engleski

Effectivness of oil-based adjuvant formulation containing PGM in rabbits

Peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDAP(wNH2)-D-Ala-D-Ala (PGM, PLIVA, Croatia) is an adjuvant active compound that is active very shortly in the mammalian organism, before it is degraded and excreted. In spite of its short action, its adjuvant properties have clearly been demonstrated, even on generation of long living specific memory. The approach of preparing depot acting adjuvant formulations containing PGM in order to prolong PGM's life in the organism, and the use of such formulations for production of specific antibodies in mice, has given very promising results so far. The aim of this study was to test the effectiveness of the water in oil in water (w/o/w) adjuvant Montanide ISA 206 (Seppic, France) formulated with PGM on specific antibody production in rabbits. Hybrid male rabbits (New Zealand x California white), weighting cca 2.5 kg, were immunised four times subcutaneously (s.c.) in the back with human immunoglobulin G (hIgG, Institute of Immunology) as an antigen, in combination with ISA 206 alone, and ISA 206 formulated with PGM. Three booster injections were given in one-month intervals. Rabbits immunised with hIgG in CFA/IFA adjuvant intramuscularly (i.m.) served as a control group. Blood was collected from lateral ear vein ten days after each booster and the presence of hIgG specific IgG determined by enzyme linked immunosorbent assay (ELISA) established in our laboratory. The relative quantity of hIgG-specific IgG was calculated by parallel line analysis using rabbit anti-hIgG immunoglobulin G preparation (Institute of Immunology) as a standard. Capability of rabbit sera to agglutinate human erythrocytes coated with human anti-D antibodies was assayed after the final booster (test of hemagglutination) and results expressed as hemagglutination titres. The results of both tests (ELISA and hemagglutination) were compared.Rabbits immunised with a novel experimental adjuvant formulation composed of ISA 206 and PGM had significantly higher level of specific antibodies at all tested points in comparison to group immunised with ISA 206 alone. What is more important, this novel adjuvant formulation given by the less painful route (s.c.) was equally effective as the CFA/IFA adjuvant given i.m.. The level of specific antibodies in rabbits immunised with ISA 206 + PGM, as well as with CFA/IFA adjuvant, increased with the first and second booster immunisation, while the third booster did not give rise to any additional increase. Rabbits immunised s.c. with ISA 206 alone reached the final level of specific antibodies already after the first booster, and this was not further increased by subsequent boosters. Sera of rabbits after the final booster were assayed for hemagglutination activity, and results obtained were in correlation with those detected by ELISA, indicating that specific antibodies produced were fully functional.Results presented in this work are the first demonstration of effectiveness of ISA206+PGM adjuvant formulation in experimental animals, and the first demonstration of effectiveness of depot acting adjuvant formulation containing PGM in the animal species other then mice. They are also a strong proof of correctness of our hypothesis that the adjuvant action of PGM could be improved in formulations that would prolong its availability in the mammalian organism.

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