TARGETING SUBDOMINANT RHOPTRY-ASSOCIATED PROTEINS OF Babesia bovis AS CANDIDATES OF A SUBUNIT-BASED VACCINE

Manuel J Rojas

doi:10.7273/000007209

Introduction: Bovine babesiosis caused by the tick-borne apicomplexan parasite Babesia bovis, poses an important threat for the development of cattle industries worldwide, and new vaccines are urgently needed. It’s been postulated that immune-subdominant (ISD) antigens are ideal targets for subunit vaccine development because they are more likely to be essential for parasite development and pathogenesis. Members of the Rhoptry Associated Protein-1 (RAP1) superfamily in B. bovis, which includes two identical gene copies of RAP1 genes and a single copy of the Rhoptry Associated Protein-1 Related Antigen (RRA), a truncated version of RAP1, are candidates for subunit vaccine development. While RAP1 is immunodominant (ID), RRA has the characteristics of an ISD antigen. Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the amino terminal (NT) of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. Also, AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Furthermore, antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence. The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. Additionally, the demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in subunit epitope-based vaccines against B. bovis. In this study, we first defined the immuno-subdominance of the well species-conserved N-terminal (NT) region of RAP-1 and RRA in young and adult bovines (n=3 and n=5 respectively) previously vaccinated with live attenuated parasites and challenged with a virulent B. bovis strain. Antibody levels were compared with the immunodominant RAP1-CT using ELISAs based on recombinant RAP1-NT, RAP1-CT, and full size RRA proteins. While both vaccinated and challenged animals demonstrated high antibody responses against the immunodominant CT region of RAP1, the antibody responses where significantly lower against the RAP1-NT and RRA antigens, confirming they are immune-subdominant. Once validated as ISD, we developed an experimental model system based on recombinant versions of RRA and RAP-1NT, to test whether immunogenicity of ISD antigens could be augmented using a FliC-based adjuvant in a cattle vaccination trial. Two groups of 6 cattle each where vaccinated either with RRA, RAP1-NT and a FliC-Emulsigen mix as adjuvant, and a control group (n=6) with the adjuvant mix alone. Vaccinations resulted in the elicitation of strong humoral immune responses against the two ISD antigens in all but one, immunized cattle. Subsequent challenge of all animals with virulent B. bovis resulted in acute babesiosis in both groups of animals. However, we found a significant delay in the average rate of decrease of hematocrit in the vaccinated group, and early monocyte response, a feature recently proposed as a correlate of protection against B. bovis, in half of the vaccinated animals. Conclusions: This study confirmed the immuno-subdominance of Rap1 NT and RRA proteins, but also, the ability of FliC to increase immunogenicity of immune-subdominant antigens. Additionally, the evaluation of the immune response against B. bovis experimental infection generated useful information towards developing future subunit vaccines against B. bovis.

TARGETING SUBDOMINANT RHOPTRY-ASSOCIATED PROTEINS OF Babesia bovis AS CANDIDATES OF A SUBUNIT-BASED VACCINE

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