State of the art in developing allergen vaccines in Cuba: prospects of novel adjuvanted vaccines.

Standardized allergen vaccines have been developed and registered as biopharmaceutical products in Cuba. Three different vaccines were obtained from the most relevant allergenic mite species: Dermatophagoides pteronvssinus, Dermatophagoides siboney, and Blomia tropicalis. Immuno-analytical methods based on murine monoclonal antibodies and human IgE antibodies were developed for assessing allergenic potency, composition, and stability. Preclinical and clinical studies showed efficacy and safety in diagnostic prick-tests and subcutaneous immunotherapy in asthmatic patients. New approaches are now undertaken in order to develop new adjuvanted formulations based on liposomes or proteoliposomes from Neisseria meningitidis, and purified allergens; aiming to overcome the drawbacks of conventional immunotherapy.

Monoclonal antibodies against Blo t 13, a recombinant allergen from Blomia tropicalis.

BACKGROUND: The recombinant allergen of Blomia tropicalis, rBlo t 13, shows 11% of IgE reactivity to sera from allergic patients. This allergen belongs to the fatty acid-binding protein family and its natural equivalent remains to be isolated. Monoclonal antibodies (MAbs) are important tools for specific determination and isolation of natural allergens as well as for characterization of recombinant proteins. METHODS: Mice were immunized with partially purified preparation of rBlo t 13 allergen expressed in the yeast Pichia pastoris. Spleen cells were fused with myeloma cells using polyethylene glycol. Hybridoma screening was performed using a direct ELISA with recombinant allergen. MAb specificity to rBlo t 13 was tested by immunoblotting. Topography of binding sites and binding of MAb to native allergen was studied by ELISA. Reactivity of MAb against allergenic extract of B. tropicalis and Dermatophagoides siboney was analyzed by ELISA inhibition. In addition, the reactivity of MAbs against rBlot 13 from Escherichia coli and P. pastoris expression was compared. RESULTS: Two MAbs, 5G3 and 3G4 with IgG1 isotype, were generated. These MAbs specifically recognized the 16-kD band, which corresponds to the molecular weight shown by rBlo t 13 on SDS-PAGE. In ELISA, the binding of 5G3 MAb to B. tropicalis and D. siboney extracts was inhibited by rBlo t 13. Both MAbs showed the highest reactivity when the allergen was expressed in P. pastoris. CONCLUSION: Two MAbs specific for Blo t 13 were obtained. These MAbs recognized the same or close epitopes on the rBlo t 13 molecule. The occurrence of homologous allergens to Blo t 13 in D. siboney is suggested by the ELISA inhibition assay.