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

DOMINGUEZ, M; ECHAIDE, I.; TORIONI DE ECHAIDE, S.; MOSQUEDA, J.; CETRÁ, B.; SUAREZ, C.; JACOBSEN FLORIN-CHRISTENSEN, M.

VETERINARY PARASITOLOGY

ELSEVIER SCIENCE BV

Año: 2010 vol. 167 p. 216 - 226

0304-4017

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.