Marie-Françoise Drumare

Capacity of purified peanut allergens to induce degranulation in a functional in vitro assay: Ara h 2 and Ara h 6 are the most efficient elicitors

Background Peanut is a most common and potent food allergen. Many peanut allergens have been characterized using, in particular, IgE‐binding studies.

Immunological and Metabolomic Impacts of Administration of Cry1Ab Protein and MON 810 Maize in Mouse

We have investigated the immunological and metabolomic impacts of Cry1Ab administration to mice, either as a purified protein or as the Cry1Ab-expressing genetically modified (GM) MON810 maize. Humoral and cellular specific immune responses induced in BALB/cJ mice after intra-gastric (i.g.) or intra-peritoneal (i.p.) administration of purified Cry1Ab were analyzed and compared with those induced by proteins of various immunogenic and allergic potencies. Possible unintended effects of the genetic modification on the pattern of expression of maize natural allergens were studied using IgE-immunoblot and sera from maize-allergic patients. Mice were experimentally sensitized (i.g. or i.p. route) with protein extracts from GM or non-GM maize, and then anti-maize proteins and anti-Cry1Ab–induced immune responses were analyzed. In parallel, longitudinal metabolomic studies were performed on the urine of mice treated via the i.g. route. Weak immune responses were observed after i.g. administration of the different proteins. Using the i.p. route, a clear Th2 response was observed with the known allergenic proteins, whereas a mixed Th1/Th2 immune response was observed with immunogenic protein not known to be allergenic and with Cry1Ab. This then reflects protein immunogenicity in the BALB/c Th2-biased mouse strain rather than allergenicity. No difference in natural maize allergen profiles was evidenced between MON810 and its non-GM comparator. Immune responses against maize proteins were quantitatively equivalent in mice treated with MON810 vs the non-GM counterpart and no anti-Cry1Ab–specific immune response was detected in mice that received MON810. Metabolomic studies showed a slight “cultivar” effect, which represented less than 1% of the initial metabolic information. Our results confirm the immunogenicity of purified Cry1Ab without evidence of allergenic potential. Immunological and metabolomic studies revealed slight differences in mouse metabolic profiles after i.g. administration of MON810 vs its non-GM counterpart, but no significant unintended effect of the genetic modification on immune responses was seen.

Trypsin resistance of the major peanut allergen Ara h 6 and allergenicity of the digestion products are abolished after selective disruption of disulfide bonds

SCOPE 2S-albumins Ara h 2 and Ara h 6 are the most widely recognized and potent allergens for peanut-allergic patients. These allergens are particularly resistant to proteolysis and the digestion products generally retain significant allergenicity. Five disulfide bridges (DB) stabilize Ara h 6 overall structure and their influence on the trypsin resistance and on the allergenicity of the digestion products was investigated. METHODS AND RESULTS Progressive disruption of each DB was performed by site-directed mutagenesis. Successful refolding of Ara h 6 variants was confirmed by circular dichroism. Trypsin resistance, IgE-binding capacity and allergenic potency, as assessed by in vitro mediator release assay with sera from peanut-allergic patients, was not affected by the deletion of the C-terminal DB at Cys(84) -Cys(124) . Additional disruption of DB at Cys(14) -Cys(71) or at Cys(73) -Cys(115) rendered Arg(16/20) or Arg(114) susceptible to trypsinolysis, respectively, but affected principally the IgE-binding capacity of Ara h 6. DB disruption at Cys(26) -Cys(58) or at Cys(59) -Cys(107) led to an extensive proteolytic degradation and a complete loss of allergenic potency of the digestion products. CONCLUSION Selective disruption of the DB stabilizing the protease-resistant core of Ara h 6 eliminated the IgE-binding capacity of the trypsin-degradation products and their ability to trigger mast cell degranulation.

Immunochemical characterisation of structure and allergenicity of peanut 2S albumins using different formats of immunoassays

Proteins of the 2S albumin family, such as Ara h2 and Ara h6, are most frequently involved in peanut allergy. We have developed a reverse enzyme allergo-sorbent test (EAST) in which total serum IgE antibodies are first captured by immobilised anti-human IgE monoclonal antibodies, and then the binding of the anti-Ara h2 and anti-Ara h6 specific IgE to the corresponding labelled allergens is measured. This reverse immunoassay was used either as a direct EAST or as an EAST inhibition assay to study the interactions of whole peanut protein extract and purified Ara h2 and Ara h6 with IgE antibodies from peanut-allergic patients. Finally, we identified some IgE-binding epitopes on Ara h6 using a format of EAST in which the protein is immobilised in a particular, well defined, manner through interactions with specific monoclonal antibodies (mAbs) coated on the micro-plates. The fine specificity of those mAbs has been characterised at the epitope level, and their binding to the allergen thus masks a known particular epitope and makes it unavailable for recognition by IgE antibodies. The reverse EAST increased the ratio specific signal/background. It avoids interferences with competitors such as anti-peanut protein IgG antibodies and allows the study of the specificity and/or affinity of the interactions between IgE antibodies and Ara h2 or Ara h6 with a higher sensitivity and accuracy than the conventional EAST. The EAST results obtained when the allergens are presented by specific mAbs suggest that the homologous molecular domain(s) in peanut 2S albumins encompass major IgE epitope(s) and are strongly involved in peanut allergenicity.

Comparative study of the adjuvanticity of Bacillus thuringiensis Cry1Ab protein and cholera toxin on allergic sensitisation and elicitation to peanut

Abstract We aimed to investigate the adjuvanticity of Cry1Ab on allergic sensitisation and elicitation to peanut in mice, in comparison with cholera toxin (CT), a known mucosal Th2 adjuvant. Balb/c mice were gavaged with phosphate buffer saline (PBS) (control), peanut protein extract (PE) alone or PE co-administered either with CT or Cry1Ab. Peanut-specific IgE, IgG1 and IgG2a and Th1/Th2/Th17 cytokine secretion by splenocytes were assayed. Gavaged mice were further challenged with PE. Markers of the immediate and late phases of the allergic reaction were assayed in bronchoalveolar lavages (BAL) fluids collected 10 minutes or 36 hours after airway challenge, respectively. Sensitisation to peanut was only observed in mice receiving PE plus CT, as demonstrated by specific IgE and IgG1 production in sera and secretion of Th2 (and Th17) cytokines by splenocytes. Following challenge, mice sensitised with PE plus CT produced leukotrienes (LT) E4 and C4, then Th2/Th17-cytokines and showed eosinophil/neutrophil influx in BAL. Interestingly, LT production, Th2/Th17 cytokine release and eosinophil influx were also significant in mice gavaged with PE plus Cry-1Ab, but not with PBS or PE alone. Cry1Ab did not demonstrate adjuvant effects on oral sensitisation to peanut. However, this study shows possible adjuvant properties of Cry1Ab on the elicitation of the allergic reaction, at least in the Balb/c mouse model and in the experimental conditions used.

Goat's milk allergy without cow's milk allergy: suppression of non‐cross‐reactive epitopes on caprine β‐casein

Goats milk (GM) allergy associated with tolerance to cows milk (CM) has been reported in patients without history of CM allergy and in CM‐allergic children successfully treated with oral immunotherapy. The IgE antibodies from GM‐allergic/CM‐tolerant patients recognize caprine β‐casein (βcap) without cross‐reacting with bovine β‐casein (βbov) despite a sequence identity of 91%. In this study, we investigated the non‐cross‐reactive IgE‐binding epitopes of βcap.

Specificity of IgE antibodies from patients allergic to goat's milk and tolerant to cow's milk determined with plasmin-derived peptides of bovine and caprine β-caseins

SCOPE Despite a sequence homology of 90% between bovine and caprine β-caseins (CN), IgE antibodies from patients allergic to goats milk (GM), but tolerant to cows milk (CM), recognize caprine β-CN without cross-reacting with bovine β-CN. We investigated this lack of cross-reactivity by evaluating the IgE-reactivity toward peptides isolated from plasmin hydolysates of bovine and caprine β-CN. METHODS AND RESULTS The IgE-binding capacity of plasmin-derived peptides was evaluated with sera from 10 CM-allergic patients and 12 GM-allergic/CM-tolerant patients. In CM-allergic patients, IgE reactivity of caprine fragments (f29-107) and (f108-207), but not (f1-28), was similar to that of the bovine counterparts. In contrast, all bovine fragments were poorly recognized by IgE antibodies from GM-allergic/CM-tolerant patients. The peptide (f29-107) was generally the most immunoreactive fragment of caprine β-CN. By using synthetic peptides, the immunodominant IgE-binding epitope recognized by most GM-allergic/CM-tolerant patients was located in the caprine domain 49-79. CONCLUSION The restricted specificity of the IgE response toward the caprine β-CN in GM-allergic/CM-tolerant patients is mainly directed against the domain 49-79, which differs from its bovine counterpart by only three amino acid substitutions.

Comparison of the immune response induced in mice experimentally sensitized with genetically modified MON810 maize vs its conventional counterpart

Background The introduction on the market of genetically modified (GM) foods has raised the question of the assessment of the potential allergenicity of the newly expressed protein (s) and of the whole GM food. We aimed at comparing the immune responses induced in mice after experimental sensitization with the insect resistant GM maize MON810 expressing the Cry1Ab protein vs its conventional counterpart.