The Plasmodium falciparum knob-associated PfEMP3 antigen is also expressed at pre-erythrocytic stages and induces antibodies which inhibit sporozoite invasion.

The expression of the pfemp3 gene and the corresponding PfEMP3 knob-associated protein in the pre-erythrocytic stages of Plasmodium falciparum was demonstrated by RT-PCR, Western blots, IFAT and IEM. The antigen was found on the surface of the sporozoite and in the cytoplasm of mature hepatic stage parasites. Immunological cross-reactivity was observed with sporozoites from the rodent malaria parasites Plasmodium yoelii yoelii and Plasmodium berghei and was exploited to assess a potential role of this protein at the pre-erythrocytic stages. Specific antibodies from immune individuals were found to inhibit P. yoelii yoelii and P. berghei sporozoite invasion of primary hepatocyte cultures. PfEMP3 should now be added to the small list of proteins expressed at the pre-erythrocytic stages of P. falciparum, and its vaccine potential now deserves to be investigated.

A primary malarial infection is composed of a very wide range of genetically diverse but related parasites.

To address the question of how many distinct parasites are injected when a mosquito bites, we have characterized isolates resulting most probably from a single sporozoite inoculum. We describe the direct and immediate cloning on hepatocyte feeder layers of a Thai and an African Plasmodium falciparum primary isolate and the characterization of 67 independent clones by four techniques totaling nine different markers. This led to three main conclusions: (a) both the phenotypic and genotypic markers revealed an unexpectedly large degree of diversity within the clones from a single isolate; (b) the clones are nonetheless genetically related; and (c) a single mosquito inoculum would most likely be sufficient to generate considerable isolate complexity in the absence of repeated exposure. This diversity, which has been greatly underestimated in previous studies, does not bode well for the development of successful malaria control means.

Plasmodium yoelii: 17-kDa hepatic and erythrocytic stage protein is the target of an inhibitory monoclonal antibody.

Infected hepatocytes are important targets for malaria vaccines. To identify Plasmodium yoelii proteins expressed in infected hepatocytes, we immunized BALB/c ByJ mice with P. yoelii liver stage schizonts and produced a panel of monoclonal antibodies (Mabs). An IgG1 Mab, navy yoelii liver stage 3 (NYLS3), had the strongest reactivity against liver stage parasites and was selected for further characterization. The Mab does not recognize P. yoelii sporozoites, but recognizes liver stage parasites within 6 hr of invasion of mouse hepatocytes and throughout the hepatic and asexual erythrocytic stages of the parasite life cycle as determined by the immunofluorescent antibody test. This Mab is species-specific, and it reacts with liver stages of P. yoelii but does not react with liver stages of other Plasmodium species. The protein recognized by this Mab is present on the parasitophorous vacuole membrane of infected hepatocytes and erythrocytes as demonstrated by immunoelectron microscopy and has a relative molecular weight of 17 kDa as demonstrated by immunoblot of an extract of infected erythrocytes. It is therefore designated P. yoelii hepatic and erythrocytic stage protein, 17 kDa or PyHEP17. When added to primary cultures of mouse hepatocytes 24 hr after inoculation with P. yoelii sporozoites, when all sporozoites have invaded hepatocytes, NYLS3 eliminates up to 98% of liver-stage parasites. Intravenous injection of NYLS3 into mice delays the onset and reduces the density of blood-stage parasitemia after sporozoite or blood-stage challenge. The P. falciparum and P. vivax homologs of PyHEP17 may therefore be important targets for vaccines designed to attack the hepatic and erythrocytic stages of the parasite life cycle.

Nitric oxide-mediated antiplasmodial activity in human and murine hepatocytes induced by gamma interferon and the parasite itself: enhancement by exogenous tetrahydrobiopterin.

Expression of inducible nitric oxide (NO) synthase has been shown to inhibit the development of several pathogens, including fungi, bacteria, parasites, and viruses. However, there is still controversy as to whether this effector mechanism can inhibit the development of human pathogens. We now report that gamma interferon (IFN-gamma) induces the elimination of Plasmodium falciparum-infected primary human hepatocytes from cultures and that the antimalarial activity is dependent on NO. Infection with the parasite alone in the absence of added IFN-gamma caused a 10-fold increase in NO formation. Both spontaneous inhibition and IFN-gamma-induced inhibition of Plasmodium yoelii-infected murine hepatocytes were increased with the addition of the NO synthase cofactor tetrahydrobiopterin, or sepiapterin, which is converted to tetrahydrobiopterin. These results indicate that under in vitro conditions the parasite itself provides a signal that triggers induction of the NO pathway in human and murine hepatocytes and that NO formation in infected hepatocytes is limited by tetrahydrobiopterin availability.

Plasmodium falciparum liver stage antigen-1 is well conserved and contains potent B and T cell determinants.

We have previously identified a Plasmodium falciparum liver stage-specific Ag (LSA-1) found to encode tandem 17 amino acid repeats harboring B cell determinants. Here we extend this study in terms of sequence analysis, protein localization, and immunologic properties. Analysis of the N- and C-terminal regions of LSA-1 from the T9/96 clone reveals high sequence conservation with LSA-1 from NF54. This 200-kDa protein is detected throughout liver schizogony and accumulates in the parasitophorous vacuole space. In our investigation of T and B cell responses to LSA-1, we have focused on both the area of the C-terminal, nonrepetitive "hinge" region and the conserved repetitive region and derived synthetic peptides. These were found to contain major B and T cell determinants. High prevalences and elevated Ab levels to LSA-1, directed primarily, although not exclusively, to the repetitive region, were detected in sera of individuals from one moderately high and two low transmission malaria-endemic areas (prevalences of 97%, 75, and 77%, respectively). In one of these low transmission areas, secretion of the cytokine IFN-gamma, known to inhibit malaria liver stages, and T cell proliferation were detected in PBMC of 22 to 48% and 6 to 20%, respectively, of individuals in response to separate LSA-1 peptides. These results complement the recent finding of conserved CTL epitopes in LSA-1 and support the assertion that immune responses to LS Ag are involved in protection against malaria pre-erythrocytic stages.

Malaria sporozoites and circumsporozoite protein bind sulfated glycans: carbohydrate binding properties predicted from sequence homologies with other lectins.

Circumsporozoite (CS) proteins, the major surface proteins of the sporozoites of the various malaria (Plasmodium) species, share a region of highly conserved sequence homology in common with sporozoite surface protein 2 (SSP2) and a group of proteins observed to specifically bind sulfated glycoconjugates. Recombinant P. yoelii CS protein was found to bind selectively to heparin-, fucoidan-, and dextran sulfate-Sepharose, but poorly to chondroitin sulfate A- or C-Sepharose. It also bound with lower affinity to a heparan sulfate biosynthesis-deficient mutant cell line compared with the wild-type. Likewise, P. berghei sporozoite invasion into hepatocytes was selectively inhibited by fucoidan, heparin, and dextran sulfate, and sporozoites bound specifically to sulfatide [galactosyl (3-SO4) beta 1-1 ceramide] coated surfaces. Sporozoite infectivity in mice was significantly inhibited by dextran sulfate 500,000 and fucoidan. Taken together, these data indicate that CS proteins bind selectively to certain sulfated glyconjugates and invasion of host hepatocytes by sporozoites, and sporozoite infectivity can be inhibited by such compounds.

Characterization of Plasmodium falciparum sporozoite surface protein 2.

Immunization of mice with Plasmodium yoelii sporozoite surface protein 2 (PySSP2) and circumsporozoite protein protects completely against P. yoelii. The amino acid sequence of PySSP2 suggested that the thrombospondin-related anonymous protein (TRAP) [Robson, K. J. H., Hall, J. R. S., Jennings, M. W., Harris, T. J. R., Marsh, K., Newbold, C. I., Tate, V. E. & Weatherall, D. J. (1988) Nature (London) 335, 79-82] is the Plasmodium falciparum homolog of PySSP2. We report data confirming that TRAP is P. falciparum SSP2 (PfSSP2). Murine antibodies against recombinant PfSSP2 identify a 90-kDa protein in extracts of P. falciparum sporozoites, recognize sporozoites and infected hepatocytes by immunofluorescence, localize PfSSP2 to the sporozoite micronemes by immunoelectron microscopy and to the surface membrane by live immunofluorescence, and inhibit sporozoite invasion and development in hepatocytes in vitro. Human volunteers immunized with irradiated sporozoites and protected against malaria develop antibody and proliferative T-cell responses to PfSSP2, suggesting that, like PySSP2, PfSSP2 is a target of protective immunity, and supporting inclusion of PfSSP2 in a multicomponent malaria vaccine.

Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates.

Circumsporozoite (CS) proteins, which densely coat malaria (Plasmodia) sporozoites, contain an amino acid sequence that is homologous to segments in other proteins which bind specifically to sulfated glycoconjugates. The presence of this homology suggests that sporozoites and CS proteins may also bind sulfated glycoconjugates. To test this hypothesis, recombinant P. yoelii CS protein was examined for binding to sulfated glycoconjugate-Sepharoses. CS protein bound avidly to heparin-, fucoidan-, and dextran sulfate-Sepharose, but bound comparatively poorly to chondroitin sulfate A- or C-Sepharose. CS protein also bound with significantly lower affinity to a heparan sulfate biosynthesis-deficient mutant cell line compared with the wild-type line, consistent with the possibility that the protein also binds to sulfated glycoconjugates on the surfaces of cells. This possibility is consistent with the observation that CS protein binding to hepatocytes, cells invaded by sporozoites during the primary stage of malaria infection, was inhibited by fucoidan, pentosan polysulfate, and heparin. The effects of sulfated glycoconjugates on sporozoite infectivity were also determined. P. berghei sporozoites bound specifically to sulfatide (galactosyl[3-sulfate]beta 1-1ceramide), but not to comparable levels of cholesterol-3-sulfate, or several examples of neutral glycosphingolipids, gangliosides, or phospholipids. Sporozoite invasion into hepatocytes was inhibited by fucoidan, heparin, and dextran sulfate, paralleling the observed binding of CS protein to the corresponding Sepharose derivatives. These sulfated glycoconjugates blocked invasion by inhibiting an event occurring within 3 h of combining sporozoites and hepatocytes. Sporozoite infectivity in mice was significantly inhibited by dextran sulfate 500,000 and fucoidan. Taken together, these data indicate that CS proteins bind selectively to certain sulfated glycoconjugates, that sporozoite infectivity can be inhibited by such compounds, and that invasion of host hepatocytes by sporozoites may involve interactions with these types of compounds.

Recombinant pseudorabies virus carrying a plasmodium gene: herpesvirus as a new live viral vector for inducing T- and B-cell immunity.

In Balb/c mice, the sterile protective immunity induced by immunization with radiation-attenuated Plasmodium yoelii sporozoites is eliminated by in vivo depletion of CD8+ T lymphocytes, suggesting that cytotoxic T lymphocytes (CTL) against malaria antigens expressed on infected hepatocytes are required for mediating this protective immunity. To produce a vaccine that would induce CTL against the P. yoelii circumsporozoite protein (CS), we constructed an attenuated pseudorabies virus (PRV) containing a gene encoding this protein. Balb/c mice that received three doses of 10(7) plaque-forming units (p.f.u.) of this vaccine intravenously at 3 week intervals developed high levels of antibodies to sporozoites (indirect fluorescent antibody titre = 4096) and CTL against a 16 amino acid epitope (SYVPSAEQILEFVKQI, amino acids 281-296) from the P. yoelii CS protein designated PYCTL1. The cytotoxic activity of the CTL was antigen-specific, MHC-restricted, and dependent on CD8+ T cells. Furthermore, these CTL eliminated P. yoelii-infected hepatocytes from in vitro culture, indicating that they recognize this peptide on the surface of infected hepatocytes. However, all nine mice that were challenged with 200 sporozoites developed a blood-stage malaria infection. We attribute this lack of protection to the great difficulty of inducing sterile immunity against this highly infectious parasite P. yoelii. We conclude that recombinant pseudorabies virus (PRV) worked successfully as a live vaccine vector to induce both antibodies and CTL, albeit non-protective in vivo, and the herpesviruses should be considered as subunit vaccines where T- and B-cell immunity is required.

IFN-gamma inhibits development of Plasmodium berghei exoerythrocytic stages in hepatocytes by an L-arginine-dependent effector mechanism.

Primary cultures of BALB/cJ hepatocytes treated with 10(3) U/ml rIFN-gamma consistently inhibited intracellular Plasmodium berghei liver schizont development by 50 to 70%. Monomethyl-L-arginine (NGMMLA), the competitive inhibitor of L-arginine as substrate for production of nitric oxides by hepatocytes, reversed the activity of IFN-gamma on these malaria-infected cells. Reversal of IFN-gamma activity by NGMMLA was dose dependent and was maximal at 0.5 mM NGMMLA. Depletion of L-arginine by addition of arginase to the culture medium blocked the capacity of IFN-gamma to inhibit parasite development in hepatocytes; addition of excess L-arginine to cultures treated with IFN-gamma in the presence of NGMMLA competitively restored IFN-gamma capacity to activate hepatocyte anti-parasite activity. TNF-alpha was neither required for IFN-gamma activity, nor effective at any concentration tested as an inhibitor of schizont development by itself in primary hepatocytes. These data strongly suggest that the action of IFN-gamma on P. berghei-infected hepatocytes is to induce the production of L-arginine-derived nitrogen oxides that are toxic for the intracellular parasite.

Monoclonal, but not polyclonal, antibodies protect against Plasmodium yoelii sporozoites.

One of the primary strategies for malaria vaccine development has been to design subunit vaccines that induce protective levels of antibodies against the circumsporozoite (CS) protein of malaria sporozoites. In the Plasmodium yoelii mouse model system such vaccines have been uniformly unsuccessful in protecting against sporozoite-induced malaria. To demonstrate that antibodies to P. yoelii CS protein could provide protection we established a passive transfer model. Passive transfer of Navy yoelii sporozoite 1 (NYS1), an IgG3 mAb against the P. yoelii CS protein, protected 100% of mice against challenge with 5000 P. yoelii sporozoites. Binding of NYS1 to sporozoites was inhibited by incubation with (QGPGAP)2, a synthetic peptide derived from the repeat region of the P. yoelii CS protein, indicating that the epitope on sporozoites recognized by this mAb was included within this peptide. The levels of antibodies to (QGPGAP)2 by ELISA, and to sporozoites by indirect fluorescent antibody test and CS precipitation reaction were similar in sera from mice that received NYS1 in passive transfer and were protected against challenge with 5000 sporozoites, and from mice that had been immunized with subunit vaccines containing (QGPGAP)2 but were not protected against challenge with 40-200 sporozoites. To determine if antibody avidity, not absolute concentration could explain the striking differences in protection, we established a thiocyanate elution assay. The results suggest that NYS1, the protective mAb, has a lower avidity for (QGPGAP)2 and for sporozoites than do the vaccine-induced antibodies. Although the results of the conventional antibody assays did not correlate with protection, sera from the protected animals inhibited sporozoite development in mouse hepatocyte cultures significantly more than did the sera from the unprotected, subunit vaccine-immunized animals, correlating with protection. The data clearly demonstrate that antibodies to the CS protein can protect against intense sporozoite infection. Improved understanding of the differences between protective mAb and nonprotective polyclonal antibodies will be important in the further development of malaria vaccines.

Cellular mechanisms of nonspecific immunity to intracellular infection: cytokine-induced synthesis of toxic nitrogen oxides from L-arginine by macrophages and hepatocytes.

Nitric oxide (NO) produced by cytokine-treated macrophages and hepatocytes plays a vital role in protective host responses to infectious pathogens. NO inhibits iron-sulfur-dependent enzymes involved in cellular respiration, energy production, and reproduction. Synthesis of L-arginine-derived nitrite (NO2-), the oxidative end product of NO, directly correlates with intracellular killing of Leishmania major, an obligate intracellular protozoan parasite of macrophages: the level of NO2- production is a quantitative index for macrophage activation. The competitive inhibitor of NO synthesis, monomethylarginine (NGMMLA), inhibits both parasite killing and NO2- production. For Leishmania, the parasite itself participates in the regulation of this toxic effector mechanism. This participation is mediated by parasite induction of tumor necrosis factor alpha (TNF alpha), an autocrine factor of macrophages: NO synthesis by interferon-gamma (IFN-gamma)-treated cells can be blocked by monoclonal antibodies to TNF alpha. NO production by IFN gamma-treated hepatocytes is of special interest in malaria infections: sporozoite-infected hepatocytes kill the intracellular malaria parasite after treatment with IFN gamma; this killing is inhibited by NGMMLA.

Irradiated sporozoite vaccine induces cytotoxic T lymphocytes that recognize malaria antigens on the surface of infected hepatocytes.

The observation that protective immunity induced by immunization with radiation attenuated Plasmodium berghei and Plasmodium yoelii sporozoites is dependent on CD8+ T lymphocytes in some strains of mice led us to speculate that immunization with sporozoites induces cytotoxic T lymphocytes (CTL) that recognize malaria antigens on the surface of malaria-infected hepatocytes. In this report we summarize a series of experiments that confirm this hypothesis. We first showed that when immune mice are challenged with live sporozoites they develop malaria-specific, CD8+ T cell-dependent infiltrates in their livers. Next we demonstrated that spleen cells from immune mice eliminate malaria infected hepatocytes from in vitro culture in an antigen specific and genetically restricted manner, indicating that these immune cells recognize malaria antigens on the surface of infected hepatocytes. Finally we defined a CTL epitope of the P. yoelii CS protein, and demonstrated that CTL against this 16-amino-acid peptide (PYCTL1) eliminate infected hepatocytes from culture in an antigenic specific, and MHC restricted manner, indicating that this 16-amino-acid peptide from the CS protein is present on the surface of the infected hepatocytes. We are currently working on constructing vaccines that induce protective CTL against PYCTL1, and identifying additional pre-erythrocytic stage targets of CTL mediated protective immunity.

Cytotoxic T cells recognize a peptide from the circumsporozoite protein on malaria-infected hepatocytes.

Irradiated malaria sporozoites can induce CD8+ T cells that are required for protection against infection. However, the parasite antigens targeted by this immune response are unknown. We have discovered a 16-amino acid epitope from the Plasmodium yoelii circumsporozoite (CS) protein that is recognized by cytotoxic T cells from immune mice. Lymphocytes stimulated with this peptide can kill P. yoelii liver stage parasites in vitro in an MHC-restricted, antigen-specific manner. Thus, epitopes from the CS protein are presented on the surface of infected hepatocytes and can be targets for T cells, even though intact CS protein has not been detected on the surface of the infected hepatocyte. A vaccine that induced CTL to parasite antigens might protect humans against malaria by eliminating liver stage parasites.

T lymphocytes from mice immunized with irradiated sporozoites eliminate malaria from hepatocytes.

When mice are immunized with radiation-attenuated sporozoites they are solidly protected against sporozoite challenge by an immune response that has been shown to require CD8+ lymphocytes in several strains of mice. The target of this CD8+ T-cell-dependent immunity has not been established. Immune BALB/c mice were shown to develop malaria-specific, CD8+ T-cell-dependent inflammatory infiltrates in their livers after challenge with Plasmodium berghei sporozoites. Spleen cells from immune BALB/c and C57BL/6 mice eliminated hepatocytes infected with the liver stage of P. berghei in vitro. The activity against infected hepatocytes is not inhibited by antibodies to interferon-gamma and is not present in culture supernatants. It is genetically restricted, an indication that malaria antigens on the hepatocyte surface are recognized by immune T-effector cells. Further subunit pre-erythrocytic stage malaria vaccine development will require identification of the antigens recognized by these T cells and a method of immunization that induces such immunity.

Evaluation of an in vitro assay aimed at measuring protective antibodies against sporozoites.

We have evaluated the in vitro biological activities of antibodies directed against sporozoites and compared them with their capacity to protect against challenge with both human and rodent malaria. The anti-Plasmodium falciparum antibodies evaluated with the test included monoclonal antibodies (MAbs) NFS1 and NFS2 as well as polyclonal antibodies contained in human hyperimmune sera directed against sporozoites of P. falciparum. The inhibitory effect of these antibodies was dependent on their concentration. However, total inhibition was not observed except occasionally with highly concentrated MAbs (10-100 micrograms/ml). Strong but also incomplete inhibition was observed with sera from humans living in hyperendemic areas. In the P. yoelii rodent system, we tested sera from mice immunized with subunit vaccines. None of these mice were protected in vivo against challenge with 40-200 sporozoites. In vitro only a sub-total inhibition was achieved (maximum 91% at 1:10 serum dilution). In contrast, we tested sera from mice that received NYS1, an IgG3 MAb, in passive transfer and were protected against challenge with 5000 sporozoites. At 1:10 dilution, 100% inhibition was achieved in vitro while IFA titres from these mice were similar to those of vaccinated mice. These data show a close correlation between in vivo and in vitro findings and thus suggest that the inhibition of liver-stage development assay (ILSDA) appears appropriate to evaluate the potential of antibodies.