A conserved motif in the immune-subdominant RAP-1 Related Antigen (RRA) of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle

Authors: ROJAS, MANUEL - Washington State University; Bastos, Reginaldo; NAVAS, JINNA - Washington State University; LAUGHERY, JACOB - Washington State University; Lacy, Paul; Suarez, Carlos

Submitted to: Frontiers in Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/10/2024
Publication Date: 4/24/2024
Citation: Rojas, M.J., Bastos, R.G., Navas, J., Laughery, J.M., Lacy, P.A., Suarez, C.E. 2024. A conserved motif in the immune-subdominant RAP-1 Related Antigen (RRA) of Babesia bovis contains a B-cell epitope recognized by antibodies from protected cattle. Frontiers in Immunology. 15 - 2024. https://doi.org/10.3389/fimmu.2024.1380660.
DOI: https://doi.org/10.3389/fimmu.2024.1380660

Interpretive Summary: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. In this study we characterized a 15 amino acid sequence (15-mer) present in the RRA proteins that is highly conserved in the members of the RAP-1 superfamily. Similarity searches indicated that the 15-mer is also present in proteins that bind ATP. Furthermore, structure modeling using AlphaFold shows that the ATP-binding proteins containing the 15-mer have a similar predicted surface structure as the RRA molecules of Babesia parasites, suggesting possible functional roles for this molecule. In addition, we demonstrate the presence of a surface exposed epitope associated with the 15-mer sequence, that is recognized by antibodies in protected cattle. Altogether, the data support inclusion of this conserved motif as a candidate in B. bovis subunit epitope-based vaccines.

Technical Abstract: Introduction: Babesia bovis, a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis, as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins. Methods and results: 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 motif located at NT of the proteins. BlastP searches indicate that the 15-amino acid motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer motif sequences on the surface of the RRAs of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in indirect ELISA, and rabbit antibodies against the 15 amino acid motif sequence react with the surface of free merozoites in immunofluorescence. Discussion and conclusion: The presence of the 15mer-like regions in dynein and ATP-binding proteins provides rationale for investigating possible functional roles for RRA. This finding regards the identification of a surface exposed B-cell epitope in the 15 amino acid motif of the B. bovis RRA, and that this B-cell epitope is recognized by sera from protected bovines. This conserved motif is proposed as a candidate epitope to be included in B. bovis subunit epitope-based vaccines.