Adjuvant-Driven Modulation of Epitope Recognition and Protective Immunity in Bm86 vaccinated Holstein-Friesian cattle.

Livestock production is vital to the economies and food security of African countries. Rising global demand for livestock-derived products intensifies the challenge of managing ticks and tick-borne diseases. This study aimed to optimize a Bm86-based vaccine for controlling Rhipicephalus microplus. Commercial Bm86-based vaccines show variable efficacy (0-100%), reflecting incomplete understanding of the antigen and the immune response elicited. To address this, homologous challenge was conducted in Holstein-Friesian calves with Bm86 formulated with Montanide™ ISA 71 VG (referred to as Montanide™) and a novel Alum-based adjuvant alternative in two separate vaccine trials. Antibody responses were determined utilising indirect ELISA. Vaccine efficacy was assessed through controlled R. microplus challenge. Immunoinformatics mapped the antigenic regions of Bm86, followed by ex vivo validation using antisera from vaccinated cattle. Both adjuvant formulations induced high levels of Bm86-specific total IgG antibodies. However, only the Montanide™ formulation induced a protective response of 88.2%, which correlated with total IgG antibody levels (r= 0.86). In contrast, the Alum-based adjuvant formulation induced low efficacy (2.3%) with a strong inverse correlation with total IgG antibodies (r= -0.95). Both formulations induced an IgG1-biased (i.e. T-helper 2) antibody response, but the Montanide™ formulation conferred a more balanced IgG1/IgG2 response. The efficacy induced by the Montanide™ formulation strongly correlated with the levels of IgG2 antibodies (r= 0.91), suggesting that a balanced Th1/Th2 response plays a key role in protection. Despite its efficacy, the Montanide™ formulation caused adverse injection site effects, highlighting the need for safer alternatives. Epitope mapping identified similar linear B-cell epitope regions recognised by total IgG antibodies induced by vaccination with both adjuvant formulations. These findings suggest that Bm86 vaccination activates broader immune pathways than previously understood, emphasizing the need for exploration of additional immune markers to improve vaccine performance.