IgE-mediated anaphylaxis implies a first exposure to an allergen leading to the production of specific IgE. These IgE bind to the high affinity receptors FcεRI on mast cells and basophils. Upon a new encounter, the allergen or a closely related substance activates mast cells and basophils by surface IgE cross-binding, which triggers degranulation of various mediators such as histamine or tryptase. This mechanism is the basis for routine anaphylaxis biological diagnosis, which encompass degranulated tryptase and histamine measurement, as well as specific IgE assessments. A true IgE-mediated allergic reaction to COVID-19 vaccines is possible, mainly based on documented PEG-mediated reactions in the literature, but seems very rare, as we'll see below.

Up to 30% of patients with clinically proven drug anaphylaxis do not have any sign of an IgE-dependent mechanism (9). Our group has demonstrated in various mice models that anaphylaxis can be triggered by a pathway involving specific IgGs that activate neutrophils (10, 11). Activated neutrophils release platelet-activating factor (PAF), a potent vasoactive lipid with effect similar to histamine. In a multicentric clinical study, we were able to confirm this mechanism in human, and showed that signs of neutrophil activation (in particular degranulation of neutrophil elastase and production of neutrophil extracellular traps) were correlated with severity in perioperative anaphylaxis patients (9).

Besides these two mechanisms, other pathways have been proposed to explain anaphylaxis that do not rely on the adaptive immune response. Since they do not require previous sensitization, these mechanisms may explain reactions observed to the first allergen exposure. Most of these mechanisms involve pharmacological activation of mast cells by the allergen. Some allergens have been described to activate the complement system, releasing C3a and C5a cleavage fragments that are able to trigger mast cell degranulation through specific receptors. These adverse effects known as CARPA have been documented with nanomedicines in experimental models but evidence in human are lacking (12). Moreover, Szebeni group also reported anti-PEG IgG-triggered complement terminal complex-mediated damage to PEGylated nanomedicines, that could decrease the efficacy of the nanomedicine and increase the toxicity via this complement activation (13).

Mast cell direct activation by positively charged substances like iodinated contrast media, quinolones, or some neuromuscular blocking agents has been described through the Mas-Related G Protein coupled Receptor X2 (MRGPRX2) (14). Interestingly, mRNA stabilization with PEG induces also a positive charge that could make this mechanism possible during COVID19 vaccine reaction. Whether basophils can also express MRGPRX2 at their surface upon activation remains controversial, but would be of great interest in assessing COVID19 vaccine-related hypersensitivity (15). However, it was recently shown that tryptase release by activated mast cells cannot discriminate between IgE- and MRGPRX2-related mechanisms, leaving yet unanswered questions concerning this interesting receptor (16).

Finally, many mediators like prostaglandins, bradykinin, serotonin or nitric oxide could mimic anaphylaxis symptoms by inducing vasodilation or bronchoconstriction, and their potential contribution to anaphylaxis is only beginning to be investigated.

The few studies carried out on the presence of anti-PEG of the IgE isotype but also IgG and IgM have been done using “in-house” techniques (21) A recent commercial ELISA was studied in 20 patients known to have experienced clinical reactions to drugs containing PEG; in this work, 4 out of these 20 patients had anti-PEG 2,000 IgE, and all had positive PEG skin tests (35). On a technical level, it is important to note the possible interference of bovine serum albumin and Tween 20, often used in ELISA; skimmed milk and an alternative detergent would probably be more appropriate reagents (35). Flow cytometric methods have also been described to assay anti-PEG IgE (36). Interestingly, Zhou et al. (21) found anti-PEG IgE and IgG in patients who had an anaphylactic reaction to products for colonoscopy preparation containing PEG 3350. It seems that some of these antibodies preexist in the general population, with a frequency of anti-PEG IgG of 5 to 9%, which could explain the manifestations observed at first administration (37).

The recent results on the frequency of anti-PEG antibodies during post-vaccination reactions are contradictory. This may be partly due to a lack of standardization of assay methods and of the gradation of the severity of allergy to PEG (38, 39). Some authors detected neither anti-PEG IgE nor IgG in post-vaccination reactions (34, 40), others found IgE and IgM but their control population was small. One of the questions is whether it would not be preferable to develop techniques to search for antibodies directed against PEG in the form of nanoparticles, or even against the vaccine itself (41). New robust tests are needed.

When hypothesizing CARPA-type mechanism, different complement activation parameters can be measured at the time of the reaction: anaphylatoxins C3a and C5a and the soluble fraction of the membrane attack complex C5b-9. In a pig experimental work, increased soluble C5b-9 levels correlated with the presence of anti-PEG IgM, after stimulation with PEGylated liposomes (32). Lim et al. (42) found increased C3a levels just after the clinical reaction in 3 patients, persisting from 48 h to one month. However, this increase was not confirmed by our group in 5 patients sampled at the time of the reaction (43). These preliminary results do not make it possible to conclude on the interest of these markers. Moreover, it is difficult to obtain a blood sample at the time of the clinical reaction, particularly in patients vaccinated outside a hospital.

To assess a possible mast cell degranulation in favor of an anaphylactic reaction induced by mRNA vaccines, histamine and tryptase assays could be informative. Very few studies report the measurement of tryptase at the time of the reaction, and they do not show any increased levels (26, 38, 42, 44, 45). Warren et al. study is the only one reporting elevated tryptase levels (between 14 and 25 μg/l for a basal tryptase between 2 and 6 μg/L) in 8 patients at time of the reaction (34). Our group reported increased histamine levels in 1 patient out of 5, within 30 min of the post-vaccination reaction, while tryptase levels were not modified (43).

Basal tryptase levels could also be of interest, even if no increased risk for reaction has been described in patients with mastocytosis (3). A few studies have shown a subnormal concentration in some patients: median of 8.5 to 12.8 μg/l, i.e. above the 95th percentiles described in the general population (46, 47). This could be in favor of gene duplication-related hyper-alpha − tryptasemia that needs to be better documented in the future (48). Moreover, the KIT D816V mutation research in the blood can be done to document mastocytosis, even in the presence of normal baseline tryptase (49).

The basophil activation test (BAT) using CD63 and/or CD203 as activation markers by flow cytometry was developed as early as January 2021 to explore immediate hypersensitivity to mRNA vaccines. Various authors tried to determine its place in the management of patients who reported reactions to drugs containing PEG before the first dose (50), or experienced reactions just after the first dose. In both cases there was an urgent need to secure vaccine injections (46).

Most of the published studies have been done on small patient series. Troelnikov et al. (50) performed BAT with PEG 2,000 nanoparticles in 3 patients known for PEG allergy and evidenced basophil activation. Labella et al. (46) found a positive BAT to PEG 2,000 and to the vaccine in 5/16 patients. Warren et al. (34) reported a positive BAT in 10/11 patients tested in the presence of PEG 2,000 DMG in the form of nanoparticles and vaccine. The frequency of patients with positive BAT is therefore very variable and could depend on the patients (already known to react to PEG or not for example) and the stimuli used ex vivo, whole vaccine and PEG nanoparticles seeming to give the highest positivity. Different allergens can be used in BAT. PEG 2,000 and PEG 2,000 DMG have been recently marketed for this test. However, as PEG contained in the vaccines is in the form of nanoparticles conjugated with lipids, some authors carried out BAT in the presence of the vaccine and/or PEG in the form of lipid nanoparticles approaching the truly potential immunogenic form (34, 50). However, in the early 2022, some authors evidenced that BAT was positive in response to vaccine alone in 50% of the patients who had COVID, and did not react during the vaccine injection (46). This information, that remains to be confirmed, must encourage to interpret BAT results with caution, in particular in patients who experienced SARS-Cov 2 infection. However, most authors agree in concluding that in the event of an anaphylactic reaction after injection of an mRNA vaccine, BAT is more frequently positive than skin tests confirming an activation mechanism which would not necessarily be IgE dependent (34, 40, 50). In our group in Paris, preliminary data in 30 patients with anaphylaxis after the first injection of a mRNA vaccine confirm that BAT can be positive while skin tests are negative (Nicaise-Roland P, Soria A et al., unpublished results). A recent Review by Eberlein et al. concluded that BAT helps elucidate allergic reactions to COVID-19 vaccines, but defining exact threshold of positivity is still needed (51).

We can thus assume that BAT is a quite simple and well-known test that needs to be further evaluated in larger well-characterized patients, with appropriate and standardized stimuli.

This test is only documented in two studies in this setting. The first one evidenced transient positive results in 3 patients who experienced a reaction (52), and the other one described positive results in 2/10 patients with positive skin tests to PEG (53).