MOLECULAR AND IMMUNOLOGICAL CHARACTERIZATIONS OF POTENTIALLY PROTECTIVE BLOOD-STAGE PLASMODIUM YOELII ANTIGENS

Abstract

As malaria remains one of the world’s fatal diseases, an effective malaria vaccine is urgently required. Our previous studies have shown that formalin-killed blood-stage Plasmodium yoelii (FKPy)-immunized mice can be protected against subsequent challenge by live P. yoelii parasites, and two putative immunogenic proteins (~39.6 kDa) were identified. To prove that these proteins are protective antigens, their sequences were determined by LC-MS/MS and a search of the malaria protein databases revealed them to be P. yoelii YIR1 and YIR4 surface proteins. Specific primers were designed to amplify regions of yir1 and yir4 genes coding for the proteins’ ectodomains, which were ligated with a pET28a expression vector and used to transfect Escherichia coli BL21-(DE3) and heterologously express recombinant (r)YIR1 (22 kDa) and rYIR4 (31 kDa). Western blotting revealed that recombinant YIR1 and rYIR4 were only mildly antigenic to mouse anti-P. yoelii immune serum. Mice immunized with rYIR1 and rYIR4, either separately or together, were not protected against challenges from live, virulent P. yoelii YM parasites. Immunization produced a minimal cell-mediated (IFN-γ and IL-4) and weak humoral (IgG and IgG2a) responses. Given that YIR1 and YIR4 are found in FKPy extract, the proteins could constitute components in the development of protein-based subunit malaria vaccines.