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)

Immunization of rodents and humans with irradiation-attenuated malaria sporozoites confers preerythrocytic stage-specific protective immunity to challenge infection. This immunity is directed against intrahepatic parasites and involves T cells and interferon y, which prevent development of exoerythrocytic stages and subsequent blood infection. The present study was undertaken to determine how protective immunity is achieved after immunization of rodent hosts with irradiated Plasmodium berghei sporozoites. We present evidence that irradiated parasites persist in hepatocytes of rats and mice for up to 6 months after immunization. A relationship between the persistence of parasites and the maintenance of protective immunity was observed. Protective immunity was abrogated in irradiated-sporozoite-immunized rats following the application of chemotherapy to remove preexisting liver parasites. Additionally, protective immunity against sporozoite challenge was established in rats vaccinated with early and late hepatic stages of irradiated parasites. These results show that irradiation-attenuated sporozoites produce persistent intrahepatic stages in vivo necessary for the induction and maintenance of protective immunity.

Plasmodium berghei sporozoites delivered by mosquito bite were more infectious to outbred CD-1 mice than were sporozoites delivered by intravenous inoculation. The route of challenge also affected vaccine efficacy. In view of these findings and the fact that mosquito bites are the natural mode of sporozoite delivery, infectious mosquito bites should be considered the challenge protocol of choice for sporozoite vaccine efficacy trials.

Despite the low susceptibility of BALB/c mice to hepatic infection by Plasmodium berghei, this animal model is routinely used to investigate the basic biology of the malaria parasite and to test vaccines and the immune response against exoerythrocytic (EE) stages derived from sporozoites. A murine model in which a large number of EE parasites are established would be useful for furthering such investigations. Therefore, we assayed six mouse strains for susceptibility to erythrocytic and hepatic infections. The administration of 50 sporozoites by intravenous inoculation was suflicient to establish erythrocytic infections in five of five C57BL/6 mice compared with 10,000 sporozoites required to infect 100%/ of BALB/c mice. To assay for hepatic infections, mice received an intravenous inoculum of 106 sporozoites, and liver sections for light microscopy and histology were obtained at 29 and 44 h postinoculation. EE parasites were visualized by immunofluorescence, using an antibody to a P.fakciparum heat shock protein. The mean number of EE parasites per 100 cm2 for C57BL/6 and A/J strains was significantly higher than that for BALB/c (2,190 260, 88 38, and 6 2, respectively). The proportion of inoculated sporozoites transforming into liver schizonts was 8.2% in C57BL/6 and <1% in C3HVHeJ, DBA/1, and Swiss CD-1/ICR mice. Nonspecific inflammatory infiltrates around EE parasites were less prevalent in liver sections from C57BL/6 mice than in those from BALB/c mice, which contributed to the decrease in developing EE stages in BALB/c mice. These data indicate that the C57BL/6-P. berghei system is preferable for investigating the biology and immunology of liver stage parasites.

The elimination of liver-stage malaria parasites by nitric oxide (NO)-producing hepatocytes is regulated by T cells. Both CD8 ؉ and CD4 ؉ T cells, which surround infected hepatocytes, are evident by 24 h after sporozoite challenge in Brown Norway rats previously immunized with irradiated Plasmodium berghei sporozoites. While the number of CD4 ؉ T cells remained the same beyond 24 h postchallenge, the number of CD8 ؉ T cells increased three-and sixfold by 31 and 44 h, respectively. This increase in the number of CD8 ؉ T cells correlated with a decrease in the number of intrahepatic parasites. In immunized rats, intrahepatic parasites were reduced in number by 31 h after sporozoite challenge and cleared from the liver by 44 h, as visualized by P. berghei-specific DNA in situ hybridization. If immunized rats were treated with aminoguanidine, a substrate inhibitor of NO synthase, at the time of challenge, liver-stage protection was blocked, as shown by the increase in parasite liver burden. Further histological examination of infected livers from immunized animals treated with aminoguanidine revealed fewer and smaller cellular infiltrates surrounding the infected hepatocytes, and the number of CD8 ؉ T cells that normally accumulate within the infiltrates was drastically reduced. Consequently, the infected hepatocytes were not cleared from the liver. We hypothesize that the early production of NO may promote the influx and/or enhance local proliferation of malaria parasite-specific CD8 ؉ T cells or a CD8 ؉ T-cell subset which is required for parasite clearance.

Immunization with irradiated-attenuated malaria sporozoites has been shown to protect both rodents and humans against a homologous sporozoite challenge. Irradiated-attenuated sporozoites retain their capacity to invade hepatocytes and transform into trophozoites without undergoing complete schizogony. As a result, the minute size of these trophozoites (4-8 microns) makes their detection by conventional microscopy difficult. An additional problem lies in obtaining sufficient quantities of exoerythrocytic stages of attenuated parasites in vivo to study their antigenic repertoire and the sequence of events that occur after immunization of hosts. We have used a previously described method of inoculating Plasmodium berghei sporozoites directly into specific liver lobes (HPBI = hepatic portal branch inoculation) to improve parasite yields. Comparing HPBI with tail vein inoculation of sporozoites in Brown Norway rats and C57BL/6 mice revealed up to a 6-fold increase in hepatic parasite yields by HPBI method. The inoculation of 3 x 10(6) irradiated sporozoites via HPBI yielded 139 +/- 2 and 69 +/- 2 exoerythrocytic parasites per cm2 of liver in Brown Norway rats and C57BL/6 mice, respectively. The HPBI method therefore not only facilitates visualization of a large number of irradiated hepatic stage parasites within the defined lobes of the liver but also provides ample numbers of parasites for immunization and for immunological analysis.

SummaryIn mid 1997 the first malaria DNA vaccine will enter clinical trials. This single gene DNA vaccine encoding the Plasmodium falcipartim circumsporozoite protein (PtCSP) will be studied for safety and immunogenicity. If these criteria are met. a multi-gene DNA vaccine designed to induce protective CDS'*" T cell responses against P. falclparum infected hepatocytes will be subsequently assessed for safety, immunogenicity and capacity to protect immunized volunteers against experimental challenge with P. falciparum sporozoiles. Our perspectives on malaria vaccine development in general,' and on a multi-gene DNA vaccine in particular,^ have been recently reviewed. Herein, we review the rationale and experimental foundation for the anticipated P. falciparum DNA vaccine trials.

Both CD8+ T cells and IFN-gamma (IFN-gamma) are important components in the regulation of inducible-nitric oxide synthase (iNOS) which contribute to liver stage anti-malarial activity in rodents immunized with irradiated sporozoites. IFN-gamma, provided by malaria-specific CD8+ T cells, stimulates liver cells to produce nitric oxide (NO) for the destruction of infected hepatocytes or the parasite within these cells. To identify the cell source of iNOS in livers from Brown Norway rats challenged with Plasmodium berghei sporozoites, we probed tissue sections with antisera that recognize iNOS and the malarial exoerythrocytic stage parasite. Immunofluorescence analysis of parasitized livers demonstrate that 1) iNOS was found in infected hepatocytes, not Kupffer or endothelial cells; and 2) a higher proportion of infected hepatocytes express iNOS in immunized rats compared with naive animals after challenge. There was no immunoreactivity to the iNOS antisera in liver sections of immunized rats 15 h after sporozoite challenge, however, iNOS activity was present in 18% of the infected hepatocytes by 24 h and reached 81% by 31 h. In contrast, &lt; 10% of the infected hepatocytes displayed iNOS activity in naive or immune animals 48 h after challenge. We also found a significant decrease in the ability of the immunized animals to express iNOS in response to sporozoite challenge by accelerating the removal of pre-existing irradiated-attenuated parasites from hepatocytes with the antimalarial drug, primaquine. Therefore, induction and maintenance of iNOS activity were dependent on intrahepatic persistence of the irradiated-attenuated parasite. These results suggest that liver-iNOS expression following sporozoite challenge is restricted to the infected hepatocyte and dependent on the presence of the irradiated-attenuated parasite in immune animals.