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Title: In silico predicted conserved B-cell epitopes in the Merozoite Surface Antigen -2 family of B. bovis are neutralization-sensitive

Author
item DOMINGUEZ, M - Instituto Nacional Tecnologia Agropecuaria
item ECHAIDE, I - Instituto Nacional Tecnologia Agropecuaria
item TORIONI DE ECHAIDE, S - Instituto Nacional Tecnologia Agropecuaria
item MOSQUEDA, J - Instituto Nacional De Investigaciones Forestales Y Agropecuarias (INIFAP)
item CETRA, B - Instituto Nacional De Investigaciones Forestales Y Agropecuarias (INIFAP)
item Suarez, Carlos
item FLORIN-CHRISTENSEN, M - Instituto Nacional Tecnologia Agropecuaria

Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/18/2009
Publication Date: 2/1/2010
Citation: Dominguez, M., Echaide, I., Torioni De Echaide, S., Mosqueda, J., Cetra, B., Suarez, C.E., Florin-Christensen, M. 2010. In silico predicted conserved B-cell epitopes in the Merozoite Surface Antigen -2 family of B. bovis are neutralization-sensitive. Veterinary Parasitology. 167(2-4):216-226.

Interpretive Summary: This manuscript describes the in silico identification of B-cell epitopes derived from the merozoite surface antigen-2 family of Babesia bovis. Synthetic peptides representing some of these epitopes are recognized by antibodies in naturally infected bovines and some are able to elicit antibodies in mice that are able to block infection of erythrocytes by the parasites in in vitro cultures, suggesting that they are parts of functionally relevant domains of these proteins. The results may be of significance for the future design of sub-unit vaccines against B. bovis.

Technical Abstract: The Merozoite Surface Antigens-2 of Babesia bovis conform a family of GPI-anchored glycoproteins located at the parasite cell surface, that contain neutralization-sensitive B-cell epitopes, thus constituting putative vaccine candidates for bovine babesiosis. It was previously shown that (i) the MSA-2 antigens of the biologically cloned Mo7 strain are encoded by four tandemly organized genes: msa-2a1, a2, b and c, and (ii) at least one allele of each of these genes is present in the Argentine R1A strain with moderate degree of polymorphism. The present work was aimed at identifying B-cell epitopes in the MSA-2 family, conserved among different B. bovis geographical isolates. To this aim, msa-2a, b and c alleles from different isolates from Argentina, USA and Mexico were amplified by PCR, cloned and sequenced. Bioinformatic analysis of the nucleotide sequences by ClustalW alignments and B-cell epitope prediction algorithms allowed the identification of several peptides containing putative conserved B-cell epitopes. Four of these peptides (KDYKTMVKFCN from msa-2a1; YYKKHIS, from msa-2b; and THDALKAVKQLIKT and ELLKLLIEA from msa-2c) were chemically synthesized, conjugated to keyhole limpet hemocyanin and used to inoculate mice to obtain immune sera. Anti-peptide antibodies recognized B. bovis merozoite (mz) extracts in an ELISA test in all cases. In addition, these sera reacted with the surface of mz of an Argentine and a Mexican B. bovis strains in immunofluorescence assays. Importantly, sera against two of the selected peptides impaired invasion of erythrocytes by in vitro cultured mz. These results show that the peptide sequences selected by bioinformatic analysis correspond to naturally occurring B-cell epitopes, conserved among Mexican and Argentine B. bovis isolates. The neutralization-sensitive B-cell epitopes identified in this study might be especially attractive candidates for the development of subunit vaccines.