Functional Conservation of P48/45 Proteins in the Transmission Stages of Plasmodium vivax (Human Malaria Parasite) and P. berghei (Murine Malaria Parasite)

Sexual-stage proteins have a distinct function in the mosquito vector during transmission and also represent targets for the development of malaria transmission-blocking vaccine. P48/45, a leading vaccine candidate, is critical for male gamete fertility and shows >50% similarity across various species of Plasmodium. We evaluated functional conservation of P48/45 in Plasmodium vivax and P. berghei with the motivation to establish transgenic P. berghei strains expressing P. vivax P48/45 (Pvs48/45) in an in vivo assay to evaluate the transmission-blocking activity of antibodies elicited by Pvs48/45. Homologous recombination was employed to target P. berghei s48/45 (pbs48/45) for knockout (KO) or for its replacement by two different forms of P. vivax s48/45 (pvs48/45) (the full-length gene and a chimeric gene consisting of pbs48/45 5′ signal and 3′ anchor sequences flanking pvs48/45). Expression of Pvs48/45 in transgenic parasites and lack of expression of any P48/45 in KO parasites were confirmed by reverse transcription-PCR (RT-PCR) and Western blotting. Both transgenic and knockout parasites revealed asexual growth kinetics in mice comparable to that seen with wild-type parasites. When employed in mosquito infection experiments, both transgenic parasite strains remained transmission competent and developed into infectious sporozoites, whereas the knockout parasites were incapable of establishing mosquito-stage infection. These results indicate the functional conservation of P48/45 protein during transmission, and the transgenic parasites generated in this study represent a valuable tool to evaluate the protective efficacy of transmission-blocking antibodies elicited by Pvs48/45-based vaccines using an in vivo mouse animal assay instead of ex vivo membrane feeding assays (MFA) relying on access to P. vivax gametocytes from infected patients.