A vaccine formulation combining rhoptry proteins NcROP40 and NcROP2 improves pup survival in a pregnant mouse model of neosporosis

• NcROP40 and NcNTPase were evaluated as new vaccine candidates against Neospora caninum.
• All vaccine candidates elicited a specific immune response against the native proteins.
• rNcROP40 + rNcROP2 and rNcROP2-vaccines partially prevented vertical transmission and pup mortality.
• A synergistic effect was observed with NcROP40 + NcROP2 in combination.

Currently there are no effective vaccines for the control of bovine neosporosis. During the last years several subunit vaccines based on immunodominant antigens and other proteins involved in adhesion, invasion and intracellular proliferation of Neospora caninum have been evaluated as targets for vaccine development in experimental mouse infection models. Among them, the rhoptry antigen NcROP2 and the immunodominant NcGRA7 protein have been assessed with varying results. Recent studies have shown that another rhoptry component, NcROP40, and NcNTPase, a putative dense granule antigen, exhibit higher expression levels in tachyzoites of virulent N. caninum isolates, suggesting that these could be potential vaccine candidates to limit the effects of infection. In the present work, the safety and efficacy of these recombinant antigens formulated in Quil-A adjuvant as monovalent vaccines or pair-wise combinations (rNcROP40 + rNcROP2 and rNcGRA7 + rNcNTPase) were evaluated in a pregnant mouse model of neosporosis. All the vaccine formulations elicited a specific immune response against their respective native proteins after immunization. Mice vaccinated with rNcROP40 and rNcROP2 alone or in combination produced the highest levels of IFN-γ and exhibited low parasite burdens and low IgG antibody levels after the challenge. In addition, most of the vaccine formulations were able to increase the median survival time in the offspring. However, pup survival only ensued in the groups vaccinated with rNcROP40 + rNcROP2 (16.2%) and rNcROP2 (6.3%). Interestingly, vertical transmission was not observed in those survivor pups immunized with rNcROP40 + rNcROP2, as shown by PCR analyses. These results show a partial protection against N. caninum infection after vaccination with rNcROP40 + rNcROP2, suggesting a synergistic effect of the two recombinant rhoptry antigens.

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