Evaluation of the IgE cross-reactions among vespid venoms. A possible approach for the choice of immunotherapy
Giovanni Passalacqua MD
Allergy and Respiratory Diseases
Department of Internal Medicine
L.go R. Benzi 10
Background: Hymenoptera venom allergy can be effectively cured with specific immunotherapy, thus the correct identification of the allergen is essential. In the case of multiple skin and serum positivities it is important to know if a cross-reaction among venoms is present. We studied by CAP-inhibition assays the degree of cross-reactivity between Vespula vulgaris and Polistes dominulus.
Methods: Serum samples were obtained from consecutive patients with a clinical history of grade III–IV reactions to hymenoptera sting and with nondiscriminative skin/CAP positivity to both Vespula and Polistes. Inhibition assays were carried out with a CAP method, incubating the sera separately with both venoms and subsequently measuring the specific immunoglobulin E (IgE) to venoms themselves.
Results: Forty-five patients (33 male, mean age 40 years, age range 12–74, total serum IgE 242 ± 168 kU/l) were included. Their specific IgE to Vespula and Polistes were 12.03 ± 5.70 kU/l and 10.7 ± 2.0 kU/l (P = NS), respectively. At the CAP-inhibition assays, in 25 patients a >75% heterologous inhibition by P. dominulus venom against V. vulgaris-specific IgE was found. In six subjects V. vulgaris venom effectively inhibited the P. dominulus-specific IgE. In the remaining 14 cases the CAP-inhibition test provided intermediate and not discriminative results.
Conclusion: In 31/45 patients, the double sensitizations to venoms were probably the result of cross-reactions and the CAP-inhibition allowed identifying the true double sensitizations. This approach may be helpful for the correct prescription of immunotherapy in the case of V. vulgaris and P. dominulus double positivity.
Hymenoptera venom hypersensitivity is sustained by a virtually ‘pure’ Immunoglobulin E (IgE)-mediated reaction, where the inflammatory process is absent or negligible. In this situation, specific immunotherapy (SIT) is highly effective, and the allergen-specificity of the vaccine plays a crucial role. In clinical practice, the identification of the causal venom for prescribing SIT is essentially based on the clinical history, the identification of the stinging insect, the skin prick (and intradermal) test and the venom-specific IgE measurement (1, 2). As a matter of fact, some patients display multiple skin and/or serum positivities, so that multiple extracts for immunotherapy are usually prescribed and administered. Obviously, this may result in increased costs, increased risks of adverse events and, possibly, in sensitization to new allergens. On the other hand, the multiple positivities may be due, at least in some cases, to cross-reactions among components of venoms that belong to different hymenoptera species (3, 4), so that vaccination with one single venom could suffice. Cross-reactions may, in fact, occur among Vespidae, (e.g. Polistes and Vespula)(5), but they have also been described between bees and Vespidae (6).
The cross-reactivity between the venoms of Vespula vulgaris and Polistes dominulus (European paper wasp) has not been investigated yet. Thus, we attempted to identify and to quantify such cross-reactivity by means of CAP-inhibition assays in patients with multiple sensitizations to those hymenoptera.
Material and methods
Consecutive adult patients, referred to our service (between June 2004 and September 2006) for hymenoptera venom allergy, and with skin/serum positivity to both Vespula and Polistes were studied. The clinical aspects and severity of reactions to stings were graded according to Mueller (7).
The routine diagnosis involved skin tests and CAP-assay. Prick tests were performed with standardized extracts at increasing concentrations from 0.01 to 100 μg/ml, whereas intradermal tests involved the injection of 0.02 ml extract at 0.001 to 1 μg/ml concentrations. The tests were carried out with extracts of Apis mellifera, Vespula sp. (Stallergènes, Milan, Italy), Polistes dominulus and Vespa crabro (Anallergo, Florence, Italy). The results were read and scored according to current recommendations (2, 8). Serum specific IgE were assayed with an automated immuno-fluorimetric method (CAP-FEIA System, Unicap 100; Phadia, Uppsala, Sweden) (9) for the same allergens listed above. Two 5 ml serum aliquots from each patient were collected and stored at −20°C for the CAP-inhibition assays, which were carried out in a single session.
The inhibition assays were performed according to Straumann et al. (6), thus a specific IgE level >1 kU/l was required. Briefly, two 100 μL aliquots of patient's serum were incubated separately for 12 h at 4°C with 200 μl of P. dominulus or Vespula venoms at increasing dilutions (0; 0.3; 3.0; 300 μg/ml). Subsequently, specific IgE against each of the venoms were determined in the samples prepared as above. The IgE calibration curve (Phadia) followed the same dilution (sample diluent Phadia), incubation times and temperatures of each sample. The CAP-inhibition test was carried out with a specific programme in UniCap 100 (Phadia). The extent of homologous (blockage of venom-specific IgE by the same venom) and heterologous (blockage of the venom-specific IgE by the other venom) inhibition was computed with the following formula: %inhibition = 100−[IgE inhibited sample (kU/l) × 100/IgE anti-venom (kU/l) at zero concentration of venom]. A percentage of heterologous inhibition ≥75% was considered strongly suggestive of cross-reactivity. In order to determine the precision of the inhibition test, two pool of sera in 12 replicates were assayed with different calibration curves at two different concentrations of IgE: 0.35 and 1.0 kU/l (6). The results read were 0.32 ± 0.033 and 0.97 ± 0.056 kU/l, respectively, with a coefficient of variation below 10%
Forty-five patients (33 male, mean age 40 years, age range 12–74 years, total serum IgE 242 ± 168 kU/l) were included. They had a consistent clinical history of hymenoptera venom allergy, with grade III–IV reactions according to Mueller (7). All these patients displayed a concomitant sensitization to Vespula and P. dominulus at skin tests and CAP assay, but the results were not discriminative. The values of specific IgE (mean ± SEM) to Vespula and Polistes were 12.03 ± 5.70 kU/l and 10.7 ± 2.0 kU/l (P = NS), respectively. Some of the subjects were also positive to honeybee or Vespa crabro, but their sensitizations were not clinically relevant or did not represent a diagnostic problem for the choice of the vaccine. The detailed results of the CAP assays are shown in Table 1.
Table 1. Hymenoptera venom-specific IgE in the patients’ population
In the majority of patients the preincubation with the venom of V. vulgaris inhibited the V. vulgaris-specific IgE, and the same happened with the venom of P. dominulus for the Polistes-specific IgE (CAP-inhibition >90%), as expected. When the assays were performed by preincubation with the heterologous venom, in 25 patients the P. dominulus venom inhibited the V. vulgaris-specific IgE by 75% or more. Based on this result, these patients were prescribed a vaccine for P. dominulus. In six subjects the opposite happened, as the preincubation with V. vulgaris venom inhibited more than 75% of the P. dominulus-specific IgE. These patients received vaccination only for Vespula. In the remaining 14 cases the CAP-inhibition test provided intermediate results and both venoms were used for vaccination. The results of the CAP-inhibition assays are summarized in Table 2.
Table 2. Percentages of inhibition at the CAP assay, computed with the following formula: % inhibition = 100−(IgE inhibited sample ×100/IgE anti-venom at zero concentration
The cross-reactivity among different allergens is quite common and occurs, in fact, with vegetables, pollens and drugs (10, 11). The same happens with hymenoptera venom, where cross-reactions have been described within different species of vespids and even between vespids and bees (5, 6). Also in the case of hymenoptera, the cross reactivity may lead to multiple diagnostic positivities (e.g. skin tests or CAP assay), with the consequent prescription of multiple vaccines, also when one single immunotherapy would be appropriate and sufficient. The problem is of particular clinical relevance with Vespidae sp In fact, in the Mediterranean regions, the specific reactivity to P. dominulus/gallicus (European paper wasp) has been reported to occur frequently (12). Thus, the correct diagnosis of allergy to one or another wasp is important for the subsequent prescription of immunotherapy. In our clinical experience, the positivity to both P. dominulus and Vespula is quite frequent, and the appropriate vaccine cannot be usually chosen on the basis of skin test and CAP assay results. For this reason we undertook this study, to identify and quantify the cross-reactivity among the two venoms. In other words, we attempted to define if a patient had a true double sensitization or if the skin and serum positivities were because of cross-reactions (13). The study was carried out by a CAP-inhibition assay, which is a very sensitive technique, largely utilized for the characterization of hymenoptera allergens (14). In this procedure, the patient's serum is incubated with the two venoms separately, then the specific IgE against the two venoms are measured. A high rate of inhibition means that the majority of the specific IgE has been bound by the venom previously added to the sample.
The results of the CAP-inhibition assays demonstrated that the venom of P. dominulus effectively bound the Vespula-specific IgE in 25 subjects (56% of cases), and the opposite happened in six patients (13%). Thus, out of 45 subjects who had been vaccinated with both venoms, 31 could receive only one immunotherapy. In this exploratory study we could not identify the exact nature of the cross-reactive epitope, although it is conceivable that part of the cross-reactivity is because of carbohydrate determinants, as previously described for honeybee and yellow jacket (15, 16). Certainly, the clinical evaluation remains the basic criteria, but in selected cases, the CAP-inhibition can be regarded as an useful tool to better detail the diagnosis and the consequent therapeutic approach.