Coordinated IgG2 and IgE responses as a marker of allergen immunotherapy efficacy.

BACKGROUND: IgG2 responses are associated with repeated antigen exposure and display highly mutated variable domains. A recent study highlighted a role of IgG2+ memory B cells and allergen-specific IgG2 levels after a 3rd consecutive pre-seasonal sublingual allergen immunotherapy (AIT) with grass pollen tablet. Herein, we aim to explore changes in allergen-specific IgG2 in individuals undergoing house dust mite immunotherapy (HDM-AIT) and explore whether the interrelationship with other humoral responses (i.e., IgG4 and IgE) may discriminate between high and low responders. METHODS: Levels of serum Dermatophagoides pteronyssinus and Dermatophagoides farinae-specific IgG2, IgG4, and IgE antibodies were measured by ELISA or ImmunoCap in a sub-group of individuals enrolled in a randomized, double-blind, placebo-controlled, sublingual AIT study evaluating the safety and efficacy of a 300 IR HDM tablet. RESULTS: After 1-year sublingual AIT, HDM-specific serum IgG2 responses increase mostly in high versus low responders and are distinctive according to the clinical benefit. Higher correlation between HDM-specific IgG2, IgE, and/or IgG4 responses is seen in subjects benefiting the most from HDM-AIT as indicated by changes in Average Total Combined Scores. More strikingly, statistically significant correlation between HDM-specific IgG2 and IgE responses is only observed in individuals stratified as high responders. CONCLUSIONS: We provide evidence for coordinated serum immune responses upon AIT in HDM-allergic subjects exhibiting high clinical benefit when compared with low responders. Assessing HDM-specific IgE, IgG2, and IgG4 in serum could be used as follow-up combined markers to support decision as to AIT continuation and/or adaptation.

Structural and Functional Characterization of the Major Allergen Amb a 11 from Short Ragweed Pollen.

Allergy to the short ragweed (Ambrosia artemisiifolia) pollen is a major health problem. The ragweed allergen repertoire has been recently expanded with the identification of Amb a 11, a new major allergen belonging to the cysteine protease family. To better characterize Amb a 11, a recombinant proform of the molecule with a preserved active site was produced in Escherichia coli, refolded, and processed in vitro into a mature enzyme. The enzymatic activity is revealed by maturation following an autocatalytic processing resulting in the cleavage of both N- and C-terminal propeptides. The 2.05-Å resolution crystal structure of pro-Amb a 11 shows an overall typical C1A cysteine protease fold with a network of molecular interactions between the N-terminal propeptide and the catalytic triad of the enzyme. The allergenicity of Amb a 11 was confirmed in a murine sensitization model, resulting in airway inflammation, production of serum IgEs, and induction of Th2 immune responses. Of note, inflammatory responses were higher with the mature form, demonstrating that the cysteine protease activity critically contributes to the allergenicity of the molecule. Collectively, our results clearly demonstrate that Amb a 11 is a bona fide cysteine protease exhibiting a strong allergenicity. As such, it should be considered as an important molecule for diagnosis and immunotherapy of ragweed pollen allergy.

Expression and characterization of natural-like recombinant Der p 2 for sublingual immunotherapy.

BACKGROUND: Recombinant allergens with a native conformation represent an alternative to natural extracts for immunotherapy and diagnostic purposes. METHODS: We produced the Der p 2 mite allergen in Pichia pastoris and Escherichia coli. After purification by cation exchange chromatography, recombinant molecules were compared to their natural counterpart based upon structural (disulfide bonds, secondary structure, thermal stability) and immunological properties (antibody reactivity, basophil and T cell activation, tolerance induction in a murine sublingual immunotherapy model). RESULTS: The Der p 2.0101 isoform was confirmed to be prevalent in Dermatophagoides pteronyssinus extracts. It was then produced as a secreted molecule in P. pastoris or refolded from E. coli inclusion bodies. The yeast-expressed rDer p 2 molecule exhibits a natural-like disulfide bridge distribution and secondary structure, whereas the E. coli-derived rDer p 2 presents some heterogeneity in cysteine bonds and a lower stability following thermal stress. The two recombinant as well as natural Der p 2 molecules exhibit comparable IgE recognition and activate basophil and CD4+ T cells. Sublingual immunotherapy of nDer p 2- sensitized mice using either one of the rDer p 2 molecules efficiently decreases airway hyperresponsiveness as well as Th2 responses. CONCLUSIONS: Natural and recombinant Der p 2 molecules produced in P. pastoris and E. coli exhibit comparable immunological properties despite distinct structural features. Natural-like cysteine pairing is a critical parameter to identify stable, well-folded and homogenous proteins appropriate for immunotherapy and diagnostic purposes.

Efficacy of sublingual vectorized recombinant Bet v 1a in a mouse model of birch pollen allergic asthma.

BACKGROUND: Second generation sublingual allergy vaccines based upon recombinant allergens combined with vector systems are being developed as an alternative to conventional allergen extracts. Herein, we evaluated the efficacy of a recombinant form of the major allergen Bet v 1a (rBet v 1a) formulated as a mucoadhesive particle in a preclinical model of birch pollen (BP) respiratory allergy. MATERIALS AND METHODS: BALB/c mice were sensitized to BP extracts by intraperitoneal injections followed by aerosol exposures. Sensitized mice underwent sublingual immunotherapy (SLIT) twice a week for eight weeks with either a BP extract or rBet v 1a formulated in amylopectin-based microparticles (MPA). SLIT efficacy was assessed using whole body plethysmography, lung histology and cell counts in broncho-alveolar lavages (BAL) as read outs. BP and/or rBet v 1a-specific T cell and antibody responses were monitored in lung and serum, respectively. IgA levels were measured in saliva. RESULTS: Mice sensitized to BP exhibit chronic airway hyperresponsiveness (AHR), lung inflammation (documented by compliance and resistance measurements), eosinophil infiltrates in BAL, as well as Bet v 1-specific Th2 biased responses. Both SLIT with soluble rBet v 1a (50µg/dose) and BP extract (equivalent to 50µg rBet v 1 per dose) lead to a significant reduction in AHR, lung eosinophilia and Th2 responses. A sub-optimal dose of 5µg of rBet v 1a displays a similar level of efficacy with a significant decrease of Th2 responses when formulated with MPA microparticles. In addition, allergen vectorization with mucoadhesive particles allows a faster reduction in AHR in sensitized animals. CONCLUSION: We demonstrate in a murine model of chronic BP respiratory allergy the efficacy of SLIT with vectorized rBet v 1a. Thus, combining recombinant allergens with mucoadhesive vector systems paves the ground for improved second generation sublingual allergy vaccines.

Improvement of sublingual immunotherapy efficacy with a mucoadhesive allergen formulation.

BACKGROUND: Sublingual immunotherapy is a noninvasive and efficacious treatment of type I respiratory allergies. A murine model of sublingual immunotherapy is needed to understand better the immune mechanisms involved in successful immunotherapy and to assess second-generation candidate vaccines. OBJECTIVE: Herein, we developed a therapeutic murine model of sublingual immunotherapy in which we document the value of mucoadhesive formulations to enhance treatment efficacy. METHODS: BALB/c mice were sublingually treated with soluble or formulated ovalbumin before or after sensitization with ovalbumin. Airways hyperresponsiveness and lung inflammation were assessed by whole-body plethysmography and lung histology, respectively. Humoral and cellular immune responses were monitored by ELISA and ELISPOT techniques. RESULTS: Prophylactic sublingual administration of ovalbumin completely prevents airways hyperresponsiveness as well as IL-5 secretion and IgE induction. Therapeutic administration of ovalbumin as a solution via either the sublingual or oral route has a limited efficacy. In contrast, sublingual application of ovalbumin formulated with maltodextrin to enhance mucosal adhesion results in a major reduction of established airways hyperresponsiveness, lung inflammation, and IL-5 production in splenocytes. This mucoadhesive formulation significantly enhances ovalbumin-specific T-cell proliferation in cervical but not mesenteric lymph nodes, and IgA production in the lungs. CONCLUSION: A mucoadhesive maltodextrin formulation of ovalbumin enhances priming of the local mucosal immune system and tolerance induction via the sublingual route. CLINICAL IMPLICATIONS: Mucoadhesive formulations offer the opportunity to improve dramatically sublingual immunotherapy in human beings, most particularly by simplifying immunization schemes.