A novel 20-kilodalton protein conserved in Babesia bovis and B. bigemina stimulates memory CD4 + T lymphocyte responses in B. bovis-immune cattle

Abstract

Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4 + T lymphocytes and IFN-γ production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4 + T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were shown previously to stimulate memory CD4 + lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20-kDa CFE fraction identified a 20-kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20-kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions shown previously to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4 + T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4 + T cell clones proliferated in response to several B. bovis strains and produced IFN-γ. BLAST analysis revealed significant similarity of the Bbo20 and Bbg20 amino acid sequences with the hsp20/α-crystallin family.