Prediction of antigenic B-cell epitopes in the members of the Merozoite Surface Antigen-2 family of Babesia bovis

DOMINGUEZ M; WILKOWSKY SILVINA; MOSQUEDA J; ECHAIDE I; CETRÁ B; SUAREZ, C; FLORIN CHRISTENSEN, M

Palermo, Italia

Congreso; II Babesia World Summit,; 2007

Instituto Zooprofilactico de Sicilia

Prediction of antigenic B-cell epitopes in the members of the Merozoite Surface Antigen-2 family of Babesia bovis Purpose of the job: Infection of bovines with the hemoparasite Babesia bovis causes important economic losses and limits cattle production in vast areas of the world suitable for the growth of its vector, Rhipicephalus (Boophilus) microplus ticks. An effective control measure is vaccination with live attenuated parasites. However, this kind of vaccines has serious disadvantages, including the need of a cold chain, a short storage life, the danger of parasite reversion of virulence, the possibility of co-transmission of other microorganisms and these vaccines are only safe in bovines younger than 1 year old. For these reasons, our aim is to identify by bioinformatics methods the presence of B-cell epitopes that could be potential vaccine candidate antigens for the development of a subunit vaccine against B. bovis. In particular, we have focused on the members of the Merozoite Surface Antigen-2 family. These are immunodominant antigens, homogenously distributed on the parasite surface and that bear neutralization sensitive B-cell epitopes. Materials and Methods: Msa-2 genes from several B. bovis geographical isolates were amplified by PCR, cloned and sequenced. Msa-2a and msa-2b genes were amplified together since they share common 5´and 3´ends. Msa-2c genes were amplified separately because they have a different 5´ region. Sequences of msa-2 a1, b and c alleles were aligned using ClustalW and conserved regions were highlighted using Boxshade. Putative B-cell epitopes were identified using the method of Kolaskar and Tongaonkar (http://bio.dfci.harvard.edu/Tools/antigenic.pl). Two peptides were selected and chemically synthesized, one of them corresponding to a putative conserved B-cell epitope in MSA-2c (peptide 1: ELLKLLIEAGGGC) and the other one to MSA-2 a/b (peptide 2: YYKKHISGGGC from MSA-2b). Both peptides were conjugated to keyhole limpet hemocyanin (KLH) to immunize mice. Recognition of Babesia bovis B-cell epitopes by murine sera was assayed by immunofluorescence using smears of the in vitro cultured RAD Mexican strain. In addition, an indirect ELISA was set up to analyze the reaction of different bovine sera with peptide 1, adjusting antigen concentration, ELISA plate type, coating buffer and blocking reagent The peptide was dissolved in DMSO, diluted in PBS and bound to Nunc MaxiSorp plates (50 ng/well). After blocking, plates were sequentially incubated with 1/100 dilutions of test sera, an adequate dilution of peroxidase-conjugated anti-bovine IgG and the colorimetric substrate OPD-H2O2,  stopped with H2SO4/H2O and absorbance was read at 490 nm. Results: Murine hyperimmune sera against peptides 1 and 2 reacted with the surface of merozoites from the Mexican RAD strain, as shown by immunofluorescence. This result indicates that the regions selected using bioinformatic tools correspond to real B-cell epitopes in the parasite. In addition, serum samples from B. bovis endemic areas of Northwestern Argentina and negative samples from a tick-free Argentine Southern province were tested using the ELISA based on peptide 1. Although preliminary, our results indicate that bovine serum from naturally and experimentally-infected bovines (n=3) recognize this peptide, since absorbancies obtained for B. bovis-infected sera were significantly higher than with uninfected ones (n=2). These results were confirmed with serum from a bovine immunized with recombinant MSA-2c (n=1). These experiments show that the selected B-cell epitope is expressed during infections of bovines with B. bovis. Preliminary ELISA results for peptide 2 showed clear recognition by one serum sample of a naturally-infected bovine, while another field sample failed to recognize this peptide. Optimal conditions for this assay are now being evaluated after which a larger number of samples will be tested. . Conclusions: Since (i) the ELISA performed showed positive results with different bovine serum samples from Argentina, and (ii) anti-peptide serum recognized the merozoite surface of a Mexican strain, we can conclude that peptide 1 corresponds to B-cell epitope that is highly conserved, at least among strains from Latin America. The same kind of ELISA test will be applied to peptide 2 in the near future. In addition, we will analyze if both B-cell epitopes are neutralization-sensitive using in vitro cultured B. bovis merozoites. Supported by CONICET, ANPCyT PICTR 2002-00054 and PICTO 08-12920; and the European Commission, INCO 003691-MEDLABAB.