Consistent, high-level, vaccine-induced protection against human malaria has only been achieved by inoculation of Plasmodium falciparum (Pf) sporozoites (SPZ) by mosquito bites. We report that the PfSPZ Vaccine--composed of attenuated, aseptic, purified, cryopreserved PfSPZ--was safe and well tolerated when administered four to six times intravenously (IV) to 40 adults. Zero of six subjects receiving five doses and three of nine subjects receiving four doses of 1.35 × 10(5) PfSPZ Vaccine and five of six nonvaccinated controls developed malaria after controlled human malaria infection (P = 0.015 in the five-dose group and P = 0.028 for overall, both versus controls). PfSPZ-specific antibody and T cell responses were dose-dependent. These data indicate that there is a dose-dependent immunological threshold for establishing high-level protection against malaria that can be achieved with IV administration of a vaccine that is safe and meets regulatory standards.

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 efficacy of a sporozoite-based malaria vaccine is tested in humans, nonhuman primates, and mice.

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CD8+ cytotoxic T lymphocytes (CTLs) are critical for protection against intracellular pathogens but often have been difficult to induce by subunit vaccines in animals. DNA vaccines elicit protective CD8+ T cell responses. Malaria-naïve volunteers who were vaccinated with plasmid DNA encoding a malaria protein developed antigen-specific, genetically restricted, CD8+ T cell-dependent CTLs. Responses were directed against all 10 peptides tested and were restricted by six human lymphocyte antigen (HLA) class I alleles. This first demonstration in healthy naïve humans of the induction of CD8+ CTLs by DNA vaccines, including CTLs that were restricted by multiple HLA alleles in the same individual, provides a foundation for further human testing of this potentially revolutionary vaccine technology.

In recent malaria sporozoite vaccine trials in humans and mice, antibodies to the sporozoite coat protein have given only modest protection against sporozoite challenge. In contrast, irradiated sporozoites can protect mice against massive sporozoite infections. Evidence suggests that immunity in these mice is mediated by T cells. To identify the mechanism of immunity, we used monoclonal antibodies specific for either the CD4 or CD8 molecule to selectively deplete sporozoite-immunized mice of T-cell subsets. Though in vivo depletion of CD4+ T cells did not reduce immunity, depletion of CD8+ T cells abolished protection. Monoclonal antibody treatment did not affect anti-sporozoite antibody levels. Our data indicate that cytotoxic T cells are critical for immunity to large numbers of sporozoites and suggest that vaccine development should be reoriented toward stimulating cellular as well as humoral immunity.In the life cycle of the malaria parasite, sporozoites pass from the mosquito through the blood of the host and invade liver cells where they develop into hepatic-stage parasites. Later, mature parasites are released from the liver to invade erythrocytes. When sporozoites are experimentally irradiated, they still invade liver cells but they are unable to mature to the stage that infects erythrocytes (1). Immunization with irradiated sporozoites can protect mice against infection with several thousand sporozoites (2). Humans can also be successfully immunized with irradiated sporozoites (3,4), but use of attenuated parasites as a vaccine is impractical. A current sporozoite vaccine strategy is to induce antibodies against the central repetitive sequence of the circumsporozoite (CS) protein, which covers the sporozoite surface (5-7). Several recent trials of such vaccines in humans (8, 9) and mice (10, 11) have had limited success. This suggests that humoral immunity may be less important than previously thought and that cellular immunity may be critical for a highly effective vaccine (10,12,13 (20). Thirty to 60 mosquitoes were anesthetized with chloroform and placed on a glass slide in a drop of medium 199 with 3% mouse serum. The abdomen was held with fine forceps, and the thorax of each mosquito was cut just anterior to the wing. The collection of upper bodies without further preparation was then spun through glass wool according to the published method. After counting in a hemacytometer, sporozoites were diluted to final concentration in medium 199 with 3% mouse serum.Antibodies. Monoclonal antibodies were produced in ascites fluid of athymic mice and of BALB/c mice treated with cortisone and irradiation (21). Anti-CD8 antibody came from the anti-Lyt2.2 hybridoma 19/178 (mouse IgG2a) (22). Anti-CD4 antibody came from the anti-L3T4 clone GK1.5 (rat IgG2b) (23). A control antibody came from the antiPlasmodium falciparum gamete clone 1B3 (mouse IgG2a) (45). Control rat immunoglobulin was from normal rat serum purchased from Accurate Chemical and Scientific (Westbury, NY). All immunoglobulins were purifie...

Immunization with irradiated sporozoites protects animals and h against malaria, and the circumsporozoite protein is a target of this protective immunity. We now report that adjuvant-free intramuscular injection of mice with plasmid DNA encding the Plasmodium yoeli circumsporozoite protein induced higher levels of antibodies and cytotoxic T lymphocytes against the P. yoel& circumsporozoite protein than did immunization with irradiated sporozoites. Mice immunized with this vaccine had an 86% reduction in liver-stage parasite burden after challenge with 5 x 10W sporozoites (>10 median infectious doses). Eiteen (68%) of 28 mice that received two or three doses of vaccine were protected against challenge with 102 sporozoites, and the protection was dependent on CD8+ T cells. These studies demonstrate the utility of plasmid DNA immunization against a nonviral infection. By obviating the requirement for peptide synthesis, expression and purification of recombinant proteins, and ad-

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