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.