Evidence Review of the Adverse Effects of COVID-19 Vaccination and Intramuscular Vaccine Administration (2024)

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7 Myocarditis, Pericarditis, and COVID-19 Vaccines This chapter describes the potential relationship between COVID-19 vaccines and myocarditis and pericarditis (see Box 7-1 for all conclusions in this chapter). BOX 7-1 Conclusions for Myocarditis and Pericarditis Conclusion 7-1: The evidence establishes a causal relationship between the BNT162b2 vaccine and myocarditis. Conclusion 7-2: The evidence establishes a causal relationship between the mRNA-1273 vaccine and myocarditis. Conclusion 7-3: The evidence is inadequate to accept or reject a causal relationship between the Ad26.COV2.S vaccine and myocarditis. Conclusion 7-4: The evidence is inadequate to accept or reject a causal relationship between the NVX-CoV2373 vaccine and myocarditis. Conclusion 7-5: The evidence is inadequate to accept or reject a causal relationship between the BNT162b2 vaccine and pericarditis without myocarditis. Conclusion 7-6: The evidence is inadequate to accept or reject a causal relationship between the mRNA-1273 vaccine and pericarditis without myocarditis. Conclusion 7-7: The evidence is inadequate to accept or reject a causal relationship between the Ad26.COV2.S vaccine and pericarditis without myocarditis. Conclusion 7-8: The evidence is inadequate to accept or reject a causal relationship between the NVX-CoV2373 vaccine and pericarditis without myocarditis. PREPUBLICATION COPY—Uncorrected Proofs 

200 VACCINE EVIDENCE REVIEW BACKGROUND Myocarditis Myocarditis is defined as inflammation of the myocardium with or without necrosis (Cooper, 2009), and the gold standard for diagnosis is based on endomyocardial biopsy and established histologic, immunologic and immunohistochemical criteria (Caforio et al., 2013; Matsumori, 2003) based on the position statement of the Working Group on Myocardial and Pericardial Diseases of the European Society of Cardiology (ESC) (Van Linthout and Tschöpe, 2018). For cases where a biopsy is not obtained, which is typical in the United States, the diagnosis can be made based on cardiac magnetic resonance imaging (MRI) (Ferreira et al., 2018). Cardiac MRI provides strong evidence for myocarditis based on a combination of T2- and T1-based markers that indicate cardiac edema as a sign of myocardial inflammation (Ferreira et al., 2018) in patients with the classical clinical findings associated with otherwise unexplained troponin elevation. No approved imaging modalities directly detect cardiac inflammation. A clinical definition of SARS-CoV-2-induced myocarditis has been proposed that includes new or worsening clinical symptoms and one or more of the following: arrhythmias on electrocardiogram, cardiac dysfunction using echocardiography, or cardiac magnetic resonance imaging indicative of myocarditis after infection (Heidecker et al., 2022; Tschöpe et al., 2021). Most individuals develop acute myocarditis symptoms within a few days to 2 weeks after a viral infection (Cooper, 2009)and lymphocytic myocarditis is the most common form of myocarditis in the post viral settings. Myocarditis has a variable presentation, ranging from subclinical disease to fatigue, chest pain, new-onset heart failure, cardiogenic shock, and sudden death (Cooper, 2009) and it is a common cause of sudden cardiac death in young adults. In cases of myocarditis following COVID-19 vaccination, signs and symptoms have been similar to those associated with other forms of myocarditis (Heidecker et al., 2022). However, the prognosis for myocarditis after COVID-19 vaccination appears to be much less severe. In a study of nearly 4 million residents of Hong Kong, only one death occurred (1%) among 104 cases of postvaccination myocarditis, compared with 84 deaths (11 percent) among 762 cases of viral infection-related myocarditis (HR 0.08, 95% CI 0.01–0.57) (Lai et al., 2022). In a surveillance study of cases of myocarditis related to COVID-19 vaccination in the United States reported to VAERS, among 484 hospitalizations there were no deaths, and evidence of ongoing myocarditis at follow-up cardiac MR was uncommon (13 percent) (Kracalik et al., 2022). Myocarditis is classified based on histological findings, including lymphocytic (the most common form in Europe and the United States), fulminant, eosinophilic, and giant cell (Caforio et al., 2013). Myocarditis can also be described by presumed causes, including viral, autoimmune, or other causes (Ball et al., 2019). Most patients with lymphocytic myocarditis recover fully, but some may develop dilated cardiomyopathy (DCM) several weeks to months later and progress to chronic heart failure that may need a heart transplant (McNamara et al., 2011; Schultheiss et al., 2019; Tschöpe et al., 2021). Fulminant, eosinophilic, and giant cell myocarditis are rare, result in a more severe clinical course, and have a greater risk of sudden death (Abston et al., 2012a; Ammirati et al., 2019; Cooper et al., 1997; Maleszewski et al., 2015). Pediatric myocarditis tends to be more fulminant (Law et al., 2021). PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 201 Pericarditis Pericarditis is defined as inflammation of the pericardium, the fibroelastic sac that surrounds the heart, according to the WHO classification (Adler et al., 2015). The major clinical manifestations and diagnostic criteria include chest pain, which is typically sharp and pleuritic, pericardial friction rub, electrocardiogram changes, and pericardial effusion (Chiabrando et al., 2020; Imazio et al., 2015). The diagnosis is typically made based on clinical signs and symptoms, which in many cases can be challenging. Acute myocarditis and pericarditis frequently co-occur in clinical practice and animal models of viral or autoimmune myocarditis and are often referred to as “myopericarditis” (primary myocarditis phenotype) or “perimyocarditis” (primarily pericarditis phenotype) depending on the primary clinical symptoms. The etiology is similar for both myocarditis and perimyocarditis, with viral infections being the predominant causative agent, including coxsackieviruses, influenza and SARS-CoV-2 (Aljohani et al., 2022; Fairweather et al., 2023a). Because of the challenges in distinguishing myocarditis alone from myocarditis with features of pericarditis, and because of the clinical and prognostic importance of myocarditis, we included both in our review of the evidence for myocarditis related to COVID-19 vaccines. Separately, we reviewed evidence for the potential effect of COVID-19 vaccines on acute pericarditis alone (i.e. pericarditis without myocarditis). In this chapter, the term pericarditis and our conclusions about pericarditis refer only to pericarditis without myocarditis. EPIDEMIOLOGY OF MYOCARDITIS AND PERICARDITIS The latest Global Burden of Disease (GBD) report estimates the worldwide age- standardized morbidity and mortality of myocarditis combined with all types of cardiomyopathy in men versus women prior to the COVID-19 pandemic to be 6.5 per 100,000 years lived with disability (YLDs) (95% UI: 4.3–9.3 per 100,000 YLDs) and 4.2 per 100,000 YLDs (95% UI: 2.8 –6.0 per 100,000 YLDs) and 148.9 per 100,000 years of life lost (YLLs) (95% UI: 120.2–168.7 per 100,000 YLLs) and 71.4 per 100,000 YLLs (95% UI: 61.0–79.9 per 100,000 YLLs), respectively. After adjustment for a 7-day risk period, estimated background or expected rates of myocarditis and pericarditis after COVID-19 vaccination in the United States are 0.2 and 1.4 per 1 million people, respectively (Pillay et al., 2022). Similar to myocarditis, men aged 16–65 have a greater risk of acute pericarditis than women (Fairweather et al., 2023a; Kytö et al., 2014). Prevalence refers to the frequency of a condition at a given point in time, which cannot be used to estimate a background rate of an event during a unit of time (e.g. incidence). For myocarditis only, the prevalence of myocarditis in individuals 35–39 years of age is 6.1 per 100,000 (95% UI: 4.2–8.7 per 100,000) in men and 4.4 per 100,000 (95% UI: 3.0–6.3 per 100,000) in women. MECHANISMS Myocarditis and Pericarditis Most of the understanding of the pathogenesis or mechanisms of myocarditis and myopericarditis comes from animal models, where pericarditis always occurs with myocarditis. Myocarditis can be caused by many infectious and noninfectious agents, such as viruses, bacteria, parasites (Trypanasoma cruzi leading to Chagas disease) and toxins, including PREPUBLICATION COPY—Uncorrected Proofs 

202 VACCINE EVIDENCE REVIEW anthracyclines, ethanol, arsenic, cocaine, and heavy metals (Jain et al., 2022). Myocarditis is often considered to result from direct damage by infections or toxins, but it may also involve autoimmune mechanisms, perhaps triggered by infections/toxins (Fairweather et al., 2001; Root- Bernstein and Fairweather, 2014; Root-Bernstein et al., 2023). The primary mouse models of myocarditis are viral, autoimmune, or both (Ciháková et al., 2004; Fairweather et al., 2012; Poli et al., 2020), and most use male mice. Regardless of the animal model, common immune mechanisms have been identified in all models that increase the severity of the condition (myocardial inflammation), particularly in males (Fairweather et al., 2023a). All models in male mice have shown that the inflammatory infiltrate during peak acute myocarditis consists of a mixed infiltrate of predominantly macrophages, with fewer T and B cells and small numbers of natural killer cells, dendritic cells, mast cells and other cell types (Ciháková et al., 2008; Frisancho-Kiss et al., 2007; Huber and Job, 1983; Liu et al., 2013). In contrast, female mice have far less cardiac inflammation (Fairweather et al., 2023a; Frisancho-Kiss et al., 2007; Huber and Job, 1983). In animal models of myocarditis, male mice have more mast cells and macrophages than females (Frisancho-Kiss et al., 2006a; Frisancho-Kiss et al., 2007; Frisancho-Kiss et al., 2009). Similar histologic findings are also observed in biopsies from patients with myocarditis (Baumeier et al., 2022; Fairweather et al., 2014, 2023b; Heidecker et al., 2022; Lüscher and Akhtar, 2022). Mechanisms that drive sex differences in young male white-genetic-background mice (i.e., BALB/c, A/J) have been well described (Fairweather et al., 2013, 2023b; Huber and Job, 1983). Increased viral myocarditis in male mice is associated with elevated numbers of mast cells and macrophages in the heart that express complement receptors (CR3, also called “CD11b,” and C3aR and C5aR) and Toll-like receptor 4 (TLR4)/inflammasome (Cooper et al., 2010; Fairweather et al., 2003; Frisancho-Kiss et al., 2007). Only white-background mice are susceptible to autoimmune myocarditis/perimyocarditis and progressing to DCM (Fairweather et al., 2001; Neu et al., 1987). This is due to higher levels of mast cells in the peritoneum, spleen, and heart that drive the immune response toward a proinflammatory and profibrotic immune response after infection or in response to self-antigens (Fairweather et al., 2004a). Evidence of the importance of the complement and TLR4/inflammasome pathways in the pathogenesis of myocarditis was recently illustrated when microRNA (in extracellular vesicles (EVs)) targeting these specific pathways was able to prevent myocardial inflammation in a viral animal model (Beetler et al., 2023a). EVs, with their receptors and content, can be proinflammatory or immunoregulatory (Beetler et al., 2023a), which is important when discussing potential vaccine mechanisms, particularly mRNA vaccines which consist of mRNA packaged in a lipid nanoparticle. Activation of the innate immune response is critical for the induction and progression of viral and autoimmune myocarditis. Key innate pathways include complement pathways, and TLR4/ inflammasome pathways are known to play a role in animal models and human myocarditis (Cooper et al., 2010; Fairweather et al., 2003, 2006; Frisancho- Kiss et al., 2007; Roberts et al., 2013; Tschöpe et al., 2017). The majority of immune cells in the heart of male mice and humans during acute myocarditis are CR3 positive activated mast cells, and macrophages (Fairweather et al., 2014; Frisancho-Kiss et al., 2007). TLR4 signaling works through the inflammasome (NLRP3) to produce the cytokines interleukin (IL)-1β and IL-18 during myocarditis in males (Fairweather et al., 2003; Tschöpe et al., 2017). IL-1β increases inflammation and remodeling, leading to cardiac fibrosis and then DCM in susceptible strains of mice (Coronado et al., 2012; Fairweather et al., 2004b). IL-18 strongly induces interferon (IFN) γ responses that drive M1 macrophages and T helper (Th)1-type immune responses in male mice with viral myocarditis and are needed to control viral replication (Frisancho-Kiss et al., 2006b; PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 203 Toldo and Abbate, 2023). TLR2 has also been found to be important in mouse models of myocarditis; TLR2 signaling can be activated by cardiac myosin antigens and promote autoimmune T helper 17 (Th17)-type immune responses that contribute to remodeling and progression to DCM in male mice (Baldeviano et al., 2010; Myers et al., 2016; Roberts et al., 2013). Susceptibility to myocarditis in animal models is associated with mast cells, which are abundantly present in allergy-prone white-background mouse strains, that promote inflammation, pericarditis, and fibrosis, leading to DCM (Abston et al., 2012b, 2013; Afanasyeva et al., 2001; Coronado et al., 2012; Fairweather et al., 2004a). Mouse strains with very few mast cells, such as C57BL/6 (B6) or B10, do not develop autoimmune myocarditis or progress to DCM after acute myocarditis (Abston et al., 2012a, 2013; Afanasyeva et al., 2001; Fairweather et al., 2004a). Mast cells are the first antigen-presenting cells to respond to virus in the autoimmune coxsackievirus B3 (CVB3) model of myocarditis in BALB/c mice, where they upregulate CD11b/CR3 and TLR4 (Frisancho-Kiss et al., 2006b, 2007). Mast cells are critical in driving macrophages to an alternatively activated M2 phenotype during acute myocarditis, where they work together to increase cardiac inflammation and remodeling/fibrosis in males (Coronado et al., 2012). Mast cell degranulation is associated with pericarditis/perimyocarditis in mice (Bruno et al., 2019, 2021; Fairweather et al., 2004a, 2006). Almost no research has examined mechanisms underlying pericarditis in the absence of myocarditis because in these animal models the two are always present together. The efficacy of colchicine in treating patients with pericarditis (Imazio et al., 2005a, 2005b, 2011, 2013, 2014) points to an important role for the NLRP3 inflammasome in its pathogenesis. The NLRP3 inflammasome cleaves caspase-1, leading to the production of IL-1β and IL-18 (Martinon et al., 2006). TLR4 produces proIL-1β and proIL-18 that caspase-1 cleaves, leading to active IL-1β and IL-18 that promote soluble ST2 and IL-6 levels, which are serum biomarkers for all forms of heart failure, including myocarditis (Coronado et al., 2019; Potere et al., 2023). Colchicine also impairs neutrophil adhesion to vascular endothelium, increases leukocytic cyclic adenosine monophosphate levels, and inhibits IL-1β and TNF production from macrophages (Potere et al., 2023). SARS-CoV-2-Associated Myocarditis and Pericarditis SARS-CoV-2 infection dramatically increased the reported incidence of myocarditis and pericarditis. The overall U.S. incidence of myocarditis from SARS-CoV-2 infection has been estimated in a study by the Centers for Disease Control and Prevention (CDC) at around 150 cases per 100,000 versus 9 cases per 100,000 in non-COVID-19 cases during the same time period (Boehmer et al., 2021). A separate study in the United States and Europe estimated 240 and 410 cases per 100,000 of definite/probable or possible myocarditis, respectively (Ammirati et al., 2022). These data indicate around a 15+-fold increased risk of developing myocarditis from SARS-CoV-2 infection compared to pre-COVID-19 rates. The signs and symptoms of COVID-19–associated myocarditis are very similar to other forms. Much like in other causes of myocarditis, immunohistochemistry performed on biopsies found a predominant infiltrate of CD68+ macrophages (CD11b is not typically assessed in clinical biopsies) with fewer T cells—the same as animal models of myocarditis (Basso et al., 2020; Heidecker et al., 2022; Lovell et al., 2022). Thus, COVID-19-associated myocarditis is histologically similar to other forms of myocarditis. PREPUBLICATION COPY—Uncorrected Proofs 

204 VACCINE EVIDENCE REVIEW SARS-CoV-2 infection causes an immune response (and sex differences in the immune response) that is very similar to that which has been found to drive myocarditis/perimyocarditis in animal models. For example, most studies of COVID-19 reported more male than female patients and higher numbers of circulating neutrophils and macrophages; female patients had more T cells (Lau et al., 2021; Takahashi et al., 2020). Male patients with COVID-19 were also reported to have higher circulating levels of ferritin, C-reactive protein (CRP), IL-6, IL-8, and IL-18 (Lau et al., 2021; Takahashi et al., 2020). COVID-19 has been documented to strongly complement and activate other innate immune pathways, such as TLR4 and the inflammasome, which leads to increased IL-1β and IL-18 levels (Amin et al., 2022; Carvelli et al., 2020; Huber et al., 2021; Toldo et al., 2021). TLR4 signaling is key in driving proinflammatory responses associated with COVID-19 and contributes to an increased Th1-type immune response because IL-18 (and IL-1β) strongly induces IFNs (Cai et al., 2000; Frisancho-Kiss et al., 2006a). T cell immunoglobulin mucin (Tim-3) is a receptor that is upregulated on mast cells and macrophages in female patients during viral myocarditis that inhibits T cell responses and is associated with increased IL-10 release from alternatively activated M2 macrophages, conferring protection (Frisancho-Kiss et al., 2007, 2009). Tim-3 and IL-10 upregulation are also observed in COVID- 19 and thought to contribute to the immunosuppressive state (Shahbazi et al., 2021). Angiotensin-converting enzyme II (ACE2) had been identified as the receptor for SARS- CoV-2 (Lan et al., 2020). The spike protein binds ACE2 and is cleaved by type II transmembrane serine protease 2 (TMPRSS2), facilitating viral entry into the cytosol, and is also required for entry into cells (Hoffmann et al., 2020), this process is detailed in Chapter 2. A number of cell types in the heart and immune cells express ACE2, including cardiomyocytes, pericytes (located around vessels in the heart), fibroblasts, endothelial cells, and macrophages and mast cells (Chen et al., 2020; Hikmet et al., 2020; Theoharides, 2021). Other accessory proteins (i.e., neuropilin-1 receptor/NRP1, CD147, integrin α5β1, and cathepsin B/L) are also needed for SARS-CoV-2 infection and found on mast cells (Theoharides, 2021). As described earlier, mast cells and macrophages are found in much higher levels in males than females. Thus, the spike protein activating mast cells by ACE2 is a potential mechanism that contributes to the development of myocarditis following COVID-19 in males (Fairweather et al., 2023b). COVID-19 Vaccine–Associated Myocarditis/Pericarditis Similar to other forms of myocarditis (Halsell et al., 2003; Roth et al., 2020), myocarditis after COVID-19 vaccination has been reported to occur most frequently in White male patients aged 16–30 and primarily in individuals under 50 (Straus et al., 2023), with few reports past age 50. The findings of a similar sex, age, and race/ethnicity for myocarditis of all types, including vaccine-associated cases, suggests similar mechanisms are at play. Most vaccine-associated cases of myocarditis were reported after vaccination with messenger ribonucleic acid (mRNA) platform vaccines, specifically after the second dose. The second dose was already known from healthy volunteers to promote a robust autoantibody, cell- mediated and innate immune response (Arunachalam et al., 2021). These vaccines contain modified mRNA that encodes the spike protein encapsulated by lipid nanoparticles (LNPs) that are similar in structure and composition to extracellular vesicles (EVs). The mRNA vaccines do not contain live or heat-inactivated virus. Cases of myocarditis were also reported after adenoviral vector SARS-CoV-2 vaccines (Husby et al, 2021), but by far most cases were associated with mRNA platforms (Diaz et al., 2021). PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 205 When biopsies were obtained from patients with myocarditis after COVID-19 vaccination, the immune infiltrate was found to resemble classic lymphocytic myocarditis, with macrophages and T and B cell infiltrates (Baumeier et al., 2022; Fairweather et al., 2023b; Heidecker et al., 2022; Lüscher and Akhtar, 2022). The authors of case reports and small case series often identified the vaccine as the probable cause because people developed myocarditis shortly after receiving it (Baumeier et al., 2022). Some studies also carefully tested for viral infections to eliminate that as a cause (Baumeier et al., 2022). That similar infiltrates occur in vaccine-associated cases of myocarditis as in that from other causes and animal models suggests a common mechanism. Evidence that mRNA vaccine components may increase complement and TLR4/inflammasome/IL-1β immune responses and activate mast cells comes from a number of studies. LNPs have been used in mRNA vaccine platforms to prevent mRNA degradation, facilitate mRNA delivery, and stimulate the immune response, but have also been linked with complement activation-related mast cell hypersensitivity reactions and TLR-mediated release of proinflammatory cytokines (Halamoda-Kenzaoui and Bremer-Hoffmann, 2018; Kauffman et al., 2016; Lamerton et al., 2022; Power et al., 2022; Samaridou et al., 2020; Seneff et al., 2022). A number of components in the mRNA LNPs, including polyethylene glycol, cholesterol, and saponin, are well known to activate mast cells and a primary reason individuals develop allergic responses (Hou et al., 2021; Tsilingiris et al., 2022). Ndeupen et al. (2021) found that the mRNA platform’s LNP component was highly inflammatory. They observed significant upregulation of gene transcripts associated with activating the TLR4/inflammasome, such as Nlrp3, IL-1β, and IL-6, and confirmed increased IL-1β and IL-6 levels in mice. Overall, these findings indicate that mRNA vaccines have contents that can activate the precise pathways known to drive myocarditis in mice in a sex- and background-specific manner (elevated in males and white-background mice with many mast cells), including complement and TLR4/inflammasome/IL-1β. Regardless of vaccine platform, all COVID vaccines include or lead to the production of the spike protein, which binds ACE2. As noted previously, ACE2 is expressed on antigen presenting cells such as mast cells and macrophages and binding may activate an innate immune response (Fairweather et al., 2023b). However, Yonker et al. (2023) found that in patients with vaccine-associated myocarditis, levels of circulating spike protein remained elevated in the blood for at least 3 weeks after vaccination, instead of the protein being quickly cleared. No healthy controls had detectable free spike protein in their serum at any time after vaccination. The patients with vaccine-associated myocarditis also had elevated serum levels of IL-1β, IL-6, and other cytokines, suggesting a persistent innate proinflammatory response (Yonker et al., 2023). Thus, it is possible that persistent free spike protein may activate mast cells not only at the site of vaccination but also at other sites, including the heart, where mast cells are located at their highest levels at vessels and along the pericardium. Spike protein has been found in biopsies from the heart of patients with myocarditis after vaccination, indicating that it can reach, and deposit in, the heart (Baumeier et al., 2022). Additionally, several studies have reported that exosomes (a type of EV) leave cells after vaccination, enter the circulation, and express the spike protein on their surface (Bansal et al., 2021; Chaudhary et al., 2021; Seneff et al., 2022). They also found that circulating spike protein– expressing exosomes increased by a factor of 12 after the second vaccination. These exosomes may activate ACE2 on mast cells and macrophages contributing to the increased incidence of myocarditis reported after the second mRNA vaccination. Animal models of many autoimmune diseases require two injections to initiate diseases (Ciháková et al., 2004). One factor that may contribute to the much higher incidence of vaccine- PREPUBLICATION COPY—Uncorrected Proofs 

206 VACCINE EVIDENCE REVIEW associated myocarditis after the second vaccination is “trained immunity,” which is used to explain how innate immune cells mount a much higher response the second time they are exposed to an antigen, as long as the second exposure is not too long after the first. It has been revealed that the TLR4/inflammasome/IL-1β pathway is critical to developing this innate immunological memory (Moorlag et al., 2018). These findings provide a potential mechanism for how COVID-19 vaccines that use spike protein to induce an immune response may activate the precise immune pathways that are known to drive myocarditis. The risk may be increased by adding the lipid layer in mRNA vaccine platforms that has additional elements that may further activate these pathways. In support of this idea, patients with myocarditis after COVID-19 vaccination with mRNA in LNP, have been found to have immune responses associated with activation of the TLR4/inflammasome/IL-1β/IL-18 pathway. TLR4 expression on mast cells and macrophages could drive this response, and it is well established in animal models of viral and autoimmune myocarditis that inhibiting this pathway using therapies, such as mesenchymal stem cells or EVs, or drugs, such as colchicine, reduce myocardial and pericardial inflammation (Beetler et al., 2023a; Fairweather et al., 2003; Miteva et al., 2018; Pappritz et al., 2022). A case series by Frustaci et al. (2022) reported three cases of severe eosinophilic myocarditis after mRNA vaccination (eosinophils are activated by mast cells) in individuals who had experienced hypersensitivity/allergic reactions to mRNA vaccines. Another case report described a patient with Still’s disease and myocarditis after an mRNA vaccine (Hugues et al., 2022). Still’s disease is associated with elevated levels of IL-1β and IL-18, and treatment with IL-1β and IL-6 inhibitors was effective (Hugues et al., 2022). Another case report examined the immune response of a patient with myocarditis after mRNA vaccination and found elevated circulating levels of IL-18 (Won et al., 2022). Another case study of vaccine-associated myocarditis cases found antibodies against IL-1R antagonist (IL-1RA) in the serum of patients with myocarditis after mRNA vaccination, indicating activation/regulation of the IL-1β receptor pathway (Thurner et al., 2022). Overall, these several reports found consistent associations with inflammasome activation. Last, one animal study reported that the COVID-19 mRNA vaccine was able to induce myopericarditis in BALB/c mice (Li et al., 2022), though these animals did not demonstrate myocardial inflammation typical of myocarditis animal models. Mast cells are most concentrated in BALB/c mice along the pericardium, where they could be activated by the spike protein, leading to pericardial inflammation. Unfortunately, these investigators did not examine mast cell numbers or activation. However, pericardial mast cell activation is well known as a major driver of myocarditis/perimyocarditis in viral animal models, as described. Evidence of activation of this pathway was observed in male BALB/c mice given mRNA vaccines that had higher levels of IL-1β, indicating TLR4/inflammasome activation (Li et al., 2022). Overall, these findings provide a possible mechanism for how mRNA vaccines (and, to a lesser extent, other platforms) may activate complement and TLR4/inflammasome pathways on mast cells and macrophages to induce myocardial and pericardial inflammation. The potential role of mast cells in effects from COVID-19 vaccines has been reviewed in Fairweather et al. (2023b) and Theoharides (2021). PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 207 COVID-19 VACCINES: CLINICAL AND EPIDEMIOLOGICAL EVIDENCE The committee considered randomized controlled trials (RCTs) and observational studies to determine the relationship between COVID-19 vaccines and myocarditis and pericarditis. BNT162b2 RCTs, BNT162b2 A Cochrane Systematic Review of 41 RCTs of COVID-19 vaccines did not report findings for myocarditis or pericarditis (Graña et al., 2022). A Brighton Collaboration systematic review of serious adverse events after mRNA vaccination (Fraiman et al., 2022) reported on myocarditis/pericarditis in two large, Phase 3 placebo-controlled RCTs of BNT162b2 1 and mRNA-1273 2 but with no imbalance in the number of events and no inference about causality or association was attempted (Fraiman et al., 2022). The number of events in the Brighton Collaboration review does not align with the more detailed review of the trial results described from Food and Drug Administration (FDA) source materials. Next, the committee reviewed myocarditis/pericarditis events from published and unpublished reports of individual RCTs of BNT162b2. The primary unpublished data sources include the FDA advisory committee, emergency use authorization (EUA), and biologic license application materials, which reported individual counts of events in each arm of each trial and details about them. These counts sometimes changed with additional follow-up and clinical review, so counts from the most recent FDA review documents were used when possible. In the Phase 2/3 RCT C4591001, among individuals aged 16+ (n = 22,030), no myocarditis or pericarditis events were observed that were considered at least possibly related (FDA, 2020a, 2020b, 2021a). In the same RCT, among individuals aged 12–15 (n = 1,131), one myocarditis event and no pericarditis events were observed (FDA, 2021b, 2021c). In this trial, one 15-year-old boy in the placebo arm crossed over to receive open-label BNT162b2 at age 16, and 3 days later, he developed myocarditis; the FDA reviewer noted a “reasonable possibility that the myopericarditis was related to the vaccine administration due to the plausible temporal relationship” (FDA, 2021c). In the Phase 2/3 RCT C4591007, among children aged 5–11 (n = 3,109), no myocarditis or pericarditis events were observed that were at least possibly related (FDA, 2021c). In that same RCT, among children 6 months to 4 years (n = 3,013), no myocarditis or pericarditis events were observed (FDA, 2022a). The count of myocarditis and pericarditis events in these trials was so low (one myocarditis event across all trial populations) that no statistical inference could be made. The lack of a clear signal for myocarditis or pericarditis in these trials effectively excludes a large average increase in risk in broad populations studied but not the possibility of a causal effect that results in one case per tens of thousands of vaccine exposures. Observational Studies, BNT162b2 Because of the large number of observational studies on myocarditis and pericarditis after mRNA vaccines, the committee next reviewed findings from systematic reviews or meta- 1 Refers to the COVID-19 vaccine manufactured by Pfizer-BioNTech under the name Comirnaty®. 2 Refers to the COVID-19 vaccine manufactured by Moderna under the name Spikevax®. PREPUBLICATION COPY—Uncorrected Proofs 

208 VACCINE EVIDENCE REVIEW analyses of observational studies. Many of the systematic reviews had serious methodological limitations, including a failure to account for different study designs and especially differences in outcome surveillance and ascertainment methods, which can vary substantially by country and across health care systems and surveillance systems within a country. Rates of myocarditis tended to be lower in passive surveillance studies (i.e., spontaneous adverse event reporting) than in those that relied on diagnosis codes from health care encounters. Although passive surveillance (i.e., pharmacovigilance) studies suffer from reporting bias and are typically considered a weaker design than epidemiological studies with a well-enumerated population base and outcome identification through health care encounters, the likelihood of nondifferential misclassification of outcomes by exposure status is also high in studies nested within health care systems, given the high level of public awareness about myocarditis as a potential harm of some COVID-19 vaccines. Some pharmacovigilance studies used the most rigorous methods to identify outcomes, such as verifying cases by reviewing medical records and applying CDC clinical criteria for myocarditis (Oster et al., 2022). Confounding by age and sex was also a serious limitation in many studies. Because of the substantial evidence of effect modification of vaccine-related myocarditis risk by sex and age and possible confounding by these factors, the committee prioritized analyses that carefully accounted for both. The most comprehensive review of epidemiologic studies of myocarditis and pericarditis related to COVID-19 mRNA vaccines was A Living Evidence Synthesis by Canada’s Strategy for Patient-Oriented Research (SPOR), which provided detailed results stratified by age and sex for each mRNA vaccine from a protocolized review of the literature. Results from an interim review of the evidence were published in 2022 (Pillay et al., 2022), and more recent updates have been posted on the SPOR website (Update #4 on March 29, 2023) (Gaudet, 2023). This review reported incidences by age groups consistent with the age eligibility criteria in the registration clinical trials of the mRNA vaccines. The main findings from SPOR (Gaudet, 2023) are summarized in Table 7-1. PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 209 TABLE 7-1 Findings from Canada’s Strategy for Patient-Oriented Research Myocarditis Male Female Age Group Cases/million Certainty Studies Cases/million Certainty Studies 6m–4y <20 Low 1 <20 Low 1 5–11y <20 Moderate 6 <20 Low 7 12–17y 13–390 Moderate 16 1-50 Very low 16 18–29y 29–157 Moderate 14 2-37 Very low 12 18–39y 8–104 Moderate 12 <20 Low 9 mRNA-1273 Risk Compared to BNT162b2 Age Group (years) RR Certainty Studies RR Certainty Studies 6m–4y Same Low 1 Same Low 1 5–11 - - 0 - - 0 12–17 Higher Low 1 Uncertain 1 18–29 2–3x Moderate 6 2–3x Moderate 5 30–39 Higher High 5 Higher Low 5 Pericarditis Age Group Cases/million Certainty Studies Cases/million Certainty Studies 6m–4y - - 0 - - 0 5–11y Uncertain Very Low 1 Uncertain Very Low 1 12–17y <20 Low 2 <20 Low 2 18–24y Uncertain Very Low 2 <20 Low 2 25–39y <20 Low 2 Uncertain Uncertain 2 NOTES: Certainty refers to certainty of the risk estimates using Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) (Siemieniuk and Guyatt, 2024). Rates are excess incidence compared to background rate. Estimated background rate after vaccination is 0.2/million for myocarditis and 1.4/million for pericarditis. <20 per million reported when incidence rates from all studies reported as low. BNT162b2 refers to the COVID-19 vaccine manufactured by Pfizer-BioNTech under the name Comirnaty®. mRNA-1273 refers to the COVID-19 vaccine manufactured by Moderna under the name Spikevax®. m: month; RR, risk ratio; y: years. SOURCE: Gaudet, 2023. The key findings from the SPOR review were that BNT162b2 and mRNA-1273 likely increased the risk of myocarditis in male adolescents (12–17) and young adults (18–39) (Gaudet, 2023). Absolute risk estimates varied across studies by an order of magnitude, but even the highest reflect a low absolute risk. Nonetheless, the absolute risk estimates in certain male age groups appear to be orders of magnitude greater than the estimated background rate in the general population, making confounding or reporting bias an unlikely explanation. The risk in women, young children, and older adults may also be elevated, but the magnitude of the risk and the certainty of this finding are lower. The evidence reflected moderate to higher certainty that the risk of myocarditis is greater with mRNA-1273 compared with BNT162b2 in the older age groups, but risk associated with BNT162b2 remained elevated. The results from this review are consistent with results from several other systematic reviews. PREPUBLICATION COPY—Uncorrected Proofs 

210 VACCINE EVIDENCE REVIEW The evidence from the SPOR review on pericarditis without myocarditis was sparse (Gaudet, 2023). For example, no studies in children aged 0–4 were identified, and only a single study included those aged 5–11. With just two studies, the certainty was low for boys and girls aged 12–17, women aged 18–24, and men aged 25–39, with an estimated absolute risk of <20 cases per million in those age–sex groups. In addition to the SPOR review, Table 7-2 summarizes findings from some of the most informative individual epidemiological studies, prioritizing studies that had large numbers of vaccine-related myocarditis cases, used an appropriate control group, attempted to address confounding by factors that may be associated with the vaccine, and reported risk estimates stratified by sex and age. These studies ascertained myocarditis events after the first or second dose of the vaccine. All but one study relied on administrative data (diagnosis codes from health care encounters) to identify cases (Goddard et al., 2022a, 2022b). Validation studies have reported that the positive predictive value of myocarditis ICD-10 diagnosis codes for validated events is approximately 70 percent (Wu et al., 2023). Therefore, bias due to outcome misclassification, which may be differential, is a potential limitation of nearly all of these population-based studies. Collectively, these studies strongly suggested an increased risk of myocarditis associated with BNT162b2, although it is likely to be lower than with mRNA-1273. PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 211 TABLE 7-2 Selected Epidemiological Studies of Risk of Myocarditis Associated with BNT162b2 Vaccinated Author Age Setting Design Confounding Outcome Interval N Cases Results (95% CI) Myocarditis only All ages: RR 3.18 (1.65–6.12) Europe Bots et al. Self- ICD 12.4 5+ (4 Self-controlled 28d 72 (2022) controlled codes million Men: countries) RR 2.78 (1.53–5.07) Aged 12–29: RR 7.78 (2.58–23.5) Myocarditis/pericarditis Goddard et Vaccinated Record 5 to 39 VSD None 7d 2.6 Higher risk for males, al. (2022a) only review 101 million second dose, and 12–15 and 16–17 age groups Myocarditis and pericarditis Goddard et Self- Record 1.5 18– 39 VSD Self-controlled 7d 41 al. (2022b) controlled review million All ages RR 6.94 (3.57–14.13) Myocarditis only Higher risk for males, Europe Karlstad et Unvaccinat ICD 15.1 second dose, and 16–24 12+ (4 Adjustment 28d 220 al. (2022) ed control codes million age groups. countries) mRNA-1273 risk greater than BNT162b2 PREPUBLICATION COPY—Uncorrected Proofs 

212 VACCINE EVIDENCE REVIEW TABLE 7-2 Continued Vaccinated Author Age Setting Design Confounding Outcome Interval N Cases Results (95% CI) Myocarditis only RR 8.1 (6.7–9.9) Higher risk for males, Le Vu et al. Case- ICD 21.2 12– 50 France Adjustment 21d 405 second dose, and 18–24 (2022) control codes million age group. mRNA-1273 risk greater than BNT162b2 Myocarditis or pericarditis RR 3.39 (2.02–5.68) Massari et Self- ICD 2.4 Higher risk for males, al. 12– 39 Italy Self-controlled 21d 74 controlled codes million second dose, and 12–17 (2022) and 18–29 age groups. mRNA-1273 risk greater than BNT162b2 Myocarditis only RR 1.57 (1.28–1.92) Patone et Self- ICD 21.0 Higher risk for males, 13+ England Self-controlled 28d 243 al. (2022a) controlled codes million second dose, and <40 age group. mRNA-1273 risk greater than BNT162b2 NOTES: BNT162b2 refers to the COVID-19 vaccine manufactured by Pfizer-BioNTech under the name Comirnaty®. mRNA-1273 refers to the COVID-19 vaccine manufactured by Moderna under the name Spikevax®. d: days; ICD: International Classification of Diseases; RR: relative risk; SC: self-controlled. SOURCES: Bots et al., 2022; Corrao et al., 2022; Goddard et al., 2022a, 2022b; Karlstad et al., 2022; Le Vu et al., 2022; Massari et al., 2022; Patone et al., 2022a. PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 213 In contrast with myocarditis, few high-quality epidemiological studies of pericarditis without myocarditis were identified. Three found no increased risk, and two found an increased risk for BNT162b2. The relative risk estimates for pericarditis in the positive studies were much lower than the corresponding relative risk estimates for myocarditis. PREPUBLICATION COPY—Uncorrected Proofs 

214 VACCINE EVIDENCE REVIEW TABLE 7-3 Selected Epidemiological Studies of Risk of Pericarditis Associated with BNT162b2 Vaccinated Author Age Setting Design Confounding Outcome Interval N Cases Results (95% CI) Bots et al. 5+ Europe (4 Self- Self- ICD 28d 12.4 244 (2022) countries) controlled controlled codes million RR 0.90 (0.67–1.21) No increased risk Corrao et 12+ Italy Cohort vs. Adjustment ICD 28d 8.9 146 No increased risk al. (2022) unvaccinated codes million Karlstad et 12+ Europe (4 Unvaccinated Adjustment ICD 28d 15.1 267 Increased risk in males al. (2022) countries) control codes million RR 1.38 (1.1–1.74) and females RR 1.47 (1.05– 2.05) Higher RR under age 40 compared to 40 and over. mRNA-1273 risk greater than BNT162b2 Le Vu et 12–50 France Case-control Adjustment ICD 21d 21.2 288 RR 2.9 (2.3–3.8). al. (2022) codes million Increased risk under age 40 in males, under age 30 if females. Patone et 13+ England Self- Self- ICD codes 28d 17.0 134No increased risk al. (2022b) controlled controlled million NOTES: BNT162b2 refers to the COVID-19 vaccine manufactured by Pfizer-BioNTech under the name Comirnaty®. mRNA-1273 refers to the COVID-19 vaccine manufactured by Moderna under the name Spikevax®. d: days; RR: relative risk; SC: self-controlled. SOURCES: Bots et al., 2022; Corrao et al., 2022; Karlstad et al., 2022; Le Vu et al., 2022; Patone et al., 2022b. PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 215 Evidence from passive surveillance studies of potential harms was also considered. An analysis of cases of myocarditis related to mRNA vaccines in the Vaccine Adverse Event Reporting System (VAERS) corroborated the findings from the epidemiological studies and was considered just as informative for the causality assessment (Oster et al., 2022). Notably, the myocarditis cases in VAERS were validated by medical record review and the application of CDC clinical criteria, and the observed reporting rates were orders of magnitude greater than the estimated background rate. Specifically, the rate of validated cases was much higher in male than in female vaccinees, higher after the second dose, and highest in the 12–15, 16–17, and 18–24 age groups. PREPUBLICATION COPY—Uncorrected Proofs 

216 VACCINE EVIDENCE REVIEW TABLE 7-4 Reports to VAERS After mRNA-Based COVID-19 Vaccination That Met the CDC’s Case Definition for Myocarditis Within a 7-Day Risk Interval per Million Doses of Vaccine Administered Reported Cases of Myocarditis Within a 7-Day Risk Interval per Million Doses of Vaccine Administered (95% CI) Expected Cases of Age Vaccination with BNT162b2 Vaccination with mRNA-1273 Myocarditis in a 7- Group Day Risk Interval (years) First Dose Second Dose First Dose Second Dose per Million Doses Males 12–15 7.06 (4.88–10.23) 70.73 (61.68–81.11) – – 0.53 (040–0.70) 16–17 7.26 (4.45–11.86) 105.86 (91.65–122.27) – – 1.34 (1.05–1.72) 18–24 3.82 (2.40–6.06) 52.43 (45.56–60.33) 10.73 (7.50–15.34) 56.31 (47.08–67.34) 1.76 (1.58–1.98) 25–29 1.74 (0.78–3.87) 17.28 (13.02–22.93) 4.88 (2.70–8.80) 24.18 (17.93–32.61) 1.45 (1.21–1.74) 30–39 0.54 (0.20–1.44) 7.10 (5.26–9.57) 3.00 (1.81–4.97) 7.93 (5.61–11.21) 0.63 (0.54–0.73) 40–49 0.55 (0.21–1.48) 3.50 (2.28–5.36) 0.59 (0.19–1.82) 4.27 (2.69–6.78) 0.78 (0.67–0.90) 50–64 0.42 (0.17–1.01) 0.68 (0.33–1.43) 0.62 (0.28–1.39) 0.85 (0.41–1.79) 0.77 (0.68–0.86) ≥65 0.19 (0.05–0.76) 0.32 (0.10–1.00) 0.18 (0.05–0.72) 0.51 (0.21–1.23) – Females 12–15 0.49 (0.12–1.98) 6.35 (4.05–9.96) – – 0.17 (0.11–0.29) 16–17 0.84 (0.21–3.37) 10.98 (7.16–16.84) – – 0.42 (0.27–0.66) 18–24 0.18 (0.03–1.31) 4.12 (2.60–6.54) 0.96 (0.31–2.96) 6.87 (4.24–11.05) 0.38 (0.30–0.49) 25–29 0.26 (0.04–1.84) 2.23 (1.07–4.69) 0.41 (0.06–2.94) 8.22 (5.03–13.41) 0.48 (0.35–0.65) 30–39 0.72 (0.32–1.60) 1.02 (0.49–2.14) 0.74 (0.28–1.98) 0.68 (0.22–2.10) 0.47 (0.39–0.57) 40–49 0.24 (0.06–0.97) 1.73 (0.98–3.05) 0.18 (0.02–1.25) 1.89 (0.98–3.63) 0.89 (0.77–1.04) 50–64 0.37 (0.15–0.88) 0.51 (0.23–1.14) 0.65 (0.31–1.36) 0.43 (0.16–1.15) 01.00 (0.89–1.13) 5≥65 0.08 (0.01–0.54) 0.35 (0.13–0.92) – 0.26 (0.08–0.81) – PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 217 TABLE 7-4 Continued NOTES: BNT162b2 refers to the COVID-19 vaccine manufactured by Pfizer-BioNTech under the name Comirnaty®. mRNA-1273 refers to the COVID-19 vaccine manufactured by Moderna under the name Spikevax®. SOURCE: Table adapted from Oster et al., 2022. PREPUBLICATION COPY—Uncorrected Proofs 

218 VACCINE EVIDENCE REVIEW mRNA-1273 RCTs, mRNA-1273 The committee reviewed myocarditis/pericarditis events from published and unpublished reports of individual RCTs of mRNA-1273. The primary unpublished sources include the FDA advisory committee, EUA, and biologic license application materials, which reported individual counts of myocarditis/pericarditis events in each arm of each trial and details about these cases. These counts sometimes changed with additional follow-up and clinical review, so counts from the most recent FDA review documents were used when possible. In the Phase 2/3 RCT P301, among individuals ages 18+ (n = 15,206), no myocarditis was observed, and two pericarditis events were observed that were considered at least possibly related (FDA, 2020c, 2022c). In the Phase 2/3 RCT P203, among individuals aged 12–17 (n = 2,486), no myocarditis or pericarditis events were observed that were judged to be at least possibly related (FDA, 2022b) In the Phase 2/3 RCT P204, among children ages 6–11 (n = 3,007), no myocarditis or pericarditis events were observed that were judged to be at least possibly related (FDA, 2023a). In the Phase 2/3 RCT P204, among children ages 6 months to 5 years (n = 4,792), no myocarditis or pericarditis events were observed that were judged to be at least possibly related (FDA, 2023a). The count of myocarditis and pericarditis events in these trials was so low (two pericarditis events across all trial populations) that no statistical inference could be made. The lack of a clear signal for myocarditis or pericarditis in these trials effectively excludes a large average increase in risk in broad populations studied but not the possibility of a causal effect that results in one case per tens of thousands of vaccine exposures. Based on the RCT evidence alone, no conclusion was made about a potential causal effect of mRNA-1273 on these outcomes. Observational Studies, mRNA-1273 As described, the Living Evidence Synthesis (Table 7-4) provided strong evidence that mRNA vaccines likely increase the risk of myocarditis, and the same age, sex, and dose trends were observed for BNT162b2 and mRNA-1273. Moreover, the evidence reflected moderate to higher certainty that the risk is greater with mRNA-1273 in the older age groups. Results from the SPOR review were consistent with results from the most informative individual epidemiological studies (Gaudet, 2023). Collectively, these studies strongly suggested an increased risk of myocarditis with mRNA-1273, which is likely to be larger compared to BNT162b2. PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 219 TABLE 7-5 Selected Epidemiological Studies of Risk of Myocarditis Associated with mRNA-1273 Vaccinated Author Age Setting Design Confounding Outcome Interval N Cases Results (95% CI) Bots et al. 5+ Europe Self- Self- ICD 28d 1.5 20 Myocarditis only (2022) (4 controlled controlled codes million countries All ages: ) RR 5.28 (1.68–16.6) Men: RR 5.80 (1.62–20.7) Men aged 12-29: RR 6.05 (1.09–33.5) Goddard et 5 to 39 VSD Vaccinate None Record 7d Myocarditis/pericarditis al. (2022a) d only review 36 949,272 Higher risk for males, second dose, and 18–29 age group Goddard et 18–39 VSD Self- Self- Record 7d 38 Myocarditis and pericarditis al. (2022b) controlled controlled review 923,711 All ages: RR 18.75 (6.73–64.94) mRNA-1273 vs. BNT162b2 RR 1.61 (1.02–2.54) Karlstad et al. 12+ Europe Unvaccina Adjustment ICD 28d 2.4 78 Myocarditis only (2022) (4 ted control codes million countries Higher risk for males, ) second dose, and aged 16– 24. mRNA-1273 risk greater than BNT162b2 PREPUBLICATION COPY—Uncorrected Proofs 

220 VACCINE EVIDENCE REVIEW TABLE 7-5 Continued Vaccinated Author Age Setting Design Confounding Outcome Interval N Cases Results (95% CI) Le Vu et al. 12–50 France Case- Adjustment ICD 21d 2.9 129 Myocarditis only (2022) control codes million RR 30 (21–43) Higher risk for males, second dose, and 18–24 age group. mRNA-1273 risk greater than BNT162b2 Massari. 12–39 Italy Self- Self- ICD 21d 456,050 40 Myocarditis or pericarditis (2022) controlled controlled codes RR 7.59 (3.26–17.65) Higher risk for males, second dose, and 18-29 age groups. mRNA-1273 risk greater than BNT162b2 Patone et al. 13+ England Self- Self- ICD 28d 1.2 51 Myocarditis only (2022a) controlled controlled codes million RR 11.76 (7.25–19.08) Higher risk for males, second dose, and <40 age group. mRNA-1273 risk greater than BNT162b2 NOTES: BNT162b2 refers to the COVID-19 vaccine manufactured by Pfizer-BioNTech under the name Comirnaty®. mRNA-1273 refers to the COVID-19 vaccine manufactured by Moderna under the name Spikevax®. d: days; ICD: International Classification of Diseases; RR: relative risk; SC: self-controlled. SOURCES: Bots et al., 2022; Goddard et al., 2022a, 2022b; Karlstad et al., 2022; Le Vu et al., 2022; Massari et al., 2022; Patone et al., 2022a. PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 221 Few high-quality epidemiological studies of pericarditis without pericarditis were identified; two found no increased risk, and three found an increased risk for mRNA-1273. The relative risk estimates for pericarditis in the positive studies were larger than the risk estimates for BNT162b2 but much lower than the corresponding ones for myocarditis. The analysis of cases of myocarditis related to VAERS also corroborated these findings (Oster et al., 2022). PREPUBLICATION COPY—Uncorrected Proofs 

222 VACCINE EVIDENCE REVIEW TABLE 7-6 Selected Epidemiological Studies of Risk of Pericarditis Associated with mRNA-1273 Vaccinated Author Age Setting Design Confounding Outcome Interval N Cases Results (95% CI) Bots et al. 5+ Europe (4 Self- Self- ICD 28d 1.5 46 No increased risk (2022) countries) controlled controlled codes million Corrao et 12+ Italy Cohort vs Adjustment ICD 28d 1.6 52 RR 1.9 al. (2022) unvaccinat codes million (95% CI: 1.5–2.5) ed Karlstad et 12+ Europe (4 Unvaccina Adjustment ICD 28d 2.4 62 Increased risk in males al. (2022) countries) ted control codes million RR 2.99 (95% CI: 2.02–4.44) Females RR 3.41 (95% CI: 1.98–5.87) Higher RR under age 40 compared to 40 and over. mRNA-1273 risk greater than BNT162b2 Le Vu et 12– France Case- Adjustment ICD 21d 2.9 54 RR 5.5 (3.3–9.0) al. (2022) 50 control codes million Patone et 13+ England Self- Self- ICD 28d 1.0 0 No increased risk al. (2022b) controlled controlled codes million NOTES: BNT162b2 refers to the COVID-19 vaccine manufactured by Pfizer-BioNTech under the name Comirnaty®. mRNA-1273 refers to the COVID-19 vaccine manufactured by Moderna under the name Spikevax®. d: days; ICD: International Classification of Diseases; RR: relative risk; SC: self-controlled. SOURCES: Bots et al., 2022; Corrao et al., 2022; Karlstad et al., 2022; Le Vu et al., 2022; Patone et al., 2022b. PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 223 Ad26.COV2.S RCTs, Ad26.COV2.S The committee reviewed myocarditis/pericarditis events from published and unpublished reports of individual RCTs of Ad26.COV2.S. 3 The primary unpublished data sources include the FDA advisory committee and EUA. In the Phase 3 RCT 3001, among individuals aged 18+ (n = 21,895), no myocarditis events were observed, and one pericarditis event was observed that was judged to be at least possibly related (FDA, 2021d, 2021e). The count of myocarditis and pericarditis events was so low (one pericarditis event) that no statistical inference could be made. The lack of a clear signal for myocarditis or pericarditis in these trials effectively excludes a large average increase in risk in broad populations studied but not the possibility of a causal effect that results in one case per tens of thousands of vaccine exposures. Based on the RCT evidence alone, no conclusion was made about a potential causal effect of Ad26.COV2.S on these outcomes. Observational Studies, Ad26.COV2.S Few epidemiological studies have attempted to evaluate whether Ad26.COV2.S is associated with myocarditis or pericarditis. The committee identified no studies that included a large number of vaccine-related myocarditis or pericarditis cases, used an appropriate control group, and attempted to address confounding by factors that may be associated with Ad26.COV2.S. For example, in Bots et al. (2022), cited in the BNT162b2 vaccine evidence review, fewer than five cases of myocarditis were reported after Ad26.COV2.S, resulting in a risk estimate that was uninformative (incidence rate ratio 1.6, 95% CI: 0.1–21.6). In the pharmacovigilance literature, the committee identified only two studies that evaluated the potential myocarditis risks associated with Ad26.COV2.S. One used a global database of spontaneous adverse event reports to evaluate a disproportionality ratio for various COVID-19 vaccines, comparing vaccine exposure in myocarditis cases to other adverse event reports (Macías Saint-Gerons et al., 2023). This study design is highly susceptible to bias and provides no information about the absolute magnitude of risk; the reporting odds ratio for Ad26.COV2.S was 1.9 (95% CI: 1.7–2.1) compared to 17 (95% CI: 16–17) for BNT162b2 and 7.6 (95% CI: 7.4–7.8) for mRNA-1273. In the second pharmacovigilance study conducted using VAERS data through February 2022, 189 cases of myopericarditis were identified, but only 52 met the CDC case definition for a validated event (Woo et al., 2023). The observed incidence was compared to background rates of myocarditis estimated in two different studies: the relative risk estimates for myopericarditis with Ad26.COV2.S were 3.2 (95% CI: 2.0–4.8) and 1.1 (95% CI: 0.7–1.7), depending on which of the two data sources was used to estimate the background rate. NVX-CoV2373 RCTs, NVX-CoV2373 The committee reviewed myocarditis/pericarditis events from published and unpublished reports of individual RCTs of NVX-CoV2373. 4 The primary unpublished data source was the EUA memorandums, which included a detailed accounting of the events observed. 3 Ad26.COV2.S refers to the COVID-19 vaccine manufactured by Janssen. 4 Refers to the COVID-19 vaccine manufactured by Novavax. PREPUBLICATION COPY—Uncorrected Proofs 

224 VACCINE EVIDENCE REVIEW In RCT 301, among individuals aged 18+ (n = 19,735), one myocarditis and one pericarditis event were judged to be at least possibly related (FDA, 2023b; Marks, 2023). In RCT 302, among individuals age 18+ (n = 7,750), one myocarditis and one pericarditis event were judged to be at least possibly related (FDA, 2023b). In RCT 301, among individuals aged 12–17 (n = 1,487), one myocarditis event and no pericarditis events were judged to be at least possibly related (FDA, 2023b). Across all of these trial populations, the number of myocarditis and pericarditis events observed (six) out of approximately 42,000 vaccine exposures raises the possibility of a signal for rare events that typically would not be observed in trials of this size. However, the number of events was inadequate for statistical inference. Based on the RCT evidence alone, no conclusion was made about a potential causal effect of NVX-CoV2373 on these outcomes. Observational Studies, NVX-CoV2373 In the United States, only 89,000 doses of NVX-CoV2373 were administered as of May 11, 2023 (CDC, 2023), approximately double the population that received the vaccine in the Phase 3 trials. The committee did not identify any epidemiological studies of the risk of myocarditis associated with this vaccine. The global pharmacovigilance study cited earlier identified 61 cases of myopericarditis, 50 of which were from Australia (Macías Saint-Gerons et al., 2023). The reported odds ratio for NVX-CoV2373 was 15 (95% CI: 11–19), compared to 17 (95% CI: 17–17) for BNT162b2 and 6.9 (95% CI: 6.8–7.1) for mRNA-1273. From Evidence to Conclusions The committee identified consistent findings of a large relative risk of myocarditis after either mRNA vaccine in numerous high-quality observational studies, an absolute risk that is orders of magnitude greater than the background rate in certain age and sex subgroups, and a plausible biological mechanism for mRNA vaccines. The strong and substantial body of evidence indicates that the risk of harm varies by age and sex, but it does not exclude the presence of a causal effect in any particular group defined by age or sex. Conclusion 7-1: The evidence establishes a causal relationship between the BNT162b2 vaccine and myocarditis. Conclusion 7-2: The evidence establishes a causal relationship between the mRNA- 1273 vaccine and myocarditis. Evidence of a clear association from any well-designed and adequately powered observational studies and compelling mechanistic evidence was lacking for both Ad26.COV2.S and NVX-CoV2373 and myocarditis. Conclusion 7-3: The evidence is inadequate to accept or reject a causal relationship between the Ad26.COV2.S vaccine and myocarditis. Conclusion 7-4: The evidence is inadequate to accept or reject a causal relationship between the NVX-CoV2373 vaccine and myocarditis. PREPUBLICATION COPY—Uncorrected Proofs 

MYOCARDITIS AND PERICARDITIS 225 In contrast to the abundance of evidence regarding the risk of myocarditis and mRNA COVID-19 vaccines, few high-quality epidemiological studies have investigated the risk of pericarditis without myocarditis. Several studies did not find an increased risk, and those that did estimated much lower relative risk of pericarditis than what was observed for myocarditis. Additionally, very few events were observed in RCTs, with uncertainty as to whether all events were related to COVID-19 vaccines. Conclusion 7-5: The evidence is inadequate to accept or reject a causal relationship between the BNT162b2 vaccine and pericarditis without myocarditis. Conclusion 7-6: The evidence is inadequate to accept or reject a causal relationship between the mRNA-1273 vaccine and pericarditis without myocarditis. Conclusion 7-7: The evidence is inadequate to accept or reject a causal relationship between the Ad26.COV2.S vaccine and pericarditis without myocarditis. Conclusion 7-8: The evidence is inadequate to accept or reject a causal relationship between the NVX-CoV2373 vaccine and pericarditis without myocarditis. PREPUBLICATION COPY—Uncorrected Proofs 

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