RESUMO
BACKGROUND Malaria is an infectious disease caused by protozoan parasites belonging to the genus Plasmodium. Human-to-human transmission depends on a mosquito vector; thus, the interruption of parasite transmission from humans to mosquitoes is an important approach in the fight against malaria. The parasite stages infectious to mosquitoes are the gametocytes, sexual stages that are ingested by the vector during a blood meal and transform into male and female gametes in the midgut. Immunity against sexual stage antigens expressed by gametocytes, gametes, and the zygote formed after fertilisation can interrupt the parasite sexual cycle in the mosquito. This transmission blocking immunity is mediated by specific antibodies ingested during the mosquito blood feed, inhibiting the parasite development in the midgut. Merozoite thrombospondin related anonymous protein (MTRAP) is a merozoite and gametocyte surface protein essential for gamete egress from erythrocytes and for parasite transmission to mosquitoes. OBJECTIVES Here, we evaluated the potential of the P. berghei MTRAP to elicit antibodies with the ability to inhibit gamete fertilisation in vitro. METHODS We expressed a soluble recombinant PbMTRAP and used it to immunise BALB/c mice. The transmission blocking activity of the anti-rPbMTRAP antibodies was tested through in vivo challenge experiments followed by in vitro conversion assays. FINDINGS Immunisations with the rPbMTRAP induced a strong antibody response and the antibodies recognised the native protein by Western Blot and IFA. Anti-rPbMTRAP present in the blood stream of immunised mice partially inhibited gamete conversion into ookinetes. CONCLUSION Our results indicate that antibodies to PbMTRAP may reduce but are not sufficient to completely block transmission.
RESUMO
Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC) protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS) protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210) was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.