Assessment of Allergic and Anaphylactic Reactions to mRNA COVID-19 Vaccines With Confirmatory Testing in a US Regional Health System

: Importance: As of May 2021, more than 32 million cases of COVID-19 have been confirmed in the United States, resulting in more than 615 000 deaths. Anaphylactic reactions associated with the Food and Drug Administration (FDA)-authorized mRNA COVID-19 vaccines have been reported. Objective: To characterize the immunologic mechanisms underlying allergic reactions to these vaccines. Design, setting, and participants: This case series included 22 patients with suspected allergic reactions to mRNA COVID-19 vaccines between December 18, 2020, and January 27, 2021, at a large regional health care network. Participants were individuals who received at least 1 of the following International Statistical Classification of Diseases and Related Health Problems, Tenth Revision anaphylaxis codes: T78.2XXA, T80.52XA, T78.2XXD, or E949.9, with documentation of COVID-19 vaccination. Suspected allergy cases were identified and invited for follow-up allergy testing. Exposures: FDA-authorized mRNA COVID-19 vaccines. Main outcomes and measures: Allergic reactions were graded using standard defi-nitions, including Brighton criteria. Skin prick testing was conducted to polyethylene glycol (PEG) and polysorbate 80 (P80). Histamine (1 mg/mL) and filtered saline (negative control) were used for internal validation. Basophil activation testing after stimulation for 30 minutes at 37 °C was also conducted. Concentrations of immunoglobulin (Ig) G and IgE antibodies to PEG were obtained to determine possible mechanisms. Results: Of 22 patients (20 [91%] women; mean [SD] age, 40.9 [10.3] years; 15 [68%] with clinical allergy history), 17 (77%) met Brighton anaphylaxis criteria. All reactions fully resolved. Of patients who underwent skin prick tests, 0 of 11 tested positive to PEG, 0 of 11 tested positive to P80, and 1 of 10 (10%) tested positive to the same brand of mRNA vaccine used to vaccinate that individual. Among these same participants, 10 of 11 (91%) had positive basophil activation test results to PEG and 11 of 11 (100%) had positive basophil activation test results to their administered mRNA vaccine. No PEG IgE was detected; instead, PEG IgG was found in tested individuals who had an allergy to the vaccine. Conclusions and relevance: Based on this case series, women and those with a history of allergic reactions appear at have an elevated risk of mRNA vaccine allergy. Immunological testing suggests non-IgE-mediated immune responses to PEG may be responsible in most individuals. Abstract IMPORTANCE As of May 2021, more than 32 million cases of COVID-19 have been confirmed in the United States, resulting in more than 615000 deaths. Anaphylactic reactions associated with the Food and Drug Administration (FDA)–authorized mRNA COVID-19 vaccines have been reported. OBJECTIVE To characterize the immunologic mechanisms underlying allergic reactions to these vaccines. DESIGN, SETTING, AND PARTICIPANTS This case series included 22 patients with suspected allergic reactions to mRNA COVID-19 vaccines between December 18, 2020, and January 27, 2021, at a large regional health care network. Participants were individuals who received at least 1 of the following International Statistical Classification of Diseases and Related Health Problems, Tenth Revision anaphylaxis codes: T78.2XXA, T80.52XA, T78.2XXD, or E949.9, with documentation of COVID-19 vaccination. Suspected allergy cases were identified and invited for follow-up allergy testing. EXPOSURES FDA-authorized mRNA COVID-19 vaccines. many of whom were vaccinated via occupational health programs in hospital settings. Hypervigilance toward adverse reactions to vaccines due to early publicized reports of vaccine-induced anaphylaxis and high rates of vaccine hesitancy may also lead to false-positive reports in VAERS. Given the high and growing prevalence of allergic disease in the general US population, public concern about possible vaccine-induced anaphylaxis risk among individuals with allergies, and the key role of vaccination in achieving herd to

vaccinate and found "no evidence that vaccination contributed to patient deaths." 2 VAERS provides valuable insights into vaccine-induced anaphylaxis; however, it has limitations.
Notably, VAERS is a passive reporting system requiring health care professionals to submit event reports that include vaccine lot numbers, which can be cumbersome to obtain and submit by treating clinicians. Additionally, the anaphylaxis case definition used by VAERS requires reactions to meet strict criteria, which can exclude mild reactions and some severe allergic reactions whose systemic involvement was limited by prompt treatment. Such treatment is more likely in health care workers who were overrepresented among the first wave of vaccinations, many of whom were vaccinated via occupational health programs in hospital settings. Hypervigilance toward adverse reactions to vaccines due to early publicized reports of vaccine-induced anaphylaxis and high rates of vaccine hesitancy may also lead to false-positive reports in VAERS. Given the high and growing prevalence of allergic disease in the general US population, public concern about possible vaccine-induced anaphylaxis risk among individuals with allergies, and the key role of vaccination in achieving herd immunity to COVID 19, it is essential that additional, comprehensive, and up-to-date clinical data be evaluated to further understand this important topic. Therefore, we hypothesized that lifethreatening reactions to the vaccine are extremely rare and that most allergic reactions to vaccines are due to non-immunoglobulin (Ig) E-mediated pathways. 3 As the global public health community expands vaccine access to include younger, more diverse populations who have historically exhibited higher rates of vaccine hesitancy, 4 it is especially critical that we better understand the mechanisms underlying vaccine-induced anaphylaxis for risk stratification and improved anaphylaxis management as well as to inform further vaccine refinement.
To those ends, this study provides clinical data, including skin prick tests (SPTs), basophil activation tests (BATs), and tryptase levels for a case series of vaccine-associated allergic reactions to mRNA COVID-19 vaccines from a large regional health system that was among the first in the United States to distribute these FDA-authorized vaccines.

Methods
This case series was designed to generate hypotheses and provide proof of concept, to recognize sentinel adverse events (allergic reactions and anaphylaxis), and to study the outcomes of new treatments (novel mRNA vaccines for COVID -19 institutional review board, and all participants provided written informed consent. This study followed the reporting guideline for case series.

Based on multiple International Statistical Classification of Diseases and Related Health
Problems, Tenth Revision (ICD-10) codes and systematic medical record review of patients with COVID-19 vaccine-associated allergic reactions, we identified those meeting prespecified criteria for suspected allergy (Figure 1). Specifically, the following search criteria were used: any patient receiving at least 1 of the following ICD-10 anaphylaxis codes between December 18, 2020, and January 26, 2021: T78.2XXA (anaphylaxis, initial encounter), T80.52XA (anaphylactic reaction due to vaccination, initial encounter), T78.2XXD (anaphylaxis, subsequent encounter), or E949.9 (vaccine or biological substance causing adverse effect in therapeutic use

BAT
Whole blood preserved in heparin, as described in Mukai et al, 6 was collected from participants.
Filtered saline was used as a negative control and anti-IgE (Bethyl Laboratories; 1 μg/mL) was used as a positive control.
Vaccine-discarded remnant material was used at 0.007 μg/μL. All stimuli were prepared in Roswell Park Memorial Institute (RPMI) medium. Basophils were gated as CD123+HLA−DR− cells, and the percentage of CD63+ basophils was quantified by flow cytometry. Control participants were also consented using the same IRB-approved protocol, and SPT and BAT assays were performed ( Table 1). Figure 2 illustrates an example of BAT results among control participants using anti-IgE (positive control), saline, and vaccine material as an activator. maximum detection cutoffs were determined as OD 405 1.0 and OD 405 1.9 for PEG IgE and PEG IgG respectively. High PEG IgG was considered for levels greater than OD 405 1.5. The blood draw for the assays performed (both BAT and Ig levels) was done at the same visit for the each participant.

Statistical Analysis
No statistical testing was performed. R version 4.0 (R Project for Statistical Computing) was used to generate descriptive statistics.  Because it is possible that the BATs were activated due to IgG (via complement activationrelated pseudoallergy [CARPA]) or IgE (via IgE-FcεRec activation), we performed standard enzymelinked immunosorbent assay to measure IgE to PEG and IgG to PEG on collected blood samples.

Between
Given that some participants had limitations with scheduling appointments for blood draws during  the COVID pandemic, sampling occurred between 0 to 78 days after the first dose of the vaccine, and high levels of IgG to PEG were detected during these periods. None of the individuals with an allergic reaction had IgE to PEG greater than the cutoff value.

Discussion
Currently, the CDC recommends that individuals with a history of allergic reaction to any mRNA COVID-19 vaccine component or who experienced a severe allergic reaction to the first dose not take either FDA-authorized mRNA vaccine. 9 The published data to date suggest that vaccination may be specifically contraindicated among patients with allergic reactions to PEG and/or P80. 9 The data presented here, collected from a large regional health center, suggest that allergic reactions from the mRNA vaccines are likely owing to PEG and non-IgE-mediated mechanisms, likely CARPA.
Of the stabilizing ingredients in the mRNA vaccine that we tested, P80 is a widely used emulsifier that can solubilize agents in foods and medicines, including vaccines. 10 Previous work has found that this nonionic detergent can induce both local and systemic allergic reactions, including both IgE-and non-IgE-mediated anaphylaxis. 11 The hydrophilic polymer known as PEG is structurally similar to P80. 12 PEG and its derivatives are common ingredients in household products, including toothpaste, cosmetics, pharmaceuticals, and foods. 13 In pharmaceuticals, PEG is often conjugated to biological therapeutics to form a depot agent, and sensitivity to PEG has been linked to IgE-mediated anaphylaxis after administration of PEG-conjugated biological therapeutics. 9,10,14-16 Interestingly, severe allergic reactions to PEG have been associated with preexisting anti-PEG antibodies induced by PEG-containing household products, 17 which may be more extensively used by women.
Polysorbates are obtained from PEG moieties but have lower molecular weights and thus may be less allergenic. 3 PEG may also be cross-reactive with polysorbates, which are present in some COVID-19 vaccines. 18,19 However, measurements of preexisting anti-PEG antibodies vary widely, with a recent literature review reporting estimates ranging from 0.2% to 72% among healthy individuals. 20 This is important because a high-molecular weight version of PEG is present in both of the FDA-authorized mRNA COVID-19 vaccines, where it helps to form a protective hydrophilic layer that sterically stabilizes the lipid nanoparticles. 21 While further work is needed to clarify the causative role of PEG and/or P80 in the anaphylactic reactions to mRNA COVID-19 vaccines observed here and elsewhere, previous reports of similar reactions to other PEG-conjugated biologics suggest that PEG 2000 is likely to be an important causative agent that warrants further study. [22][23][24] While allergy and/or anaphylaxis to FDA-authorized mRNA vaccines appear to be rare in all demographic groups, based on the present case series, women and those with a previous history of allergic reactions appear to have elevated risk. This is consistent with previous epidemiological data, which has found that approximately 85% of vaccine anaphylaxis cases had a history of prior allergic disease and that women are at a greater risk than men. 25,26 Although our SPTs and BATs are researchbased only, our data suggest a non-IgE-mediated immune pathway may be responsible for most reactions, possibly via complement activation through plasma immune complexes with the vaccine material or its components. 5 This might explain the differences we observed between the SPT and whole blood BAT results, given that such PEG immune complexes likely exist in the blood more than the skin.
Future clinical trials in atopic populations-such as the ongoing National Institute of Allergy and Infectious Disease-supported phase 2 trial, Systemic Allergic Reactions to SARS-CoV-2 Vaccination (NCT04761822)-will help to elucidate mechanisms, assist with guidelines to better assess vaccine allergy risk, and inform ongoing vaccine development, such as recently announced booster shots under development to protect against COVID-19 variants. Data suggest that patients who experience allergy to mRNA vaccines, as well as those who do not experience adverse effects after vaccination, still retain relative protection against SARS-CoV-2 infection. 27,28 Given the demonstrated safety and real-world effectiveness of these mRNA vaccines, 29 efforts to characterize and encourage reasoned consideration of the relative risks and benefits associated with COVID-19 vaccination among patients with higher risk of vaccine allergy can also help to advance mass vaccination campaigns, including ongoing efforts to address vaccine hesitancy. For example, when considering the risks associated with COVID-19 vaccination, it is important to note that an estimated 2% to 5% of the US population have experienced anaphylaxis, most commonly to medication, food, or insect stings. 30 However, fatal anaphylaxis is exceedingly rare, with a recent review 30 estimating an annual incidence of fatal drug-induced anaphylaxis at lower than death due to lightning strike in the general population. In contrast, COVID-19 has killed more than 615 000 US residents and made millions ill-some for many months, with a subset who may continue to experience long-term adverse health effects. 31,32 Moreover, allergic reactions are highly treatable, and even severe anaphylaxis usually can be promptly mitigated with appropriate preparation and medication, as all patients in the present case series experienced; each of their allergic reactions resolved.

Limitations
This study has limitations. It is important to note that our data should not be generalized for the purposes of epidemiology of allergies to vaccines because this is a single-site study, evaluated over a limited time period, which did not incorporate a population-based sampling frame. Specific care should be taken when comparing these findings with previous reports of VAERS data 1,2 given that the case definition used here was not intended only to identify severe allergic reactions but rather to identify cases of suspected mRNA vaccine allergy for mechanistic clinical follow-up.

Conclusions
In this study, women and those with a previous history of allergic reactions appeared to have a higher risk of developing mRNA vaccine allergy. SPT and BAT results to whole vaccine and PEG suggest a non-IgE-mediated immune response to PEG may be responsible. In the future, testing at baseline and longitudinal measurement of IgG PEG, BATs, and other molecules will be important to further test mechanisms. If confirmed by more systematic future investigations, these findings highlight potential opportunities for patient risk stratification and for alternatives in vaccine manufacturing; furthermore, they can inform ongoing mRNA vaccine development, including that of possible COVID-19 booster shots to protect against emerging disease variants.