, a recent study showed an increased risk of neurological complications in COVID-19 vaccine recipients (which was nevertheless lower than the risk in COVID-19 patients) [

34.

]. The molecular basis of these AEs remains largely unknown. We postulate that, since most (if not all) of them are also apparent in severe COVID-19 [31., they may be related to acute inflammation caused by both the virus and the vaccine, as well as in the common denominator between the virus and the vaccine, namely, the SARS-CoV-2 S protein (Box 1). The vaccine-encoded antigen (S protein) is stabilized in its prefusion form in the BNT162b2 and mRNA-1273 vaccines [1920.

]; it is therefore plausible that, if entering the circulation and distributing systemically throughout the human body (Figure 2), it can contribute to these AEs in susceptible individuals.

Figure 2
Figure 2Schematic of the vasculature components showing vaccination-produced S protein/subunits/peptide fragments in the circulation, as well as soluble or endothelial cell membrane-attached angiotensin-converting enzyme 2 (ACE2).Show full captionView Large ImageDownload Hi-res imageDownload (PPT)

There is also evidence that ionizable lipids within LNPs can trigger proinflammatory responses by activating Toll-like receptors (TLRs) [40.

]. A recent report showed that LNPs used in preclinical nucleoside-modified mRNA vaccine studies are (independently of the delivery route) highly inflammatory in mice, as evidenced by excessive neutrophil infiltration, activation of diverse inflammatory pathways, and production of various inflammatory cytokines and chemokines [41.

]. This finding could explain the LNPs’ potent adjuvant activity, supporting the induction of robust adaptive immune responses [24.

]. Interestingly, inflammatory responses can be exacerbated on a background of pre-existing inflammatory conditions, as was recently shown in a mouse model after administration of mRNA–LNPs [42.

]; this effect was proven to be specific to the LNP, acting independently of the mRNA cargo.Although chemical modifications in the RNA molecules used in vaccines (detailed earlier) are intended to decrease TLR sensing of external single-stranded RNAs (and thus proinflammatory signals), there is some evidence that modified uracil residues do not completely abrogate TLR detection of the mRNA; also, while efforts are made to reduce double-stranded (ds) RNA production, there may be small amounts of dsRNA that can occasionally get packaged within mRNA vaccines [26.].

In this context, frequent booster immunizations may increase the frequency and/or the severity of the reported AEs.

Vaccine-encoded antigen distribution in the human body and possible interactions with human proteins

Following vaccination, a cell may present the produced S protein (or its subunits/peptide fragments) to mobilize immune responses or be abolished by the immune system (e.g., cytotoxic T cells) [25.

]. Consequently, the debris produced, or even the direct secretion (including shedding) of the antigen by the transfected cells, may release large amounts of the S protein or its subunits/peptide fragments to the circulation (Figure 1) [19.20.

]. The anti-SARS-CoV-2 vaccine mRNA-containing LNPs are injected into the deltoid muscle and exert an effect in the muscle tissue itself, the lymphatic system, and the spleen, but can also localize in the liver and other tissues [21.2243.44.

] from where the S protein or its subunits/peptide fragments may enter the circulation and distribute throughout the body. It is worth mentioning that liver localization of LNPs is not a universal property of carrier nanoparticles, as specific modifications in their chemistry can retain immunogenicity with minimal liver involvement [43.45.

]. In line with a plausible systemic distribution of the antigen, it was found that the S protein circulates in the plasma of the BNT162b2 or mRNA-1273 vaccine recipients as early as day 1 after the first vaccine injection [46.

]. Reportedly, antigen clearance is correlated with the production of antigen-specific immunoglobulins or may remain in the circulation (e.g., in exosomes) for longer periods [4748. ], providing one reasonable explanation (among others) for the robust and durable systemic immune responses found in vaccinated recipients [49.50.

]. Therefore, there is likely to be an extensive range of expected interactions between free-floating S protein/subunits/peptide fragments and ACE2 circulating in the blood (or lymph), or ACE2 expressed in cells from various tissues/organs (Figure 2) [14.15.

,16. This notion is further supported by the finding that in adenovirus-vectored vaccines (Box 2), the S protein produced upon vaccination has the native-like mimicry of SARS-CoV-2 S protein’s receptor binding functionality and prefusion structure [51.].

Additional interactions with human proteins in the circulation, or even the presentation to the immune system of S protein antigenic epitopes [52. mimicking human proteins (molecular mimicry) may occur [53.54.55.56.

]. Reportedly, some of the near-germline SARS-CoV-2-NAbs against S receptor-binding domain (RBD) reacted with mammalian self-antigens [57.

], and SARS-CoV-2 S antagonizes innate antiviral immunity by targeting multiple pathways controlling interferon (IFN) production [58.

]. Also, a sustained elevation in T cell responses to SARS-CoV-2 mRNA vaccines has been found (data not yet peer-reviewed) in patients who suffer from chronic neurologic symptoms after acute SARS-CoV-2 infection as compared with healthy COVID-19 convalescents [59.

]. Given the reported (rare) neurological AEs following vaccination, it was suggested that further studies are needed to assess whether antibodies against the vaccine-produced antigens can cross-react with components of the peripheral nerves [34.

]. Further concerns include the possible development of anti-idiotype antibodies against vaccination-induced antibodies as a means of downregulation; anti-idiotype antibodies – apart from binding to the protective neutralizing SARS-CoV-2 antibodies – can also mirror the S protein itself and bind ACE2, possibly triggering a wide array of AEs [60.

]. Worth mentioning is a systems vaccinology approach (31 individuals) of the BNT162b2 vaccine (two doses) effects, where anticytokine antibodies were largely absent or were found at low levels (contrary to findings in acute COVID-19 [61.62.

]), while two individuals had anti-interleukin-21 (IL-21) autoantibodies, and two other individuals had anti-IL-1 antibodies [63.

]. In this context, anti-idiotypic antibodies can be particularly enhanced after frequent boosting doses that trigger very high titers of immunoglobulins [64.

]. Frequent boosting doses may also become a suboptimal approach as they can imprint serological responses toward the ancestral Wuhan-Hu-1 S protein, minimizing protection against novel viral S variants [65.66.].

The potential interaction at a whole-organism level of the native-like S protein and/or subunits/peptide fragments with soluble or cell-membrane-attached ACE2 (Figure 2) can promote ACE2 internalization and degradation 67.68.]. In support of this, soluble ACE2 induces receptor-mediated endocytosis of SARS-CoV-2 via interaction with proteins related to the RAS [69.

]. Prolonged loss or reduced ACE2 activity may result in extensive destabilization of the RAS which may then trigger vasoconstriction, enhanced inflammation, and/or thrombosis due to unopposed ACE and angiotensin-2 (ANG II)-mediated effects (Figure )[13.. Indeed, decreased ACE2 expression and/or activity contributes, among other things, to the development of ANG II-mediated hypertension in mice, indicating vasculature dysfunction [67.]. The baseline expression levels of ACE2 in endothelial cells, or its induced expression levels upon stimulation from other tissue-resident cells, along with the potential of endothelial cells to shed ACE2 to the circulation, or their sensitivity to SARS-CoV-2 infection is debatable [0.71.72.73.]. Nonetheless, even relatively low ACE2 expression levels in endothelial cells (e.g., compared to levels in epithelial cells) [15.16.70.71.

], along with the high expression levels of ACE2 in other cell types of the vasculature (e.g., heart fibroblasts/pericytes) [

15.

,

74.

], indicate that the vasculature can be sensitive to free-floating S protein or its subunits/peptide fragments (Figure 2). These effect(s), especially in capillary beds, and the prolonged antigen presence in the circulation [

46.

47.

48.

], along with the systemic excessive immune response to the antigen, can then trigger sustained inflammation (discussed later) which can injure the endothelium, disrupting its antithrombogenic properties in multiple vascular beds.

The SARS-CoV-2 S protein-induced effects in mammalian cells or model organisms

Reportedly, intravenous (i.v.) injection of the S1 subunit in mice results in its localization in endothelia of mice brain microvessels showing colocalization with ACE2, caspase-3, IL-6, tumor necrosis factor α (TNF-α), and C5b-9; it was thus suggested that endothelial damage is a central part of SARS-CoV-2 pathology which may be induced by the S protein alone [75.

]. Also, the S1 subunit (or recombinant S1 RBD) impaired endothelial function via downregulation of ACE2 [76.

] and induced degradation of junctional proteins that maintain endothelial barrier integrity in a mouse model of brain microvascular endothelial cells or cerebral arteries; this latter effect was more enhanced in endothelial cells from diabetic versus normal mice [7.]. Similarly, the S1 subunit decreased microvascular transendothelial resistance and barrier function in cultured human pulmonary cells [78.]. Further, S protein disrupted human cardiac pericytes function and triggered increased production of proapoptotic factors in pericytes, causing endothelial cells death [79.

]. In support of this, administration of the S protein promoted dysfunction of human endothelial cells as evidenced by, for example, increased expression of the von Willebrand factor [80.

]. Other reports indicate that S1 can directly induce coagulation by competitive binding to both soluble and cellular heparan sulfate/heparin (an anticoagulant) [81.82.83.84.

], while cell-free hemoglobin, as a hypoxia counterbalance, cannot attenuate disruption of endothelial barrier function, oxidative stress, or inflammatory responses in human pulmonary arterial endothelial cells exposed to S1 [85.

]. Consistently, S protein binds fibrinogen (a blood coagulation factor), and S protein virions have been found to enhance fibrin-mediated microglia activation (data not yet peer-reviewed) and induce fibrinogen-dependent lung pathology in mice [86.

], while S1 binding to platelets’ ACE2 triggers their aggregation [84.

]. Interestingly, both the ChAdOx1 (AstraZeneca) and BNT162b2 vaccines can elicit antiplatelet factor 4 (anti-PF4) antibody production even in recipients without clinical manifestation of thrombosis [87.].Intriguingly, the S protein increases human cell syncytium formation [88.89.], triggering pyroptosis of syncytia formed by fusion of S and ACE2-expressing cell90.

]. Also, in cells or mouse experimental models, it was shown that S removes lipids from model membranes and interferes with the capacity of high-density lipoprotein to exchange lipids [91.], inhibits DNA damage repair processes [92.

], and induces Snail-mediated epithelial–mesenchymal transition marker changes and lung metastasis in a breast cancer mouse model [93.].

In support of the possibility that there is a wide range of S protein binders, Aβ142 (the 42 amino acid form of amyloid β in cerebrospinal fluid) was found to bind with high affinity to the S1 subunit and ACE2 [

. Aβ1–42 strengthened the binding of S1 to ACE2 and increased viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection mouse model. Data from this surrogate mouse model with IV inoculation of Aβ1–42 showed that the clearance of Aβ1–42 in the blood was dampened in the presence of the extracellular domain of the S protein trimers [94.

]. Given the wide ACE2 expression in human brain [95.

], a study of particular interest showed that IV-injected radioiodinated S1 (I-S1) readily crossed by adsorptive transcytosis the blood–brain barrier in male mice, was taken up by brain regions, and entered the parenchymal brain space. I-S1 was also taken up by the lung, spleen, kidney, and liver; intranasally administered I-S1 also entered the brain, although at lower levels than after i.v. administration [96.

]. Similarly, S1 was found to disrupt the blood–brain barrier integrity at a 3D blood–brain barrier microfluidic model [97.

]. In support of this, biodistribution studies of the mRNA–LNP platform by Moderna in Sprague Dawley rats revealed the presence of low levels of mRNA in the brain, indicating that the mRNA–LNPs can cross the blood–brain barrier [22.].

Finally, it was recently reported that human T cells express ACE2 at levels sufficient to interact with the S protein [98.

], while it had been shown previously that SARS-CoV-2 uses CD4 to infect T helper lymphocytes, and that S promotes a proinflammatory activation profile on the most potent antigen-presenting cells (APCs) (i.e., human dendritic cells) [99.

]. If these observations are confirmed, they may explain a SARS-CoV-2 vaccination-mediated AE, namely, reactivation of varicella zoster virus [100101..

S protein-induced proinflammatory responses and unique gene expression signatures following vaccination

Reportedly, S protein (apart from the LNP–mRNA platform discussed earlier) mediates proinflammatory and/or injury (of different etiology) responses in various human cell types [102.103.104.

, and ACE2-mediated infection of human bronchial epithelial cells with S protein pseudovirions induced inflammation and apoptosis [105.

]. Also, S protein promoted an inflammatory cytokine IL-6/IL-6R-induced trans signaling response and alarmin secretion in human endothelial cells, along with increased oxidative stress, induction of inflammatory paracrine senescence, and higher levels of leucocyte adhesion [106.

]. Other reports indicate that S protein triggers an inflammatory response signature in human corneal epithelial cells [107.

], increases oxidative stress and DNA ds breaks in human peripheral-blood mononuclear cells (PBMCs) postvaccination [108.

], and binds to lipopolysaccharide, boosting its proinflammatory activity [109.110.

]. Furthermore, S protein induces neuroinflammation and caspase-1 activation in BV-2 microglia cells [111.] and blocks neuronal firing in sensory neurons [112.

]. The S protein-induced systemic inflammation may proceed via TLR2-dependent activation of the nuclear factor κB (NF-κB) pathway [113.

], while structure-based computational models showed that S protein exhibits a high-affinity motif for binding T cell receptors (TCRs), and may form a ternary complex with histocompatibility complex class II molecules; indeed, analysis of the TCR repertoire in COVID-19 patients showed that those with severe hyperinflammatory disease exhibit TCR skewing consistent with superantigen (S protein) activation [114.

]. In in vivo mouse models, S protein activated macrophages and contributed to induction of acute lung inflammation [115.

], while intratracheal instillation of the S1 subunit in transgenic mice overexpressing human ACE2 induced severe COVID-19-like acute lung injury and inflammation. These effects were milder in wild-type mice, indicating the phenotype dependence on human ACE2 expression [78.

]. Consistently, the S1 subunit has been found to act as a PAMP that, via pattern recognition receptor engagement, induces viral infection-independent neuroinflammation in adult rats [116..

These observations correlate with the finding of a systemic inflammatory signature after the first BNT162b2 vaccination which was accompanied by TNF-α and IL-6 upregulation after the second dose [117.

]; these effects may also relate to a proinflammatory action of the mRNA–LNP platform (see earlier). In a thorough systems vaccinology study of the BNT162b2 mRNA vaccine effects, younger participants tended to have greater changes in monocyte and inflammatory modules 1 day after the second dose, whereas older individuals had increased expression of B and T cell modules. Moreover, single-cell transcriptomics analysis at the same time point revealed the emergence of a unique myeloid cell cluster out of 18 cell clusters identified in total. This cell cluster does not represent myeloid-derived suppressor cells, it expressed IFN-stimulated genes and was not found in COVID-19 infection; also, it was similar to an epigenetically reprogrammed monocyte population found in the blood of donors being vaccinated with two doses of an influenza vaccine [63.

. Whether epigenetic reprogramming underlies the enhanced IFN-induced gene response in C8 cells after secondary BNT162b2 vaccination remains to be clarified. Finally, a comparison between the BNT162b2 vaccine-induced gene expression signatures at day 7 post-prime (d7PP) and post-boost (d7PB) doses and that of other vaccine types (e.g., inactivated or live-attenuated vaccines) exhibited weak correlation both between d7PP and d7PB as well as with other vaccines [63.]. These findings suggest the evolution of novel genomic responses after the second dose and, more importantly, the unique biology of mRNA vaccines versus other more conventional platforms. Of particular interest is also the report of a cytokine release syndrome (CRS) – an extremely rare immune-related AE of immune checkpoint inhibitors – post-BTN162b2 vaccination in a patient with colorectal cancer on longstanding anti-programmed death 1 (PD-1) monotherapy; the anti-PD1 blockade-mediated CRS was evidenced by increased inflammatory markers, thrombocytopenia, elevated cytokine levels, and steroid responsiveness [118.]. These proinflammatory effects could be particularly pronounced in the elderly, since recent data revealed that senescent cells become hyperinflammatory in response to the S1 subunit, followed by increased expression of viral entry proteins and reduced antiviral gene expression in nonsenescent cells through a paracrine mechanism [119.].

The need to investigate the molecular basis of vaccination-induced AEs

Anti-SARS-CoV-2 mRNA vaccines induce durable and robust systemic immunity, and thus their contribution in mitigating the COVID-19 pandemic and saving thousands of lives is beyond doubt. This technology has several advantages over conventional vaccines [120.

] and opens a whole new era for the development of novel vaccines against various infectious and other diseases, including cancer. Based on currently available molecular insights (mostly in cell-based assays and model organisms), we hypothesize that the rare AEs reported following vaccination with S protein-encoding mRNA vaccines may relate to the nature and binding profile of the systemically circulating antigen(s) (Figure 1Figure 2), although the contribution of the LNPs and/or the delivered mRNA is likely also significant [ 24.26,41.

]. Therefore, the possibility of subclinical organ dysfunction in vaccinated recipients which could increase the risk, for example, of future (cardio)vascular or inflammatory diseases should be thoroughly investigated. Given that severe COVID-19 correlates with older age, hypertension, diabetes, and/or cardiovascular disease, which all share a variable degree of ACE2 signaling deregulation, additional ACE2 downregulation induced by vaccination may further amplify an unbalanced RAS. Regarding localization of LNPs in the liver and consequent antigen expression, it is worth mentioning that the liver is continuously exposed to a multitude of self and foreign antigens and can mount efficient immune responses against pathogens as it hosts convectional APCs (e.g., dendritic cells, B cells, and Kupfer cells). Additional liver cell types – such as liver sinusoidal endothelial cells, hepatic stellate cells, and hepatocytes – also have the capacity to act as APCs [121.

]. It is plausible, though as yet unproven, that as the S protein is produced in liver cells, both conventional and unconventional APCs may prime adaptive but also innate immune responses in the liver’s immunological niche. Despite the liver’s major tolerogenic role [

22.

], the sustained expression of S protein-related antigens (Figure 1) can potentially skew the immune response towards autoimmune-like tissue damage, as in the observed cases of autoimmune hepatitis following vaccination

]. It therefore merits further investigation whether LNPs can transfect any other nonimmunological body tissues bearing cells that can act as unconventional APCs, thus inducing a sustained immune response but also a self-response, as in cases of chronic viral infections [

Concluding remarks

Although the benefit–risk profile remains strongly in favor of COVID-19 vaccination for the elderly and patients with age-related or other underlying diseases, an understanding of the molecular–cellular basis of the anti-SARS-CoV-2 mRNA vaccine-induced AEs is critical for the ongoing and future vaccination and booster campaigns. In parallel, the prospective pharmacovigilance and long-term monitoring (clinical/biochemical) of vaccinated recipients versus matched controls should evolve in well-designed clinical trials. Moreover, the use of alternative chemistries for LNPs, and of S protein in its closed form (not prone to ACE2 binding) [126.

], along with the use of SARS-CoV-2 nucleocapsid protein or solely the S RDB, may be valuable alternatives for refined, next-generation mRNA vaccines. Finally, as we essentially do not know for how long and at what concentration the LNPs and the antigen(s) remain in human tissues or the circulation of poor vaccine responders, the elderly, or children (see Outstanding questions), and given the fact that cellular immunity likely persists despite reduced in vitro neutralizing titers [28.

], boosting doses should be delivered only where the benefit–risk profile is clearly established.

Overall, parallel to the ongoing research on the most challenging topics of SARS-CoV-2 biology, evolving dynamics and adaptation capacity to human species (i.e., transmission–infection rate and disease severity), the basic and clinical research (see Outstanding questions) aiming to understand the molecular–cellular basis of the rare AEs of the existing first-generation mRNA vaccines should be accelerated as an urgent and vital public health priority.

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Adverse effects of COVID-19 mRNA vaccines: the spike hypothesis

Authors: Ioannis P. Trougakos,1,⁎ Evangelos Terpos,2 Harry Alexopoulos,1 Marianna Politou,3 Dimitrios Paraskevis,4 Andreas Scorilas,5 Efstathios Kastritis,2 Evangelos Andreakos,6 and Meletios A. Dimopoulos2 Trends Mol Med. 2022 Jul; 28(7): 542–554. Publishedonline2022Apr21. doi: 10.1016/j.molmed.2022.04.007PMCID: PMC9021367PMID: 35537987

Abstract

Vaccination is a major tool for mitigating the coronavirus disease 2019 (COVID-19) pandemic, and mRNA vaccines are central to the ongoing vaccination campaign that is undoubtedly saving thousands of lives. However, adverse effects (AEs) following vaccination have been noted which may relate to a proinflammatory action of the lipid nanoparticles used or the delivered mRNA (i.e., the vaccine formulation), as well as to the unique nature, expression pattern, binding profile, and proinflammatory effects of the produced antigens – spike (S) protein and/or its subunits/peptide fragments – in human tissues or organs. Current knowledge on this topic originates mostly from cell-based assays or from model organisms; further research on the cellular/molecular basis of the mRNA vaccine-induced AEs will therefore promise safety, maintain trust, and direct health policies.

Fighting the COVID-19 pandemic with SARS-CoV-2 S protein-encoding mRNA vaccines

COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Box 1 ) and has resulted in millions of deaths worldwide. Nevertheless, for the majority of SARS-CoV-2-infected individuals, COVID-19 will remain asymptomatic or only mildly symptomatic [1,2]. Although SARS-CoV-2 may also circulate in the gastrointestinal tract [3], being a respiratory virus, the virus itself or its related antigens will not, in most cases, impact tissues and organs other than the respiratory system (RS) (Box 1) [4.5.6.]. In patients with severe disease, infection of airway and lung tissues may cause pneumonia and excessive inflammation which can lead to acute respiratory distress syndrome (ARDS) (see Glossary) (Box 1) [7.8.9.10.]. ARDS may then lead to organ damage beyond the RS because of micro-/macro-thromboembolism, hyperinflammation, aberrant complement activation, or extended viremia [7.8.9.10.11.12.13.]. This may be due to the broad expression of its receptor angiotensin-converting enzyme 2 (ACE2) in several cell types and tissues [14.15.16.] which results in an expanding tropism of SARS-CoV-2 for various critical organs (heart, pancreas, kidneys, etc.). If systemic collapse and death are avoided, the postulated direct virus ‘attack’ – or indirect effects due to cytokine storm [10,13] or imbalance of the renin–angiotensin system (RAS) [13] – causing multiorgan damage, possibly foster systemic defects which cause a chronic condition (referred to as long COVID-19) which is independently associated with the severity of the initial illness [17].

Box 1

SARS-CoV-2 infection of human cells

SARS-CoV-2 infection of human cells proceeds via its binding to the cell surface protein ACE2 through the RBD of its protruding S glycoprotein [127] which remains in a metastable prefusion state through the association of subunits 1 (S1) and 2 (S2) via noncovalent interactions [18,19]; the infection process is also facilitated by host proteases [127,128]. In most of SARS-CoV-2-infected carriers the virus is contained in the upper RS, resulting in either no symptoms or mild symptoms [1,2]. A minority will require hospitalization; this is due to severe symptoms which develop due to extensive inflammation, a process often referred to as a ‘cytokine storm’, causing ARDS which may be accompanied by viremia and can lead to systemic multiorgan collapse [7.8.9.10.]. The risk for severe COVID-19 increases significantly with age or pre-existing comorbidities [1,2,129], and younger individuals have a substantially lower risk – even compared to influenza infection [129] – for developing severe COVID-19 [130,131]. It has been postulated that higher pediatric innate interferon responses restrict viral replication and disease progression [132]. In a recent trial, in which young people were intentionally exposed to a low dose of SARS-CoV-2, nearly half of the participants did not become infected, some were asymptomatic, and those who developed COVID-19 reported mild to moderate symptoms, including sore throats, runny noses, sneezing, and loss of sense of smell and taste; fever was less common, and no one developed a persistent cough [133].

SARS-CoV-2 infection in healthy individuals triggers innate as well as adaptive immune system responses, that is, CD4+ and CD8+ T cells and antibodies, including neutralizing antibodies (NAbs) produced by terminally differentiated B cells, which altogether suppress the extent of infection [132,134,135]. As SARS-CoV-2 initially infects the upper RS, defensive immune responses start to develop at respiratory mucosal surfaces, and this is followed by systemic immunity [136,137]. These immune responses are age- and gender-dependent and may either mount poorly in a background of genetic causes and pre-existing morbidities, or become very intense and essentially uncontrolled in severe disease leading to ARDS and systemic failure [11.12.13.].

Following an unprecedented effort of biomedical research and mobilization of resources, two mRNA vaccines – namely BNT162b2 (ComirnatyTM) from Pfizer-BioNTech and the mRNA-1273 of Moderna (encoded antigen: SARS-CoV-2 S protein of the Wuhan-Hu-1 strain) [18.19.20.] – were the first to receive FDA emergency use authorization. In mRNA vaccines, which are characterized by relatively rapid prototyping and manufacturing on a large scale, the S protein-encoding mRNA is delivered via lipid nanoparticles (LNPs) to human cells that produce the mature viral protein or related antigens (Figure 1 , Key figure), which can exhibit a rather wide tissue/organ distribution (discussed later) [20.21.22.]. In addition to the plausible proinflammatory role of LNPs (evidenced also from reported immediate allergic reactions) [23,24] and of packaged mRNA – which has nonetheless been engineered by a replacement of uridine with pseudouridine [20,25,26] so as not to trigger innate immunity through pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) receptors – we surmise that vaccination-mediated adverse effects (AEs) can be attributed to the unique characteristics of the S protein itself (antigen) either due to molecular mimicry with human proteins or as an ACE2 ligand.

Figure 1

Figure 1

Key figure. Antigen expression–localization following cell transfection with spike (S) protein mRNA-containing lipid nanoparticles (LNPs) used in anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines.

Following LNP internalization and mRNA release, the authentic viral signal peptide (as in the Pfizer–BioNTech and Moderna vaccines) drives antigen production in the lumen of the endoplasmic reticulum (ER) where it adopts its natural transmembrane localization via subunit 2 (S2) anchoring. After sorting in the trans Golgi network (TGN), S protein acquires its final position in the transfected human cell membrane, where S1 is exposed to the extracellular space (i.e., may face circulation). Although the extent of antigen expression per cell remains unknown, it is reasonable to assume that this process results in rather extended decoration of transfected cells with S protein. Furin-mediated proteolytic cleavage (as in SARS-CoV-2-infected cells) in the absence of a mutated S1/S2 furin cleavage site at the TGN may result in shedding of cleaved S1 and conversion of S2 into its postfusion structure (S2*). Antigen sorting and trafficking may also induce the release of S protein-containing exosomes. The events shown will occur in the apical and/or basolateral surfaces of polarized (e.g., epithelial) cells. The Pfizer–BioNTech and Moderna constructs do not contain a mutated S1/S2 furin cleavage site. Further research will clarify the impact of the S1/S2 subunits stabilizing D614G (or other) mutation or of a mutated furin cleavage site in antigen distribution, the immunogenicity of the vaccine, and induced adverse events (AEs). Also shown are dendritic cells (professional antigen-presenting cells, APCs) engulfing circulating antigens, and antibody-mediated binding of B cells to cell-anchored antigens.

As delivered mRNAs can theoretically trigger the production of distinct antigens that can distribute systemically [20], they are radically different from conventional platforms (i.e., inactivated whole-virus vaccines or even protein-subunit nanoparticle vaccines) (Box 2 ) where the produced antigen and its distribution are more predictable. As all COVID-19 vaccines rely on the S protein of the original Wuhan-Hu-1 strain [19,20], the differences across different vaccination platforms thus far reported (Box 2) may relate to the various vectors and formulations and/or the S protein constructs employed.

Box 2

Other types of COVID-19 vaccine

In viral vector vaccines, the S protein coding information is delivered via a replication-deficient adenoviral vector system that contains an encoding dsDNA. In this case, transcripts from adenoviral vectors are generated in the cell nucleus. Here, a major reported AE is immune thromboembolism (including cerebral venous sinus thrombosis) in various organs, probably through excessive innate immune system and endothelial activation [138]. Apart from the S protein itself, AEs can be also attributed to background expression of remaining adenoviral genes or to persisting adenovirus-vector DNA in a transcriptionally active form. Further concerns are the presence of other contaminant proteins, remnants of the vaccine production line, and to pre-existing antivector immunity [20]; this last issue does not apply to the recombinant ChAdOx1-S (Oxford–AstraZeneca) vaccine which employs a nonhuman adenovirus vector. More importantly, the infectious cycle of SARS-CoV-2 takes place exclusively in the cytoplasm, and thus there has been no evolutionary pressure against the presence of splice donor and acceptor sites in its genes. This is a major difference from mRNA vaccines that function in the cytoplasm, since various spliced transcripts from adenoviral vectors can be generated in the cell nucleus [56].

In protein subunit nanoparticle vaccines (e.g., NVX-CoV2373), the S protein is harvested in a cell culture system, purified, and delivered as a trimer via a nanoparticle assembly in an adjuvant. Although preliminary trials indicate that these vaccines can trigger robust immunity [139], reports on AEs are still scarce due to the limited amount of vaccination data.

Finally, in conventional vaccines, the whole virus is inactivated and inoculated using an appropriate adjuvant [26]. A significant benefit is that whereas in the previously discussed technologies the S protein is the sole source of immunogenic epitopes, in this case a wide repertoire of epitopes in other viral proteins is presented. Possible disadvantages include lower immunogenicity, production issues, AEs due to used adjuvant(s) (e.g., aluminum hydroxide), as well as issues that relate to incomplete inactivation of the virus. Given that these vaccines have not reached mass production, reports on possible AEs do not exist.

Anti-SARS-CoV-2 mRNA vaccines and their reported adverse effects

Both the BNT162b2 and mRNA-1273 vaccines are administered intramuscularly and mobilize robust and likely durable innate, humoral, and cellular adaptive immune responses [27.28.29.30.]. Existing data on the available mRNA vaccines are mostly limited to serological analyses. Nonetheless, beyond the assessment of immune responses, the understanding of the safety profile of these vaccines is critical to ensure safety, maintain trust, and inform policy. Reportedly, mRNA vaccines are in general well tolerated, with very low frequencies of associated severe postimmunization AEs. Although rare, AEs include serious clinical manifestations such as acute myocardial infarction, Bell’s palsycerebral venous sinus thrombosisGuillain–Barré syndrome, myocarditis/pericarditis (mostly in younger ages), pulmonary embolism, stroke, thrombosis with thrombocytopenia syndrome, lymphadenopathy, appendicitis, herpes zoster reactivation, neurological complications, and autoimmunity (e.g., autoimmune hepatitis and autoimmune peripheral neuropathies [31.32.33.34.]) (see Clinician’s corner). Apart from AEs documented in clinical trials, most of the syndromes or isolated manifestations have been reported in multicenter or even nationwide retrospective observational studies and case series. Although correlation does not necessarily mean causation, active monitoring and awareness regarding reported postvaccination AEs are essential. Importantly, these associated AEs are significantly less frequent than analogous or additional serious AEs induced after severe COVID-19 [31,32,34]. Some vaccine-induced AEs (e.g., myocardial infarction, Guillain–Barré syndrome) were found to increase with age, while others (e.g., myocarditis, anaphylaxis, appendicitis) were more common in younger people [35,36]. Although myocarditis cases are rather rare, in a study of US military personnel the number was higher than expected among males after a second vaccine dose [37]; similarly, the rate of postvaccination cardiac AEs was higher in young boys following the second dose [38,39]. Finally, a recent study showed an increased risk of neurological complications in COVID-19 vaccine recipients (which was nevertheless lower than the risk in COVID-19 patients) [34]. The molecular basis of these AEs remains largely unknown. We postulate that, since most (if not all) of them are also apparent in severe COVID-19 [31], they may be related to acute inflammation caused by both the virus and the vaccine, as well as in the common denominator between the virus and the vaccine, namely, the SARS-CoV-2 S protein (Box 1). The vaccine-encoded antigen (S protein) is stabilized in its prefusion form in the BNT162b2 and mRNA-1273 vaccines [19,20]; it is therefore plausible that, if entering the circulation and distributing systemically throughout the human body (Figure 2 ), it can contribute to these AEs in susceptible individuals.

Figure 2

Figure 2

Schematic of the vasculature components showing vaccination-produced S protein/subunits/peptide fragments in the circulation, as well as soluble or endothelial cell membrane-attached angiotensin-converting enzyme 2 (ACE2).

(A,B) Parallel to immune system activation, circulating S protein/subunits/peptide fragments (B) binding to ACE2 may occur not only to ACE2-expressing endothelial cells, but also in multiple cell types of the vasculature and surrounding tissues due to antigen diffusion (e.g., in fenestrated or discontinuous capillary beds) (A, red arrows). These series of molecular events are unlikely for any severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related antigen in the absence of severe coronavirus disease 2019 (COVID-19), where SARS-CoV-2 is contained in the respiratory system. In (C) the two counteracting pathways of the renin–angiotensin system (RAS), namely the ‘conventional’ arm, that involves ACE which generates angiotensin II (ANG II) from angiotensin I (ANG I), and the ACE2 arm which hydrolyzes ANG II to generate angiotensin (1–7) [ANG (1–7)] or ANG I to generate angiotensin (1–9) [ANG (1–9)] are depicted. ANG II binding and activation of the ANG II type 1 receptor (AT1R) promotes inflammation, fibrotic remodeling, and vasoconstriction, whereas the ANG (1–7) and ANG (1–9) peptides binding to MAS receptor (MASR) activate antifibrotic, anti-inflammatory pathways and vasodilation. Additional modules of the RAS (i.e., renin and angiotensinogen, AGT) are also shown. Abbreviation: AT1R, angiotensin II type 1 receptor.

Clinician’s corner

Given the plethora of existing data on the available mRNA vaccines, a major ‘known’ is that in the short-term mRNA vaccines are well tolerated by the recipient, and that they can induce a robust immune response and therefore provide prolonged protection against severe COVID-19 (including emerging variants of concern); thus, vaccination remains a major tool for mitigating the COVID-19 pandemic and saving thousands of lives.

It is well established that the risk for severe COVID-19 increases with age or pre-existing comorbidities. Given the ‘unknowns’ discussed herein, boosting doses in healthy children and even adolescents should be delivered only if the benefit–risk profile is clearly established.

Multidisciplinary clinical and basic research aiming at understanding the cellular–molecular basis of the COVID-19 mRNA vaccine-induced AEs – along with active pharmacovigilance and long-term recording in the clinical setting of reported AEs in vaccinated recipients – are critical components for improving vaccines, guaranteeing safety, maintaining trust, and directing health policies.

The technology of the mRNA vaccines will continue to evolve as it opens up a whole new era of novel applications for large-scale development of new vaccines against various infectious and other diseases, including cancer.

There is also evidence that ionizable lipids within LNPs can trigger proinflammatory responses by activating Toll-like receptors (TLRs) [40]. A recent report showed that LNPs used in preclinical nucleoside-modified mRNA vaccine studies are (independently of the delivery route) highly inflammatory in mice, as evidenced by excessive neutrophil infiltration, activation of diverse inflammatory pathways, and production of various inflammatory cytokines and chemokines [41]. This finding could explain the LNPs’ potent adjuvant activity, supporting the induction of robust adaptive immune responses [24]. Interestingly, inflammatory responses can be exacerbated on a background of pre-existing inflammatory conditions, as was recently shown in a mouse model after administration of mRNA–LNPs [42]; this effect was proven to be specific to the LNP, acting independently of the mRNA cargo.

Although chemical modifications in the RNA molecules used in vaccines (detailed earlier) are intended to decrease TLR sensing of external single-stranded RNAs (and thus proinflammatory signals), there is some evidence that modified uracil residues do not completely abrogate TLR detection of the mRNA; also, while efforts are made to reduce double-stranded (ds) RNA production, there may be small amounts of dsRNA that can occasionally get packaged within mRNA vaccines [26].

In this context, frequent booster immunizations may increase the frequency and/or the severity of the reported AEs.

Vaccine-encoded antigen distribution in the human body and possible interactions with human proteins

Following vaccination, a cell may present the produced S protein (or its subunits/peptide fragments) to mobilize immune responses or be abolished by the immune system (e.g., cytotoxic T cells) [25]. Consequently, the debris produced, or even the direct secretion (including shedding) of the antigen by the transfected cells, may release large amounts of the S protein or its subunits/peptide fragments to the circulation (Figure 1) [19,20]. The anti-SARS-CoV-2 vaccine mRNA-containing LNPs are injected into the deltoid muscle and exert an effect in the muscle tissue itself, the lymphatic system, and the spleen, but can also localize in the liver and other tissues [21,22,43,44] from where the S protein or its subunits/peptide fragments may enter the circulation and distribute throughout the body. It is worth mentioning that liver localization of LNPs is not a universal property of carrier nanoparticles, as specific modifications in their chemistry can retain immunogenicity with minimal liver involvement [43,45]. In line with a plausible systemic distribution of the antigen, it was found that the S protein circulates in the plasma of the BNT162b2 or mRNA-1273 vaccine recipients as early as day 1 after the first vaccine injection [46]. Reportedly, antigen clearance is correlated with the production of antigen-specific immunoglobulins or may remain in the circulation (e.g., in exosomes) for longer periods [47,48], providing one reasonable explanation (among others) for the robust and durable systemic immune responses found in vaccinated recipients [49,50]. Therefore, there is likely to be an extensive range of expected interactions between free-floating S protein/subunits/peptide fragments and ACE2 circulating in the blood (or lymph), or ACE2 expressed in cells from various tissues/organs (Figure 2) [14.15.16.]. This notion is further supported by the finding that in adenovirus-vectored vaccines (Box 2), the S protein produced upon vaccination has the native-like mimicry of SARS-CoV-2 S protein’s receptor binding functionality and prefusion structure [51].

Additional interactions with human proteins in the circulation, or even the presentation to the immune system of S protein antigenic epitopes [52] mimicking human proteins (molecular mimicry) may occur [53.54.55.56.]. Reportedly, some of the near-germline SARS-CoV-2-NAbs against S receptor-binding domain (RBD) reacted with mammalian self-antigens [57], and SARS-CoV-2 S antagonizes innate antiviral immunity by targeting multiple pathways controlling interferon (IFN) production [58]. Also, a sustained elevation in T cell responses to SARS-CoV-2 mRNA vaccines has been found (data not yet peer-reviewed) in patients who suffer from chronic neurologic symptoms after acute SARS-CoV-2 infection as compared with healthy COVID-19 convalescents [59]. Given the reported (rare) neurological AEs following vaccination, it was suggested that further studies are needed to assess whether antibodies against the vaccine-produced antigens can cross-react with components of the peripheral nerves [34]. Further concerns include the possible development of anti-idiotype antibodies against vaccination-induced antibodies as a means of downregulation; anti-idiotype antibodies – apart from binding to the protective neutralizing SARS-CoV-2 antibodies – can also mirror the S protein itself and bind ACE2, possibly triggering a wide array of AEs [60]. Worth mentioning is a systems vaccinology approach (31 individuals) of the BNT162b2 vaccine (two doses) effects, where anticytokine antibodies were largely absent or were found at low levels (contrary to findings in acute COVID-19 [61,62]), while two individuals had anti-interleukin-21 (IL-21) autoantibodies, and two other individuals had anti-IL-1 antibodies [63]. In this context, anti-idiotypic antibodies can be particularly enhanced after frequent boosting doses that trigger very high titers of immunoglobulins [64]. Frequent boosting doses may also become a suboptimal approach as they can imprint serological responses toward the ancestral Wuhan-Hu-1 S protein, minimizing protection against novel viral S variants [65,66].

The potential interaction at a whole-organism level of the native-like S protein and/or subunits/peptide fragments with soluble or cell-membrane-attached ACE2 (Figure 2) can promote ACE2 internalization and degradation [67,68]. In support of this, soluble ACE2 induces receptor-mediated endocytosis of SARS-CoV-2 via interaction with proteins related to the RAS [69]. Prolonged loss or reduced ACE2 activity may result in extensive destabilization of the RAS which may then trigger vasoconstriction, enhanced inflammation, and/or thrombosis due to unopposed ACE and angiotensin-2 (ANG II)-mediated effects (Figure 2) [13]. Indeed, decreased ACE2 expression and/or activity contributes, among other things, to the development of ANG II-mediated hypertension in mice, indicating vasculature dysfunction [67]. The baseline expression levels of ACE2 in endothelial cells, or its induced expression levels upon stimulation from other tissue-resident cells, along with the potential of endothelial cells to shed ACE2 to the circulation, or their sensitivity to SARS-CoV-2 infection is debatable [70.71.72.73.]. Nonetheless, even relatively low ACE2 expression levels in endothelial cells (e.g., compared to levels in epithelial cells) [15,16,70,71], along with the high expression levels of ACE2 in other cell types of the vasculature (e.g., heart fibroblasts/pericytes) [15,74], indicate that the vasculature can be sensitive to free-floating S protein or its subunits/peptide fragments (Figure 2). These effect(s), especially in capillary beds, and the prolonged antigen presence in the circulation [46.47.48.], along with the systemic excessive immune response to the antigen, can then trigger sustained inflammation (discussed later) which can injure the endothelium, disrupting its antithrombogenic properties in multiple vascular beds

The SARS-CoV-2 S protein-induced effects in mammalian cells or model organisms

Reportedly, intravenous (i.v.) injection of the S1 subunit in mice results in its localization in endothelia of mice brain microvessels showing colocalization with ACE2, caspase-3, IL-6, tumor necrosis factor α (TNF-α), and C5b-9; it was thus suggested that endothelial damage is a central part of SARS-CoV-2 pathology which may be induced by the S protein alone [75]. Also, the S1 subunit (or recombinant S1 RBD) impaired endothelial function via downregulation of ACE2 [76] and induced degradation of junctional proteins that maintain endothelial barrier integrity in a mouse model of brain microvascular endothelial cells or cerebral arteries; this latter effect was more enhanced in endothelial cells from diabetic versus normal mice [77]. Similarly, the S1 subunit decreased microvascular transendothelial resistance and barrier function in cultured human pulmonary cells [78]. Further, S protein disrupted human cardiac pericytes function and triggered increased production of proapoptotic factors in pericytes, causing endothelial cells death [79]. In support of this, administration of the S protein promoted dysfunction of human endothelial cells as evidenced by, for example, increased expression of the von Willebrand factor [80]. Other reports indicate that S1 can directly induce coagulation by competitive binding to both soluble and cellular heparan sulfate/heparin (an anticoagulant) [81.82.83.84.], while cell-free hemoglobin, as a hypoxia counterbalance, cannot attenuate disruption of endothelial barrier function, oxidative stress, or inflammatory responses in human pulmonary arterial endothelial cells exposed to S1 [85]. Consistently, S protein binds fibrinogen (a blood coagulation factor), and S protein virions have been found to enhance fibrin-mediated microglia activation (data not yet peer-reviewed) and induce fibrinogen-dependent lung pathology in mice [86], while S1 binding to platelets’ ACE2 triggers their aggregation [84]. Interestingly, both the ChAdOx1 (AstraZeneca) and BNT162b2 vaccines can elicit antiplatelet factor 4 (anti-PF4) antibody production even in recipients without clinical manifestation of thrombosis [87].

Intriguingly, the S protein increases human cell syncytium formation [88,89], triggering pyroptosis of syncytia formed by fusion of S and ACE2-expressing cells [90]. Also, in cells or mouse experimental models, it was shown that S removes lipids from model membranes and interferes with the capacity of high-density lipoprotein to exchange lipids [91], inhibits DNA damage repair processes [92], and induces Snail-mediated epithelial–mesenchymal transition marker changes and lung metastasis in a breast cancer mouse model [93].

In support of the possibility that there is a wide range of S protein binders, Aβ1  42 (the 42 amino acid form of amyloid β in cerebrospinal fluid) was found to bind with high affinity to the S1 subunit and ACE2 [94]. Aβ1  42 strengthened the binding of S1 to ACE2 and increased viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection mouse model. Data from this surrogate mouse model with IV inoculation of Aβ1  42 showed that the clearance of Aβ1  42 in the blood was dampened in the presence of the extracellular domain of the S protein trimers [94]. Given the wide ACE2 expression in human brain [95], a study of particular interest showed that IV-injected radioiodinated S1 (I-S1) readily crossed by adsorptive transcytosis the blood–brain barrier in male mice, was taken up by brain regions, and entered the parenchymal brain space. I-S1 was also taken up by the lung, spleen, kidney, and liver; intranasally administered I-S1 also entered the brain, although at lower levels than after i.v. administration [96]. Similarly, S1 was found to disrupt the blood–brain barrier integrity at a 3D blood–brain barrier microfluidic model [97]. In support of this, biodistribution studies of the mRNA–LNP platform by Moderna in Sprague Dawley rats revealed the presence of low levels of mRNA in the brain, indicating that the mRNA–LNPs can cross the blood–brain barrier [22].

Finally, it was recently reported that human T cells express ACE2 at levels sufficient to interact with the S protein [98], while it had been shown previously that SARS-CoV-2 uses CD4 to infect T helper lymphocytes, and that S promotes a proinflammatory activation profile on the most potent antigen-presenting cells (APCs) (i.e., human dendritic cells) [99]. If these observations are confirmed, they may explain a SARS-CoV-2 vaccination-mediated AE, namely, reactivation of varicella zoster virus [100,101]

S protein-induced proinflammatory responses and unique gene expression signatures following vaccination

Reportedly, S protein (apart from the LNP–mRNA platform discussed earlier) mediates proinflammatory and/or injury (of different etiology) responses in various human cell types [102.103.104.], and ACE2-mediated infection of human bronchial epithelial cells with S protein pseudovirions induced inflammation and apoptosis [105]. Also, S protein promoted an inflammatory cytokine IL-6/IL-6R-induced trans signaling response and alarmin secretion in human endothelial cells, along with increased oxidative stress, induction of inflammatory paracrine senescence, and higher levels of leucocyte adhesion [106]. Other reports indicate that S protein triggers an inflammatory response signature in human corneal epithelial cells [107], increases oxidative stress and DNA ds breaks in human peripheral-blood mononuclear cells (PBMCs) postvaccination [108], and binds to lipopolysaccharide, boosting its proinflammatory activity [109,110]. Furthermore, S protein induces neuroinflammation and caspase-1 activation in BV-2 microglia cells [111] and blocks neuronal firing in sensory neurons [112]. The S protein-induced systemic inflammation may proceed via TLR2-dependent activation of the nuclear factor κB (NF-κB) pathway [113], while structure-based computational models showed that S protein exhibits a high-affinity motif for binding T cell receptors (TCRs), and may form a ternary complex with histocompatibility complex class II molecules; indeed, analysis of the TCR repertoire in COVID-19 patients showed that those with severe hyperinflammatory disease exhibit TCR skewing consistent with superantigen (S protein) activation [114]. In in vivo mouse models, S protein activated macrophages and contributed to induction of acute lung inflammation [115], while intratracheal instillation of the S1 subunit in transgenic mice overexpressing human ACE2 induced severe COVID-19-like acute lung injury and inflammation. These effects were milder in wild-type mice, indicating the phenotype dependence on human ACE2 expression [78]. Consistently, the S1 subunit has been found to act as a PAMP that, via pattern recognition receptor engagement, induces viral infection-independent neuroinflammation in adult rats [116].

These observations correlate with the finding of a systemic inflammatory signature after the first BNT162b2 vaccination which was accompanied by TNF-α and IL-6 upregulation after the second dose [117]; these effects may also relate to a proinflammatory action of the mRNA–LNP platform (see earlier). In a thorough systems vaccinology study of the BNT162b2 mRNA vaccine effects, younger participants tended to have greater changes in monocyte and inflammatory modules 1 day after the second dose, whereas older individuals had increased expression of B and T cell modules. Moreover, single-cell transcriptomics analysis at the same time point revealed the emergence of a unique myeloid cell cluster out of 18 cell clusters identified in total. This cell cluster does not represent myeloid-derived suppressor cells, it expressed IFN-stimulated genes and was not found in COVID-19 infection; also, it was similar to an epigenetically reprogrammed monocyte population found in the blood of donors being vaccinated with two doses of an influenza vaccine [63]. Whether epigenetic reprogramming underlies the enhanced IFN-induced gene response in C8 cells after secondary BNT162b2 vaccination remains to be clarified. Finally, a comparison between the BNT162b2 vaccine-induced gene expression signatures at day 7 post-prime (d7PP) and post-boost (d7PB) doses and that of other vaccine types (e.g., inactivated or live-attenuated vaccines) exhibited weak correlation both between d7PP and d7PB as well as with other vaccines [63]. These findings suggest the evolution of novel genomic responses after the second dose and, more importantly, the unique biology of mRNA vaccines versus other more conventional platforms. Of particular interest is also the report of a cytokine release syndrome (CRS) – an extremely rare immune-related AE of immune checkpoint inhibitors – post-BTN162b2 vaccination in a patient with colorectal cancer on longstanding anti-programmed death 1 (PD-1) monotherapy; the anti-PD1 blockade-mediated CRS was evidenced by increased inflammatory markers, thrombocytopenia, elevated cytokine levels, and steroid responsiveness [118]. These proinflammatory effects could be particularly pronounced in the elderly, since recent data revealed that senescent cells become hyperinflammatory in response to the S1 subunit, followed by increased expression of viral entry proteins and reduced antiviral gene expression in nonsenescent cells through a paracrine mechanism [119]

The need to investigate the molecular basis of vaccination-induced AEs

Anti-SARS-CoV-2 mRNA vaccines induce durable and robust systemic immunity, and thus their contribution in mitigating the COVID-19 pandemic and saving thousands of lives is beyond doubt. This technology has several advantages over conventional vaccines [120] and opens a whole new era for the development of novel vaccines against various infectious and other diseases, including cancer. Based on currently available molecular insights (mostly in cell-based assays and model organisms), we hypothesize that the rare AEs reported following vaccination with S protein-encoding mRNA vaccines may relate to the nature and binding profile of the systemically circulating antigen(s) (Figure 1Figure 2), although the contribution of the LNPs and/or the delivered mRNA is likely also significant [24,26,41]. Therefore, the possibility of subclinical organ dysfunction in vaccinated recipients which could increase the risk, for example, of future (cardio)vascular or inflammatory diseases should be thoroughly investigated. Given that severe COVID-19 correlates with older age, hypertension, diabetes, and/or cardiovascular disease, which all share a variable degree of ACE2 signaling deregulation, additional ACE2 downregulation induced by vaccination may further amplify an unbalanced RAS. Regarding localization of LNPs in the liver and consequent antigen expression, it is worth mentioning that the liver is continuously exposed to a multitude of self and foreign antigens and can mount efficient immune responses against pathogens as it hosts convectional APCs (e.g., dendritic cells, B cells, and Kupfer cells). Additional liver cell types – such as liver sinusoidal endothelial cells, hepatic stellate cells, and hepatocytes – also have the capacity to act as APCs [121]. It is plausible, though as yet unproven, that as the S protein is produced in liver cells, both conventional and unconventional APCs may prime adaptive but also innate immune responses in the liver’s immunological niche. Despite the liver’s major tolerogenic role [122], the sustained expression of S protein-related antigens (Figure 1) can potentially skew the immune response towards autoimmune-like tissue damage, as in the observed cases of autoimmune hepatitis following vaccination [123,124]. It therefore merits further investigation whether LNPs can transfect any other nonimmunological body tissues bearing cells that can act as unconventional APCs, thus inducing a sustained immune response but also a self-response, as in cases of chronic viral infections [125

Concluding remarks

Although the benefit–risk profile remains strongly in favor of COVID-19 vaccination for the elderly and patients with age-related or other underlying diseases, an understanding of the molecular–cellular basis of the anti-SARS-CoV-2 mRNA vaccine-induced AEs is critical for the ongoing and future vaccination and booster campaigns. In parallel, the prospective pharmacovigilance and long-term monitoring (clinical/biochemical) of vaccinated recipients versus matched controls should evolve in well-designed clinical trials. Moreover, the use of alternative chemistries for LNPs, and of S protein in its closed form (not prone to ACE2 binding) [126], along with the use of SARS-CoV-2 nucleocapsid protein or solely the S RDB, may be valuable alternatives for refined, next-generation mRNA vaccines. Finally, as we essentially do not know for how long and at what concentration the LNPs and the antigen(s) remain in human tissues or the circulation of poor vaccine responders, the elderly, or children (see Outstanding questions), and given the fact that cellular immunity likely persists despite reduced in vitro neutralizing titers [28], boosting doses should be delivered only where the benefit–risk profile is clearly established.

Outstanding questions

What are the localization pattern, transfection efficacy, and clearance rates of the mRNA vaccine LNPs in the human body?

Can we refine LNP chemistry towards retaining transfection efficacy and at the same time assuring on-demand tissue distribution?

Do the adverse inflammatory reactions noted postvaccination also relate – and if yes, to what extent – to LNPs and/or the mRNA used in mRNA vaccines?

What are the mechanistic details of antigen expression, processing, and cellular localization following cell transfection with the LNPs?

What would the impact be of excessive ‘decoration’ of nonprofessional antigen-presenting transfected human (e.g., liver) cells with transmembrane S protein?

Does the antigen or related subunits‐peptide fragments leak into the circulation, and if so, in which form, at what concentration, and for how long? Is there any association with the vaccine-mediated immune responses?

Is the probable binding of the antigen to ACE2 in the vasculature accountable for the cardiovascular, metabolic, or other (e.g., inflammation-related) reported AEs?

Does the antigen cross the blood–brain barrier?

Is there any noteworthy molecular mimicry (especially of the major antigenic sites) between the S protein and the human proteome?

What is the profile of mucosal immunity induced by the mRNA COVID-19 vaccines?

It is the case that vaccination-mediated immunity (two doses) against the used ancestral antigen (Wuhan-Hu-1 S protein) wanes over time, or do we simply partially lose protection due to evolutionary leaps of the S protein (e.g., at the Omicron variant)? In that case, do we really need boosting doses with the same antigen?

Does boosting, apart from raising antibody titers, also promote antibody diversification?

What would be the profile of immune responses and AEs following mRNA-guided expression of the S protein in its closed form (a form not prone to ACE2 binding)?

Alt-text: Outstanding questions

Overall, parallel to the ongoing research on the most challenging topics of SARS-CoV-2 biology, evolving dynamics and adaptation capacity to human species (i.e., transmission–infection rate and disease severity), the basic and clinical research (see Outstanding questions) aiming to understand the molecular–cellular basis of the rare AEs of the existing first-generation mRNA vaccines should be accelerated as an urgent and vital public health priority.

Glossary

Acute respiratory distress syndrome (ARDS)a life-threatening condition in which fluid builds up in the lungs, interfering with the gas exchange function and preventing oxygenation of the blood and organs.
Adverse effect (AE)an undesired effect of a medication or clinical intervention with potentially harmful consequences.
Angiotensin-converting enzyme 2 (ACE2)an enzyme involved in the homeostatic regulation of circulating angiotensin I and angiotensin II levels by converting them to angiotensin (1–9) and angiotensin (1–7) peptides respectively.
Bell’s palsyan idiopathic episode of facial muscle weakness or paralysis on one side of the face. This condition results from dysfunction of the seventh cranial nerve (the facial nerve).
Cerebral venous sinus thrombosisa rare blood-clotting event that occurs in the venous sinuses of the brain and prevents blood from draining out of the brain. As a result, pressure builds up and can lead to swelling and hemorrhage.
Cytokine storma characteristic of COVID-19 (or other disease) where abnormally high levels of circulating cytokines are produced and contribute to disease severity.
Guillain–Barré syndromea rare, autoimmune neurological disorder in which the body’s immune system erroneously attacks the peripheral nerves, causing muscle weakness and, if left untreated, paralysis.
Long COVID-19a term that refers to a range of new, returning, or ongoing symptoms that persist beyond the initial phase of a SARS-CoV-2 infection.
Molecular mimicrythe process in which an immune response against a foreign antigen is inadvertently also directed against a self-antigen that closely resembles the triggering foreign antigen.
Receptor-binding domain (RBD)the part of a binding protein (e.g., in SARS-CoV-2 S protein) used to anchor the protein to its receptor.
Renin–angiotensin system (RAS)a system that is critical in the physiological regulation of (among others) neural, gut, cardiovascular, blood pressure, and kidney functions, as well as fluid and salt balance. It involves the enzyme renin which catalyzes the production of angiotensin I.
Serological analysisany analysis performed with blood serum, usually focusing on measuring antibody levels.
Syncytiuma cell with multiple nuclei resulting from multiple fusions of uninuclear cells.
Viremiathe detection of replication-competent viral particles in the bloodstream.

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1 in 780 German Children Under 5 REQUIRES HOSPITALIZATION Due to Severe Adverse Event Following Pfizer’s mRNA COVID shots

BAuthors: Jim Hoft October 20, 2022 JAMA

According to the findings of German research, one in every 700 children under the age of five who received the Pfizer mRNA Covid vaccine was hospitalized with severe adverse events (SAE), and one in every 200 children had ‘symptoms that were currently ongoing and thus of unknown significance.’

The study, “Comparative Safety of the BNT162b2 Messenger RNA COVID-19 Vaccine vs Other Approved Vaccines in Children Younger Than 5 Years,” was published in JAMA on Tuesday, two days before the CDC’s Advisory Committee on Immunization Practices voted to recommend COVID-19 to be included in the 2023 childhood immunization schedule.

Participants in this retrospective cohort study were German parents or caregivers who had enrolled their children in a Covid-19 vaccination program at 21 outpatient care facilities. The survey used in the study was conducted in a secure online environment. From April 14th, 2022, till May 9th, 2022, a total of 19 000 email addresses were contacted using data from vaccine registration databases.

It concluded that the symptoms reported after Pfizer vaccination were “comparable overall” to those for other vaccines. Let’s see.

  • Any symptoms: 62% higher
  • Musculoskeletal (muscles and bones) symptoms: 155% higher
  • Dermatologic (skin) symptoms: 118% higher
  • Otolaryngologic (ears, nose and throat) symptoms: 537% higher
  • Cardiovascular (heart etc.): 36% higher
  • Gastrointestinal (stomach etc.): 54% higher

It calls these “modestly elevated.” (Note that not all are statistically significant and some confidence intervals are wide, see below.)

In 0.5% of the children (40 of 7,806) symptoms were “currently ongoing and thus of unknown significance”. This is in a study with a 2-4 month follow-up period. That means 0.5% of children had an adverse effect that lasted for weeks or months. In two cases (0.03%), symptoms were confirmed to have lasted longer than 90 days.

Ten children were hospitalised with reported serious adverse events (SAEs), compared to zero with the other vaccines. This reported as 0.1%, as it is out of 7,806. However, the study also states that no hospitalisations were reported for children administered the low dosage of 3 μg. Since it also tells us that 6,033 children received at least one dose of over 3 μg (or unknown dosage), the rate in the relevant cohort is closer to 0.2%, or around one in 500.

Four of the hospitalisations were for cardiovascular injury; one child was hospitalised after both doses for this reason. Four were pulmonary (lung) related. Symptoms of the hospitalised children lasted an average of 12.2 days and a maximum of 60 days. None reported a myocarditis diagnosis. Mercifully, no deaths were reported in this relatively small sample.

The mortality rate in under-20s has been shown to be 0.0003%. The figure for under-fives will be even lower. But even if we unrealistically assume this is the mortality rate for under-fives and the vaccines reduce it to zero, this still means that at least 500 children are hospitalised for every life the vaccines save. In reality the ratio will be much worse than this.

On Wednesday, The Gateway Pundit reported that the CDC’s Advisory Committee on Immunization Practices voted to include the COVID-19 vaccine as part of the Vaccines for Children (VFC) Program.

The Vaccines For Children (VFC) program is a federally funded program that provides vaccines at no cost to children who might not otherwise be vaccinated because of their inability to pay, according to the CDC.

The CDC buys vaccine at a discounted rate for distribution to registered VFC providers. Children who are eligible* for VFC vaccines are entitled to receive those vaccines recommended by the Advisory Committee on Immunization Practices (ACIP).

The advisory committee voted 15-0, without objection.

On Thursday, the CDC’s Advisory Committee on Immunization Practices voted to recommend COVID-19 to be included in the 2023 childhood immunization schedule in 15 unanimous votes.

25% Of People Who Received Covid-19 Vaccination Missed Work Or Reported A “Serious Event” Affecting Their Normal Life Functions, According To CDC Data

Authors: NICOLE DOMINIQUE· Oct 5th 2022

Official data from the CDC has been released due to court orders, as stated by lawyer Aaron Siri. The findings show that 25% of people who got the shot (from a database of 10 million) couldn’t perform normal activities and had to miss work or school afterward.

Lawyer Aaron Siri has successfully obtained reports from the CDC after the Informed Consent Action Network sued the organization twice. The court order required the CDC to release crucial information on the vaccine’s safety. The data is gathered from 10 million individuals who utilized the CDC’s “v-safe” program, a smartphone-based tool where recipients of the Covid-19 vaccine can go for health check-ins. The tool allows people to go on their smartphone and provide information on how they’re feeling post-shot. The newly released data is eye-opening. According to the official CDC data shared by Siri, about 1.2 million people were unable to perform regular activities, 1.3 million had to miss work or school, and another 800,000 people required medical care after getting the vaccine. A total of 3,353,110 recipients were negatively impacted by the jab.

Siri appeared on Fox to talk about the lengthy process of attaining the documents. It took 463 days to receive the data, and Siri believes the CDC could have provided the information in a matter of minutes. “Why did it take numerous legal demands, multiple appeals – two lawsuits in fact – before the CDC finally handed over the v-safe data?” Siri asks.

These findings are very concerning; for years, the vaccine was advertised as “safe” and “effective.” Siri said, “A big reason that they pushed the Covid vaccine is [because] they said, ‘look, not everybody is gonna get – you know – seriously injured by Covid, but for many, it’ll prevent them from having symptoms, being hospitalized, missing work.’ Well, now that we have the data, we could see that getting the vaccine caused 25% of people who got the shot – within this data set of 10 million people – to miss work, to have some serious event affecting their normal life functions.

So far, 68.4% of the U.S. population has been fully vaccinated (as stated by Our World in Data). It’s difficult to determine just how many people have been negatively affected by the vaccine since the information on it seems to be suppressed. The CDC has not yet addressed the released documents, and the information is not available on their website. 

Adverse effects of COVID-19 vaccines and measures to prevent them

Authors: Kenji Yamamoto  Virology Journal volume 19, Article number: 100 (2022) 

Abstract

Recently, The Lancet published a study on the effectiveness of COVID-19 vaccines and the waning of immunity with time. The study showed that immune function among vaccinated individuals 8 months after the administration of two doses of COVID-19 vaccine was lower than that among the unvaccinated individuals. According to European Medicines Agency recommendations, frequent COVID-19 booster shots could adversely affect the immune response and may not be feasible. The decrease in immunity can be caused by several factors such as N1-methylpseudouridine, the spike protein, lipid nanoparticles, antibody-dependent enhancement, and the original antigenic stimulus. These clinical alterations may explain the association reported between COVID-19 vaccination and shingles. As a safety measure, further booster vaccinations should be discontinued. In addition, the date of vaccination should be recorded in the medical record of patients. Several practical measures to prevent a decrease in immunity have been reported. These include limiting the use of non-steroidal anti-inflammatory drugs, including acetaminophen to maintain deep body temperature, appropriate use of antibiotics, smoking cessation, stress control, and limiting the use of lipid emulsions, including propofol, which may cause perioperative immunosuppression. In conclusion, COVID-19 vaccination is a major risk factor for infections in critically ill patients.

Dear Editor,

The coronavirus disease (COVID-19) pandemic has led to the widespread use of genetic vaccines, including mRNA and viral vector vaccines. In addition, booster vaccines have been used, but their effectiveness against the highly mutated spike protein of Omicron strains is limited. Recently, The Lancet published a study on the effectiveness of COVID-19 vaccines and the waning of immunity with time [1]. The study showed that immune function among vaccinated individuals 8 months after the administration of two doses of COVID-19 vaccine was lower than that among unvaccinated individuals. These findings were more pronounced in older adults and individuals with pre-existing conditions. According to the European Medicines Agency’s recommendations, frequent COVID-19 booster shots could adversely affect the immune response and may not be feasible [2]. Several countries, including Israel, Chile, and Sweden, are offering the fourth dose to only older adults and other groups rather than to all individuals [3].

The decrease in immunity is caused by several factors. First, N1-methylpseudouridine is used as a substitute for uracil in the genetic code. The modified protein may induce the activation of regulatory T cells, resulting in decreased cellular immunity [4]. Thereby, the spike proteins do not immediately decay following the administration of mRNA vaccines. The spike proteins present on exosomes circulate throughout the body for more than 4 months [5]. In addition, in vivo studies have shown that lipid nanoparticles (LNPs) accumulate in the liver, spleen, adrenal glands, and ovaries [6], and that LNP-encapsulated mRNA is highly inflammatory [7]. Newly generated antibodies of the spike protein damage the cells and tissues that are primed to produce spike proteins [8], and vascular endothelial cells are damaged by spike proteins in the bloodstream [9]; this may damage the immune system organs such as the adrenal gland. Additionally, antibody-dependent enhancement may occur, wherein infection-enhancing antibodies attenuate the effect of neutralizing antibodies in preventing infection [10]. The original antigenic sin [11], that is, the residual immune memory of the Wuhan-type vaccine may prevent the vaccine from being sufficiently effective against variant strains. These mechanisms may also be involved in the exacerbation of COVID-19.

Some studies suggest a link between COVID-19 vaccines and reactivation of the virus that causes shingles [1213]. This condition is sometimes referred to as vaccine-acquired immunodeficiency syndrome [14]. Since December 2021, besides COVID-19, Department of Cardiovascular Surgery, Okamura Memorial Hospital, Shizuoka, Japan (hereinafter referred to as “the institute”) has encountered cases of infections that are difficult to control. For example, there were several cases of suspected infections due to inflammation after open-heart surgery, which could not be controlled even after several weeks of use of multiple antibiotics. The patients showed signs of being immunocompromised, and there were a few deaths. The risk of infection may increase. Various medical algorithms for evaluating postoperative prognosis may have to be revised in the future. The media have so far concealed the adverse events of vaccine administration, such as vaccine-induced immune thrombotic thrombocytopenia (VITT), owing to biased propaganda. The institute encounters many cases in which this cause is recognized. These situations have occurred in waves; however, they are yet to be resolved despite the measures implemented to routinely screen patients admitted for surgery for heparin-induced thrombocytopenia (HIT) antibodies. Four HIT antibody-positive cases have been confirmed at the institute since the start of vaccination; this frequency of HIT antibody-positive cases has rarely been observed before. Fatal cases due to VITT following the administration of COVID-19 vaccines have also been reported [15].

As a safety measure, further booster vaccinations should be discontinued. In addition, the date of vaccination and the time since the last vaccination should be recorded in the medical record of patients. Owing to the lack of awareness of this disease group among physicians and general public in Japan, a history of COVID-19 vaccination is often not documented, as it is in the case of influenza vaccination. The time elapsed since the last COVID-19 vaccination may need to be considered when invasive procedures are required. Several practical measures that can be implemented to prevent a decrease in immunity have been reported [16]. These include limiting the use of non-steroidal anti-inflammatory drugs, including acetaminophen, to maintain deep body temperature, appropriate use of antibiotics, smoking cessation, stress control, and limiting the use of lipid emulsions, including propofol, which may cause perioperative immunosuppression [17].

To date, when comparing the advantages and disadvantages of mRNA vaccines, vaccination has been commonly recommended. As the COVID-19 pandemic becomes better controlled, vaccine sequelae are likely to become more apparent. It has been hypothesized that there will be an increase in cardiovascular diseases, especially acute coronary syndromes, caused by the spike proteins in genetic vaccines [1819]. Besides the risk of infections owing to lowered immune functions, there is a possible risk of unknown organ damage caused by the vaccine that has remained hidden without apparent clinical presentations, mainly in the circulatory system. Therefore, careful risk assessments prior to surgery and invasive medical procedures are essential. Randomized controlled trials are further needed to confirm these clinical observations.

In conclusion, COVID-19 vaccination is a major risk factor for infections in critically ill patients.

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A Case Report: Multifocal Necrotizing Encephalitis and Myocarditis after BNT162b2 mRNA Vaccination againstCOVID-19

Authors: Michael Mörz

Abstract:

The current report presents the case of a 76-year-old man with Parkinson’s disease (PD)who died three weeks after receiving his third COVID-19 vaccination. The patient was first vac-cinated in May 2021 with the ChAdOx1 nCov-19 vector vaccine, followed by two doses of theBNT162b2 mRNA vaccine in July and December 2021. The family of the deceased requested anautopsy due to ambiguous clinical signs before death. PD was confirmed by post-mortem exami-nations. Furthermore, signs of aspiration pneumonia and systemic arteriosclerosis were evident. However, histopathological analyses of the brain uncovered previously unsuspected findings, including acute vasculitis (predominantly lymphocytic) as well as multifocal necrotizing encephalitis of unknown etiology with pronounced inflammation including glial and lymphocytic reaction. In the heart, signs of chronic cardiomyopathy as well as mild acute lympho-histiocytic myocarditis and vasculitis were present. Although there was no history of COVID-19 for this patient, immunohistochemistry for SARS-CoV-2 antigens (spike and nucleocapsid proteins) was performed. Surprisingly, only spike protein but no nucleocapsid protein could be detected within the foci of inflammation in both the brain and the heart, particularly in the endothelial cells of small blood vessels. Since no nucleocapsid protein could be detected, the presence of spike protein must be ascribed to vaccination rather than to viral infection. The findings corroborate previous reports of encephalitis and myocarditis caused by gene-based COVID-19 vaccines.

1. Introduction

The emergence of the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) in 2019 with the subsequent worldwide spread of COVID-19 gave rise to a perceived need for halting the progress of the COVID-19 pandemic through the rapid development and deployment of vaccines. Recent advances in genomics facilitated gene-based strategies for creating these novel vaccines, including DNA-based nonreplicating viral vectors, and mRNA-based vaccines, which were furthermore developed on an aggressively shortened timeline [1–4].The WHO Emergency Use Listing Procedure (EUL), which determines the acceptability of medicinal products based on evidence of quality, safety, efficacy, and performance[5], permitted these vaccines to be marketed as soon as 1–2 years after development had begun. Published results of the phase 3 clinical trials described only a few severe side effects [2,6–8]. However, it has since become clear that severe and even fatal adverse events may occur; these include in particular cardiovascular and neurological manifestations [9–13]. Clinicians should take note of such case reports for the sake of early detection and management of such adverse events among their patients. In addition , a thorough post-mortem examination of deaths in connection with COVID-19 vaccination should be considered in ambiguous circumstances, including histology. This report presents the case of a senior aged 76 years old, who had received three doses overall of two different COVID-19 vaccines, and who died three weeks after the second dose of the mRNA-BNT162b-vaccine. Autopsy and histology revealed unexpected necrotizing encephalitis and mild myocarditis with pathological changes in small blood vessels. A causal connection of these findings to the preceding COVID-19 vaccination was established by immunohistochemical demonstration of SARS-CoV-2 spike protein. The methodology introduced in this study should be useful for distinguishing between causation by COVID-19 vaccination or infection in ambiguous cases.

2. Materials and Methods

2.1. Routine Histology

Formalin-fixed tissues were routinely processsed and paraffin-embedded tissueswere cut into 5μm sections and stained with hematoxylin and eosin (H&E) for histo-pathological examination.

2.2. Immunohistochemistry

 Immunohistochemical staining was performed on the heart and brain, using a fullyautomated immunostaining system (Ventana Benchmark, Roche). An antigen retrieval(Ultra CC1, Roche Ventana) was used for every antibody. The target antigens and dilution factors for the antibodies used are summarized in Table 1. Incubation with the primary antibody was carried out for 30 min in each case. Tissues fromSARS-CoV-2-positive COVID-19 patients were used as a control for the antibodies against SARS-CoV-2-spike and nucleocapsid (Figure 1). Cultured cells that had been transfected in vitro (see hereafter) served as a positive control for the detection of vaccine-induced spike protein expression and as a negative control for the detection of nucleocapsid protein. The slides were examined with a light microscope (Nikon ECLIPSE80i) and representative images were captured by the camera system Motic MP3.

Table 1.

Primary antibodies used for immunohistochemistry. Tissue sections were incubated 30min with the antibody in question, diluted as stated in the table.

Target Antigen Manufacturer Clone Dilution Incubation Time

CD3 (expressed by T-Lymphocytes) cytomed ZM-45 1:200 30 minCD68 (expressed by monocytic cells) DAKO PG-M1 1:100 30 minSARS-CoV-2-Spike subunit 1 ProSci 9083 1:500 30 minSARS-CoV-2-Nucleocapsid ProSci 35–720 1:500 30 min

Vaccines 2022, 10 , 1651 3 of 17

Figure 1.

Nasal smear from a person with acute symptomatic SARS-CoV-2-infection (confirmed byPCR). Note the presence of ciliated epithelium. Immunohistochemistry for two SARS-CoV-2antigens (spike and nucleocapsid protein) revealed a positive reaction for both as to be expectedafter infection. (a) Detection of the spike protein. Positive control for spike subunit 1 SARS-CoV-2protein detection. Several ciliated epithelia of the nasal mucosa show brownish granular deposits of DAB (red arrow). Compared to nucleocapsid, the DAB-granules are fewer and less densely packed granular deposits of DAB. (b) Detection of nucleocapsid protein. Positive control for nucleocapsid SARS-CoV-2 protein detection. Several ciliated epithelia of the nasal mucosa show dense brownish granular deposits of DAB in immunohistochemistry (examples red arrows).Compared to spike detection, the granules of DAB are finer and more densely packed.Magnification: 400x.

2.3. Preparation of Positive Control Samples for the Immunohistochemical Detection of theVaccine-Induced Spike Protein

Cell culture and transfection: Ovarian cancer cell lines (OVCAR-3 and SK-OV3, CSL cell Lines Service, Heidelberg, Germany) were grown to 70% confluence in flat bottom 75cm2

 cell culture flasks (Cell star) in DMEM/HAMS-F12 medium supplemented with Glutamax (Sigma-Aldrich, St. Louis, MO, USA), 10% FCS (Gibco, Shanghai, China) and Gentamycin (final concentration 20 μg/mL, Gibco), at 37 °C, 5% CO2

 in a humidified cell incubator. For transfection, the medium was completely removed, and cells were incubated for 1 h with 2 mL of fresh medium containing the injection solutions directly from the original bottles, diluted 1:500 in the case of BNT162b2 (Pfizer/Biotech), and 1:100in cases of mRNA-1273 (Moderna), Vaxzevria (AstraZeneca), and Jansen (COVID-19vaccine Jansen). Then, another 15 mL of fresh medium was added to the cell cultures and cells were grown to confluence for another 3 days. Preparation of tissue blocks from transfected cells: The cell culture medium was removed from transfected cells, and the monolayer was washed twice with PBS, then trypsinized by adding 1 mL of 0.25% Trypsin-EDTA (Gibco), harvested with 10 mL of PBS/10% FCS, and washed 2× with PBS and centrifugation at 280×g  for 10 min each. Cell pellets were fixed overnight in 2 mL in PBS/4% Formalin at 8 °C and then washed in PBmm Sonce. The cell pellets remaining after centrifugation were suspended in 200 μ

L PBS each,mixed with 400

μ

L 2% agarose in PBS solution (precooled to around 40 °C), and immediately transferred to small (1 cm) dishes for fixation. The fixed and agarose-embedded cell pellets were stored in 4% Formalin/PBS till subjection to routine paraffin embedding in parallel to tissue samples.

2.4. Case Presentation and Description

2.4.1. Clinical History This report presents the case of a 76-year-old male with a history of Parkinson’s disease (PD) who passed away three weeks after his third COVID-19 vaccination. On the day of his first vaccination in May 2021 (ChAdOx1 nCov-19 vector vaccine), he experienced pronounced cardiovascular side effects, for which he repeatedly had to consult his doctor. After the second vaccination in July 2021 (BNT162b2 mRNA vaccine/Comirnaty), the family noted obvious behavioral and psychological changes(e.g., he did not want to be touched anymore and experienced increased anxiety, lethargy, and social withdrawal even from close family members). Furthermore, therewas a striking worsening of his PD symptoms, which led to severe motor impairment and a recurrent need for wheelchair support. He never fully recovered from these side effects after the first two vaccinations but still got another vaccination in December 2021.Two weeks after the third vaccination (second vaccination with BNT162b2), he suddenly collapsed while taking his dinner. Remarkably, he did not show coughing or any signs of food aspiration but just fell down silently. He recovered from this more or less, but one week later, he again suddenly collapsed silently while taking his meal. The emergency unit was called, and after successful, but prolonged resuscitation attempts

Vaccines 2022  ,10, 1651 4 of 17

(over one hour), he was transferred to the hospital and directly put into an artificial co-ma but died shortly thereafter. The clinical diagnosis was death due to aspirationpneumonia. According to his family, there was no history of a clinical or laboratorydiagnosis of COVID-19 in the past.2.4.2. AutopsyThe autopsy was requested and consented to by the family of the patient because ofthe ambiguity of symptoms before his death. The autopsy was performed according tostandard procedures including macroscopic and microscopic investigation. Gross braintissue was prepared for histological examination including the brain (frontal cortex,Substantia nigra, and Nucleus ruber) as well as the heart (left and right ventricular car-diac tissue).

3. Results

3.1. Autopsy Findings

Anatomical Specifications: Body weight, height, and specifications of body organswere summarized in Table 2

Table 2.

Anatomical Specifications.

Item Measure Body weight 60 kg Hight 175 cmHeart weight 410 g Brain weight 1560 gL iver weight 1500 

Brain: A macroscopic examination of brain tissue revealed a circumscribed seg-mental cerebral parenchymal necrosis at the site of the right hippocampus. Substantianigra showed a loss of pigmented neurons. Microscopically, several areas with lacunarnecrosis were detected with inflammatory debris reaction on the left frontal side (Figure2). Staining of Nucleus ruber with H&E showed neuronal cell death, microglia, and lymphocyte infiltration (Figure 3). Furthermore, there were microglial and lymphocytic reactions as well as predominantly lymphocytic vasculitis, sometimes with mixed infiltrates including neutrophilic granulocytes (Figure 4) in the frontal cortex, para ventricu-lar, Substantia nigra, and Nucleus ruber on both sides. In some places with inflammatory changes in brain capillaries, there were also signs of apoptotic cell death within the endothelium (Figure 4). Meninges’ findings were unremarkable. The collective findings were suggestive of multifocal necrotizing encephalitis. Furthermore, chronic arterio-sclerotic lesions of varying degrees were noted in large brain vessels, which are described in detail in section “Vascular system”. Parkinson’s disease (PD): Macroscopic and histological examination of brain tissue revealed bilateral pallor of the substantia nigra with loss of pigmented neurons. In addition, pigment-storing macrophages as well as scattered neuronal necrosis with glial de bris reaction were noted. These findings were suggestive of PD, confirming the clinical diagnosis. Thoracic cavity: An examination of the chest showed a funnel-shaped chest with serial rib fractures (extending from the second to fifth ribs on the right, and from the second to sixth ribs on the left); which is a common picture of a patient who underwent cardiopulmonary resuscitation. An endotracheal tube was properly inserted. There was evidence of regular placement of a central venous catheter in the left femoral vein. There was evidence of regular placement of an arterial catheter in the left radial artery. The

urinary catheter was inserted as well. There was a 9 cm long skin scar on the front of theright shoulder.

Lungs: Macroscopical lung examination revealed cloudy secretion and purulentspots with notably brittle parenchyma. The pleura showed bilateral serous effusion,amounting to 450 mL of fluid on the right side and 400 mL on the left side. Bilateralmucopurulent tracheobronchitis was evident with copious purulent secretion in the tra-chea and bronchi. Bilateral chronic destructive pulmonary emphysema was detected.Bilateral bronchopneumonia was noted in the lower lung lobes at multiple stages of de-velopment and lobe-filling with secretions and fragile parenchyma. Furthermore, chronicarteriosclerotic lesions of varying degrees were noted, which are described in detail in thesection “Vascular system”.Heart: Macroscopic cardiac examination revealed manifestations of acute andchronic cardiovascular insufficiency, including ectasia of the atria and ventricles. Fur-thermore, left ventricular hypertrophy was noted (wall thickness: 18 mm, heart weight:410 g, body weight: 60 kg, height: 1.75 m). There was evidence of tissue congestion(presumably due to cardiac insufficiency) in the form of pulmonary edema, cerebraledema, brain congestion, chronic hepatic congestion, renal tissue edema, and pituitarytissue edema. Moreover, there was evidence of shock kidney disorder. Histological ex-amination of the heart revealed mild myocarditis with fine-spotted fibrosis and lym-pho-histiocytic infiltration (Figure 5). Furthermore, there were chronic arterioscleroticlesions of varying degrees, which are described in detail under “Vascular system”. Inaddition to these, there were more acute myocardial and vascular changes in the heart.They consisted of mild signs of myocarditis, characterized by infiltrations with foamyhistiocytes and lymphocytes as well as hypereosinophilia and some hypercontraction ofcardiomyocytes. Furthermore, mild acute vascular changes were observed in the capil-laries and other small blood vessels of the heart. They consisted of mild lym-pho-histiocytic infiltrates, prominent endothelial swelling and vacuolation, multifocalmyocytic degeneration and coagulation necrosis as well as karyopyknosis of single en-dothelial cells and vascular muscle cells (Figure 5). Occasionally, adhering plasma coag-ulates/fibrin clots were present on the endothelial surface, indicative of endothelialdamage (Figure 5).Vascular system (large blood vessels): The pulmonary arteries showed ectasia andlipidosis. The kidney showed slight diffuse glomerulosclerosis and arteriosclerosis withrenal cortical scars (up to 10 mm in diameter). The findings are suggestive of generalizedatherosclerosis and systemic hypertension. Major arteries including the aorta and its branches as well as the coronary arteries showed variable degrees of arteriosclerosis andmild to moderate stenosis. Furthermore, examination revealed mild nodular arterioscle-rosis of cervical arteries. Ascending aorta, aortic arch, and thoracic aorta showed mod-erate, nodular, and partially calcified arteriosclerosis. The cerebral basilar artery showedmild arteriosclerosis. Nodular and calcified arteriosclerosis were of high grade in theabdominal aorta and iliac arteries and moderate grade with moderate stenosis in theright coronary arteries. Coronary artery examination showed variable degrees of arteri-osclerosis and stenosis more on the left coronary arteries. The left anterior descendingcoronary artery (the anterior interventricular branch of the left coronary artery; LAD)showed high-grade and moderately stenosed arteriosclerosis. The arteriosclerosis andstenosis of the left circumflex artery (the circumflex branch of the left coronary artery)were mild.

Mild cerebral basal artery sclerosis. High-grade nodular and calcified arteriosclerosis of the abdominal aorta and the iliac arteries. Moderate stenosed arteriosclerosis of the right coronary artery. Lymphocytic periarteritis was detected as well

Vaccines 2022,10, 1651 6 of 17

Figure 2.

Frontal brain. Already in the overview image (

a), prominent vacuolations with increased parenchymal cellularity are evident, indicative of degenerative and inflammatory processes. At higher magnification (b acute brain damage is visible with diffuse and zonal neuronal and glial cell death, activation of microglia, and inflammatory infiltration by granulocytes and lymphocytes.1: neuronal deaths (cells with red cytoplasm); 2: microglial proliferation; 3: lymphocytes. H&Estain. Magnification 40× ) and 200× (b).

Figure 3.

Brain, Nucleus ruber. In the overview image (a0 note pronounced focal necrosis with increased cellularity, indicative of ongoing inflammation and glial reaction. At higher magnification (b, death of neuronal cells is evident and associated with an increased number of glial cells. Note activation of microglia and presence of inflammatory cell infiltrates, predominantly lymphocytic. 1: neuronal death with hypereosinophilia and destruction of cell nucleus with signs of karyolysis (nuclear content being distributed into the cytoplasm 2: microglia (example); 3:lymphocyte (example). H&E stain. Magnification 40× ( and 400× (b).

Vaccines

2022

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Figure 5.

Heart left ventricle. (

a

): Mild lympho-histiocytic myocarditis.Pronounced interstitialedema (7) and mild lympho-histiocytic infiltrates (2 + 4). Signs of cardiomyocytic degeneration (5)with cytoplasmic hypereosinophilia and single contraction bands. (

d

): Arteriole with signs of acutedegeneration and associated inflammation, associated by lymphocytic infiltrates (2) within thevascular wall, endothelial swelling and vacuolation (3), and vacuolation of vascular myocytes withsigns of karyopyknosis (1). Within the vascular lumen (

d

), note plasma coagulation/fibrin clotsadhering to the endothelial surface, indicative of endothelial damage. 1: pyknotic vascular myo-cytes, 2: lymphocytes, 3: swollen endothelial cells, 4: macrophages, 5: necrotic cardiomyocytes, 6:eosinophilic granulocytes, 7 (blue line): interstitial edema. H&E stain. Magnification: 200x (a) and(c), 40×(b), and detailed enlargement (d).

.2. Other Findings

Oral cavity: tongue bite was detected with bleeding under the tongue muscle(tongue bite is common with epileptic seizures).-

Adrenal glands: bilateral mild cortical hyperplasia.-

Colon: the elongated sigmoid colon was elongated with fecal impaction.-

Kidneys: slight diffuse glomerulosclerosis and arterio-sclerosis, renal corticalscars (up to 10 mm in diameter), bilateral mild active nephritis and urocystitis aswell as evidence of shock kidney disorder.-

Liver: slight lipofuscinosis.-

Spleen: mild acute splenitis.-

Stomach: mild diffuse gastric mucosal bleeding.-

Thyroid gland: bilateral nodular goiter with chocolate cysts (up to 0.5 cm indiameter).-

Prostate gland: benign nodular prostatic hyperplasia and chronic persistentprostatitis.

3.3. Immunohistochemical Analyses

Immunohistochemical staining for the presence of SARS-CoV-2 antigens (spike protein and nucleocapsid) was studied in the brain and heart. In the brain, SARS-CoV-2spike protein subunit 1 was detected in the endothelia, microglia, and astrocytes in thenecrotic areas (Figures 6 and 7). Furthermore, spike protein could be demonstrated in theareas of lymphocytic periarteritis, present in the thoracic and abdominal aorta and iliac branches, as well as a cerebral basal artery (Figure 8). The SARS-CoV-2 subunit 1 was found in macrophages and in the cells of the vessel wall, in particular the endothelium(Figure 9), as well as in the Nucleus ruber (Figure 10). In contrast, the nucleocapsid pro-tein of SARS-CoV-2 could not be detected in any of the corresponding tissue sections(Figures 11 and 12). In addition, SARS-CoV-2 spike protein subunit 1 was detected in the cardiac endothelial cells that showed lymphocytic myocarditis (Figure 13). Immuno-histochemical staining did not detect the SARS-CoV-2 nucleocapsid protein (Figure 14)

3.4. Autopsy-Based Diagnosis

The 76-year-old deceased male patient had PD, which corresponded to typicalpost-mortem findings. The main cause of death was recurrent aspiration pneumonia. Inaddition, necrotizing encephalitis and vasculitis were considered to be major contribu-tors to death. Furthermore, there was mild lympho-histiocytic myocarditis with fi-ne-spotted myocardial fibrosis as well as systemic arteriosclerosis, which will have alsocontributed to the deterioration of the physical condition of the senior.The final diagnosis was abscedating bilateral bronchopneumonia (J18.9), Parkin-son’s disease (G20.9), necrotic encephalitis (G04.9), and myocarditis (I40.9).Immunohistochemistry for SARS-CoV-2 antigens (spike protein and nucleocapsid)revealed that the lesions with necrotizing encephalitis as well as the acute inflammatorychanges in the small blood vessels (brain and heart) were associated with abundant de-posits of the spike protein SARS-CoV-2 subunit 1. Since the nucleocapsid protein ofSARS-CoV-2 was consistently absent, it must be assumed that the presence of spike pro-tein in affected tissues was not due to an infection with SARS-CoV-2 but rather to thetransfection of the tissues by the gene-based COVID-19-vaccines. Importantly, spikeprotein could be only demonstrated in the areas with acute inflammatory reactions(brain, heart, and small blood vessels), in particular in endothelial cells, microglia, andastrocytes. This is strongly suggestive that the spike protein may have played at least acontributing role to the development of the lesions and the course of the disease in thispatient.

4. Discussion

This is a case report of a 76-year-old patient with Parkinson’s disease (PD) who diedthree weeks after his third COVID-19 vaccination. The stated cause of death appeared to be a recurrent attack of aspiration pneumonia, which is indeed common in PD [14,15].However, the detailed autopsy study revealed additional pathology, in particular necrotizing encephalitis and myocarditis. While the histopathological signs of myocarditis were comparatively mild, the encephalitis had resulted in significant multifocal necrosis and may well have contributed to the fatal outcome. Encephalitis often causes epileptic seizures, and the tongue bite found at the autopsy suggests that it had done so in this case. Several other cases of COVID-19 vaccine-associated encephalitis with status epilepticus have appeared previously [16–18].The clinical history of the current case showed some remarkable events in correlation to his COVID-19 vaccinations. Already on the day of his first vaccination in May2021 (ChAdOx1 nCov-19 vector vaccine), he experienced cardiovascular symptoms, which needed medical care and from which he recovered only slowly. After the second vaccination in July 2021 (BNT162b2 mRNA vaccine), the family recognized remarkable behavioral and psychological changes and a sudden onset of marked progression of hisPD symptoms, which led to severe motor impairment and recurrent need for wheel chairs upport. He never fully recovered from this but still was again vaccinated in December2021. Two weeks after this third vaccination (second vaccination with BNT162b2), he suddenly collapsed while taking his dinner. Remarkably, he did not show any coughing or other signs of food aspiration but just fell from his chair. This raises the question of whether this sudden collapse was really due to aspiration pneumonia. After intense resuscitation, he recovered from this more or less, but one week later, he again suddenlycollapsed silently while taking his meal. After successful but prolonged resuscitation at-tempts, he was transferred to the hospital and directly set into an artificial coma but died shortly thereafter. The clinical diagnosis was death due to aspiration pneumonia. Due to his ambiguous symptoms after the COVID-vaccinations the family asked for an autopsy. Based on the alteration pattern in the brain and heart, it appeared that the small blood  vessels were especially affected, in particular, the endothelium. Endothelial dysfunction is known to be highly involved in organ dysfunction during viral infections, as it induces a pro-coagulant state, microvascular leak, and organ ischemia [19,20]. This is also the case for severe SARS-CoV-2 infections, where a systemic exposure to the virus and its spike protein elicits a strong immunological reaction in which the endothelial cells play a crucial role, leading to vascular dysfunction, immune-thrombosis, and inflammation [21].Although there was no history of COVID-19 for this patient, immunohistochemistry for SARS-CoV-2 antigens (spike and nucleocapsid proteins) was performed. Spike pro-tein could be indeed demonstrated in the areas of acute inflammation in the brain (par-ticularly within the capillary endothelium) and the small blood vessels of the heart. Re-markably, however, the nucleocapsid was uniformly absent. During an infection with thevirus, both proteins should be expressed and detected together. On the other hand, thegene-based COVID-19 vaccines encode only the spike protein and therefore, the presenceof spike protein only (but no nucleocapsid protein) in the heart and brain of the currentcase can be attributed to vaccination rather than to infection. This agrees with the pa-tient’s history, which includes three vaccine injections, the third one just 3 weeks beforehis death, but no positive laboratory or clinical diagnosis of the infection.Discrimination of vaccination response from natural infection is an important ques-tion and had been addressed already in clinical immunology, where the combined ap-plication of anti-spike and anti-nucleocapsid protein-based serology was proven as auseful tool [22]. In histology, however, this immunohistochemical approach has not yet been described, but it is straightforward and appears to be very useful for identifying thepotential origin of SARS-CoV-2 spike protein in autopsy or biopsy samples. Where addi-tional confirmation is required, for instance in a forensic context, rt-PCR methods might be used to ascertain the presence of the vaccine mRNA in the affected tissues [23,24].Assuming that, in the current case, the presence of spike protein was indeed driven by the gene-based vaccine, then the question arises whether this was also the cause the accompanying acute tissue alterations and inflammation. The stated purpose of the gene-based vaccines is to induce an immune response against the spike protein. Such an immune response will, however, not only results in antibody formation against the spike protein but also lead to direct cell- and antibody-mediated cytotoxicity against the cells expressing this foreign antigen. In addition, there are indications that the spike protein on its own can elicit distinct toxicity, in particular, on pericytes and endothelial cells of blood vessels [25,26]

While it is widely held that spike protein expression, and the ensuing cell and tissuedamage will be limited to the injection site, several studies have found the vaccinemRNA and/or the spike protein encoded by it at a considerable distance from the injec-tion site for up to three months after the injection [23,24,27–29]. Biodistribution studies inrats with the mRNA-COVID-19 vaccine BNT162b2 also showed that the vaccine does notstay at the injection site but is distributed to all tissues and organs, including the brain[30]. After the worldwide roll-out of COVID-19 vaccinations in humans, spike proteinhas been detected in humans as well in several tissues distant from the injection site(deltoid muscle): for instance in heart muscle biopsies from myocarditis patients [28],within the skeletal muscle of a patient with myositis [23] and within the skin, where itwas associated with a sudden onset of Herpes zoster lesions after mRNA-COVID-19vaccination [29].The underlying diagnosis in this patient was Parkinson’s disease, and one may askwhat role, if any, this condition had played in the causation of the encephalitis, and themyocarditis detected at post-mortem examination. PD had been long-standing in thecurrent case, whereas the encephalitis was acute. Conversely, there is no plausiblemechanism and no case report of PD causing secondary necrotizing encephalitis. On theother hand, numerous cases have been reported of autoimmune encephalitis and en-cephalomyelitis after COVID-19 vaccination [12,31]. Autoimmune diseases in organsother than the CNS have been reported as well, for example, a striking case of a patientwho after mRNA vaccination suffered multiple autoimmune disorders all at once—acutedisseminated encephalomyelitis, myasthenia gravis, and thyroiditis [32]. In the case re-ported here, it may be noted that the spike protein was primarily detected in the vascularendothelium and sparsely in the glial cells but not in the neurons. Nevertheless, neuronalcell death was widespread in the encephalitic foci, which suggests some contribution ofimmunological bystander activation, i.e., autoimmunity, to the observed cell and tissuedamage.A contributory role of PD in the development of cardiomyopathy is indeed docu-mented and cannot be ruled out with absolute certainty. However, inflammatory myo-cardial changes with pathological alterations in small blood vessels as seen in the currentcase are uncommon. Instead, the most prominent cause of cardiac failure in PD patientsis rather due to cardiac autonomic dysfunction [33,34]. PD seems well to be significantlyassociated with increased left ventricular hypertrophy and diastolic dysfunction [34]. Inthe current case, ventricular dilatation and hypertrophy were present but seem ratherrelated to manifest signs of chronic hypertension. In contrast, myocardial inflammatoryreactions had been well-linked to gene-based COVID-19 vaccinations in numerous cases[9,35–37]. In one case, the spike protein of SARS-CoV-2 could also be demonstrated byimmunohistochemistry in the heart of vaccinated individuals [28].

5. Conclusions

Numerous cases of encephalitis and encephalomyelitis have been reported in con-nection with the gene-based COVID-19 vaccines, with many being considered causally related to vaccination [31,38,39]. However, this is the first report to demonstrate the presence of the spike protein within the encephalitic lesions and to attribute it to vac-cination rather than infection. These findings corroborate a causative role of thegene-based COVID-19 vaccines, and this diagnostic approach is relevant to potentially vaccine-induced damage to other organs as well

References

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Sultana, J.; Mazzaglia, G.; Luxi, N.; Cancellieri, A.; Capuano, A.; Ferrajolo, C.; de Waure, C.; Ferlazzo, G.; Trifirò, G. Potentialeffects of vaccinations on the prevention of COVID-19: Rationale, clinical evidence, risks, and public health considerations.Expert Rev. Vaccines2020

 19 919–936. https://doi.org/10.1080/14760584.2020.1825951.3.WHO. COVID-19 Vaccine Tracker and Landscape. Available online:https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines (accessed on 2 June 2022).4.

Lurie, N.; Saville, M.; Hatchett, R.; Halton, J. Developing Covid-19 Vaccines at Pandemic Speed.N. Engl. J. Med.202

82,1969–1973. https://doi.org/10.1056/NEJMp2005630.5.World Health Organization (WHO). Diagnostics Laboratory Emergency Use Listing. Available online:https://www.who.int/teams/regulation-prequalification/eul (accessed on 2 June 2022).6.Baden, L.R.; El Sahly, H.M.; Essink, B.; Kotloff, K.; Frey, S.; Novak, R.; Diemert, D.; Spector, S.A.; Rouphael, N.; Creech, C.B.; etal. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine.N. Engl. J. Med.2021384, 403–416.https://doi.org/10.1056/NEJMoa2035389.7.O’Reilly, P. A phase III study to investigate a vaccine against COVID-19.ISRCTN 2020https://doi.org/10.1186/ISRCTN89951424.8.

Polak, S.B.; Van Gool, I.C.; Cohen, D.; von der Thüsen, J.H.; van Paassen, J. A systematic review of pathological findings inCOVID-19: A pathophysiological timeline and possible mechanisms of disease progression.,Mod. Pathol.2020 3

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oticMP3

Detection of Messenger RNA COVID-19 Vaccines in Human Breast Milk

Authors: Nazeeh Hanna, MD1Ari Heffes-Doon, MD1Xinhua Lin, PhD2et alClaudia Manzano DeMejia, MD2Bishoy Botros, BS2;  Ellen Gurzenda, BS2Amrita Nayak, MD1 JAMA Pediatric Published online September 26, 2022. doi:10.1001/jamapediatrics.2022.3581

Vaccination is a cornerstone in fighting the COVID-19 pandemic. However, the initial messenger RNA (mRNA) vaccine clinical trials excluded several vulnerable groups, including young children and lactating individuals.1 The US Food and Drug Administration deferred the decision to authorize COVID-19 mRNA vaccines for infants younger than 6 months until more data are available because of the potential priming of the children’s immune responses that may alter their immunity.2 The Centers for Disease Control and Prevention recommends offering the COVID-19 mRNA vaccines to breastfeeding individuals,3 although the possible passage of vaccine mRNAs in breast milk resulting in infants’ exposure at younger than 6 months was not investigated. This study investigated whether the COVID-19 vaccine mRNA can be detected in the expressed breast milk (EBM) of lactating individuals receiving the vaccination within 6 months after delivery.

Methods

This cohort study included 11 healthy lactating individuals who received either the Moderna mRNA-1273 vaccine (n = 5) or the Pfizer BNT162b2 vaccine (n = 6) within 6 months after delivery (Table 1). Participants were asked to collect and immediately freeze EBM samples at home until transported to the laboratory. Samples of EBM were collected before vaccination (control) and for 5 days postvaccination. A total of 131 EBM samples were collected 1 hour to 5 days after vaccine administration. Extracellular vesicles (EVs) were isolated in EBM using sequential centrifugation, and the EV concentrations were determined by ZetaView (Analytik) (eMethods in the Supplement). The presence of COVID-19 vaccine mRNA in different milk fractions (whole EBM, fat, cells, and supernatant EVs) was assayed using 2-step quantitative reverse transcriptase–polymerase chain reaction. The vaccine detection limit was 1 pg/mL of EBM (eMethods in the Supplement).

Results

Of 11 lactating individuals enrolled, trace amounts of BNT162b2 and mRNA-1273 COVID-19 mRNA vaccines were detected in 7 samples from 5 different participants at various times up to 45 hours postvaccination (Table 2). The mean (SD) yield of EVs isolated from EBM was 9.110 (5.010) particles/mL, and the mean (SD) particle size was 110.0 (3.0) nm. The vaccine mRNA appears in higher concentrations in the EVs than in whole milk (Table 2). No vaccine mRNA was detected in prevaccination or postvaccination EBM samples beyond 48 hours of collection. Also, no COVID-19 vaccine mRNA was detected in the EBM fat fraction or the EBM cell pellets.

Discussion

The sporadic presence and trace quantities of COVID-19 vaccine mRNA detected in EBM suggest that breastfeeding after COVID-19 mRNA vaccination is safe, particularly beyond 48 hours after vaccination. These data demonstrate for the first time to our knowledge the biodistribution of COVID-19 vaccine mRNA to mammary cells and the potential ability of tissue EVs to package the vaccine mRNA that can be transported to distant cells. Little has been reported on lipid nanoparticle biodistribution and localization in human tissues after COVID-19 mRNA vaccination. In rats, up to 3 days following intramuscular administration, low vaccine mRNA levels were detected in the heart, lung, testis, and brain tissues, indicating tissue biodistribution.4 We speculate that, following the vaccine administration, lipid nanoparticles containing the vaccine mRNA are carried to mammary glands via hematogenous and/or lymphatic routes.5,6 Furthermore, we speculate that vaccine mRNA released into mammary cell cytosol can be recruited into developing EVs that are later secreted in EBM.

The limitations of this study include the relatively small sample size and the lack of functional studies demonstrating whether detected vaccine mRNA is translationally active. Also, we did not test the possible cumulative vaccine mRNA exposure after frequent breastfeeding in infants. We believe it is safe to breastfeed after maternal COVID-19 vaccination. However, caution is warranted about breastfeeding children younger than 6 months in the first 48 hours after maternal vaccination until more safety studies are conducted. In addition, the potential interference of COVID-19 vaccine mRNA with the immune response to multiple routine vaccines given to infants during the first 6 months of age needs to be considered. It is critical that lactating individuals be included in future vaccination trials to better evaluate the effect of mRNA vaccines on lactation outcomes.

References

1.Van Spall  HGC.  Exclusion of pregnant and lactating women from COVID-19 vaccine trials: a missed opportunity.   Eur Heart J. 2021;42(28):2724-2726. doi:10.1093/eurheartj/ehab103PubMedGoogle ScholarCrossref

2.US Food and Drug Administration. Coronavirus (COVID-19) update: FDA authorizes Moderna and Pfizer-BioNTech COVID-19 vaccines for children down to 6 months of age. Released June 17, 2022. https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-moderna-and-pfizer-biontech-covid-19-vaccines-children

3.Centers for Disease Control and Prevention. COVID-19 vaccines while pregnant or breastfeeding. Accessed March 8, 2021. https://www.cdc.gov/coronavirus/2019-ncov/vaccines/recommendations/pregnancy.html.

4.European Medicines Agency. Assessment report: COVID-19 vaccine Moderna. Published March 11, 2021. http://www.ema.europa.eu/en/documents/assessment-report/spikevax-previously-covid-19-vaccine-moderna-epar-public-assessment-report_en.pdf.

5.Pardi  N, Tuyishime  S, Muramatsu  H,  et al.  Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes.   J Control Release. 2015;217:345-351. doi:10.1016/j.jconrel.2015.08.007PubMedGoogle ScholarCrossref

6.Bansal  S, Perincheri  S, Fleming  T,  et al.  Cutting edge: circulating exosomes with COVID spike protein are induced by BNT162b2 (Pfizer-BioNTech) vaccination prior to development of antibodies: a novel mechanism for immune activation by mRNA vaccines.   J Immunol. 2021;207(10):2405-2410. doi:10.4049/jimmunol.2100637PubMedGoogle ScholarCrossref