Immunogenicity of Phleum pratense depigmented allergoid vaccines: experimental study in rabbits.

BACKGROUND: Immunogenicity studies are based on accurate preclinical and clinical assessment of pharmaceutical products. The immunogenicity of modified allergen vaccines has not been fully elucidated, and the mechanisms involved are not well understood. Animal and human models have recently shown that depigmented allergoids induce specific immunoglobulin (Ig) G against individual allergens, thus supporting the clinical efficacy of these vaccines. OBJECTIVE: The aim of this study was to investigate the production of specific IgG against individual antigens and their isoforms in rabbits injected with depigmented allergoid extracts of Phleum pratense pollen. METHODS: Two New Zealand rabbits were immunized with depigmented-polymerized extracts adsorbed onto aluminum hydroxide (Depigoid) of P pratense. Rabbits were injected 3 times (35 microg Phl p 5). Specific IgG titers against native, depigmented, and depigmented-polymerized extracts and individual allergens (rPhl p 1 and rPhl p 5a) were analyzed by direct enzyme-linked immunosorbent assay. The capacity of these synthesized antibodies to recognize individual native and depigmented allergens and different isoforms was evaluated by immunoblot and 2-D analysis. RESULTS: All rabbits produced high titers of specific IgG against the 3 extracts. Rabbits injected with depigmented allergoids produced similar specific antibody titers against native, depigmented, and depigmented-polymerized extracts. Serum samples recognized individual allergens and their isoforms in the nonmodified extracts. CONCLUSION: Vaccines containing depigmented allergoid extracts of P pratense induce immunogenicity in vivo. The antibodies produced after injection of these extracts clearly recognized allergens and different isoforms in their native configuration.

Comparison of skin-prick test assay and reverse enzyme immunoassay competition (REINA-C) for biological activity of allergens.

An in vitro technique for measuring the relative potency of allergenic extracts has been developed and compared with the dose-response skin-prick test. The in vitro technique is a modification of an ELISA based on the method of antigen capture using polystyrene microtitre wells as solid phase (REINA-competition) and is also based on a dose-response. Both methods have been applied to allergen extracts of Dermatophagoides pteronyssinus, Phleum pratense, Secale cereale, Lolium perenne, Plantago lanceolata, Artemisia vulgaris, Salsola kali and Parietaria judaica. For each allergen, the slope of the regression log-log dose-response lines displayed by skin-prick test and REINA-C does not show a statistically significant difference, being parallel (P < 0.001), and indicating that the allergen-ligand binding kinetics are identical in both methods.

Estimation of IgE antibodies to the common allergens by reverse enzyme immunoassay. Comparison with the radioallergosorbent test.

A reverse enzyme immunoassay (REINA) is described, in which polystyrene microtiter wells are sensitized with murine monoclonal anti-human IgE, and then sequentially allowed to react with patient's serum, peroxidase-labeled allergens and substrate. The results obtained with the sera of patients allergic to Lolium perenne grass pollen, the tree pollens of Betula alba and Olea europea, the epithelia of cat and dog, the mite Dermatophagoides pteronyssinus, or to the foodstuffs cow's milk, chicken eggwhite or peanut were compared with the analytical data from the ratio allergosorbent test (RAST). The results show a good correlation between these two laboratory techniques.