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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 ...

To discover how nitric oxide (NO) synthesis is controlled in different tissues as cells within these tissues combat intracellular pathogens, we examined three distinctively different experimental murine models designed for studying parasite-host interactions: macrophage killing of Leishmania major; nonspecific protection against tularemia (Francisella tularensis) by Mycobacterium bovis (BCG); and specific vaccine-induced protection against hepatic malaria with Plasmodium berghei. Each model parasite and host system provides information on the source and role of NO during infection and the factors that induce or inhibit its production. The in vitro assay for macrophage antimicrobial activity against L. major identified cytokines involved in regulating NO-mediated killing of this intracellular protozoan. L. major induced the production of two competing cytokines in infected macrophages: (1) the parasite activated the gene for tumor necrosis factor (TNF), and production of TNF protein was enhanced by the presence of interferon-gamma (IFN-gamma). TNF then acted as a autocrine signal to amplify IFN-gamma-induced production of NO; and (2) the parasite upregulated production of transforming growth factor-beta (TGF-beta), which blocked IFN-gamma-induced production of NO. Whether parasite-induced TNF (parasite destruction) or TGF-beta (parasite survival) prevailed depended upon the presence and quantity of IFN-gamma at the time of infection. The relationship between NO production in vivo and host resistance to infection was demonstrated with M. bovis (BCG).(ABSTRACT TRUNCATED AT 250 WORDS)

Type-specific monoclonal antibodies prepared against the four dengue (DEN) virus serotypes were evaluated for their ability to identify low-passage human and mosquito isolates from Jamaica and West Africa by an indirect immunofluorescence assay. Serotyped human isolates from Jamaican dengue fever patients included 12 DEN-1, two DEN-2, and five DEN-4 viruses. Viruses from West Africa included 84 DEN-2 mosquito strains as well as two DEN-1 and one DEN-2 from humans. Results obtained using the immunofluorescence assay were consistent with virus identifications obtained using the more classical but costly and time-consuming plaque-reduction neutralization test. More viral isolates and higher virus yields were obtained using the C6/36 clone of Aedes albopictus cells rather than LLC-MK2 (monkey kidney) cells. Dengue type-specific monoclonal antibodies detected prototype viral antigens 24-48 hours postinfection in C6/36 cells. This is the first time that monoclonal antibodies have been used to serotype low-passage flavivirus isolates.

Extraintestinally invasive Escherichia coli (EC) that possess both a complete LPS and K1 capsule evade both complement-mediated bacteriolysis and neutrophil-mediated killing. Since C3H/HeJ mice that are hyporesponsive to LPS were uniquely susceptible to lethal infection with EC of this phenotype, we speculated there was an LPS-initiated host defense mechanism against this pathogenic phenotype. The LPS-normoresponsive C3H/HeN as well as the C3H/HeJ mice cleared these EC from the circulation within 4 h of intravenous administration. Whereas electron micrographs of the liver demonstrated these EC undergoing degeneration within the phagolysosomes of of both macrophages and Kupffer cells of C3H/HeN mice, these EC replicated within these cells of the C3H/HeJ mice. Restoration of anti-EC activity of C3H/HeJ mice occurred with activation of Kupffer cells and peritoneal macrophages in vivo with BCG and in vitro with IFN-y, but not with LPS. Pretreatment of C3H/ HeJ mice with a combination of recombinant murine IL-1 and TNF-a also restored the killing of K1 +-EC but did not enhance the killing of a K1--EC mutant. These data are consistent with the hypothesis that (a) there is no intrinsic inability of C3H/HeJ phagocytes to kill EC, but (b) an LPS-initiated, cytokine-mediated host defense mechanism is required for such killing. These studies emphasize the importance of bacterial surface characteristics in the interaction with specific host defenses. (J. Clin. Invest. 1995.

NYVAC-based vaccinia virus recombinants expressing the circumsporozoite protein (CSP) were evaluated in the Plasmodium berghei rodent malaria model system. Immunization of mice with a NYVAC-based CSP recombinant elicited a high level of protection (60 to 100%). Protection did not correlate with CS repeat-specific antibody responses and was abrogated by in vivo CD8 ؉ T-cell depletion. Protection was not enhanced by modification of the subcellular localization of CSP. These results suggest the potential of poxvirus-based vectors for the development of vaccine candidates for human malaria.

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The circumsporozoite protein (CSP) is a target for effector Ab and cell mediated immunity against malaria parasites; DNA vaccination can induce both types of effector response. The immunogenicity and efficacy of two DNA plasmids expressing different amounts of Plasmodium berghei CSP were evaluated by immunizing BALB/c mice i.m. or epidermally and by varying the number of immunizations (one to three doses) and the interval between immunizations. Expanding the interval gave the strongest effect, increasing efficacy and antibody boosting, and, in the case of epidermal vaccination, promoting a switch in CSP-specific IgG isotypes from IgG1 to a balance with IgG2a. The strongest humoral immune response and the greatest level of protection were induced by vaccinating epidermally with high expresser plasmid, using a gene gun to administer three doses at 6-wk intervals. For this group, the mean, repeat-specific, prechallenge antibody titer among mice not infected after challenge was significantly higher than that in infected mice, but the mean prechallenge titers for antibody reactive with whole sporozoites were not significantly different. The interval-dependent induction of IgG2a antibodies by epidermal vaccination contradicts the widely held belief that antibody responses induced by this method are restricted to those that are Th2 dependent.