Toxicologic Pathology Forum: mRNA Vaccine Safety–Separating Fact From Fiction

Abstract

SARS-CoV-2 spread rapidly across the globe, contributing to the death of millions of individuals from 2019 to 2023, and has continued to be a major cause of morbidity and mortality after the pandemic. At the start of the pandemic, no vaccines or anti-viral treatments were available to reduce the burden of disease associated with this virus, as it was a novel SARS coronavirus. Because of the tremendous need, the development of vaccines to protect against COVID-19 was critically important. The flexibility and ease of manufacture of nucleic acid–based vaccines, specifically mRNA-based products, allowed the accelerated development of COVID-19 vaccines. Although mRNA-based vaccines and therapeutics had been in clinical trials for over a decade, there were no licensed mRNA vaccines on the market at the start of the pandemic. The rapid development of mRNA-based COVID-19 vaccines reduced serious complications and death from the virus but also engendered significant public concerns, which continue now, years after emergency-use authorization and subsequent licensure of these vaccines. This article summarizes and addresses some of the safety concerns that continue to be expressed about these vaccines and their underlying technology.

Introduction

Rapid COVID-19 vaccine development using mRNA technology not previously used in licensed vaccines created a news and social media tempest that was often fueled by misinformation and scientific misunderstanding, which ranged from the absurd to the dangerous. This fear-fueled public discourse reduced the uptake of effective COVID-19 mRNA vaccines, which had a good safety profile. The manipulation of public fear likely contributed to unnecessary COVID-19-related deaths ⁴¹ and engendered support for the anti-vaccine movement generally. ⁴³

One of the most absurd theories floated in online platforms was that the COVID-19 vaccine contained microchips (putatively of Microsoft origin), which would enable tracking of peoples’ movements and might even collect biometric data (https://revealnews.org/article/where-did-the-microchip-vaccine-conspiracy-theory-come-from-anyway/; accessed July 22, 2024). The link to Microsoft and Bill Gates in this conspiracy theory was likely based on extensive vaccine grant funding from the Bill & Melinda Gates Foundation. ⁵⁴

However, the obviously absurd conspiracy theories were not the most harmful. Rather, the most damaging misinformation was built on poorly performed studies, distortion, and misinterpretation of nonclinical study data by non-toxicologists and/or non-toxicological pathologists (including by some clinicians), and manipulation of biological plausibility claims (i.e., theoretical vs demonstrated risks). In an effort to share knowledge about the newly emerged SARS-CoV-2 virus rapidly, virus-related research data were placed in the public domain prior to peer review. Poorly performed nonclinical and clinical studies and inaccurately interpreted, inadequately vetted data on COVID-19 disease, therapeutics, and vaccines were publicly available in non-peer reviewed, pre-publication scientific venues (BioRxiv and Medrxiv) as well as in peer-reviewed scientific journals. ⁸³ In some cases, incorrect information was used to inform treatment. For example, the clinical use of the FDA approved antiparasitic drug ivermectin as an effective treatment for COVID-19 was reported in peer-reviewed manuscripts (cited manuscript Bryant et al ¹⁵ has been retracted).^16,66 This story was picked up by the media, and the information was widely disseminated (e.g., https://www.nbcmiami.com/news/local/local-doctor-tries-new-coronavirus-drug-treatment/2219465/;  https://www.foxnews.com/health/head-lice-drug-possible-coronavirus-treatment-report; accessed July 22, 2024). Ultimately, follow-on retrospective and prospective studies demonstrated that ivermectin had neither a positive nor negative effect on COVID-19 prevention or disease course and, thus, the benefit-risk of this application of ivermectin was negative.^60,70 Another example was the reported benefit of chloroquine and hydroxychloroquine as a treatment for COVID-19. Clinical publications supporting the potential of these compounds in the COVID-19 treatment^23,59 (Chen et al, remains as a non-peer-reviewed preprint) received extensive public attention, and the therapy was promoted widely (e.g., https://abcnews.go.com/Health/chloroquine-malaria-drug-treat-coronavirus-doctors/story?id=69664561;  https://www.foxnews.com/politics/hydroxychloroquine-helped-save-coronavirus-study; accessed July 22, 2024). In March 2020, the FDA approved emergency use authorization for chloroquine and hydroxychloroquine in the treatment of severe COVID-19 disease. However, subsequent nonclinical studies in hamster and macaque models demonstrated no benefit of chloroquine on SARS-CoV-2 infection, ⁷⁷ and numerous clinical publications reported that there was no therapeutic value in humans.^7,27 The emerging scientific data resulted in the FDA retracting its emergency use authorization for the treatment of severe COVID-19 disease in June of 2020, less than 3 months after its authorization. The conflicting information from scientific publications, the news media, and government agencies contributed to public distrust of science and the government regarding COVID-19 prevention and treatment. As of July 2024, there have been 432 retracted papers related to SARS-CoV-2 and SARS-CoV-2 vaccines and therapeutics (Retracted coronavirus [COVID-19] papers—Retraction Watch; accessed July 22, 2024). For comparison, review of PubMed data identified <40 retractions of research publications on human influenza viruses (as of February 2024).

Considerations on mRNA Vaccine Concerns

Nonclinical Vaccine Data Included in Emergency Use Authorizations

Nonclinical data from the mRNA-lipid nanoparticle (LNP) COVID-19 vaccines tested in non-human primates, rats, and rabbits were made publicly available at the time of emergency use authorization by various global regulatory bodies (e.g., European Public Assessment Reports [EPAR]; UK Summary of the Public Assessment Report [SPAR], and the Japanese Pharmaceutical and Medical Devices Agency [PMDA] Report on the Deliberation results). Unfortunately, data from these reports were often interpreted out of context or fully misinterpreted to insinuate greater risk from vaccine exposure than was accurate. For example, using these publicly available reports, some researchers have hypothesized that the vaccine causes liver damage in nonclinical species, ² a conclusion that is not supported by evidence. ⁷⁴ In addition, the PMDA report, which included biodistribution information on the lipid components of the Pfizer vaccine LNP, was incorrectly used to support theories that the vaccine would cause infertility (see section Female Infertility and Abortion). ⁸⁰

Objections to the mRNA-LNP vaccines also derived from the assertion that they contained fetal cells. In June 2022, Factcheck.org published an article entitled: COVID-19 Vaccines Don’t Contain Fetal Tissue (https://www.factcheck.org/2022/07/scicheck-covid-19-vaccines-dont-contain-fetal-tissue/). The mRNA-LNP vaccines are manufactured in a completely cell-free system and contain no human or animal cells or residual cellular materials. ⁷⁶ However, in vitro studies during COVID-19 vaccine development and testing did utilize the adenovirus-transformed human embryonic kidney cell line, HEK-293T. The progenitors of these cells were originally isolated from a fetus (unclear if stillborn or medically aborted) in the early 1970s in the Netherlands. ³⁵ This cell line is widely used in research in both academic and biopharmaceutical settings. ⁸⁶

Vaccine Adverse Event Reporting System

Interpreting the confusing and highly inflammatory information published in anti-vaccine forums requires an understanding of the Vaccine Adverse Event Reporting System (VAERS). VAERS is a publicly available and searchable database, which was established as a collaboration between the CDC and FDA in 1990. The database contains a collection of reports of post-vaccination health incidents, with the same incidents occasionally reported two or more times. Much of the information uploaded into VAERS is not assessed for causality, and information from reports that are made available to the public is generally limited. Therefore, VAERS includes reports of post-vaccine events regardless of whether the events were caused by vaccination. VAERS should only be used for signal detection, not validation, because many potential signals derived from the database are ultimately not confirmed.

The intention of VAERS is to identify potential post-marketing adverse events (AE) associated with a vaccine. For example, in 1999, the database flagged a possible relationship between vaccination with a rotavirus gastroenteritis vaccine, RotaShield, and intussusception. To verify causality, the CDC and FDA initiated two large investigations; these studies confirmed a transiently increased risk of intussusception in infants after vaccination. ¹⁹ RotaShield was subsequently voluntarily removed from the market by the manufacturer. ²⁰ The system also flagged a potential risk of myocarditis after vaccination with the mRNA-LNP COVID-19 vaccines produced by Pfizer-BioNTech and Moderna. The myocarditis risk also was subsequently verified, and the risk was added to the vaccines’ labels.

Currently, there are over 1.5 million mRNA-LNP COVID-19 post-vaccination AE and over 30,000 deaths reported in VAERS, of which >19,000 were reported in the United States. Such large numbers have helped to cast doubt on the safety of COVID-19 vaccines. However, these data can be readily taken out of context, as the reports of AEs or deaths from the vaccine are often not verified, the AEs are not reported in the context of the number of vaccine doses administered, and VAERS does not provide information on vaccine benefits. Any death after COVID-19 vaccination may be reported in VAERS, regardless of cause. After review of available details (and data are often limited) on medical history, death certificates, and autopsy findings from these reported deaths, the CDC and FDA have identified no causality between mRNA-LNP COVID-19 vaccine administration and mortality (https://www.cdc.gov/coronavirus/2019-ncov/vaccines/safety/adverse-events.html; accessed July 22, 2024).

mRNA-LNP COVID-19 Vaccine Reactogenicity Profile

The most common clinical complaints after immunization with the mRNA-LNP COVID-19 vaccines are related to innate immune responses: injection site pain, swelling and redness, fatigue, muscle pain, fever, and headache. Many of these findings are anticipated vaccine responses (reactogenicity), and several were identified in the nonclinical toxicity studies, including transient swelling and redness of the injection site and low-grade fever. A less common finding in humans is lymphadenopathy, typically in the lymph nodes draining the injection site. Lymphadenopathy is commonly identified in nonclinical species in the draining lymph nodes after vaccination generally, and this was the case after immunization with the mRNA-LNP vaccines.^8,69

Currently, the data suggest that the lipid component of the mRNA-LNP vaccine is largely responsible for reactogenicity as well as adjuvanticity (ability to enhance the immune response to the antigen), as administration of empty LNPs will result in robust innate immune responses similar to those elicited by mRNA-LNP comparators. ⁹⁰ The robust adjuvant-driven response enhances the immune response to the vaccine-encoded antigen, an effect that is particularly valuable in older adults, who typically have less of an adaptive immune response to vaccines than do younger individuals and who also typically experience less reactogenicity. Currently, studies are underway to determine if LNP adjuvanticity and reactogenicity are coupled; ideally, adjuvanticity of LNPs can be maintained while reactogenicity is attenuated.

Anaphylaxis and Hypersensitivity Reactions

In December 2020, after the mRNA-LNP COVID vaccines were authorized for clinical use, the first doses were administered to healthcare workers in the United Kingdom. On the first day of vaccination, immediately after dosing, 2 healthcare workers developed acute allergic-like reactions that required medical intervention. Shortly after that event, 2 healthcare workers in the United States developed allergic reactions immediately after dosing, and these cases were also highly publicized. ⁸² Because these serious adverse events (SAE) were not identified in either nonclinical studies or clinical trials (which included more than 40,000 people), media attention was intensely focused on the vaccine’s safety. Because anaphylaxis after immunization is exceptionally rare (~1-2 per million doses), ⁵⁸ it generally is not observed during clinical trials. Further, clinical trials of vaccines exclude individuals with a history of hypersensitivity to vaccine or vaccine components. However, in the context of concerns on the pace at which the mRNA-LNP vaccines were approved for emergency use, these hypersensitivity reactions fueled the narrative that the vaccine was unsafe and inadequately tested. Soon, the news was rife with stories of people who claimed to have had hypersensitivity reactions to the vaccine. Very few of these individuals had true hypersensitivity reactions, and we now know that the incidence is around 5 events per 1 million doses (https://www.cdc.gov/coronavirus/2019-ncov/vaccines/safety/adverse-events.html; accessed July 22, 2024). Likely, many of those reporting hypersensitivity reactions during the initial roll-out were having vaso-vagal responses or developed psychosomatic symptoms because of the intense media attention to this rare but distressing SAE. ⁴⁵

Ultimately, the cause for the hypersensitivity reactions was determined to be immune responses against the PEGylated (PEG) lipid in the LNP. Nonclinical studies would not have identified this response, as animals used in toxicology studies are not exposed to PEG. ⁴⁷ The anti-PEG responses appear to be mostly IgG/IgM-mediated, rather than classical IgE-mediated anaphylactic responses. Because PEG is in many products such as toothpaste, shampoo and cosmetics, many individuals have anti-PEG antibodies, ⁴⁷ though few develop anaphylactic reactions. In fact, anaphylactic reactions to the COVID-19 vaccines are less common than those reported for several other vaccines. A recent publication comparing the rates of vaccine-associated anaphylaxis reported that anaphylactic responses to the COVID-19 mRNA-LNP vaccines were less than those associated with non-mRNA-based vaccines against tick-borne encephalitis, rabies, measles-mumps-rubella, and hepatitis A. ⁴⁴

Myocarditis and Pericarditis

Although no heart lesions were identified in nonclinical safety studies, ⁷⁴ increased risk of symptomatic myocarditis and pericarditis was identified clinically after vaccination with the mRNA-LNP COVID-19 vaccines, with the highest incidence in males less than 30 years of age.^60,92 In 12- to 17-year-old males, the estimated risk is 7-8 in 100,000, and in 25- to 29-year-old males, it is around 1.6 in 100,000. There is little increased risk to females in those age groups. In contrast, the risk of myocarditis associated with SARS-CoV-2 infection is approximately 16-fold higher than in uninfected individuals. ¹⁰ Interestingly, non-vaccine-related myocarditis is most frequently identified in males less than 35 years of age, with an incidence of 1.8 per 100,000 boys aged 15-18 years of age. Often, no etiology is identified, but when it is, it is typically the result of a virus, bacterium, or toxin. Testosterone has been identified as a primary contributing factor. ⁴⁸

mRNA-LNP vaccine-associated myocarditis and pericarditis usually occur within 2 weeks after the first or second dose. Data suggest that there is a vaccine dose-response in the incidence of myocarditis and pericarditis. ⁶⁸ As in the case of non-vaccine causes, vaccine-associated myocarditis, pericarditis, or myopericarditis is generally self-limiting and is treated symptomatically. To date, no microbial or autoimmune etiologies have been identified, and there has been no evidence of chronic heart disease developing in affected individuals. ⁶⁷

Because the severity of post-vaccine myocarditis and pericarditis is limited, there is rarely a need for invasive endomyocardial biopsies. As a result, there is little histology data to help understand the pathogenesis. Furthermore, the histology data that have been published have wide variation in the types of cellular infiltrates reported, which may be due, in part, to limited sampling. Some papers have demonstrated T-cell and macrophage infiltrates^4,65,94; another has identified neutrophils and macrophages ²⁴ ; and one case of fulminant necrotizing eosinophilic myocarditis has been reported. ⁵ The presence of cardiomyocyte necrosis in these lesions has been variably reported. ⁴

Theories regarding the cause of vaccine-associated myocarditis have included molecular mimicry to spike-like peptides expressed in the heart, vaccine distribution to the heart with local expression and subsequent targeting by the immune system, hypersensitivity responses, and anti-idiotype antibodies to the spike.^12,88 However, the SARS-CoV-2 S glycoprotein has not been identified in heart biopsies from individuals with vaccine-associated myocarditis. Given the notable immune activation induced by the vaccine, it is possible that vaccination results in an exacerbation of underlying subclinical myocarditis in at-risk populations. One published study using a mouse model suggested that inadvertent IV vaccine administration causes myocarditis. ⁴⁹ Although this publication made headlines, its conclusions on microscopic findings in the heart were incorrectly interpreted by individuals who are not veterinary pathologists and who may be unfamiliar with background findings in mice and artifacts of tissue handling and processing. The lesions identified in the heart in this study were not, in fact, due to the vaccine. Rather, the lesions were consistent with chronic dystrophic cardiac calcification, which is reported in mice, including in the strain of mice in this study (BALB/c). ³¹ Additionally, the microscopic lesions shown in the images in the publication appeared to reflect handling artifacts rather than pathological changes. In sum, inadvertent IV administration of the vaccine does not cause myocarditis in mice. Additional nonclinical studies in mouse models of autoimmune myocarditis and immune modulation by immune checkpoint inhibitors have not revealed any association between COVID-19 mRNA-LNP vaccination and increased risk for inflammatory heart lesions except in mice administered anti-CTLA4 antibodies. ⁹⁶

Guillain-Barré Syndrome (GBS)

Guillain-Barré syndrome (GBS) is a rare immune-mediated polyneuropathy caused by antibodies that cross react with gangliosides in axons and myelin sheaths, causing muscle weakness and sometimes paralysis in humans. The majority of GBS cases has been reported after infections ⁸⁹ and less commonly after vaccination with a number of prophylactic vaccines, the most reported being influenza vaccines.^37,71,89 However, many published studies examining the risk of GBS after influenza vaccination (as well as after other vaccinations) have found little to no increased risk. Moreover, the risk of GBS is higher in association with virus infection than with vaccination. ⁸⁹ There is no identified increased risk of GBS after immunization with the Pfizer-BioNTech or Moderna mRNA-LNP COVID-19 vaccines. ³⁸

Increased Risk of Liver Injury

Concerns have arisen around the risk of liver injury from the mRNA-LNP vaccines.^32,79 In part, the concerns were based on biodistribution data in mice that demonstrated rapid and notable distribution to the liver after intramuscular (IM) administration of an LNP-formulated mRNA that is similar to the COVID-19 vaccine but that expresses luciferase rather than the Spike protein. Peak bioluminescence in the liver in mice occurred 6 hours after administration and returned to background levels within 48 hours. ²¹ In another study, mRNA expression was identified in the liver of rats after IM injection of vaccine and was cleared within 3 days. ²² Evaluation of the biodistribution of the lipid components of the LNP using radioactively labeled lipid demonstrated that the greatest concentration was at the injection site, with clearance from most tissues within 48-72 hours. Very low levels of LNP lipid were identified at 7 days after administration, primarily at the injection site, spleen, and liver (21.5% of the injected dose).^21,25 It should also be noted that the doses of mRNA-LNP materials administered to mice and rats were, on a mg/kg basis, ~300x those administered to humans (based on a 30 µg mRNA vaccine dose in an 80 kg human).

Distribution of mRNA-LNP vaccine components to the liver has led to concerns about the impact of the mRNA-LNP vaccines on liver function. Reports of liver injury, such as individual hepatocyte apoptosis/necrosis, have been reported with non-vaccine RNA-LNP therapeutics administered intravenously. ⁸¹ However, some high-profile publications have inaccurately reported nonclinical safety study data from mRNA-LNP COVID-19 vaccines. For example, one publication reported that COVID-19 mRNA-LNP vaccines cause hepatocellular damage, as evidenced by increased serum liver enzymes and liver weights in nonclinical safety studies. ² Published Pfizer nonclinical data obtained with the mRNA-LNP COVID-19 vaccine ⁷⁴ did not identify liver weight increases or elevation in the liver enzymes alanine aminotransferase (ALT) or aspartate aminotransferase (AST) to indicate liver injury. ⁷⁴ Studies in rats did identify minimal transient hepatocellular vacuolation, which was interpreted to reflect uptake of lipid from the LNP. Initial studies identified elevations in gamma-glutamyl transferase (GGT), but this finding was not identified in subsequent studies. Serum GGT elevations in rodents are considered a biomarker for hepatic bile duct injury. ³⁴ As no evidence of biliary injury was identified microscopically, and the data could not be duplicated, the finding was attributed to sample collection and handling in the initial study. ⁷⁴

Several papers have suggested a clinical relationship between autoimmune hepatitis and the COVID-19 mRNA-LNP vaccines,^{3,32,79,87,95} However, the European Medicines Agency (EMA) Pharmacovigilance Risk Assessment Committee has concluded that there is no relationship between the COVID-19 mRNA vaccines and autoimmune hepatitis risk (https://www.ema.europa.eu/en/news/meeting-highlights-pharmacovigilance-risk-assessment-committee-prac-4-7-april-2022; https://www.fdanews.com/articles/207373-prac-finds-no-link-between-autoimmune-hepatitis-and-mrna-based-covid-19-vaccines?v=preview; accessed July 22, 2024). ⁹³ Clinical studies suggest that the COVID-19 vaccines help protect against liver injury related to infection with SARS-CoV-2. ⁷²

Female Infertility and Abortion

Very early in the development of COVID-19 vaccines that use the Spike antigen, two scientists raised a concern on a German website that the vaccines could cause infertility in women and petitioned the EMA to stop COVID-19 vaccine clinical trials (https://www.regulations.gov/comment/CDC-2020-0121-0181;  https://2020news.de/en/dr-wodarg-and-dr-yeadon-request-a-stop-of-all-corona-vaccination-studies-and-call-for-co-signing-the-petition/; accessed July 22, 2024). This story exploded on social media and caused significant concerns among women of childbearing age. ⁸⁰ The stated risk of infertility was related to purported cross-reacting immune responses between SARS-CoV-2 sequences and the human protein syncytin-1. Syncytin-1 is expressed in a wide assortment of tissues, including in syncytiotrophoblasts, and is essential for the formation of the placenta. ⁷³ This led to a concern that immunization targeting SARS-CoV-2 spike protein antigens would elicit immune responses against syncytin-1 and cause immune-mediated abortion and infertility (https://www.reuters.com/investigates/special-report/health-coronavirus-vaccines-skeptic/; accessed July 22, 2024). The SARS-CoV-2 S glycoprotein antigens encoded by the RNAs in the Pfizer-BioNTech and Moderna COVID-19 vaccines both have a 5 amino acid motif that shares 4 amino acids with syncytin-1 (sequence VVNQN in SARS-CoV-2 vs VVLQN in syncytin-1). This sequence in syncytin-1 is not available to antibodies, as it is in the protein’s transmembrane domain. Further, the sequence is too short to, by itself, constitute a T-cell epitope. ⁵²

Based on a nonclinical rat biodistribution study that identified LNP lipid at <0.1% of the total dose in the ovary, the possibility of anti-spike immune responses directed against protein expressed by mRNA vaccines in the ovary was raised. Specifically, a social media post on platform X (https://twitter.com/erin_bsn/status/1399036029059796993; accessed July 22, 2024) included images from the Pfizer-BioNTech PMDA emergency use authorization filing, and biodistribution to the ovary was highlighted (for more information on this misinformation string, see https://www.respectfulinsolence.com/2021/06/14/covid-19-vaccines-and-female-infertility-a-lie-that-never-dies/; accessed July 22, 2024). In fact, the data only demonstrated distribution of a labeled lipid, not of vaccine mRNA, and there was no evidence that the lipid-induced inflammation or that spike protein was expressed and resulted in immune-mediated cell injury. ⁷⁴ Similar biodistribution data were reported by Ci et al. ²⁶ Nonclinical developmental and reproductive toxicity studies ¹¹ and real-world clinical data have supported the vaccine’s safety in women of childbearing potential, including in pregnant women (https://www.fda.gov/media/155931/, summary basis of approval for Spikevax; accessed July 22, 2024).^61,62 Importantly, during the pandemic, women administered the mRNA COVID-19 vaccines had a lower incidence of pregnancy complications and fetal deaths than unvaccinated women, ²⁶ thus underscoring that the vaccine’s use during pregnancy had a favorable benefit-risk balance.

Increased Risk for Ischemic Stroke After Bivalent Vaccination

The release of Pfizer-BioNTech mRNA-LNP bivalent COVID-19 vaccine was reported to have a statistically significant signal (one-sided P < .005) for increased risk of ischemic stroke for those over the age of 65 years in days 1 to 21 after vaccination, as compared to days 22 to 42 after vaccination. This finding was not identified with the Moderna mRNA-LNP bivalent COVID-19 vaccine. The signal was consistently evident over the first 7 weeks after the vaccine roll-out and in individuals who had concomitantly received a high-dose influenza vaccine. However, after the 7-week timepoint, the risk signal disappeared. Detailed analysis revealed that the apparent increase in risk in days 1 to 21 after vaccination was the result of an unusually low incidence of ischemic stroke 22 to 42 days after vaccination in vaccinated individuals. Importantly, there was no increased risk of ischemic stroke in the first 21 days after vaccination when compared to age-matched non-vaccinated individuals (FDA 178th Meeting of Vaccines and Related Biological Products Advisory Committee,  1/26/2023;  https://www.youtube.com/watch?v=ZjULNuSYfd0; accessed July 22, 2024).

Other considerations included potential bias from the small number of individuals vaccinated for both COVID-19 and influenza who developed ischemic stroke. Other suggested potential confounding effects discussed at that meeting included the possibility that early adopters of the bivalent vaccine were in the higher cardiovascular risk group. Regardless, there is no evidence of an increased risk of ischemic stroke in adults 65 years of age or older after administration of the mRNA-LNP COVID-19 vaccines.

Vascular Incidents and Thrombocytopenia

Some concerns have been raised related to an increased risk of vascular incidents or thrombocytopenia with the COVID-19 mRNA vaccines https://www.factcheck.org/2022/12/scicheck-social-media-posts-misrepresent-fdas-covid-19-vaccine-safety-research/; accessed July 22, 2024). However, unlike the adenovirus-based COVID-19 vaccines, no increased risk has been identified with the COVID-19 mRNA vaccines. Adenovirus vector-based COVID-19 vaccines have been widely reported to increase the risk of thrombotic thrombocytopenia syndrome (TTS), particularly in women of childbearing age. This phenomenon is termed vaccine-induced thrombotic thrombocytopenia (VITT). TTS and VITT commonly present as deep vein thrombosis, cerebral venous sinus thrombosis, or pulmonary embolism.^33,36 VITT occurs ~5 to 30 days after vaccination at an incidence of up to 16 cases per million doses (ChAdOx1 nCoV-19) ⁴⁰ and is associated with high serum levels of anti-platelet factor 4 (PF4) antibodies. The anti-PF4 antibodies bind to and activate platelets. It has been reported that up to 5-6% of donated blood from healthy individuals have anti-PF4 antibodies, suggesting a high incidence within the population. ⁴² Venous thromboembolism has been reported to occur unassociated with vaccination in 1-2 individuals per million ⁵¹ annually. For this reason, the observed occurrence of venous thromboembolism within days to weeks of Adenovirus vector-based COVID-19 vaccination is considered relatively high. Although there is a case report of TTS with anti-PF4/heparin IgG after vaccination with an mRNA-LNP COVID-19 vaccine, no clinical signal for VITT has been identified. ⁶⁴

Development of “COVID Toes” and Cosmetic Filler Reactions With mRNA-LNP COVID-19 Vaccination

The term “COVID toes/fingers” refers to the development of cutaneous lesions of the fingers and toes, primarily in children, after exposure to SARS-CoV-2. These lesions resemble acral pernio (aka chilblains). A cause for this lesion is not clear but it has been hypothesized to be a manifestation of a robust immune response to SARS-CoV-2. ⁴⁶ There are case reports that suggest a link between the development of COVID toes and fingers and vaccination with COVID-19 vaccines, including adenoviral-vectored and mRNA-LNP vaccines.^1,56 Although these lesions may reflect an immune response to the spike protein, they are more likely related to the immune activation that follows immunization.

Facial swelling following mRNA-LNP COVID-19 vaccine administration has been reported in patients who had prior use of injectable cosmetic fillers. This finding was identified in the clinical trials for the Moderna mRNA vaccine. These responses may be a delayed hypersensitivity reaction to the filler because of immune activation by the vaccine. Such responses have also been identified after viral infections and flu vaccination. ⁸⁴ Most fillers contain PEG. Thus, anti-PEG immune responses may contribute to the AE associated with cosmetic fillers. Fortunately, these events are rare, minor, and self-limiting, and they should not limit vaccination against SARS-CoV-2.

mRNA Integration Into the Genome

The concern that the mRNA vaccine sequences might be reverse transcribed into the genome of those vaccinated has circulated in some scientific and non-scientific circles. Although retroviral reverse-transcriptase has been discussed as a possible source of integration of vaccine mRNA sequences into the genome, there is no evidence that such events have occurred. Vaccine mRNA could, theoretically, be reverse transcribed and potentially inserted in the genome via long interspersed element-line 1 (L1) retroelements. L1 is the only independently functioning retrotransposon in humans. Although retrotransposition-competent L1s (RC-L1) preferentially transpose their own (L1) mRNA in cis, they can theoretically target any mRNA with a polyA tail in trans. ³⁰ Retrotransposition in trans may occur with viruses and with endogenous mRNAs.

A recent publication in PNAS demonstrated that Pfizer-BioNTech COVID 19 vaccine exposure increased L1 expression in human liver cancer cells in vitro and that vaccine cDNA could be identified within 6 hours of exposure. ² Attention to this article engendered fear that getting the mRNA vaccine would permanently change host DNA, and potentially risk functional mutations (Science blog debunking SARS-COV-2 integration into genome https://www.science.org/content/blog-post/integration-human-genome; accessed July 22, 2024). The studies that triggered these concerns were performed in a cancer cell line; L1 expression is higher in cancer cells compared to non-cancer cells, ⁵⁵ and L1 retroelements acting in trans (i.e., acting on non-L1 mRNAs) are very uncommon. ⁹¹ Currently, there is no evidence that mRNA vaccine sequences have been incorporated into the genomes of vaccine recipients. Further, the low incidence of L1 retroelements acting in trans on non-L1 mRNA, low L1 expression in non-cancer cells, low exposure of cells to the vaccine mRNA, and cellular degradation of vaccine mRNA suggest a very low risk for retrotransposition of mRNA from the vaccines into the genome.

Prolonged Vaccine mRNA and Spike Antigen Expression in Tissues

Recent publications have identified the persistence of mRNA-LNP vaccine mRNA and spike antigen in the plasma and draining lymph nodes of humans after vaccination. This persistence is consistent with nonclinical study data on the biodistribution of mRNA-LNP mRNA and its antigen product in mice, rats, and rabbits. Nonclinical studies assessing biodistribution were evaluated using an mRNA-LNP in which luciferase sequences replaced the spike nucleic acid sequences in the mRNA. Using RT-qPCR, mRNA was cleared from the injection site and draining lymph nodes by 7 days after administration, but was identified in the plasma up to 14 days after administration in rabbits and rats.^14,25 The delivered mRNA was also present in an assortment of other tissues, but mostly only up to ~24 to 72 hours after administration. The mRNA-expressed luciferase protein was evident at the injection site/draining lymph nodes up to 9 days after administration in mice. ²¹ Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), the protein was detected at very low levels in most tissues at all timepoints, except liver, where initial protein levels were high, but rapidly dropped off and were at negligible levels at 7 days after administration.^14,25 The liver data for mRNA and protein expression are consistent with the clearance kinetics of the lipid. ²⁵ The retention of antigen in the draining lymph nodes after vaccination is believed to be beneficial for effective anti-microbial adaptive immune responses.^39,63

In humans, one study in patients with chronic hepatitis C found that, after administration of a COVID-19 mRNA-LNP vaccine, RNA sequence fragments could be demonstrated by RT-qPCR in the plasma of 9% of the individuals tested (n = 108) from 1 to 28 days after vaccination. ¹⁸ A proteomic study using mass spectrometry to identify vaccine-specific PP-spike fragments demonstrated vaccine antigen fragments in the blood of some individuals longer than 30 days after mRNA-LNP vaccination. ¹³ Vaccine-specific spike proteins have been reported in circulating exosomes in people, peaking at 14 days after vaccination and dropping to negligible levels by 16 weeks after vaccination. ⁹ In draining lymph nodes, core biopsies from individuals vaccinated with a COVID-19 mRNA-LNP vaccines have demonstrated both vaccine RNA and vaccine antigen up to 60 days after vaccination in some individuals using in situ hybridization and immunohistochemistry, respectively. The vaccine RNA was localized primarily within germinal centers, while the antigen was identified around germinal centers, putatively within dendritic cells. ⁷⁵ The authors suggested that this vaccine retention contributed to strong T-cell immunity against COVID-19.

In humans, it is well known that vaccine virus shedding occurs after immunization with modified live attenuated viral vaccines, such as the widely used measles, mumps, rubella vaccine and the vaccinia virus vaccine. Measles attenuated virus vaccine shedding has been detected in the urine up to 2 weeks after vaccination ⁷⁸ and in the lungs up to 800 days after vaccination. ⁵⁷ Retention of antigen has also been reported for protein subunit-based vaccines in nonclinical studies. In fact, it has been demonstrated that antigen is specifically retained within the germinal center of lymph nodes, and that this retention is important to robust immune responses. ⁶ In mice, antigen retention in the draining lymph nodes after vaccination may be as long as 5 weeks. Duration of retention depends on the physicochemical characteristics of the protein and the adjuvant with which it is formulated.^17,29,50 For example, antigen formulated with the adjuvant MF59, which is a component of some influenza vaccines, is evident in the draining lymph nodes of mice up to 7 days after administration (duration of the study). Another study in mice demonstrated vaccine antigen retention in the draining lymph nodes up to 3 weeks after administration. ⁸⁵ To date, no studies of antigen retention for licensed protein subunit vaccines have been performed in humans. Given the consistent retention of protein antigens in nonclinical species, it is likely that this also occurs in humans.

In summary, retention of antigen within the germinal centers of lymphoid organs is considered a desirable characteristic of a vaccine. Researchers have proposed that this prolonged exposure to vaccine antigens is important in generating a strong humoral and cellular immune response to protect against pathogens.^{6,13,28,53,75} Therefore, the presence and retention of vaccine mRNA and antigen within draining lymph nodes in individuals administered mRNA-LNP COVID-19 vaccines is likely beneficial in that it may contribute to the robust immune response the vaccine generates for protection against COVID-19.

Conclusion

Over the 3 years since the first mRNA-LNP vaccine against COVID-19 was authorized, much of the angst about the risks has diminished. However, the mythology of danger remains pervasive through social media and even within the medical community. Although there are clearly identified risks associated with the vaccines, such as myo- and pericarditis in young men, the overall risks are remarkably low, and the value of vaccination in reducing COVID-19-associated illness and death has far outweighed these risks. As mRNA vaccine technology evolves, modifications are likely to be made to decrease vaccine-associated AE by reducing reactogenicity and systemic immune stimulation, while retaining antigen-specific immunogenicity. Through the pandemic response, mRNA vaccine technology has already improved human health, and the technology has the potential to accomplish further, future improvements in human and animal health. We in the scientific and medical community should continue to educate the public and our colleagues on the value of vaccination and to continue to separate truth from fiction.