Malaria transmission-blocking vaccination can effectively reduce and/or eliminate transmission of parasites from the human host to the mosquito vector. The immunity achieved by inducing an antibody response to surface antigens of male and female gametes and parasite stages in the mosquito. Our laboratory has developed DNA vaccine constructs, based on Pfs25 (a Plasmodium falciparum surface protein of 25 kDa), that induce a transmission-blocking immune response in mice (C. A. Lobo, R. Dhar, and N. Kumar, Infect. Immun. 67:1688-1693, 1999). To evaluate the safety, immunogenicity, and efficacy of the Pfs25 DNA vaccine in nonhuman primates, we immunized rhesus macaques (Macaca mulatta) with a DNA vaccine plasmid encoding Pfs25 or a Pfg27-Pfs25 hybrid or with the plasmid (empty plasmid) alone. Immunization with four doses of these DNA vaccine constructs elicited antibody titers that were high but nonetheless unable to reduce the parasite's infectivity in membrane feeding assays. Further boosting of the antibody response with recombinant Pfs25 formulated in Montanide ISA-720 increased antibody titers (30-fold) and significantly blocked transmission of P. falciparum gametocytes to Anopheles mosquitoes (ϳ90% reduction in oocyst numbers in the midgut). Our data show that a DNA prime-protein boost regimen holds promise for achieving transmissionblocking immunity in areas where malaria is endemic and could be effective in eradicating malaria in isolated areas where the level of malaria endemicity is low.Plasmodium falciparum, one of the deadliest of the malariacausing species, continues to threaten humans, especially children and pregnant women, in many parts of the world (8). The available drugs and the vector control campaigns used to date have not had a significant impact on the transmission of malaria from humans to mosquitoes. When a female Anopheles mosquito bites an infected human, the male and female gametocytes (formed during the erythrocytic phase of the malaria life cycle) are taken up in the blood meal and rapidly undergo gametogenesis and fertilization. Oocysts and eventually infective sporozoites are formed, thus completing parasite development. It has been shown that the crucial link for malaria transmission, i.e., infectivity of male and female gametocytes, can be blocked in the mosquito vector by antibodies directed against sexual-stage-specific surface antigens when they are ingested along with the parasites in the blood meal (5, 12, 18). It is believed that transmission-blocking immunity will play a significant role in reducing the emergence of vaccine-resistant strains. Such strains could be selected by vaccines targeting erythrocytic asexual forms. Likewise, spread of drug resistance could be diminished by reducing overall malaria transmission (4).P. falciparum zygote-ookinete surface protein 25 (Pfs25) is one of the most promising candidates identified so far for the development of P. falciparum transmission-blocking vaccines. Pfs25 (a 25-kDa surface protein) is expressed at the onset of gametogenesis ...