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...

vaccine ͉ dendritic cell ͉ cross-presentation ͉ cellular immunity

There is a remarkable heterogeneity in the functional profile (quality) of T cell responses. Importantly, the magnitude and/or quality of a response required for protection may be different depending on the infection. Here, we assessed the capacity of different Toll like receptor (TLR)-binding compounds to influence T helper cell (Th)1 and CD8+ T cell responses when used as adjuvants in nonhuman primates (NHP) with HIV Gag as a model antigen. NHP were immunized with HIV Gag protein emulsified in Montanide ISA 51, an oil-based adjuvant, with or without a TLR7/8 agonist, a TLR8 agonist, or the TLR9 ligand cytosine phosphate guanosine oligodeoxynucleotides (CpG ODN), and boosted 12 wk later with a replication-defective adenovirus-expressing HIV-Gag (rAD-Gag). Animals vaccinated with HIV Gag protein/Montanide and CpG ODN or the TLR7/8 agonist had higher frequencies of Th1 responses after primary immunization compared to all other vaccine groups. Although the rAD-Gag boost did not elevate the frequency of Th1 memory cytokine responses, there was a striking increase in HIV Gag-specific CD8+ T cell responses after the boost in all animals that had received a primary immunization with any of the TLR adjuvants. Importantly, the presence and type of TLR adjuvant used during primary immunization conferred stability and dramatically influenced the magnitude and quality of the Th1 and CD8+ T cell responses after the rAD-Gag boost. These data provide insights for designing prime-boost immunization regimens to optimize Th1 and CD8+ T cell responses.

SummaryTwo field studies in Kenya and an experimental challenge study in the USA were done to assess the accuracy of a dipstick antigen-capture assay based on qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP-2) in peripheral blood for diagnosis of P falciparum infection.In these studies, the assay was 96·5-100% sensitive for detection of greater than 60 P falciparum asexual parasites/ µL blood, 70-81% sensitive for 11-60 parasites/µL blood, and 11-67% sensitive for 10 parasites or less/µL blood. Specificity was 95% (95% Cl 85-105%; n = 20) among naive American volunteers, 98% (96-101%; n=112) among volunteers exposed to the bite of P falciparium-infected mosquitoes, and 88% (84-92%; n=285) among Kenyans living in an area with holoendemic malaria. Our results also indicated that PfHRP-2 antigen was not detectable in blood 6 days after initiation of curative chemotherapy, and suggest that such circulating antigens rarely lead to false-positive tests.The dipstick assay's sensitivity, specificity, simplicity, and speed may make it an important tool in the battle against malaria.

When a person is infected with malaria by the bite of a mosquito, sporozoites travel through the bloodstream and enter hepatocytes. The parasite develops within the liver cell for days to weeks, and during this period there are no symptoms. Only after the liver stage parasites mature, and merozoites are released to invade red blood cells, do the fevers, chills and lethal complications of malaria begin. Thus, a vaccine aimed at sporozoites or liver stage parasites could eliminate malaria infections before clinical disease appears.Immunization with radiation-attenuated sporozoites can protect rodents and humans against live sporozoite challenge (1, 2). One hypothesis is that this protective immunity is mediated by cytotoxic T cells directed against malaria antigens on the infected hepatocyte . Several recently published experiments give support to this idea. Immune animals became susceptible to sporozoite infection if they were depleted of their CD8+ T cells (3, 4), but the antigens recognized by these cells and their site and mode of action were not determined. Infiltrates with CD8+ and CD4' T cells and other cellular elements were found in the livers of immune animals after challenge with sporozoites and these infiltrates formed after the transfer of immune CD8 + cells (5). Unfractionated spleen cells from immune animals eliminated malaria-infected hepatocytes from in vitro culture (5), but the active cell types and the targeted parasite antigens were not identified . A very recent study showed that mice were protected by adoptive transfer of cloned CD8+ T cells specific for an epitope on the Plasmodium berghei circumsporozoite (CS) 1 protein (6). However, it was not shown that these cells were attacking infected hepatocytes directly, or releasingThe opinions and assertions herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the U. S. Navy or the naval service at large.

Primaquine was tested as a prophylactic drug against Plasmodium falciparum in a region in western Kenya in which malaria is holoendemic. Children 9-14 years old were randomized to receive regimens of daily primaquine, daily doxycycline, daily proguanil plus weekly chloroquine, daily vitamin plus weekly mefloquine, or daily vitamin alone. Primaquine, doxycycline, and mefloquine were equally effective in preventing both symptomatic and asymptomatic malarial infections. Chloroquine plus proguanil was the least effective regimen. There was no toxicity from daily primaquine during the 11 weeks of the study. Findings show that primaquine can be successfully used as a causal prophylactic regimen against falciparum malaria in western Kenya; chloroquine plus proguanil was not as efficacious as the three other preventive regimens; most Kenyan children receiving standard doses of mefloquine and doxycycline had lower than expected serum trough drug levels; and some volunteers with adequate mefloquine or doxycycline levels at trough developed asymptomatic parasitemias and clinical malaria.

Live attenuated malaria vaccines are more potent than the recombinant protein, bacterial or viral platform vaccines that have been tested, and an attenuated sporozoite vaccine against falciparum malaria is being developed for humans. In mice, attenuated malaria sporozoite vaccines induce CD8+ T cells that kill parasites developing in the liver. We were curious to know if CD8+ T cells were also important in protecting primates against malaria. We immunized 9 rhesus monkeys with radiation attenuated Plasmodium knowlesi sporozoites, and found that 5 did not develop blood stage infections after challenge with live sporozoites. We then injected 4 of these protected monkeys with cM-T807, a monoclonal antibody to the CD8 molecule which depletes T cells. The fifth monkey received equivalent doses of normal IgG. In 3 of the 4 monkeys receiving cM-T807 circulating CD8+ T cells were profoundly depleted. When re-challenged with live sporozoites all 3 of these depleted animals developed blood stage malaria. The fourth monkey receiving cM-T807 retained many circulating CD8+ T cells. This monkey, and the vaccinated monkey receiving normal IgG, did not develop blood stage malaria at re-challenge with live sporozoites. Animals were treated with antimalarial drugs and rested for 4 months. During this interval CD8+ T cells re-appeared in the circulation of the depleted monkeys. When all vaccinated animals received a third challenge with live sporozoites, all 5 monkeys were once again protected and did not develop blood stage malaria infections. These data indicate that CD8+ T cells are important effector cells protecting monkeys against malaria sporozoite infection. We believe that malaria vaccines which induce effector CD8+ T cells in humans will have the best chance of protecting against malaria.