TpUB05, a Homologue of the Immunodominant Plasmodium falciparum Protein UB05, Is a Marker of Protective Immune Responses in Cattle Experimentally Vaccinated against East Coast Fever.

Abstract

INTRODUCTION: East Coast fever, a devastating disease of cattle, can be controlled partially by vaccination with live T. parva sporozoites. The antigens responsible for conferring immunity are not fully characterized. Recently it was shown that the P. falciparum immunodominant protein UB05 is highly conserved in T. parva, the causative agent of East Coast fever. The aim of the present investigation was to determine the role of the homologue TpUB05 in protective immunity to East Coast fever. METHODS: The cloning, sequencing and expression of TpUB05 were done according to standard protocols. Bioinformatics analysis of TpUB05 gene was carried out using algorithms found in the public domain. Polyclonal antiserum against recombinant TpUB05 were raised in rabbits and used for further analysis by Western blotting, ELISA, immunolocalization and in vitro infection neutralization assay. The ability of recombinant TpUB05 (r-TpUB05) to stimulate bovine PBMCs ex-vivo to produce IFN-γ or to proliferate was tested using ELISpot and [3H]-thymidine incorporation assays, respectively. RESULTS: All the 20 cattle immunised by the infection and treatment method (ITM) developed significantly higher levels of TpUB05 specific antibodies (p<0.0001) compared to the non-vaccinated ones. Similarly, r-TpUB05 highly stimulated bovine PMBCs from 8/12 (67%) of ITM-immunized cattle tested to produce IFN-γ and proliferate (p< 0.029) as compared to the 04 naїve cattle included as controls. Polyclonal TpUB05 antiserum raised against r-TpUB05 also marginally inhibited infection (p < 0.046) of bovine PBMCs by T. parva sporozoites. In further experiments RT-PCR showed that the TpUB05 gene is expressed by the parasite. This was confirmed by immunolocalization studies which revealed TpUB05 expression by schizonts and piroplasms. Bioinformatics analysis also revealed that this antigen possesses two transmembrane domains, a N-glycosylation site and several O-glycosylation sites. CONCLUSION: It was concluded that TpUB05 is a potential marker of protective immunity in ECF worth investigating further.