Inhibition of Plasmodium yoelii blood-stage malaria by interferon alpha through the inhibition of the production of its target cell, the reticulocyte.

The effect of a recombinant hybrid human interferon alpha (IFN-alpha) (which cross-reacts with murine cells) on C57BL/6 mice infected with Plasmodium yoelii sporozoites or parasitized erythrocytes was determined. IFN-alpha did not inhibit the development of the parasite in the liver, but it did reduce the blood parasite load and the hepatosplenomegaly induced by the infection in mice injected with blood-stage parasites. The extent of anemia in IFN-alpha-treated and control mice was similar, despite the lower parasite load in the IFN-alpha-treated mice. The reduced blood parasite load in IFN-alpha-treated mice was associated with reduced erythropoiesis and reticulocytosis. As reticulocytes are the preferred target cells for the strain of P yoelii used (P yoelii yoelii 265 BY), it was postulated that the inhibition of reticulocytosis in IFN-alpha-treated mice was causally related to the observed decreased blood parasite load. This was supported by the finding that IFN-alpha inhibited a different strain of P yoelii (17X clone A), which also displays a tropism for reticulocytes, but not a line of Plasmodium vinckei petteri, which infects only mature red blood cells. As human malaria species also display different tropism for reticulocytes, these findings could be relevant for people coinfected with multiple Plasmodium species or strains or coinfected with Plasmodium and virus. (Blood. 2001;97:3966-3971)

The effects of subcurative doses of chloroquine on Plasmodium vinckei petteri gametocytes and on their infectivity to mosquitoes.

The effects of subcurative doses of chloroquine on rodent and human Plasmodium transmission to the mosquito have been studied by several authors who showed a short-term (12 h) enhancement of gametocyte infectivity by the drug, restricted to chloroquine-resistant strains, and a long term (4-6 days) enhancement of gametocytogenesis of chloroquine-sensitive strains of Plasmodium chabaudi. We investigated both short- and long-term effects of chloroquine on Plasmodium vinckei petteri, a chloroquine-sensitive rodent Plasmodium strain. Chloroquine treatment reduced the index of gametocytogenesis to 73% (5 mg/kg) and 55% (2.5 mg/kg) of controls, on day 6 post-infection (p.i.). The reduction was statistically significant with 5 mg/kg chloroquine. However, the reduction of gametocyte numbers did not affect the transmission capabilities of the strain. Our experiments showed that doses of 1 mg/kg chloroquine had no effect on the oocyst counts, 12 h post-administration to mice. A statistically non-significant 61% reduction of oocyst numbers was observed in mosquitoes fed on mice treated with 5 mg/kg chloroquine. The effect of 5 mg/kg chloroquine administration on the infectivity of gametocytes to mosquitoes fed 1 h post-treatment was also investigated. An overall 41% reduction of oocyst numbers was observed. This immediate effect was statistically significant in 73% of the mice. These results are consistent with the hypothesis that the short-term enhancing effect of chloroquine on transmission is restricted to the drug-resistant strains of Plasmodium.

Rodent malaria prophylaxis by transdermal delivery of primaquine.

The efficacy of a new transdermal delivery system of primaquine in order to obtain causal prophylaxis against sporozoite-induced Plasmodium yoelii infection was evaluated. A single administration of a 1.0 cm2 transdermal delivery system containing 5.0 mg of primaquine was able to protect 100% of treated mice. This result suggests that the transdermal route may be a very interesting approach for malaria prophylaxis and should encourage further studies in order to determine the absolute bioavailability of the drug as well as its dose-effect relationship.

Immunization with a recombinant C-terminal fragment of Plasmodium yoelii merozoite surface protein 1 protects mice against homologous but not heterologous P. yoelii sporozoite challenge.

It has been reported previously that immunization with recombinant protein containing the two epidermal growth factor (EGF)-like modules from merozoite surface protein 1 (MSP-1) of Plasmodium yoelii (strain YM) protects mice against a lethal blood-stage challenge with the same parasite strain. Since MSP-1 is expressed in both liver- and blood-stage schizonts and on the surface of merozoites, we evaluated the effectiveness of immunization with recombinant proteins containing either the individual or the two combined EGF-like modules in producing a protective response against a sporozoite challenge. The recombinant protein expressing the combined EGF-like modules of the YM strain protected mice against a homologous sporozoite challenge, and sterile protection, as defined by the absence of detectable blood-stage parasites, was observed in the majority of the mice. In contrast, mice immunized with recombinant P. yoelii YM MSP-1 were not protected against a heterologous challenge with sporozoites from strain 265 BY of P. yoelii. The lack of protection may be explained by differences identified in the amino acid sequences of MSP-1 for the two strains. A recombinant protein containing the two EGF-like modules of MSP-1 from P. yoelii 265 BY was produced and used to immunize mice. These mice were protected against a homologous challenge with sporozoites of P. yoelii 265 BY. The results suggest that a recombinant MSP-1 has potential as a vaccine against malaria, but its efficacy may be limited by sequence polymorphism and selection of variants.

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.

Linear and multiple antigen peptides containing defined T and B epitopes of the Plasmodium yoelii circumsporozoite protein: antibody-mediated protection and boosting by sporozoite infection.

We previously reported the identification of a T cell epitope in the N-terminal part of the circumsporozoite protein (CSP) of Plasmodium yoelii yoelii (Pyy). CD4+ T cell clones derived from mice immunized with a 21-mer peptide (amino acids 59-79, referred to as Py1) containing this epitope confer complete protection after passive transfer in mice. These clones proliferate in vitro in the presence of a 13-mer peptide (amino acids 59-71, referred to as Py1T). This shorter peptide was found to behave as a Th epitope in vivo, allowing overcoming of the genetic restriction for production of anti-repeat antibodies in BALB/c mice, when cross-linked to three (QGPGAP) repeats of the Pyy CSP. In this study, we report protection in BALB/c mice, against a challenge with Pyy sporozoites after immunization with linear and multiple antigen peptides containing Py1T as T epitope and three repeats QGPGAP (Py3) as B epitope. Multiple antigen peptide (MAP4-Py1T-Py3)-induced immunity was shown to be more effective than immunity induced by the linear form of the conjugate (Py1T-Py3), protecting against challenges with higher numbers of sporozoites. In both cases, levels of anti-repeat antibodies were strongly correlated with anti-parasite antibodies and protection. When tested in vitro, sera from mice immunized with the protective constructs strongly inhibited Pyy liver stages, while lymph node T cells displayed no cytotoxicity. In vivo, depletion of CD4+ or CD8+ T cells did not affect protection. Furthermore, MAP4-Py1T-Py3-immunized mice were not protected against a challenge with P. yoelii nigeriensis sporozoites, a parasite which has the same Py1T sequence but differs from Pyy in its repeated sequence. These results demonstrate that anti-repeat antibodies raised by immunization with the linear or the MAP form are exclusively responsible for the protection. Furthermore, this antibody response is boosted by a sporozoite challenge, allowing protection against a second challenge.

Enhanced gametocyte formation by Plasmodium chabaudi in immature erythrocytes: pattern of production, sequestration, and infectivity to mosquitoes.

The objective of this study was to investigate the chronobiology and infectivity of the gametocytes of Plasmodium chabaudi chabaudi. In order to increase the production of gametocytes, mice were treated with phenylhydrazine to induce a hyper-reticulocytosis. The authors observed an important stimulation of gametocytogenesis. Gametocytes were seen as soon as the second day postinoculation and were produced periodically at each schizogony, every 24 hr. The gametocytic developmental cycle lasted 60 hr and consisted of 4 successive stages: stage 0 at 36 hr, from merozoite invasion, stage I at 42 hr, stage II at 48 hr, and stage III at 54 hr. An important fraction of stage II was sequestered in small peripheral capillaries. The numbers of oocysts in the mosquitoes fed on phenylhydrazine-treated mice were larger than in controls. When mosquitoes were fed at different times of the day, circadian differences in the oocyst counts were not statistically significant. However, stage II was considered to be probably the most infective stage because, like the infective gametocyte stage of other species of murine malaria, it is sequestered in the peripheral capillaries. In contrast with Plasmodium vinckei, there is no peak of infectivity at the time of sequestration of the infective stage; this is probably due to the inhibitory effect of the schizogony occurring at this time.

Synchronized Plasmodium yoelii yoelii: pattern of gametocyte production, sequestration and infectivity.

The chronobiology of the gametocytes of P. yoelii was studied in Percoll-glucose synchronized infection in the mouse. The gametocyte developmental cycle consisted of 4 successive stages: stage 0 maturation took 27 hours from merozoite invasion, stage 0 to stage 1 lasted 6 hours, stage I to stage II and stage II to stage III lasted 3 hours each. Stage 0 gametocytes were found to sequester in small peripheral capillaries, and the number of oocysts in mosquitoes was related to the number of stage 0 gametocytes ingested.

The gametocytes of Plasmodium vinckei petteri, their morphological stages, periodicity and infectivity.

Gametocyte production by P. vinckei petteri was cyclic, occurring at each schizogony every 24 h. They matured in 27 h from merozoite to type 0 microgametocyte, in 3 h from type 0 to type I, 6 h from type I to type II and 3 h from type II to type III. Transmission experiments showed that the time of maximum infectivity was midday when mice were inoculated at midnight, and midnight when mice were inoculated at midday. In all instances, maximum infectivity coincided with a peak in intensity by type II microgametocytes, a relationship confirmed by multiple correspondence analysis. The proportion of type II microgametocytes was higher in the mosquitoes blood meal than in smears of tail blood of mice, suggesting a sequestration phenomenon with this stage.

A method for the quantitative assessment of malaria parasite development in organs of the mammalian host.

A non-radioactive PCR method was developed to quantify the development of malaria parasites in the infected host. This was achieved by using Plasmodium genus-specific primers corresponding to the parasite's small subunit ribosomal RNA genes. The quantification of the PCR product was performed by high performance liquid chromatography, and calibration curves were obtained by amplification from defined quantities of purified Plasmodium genomic DNA. Using this method, it was possible to quantify development of P. berghei and P. yoelii blood-stage parasites from blood and brain samples of infected mice, and of hepatic stage parasites, from liver samples of mice infected with different numbers of sporozoites.

Iron overload increases hepatic development of Plasmodium yoelii in mice.

Iron overload in BALB/c mice by treatment with ferric ammonium citrate promotes the hepatic development of Plasmodium yoelii in vivo and in vitro. This was the result of increased penetration of the parasite into hepatocytes since no effect was observed on parasite transformation or maturation. These results could explain why in endemic regions iron supplementation led, in certain studies, to an increase in clinical episodes of malaria and in the prevalence of malaria infection.

Effects of iron deficiency on the hepatic development of Plasmodium yoelii.

Iron deficiency through an iron-deficient diet and through an inactivation of hepatic xanthine dehydrogenase (XD) was shown to modulate Plasmodium yoelii sporozoite development in hepatocytes. When mice that are on iron-deficient diet were challenged with sporozoites, enhancement of hepatic stage development was observed, resulting in the earlier appearance of blood parasites. In contrast, inhibition of parasite hepatic development was noticed when iron deficiency was the result of hepatic XD inactivation. In vitro studies have shown that diet induced iron-depletion increases penetration of sporozoites into liver cells while inactivation of hepatic XD resulted in an inhibition of both sporozoite penetration and schizont maturation. Moreover, inhibition of heme synthesis (which requires intrahepatic iron) also led to an inhibition of parasite development.

Effect of fatty acid treatment in cerebral malaria-susceptible and nonsusceptible strains of mice.

Cerebral malaria-susceptible (C57BL/6) mice infected with Plasmodium berghei ANKA (PbA) developed low parasitemia and died from typical neurological symptoms between 8 to 10 days after infection. In contrast, nonsusceptible (BALB/c) mice developed high peripheral blood parasitemia and died 22-24 days later without neurological implications. Daily injections of fatty acids (FA) during the first 3 days after infection protected C57BL/6 mice from cerebral symptoms but had no effect on BALB/c mice. Thus, treated C57BL/6 mice developed hyperparasitemia and died 25 days after infection, like BALB/c mice. Red blood cells from C57BL/6 control mice were found to be more resistant to lysis by linoleic acid than those of BALB/c mice. Three days following infection with PbA, these differences disappeared. Treatment with FA prevented these changes. We concluded that the host's cells were altered soon after infection and that the nature and degree of alterations depended on the mouse strain, thus determining the eventual outcome of the infection. Likewise, the effects of FA might not be directed against the parasite but rather seem to act early after infection on these parasite-induced modifications of host cells.

Protective immunity against malaria: cellular changes in the liver vary according to the method of immunization.

Characterization of cells present in the extravascular compartment of murine liver was performed after different immunization procedures against the malaria parasite Plasmodium yoelii. Mice were immunized with live or irradiated sporozoites or with parasitized erythrocytes. Whatever the immunization protocol used, the mice were protected against a sporozoite challenge but each immunization procedure induced a specific profile of cell types. Immunization with irradiated sporozoite induce a significant increase in CD8+ lymphocytes, parasitized erythrocytes stimulates production of monocytes/macrophages and CD8+ lymphocytes while, after live sporozoites immunization, polymorphonuclear cells, macrophages/monocytes, B cells and a range of T cell subsets were increased in number.

Characterization of liver lymphomyeloid cells in mice infected with Plasmodium yoelii sporozoites.

We have isolated, characterized and quantified the immunocompetent cells present in the extravascular hepatic compartment at various stages after Plasmodium yoelii malaria infection with sporozoites. Cytological analyses revealed a predominantly lymphoid population. In mice with a primary infection, the predominant cells were CD4+, CD8+ and B lymphocytes. In fully protected mice, CD3+ CD4- CD8- and polymorphonuclear cells, particularly eosinophils, were most common. The significance of changes in subpopulations is discussed in relation to antigen presentation and host-protective mechanisms.

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.

In vivo induction of the nitric oxide pathway in hepatocytes after injection with irradiated malaria sporozoites, malaria blood parasites or adjuvants.

The mechanisms responsible for malarial immunity induced by repetitive injections of X-irradiated sporozoites have not been fully established. We demonstrate here that a single injection of irradiated sporozoites induced, as soon as 24 h after, a non-permissive state to hepatocyte reinfection with sporozoites in vitro. The same effect was observed when malarial blood forms, irradiated promastigotes of Leishmania infantum, adjuvants (muramyl dipeptide, poly acidylic uridylic) or interferon-gamma was injected. Activation of the nitric oxide (NO) pathway in the hepatocyte by these factors was found to be responsible for hepatocyte refractory status. Additionally, this metabolic pathway is involved in protection given by repeated injections of irradiated sporozoites since protection could be reversed by treating mice at the time of sporozoite challenge with a competitive inhibitor (NG-monomethyl-L-arginine) of the NO pathway. These results suggest that, in view of an antisporozoite vaccine, further studies are needed to find out how to activate specifically a long-lasting nonspecific immune response.

Effector functions of circumsporozoite peptide-primed CD4+ T cell clones against Plasmodium yoelii liver stages.

Previously, CD4+ T cell lines and clones were isolated after immunization of BALB/c and C57BL/6 mice with the Py1 peptide, a 21-mer synthetic peptide corresponding to a N-terminal segment of the circumsporozoite protein of Plasmodium yoelii. The clones were separated into the Th1 and Th2 subsets on the basis of lymphokine production. It was observed that immunization with the Py1 peptide induced preferentially Th1 cells in BALB/c and Th2 cells in C57BL/6 mice. These clones were then tested for their cytolytic ability in vitro. Some of the clones from BALB/c and C57BL/6 mice eliminated liver stage parasites from cultured hepatocytes in a MHC restricted manner. Nevertheless, none of these clones was able to lyse Py1 peptide-pulsed target cells. It was also found that two clones could protect BALB/c mice against a sporozoite challenge. These results provide evidence that CD4+ T cells, induced after priming with a defined peptide, could participate in the effector mechanisms against malaria liver stages.

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.

Activity of dihydrofolate reductase inhibitors on the hepatic stages of Plasmodium yoelii yoelii in vitro.

The effects of the dihydrofolate reductase inhibitors proguanil and chlorproguanil, their active metabolites cycloguanil and chlorcycloguanil, and pyrimethamine, against the hepatic stages of Plasmodium yoelii yoellii were investigated in cultured BALB/c mouse hepatocytes. Proguanil was inactive at concentrations of 10(-8) M, whereas the other compounds were fully active at this and lower concentrations. Chlorcycloguanil was the most active compound and almost completely inhibited schizont development in concentrations as low as 10(-12) M.