key: cord-0765082-fm73ly1x
authors: Risma, Kimberly A.; Edwards, Kathryn M.; Hummell, Donna S.; Little, Frederic F.; Norton, Allison E.; Stallings, Amy; Wood, Robert A.; Milner, Joshua
title: Potential Mechanisms of Anaphylaxis to COVID-19 mRNA Vaccines
date: 2021-04-20
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2021.04.002
sha: 060ccbdbbf31e14bfd07662ca3b19f9e17458339
doc_id: 765082
cord_uid: fm73ly1x

Anaphylaxis to vaccines is historically a rare event. The Coronavirus Disease 2019 (COVID-19) pandemic drove the need for rapid vaccine production applying a novel antigen delivery system: mRNA vaccines packaged in lipid nanoparticles (LNP). Unexpectedly, public vaccine administration led to a small number of severe allergic reactions with resultant substantial public concern, especially within atopic individuals. We reviewed the constituents of the mRNA LNP vaccine and considered several contributors to these reactions: 1) contact system activation by nucleic acid, 2) complement recognition of the vaccine activating allergic effector cells, 3) pre-existing antibody recognition of polyethylene glycol (PEG), a LNP surface hydrophilic polymer, and 4) direct mast cell activation, coupled with potential genetic or environmental predispositions to hypersensitivity. Unfortunately, measurement of anti-PEG antibodies in vitro is not clinically available, and the predictive value of skin testing to PEG components as a COVID-19 mRNA vaccine-specific anaphylaxis marker is unknown. Even less is known regarding the applicability of vaccine use for testing (in vitro/vivo) to ascertain pathogenesis or predict reactivity risk. Expedient and thorough research-based evaluation of patients who have suffered anaphylactic vaccine reactions and prospective clinical trials in putative at-risk individuals are needed to address these concerns during a public health crisis.

Abstract: 39 Anaphylaxis to vaccines is historically a rare event. The Coronavirus Disease 2019 (COVID- 40 19) pandemic drove the need for rapid vaccine production applying a novel antigen delivery 41 system: mRNA vaccines packaged in lipid nanoparticles (LNP). Unexpectedly, public 42 vaccine administration led to a small number of severe allergic reactions with resultant 43 substantial public concern, especially within atopic individuals. We reviewed the 44 constituents of the mRNA LNP vaccine and considered several contributors to these 45 reactions: 1) contact system activation by nucleic acid, 2) complement recognition of the 46 vaccine activating allergic effector cells, 3) pre-existing antibody recognition of polyethylene 47 glycol (PEG), a LNP surface hydrophilic polymer, and 4) direct mast cell activation, coupled 48 with potential genetic or environmental predispositions to hypersensitivity. Unfortunately, 49 measurement of anti-PEG antibodies in vitro is not clinically available, and the predictive 50 value of skin testing to PEG components as a COVID-19 mRNA vaccine-specific 51 anaphylaxis marker is unknown. Even less is known regarding the applicability of vaccine 52 use for testing (in vitro/vivo) to ascertain pathogenesis or predict reactivity risk. Expedient 53 and thorough research-based evaluation of patients who have suffered anaphylactic 54 vaccine reactions and prospective clinical trials in putative at-risk individuals are needed to 55 address these concerns during a public health crisis. (11)). 119

Previous investigations into the immune mechanisms of vaccine-associated anaphylaxis have 120

focused on the presence of gelatin, latex, egg protein, and more recently on a widely utilized 121 surfactant, polysorbate 80 (PS80), present in numerous vaccines (12) . However, since none of 122 these excipients were included in the Pfizer BioNTech COVID-19 mRNA vaccine (Table 1 , 123 Figure 1 ) and no cases of anaphylaxis had been observed in the large phase 2/3 clinical trials, 124 (7) (8) this occurrence was unexpected. 125

The public reports of these reactions and early precautionary guidance in patients with a history 126 of severe allergic reactions substantially alarmed our patients. It is incumbent upon the allergy 127 community to respond to these concerns. Recommendations for reasonable clinical 128 management have been published (13) at a time when we have very limited understanding of 129 the nature of these reactions; there also emerge a series of research-based questions which are 130 critical to answer. 131

The occurrence of anaphylaxis upon first exposure to the COVID-19 vaccine implies either pre-133 existing, antibody-mediated immunity (allergic) or a pseudo-allergic response independent of 134 previous exposure. Although anaphylaxis related to known allergens is best understood via the 135 classic paradigm of crosslinking IgE bound to Fcε receptors on mast cells and basophils, non-136 classical pathways such as antibody-dependent activation of complement or IgG mediated mast 137 cell/granulocyte/platelet/basophil activation via Fcγ receptors have been described in animal 138 models and in allergic responses to medications in humans (14) (15) (16) (17) (18) . Additionally, 139 a variety of pseudo-allergic mechanisms that lead to direct activation/degranulation of mast cells 140

(through G protein coupled receptors or complement activation) or mast cell independent 141 mechanisms (stimulation of bradykinin production) causing vascular leak have been described 142 (14, 15) . These mechanisms are summarized in Figure 2 and discussed in consideration of the 143 COVID-19 vaccines based on what is known about the components of the vaccine. 144 J o u r n a l P r e -p r o o f

Naked RNA is inherently pro-inflammatory due to its ability to bind pathogen-associated 146 molecular pattern receptors, and by its negative charge, RNA may directly activate proteins in 147

the contact system (19) (20) . Exogenous nucleic acids activate factor XII of this system and lead 148

to the subsequent production of bradykinin, causing angioedema and/or anaphylactoid 149

reactions. To decrease reactivity and protect the nucleic acid from degradation, the mRNA in 150

the COVID-19 vaccines have been chemically modified and packaged in "stealth" lipid 151 nanoparticles (LNP) (1), Figure 1 . Since the LNPs encapsulate the mRNA and are rapidly 152 endocytosed into phagocytic cells, the mRNA payload is less likely to be the primary stimulus 153

for the injection reactions, unless the stability of the LNP vesicle has been disrupted. The latter 154 may occur during freeze/thaw cycles prior to vaccination. By design, the LNP is disrupted when 155 the vaccine payload is phagocytosed to the endosome, allowing the mRNA to escape to the 156 cytoplasm. 157

To further our understanding of vaccine reactions, the extent to which the mRNA may be 158

liberated acutely upon injection, should be examined. Measuring intact and cleaved high 159 molecular weight kininogen in blood samples after a vaccine reaction may help determine 160 whether the contact system pathway is activated during these acute events (21). Assessments 161

will require a prospective approach to capture rare events, although mild reactions may also be 162

informative. Animal models would certainly be useful. 163

The direct activation of mast cells or basophils leads to of degranulation via a variety of 165 receptors including opioid receptors, Mas-related G protein coupled receptor X2 (MRGPRX2) 166

receptors, and other yet to be defined receptors for contrast agents (14, 15) . As mast cells are 167

poised to respond to pathogen danger signals, it is feasible that connective tissue mast cells in 168 the muscle may degranulate in response to interaction with the LNP. A recent publication 169 described efficient transfection of human mast cells using an LNP delivery system (22) 

The LNP is composed of an ionizable lipid bearing a positive charge at low pH that neutralizes 182 the negative charge of the mRNA ( Figure 1 , Table 1 ), reviewed in (1), (24) . Additionally, the LNP 183

includes neutral lipids and cholesterol which self-assemble into a core lipid structure with a 184

surface layer that mimics a cell membrane. Finally, the LNP incorporates a phospholipid 185 conjugated to polyethylene glycol (PEG) to increase hydrophilicity of the LNP surface and to 186

provide stability to the mRNA carrier. Historically, PEG has been utilized to decrease the 187 immunogenicity of proteins and nucleic acids administered as pharmaceuticals (25) . 188 J o u r n a l P r e -p r o o f Doxorubicin was the first pharmaceutical delivered in a pegylated liposome (Doxil) to be FDA 189 approved in 1995. Liposomal preparations containing doxorubicin without PEG were rapidly 190 cleared by the reticular endothelial system, limiting utility (26) . Inclusion of 5% molar PEG led to 191 substantially improved stability. However, reports of immediate hypersensitivity reactions to 192

Doxil followed in 1996 (27) . Pseudo-allergic reactions to Doxil were also subsequently 193 demonstrated in porcine models, and were labeled as Complement Activation Related Pseudo-194

allergic Reactions (CARPA) (28) . Doxil infusions led to the production of anaphylatoxins C3a 195

and C5a that activated mast cells, resulting in severe hypotension and pulmonary hypertension 196 in pigs. Humans experiencing infusion reactions to Doxil also showed evidence of complement 197 activation, assessed by measurement of sC5b-9 in patient serum 10 minutes after infusion (29) . 198

These patients were not known to have pre-existing antibodies against PEG (30) suggesting 199 that the Doxil liposomes directly triggered their alternative pathway of complement. Anti-PEG antibodies have also been identified in individuals given PEG-asparaginase for 219 chemotherapy and high titer, pre-existing antibodies have been associated with adverse 220

reactions upon first infusions in children with leukemia (34, 35) . The proposed mechanism is a 221 non-classical pathway-whereby IgM (or potentially IgG) activates complement and mast cells 222 degranulate in response to C3a and/or C5a anaphylatoxins. Alternatively, IgG could bind to Fcγ 223 receptors on granulocytes and/or platelets, leading to secretion of serotonin, cytokines, and 224

platelet activation factor, with subsequent vascular leak. Mast cells may degranulate in 225

response to crosslinked IgG as demonstrated in vitro (36) . It is also possible that these infusion 226

reactions are IgE mediated, although anti-PEG IgE were not evaluated in these trials. 227

Infusion reactions reported for other PEG-containing liposomes have limited clinical usage. For 228 example, PEGylated liposomes were evaluated for delivery of RNA aptamers, but phase 2/3 229 trials were halted due to an unacceptably high rate of anaphylaxis occurring upon first exposure, 230 associated with pre-existing anti-PEG antibody (37, 38) . Both IgM and IgG anti-PEG antibodies 231

were documented in these patients; tryptase was elevated in 6/11 patients with severe 232 J o u r n a l P r e -p r o o f reactions; and complement C3a was also elevated at 90 minutes. Unfortunately, the authors did 233 not report if both the C3a and tryptase elevation occurred in the same patients (37) . 234

Studies are urgently needed that prospectively and retrospectively measure antibodies (IgM, 235

IgG and IgE) against PEG. Unfortunately, anti-PEG antibody (IgG, IgM, and IgE) measurements 236

are not yet available for routine clinical testing. A gold standard enzyme-linked immunosorbent 237 assay (ELISA) has not been established (39), which likely explains the reported differences in 238 measurement of anti-PEG antibodies in healthy volunteers, ranging from 5% to 70 % depending 239 upon the assay and the cutoffs utilized by individual research labs (34, 35, 37, 40, 41) . 240

As a side note, although the existence of pre-existing IgM and/or IgG antibodies against the 241 LNP may adversely lead to non-classical allergic reactions, they may also lead to enhanced 242 efficacy of the vaccine. Pre-existing IgG and IgM may enhance dendritic cell (DC) uptake of 243 LNP through Fc receptors or complement receptors on DC (Figure 3 ) leading to increased 244 delivery of mRNA to the cytoplasm, increased spike protein expression, and the capacity for 245 enhanced presentation to T cells. The data from phase 2/3 trials of COVID mRNA vaccines 246 reveal remarkable efficacy, preventing 94-95% of infections (7) implies T cell engagement. Due to its "inert" biochemical properties (25) , environmental 274 exposure to unconjugated PEG would not be expected to lead to immunogenic, PEG-hapten-275 J o u r n a l P r e -p r o o f carrier proteins. As such, most anti-PEG antibodies are theorized to arise from T independent B 276 cell production of IgM and IgG (31) and the formation of IgE would be predictably rare. This 277 uncommon immunologic occurrence could certainly account for the rarity of the current vaccine 278

reactions, whereas pseudo-allergic or non-classical allergic reactions may be anticipated to 279 occur more frequently. 280

Most recently IgE against PEG was detected in the blood of a patient suffered immediate 281 reactions to each of three different medications containing PEG --Definity liposomes, oral bowel 282

prep, and steroid injection (47) . Anti-PEG IgE was measured by two independent 283

immunoassays-chemiluminescent-based and dual cytometric bead assays (40, 47) . Zhou et al 284

showed that 6 additional patients with a history of reactions to HMW PEG had detectable IgE 285 (40, 47) ). Interestingly, IgE was also identified in 2 of 2091 serum samples when screening 286

healthy controls, suggesting that allergic sensitization may be more common than expected 287 (40); however, these blood samples were not tested for their capacity to trigger basophil or mast 288 cell activation. 289

Previous case reports of individuals with a history of PEG allergy have shown variable results 290

with the basophil activation test (BAT), using HMW PEG or PS80 as an allergen (43) (48, 49) .

Although the BAT can be an extremely helpful flow cytometry assay to document both the 292 reactivity and sensitivity of basophils to allergens in vitro, it is not yet available as a clinical test 293 (50) . The BAT is certainly useful in research studies, with 2 main limitations -the need for 294

testing fresh blood and the finding of non-reactive basophils in up to 20% of individuals. These 295 limitations can be overcome using a mast cell activation test (MAT), more recently described by 296

applying patient serum or plasma to healthy donor blood derived mast cells or immortalized 297 human mast cells and measuring degranulation by flow cytometry (51) (52). The advantage of 298 the MAT is that blood samples can be frozen and shipped to a research lab and the cultured 299 mast cells may be confirmed to degranulate prior to experimentation. 300

A key set of experiments for evaluating COVID-19 mRNA vaccine reactions is the use of the 301 BAT and/or MAT assays to determine whether the vaccine activates patient basophils or donor 302 mast cells directly as outlined above or activates only in the presence of serum from the 303 affected individual. The latter implying a mechanism of IgE-mediated degranulation, readily 304 tested by blocking IgE. 305

Genetic and environmental modifiers of mast cell activation in patients with vaccine reactions 307 may also be considered. It should be noted that the individuals experiencing anaphylactic 308

reactions to the COVID-19 mRNA vaccines have been strikingly female (9) (10). Drug allergy 309

and drug induced anaphylaxis is more common in adult females than males, with this difference 310 emerging after puberty (reviewed by (53)). Few studies have examined these differences in 311 drug allergy. The skewing of the allergic response to the COVID mRNA vaccine towards the 312 female sex may be secondary to estrogen effects in promoting a TH2 response, or conversely, 313 testosterone and progesterone's known role in diminishing TH2 responses (54) (55) . 314

Additionally sex hormones may influence mast cell degranulation; although estrogen is thought 315

to be stimulatory, studies demonstrate that progesterone suppresses histamine release from 316 mast cells (55) (56) .Estrogen has also been demonstrated to increase endothelial nitric oxide 317 synthase activity, enhancing the severity of anaphylaxis in murine studies (57) . An investigation 318 J o u r n a l P r e -p r o o f into the discrepant role of sex hormones in this setting is critical for understanding the 319 pathogenesis and potentially developing tools to screen for or prevent reactions. 320

An interesting observation is that atopic individuals also appear to be over-represented in those 321

suffering anaphylaxes to the COVID mRNA vaccines (9) (10) . The common past histories of 322 allergic reactions in those who have COVID-19 vaccine anaphylaxis need to be carefully 323 curated to determine the type of reaction and associated with triggers. This inquiry might point 324

to a predisposition for hyperresponsiveness to direct mast cell activation via these pathways. 325

Another host factor that may impact the likelihood of anaphylaxis is stress, particularly relevant 326

during a global pandemic. Corticotropin releasing hormone (CRH) and neurotensin are secreted 327 by neurons in response to acute and chronic stress and they lower the threshold for mast cell 328 degranulation (58) . Substance P is also released by neurons adjacent to mast cells and leads to 329 degranulation during a stress response (59). Finally, the use of opiates or non-steroidal anti-330 inflammatory drugs may enhance mast cell activation and/or vascular responsiveness (14) (15), 331

thus emphasizing the importance of a detailed history of medications taken prior to vaccination. 332

In addition to evaluating mechanisms and modifiers of anaphylaxis, predisposing disease 333

conditions should be explored. Mastocytosis and other forms of clonal mast cell expansion can 334 present with anaphylaxis alone, without any other associated comorbidities. This is best 335 described in the context of hymenoptera venom hypersensitivity (60) can be performed to identify if the vaccine anaphylaxis population is enriched for those with 355 HATS, and a BST >8 ng/ml can be highly suggestive as well. Additionally, the recent report of a 356 rare missense mutation in KARS provides an example of a rare monogenic predisposition to 357 severe anaphylaxis (67) . Similar findings may be noted, whether in KARS or other rare, yet 358 undiscovered variants, in this patient cohort. Whole genome sequencing of individuals with 359 reactions would be critical to identify known or novel rare variants associated with this unique 360 hypersensitivity/anaphylaxis. 361 J o u r n a l P r e -p r o o f

The high efficacy of mRNA LNP vaccination against COVID-19 disease in Phase 2/3 clinical 363 trials and the rapid successful mobilization of a useful vaccine suggests the use of this 364

technology is likely to revolutionize future vaccine approaches. The ability to generate a 365 pandemic vaccine in less than a year for mass production is extraordinary, particularly when 366 directed against RNA viruses which undergo continuous mutation. Thus, it will be prudent to 367 learn from the current worldwide vaccination efforts-not only to understand the mechanisms of 368

anaphylaxis, but to develop strategies to identify risk factors for immediate reactions, identify 369 sensitive and specific mechanisms for diagnosis and risk stratification for future vaccination. 370

Due to limited availability of clinical tools to assess for allergic responses to vaccines and the 371 likelihood that non classical allergic responses and/or pseudo-allergic responses contribute to 372 COVID-19 mRNA vaccine reactions, research studies are imperative. 373 

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