key: cord-0893611-o7ldrcru
authors: Eberlein, Bernadette; Mathes, Sonja; Fischer, Jörg; Darsow, Ulf; Biedermann, Tilo; Brockow, Knut
title: Do basophil activation tests help elucidate allergic reactions to the ingredients in COVID‐19 vaccines?
date: 2022-03-25
journal: Allergy
DOI: 10.1111/all.15278
sha: aea19fcb921c35acff0d91f65b22a5c1201287f5
doc_id: 893611
cord_uid: o7ldrcru

The worldwide use of COVID‐19 vaccines has shown that immediate allergic reactions to the ingredients are rare but should be clarified by means of an allergological work‐up. This review aims to highlight the current state of knowledge and possible pathogenesis based on the literature published to date. In addition to recording a detailed history and performing skin tests, cellular tests (basophil activation or basophil histamine release test) by using the vaccines or modified compounds containing polyethylene glycol (PEG), rather than unmodified PEGs, have proven to be particularly helpful. Negative results with vaccines seem to indicate tolerance. Details of the performance of these cellular tests with different vaccines, PEGs of different molecular weights, other ingredients of the vaccines, as well as other PEGylated drugs, and the results in the context of COVID‐19 vaccination of various working groups worldwide are summarized.

this review is to investigate whether these tests are helpful in the work-up and investigation of immediate-type allergic reactions to COVID-19 vaccine ingredients.

For advanced diagnostics of immediate-type allergic reactions, cellular in vitro tests can be used; these predominantly prove the sensitization of basophils. The tests use the detection of mediators or cellular antigens that are measurable upon successful activation.

Enriched blood leukocytes or whole blood is incubated with allergens or other triggers. The surface markers expressed after allergen stimulation or the mediators released by basophils usually serve as an indirect measure of cellular-bound specific IgE. However, IgEindependent stimuli also elicit basophil activation.

The tests are useful if problems arise in conventional diagnostics, either in interpretation (for example, in the case of contradictory results) or performance (for example, when skin tests for eczema or symptomatic dermographism are not feasible or evaluable). Furthermore, for rare allergens, the determination of sIgE antibodies may not be possible. In addition, provocation testing may not be feasible due to the pharmacological properties of drugs, the severity of the reported reaction, or ethical concerns (for example, risk of re-sensitization). 6,7

Cellular tests should be performed with fresh cells since a loss of activity can be expected after 4 h. However, storage of the cells for a maximum of 24 h is acceptable because EDTA blood is sufficiently stable and therefore shipping is possible. Depending on the test protocol, whole blood or enriched leukocyte suspensions can be used. 6 Soluble and non-cytotoxic substances can be used as allergens.

With regard to the PEG derivatives, vaccines and other drugs listed in Tables 1 and 2 , there were no indications of toxicity due to the substances used. These substances should be used at different concentrations. In most studies, two to six concentrations were used; two studies used only one concentration. 8, 9 A negative control and positive controls (IgE-dependent and/or IgE-independent stimuli) must be included. To rule out non-specific activation with particular allergens, non-sensitized control subjects should be tested. Both aspects were considered in most of the studies listed in Table 2 . Basophils from approximately 5%-15% of cell donors cannot be activated after IgE-mediated stimulation (non-responders). In such cases, the tests are false negative. 6, 7 In this overview, five non-responders have been reported. 10, 11 The basophil histamine release test (BHRT) established by Lichtenstein's group in the 1970s is based on the measurement of the preformed mediator histamine released from the granules of basophils. It can be measured spectrofluorometrically, enzymatically or radioimmunologically. Histamine release from individual samples is usually expressed as a percentage of the total histamine concentration or measured in ng/ml. 12 In addition to the direct incubation of basophils with allergens, the incubation of serum from allergic patients with IgE-depleted donor basophils is also possible (passive sensitization of basophil granulocytes). 6, 7 This method was applied to BHRT in two studies with the substances used here. 11, 13 Over the last decades, the use of the basophil activation test has increased compared to the BHRT due to the faster analysis by flow cytometry, and histamine being unstable and difficult to reliably detect. The determination of basophil activation is based on flow cytometric detection of activation markers on basophils. For IgEmediated reactions, the markers CD63 and CD203c have been used.

In the presented studies, CD63 was more often used as an activation marker than CD203c. CD63, a component of granule membranes, is not a basophil-specific marker and is expressed in other blood cells.

Therefore, further labelling is required to identify basophils. Possible markers include anti-CCR3, anti-IgE, anti-CRTH2 (excluding CD3positive cells), CD203c and anti-CD123 (excluding HLA-DR-positive cells). This was the main difference between the tests used. CD203c is a basophil-specific marker that is constitutively expressed. Because the use of different identification markers has little influence on the results, this is not explicitly listed in Tables 1 and 2. CD203c and CD63 are upregulated after IgE receptor aggregation, but have partially different metabolic pathways and follow different kinetics. Interleukin-3 potentiates allergen-induced CD63 expression without itself upregulating CD63, whereas it increases CD203c expression even in the absence of allergen ('priming' marker). The results of basophil activation tests are usually expressed as percent activated basophils, and occasionally as mean fluorescence intensity (MFI). Threshold values or stimulation indices are given for the individual allergens in the commercially available tests; otherwise, they must be calculated using receiver operator characteristic (ROC) curves. 6 The adenovirus vector-based COVID-19 vaccines contain the chimpanzee adenovirus vector including the gene of the glycoprotein spike (S) antigen of SARS-CoV-2, L-histidine, L-histidine hydrochloride monohydrate, magnesium chloride hexahydrate, polysorbate 80 (E 433), ethanol, sucrose, sodium chloride and disodium edetate (Vaxzevria, AstraZeneca; AZ-vaccine), or recombinant, replication-incompetent adenovirus type 26 encoding a stabilized variant of the SARS-CoV-2 spike (S) protein, citric acid monohydrate, trisodium citrate dihydrate, ethanol, 2-hydroxypropylβ-cyclodextrin (HBCD), polysorbate 80 and sodium chloride (Janssen COVID-19 Vaccine, Johnson and Johnson; J-vaccine). 15, 16 The inactivated Chinese SARS-CoV-2 vaccine (CoronaVac, Sinovac; S-vaccine) contains inactivated SARS-CoV-2 virus, aluminium hydroxide, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate and sodium chloride. 17 An overview of allergies and COVID-19 vaccines including possible triggers can be found in an ENDA/EAACI position paper. 18 Among the ingredients in COVID-19 vaccines, PEGs are deemed to be the possible culprit of anaphylactic reactions. Cross-reactivity with polysorbate 80 has been discussed. 1, 3, 4, 19 For this reason, these compounds in particular were investigated in cellular tests. Among other excipients with allergenic potential in COVID-19 vaccines, disodium EDTA and trometamol are mentioned, but they are not among the tested substances listed in Table 2 . 15 

Before the introduction of the COVID-19 vaccine, cellular tests (either BHRT or BAT) were performed in 10 patients with PEG or polysorbate 80 allergy (BHRT: n = 2; BAT: n = 8) with positive results in five cases. 13, 20, 21, 22, 23, 24, 25, 26 Only in two manuscripts, data about controls with negative results were published. 13,24

Positive results were found with the culprit substances containing PEG 3350, PEG 4000, PEG 6000 and PEG 8000, as well as those with PEG 1500, PEG 3350, PEG 4000 and PEG 6000. In one case, BAT with polysorbate 80 was also positive. For details see Table 1 .

Unfortunately, details of the concentrations used were not always provided. However, information about PEG 3350 used at 10% and PEG 6000 at 100%, 13, 20 PEG 3000 at 100%, 20 PEG 6000 at 1:100 000 dilution, 23 PEG 4000 at 1%, 24 as well as drugs containing PEG 4000 in different dilutions 25, 26 can be found in the cases with positive results. In another case, PEG 1500 and PEG 6000 were used at concentrations from 5 ng/ml to 500.000 ng/ml and PEG 4000 at concentrations ranging from 1.5 ng/ml to 500 mg/ml. 26 Polysorbate 80 was positive at 0.02 mg/ml in one case. 24 

%CD63 or %CD203c activation ranged from 14.9% to 75.9%, and maximum histamine release was 25 ng/ml. For details see Table 1 .

Detailed studies involving preincubation with monovalent ethylene glycol, diethylene glycol and omalizumab, which bound IgE antibodies, and a passive-positive BHRT demonstrated an IgE-dependent mechanism in one case. 13 

In 2021, significantly more cellular tests and components were used in individuals with planned COVID-19 vaccinations and suspected allergy to the ingredients of the vaccines to clarify suspected allergic reactions after COVID-19 vaccinations. In summary, 31 positive results were reported (Table 2 ).

Published data from over 100 patients are available to date, with BAT used predominantly. Eighteen patients with a diagnosed PEG allergy and 91 patients with suspected allergic reactions to COVID-19 vaccination were assessed. In addition, results from approximately 50 controls were available. For details see Table 2 . Other components used were polysorbate 80, polysorbate 20, poloxamer 407 and SARS-CoV-2 spike peptides.

In most studies, BNT-vaccine was used in the BAT at four concentrations ranging from 0.05 µg/ml to 10 µg/ml, 27 0.01 µg/ml to 10 µg/ml 28 or 0.18 µg/ml to 22.7 µg/ml. 26 In two studies, only one concentration for mRNA vaccines was used (0.007 µg/µl and 1 µl, respectively). 8, 9 Best positive results were found for concentrations around 10 µg/ml. In BHRT, the BNT-vaccine was used at six concentrations. 29 M-vaccine was used in only two studies with a concentration of 0.007 µg/µl 8 or a range from 0.36 µg/ml to 45.45 µg/ml. 26 In BHRT, M-vaccine was used at six concentrations. 29 AZ-vaccine was used in a series of dilutions ranging from 1:2000 to 1:10 27 or from undiluted to 1:125 (division by 4.4 to calculate the final concentration). 26 In BHRT, AZ-vaccine was used at six concentrations. 29 The J-vaccine was used in only one study, ranging from undiluted to 1:125 dilution (division by 4.4 to calculate the final concentration). 26 S-vaccine was used in dilutions of 1:10 and 1:100. PEGylated Doxorubicin 2000-3500 was used in two studies, concentration ranging from 1 µg/ml to 10 µg/ml. 10,27

Polyethylene glycol 200, PEG 400 and PEG 600 were used at a concentration of 5 mg/ml in one study 27 in the BAT, PEG 300 and PEG 3000 at concentrations of 0.0001 mg/ml to 10 mg/ml in the BHRT. 11 PEG 3350 was used in the BAT at concentrations ranging from 0.6 mg/ml to 15 mg/ml, 26 PEG 4000 at concentrations from 0.08 mg/ml to 4 mg/ml 31 or 0.036 mg/ml to 15 mg/ml, 26 and PEG 6000 at concentrations from 0.6 mg/ml to 15 mg/ml. 26 PEG 3350, PEG 6000 and PEG 20,000 were used at concentrations of 0.0001 mg/ml to 10 mg/ml in the BHRT. 11 

Polysorbate 80 was used in two studies at concentrations of 1 µg/µl 8 and 22.7 mg/ml (0.23%) and 2.3 mg/ml (0.023%). 26 Polysorbate 20 and Poloxamer 407 were only used in the BHRT. 11,29 SARS-CoV-2 spike peptides were used at dilutions of 1:100 and 1:1000. 30

Vaccines: In clearly diagnosed PEG-allergic patients, maximal CD63% activation in one study with three patients was 51%, 64.2% and 82.1%, 27 and 21.3%, 34.4% and 37.2% in another study with the BNT-vaccine. 26 In one study with BNT-vaccine, the results in patients supposed to be allergic were expressed in stimulation indices (SI) with values of 2.88, 3.1, 3.19 and 4.79. 28 With the M-vaccine in clearly diagnosed PEG patients, values of 16.1%, 20.5%, 34.8% and 41.8% were found. 26 In mRNA-vaccine-allergic patients, a study with 13 patients revealed values from 9% to 56%, but it was not indicated which of the two mRNA vaccines was used in each individual case. 8 In another case, the value for the vaccine in the BAT was 23.3%. 9 A study of one vaccine-reactive patient with values >15 ng/ml histamine release using BNT-vaccine, M-vaccine and AZ-vaccine was published. 29 Polyethylene glycol 2000 and derivatives: PEGylated doxorubicin was found to be positive in three PEG-allergic patients with maximal values of 22.3%, 31.6% and 35.4%. 27 In 12 out of 13 mRNA-vaccine suspected allergic patients, DMG-PEG 2000 induced values from 10% to 73%. 8 PEG 2000 SI of 3.1. and 4.57 were found in two BNT-vaccine suspected allergic patients. 28 For PEG 4000, maximal CD63% activations were 14.79% and 16.2% (one patient with reaction to BNT-vaccine and one PEG-allergic patient, respectively) and 35.8% for PEG 6000 in one PEG-allergic case. 26, 31 In the BHRT, four PEG patients were positive for other PEGs, one for PEG 20 000, one for PEG 3000, 6000 and 20 000 (additionally also for poloxamer 407), one for PEG 3350 and 6000 and one for PEG 3000 and 6000. 11 Polysorbate 80 was negative in 19 PEG-confirmed patients. 8, 11, 26 The details can be found in Table 2 , with a summary of the most important results in Table 3 . 

Nevertheless, it can be observed that in PEG-allergic patients, cellular tests with mRNA-vaccine showed clear positive results. The BNTvaccine contains 50 µg/dose PEG lipids. 5 The best concentration of the whole BNT-vaccine giving positive results in the BAT was around 10 µg/ml tozinameran. 8 Polysorbate 80 was negative in recent cellular tests in PEGallergic patients (Table 2 ). Therefore, cross-sensitization does not seem to be relevant.

Positive results with mRNA vaccines in cellular tests seem to indicate a PEG allergy, but confirmation with provocation tests was only performed in a minority of studies. It was assumed that the PEG conformation on the surface of nanoparticles results in an increased avidity augmenting IgE-crosslinking on the surface of basophils. 27 Furthermore, it was postulated that only repetitive presentation of the structure in the form of a polymer chain induces a biological response. 13 Due to previous and actual experiments in this context, an IgE-mediated mechanism for PEG allergy can be assumed. In a PEG-allergic patient, Wenande et al. (2013) showed that passive sensitization of IgE-stripped donor basophils with patient serum, and subsequent challenge with PEG 6000 and TA B L E 3 Summary of substances most often positive in cellular tests in patients with suspected allergy to PEG or COVID-19 vaccines (for details, see Table 2 ) 13 In a basophil histamine release inhibition study, PEG 3350 and PEG 6000-induced histamine release were abolished by preincubation with a monomer or dimer. This inhibition appeared to be antigen-specific, as anti-IgE-induced histamine release remained unchanged after preincubation with the monomer as well as dimer. These results strongly indicate that serum factors in the patient's blood, possibly IgE antibodies, may bind monovalent ethylene glycol. 13 Similar results were found in mice sensitized to PEGylated asparaginase (PEG-MW = 5 kDa) and pre-treated with PEG 400 Da. 32 Wortmannin experiments also confirmed that basophil activation by PEG or BNT-vaccine was mediated by IgE. 28 Some authors have claimed that a non-IgE-mediated mechanism is the cause of basophil activation, because IgE against PEG could not be found in the serum. 10, 28 The lack of detection could be due to methodological problems in these assays, because a new dual cytometric bead assay (using PEGylated products) was able to demonstrate that samples of patients with PEG-associated anaphylaxis were clearly positive for anti-PEG-IgE. 33 Direct basophil activation by the vaccines as a mechanism can possibly be excluded because of the vast majority of negative results in controls (Table 2) .

The definition of a threshold for positivity in the BAT was very different in the various studies, ranging from >4% to >15% with different 'other' conditions (>25% of the positive controls or use of a stimulation index). ROC curves were performed in one study with PEG 2000

and BNT-vaccine but were based on a very low number of positive results. 28 Even if in most studies with these vaccines, vaccinated and unvaccinated controls were negative in the cellular tests, one problem was the positive results observed in half of the controls due to a previous SARS-CoV-2 infection (vaccinated and non-vaccinated) in one study. 28 In the BHRT, a marginally positive result was also found in a control with a previous SARS-CoV-2 infection. 29 

The authors declare no conflict of interest. 

None. 

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Do basophil activation tests help elucidate allergic reactions to the ingredients in COVID-19 vaccines?