Identification, characterization and testing of geographically conserved Babesia bovis vaccine antigen candidates

Abstract

During the development of this project, we selected four potential B. bovis antigens for a subunit vaccine to prevent the clinical signs of acute bovine babesiosis. Selection of the target antigens was based on: (1) profile of expression in parasite blood stages; (2) prediction for protein location on the parasite surface and/or on the surface of infected red blood cells; and (3) target conservation between US and Israeli strains of B. bovis. Following these criteria, the B. bovis targets BBOV_IV009170, BBOV_III007410, BBOV_II001790, and BBOV_III008720 were selected. Full-length genomic sequences of these four targets were compared between Israeli and US strains of B. bovis. Four Israeli parasite strains/isolates were selected for gene conservation analysis: sample ID # 3330 (from Refet Netofa); sample ID # 2673 (Refet Netofa); Sample ID Newe Yaar - Shahar; and the Israeli B. bovis cultured live vaccine 2S61411. The reference B. bovis Texas stain was also used in the analysis. Sequencing analysis showed considerable levels of conservation ranging from 72.2-91.1% and 84.6-94.1% of amino acid identity and similarity, respectively, by comparing Israeli and US B. bovis strains. Recombinant version of the four target antigens were produced in E. coli, purified by nickel columns, and used for antigenic and immunogenic analyses. Antigenic analysis revealed that B. bovis-infected cattle develop high antibody titers to BBOV_III008720. In contrast, no antibodies against BBOV_IV009170, BBOV_III007410, and BBOV_II001790 were detected in sera from infected cattle. Vaccine trial of the four antigens in cattle is currently being carried out. However, preliminary data show that after three inoculations all vaccinated animals (n=5) seroconverted to all four B. bovis antigens. At the time of the preparation of this report, vaccinated and control animals are being challenged with a virulent B. bovis strain from Israel to evaluate protection. Results from the vaccination/trial experiment will set the future directions of this work. Considering the overall results from this project, two manuscripts are currently in preparation. A manuscript currently in preparation will describe the bioinformatics analysis and antigenic evaluation of the four transmembrane B. bovis proteins as potential targets for a subunit vaccine. A second manuscript will report the results from the vaccination/challenge trial. We believe that we have successfully accomplished our objective in this project. By developing this work, we have identified, expressed, and tested conserved antigens for a potential subunit vaccine against B. bovis, and have advanced the state-of-the-art concerning the development of an efficient and sustainable control strategy to bovine babesiosis.