|Johnson, W Carl - Carl|
Submitted to: Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/31/2011
Publication Date: 4/18/2011
Citation: Suarez, C.E., Laughery, J., Bastos, R., Johnson, W.C., Norimine, J., Asenzo, G., Brown, W.C., Florin-Christensen, M., Goff, W.L. 2011. A novel neutralization sensitive and subdominant RAP-1-related antigen (RRA) is expressed by Babesia bovis merozoites. Parasitology. 10.1017/S0031182011000321. Interpretive Summary: The rhoptry associated protein-1 is widely conserved in all known Babesias, it is considered as a leading subunit vaccine candidate, and is widely used as a component of serologic diagnostic methods. In this study, we report the occurrence of a novel rap-1 related gene in the genome of B. bovis, termed rap-1 related antigen (RRA). The rra gene is expressed in B. bovis merozoites in smaller amounts compared to RAP-1 and is poorly immunogenic, thus, in contrast to RAP-1, it is considered as a subdominant antigen. However, this study demonstrates that antibodies against RRA can neutralize B. bovis invasion of erythrocytes in in vitro cultures, thus suggesting that RRA might have a role in invasion or egression of erythrocytes. Taken together the data suggests that RRA may be a candidate for developing vaccines which could potentially elicit a neutralizing antibody response in vivo.
Technical Abstract: Objective: The Babesia bovis genome encodes a rap-1 related gene denominated RAP-1 related antigen (RRA). In this study, we analyzed the pattern of expression, immunogenicity and functional relevance of RRA. Methods: Phylogenetic analysis was performed using the program Phylip. Expression of rra was analyzed by Northern blots, RT-PCR, immunoprecipitation, Western blots and immunofluorescence. RRA antigenicity was tested by T-cell proliferation and Western blot analysis, and functional relevance was determined in an in vitro neutralization assay. Results: RRA is more closely related to RAP-1b of Babesia bigemina than to B. bovis RAP-1, and it is highly conserved among distinct strains. Transcriptional analysis suggests lower amounts of rra transcripts compared to rap-1. Immunoprecipitation of metabolically labeled B. bovis proteins with antibodies against synthetic peptides representing predicted antigenic regions of RRA confirmed the expression of a ~43 kDa RRA in cultured merozoites. Antibodies present in B. bovis hyperimmune sera, but not in field infected cattle sera, reacted weakly with recombinant RRA, and no significant stimulation was obtained using recombinant RRA as antigen in T-cell proliferation assays, indicating that RRA is a subdominant antigen. Antibodies against RRA synthetic peptides reacted with merozoites using immunofluorescence, and are able to significantly inhibited erythrocyte invasion in in vitro neutralization tests, suggesting functional relevance for parasite survival. Conclusion: B. bovis express a novel subdominant RAP-1-like molecule that may contribute to erythrocyte invasion and/or egression by the parasite.