During 1989-1999, 11 volunteers were immunized by the bites of 1001-2927 irradiated mosquitoes harboring infectious sporozoites of Plasmodium falciparum (Pf) strain NF54 or clone 3D7/NF54. Ten volunteers were first challenged by the bites of Pf-infected mosquitoes 2-9 weeks after the last immunization, and all were protected. A volunteer challenged 10 weeks after the last immunization was not protected. Five previously protected volunteers were rechallenged 23-42 weeks after a secondary immunization, and 4 were protected. Two volunteers were protected when rechallenged with a heterologous Pf strain (7G8). In total, there was protection in 24 of 26 challenges. These results expand published findings demonstrating that immunization by exposure to thousands of mosquitoes carrying radiation-attenuated Pf sporozoites is safe and well tolerated and elicits strain-transcendent protective immunity that persists for at least 42 weeks.

The gene for the circumsporozoite (CS) protein of Plasmodium falciparum has been cloned and its nucleotide sequence determined. The gene encodes a protein of 412 amino acids as deduced from the nucleotide sequence. The protein contains 41 tandem repeats of a tetrapeptide, 37 of which are Asn-Ala-Asn-Pro and four of which are Asn-Val-Asp-Pro. Monoclonal antibodies against the CS protein of Plasmodium falciparum were inhibited from binding to the protein by synthetic peptides of the repeat sequence. The CS protein of Plasmodium falciparum and the CS protein of a simian malaria parasite, Plasmodium knowlesi, have two regions of homology, one of which is present on either side of the repeat. One region contains 12 of 13 identical amino acids. Within the nucleotide sequence of this region, 25 of 27 nucleotides are conserved. The conservation of these regions in parasites widely separated in evolution suggests that they may have a function such as binding to liver cells and may represent an invariant target for immunity.

Twenty malaria-naive volunteers received a recombinant Plasmodium falciparum malaria vaccine (RTS,S) containing 19 NANP repeats and the carboxy terminus (amino acids 210-398) of the circumsporozoite (CS) antigen coexpressed in yeast with hepatitis B surface antigen. Ten received vaccine adjuvanted with alum, and 10 received vaccine adjuvanted with alum plus 3-deacylated monophosphoryl lipid A (MPL). Both formulations were well tolerated and immunogenic. MPL enhanced CS antibody levels (measured by ELISA, immunofluorescence, and inhibition of sporozoite invasion assays). After sporozoite challenge, 6 of 6 in the alum group and 6 of 8 in the alum-MPL group developed patent malaria. Protected subjects had higher levels of CS antibody titers on day of challenge than did nonprotected subjects. After immunization, 1 protected subject had increased cytotoxic T lymphocyte activity against CS and recall of memory T cell responses to RTS,S and selected CS.

A double antibody micro enzyme-linked immunosorbent assay (ELISA) for identifying Plasmodium falciparum sporozoites in mosquitoes is described. Using monoclonal antibodies made against South American P. falciparum sporozoites, the ELISA was able to detect and identify sporozoite antigens of South American and Asian origins in extracts of dried infected mosquitoes.

SummaryExposure of BALB/c mice to mosquitoes infected with irradiated Plasmodium berghei confers protective immunity against subsequent sporozoite challenge. Immunized mice challenged with viable sporozoites develop parasitemia when treated orally with substrate inhibitors of nitric oxide synthase (NOS). This suggests that the production of nitric oxide (NO) prevents the development of exoerythrocytic stages of malaria in liver. Liver tissue from immunized mice expressed maximal levels of mKNA for inducible NOS (iNOS) between 12 and 24 h after challenge with sporozoites. Intraperitoneal injection of neutralizing monoclonal antibody against interferon 3/(IFN-3') or in vivo depletion of CD8 + T cells, but not CD4 + T cells, at the time of challenge blocked expression of iNOS mlLNA and ablated protection in immunized mice. These results show that both CD8 § T cells and IFN-~/are important components in the regulation of iNOS in liver which contributes to the protective response of mice immunized with irradiated malaria sporozoites. IFN-3,, likely provided by malaria-specific CD8 + T cells, induces liver cells, hepatocytes and/or Kupffer cells, to produce NO for the destruction of infected hepatocytes or the parasite within these cells. W ithin minutes after an infected Anopheles mosquito bites the vertebrate host, malaria sporozoites migrate to the liver and invade hepatocytes. There, the parasite matures, and after several days the infected hepatocytes lyse, releasing thousands of merozoites. Once in circulation, the parasite infects erythrocytes causing parasitemia. Prior exposure to irradiated sporozoites confers protective immunity (1, 2). This immunity is directed against liver stage malaria, and does not protect against the blood stage malaria.CD8 + T cells and IFN-3' are required for protective immunity to sporozoite challenge. In vivo depletion of CD8 § T cells or neutralization of IFN-3~ blocks induction of effector activity at the hepatic stage, resulting in parasitaemia (3-5). In vitro studies show that IFN-qr kills parasites by stimulating malaria-infected hepatocytes to produce nitric oxide (NO), and the addition of monomethyl-r-arginine (NGMMLA), a substrate inhibitor of nitric oxide synthase (NOS), to primary cultures of mouse hepatocytes reversed the antiparasitic effects of IFN-'y (6, 7). Human hepatocytes also respond to IFN-3' for NO production (8). As to whether human hepatocytes exhibit antimalaria activity when stimulated to produce NO, remains to be examined.At present, the antimalaria effector mechanism triggered by sporozoites in immunized animals is not fully understood. Presumably malaria-specific CD8 + T cells act directly against infected hepatocytes by recognizing malaria antigen on the cell surface (i.e., induction of CTLs) or malaria-specific lymphocytes release cytokines, such as IFN-3,, upon parasite stimulation, which induces an antimalarial response (3)(4)(5)(9)(10)(11)(12). The relationship between CD8 + T cells, IFN-% and NO-mediated protection in sporozoite-immunized mice was ...