Helper T-cell epitopes encoded by the Babesia bigemina rap-1 gene family in the constant and variant domains are conserved among parasite strains

Among important candidates far babesial vaccines are apical complex proteins, including rhoptry-associated protein 1(RAP-1) from Babesia bovis and B. bigemina, which have been shown to induce partial immunity. Four variant B. bigemina rap-l transcripts identified in a clone of the Mexico strain have highly conserved sequence in the central region but vary in sequence at the amino and carboxy termini (NT and CT) of the predicted proteins, resulting in different combinations of NT and CT domains in the individual gene products. Cattle were immunized with native protein consisting of the RAP-alpha 1 variant, which contains NT-1 and CT-1 domains, and T-cell responses were characterized. We previously reported the identification of two T helper (Th) cell epitopes in B. bigemina RAP-1 alpha 1 protein (L. Hotzel, W. C. Brown, T. F. McElwain, S. D. Rodriguez, and G. H. Palmer, Mol. Biochem. Parasitol. 81:89-99, 1996). One epitope mapped to the constant domain of RAP-1 (amino acids [aa] 144 to 187), and one mapped to the CT-1 variable domain (aa 386 to 480). Th1-like clones responding to these epitopes proliferated differentially to different strains of B. bigemina, raising the possibilities that the T-cell epitopes may vary antigenically and that CT-I may be differentially expressed with respect to the other RAP-I CT domains in the different strains. In this report, we definitively map the T-cell epitope identified in the constant domain of RAP-1 to aa 159 to 187 (FVVSLLKKNVVRDPESNDVENFASQYFYM) and show that the predicted amino acid sequence is completely conserved among seven strains. The T-cell epitope in the CT-1 domain was mapped to aa 436 to 465 (VNSEKVDADDAGNAETQQLPDAENEVRADD), which is also completely conserved among eight strains of B. bigemina. We further show that the RAP-1 alpha 1-immunized cattle were protected against homologous B. bigemina challenge, thus suggesting an association between protective immunity and the helper T-cell response against the two epitopes. The immunogenic and highly conserved nature of these T-cell epitopes and their ability to stimulate functionally relevant Th cells that express gamma interferon support their inclusion in a vaccine.