Comparative evaluation of protective efficacy of experimental inactivated vaccines against haemorrhagic septicaemia.

Developing an effective vaccine for haemorrhagic septicaemia (HS) in cattle and buffaloes is urgently needed. While preferred for their safety, achieving sufficient, cross-protective, and long-lasting immunity is still challenging when administering inactivated vaccines. This study aimed to assess the efficacy of four inactivating components comprising three inactivating agents: (1) Binary ethylenimine (BEI), (2) Formalin, (3) a combination of BEI and Formalin, and (4) Hydrogen peroxide (H2O2), in inactivating Pasteurella multocida to enhance HS vaccine potency. H2O2 demonstrated the fastest inactivation kinetics, killing Pasteurella multocida within a few minutes. The combination of BEI and Formalin showed relatively rapid inactivation compared to Formalin or BEI alone. BEI, known for targeting nucleic acids with minimal impact on protein integrity, showed promise but required higher concentrations (8 mM) and a longer duration (16 h) for complete inactivation. Based on the inactivation kinetics, 8 mM BEI, 0.1% Formalin, 8 mM BEI + 0.1% Formalin, and 1% H2O2 were chosen for the experimental vaccine formulations. The inactivated Pasteurella multocida were adjuvanted with Montanide ISA-201 oil adjuvant and evaluated in mice model. All vaccine formulations elicited protective responses of over 6.5 log10 units, indicating the high potency of the vaccine formulations. Notably, the vaccine prepared with an H2O2 inactivating agent elicited protection of > 8 log10 units. Our study concludes that H2O2, with its rapid inactivation kinetics and safety profile, presents a promising alternative inactivating agent for HS vaccine development. Future studies should evaluate the protective efficacy of H2O2-inactivated vaccines in target animals.