Hepatic phase of malaria is the target of cellular mechanisms induced by the previous and the subsequent stages. A crucial role for liver nonparenchymal cells.

Both the sporozoites and the erythrocytic stages can modulate the hepatic phase by cytokines, notably IFN-gamma, TNF and IL-6, either directly or as a result of a cascade of events, and by MHC-restricted and antibody-dependent cell-mediated cytotoxicity. The role played by CD8+ T cells in inducing protective immunity against pre-erythrocytic stages is clearly established. The potential interest of triggering peptide-primed CD4+ T cells has to be considered regarding protection. Indeed, CD4+ T cells induced by the non-repetitive part of the CS protein of Plasmodium yoelii are protective, by eliminating malaria from hepatocytes. The crucial role of the liver NPC has to be emphasized, their participation in TNF schizonticidal effect and in ADCC mechanisms being strongly supported by our data.

Activity of human volunteer sera to candidate Plasmodium falciparum circumsporozoite protein vaccines in the inhibition of sporozoite invasion assay of human hepatoma cells and hepatocytes.

Sera from human volunteers immunized with either synthetic peptide (NANP)3-TT or recombinant protein R32tet32 Plasmodium falciparum CS vaccines were tested in the inhibition of sporozoite invasion (ISI) assays using human hepatoma (HepG2-A16) cells or primary human hepatocytes. Sera or purified immunoglobulin (Ig) from volunteers who were completely protected against P. falciparum sporozoite challenge had higher ISI activity than sera from non-protected volunteers, or the highest titre endemic serum. However, Ig from protected and non-protected volunteers did not block sporozoite invasion of human hepatocytes, suggesting that P. falciparum sporozoites invade hepatocytes by mechanisms which differ from those concerned with invasion of HepG2-A16 cells.

Hepatic stages of malaria: specific and non-specific factors inhibiting the development.

Protection against pre-erythrocytic stages of malaria is possible, as demonstrated by the resistance obtained by immunizing with irradiated sporozoites. However, the involved mechanisms are more numerous and intricate than previously believed. Recently, the hepatic stage, rather than the sporozoite stage, has been seen as the target of immune attack.

Non specific resistance against malaria pre-erythrocytic stages: involvement of acute phase proteins.

Levels of different acute phase proteins were compared in sera from parasitaemic and non-parasitaemic women living in a Plasmodium falciparum endemic area of Thailand. The ability of their sera to interfere with hepatic stage development of the parasite was examined. Correlations were found between levels of alpha-1 antitrypsin, alpha-2 macroglobulin, hemopexin and the potential of sera to block hepatocyte invasion by the sporozoite.

Genetic control of parasite clearance leads to resistance to Plasmodium berghei ANKA infection and confers immunity.

Unprecedented cure after infection with the lethal Plasmodium berghei ANKA was observed in an F2 progeny generated by intercrossing the wild-derived WLA and the laboratory C57BL/6 mouse strains. Resistant mice were able to clear parasitaemia and establish immunity. The observed resistance was disclosed as a combinatorial effect of genetic factors derived from the two parental strains. Genetic mapping of survival time showed that the WLA allele at a locus on chromosome 1 (colocalizing with Berghei resistance 1 (Berr1), a locus associated with resistance to experimental cerebral malaria) increases the probability to resist early death. Also, the C57Bl/6 allele at a novel locus on chromosome 9 (Berr3) confers overall resistance to this lethal Plasmodium infection. This report underlines the value of using wild-derived mouse strains to identify novel genetic factors in the aetiology of disease phenotypes, and provides a unique model for studying parasite clearance and immunity associated with malaria.

Inhibitory activity of IL-6 on malaria hepatic stages.

Addition of recombinant interleukin-6 (IL-6) to Plasmodium yoelii hepatic cultures resulted in a specific dose-dependent inhibition of parasite development. Time course experiments showed that, without any direct effect on free sporozoites, IL-6 exerts its action during both the early phase of infection and during the subsequent maturation of the schizonts. Elicitation of the oxidative burst appears to be one mechanism by which IL-6 interferes with the development of hepatic phase. Catalase and superoxide dismutase, two scavengers of hydrogen peroxide and superoxide anions, reversed the IL-6 mediated parasiticidal activity.

Inflammatory status and preerythrocytic stages of malaria: role of the C-reactive protein.

In the acquisition of protection against malaria, the role played by nonspecific factors, some being part of the cascade effect of cytokines, has to be considered. The C-reactive protein, a major acute phase reactant secreted by interleukin-1 stimulated hepatocytes, has an effect on the hepatic development of Plasmodia, both by preventing penetration of the sporozoite into the hepatocyte and by blocking parasite division through an antibody-like effect. This latter effect confirms the potential interest of targeting the uninuclear form of the parasite. Nevertheless, C-reactive Protein alone does not account for all the effects of the inflammatory response, other reactants from both serum and hepatocytes are also involved.

T cell response in malaria pathogenesis: selective increase in T cells carrying the TCR V(beta)8 during experimental cerebral malaria.

To characterize the T cells involved in the pathogenesis of cerebral malaria (CM) induced by infection with Plasmodium berghei ANKA clone 1.49L (PbA 1.49L), the occurrence of the disease was assessed in mice lacking T cells of either the alphabeta or gammadelta lineage (TCRalphabeta(-/-) or TCRgammadelta(-/-)). TCRgammadelta(-/-) mice were susceptible to CM, whereas all TCRalphabeta(-/-) mice were resistant, suggesting that T cells of the alphabeta lineage are important in the genesis of CM. The repertoire of TCR V(beta) segment gene expression was examined by flow cytometry in B10.D2 mice, a strain highly susceptible to CM induced by infection with PbA 1.49L. In these mice, CM was associated with an increase of T cells bearing the V(beta)8.1, 2 segments in the peripheral blood lymphocytes. Most V(beta)8.1, 2(+) T cells from peripheral blood lymphocytes of the mice that developed CM belonged to the CD8 subset, and exhibited the CD69(+), CD44(high) and CD62L(low) phenotype surface markers. The link between the increase in V(beta)8.1, 2(+) T cells and the neuropathological consequences of PbA infection was strengthened by the observation that the occurrence of CM was significantly reduced in mice treated with KJ16 antibodies against the V(beta)8.1 and V(beta)8.2 chains, and in mice rendered deficient in V(beta)8.1(+) T cells by a mouse mammary tumor virus superantigen.

Pre-erythrocytic stages of plasmodia. Role of specific and nonspecific factors.

Protection against pre-erythrocytic stages of malaria is possible as demonstrated by the generation of resistance after immunization with irradiated sporozoites. However, mechanisms involved are more numerous and intricate than previously believed and it progressively appears that the role of the presumed target, the sporozoite, might be negligible compared with that of the hepatic stage. The comparative use of in vivo and in vitro models clearly demonstrates that the intrahepatocytic parasite can be the target of antibodies, cytokines, phagocytic and cytotoxic cells, nonspecific factors--mechanisms in part induced by the previous or subsequent developmental stages.

Hepatic phase of malaria: a crucial role as "go-between" with other stages.

Besides potential interest in itself, the hepatic stage of malaria might play a crucial role as "go-between" with other stages. When present in the parasitophorous vacuole, antibodies induced by both sporozoite and erythrocytic stages efficiently disturb hepatic development of the parasite. Likewise previous and ensuing erythrocytic stages can modulate the "shielded" phase by cytokines, directly or as a result of a cascade of events, and by MHC-restricted or antibody-dependent cytotoxic mechanisms.

IL-6 induced by IL-1 inhibits malaria pre-erythrocytic stages but its secretion is down-regulated by the parasite.

The capacity of IL-6 to mediate the antiparasitic activity of IL-1 on intrahepatic development of malaria parasite was demonstrated. The comparisons of IL-6 levels in infected and noninfected hepatocyte cultures, either purified or enriched with nonparenchymal cells and stimulated by IL-1 or IL-6, indicate that subtle interactions exist between intrahepatocytic development of Plasmodium yoelii and liver synthesis of IL-6. During its intrahepatic multiplication, the parasite causes a decline in IL-6 production. IL-6 mRNA was not detected in the livers of infected mice during development of either hepatic or blood stage parasites although IL-6 activity was found in the sera during both stages.

Evidence for superantigenic activity during murine malaria infection.

TCR V beta usage was examined in C57BL/6 mice infected with Plasmodium yoelii. In addition to a polyclonal T cell activation, already described, a superantigenic-like activity was observed during the acute infection. This superantigenic activity induces a preferential deletion without prior expansion of CD4+ and CD8+ T cells bearing the TCR V beta 9 segment. The superantigen could be released by the parasite at different stages of its development since the deletion of V beta 9+ T cells was observed in blood and lymph nodes of mice infected either with sporozoites or with erythrocytic stages. Injection of sporozoite or parasitized erythrocytes to newborn mice led to a deletion and anergy of peripheral V beta 9+ T cells, without affecting thymic T cell populations. These observations suggest that the superantigen is released at very low concentrations during parasite development. The role of such parasite superantigenic activity in infectivity can be underlined by the observation that congenic BALB.D2 Mis1a mice lacking V beta 9 T cells are more susceptible to infection by P. yoelii.

TNF inhibits malaria hepatic stages in vitro via synthesis of IL-6.

We examined the capacity of murine recombinant tumor necrosis factor (rmTNF) to induce an inhibitory effect at the hepatic stage on malaria induced by Plasmodium yoelii sporozoites. When injected three times, 1.0 micrograms of rmTNF was found to protect 78% of mice against a sporozoite challenge. In contrast, whatever the dose and the schedule of administration, no inhibition was observed when purified hepatocyte cultures were infected with P. yoelii. The addition of non-parenchymal hepatic cells to hepatocyte cultures restored the capacity of TNF to modulate hepatic stage development, leading to up to 44% inhibition. Antibodies to interleukin 6 reversed the anti-parasite activity in the co-culture system.

In vitro activity of CD4+ and CD8+ T lymphocytes from mice immunized with a synthetic malaria peptide.

In previous work, a T-helper epitope was mapped within the circumsporozoite protein of the murine malaria parasite Plasmodium yoelii. A 21-mer synthetic peptide corresponding to this epitope (amino acid positions 59-79; referred to as Py1) induced a specific T-cell proliferation in BALB/c and C57BL/6 mice and provided help for the production of antibodies to peptides from the repetitive region, (Gln-Gly-Pro-Gly-Ala-Pro)n, of the P. yoelii circumsporozoite protein when mice were immunized with the Py1 peptide conjugated to the repetitive peptide. Experiments were then designed to study the in vitro antiparasite efficacy of T cells elicited in vivo by peptide immunization. T-cell activity was evaluated on cultured hepatic stages of P. yoelii. Peptide immunizations led to the preferential activation of CD8+ T cells in BALB/c mice and of both CD4+ and CD8+ T cells in C57BL/6 mice. Parasite elimination was mediated directly by these cells and did not seem to be dependent on lymphokine secretion. These data suggest that peptide-primed CD4+ T cells as well as CD8+ T cells could be cytolytic for the hepatic phase of malaria parasites. The fact that the same peptide could activate different lymphocyte populations, depending on the strain of mouse, highlights the importance of a better understanding of the fine mechanisms behind the immune responses to synthetic peptides being tested for malaria vaccine development.

L-arginine-dependent destruction of intrahepatic malaria parasites in response to tumor necrosis factor and/or interleukin 6 stimulation.

There is growing evidence that cytokines (interleukin [IL] 1, IL 6, interferon-gamma, tumor necrosis factor [TNF]) directly or indirectly interfere with the intrahepatic development of malaria parasites. Recent work in our laboratory clearly showed that TNF can affect the hepatic development of parasites via IL 6 secreted by liver nonparenchymal cells. The possible participation of an L-arginine-dependent effector mechanism has been studied to explain the TNF/IL 6-induced inhibition. We thus investigated if NGmonomethyl-L-arginine and N omega-nitro-L-arginine, two specific inhibitors of inorganic nitrogen oxide synthesis from L-arginine, were able to affect the inhibitory effect of TNF and/or IL 6 in co-cultures. At 0.1 and 0.5 mM both L-arginine analogues reversed the inhibitory effect of these cytokines. An interesting observation is that L-arginine analogues enhance schizont development in the absence of prior cytokine contact. This result indicates an hepatic basal L-arginine-dependent anti-parasitic activity which might explain the existence of self-degenerating hepatic forms as previously reported.

C-reactive protein protects against preerythrocytic stages of malaria.

We previously reported that low doses of interleukin-1 strongly inhibited in vitro development of the hepatic stages of Plasmodium falciparum and P. yoelii. Among several hypotheses, we considered the role of C-reactive protein (CRP), a major acute-phase reactant whose concentration increases markedly in infectious disorders. We demonstrated that human hepatocytes cultured in the presence of interleukin-1 released, as early as 30 min after stimulation, an increased amount of CRP. We then established that CRP bound to the P. falciparum and P. yoelii sporozoite surface membranes, probably via a phosphorylcholine binding site. Experiments in which CRP was added to rat hepatocyte monolayers during or after inoculation confirmed that the target of the CRP-mediated inhibition was at the very early phase of infection. These in vitro functional activities were confirmed in an in vivo model; rats with increased levels of CRP in serum following an injection of turpentine oil were found to be largely protected against an inoculation of P. yoelii sporozoites. The same results were observed in animals inoculated with sporozoites previously incubated in purified CRP or in sera of rats pretreated with turpentine oil. The latter effect was inhibited after incubation of serum from turpentine-injected rats with anti-CRP serum.

Cloned lines of Plasmodium berghei ANKA differ in their abilities to induce experimental cerebral malaria.

Infection with Plasmodium berghei ANKA is usually lethal. The parasite causes in some mouse strains a neurovascular syndrome, experimental cerebral malaria (ECM), involving immunopathological reactions. The effects on the development of ECM of the mouse genetic background have been clearly demonstrated, but nothing is known about the effects of the clonal diversity of the parasite. We showed that various cloned lines derived from a polyclonal line of P. berghei ANKA caused ECM but that the extent of ECM induction was dependent on the amount of inoculum. Subtle differences in ECM characteristics (survival time and hypothermia) were also observed. We also confirmed, using the 1.49L cloned line, that the mouse genetic background strongly affects ECM.

Liver CD4-CD8- NK1.1+ TCR alpha beta intermediate cells increase during experimental malaria infection and are able to exhibit inhibitory activity against the parasite liver stage in vitro.

Experimental infection of C57BL/6 mice by Plasmodium yoelii sporozoites induced an increase of CD4-CD8- NK1.1+ TCR alpha beta int cells and a down-regulation of CD4+ NK1.1+ TCR alpha beta int cells in the liver during the acute phase of the infection. These cells showed an activated CD69+, CD122+, CD44high, and CD62Lhigh surface phenotype. Analysis of the expressed TCRV beta segment repertoire revealed that most of the expanded CD4-CD8- (double-negative) T cells presented a skewed TCRV beta repertoire and preferentially used V beta 2 and V beta 7 rather than V beta 8. To get an insight into the function of expanded NK1.1+ T cells, experiments were designed in vitro to study their activity against P. yoelii liver stage development. P. yoelii-primed CD3+ NK1.1+ intrahepatic lymphocytes inhibited parasite growth within the hepatocyte. The antiplasmodial effector function of the parasite-induced NK1.1+ liver T cells was almost totally reversed with an anti-CD3 Ab. Moreover, IFN-gamma was in part involved in this antiparasite activity. These results suggest that up-regulation of CD4-CD8- NK1.1+ alpha beta T cells and down-regulation of CD4+ NK1.1+ TCR alpha beta int cells may contribute to the early immune response induced by the Plasmodium during the prime infection.

Inhibitory activity of interferons and interleukin 1 on the development of Plasmodium falciparum in human hepatocyte cultures.

We have studied the effect of natural and recombinant human interferons (HuIFN-alpha, -beta, and -gamma), and interleukin 1 (IL-1) on development of sporozoites of Plasmodium falciparum in cultures of functional hepatocytes. HuIFN-gamma inhibits hepatic schizogony of P. falciparum at very low concentrations (0.1 to 10 international units/ml), the target being the hepatocyte. Application after sporozoite inoculation is effective, suggesting an intracellular mechanism. There is also an 84% inhibition after application from 4 to 6 days following inoculation so that by day 6, there was a disappearance of a significant number of schizonts previously present at day 4, indicating more than a parasitostatic effect, and probably a postassembly action. HuIFN-alpha and -beta were effective, but only at 1000-fold higher concentrations than HuIFN-gamma. IL-1 (5 U/ml) also inhibited hepatic development of P. falciparum sporozoites; however, IL-1 treatment was effective only when applied before sporozoite inoculation.

A malaria heat-shock-like determinant expressed on the infected hepatocyte surface is the target of antibody-dependent cell-mediated cytotoxic mechanisms by nonparenchymal liver cells.

Cultured hepatic stages of Plasmodium falciparum and P. yoelii and with a monoclonal antibody recognizing a C-terminal fragment of the P. falciparum heat-shock-like protein (Pfhsp70) revealed that synthesis of this antigen first occurs during intrahepatic development of the parasite, at the two nuclei stage. Using a variety of techniques, including scanning electron microscopy, we observed that this antigenic determinant was expressed on the infected hepatocyte membrane. Its participation in antibody-dependent cell-mediated cytotoxicity was investigated. While no effect was obtained with peripheral blood cells, we found that 25% of the schizonts were specifically lysed when using spleen cells at a killer/target ratio of 30/1. More interestingly, with nonparenchymal liver cells, up to 50% of the hepatic parasites disappeared with a killer/target ratio of 10/1.