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An understanding of the nature of the immune response to asexual erythrocytic stages of malaria parasites will facilitate vaccine development by identifying which responses the vaccine should preferentially induce. The present study examined and compared the immune responses of NIH mice in either single or mixed infections with avirulent (DK) or virulent (DS) strains of Plasmodium chabaudi adami using the ELISA test for detecting and measurement of cytokines and antibody production. In both single and mixed infections, the study showed that both cell- and antibody-mediated responses were activated. In all experiments, an early relatively high level of IFN-gamma and IgG2a during the acute phase of the infection, and later elevation of IL-4 and IgG1, suggested that there was a sequential Th1/Th2 response. However, in the avirulent DK strain infection a stronger Th1 response was observed compared to the virulent DS strain-infection or in mixed infections. In the virulent DS infection, there was a stronger Th2 response compared to that in the DK and mixed infections. The faster proliferation rate of the virulent DS strain compared to the DK strain was also evident.

Interactions between antigen-presenting dendritic cells (DCs) and T cells are essential for the induction of an immune response. However, during malaria infection, DC function is compromised and immune responses against parasite and heterologous antigens are reduced. Here, we demonstrate that malaria infection or the parasite pigment hemozoin inhibits T cell and DC interactions both in vitro and in vivo, while signal 1 intensity remains unaltered. This altered cellular behaviour is associated with the suppression of DC costimulatory activity and functional T cell responses, potentially explaining why immunity is reduced during malaria infection.

A comparison of Plasmodium chabaudi AS infection in BALB/c and BALB/c IgM-deficient mice demonstrated a protective role for IgM during infection. IgM-/- mice, unlike microMT mice, display competent B cell humoral immune responses. Increased susceptibility of IgM-/- mice was demonstrated by increased mortality, an advanced ascending infection and higher peak parasitaemia, as well as enhanced anaemia and weight loss compared with wild-type mice. The recrudescent parasitaemias were also higher in the IgM-/- mice. Early specific IgM production in P. chabaudi-infected wild-type mice was followed by IgG1 and IgG2a production, while IgG1 and IgG2a production in IgM-/- mice was preceded by specific IgD production. No protective role for natural IgM against P. chabaudi AS infection was detected as passive transfer of naïve WT serum into IgM-/- mice did not alter the disease outcome or reduce parasite numbers. Passive transfer of WT antiserum, containing predominantly specific IgM, into IgM-/- mice delayed the ascending parasitaemia and reduced mortality. Similarly, coating parasitized red blood cells with WT antiserum, but not IgM-/- antisera, prior to infection also slightly delayed the ascending acute parasitaemia. Specific IgM therefore plays an important role in the limitation of parasite replication during asexual erythrocytic P. chabaudi AS infection.

The first genome survey sequencing of the rodent malaria parasite Plasmodium chabaudi is presented here. In 766 sequences, 131 putative gene sequences have been identified by sequence similarity database searches. Further, 7 potential gene families, four of which have not previously been described, were discovered. These genes may be important in understanding the biology of malaria, as well as offering potential new drug targets. We have also identified a number of candidate minisatellite sequences that could be helpful in genetic studies. Genome survey sequencing in P. chabaudi is a productive strategy in further developing this in vivo model of malaria, in the context of the malaria genome projects.

IFNgamma receptor (IFNgammaR) deficient mice and IL-4 deficient mice were infected with blood-stage Plasmodium chabaudi AS in order to analyse the role of Th1 (IFNgamma) and Th2 (IL-4)-associated cytokines in the development of protective immunity to the parasite. A high mortality rate and failure to reduce the primary parasitaemia to subpatent levels was observed in the IFNgammaR deficient mice. IL-4 deficient mice controlled a primary P. chabaudi AS infection in a similar manner to control mice and no mortality was observed. IFNyR deficient mice had a reduction in parasite-specific IgG and a significantly increased level of total IgE compared to control mice. There was no reduction in the level of parasite-specific IgG in IL-4 deficient mice. Cytological analysis of the cells present in the spleen and liver during the primary parasitaemia revealed a reduction in the numbers of lymphocytes, monocytes and polymorphonuclear (PMN) cells in the liver at the peak of parasitaemia in both IFNgammaR deficient mice and IL-4 deficient mice compared to control mice. Adoptive transfer studies demonstrated that cells isolated from the liver at day 11 post-infection could confer some protective immunity to P. chabaudi AS infection.

Protective immune mechanisms to the asexual erythrocytic stages of the malaria parasite Plasmodium chabaudi AS strain include antibody-independent mechanisms. Nitric oxide (NO) is produced during the infection and indirect evidence suggests that it can contribute to the antiparasitic mechanisms. We examined the effect of an NO producer, S-nitroso-acetyl-penicillamine (SNAP), on the growth and survival in vitro of P. chabaudi AS, P. berghei and P. falciparum. Growth of the parasites was monitored by the uptake of tritiated hypoxanthine and, in the case of P. falciparum, by morphological examination in stained blood smears. DL-penicillamine and sodium nitrite, as controls, had no inhibitory activity at the concentrations used. The results showed that at SNAP concentrations of approximately 182 microM and above NO was cytotoxic to P. falciparum but, at lower concentrations, there was a cytostatic effect and some parasites resumed growth and division after NO production had ceased. Rings were less susceptible to NO effects than later stages in the asexual cycle. The antimalarial activity of NO from SNAP also extended to the rodent parasites but, under the experimental conditions used, they were less sensitive than the human species. In the cultures of P. chabaudi, increasing the numbers of noninfected erythrocytes present did not diminish the antimalarial activity of SNAP, suggesting that here at least haemoglobin was not scavenging NO significantly.

Plasmodium chabaudi infection of mice provides an excellent model for examining acquired immunity to the blood-borne stage of malaria infection. CD4+ T-cell receptor (TCR) alphabeta-bearing T lymphocytes play a critical role in mediating protection, ascribed to both T helper (Th) 1 and Th2 subsets. One factor that may influence the Th1/Th2 cell balance is infective dose. In this study, we found that the size of the infective dose of P. chabaudi, and thus the level of antigen presented to the immune system, correlated with the balance of responder CD4+ T-cell phenotypes. Increasing the infective dose in a resistant mouse strain enhanced the Th1 cytokine (interferon-gamma; IFN-gamma) response and reduced the Th2 cytokine (interleukin-4; IL-4) response. In contrast, increasing the infective dose in a susceptible mouse strain led to a prominent and accelerated up-regulation of IL-4 production. These data show that the dose of antigen can significantly affect the balance between Th1- and Th2-mediated immune functions during infection of the mammalian host with blood-stage malaria parasites. This demonstration that parasite numbers may modulate CD4+ T-cell regulation has novel implications for the successful implementation of antimalarial vaccination and chemotherapeutic strategies.

P. chabaudi AS strain in laboratory mice provides an accessible and useful model for investigating antigenic variation in malaria parasites. Evidence that P. chabaudi AS undergoes antigenic variation is summarized. A live indirect fluorescent test (IFAT) detects a variable antigen on the surface of schizont-infected erythrocytes. Five different variable antigen types (VATS) (detected using the live IFAT) are described from a cloned mosquito transmitted parent population. Even during the rising primary parasitaemia VATS switch at high and variable rates (1.2-1.6%). Work towards cloning genes coding for the variable antigen is briefly summarized. Acquired immunity to blood-stage P. chabaudi AS is initially mediated through Th1 CD4+ T cells and after the primary parasitaemia there is a switch to Th2 CD4+ T cells. Acquired immune effector mechanisms are discussed in the context of antigenic variation by the parasite.

We have shown previously that at the time of peak primary parasitaemia of P. chabaudi infection in NIH mice, significant levels of nitric oxide are produced, detectable as nitrate in the serum, and that these contribute to the protective immune response to infection. Here, we demonstrate that following reinfection, mice show a markedly diminished ability to produce nitrate. However, if mice are treated with L-NG-monomethyl arginine specifically to block nitric oxide metabolism during the primary infection, and are then reinfected, production of nitrate is restored to levels approaching those attained at peak primary parasitaemia. These experiments, together with others we have reported, indicate that whereas nitric oxide appears to play a significant role in control of the primary parasitaemia of P. chabaudi infection, it performs no such function during subsequent patent parasitaemias. Furthermore, they suggest that factors as yet unknown may regulate nitric oxide activity during malaria infection, such that under normal circumstances its production comes under strict control. This is exemplified by the observation that after the burst of nitric oxide activity that coincides with peak primary parasitaemia, there follows a prolonged period of immunological tolerance during which nitrate levels remain low even at secondary challenge infection. This tolerized state is lifted only several months after initial infection, when the nitric oxide activity at reinfection appears to correlate with the size of the parasite challenge and the presence of a patent parasitaemia. The implications of these findings for protective immunity to malaria, malarial immunosuppression, and immunoregulation in general, are discussed.

In mice depleted of B cells from birth by treatment with anti-immunoglobulin M(mu) antibodies, progression from a Th1- to a Th2-regulated immune response during primary infection with Plasmodium chabaudi chabaudi fails to occur. While Th1-type immunity limits parasitemia, in the absence of B cells, chronic low-grade infections persist. Here, we show that reconstituting immune, and to a lesser extent naive, B cells to mice rendered deficient in B-cell function through anti-immunoglobulin M(mu) pretreatment restores the CD4+ T-cell response to the Th2 type later in P. c. chabaudi infection and with it the capacity to eliminate infection. This finding provides clear evidence that B cells are required for switching the balance of immune regulation between CD4+ T cells from Th1 to Th2 during P.c. chabaudi infection and supports the concept that B cells, through antibody production, are needed for effective antimalarial immunity.

The induction of T-helper cell subsets during the course of blood stage Plasmodium chabaudi chabaudi infection was compared in immunologically intact NIH mice and mice that were depleted of B cells from birth by treatment with anti-mu antibodies. For intact mice, in which the acute primary parasitemia peaked 10 days following infection, purified splenic CD4+ T cells recovered during the ascending parasitemia produced high levels in vitro of interleukin 2 (IL-2) (peak levels on day 10) and gamma interferon (IFN-gamma) (peak levels on day 7). Sera collected from these mice at around this time contained relatively high levels of P. c. chabaudi-specific immunoglobulin 2a (peak levels on day 12), and serum nitric oxide activity was significantly elevated at peak parasitemia. During the descending primary parasitemia, production of IFN-gamma and IL-2 decreased, while levels of IL-4 and IL-10 produced by splenic CD4+ T cells were significantly raised from the time at which subpatency was recorded (day 17) and persisted for at least 50 days. This was concomitant with a significant increase in levels of parasite-specific immunoglobulin G1, which peaked at around the time of recrudescence. Thus, in normal mice, sequential appearance of Th1 and Th2 responses was observed. In contrast, in B-cell-depleted mice, recovery from acute primary parasitemia was followed by a persistent patent infection which did not drop below 0.1% for at least 75 days after initiation of infection. These mice were unable to mount a significant Th2 response, manifest as an enduring inability of splenic CD4+ T cells to produce significant levels of IL-4 and IL-10. IL-2 and IFN-gamma levels remained significantly elevated throughout the 50-day observation period, and there was sustained production of nitric oxide. These data show that immune responses mediated by CD4+ T cells of the Th1 subset are capable of limiting infection beyond the initial acute phase, but that they do not eliminate parasitemia. Furthermore, as the progression from a Th1-regulated to a Th2-regulated immune response fails to occur in B-cell-depleted mice, the data suggest that B cells are required for the downregulation of Th1-mediated and/or the generation of Th2-mediated protective immunity to P. c. chabaudi.

The rates of switching between expression of variable antigen types (VATS) have been investigated in vivo in a cloned line of Plasmodium chabaudi chabaudi. VAT-specific hyperimmune sera combined with an immunogold-silver staining technique were used to detect VATS, and five estimates of VAT-specific switching rates were determined for three of them. VAT-specific switching rates were consistent for each VAT and differed between VATS in the range 1.3 x 10(-2) to 4.3 x 10(-4) switches per schizont per generation. This variation suggests that hierarchical expression of VATS in an infection may be determined by switch rates. A minimum estimate of the overall switching rate was determined by summation of the VAT-specific rates in each of two experiments. In both cases the results showed that at least 1 in every 80 schizonts switched VAT expression every generation. This is the first report of antigenic switching rates for malaria parasites measured in vivo in the presence of minimal specific immune pressure, and is the first to show that VAT-specific switching rates vary between VATS. We conclude that switching is rapid and spontaneous, and is regulated, at least in part, by the VATS involved.

The expression of selected interleukin receptors by cloned CD4+ T cells specific for the murine malaria parasite Plasmodium chabaudi chabaudi (P. chabaudi) representative of the T-helper (Th) 1 and Th2 subsets was examined. Both sets of clones expressed receptors for those interleukins for which they had a growth factor requirement in vitro. Each Th1 clone expressed receptors for, and was responsive to, interleukin (IL)-2 and IL-4, while each Th2 clone expressed receptors for, and was responsive to, IL-2, IL-4 and IL-1. IL-1 receptor (IL-1R) expression by the Th1 clones was either negligible or could not be detected. The disparity in expression of IL-1R by the Th1 and Th2 clones was more clear-cut than has been previously reported and IL-1R provided a definitive phenotypic marker for clones of the Th2 subset. Should IL-1R expression prove to be a feature of other Th2 cells cultured long-term in vitro, this will be invaluable for investigations involving the phenotyping, depletion or selection of CD4+ T cells of either Th1 or Th2 subset.

The distribution of Plasmodium chabaudi chabaudi AS among normocytes and reticulocytes in the peripheral blood of NIH mice undergoing a primary infection was determined from brilliant cresyl blue/Giemsa's stained thin blood films. During the early stages of infection in normal mice, parasites were found exclusively in normocytes. The presence of parasites in reticulocytes was limited to a period of severe immune pressure on the parasites, peak parasitaemia and the ensuing "crisis" phase, at which time the rapid production of new erythrocytes in response to the anaemia in these mice resulted in a high reticulocyte count. Later, during the recrudescence, parasites inhabited normocytes only. Thus, in immunologically competent animals, P. c. chabaudi AS showed no absolute preference for either mature or immature erythrocytes. In marked contrast, in chronically infected CD4+ T-cell-depleted mice, this malaria parasite apparently displayed a pronounced predilection for reticulocytes. During an unremitting parasitaemia of 2.8%-3.9% during days 13-60 post infection, all parasites were found in reticulocytes, even though these comprised only 25% of the total erythrocyte count. The possible reasons for this reversal in preference for the type of erythrocyte inhabited by asexual malaria parasites are discussed.

The host protective immune response to blood stage malaria infection was studied using Plasmodium chabaudi chabaudi (P.chabaudi) in NIH mice. It has been shown previously that CD4+ cells are critically required for protection against erythrocytic infection. Mice lacking a functional CD4+ cell compartment suffer unremitting patent primary parasitemias for at least 60 days after infection. Here, we report that the adoptive transfer of eight P. chabaudi-specific CD4+ T cell clones of either the Th1 or Th2 type to mice rendered CD4-depleted by adult thymectomy and anti-CD4 monoclonal antibody therapy fully restored the ability of recipients to control challenge infection. Control Th1 and Th2 clones specific for an unrelated antigen, ovalbumin, were unable to confer a comparable level of protection in CD4-depleted mice, even though they received regular doses of the antigen. These data demonstrate that protective immunity to asexual P. chabaudi parasites can be mediated through immune CD4+ T cell clones of either the Th1 or the Th2 subset.

CD4+ T cells play a major role in protective immunity against the blood stage of malaria, but the mechanism of protection is unclear. By adoptive transfer of cloned T cell lines, direct evidence is provided that both TH1 and TH2 subsets of CD4+ T cells can protect mice against Plasmodium chabaudi chabaudi infection. TH1 cells protect by a nitric oxide-dependent mechanism, whereas TH2 cells protect by the enhancement and accelerated production of specific immunoglobulin G1 antibody.

Splenic T-lymphocyte lines were established in vitro from Plasmodium chabaudi-infected NIH mice on days 16 and 20 of a primary infection, and from mice after two or three infections. Each line responded specifically to stimulation with a lysed soluble extract of P. chabaudi-infected erythrocytes (pRBC), and displayed a CD4+ (L3T4+) surface phenotype. Both the day 16 and 20 cell lines, when stimulated in vitro, secreted interleukin-2 (IL-2) and gamma-interferon (IFN-gamma), indicative of their belonging to the T helper 1 (Th1) CD4+ subset. In contrast, both lines derived from reinfected mice secreted interleukin-4 (IL-4) and provided helper activity in antibody production to P. chabaudi in vitro, and thereby had the characteristics of Th2 cells. All four T-cell lines provided significant protection to naïve mice infected with P. chabaudi. In immunocompromised mice, the day 16 T-cell line was as protective as in naïve mice whereas the cell line from mice infected twice required the additional transfer of mature naïve splenic B cells to provide protection comparable to that seen in the immunocompetent naïve recipients. The results establish that protective immunity to P. chabaudi may be associated with the induction of CD4+ T cells of either the Th1 or Th2 subset which confer protection against this malaria parasite by mechanisms independent of, and dependent upon, B-cell involvement, respectively.

Previous results, using a passive transfer assay, have shown that recrudescences of Plasmodium chabaudi chabaudi AS strain are antigenically different from the infecting parental population and also that the recrudescence appears to be a mix of antigenic types. This present study examines further these recrudescent populations using an indirect fluorescent antibody test on live, schizont-infected red blood cells. This analysis shows that ten clones derived from a recrudescence are all antigenically different from the parent population and that some are different from each other. The use of this method to examine the antigenic types of recrudescent clones after transmission through mosquitoes also demonstrates a resulting change in antigenicity. Such results showing a link between mosquito transmission and varying antigenicity may have important implications in terms of immunity and vaccine development.

Protective immunity to asexual malaria parasites appears to be mediated predominantly by the CD4+ subset of T lymphocytes. To examine the role of this T-cell population in the immune response to the murine malaria parasite Plasmodium chabaudi, CD4+ clones derived from infected mice were raised and propagated in vitro. Analysis of the reactivity of clones responsive to parasite antigen demonstrated that the CD4+ cell response is heterogeneous and is consistent with the idea of two functionally distinct CD4+ subsets. Those populations derived early during primary infection secreted interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) upon antigenic stimulation in vitro, i.e. they had a cytokine repertoire typical of the delayed-type inflammatory T-helper 1 (Th1) CD4+ subset. In contrast, cells taken after clearance of a secondary infection produced IL-4 and acted as effective helper cells for anti-malarial antibody (Ab) synthesis in vitro, and thereby had the characteristics of Th2 cells. The appearance in vivo of Th1 and then Th2 clones specific for P. chabaudi-parasitized erythrocytes (pRBC) supports the proposal from limiting culture analyses that for this malaria parasite resolution of primary parasitaemia is predominantly through the action of cytokines rather than Ab, and that final clearance requires helper cells and specific immunoglobulin.