key: cord-0839198-4833e9d9
authors: Maron, Jenny S; Conroy, Michelle; Naranbai, Vivek; Samarakoon, Upeka; Motazedi, Tina; Farmer, Jocelyn R; Freeman, Esther; Banerji, Aleena; Bartsch, Yannic C; Gregory, David J; Poznansky, Mark C; Alter, Galit; Blumenthal, Kimberly G
title: Differential SARS-CoV-2 Antibody Profiles after Allergic Reactions to mRNA COVID-19 Vaccine
date: 2022-03-23
journal: J Infect Dis
DOI: 10.1093/infdis/jiac107
sha: 3b934eff720e8359a46d7c91db80cc3b79e5cc6f
doc_id: 839198
cord_uid: 4833e9d9

Allergic symptoms after mRNA COVID-19 vaccines occur in up to 2% of recipients. Compared to nonallergic controls (n=18), individuals with immediate allergic reactions to mRNA COVID-19 vaccines (n=8) mounted lower IgG1 to multiple antigenic targets in SARS-CoV-2 spike following vaccination, with significantly lower IgG1 to full-length spike (p=0.04). Individuals with immediate allergic reactions to mRNA COVID-19 vaccines bound Fcγ-Receptors similarly to non-allergic controls. Although there was a trend towards an overall reduction in opsonophagocytic function in individuals with immediate allergic reactions compared to non-allergic controls, allergic patients produced functional antibodies exhibiting a high ratio of opsonophagocytic function to IgG1 titer.

M a n u s c r i p t 

There have been more than 555 million Coronavirus disease 19 (COVID-19) vaccine doses administered in the US to date, largely with the messenger RNA (mRNA) vaccines from Pfizer-BioNTech (BNT162b2, Comirnaty®) or Moderna (mRNA-1273, Spikevax®). 1 Shortly after the initial vaccination roll-out, reports of anaphylaxis and allergic reactions began. 2 Allergic reactions have now been reported in up to 2% of individuals after mRNA COVID-19 vaccination, with mRNA vaccine anaphylaxis incidence confirmed in 8 to 250 cases per million. 3, 4 Limited serologic studies in mRNA COVID-19 vaccine-allergic individuals have assessed for antibodies to the vaccine or its excipients in order to begin to elucidate the mechanism(s) of these reactions. 5 However, with allergic symptoms after vaccination resulting in incomplete COVID-19 vaccination, 6,7 we sought to assess SARS-CoV-2 antibody quantities, Fc-receptor binding, and antibody functions in individuals with mRNA vaccine allergic reactions.

Massachusetts General Hospital (MGH) Allergy/Immunology patients with history of recent (<2 months), immediate onset (<6 hours) allergic reactions after mRNA COVID-19 vaccine from Pfizer-BioNTech or Moderna were prospectively identified, consented, and enrolled in this study by allergy specialists (MC, TM, AB, KGB). We matched mRNA vaccine-allergic patients to non-allergic (i.e., vaccine tolerant) controls, enrolled through a separate MGH study, 8 A c c e p t e d M a n u s c r i p t

The relative titers of antigen-specific IgG subclasses, antibody isotypes, and FcR binding in the human plasma samples were analyzed with a customized multiplexed Luminex assay, as previously described. 9 SARS-CoV-2 wild type (WT) spike (S) (purchased from Lake Pharma), Receptor Binding 

The Antibody-Dependent Neutrophil Phagocytosis (ADNP) activity assay using isolated primary human neutrophils was performed as described previously. 10 In brief, immune complexes were formed by incubating biotinylated SARS-CoV-2 WT S antigen (purchased from Lake Pharma) coupled to 1.0 m yellow-green, fluorescent neutravidin-labeled microspheres (Thermo Fisher Scientific) with human serum, diluted 1:50 in PBS, for 2 hours at 37 ºC. White blood cells were isolated from whole blood of two healthy donors, collected by the RI, as experimental replicates. Red 

Monocyte THP-1 cell-line-mediated phagocytosis assay was performed as described previously. 11 Briefly, immune complexes were formed by incubating 1.0 m yellow-green fluorescent, neutravidinlabeled microspheres (Thermo Fisher Scientific) coupled biotinylated SARS-CoV-2 WT S antigen (purchased from Lake Pharma) and human serum diluted 1:100 in PBS for 2 hours at 37 ºC in duplicate. THP-1 monocytes (ATCC TIB-202) were added to immune complexes at 250,000 cells/well in R10 media (RPMI-1640, Sigma) supplemented with 10% FBS, 2 mM L-gluthamine, 100 U/ml penicillin/streptomycin, 20 mM HEPES, and 50 M beta-mercaptoethanol. Cells were incubated with immune complexes for 16 h at 37 ºC, 5% CO 2 , fixed with 4% paraformaldehyde, and resuspended in PBS for flow cytometric acquisition (iQue, Sartorius). The phagoscore was calculated by dividing the product of % bead-positive cells and bead-positive median fluorescence intensity by M a n u s c r i p t 9

Data were analyzed with GraphPad Prism 9.2.0 Software. Univariable comparisons between groups used non-parametric, two-sided Mann-Whitney test with p<0.05 was considered significant.

Allergic individuals were all female with mean age 40 years (Standard Deviation 16 years); 6 (75%) had reactions to Pfizer-BioNTech (BNT162b2) and 2 (25%) had reactions to Moderna (mRNA-1273, Table 1 Table 1 ).

The mRNA vaccine-allergic individuals mounted significantly lower IgG1 titers against full-length SARS-CoV-2 S antigen (p=0.04) with lower trends in IgG1 against the RBD and NTD sub-domains following vaccination that did not reach statistical significance (Figure 1A) . In contrast, similar median IgG3 (Figure 1A) , IgM, and IgA titers and median Fc-receptor binding to SARS-CoV-2 spike were observed between the allergic and non-allergic groups (Supplementary Figure 1) . There was a trend towards a reduced median phagocytic function in SARS-CoV-2 S-directed THP-1 monocyte-mediated cellular or neutrophil opsonophagocytic functions in the allergic group that did A c c e p t e d M a n u s c r i p t 10 not reach statistical significance ( Figure 1B) . However, allergic individuals had higher median opsonophagocytic effector functions per IgG1 than non-allergic controls ( Figure 1C ). This suggests that the S-specific antibodies produced by vaccine-allergic individuals are capable of inducing ADCP and ADNP and that their antibody response may compensate for reduced IgG1 titer with higher phagocytic function per IgG (Figure 1C) . The relatively higher magnitude of S-specific FcR3B binding in allergic individuals normalized across all spike-specific antibody features (Figure 1D) , may contribute to the high ratio of phagocytosis to IgG1 as FcR3B is capable of neutrophil activation. 12 Overall, allergic individuals have differential S-and RBD-specific antibody profiles compared to the uniform magnitude of the non-allergic vaccine recipient group (Figure 1D) . In figure   1D , the normalized anti-S antibody features across allergic and non-allergic groups show lower IgG subclass titers in the allergic group, indicated by the size of the petal corresponding to IgG 1 , IgG 2 , IgG 3 , and IgG 4 , with statistically significant differences only in anti-S IgG1 at the univariate level.

Repeating the experiments without Patient 2 (prior SARS-CoV-2 infection) or Patient 8 (received two vaccine doses) did not alter these findings (Supplemental Figure 2) .

We observed a significant reduction of anti-S IgG1 antibodies in mRNA COVID-19 vaccineallergic individuals versus mRNA COVID-19 vaccine-non-allergic individuals. We also identified an overall reduction in antibody-mediated opsonophagocytic functions in allergic vaccine recipients compared to non-allergic. Prior data suggests that the functional quality of the humoral immune response is a correlate of vaccine-induced immunity, with S-specific antibodies as immune correlates of mRNA-1273 vaccine-induced immunity and Fc-mediated functions in protection against SARS-CoV-2. 13, 14 The two mRNA COVID-19 vaccines authorized for use require two doses with booster vaccinations recommended. 7 Although only severe and immediate-onset mRNA COVID-19 vaccine allergic reactions contraindicates additional doses, 7,15 any allergic symptoms after vaccination may A c c e p t e d M a n u s c r i p t 11 result in incomplete vaccination, 6 jeopardizing individual protection and population immunity. Our finding that individuals with allergic reactions to mRNA COVID-19 vaccine exhibit differential SARS-CoV-2 S-and RBD-directed antibody profiles with lower IgG1 and an overall reduced trend in antibody-mediated opsonophagocytic function directed against SARS-CoV-2 S, therefore, may have important implications for vaccine efficacy and/or durability in the allergic population.

Our study was a small, single center pilot study. While we matched on key demographic and vaccine factors likely to influence antibody response, residual confounding may be present.

Comprehensive clinical data, such as medical comorbidities and detailed medication exposures, were not collected similarly in cases and controls. However, allergic cases were not prescribed systemic immunosuppressive medications and regularly prescribed corticosteroids included inhaled corticosteroids for two allergic patients. Additionally, only two allergic cases received corticosteroids as part of their vaccine reaction treatment. Given that all individuals with mRNA COVID-19 vaccine allergy also had strong allergic histories, but control patients infrequently had allergic histories, future studies must distinguish whether this humoral immune pattern is associated with mRNA vaccine allergic individuals or is associated with the allergic host more generally. Although the vaccine allergy cases included in this study were all women, women comprise the majority of the mRNA vaccine allergy cases to date. 4, 5, 15 Although allergy is a clinical diagnosis, all cases were diagnosed by allergy specialists.

These findings suggest potential quantitative deficits in COVID-19 protection in individuals with mRNA COVID-19 vaccine allergy. While larger confirmatory studies are needed, these initial data support the need for additional immunologic investigations in individuals with allergic responses and clinical and population efforts to assist mRNA vaccine-allergic individuals in completing and optimizing their COVID-19 vaccination protection. M a n u s c r i p t 

Department of Health & Human Services

Pfizer's vaccine raises allergy concerns

The Risk of Allergic Reaction to SARS-CoV-2 Vaccines and Recommended Evaluation and Management: A Systematic Review, Meta-Analysis, GRADE Assessment, and International Consensus Approach

Acute Allergic Reactions to mRNA COVID-19 Vaccines

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

Allergic symptoms after mRNA COVID-19vaccination and risk of incomplete vaccination

Interim Clinical Considerations for Use of COVID-19 Vaccines Currently Approved or Authorized in the United States

Immunogenicity of mRNA-1273, BNT162b2 and Ad26

Route of immunization defines multiple mechanisms of vaccine-mediated protection against SIV

A versatile high-throughput assay to characterize antibodymediated neutrophil phagocytosis

A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples

FcgammaRIIA and FcgammaRIIIB mediate nuclear factor activation through separate signaling pathways in human neutrophils

Immune correlates analysis of the mRNA-1273 COVID-19 vaccine efficacy clinical trial

Fab and Fc contribute to maximal protection against SARS-CoV-2 following NVX-CoV2373 subunit vaccine with Matrix-M vaccination

Safety Evaluation of the Second Dose of Messenger RNA COVID-19 Vaccines in Patients With Immediate Reactions to the First Dose

Figure 1. Vaccine allergic and non-allergic antibody profiles to SARS-CoV-2 spike protein

Lines in each box represent minimum, lowerquartile, median, upper-quartile, and maximum values of each group. B.) Violin plots of S-specific ADCP or ADNP reported as phagocytic score. Dashed lines represent the lower and upper quartiles. Solid line indicates the median. C.) Box plots showing the ratio of S-specific ADCP or ADNP to IgG1 binding levels. Lines indicate minimum

Patients 4 and 6, who received corticosteroids for allergic reaction treatment, are represented as triangle symbols. Patient 2, with prior history of SARS-CoV-2 infection, is represented as a square symbol. Patient 8, who received two vaccine doses, is represented as a star. D.) Flower plots summarizing SARS-CoV-2 S-specific antibody profiles of allergic and non-allergic groups

ADNP, antibody-dependent neutrophil phagocytosis; S, Spike; RBD, receptor binding domain; NTD, N-terminal domain

A c c e p t e d M a n u s c r i p t