A novel influenza vector-based vaccine expressing ESAT-6 and TB10.4 confers immunity and protection against Bovine tuberculosis in guinea pigs and calves

Background and Aim: Bovine tuberculosis (bTB), caused by Mycobacterium bovis, remains a significant zoonotic and eco­nomic threat globally. Despite the long-standing use of the Bacillus Calmette-Guérin (BCG) vaccine, its inconsistent efficacy and interference with surveillance tests underscore the need for alternative approaches. This study evaluated the safety, immunogenicity, and protective efficacy of a novel influenza vector-based vaccine expressing M. bovis antigens ESAT-6 and TB10.4, formulated with or without an adjuvant. Materials and Methods: Recombinant influenza A viruses expressing ESAT-6 and TB10.4 were constructed using reverse genetics and incorporated into vaccine formulations. Guinea pigs and calves were immunized with adjuvanted and non-adjuvanted formulations, followed by challenge with a virulent M. bovis strain. Safety was assessed through clinical observation and histopathology. Immune responses were monitored using interferon-gamma (IFNγ) enzyme-linked immu­nosorbent assay, and protection was evaluated through organ damage indices, bacterial load, and survival rates over a 12-month period. Results: Both formulations were safe and well-tolerated in guinea pigs and calves, with no adverse clinical signs. The non-adjuvanted vaccine induced the highest and most sustained IFNγ response, peaking between 2 and 5 months post-vac­cination. In guinea pigs, the protection index reached +0.60 lg in the non-adjuvanted group versus +0.2 lg in the adjuvanted group. In calves, lung bacterial load was reduced to 1.83–1.93 lg colony-forming unit (CFU) in vaccinated animals compared with 5.8 lg CFU in unvaccinated controls. Histopathological examination confirmed minimal tissue damage in the vaccinated groups. Both vaccine formulations demonstrated protective efficacy equivalent to or better than BCG, with the non-adju­vanted version showing superior performance. Conclusion: This novel influenza vector-based vaccine expressing ESAT-6 and TB10.4 antigens elicits strong, long-lasting cellular immunity and provides significant protection against M. bovis infection in guinea pigs and calves. The adjuvant-free formulation demonstrated higher immunogenicity, simplified production, and minimal adverse reactions, positioning it as a promising alternative to BCG for bTB control in livestock.