Evaluation of Liposome, Heat-Killed Mycobacterium w, and Alum Adjuvants in the Protection Offered by Different Combinations of Recombinant HA, NP proteins, and M2e Against Homologous H5N1 Virus.

Continued evolution of highly pathogenic H5N1 viruses causing high mortality in humans obviates need for broadly cross-reactive vaccines. For this, hemagglutinin (HA) inducing specific protective antibodies, highly conserved nucleoprotein (NP), and ectodomain of matrix (M2e) protein, either singly or in combination, were evaluated in BALB/c mice. Recombinant HA and NP (baculovirus system) and M2e (synthetic peptide) and 3 adjuvants, that is, liposomes, Mw (heat killed Mycobacterium w), and alum were utilized for the homologous virus challenge. Additional immunogens included liposome-encapsulated HA/NP proteins and corresponding DNAs. Mice groups received two doses of respective formulations given at 3-week intervals and challenged intranasally with 100LD50 of H5N1 virus strain. Dynamics of weight loss, lung viral load, titres of IgG-anti-HA, NP, and M2e antibodies (ELISA), and IgG-subtype analysis was done. Two doses of all the formulations led to 100% seroconversion against the immunogens evaluated (100% seroconversion after the first dose in majority). Antibody titres against the components were dependent on the adjuvant and combination. HA-driven Th2 response with all the adjuvants, balanced Th1/Th2 response to NP protein, and Th2-bias with alum were noted. Low anti-M2e antibody titres did not allow subtype analysis. On challenge, complete protection was observed with Mw-HA, alum-HA+NP, Lipo-HA+NP+M2e, alum-HA+NP+M2e, and HA-DP formulations with 12-fold, 8-fold, 720-fold, 17-fold, and no reduction, respectively, in lung viral load. In conclusion, the results identify several adjuvant-immunogen combinations conferring 100% protection in mice that need further evaluation in higher animals.