Protective immunity to malaria: studies with cloned lines of Plasmodium chabaudi and P. berghei in CBA/Ca mice. I. The effectiveness and inter-and intra-species specificity of immunity induced by infection

CBA/Ca mice were immunized by infection with cloned lines of Plasmodium berghei (isolates ANKA, KSP-11). Plasmodium chabaudi chabaudi (AS, CB) or Plasmodium chabaudi adami (DS) and then challenged with either homologous or heterologous parasites. Protective responses were assessed in immune mice relative to the controls by their ability to (i) extend the time taken for the mean parasitaemia to reach a predetermined level (1% or 0.1%) (ii) reduce peak parasitaemia (iii) resolve the parasitaemia sooner and/or (iv) control or eliminate recrudescences. At both the inter- and intra-species level, immunity appeared largely specific for the cloned line inducing it. At the interspecies level marginally effective cross-immunity was sometimes evident, thus P. berghei KSP-11 immune mice displayed some immunity against P.c. chabaudi AS, although immunity to this parasite was relatively ineffective against P. berghei ANKA or KSP-11. Cross-immunity was more apparent between the subspecies P.c. adami and P.c. chabaudi and between cloned lines of the latter parasite derived from the AS and CB isolates. These data reflect considerable inter- and intra-species structural and immunogenic differences in certain antigens of parasitized erythrocytes and merozoites, which have been identified in a number of murine malarias and associated with protective immunity. Similar differences recently identified in the equivalent antigens of the human parasite P. falciparum may therefore have important implications for protective immunity in man.     

Protective immunity to malaria. Studies with cloned lines of Plasmodium chabaudi chabaudi and P. berghei in CBA/Ca mice. II. The effectiveness and inter- or intra-species specificity of the passive transfer of immunity with serum

Serum was obtained from CBA/Ca mice infected, reinfected or superinfected with parasites taken one or two syringe passages from cryopreserved reference stabilates derived from cloned lines of the AS or CB isolates of P.c. chabaudi. Serum was also collected from mice superinfected with parasites derived from a cloned line of P. berghei KSP-11. When injected into normal syngeneic recipients subsequently challenged with homologous or heterologous parasites, these sera mediated some or all of the following modifications to the breakthrough parasitaemias which invariably occurred (i) an extension of the pre-patent period (ii) an extension of the time taken for the parasitaemia to reach 2% (iii) a reduction of peak parasitaemia (iv) protraction of the initial peak of parasitaemia. These modifications were particularly evident with serum from superinfected mice and to a lesser extent with serum from animals reinfected once after recovery from a primary infection. Serum taken during the course of such a primary infection produced extended pre-2% periods, other effects being only marginal. Serum mediated modifications produced by reinfection and superinfection serum appeared largely species-specific with a limited degree of cross-reactivity. Intraspecific specificity was also apparent with serum from P.c. chabaudi AS or CB reinfected or superinfected mice, although marginal cross-immunity was again observed. When analysed by the fluorescent antibody technique on smears of methanol fixed parasitized erythrocytes, reinfection and superinfection sera were almost totally cross-reactive both within and across species. Preliminary evidence that parasites breaking through the effects of these sera may constitute a phenotypic antigenic variant is presented and possible mechanisms for the parasitaemia modifying effects of the various sera discussed.     

Protective immunity to malaria: studies with cloned lines of rodent malaria in CBA/Ca mice. IV. The specificity of mechanisms resulting in crisis and resolution of the primary acute phase parasitaemia of. Plasmodium chabaudi chabaudi and P. yoelii yoelii

Low numbers of parasites from cloned lines of the rodent malaria parasites, Plasmodium chabaudi chabaudi AS and P. yoelii yoelii A, injected into CBA/Ca mice produce acute but usually self-limiting infections. During crisis, i.e. 1-2 days after peak parasitaemia, 'pre-immune' mice experiencing such 'background' infections were reinfected intravenously with homologous parasites or parasites of heterologous strains or species. P. c. chabaudi AS pre-immune mice controlled an AS challenge with essentially the same kinetics as the background infection. Reinfection of AS pre-immune mice with the heterologous (CB and IP-PCI) P. c. chabaudi strains or P. chabaudi adami DS had little effect on the initial growth of these parasites, although eventually the parasitaemia was controlled. In contrast, a partial inhibitory effect on the growth of P. vinckei lentum DS was evident. Challenge with the non-lethal (A) or lethal (YM) variants of P. y. yoelii resulted in an increase in both the growth and virulence of these parasites. P. y. yoelii A pre-immune mice controlled a homologous challenge, but were less effective at controlling the YM variant. In addition, they were unable to clear rapidly a P. c. chabaudi AS or P. v. lentum DS challenge. Both the multiplication and virulence of P. berghei ANKA were enhanced. These findings demonstrate that resolution of the primary acute parasitaemia in P. c. chabaudi AS- and P. y. yoelii A-infected mice is predominantly mediated by species- and strain-specific mechanisms.     

Early appearance of variant parasites in Plasmodium chabaudi infections

Previous studies have shown that the recrudescence parasitaemias seen in mice infected with Plasmodium chabaudi AS strain are antigenically different from the infecting parent population. Antigenic differences between recrudescent and parent populations were demonstrated in a passive transfer assay. In the present study, using the same assay system, it has been shown that in some mice, variant parasites (i.e. different from the parent population) can be detected at a time when the primary parasitaemia is still patent but in remission. This is the first report in Plasmodium of variant parasites being detected during the course of a patent primary parasitaemic episode of an infection initiated with a cloned line.     

Lactobaciilus casei ssp. rhamnosus enhances non specific protection against Plasmodium chabaudi AS in mice

OBJECTIVE: To evaluate the capacity of Lactobacillus casei ssp. rhamnosus to enhance resistance against Plasmodium chabaudi chabaudi AS. MATERIAL AND METHODS: NIH mice were IP injected with viable lactobacillus casei seven days (LC1 group) or 7 and 14 days (LC2 group) before the challenge (day 0) with Plasmodium chabaudi parasitized red blood cells (pRBC). Control mice were inoculated with pRBC only. When parasitaemia was resolved, naive mice were injected with spleen cells from each group. The parasitaemia was measured. Nitric oxide (NO*) in serum was determined. RESULTS: Mice from the LC1 group presented a reduction in parasitaemia, with a prepatent period of five days, parasitaemia lasted 11 days, and the peak was (36.3 % pRBC) on the 12th day post-infection. Mice from the LC2 group showed a prepatent period of five days, parasitaemia lasted eight days, and the peak (30 % pRBC) was of on the 11th day. In the control, the prepatent period was three days, the parasitaemia lasted 15 days, and the peak (51% pRBC) was on day nine. Mice inoculated with spleen cells from the LC2 group showed a prepatent period of 21 days, parasitaemia lasted seven days, and the peak (13.5% pRBC) was on the 26th day. CONCLUSION: L. casei enhanced nonspecific resistance to P. chabaudi, as indicated by longer prepatent periods, reduced parasitaemia, and reduction in the viability of the parasites recovered from the spleen of infected mice, along with high concentrations of NO* in serum.     

Mice immunized with a synthetic peptide construct corresponding to an epitope present on a Plasmodium falciparum antigen are protected against Plasmodium chabaudi challenge

Inhibitory monoclonal antibody (MoAb) 8E7/55 recognizes a parasitophorous vacuole membrane (PVM) antigen in Plasmodium falciparum. Previous studies have identified the epitope, DNNLVSGP, recognized by the MoAb. A synthetic peptide containing this sequence was synthesized and coupled to diphtheria toxoid (DT) and was found capable of generating antibodies when used as an immunogen in mice which recognize the native antigen exp-1. In this study we demonstrate the ability of the MoAb and antisera generated against the peptide construct to recognize a 54 kD PVM antigen in Plasmodium chabaudi. The P. chabaudi antigen is synthesized in trophozoites and released to the surrounding culture media outside the parasitized erythrocyte. Mice immunized with the peptide conjugate are protected when challenged with a lethal strain of P. chabaudi. Protection in the mice correlated with the antibody titre prior to challenge. If the PVM antigen from P. chabaudi is a homologue of exp-1 from P. falciparum, then these experiments may provide a guide to the antibody titres required in human trials before antibody mediated protection could be expected. The discovery that a PVM localized antigen is secreted into the surrounding in vitro culture media provides us with a valuable model system for further investigation of protein trafficking pathways in malaria-infected erythrocytes.     

Acute virulent infection with Plasmodium chabaudi does not impair the generation of a protective immune response

We have investigated whether a protective immune response occurred in mice infected with a virulent cloned strain of Plasmodium chabaudi. Animals inoculated intravenously with 10(7) parasitized erythrocytes (PE) showed an exponentially increasing parasitaemia and died by day 6 of the infection, presenting a pronounced anaemia. Smaller inocula produced a longer pre-patent period but did not change the lethal course of infection, since mice injected with 100 parasites died on day 12. When anaemia was compensated for by red blood cell (RBC) transfusion, infected mice recovered and thereafter exhibited a strong immunity, comparable to that of mice immunized by a drug-controlled infection. The immune response was P. chabaudi specific, as the mice were fully susceptible to a challenge by P. yoelii. Three transfusions of 5 x 10(9) RBC per mouse at 2-day intervals were necessary before all the animals were able to control the infection. Transfusion of a larger number of RBC resulted in a lower anaemia and a delay in reticulocytaemia but, paradoxically, the expression of the immune response was delayed. Three transfusions of 1.2 x 10(10) RBC enabled three out of eight mice to survive the infection, while six transfusions enabled all the mice to survive. The data suggest that parasitized immature RBC could play an important role in triggering the protective immune response.     

Parasite-specific IgM plays a significant role in the protective immune response to asexual erythrocytic stage Plasmodium chabaudi AS 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 µMT 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.     

Antigenic variation during malaria infection—the contribution from the murine parasite Plasmodium chabaudi

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.     

The course of Plasmodium chabaudi chabaudi infections in interferon-gamma receptor deficient mice

Interferon-gamma receptor (IFN-γR) deficient mice parasitized with blood-stage Plasmodium chabaudi chabaudi were used to assess the anti-malarial activity of interferon-gamma (IFN-γ). There was no significant difference in the parasitaemia between the two types of mice during the first peak of parasitaemia. However, IFN-γR deficient mice displayed an increased leucocytosis and a high mortality rate, whereas all of the wild type mice survived. IFN-γR deficient mice, unlike wild type mice, developed a pronounced second parasitaemia peak, 9 to 11 days after the first one, with a parasitaemia of up to 65% associated with mortality. Furthermore, increased serum levels of nitric oxide (NO) were only found in wild type mice at the peak of parasitaemia, whereas it remained at background levels in IFN-γR deficient mice. Parasite-specific antibody production was not significantly different in IFN-γR deficient mice, as compared to wild type mice. In addition, both wild type and IFN-γR deficient mice were equally protected upon reinfection. These results indicate a delayed development of protective immunity and imply a crucial function for the IFN-γR in the control of blood stage malaria during the initial three weeks of infection.     

Parasitaemia levels in Plasmodium chabaudi infected‐mice modify IFN‐γ and IL‐10 expression after a homologous or heterologous challenge

CB6F1 mice infected with the nonlethal Plasmodium chabaudi chabaudi AS suffer parasitaemia levels up to 40% (full parasitaemia, FP) and develop both homologous and heterologous (against the lethal Plasmodium yoelii 17XL) protective immunity. However, if mice are treated with anti‐malarial drug when parasitaemia is below 10% (low parasitaemia, LP), they only develop homologous immunity. For the better understanding of this interesting dissociation related to the degree of parasitaemia, in this work, we studied the genetic expression of some cytokines. We found that during primary parasitaemia both FP and LP mice showed at first a TNF‐α, IL‐2 and IFN‐γ response which is followed by an IL‐4 and IL‐10 response. When FP and LP mice were challenged with either the homologous (FP + AS and LP + AS mice) or the heterologous parasite (FP + 17XL and LP + 17XL mice), we observed that LP + 17XL mice, which failed to develop heterologous immunity and succumbed to the challenge, showed a stronger IFN‐γ and a weaker IL‐10 expression than FP + 17XL mice, which developed heterologous immunity and survived the challenge. The importance and the possible implications of these findings are discussed.     

夏氏疟原虫感染早期易感和抵抗小鼠树突状细胞细胞因子的分泌特点

目的探讨在夏氏疟原虫(Plasmodiumchabaudi chabaudi AS,P.c.chabaudi AS)感染早期,树突状细胞(Den-dritic cells,DCs)调控Th1细胞免疫应答的相关机制。方法用P.c.chabaudi AS感染易感型DBA/2和抵抗型BALB/c小鼠,计数红细胞感染率;感染前和感染后3、5、8d制备脾细胞悬液,磁珠分选、纯化DCs并体外培养;ELISA方法检测DCs培养上清中IL-12p40、IL-10和TGF-β1的水平。结果 DBA/2小鼠DCs培养上清中IL-12p40的水平在感染后3-8天明显升高,BALB/c小鼠DCs培养上清中IL-12p40的水平在感染后5-8d出现有意义的升高,但其水平均明显低于DBA/2小鼠;两种小鼠DCs培养上清中IL-10与TGF-β1的分泌水平在感染后5-8d明显升高,但DBA/2小鼠DCs培养上清中IL-10与TGF-β1的分泌水平明显低于BALB/c小鼠。结论 P.c.chabaudi AS感染早期,DBA/2和BALB/c小鼠在DCs细胞因子的分泌模式上存在显著差异。     

A single gene copy merozoite surface antigen and immune evasion?

During the course of chronic malaria infection antigenic variants of a parasite antigen are expressed and exposed on the surface of infected erythrocyte membranes. There also exists a number of apparently invariant single gene copy blood-stage antigens, exposed or non-exposed, which have been shown to afford immunity under experimental conditions. To determine why the host, presented with invariant 'protective' antigens, is unable to control infections effectively, immunity to a representative single gene copy antigen, the merozoite surface protein 1 (MSP1) was investigated in Plasmodium chabaudi chabaudi AS, a murine model of chronic malaria. Immunization with monoclonal antibody affinity purified native MSP1 resulted in enhanced control of parasitaemia on challenge, irrespective of the parasite inoculum size; challenge with a single parasite, however, suggested that expansion of resistant parasite subpopulations was not occurring. Challenge of mice immunized with recombinant fusion proteins encoding N- or C-terminal regions of the P.c. chabaudi AS MSP1 produced inconsistent effects, often parasitaemias were indistingishable from controls despite significant anti-MSP1 antibody responses. The not unlikely contamination of MSP1 native preparations with erythrocyte (E) components was considered. Immunization with a mixture of the MSP1 C-terminus recombinant polypeptide and a Triton X-100 solubilized lysate of normal E resulted in enhanced control of parasitaemia, however, no effect was seen after administration of either component on its own. Co-immunization of E with the N-terminus polypeptide reversed the inhibition seen, on this occasion with this construct alone.     

Mouse splenic CD4+ and CD8+ T cells undergo extensive apoptosis during a Plasmodium chabaudi chabaudi AS infection

The presence and phenotype of apoptotic lymphocytes was studied in spleen cell suspensions taken from CB6F1 mice infected with Plasmodium chabaudi chabaudi AS. High levels of apoptotic cells were found, associated with high parasitaemias and splenomegaly. This was also accompanied by expansion and disarray of spleen white pulp. Apoptosis levels lowered when parasitaemia was cleared, but were still higher than in normal mice. At this time, the spleen was diminishing in size and the white pulp was contracting and rearranging. When parasitaemia was patent, the cells most affected by apoptosis were CD4+ T cells followed by CD8+ T cells, and to a lesser extent B220+ B cells. When parasitaemia was cleared, CD8+ T cells and B220+ B cells returned to basal levels of apoptosis, while CD4+ T cells still had higher apoptosis levels than normal mice. A similar pattern of lymphocyte subpopulation apoptosis was found in infected BALB/c mice, despite the fact that, for this mouse model, it has been reported that B cells are the cells that are most affected by apoptosis. We consider that the high levels of apoptosis in CD4+ T cells when parasitaemias are still high are not easily explained by a normal mechanism of down regulation of the immune response.     

Protective immune response to Plasmodium chabaudi, developed by mice after drug controlled infection or vaccination with parasite extracts: analysis of stage specific antigens from the asexual blood cycle

Summary The protective immune response to asexual blood infection by Plasmodium chabaudi was studied in mice immunized either by drug controlled infection or by vaccination with preparations of merozoïtes or free parasites at different stages of development. Animals immunized by the first method developed a sterile immunity. The passive transfer of their serum protected naive recipients from the lethal development of the infection, but affected only moderately the initial course of the parastiaemia. Animals immunized with either ring, schizont or merozoi'te preparations exhibited a limited but significant resistance to infection: when challenged with 10⁶ parasites of the homologous strain they exhibited a reduced parasitaemia as compared to control mice, and in addition, 50% of them recovered from the infection. Immunochemical analysis of parasite antigens showed that a family of high molecular weight proteins synthesized essentially at the schizont stage and conserved in the merozoites are important immunogens. Quantitative rather than qualitative differences were observed in the pattern of parasite proteins immunoprecipitated by serum of animals exhibiting sterile immunity or moderate protective immunity. A schizont specific polypeptide of mol. wt 82 Kd which is found in the surface of the merozoite is preferentially immunoprecipited by serum from animals exhibiting sterile immunity.     

Recombinant mouse IL-6 boosts specific serum anti-plasmodial IgG subtype titres and suppresses parasitaemia in Plasmodium chabaudi chabaudi infection

C57BL/6 mice which were treated with recombinant mouse interleukin-6 (rmIL-6), 75 ng in 0.2 ml saline, i.p., 24 h before inoculation with Plasmodium chabaudi chabaudi and on days one, two and three could not control the primary acute parasitaemia but were able to suppress secondary parasitaemia significantly, peak of 3% and to reduce parasitaemia to subpatent levels within three weeks. Control C57BL/6 mice, which received saline injections only, developed two identical peaks of parasitaemia of at least 21% each, typical of a primary P. chabaudi chabaudi infection and cleared parasitaemia by day 28 post-inoculation. Serum levels of IL-6 measured in the first week by enzyme-linked immunosorbent assay were significantly higher (two-fold) in recipients compared to control mice. Anti-plasmodial IgG1, 2a and 2b titres were four to 16 times higher in rmIL-6 recipients than in the control mice. Reduction of parasitaemia to subpatency during the secondary phase of P. chabaudi chabaudi infection in C57BL/6 mice is primarily antibody-mediated and rmIL-6 accelerates this process by boosting the levels of the required specific anti-plasmodial IgG subclasses of antibodies.     

Lymphotoxin alpha and tumour necrosis factor are not required for control of parasite growth, but differentially regulate cytokine production during Plasmodium chabaudi chabaudi AS infection

Tumour necrosis factor (TNF) plays important roles in the pathogenesis of severe malaria, as well as in the generation of immune responses against malaria parasites. However, far less is known about the role of the closely related TNF family member lymphotoxin alpha (LTalpha) during malaria. We have used mice deficient in either TNF or LTalpha, as well as chimeric mice generated using donor bone marrow from these animals, to study the roles of these cytokines following Plasmodium chabaudi chabaudi AS infection. TNF and LTalpha were not required for the resolution of P. chabaudi chabaudi AS blood-stage infection. However, LTalpha, but not TNF, was necessary for early IFNgamma production and the regulation of IFNgamma production later in infection. A similar delay to that found for IFNgamma production was also observed for TNF production in LTalpha-deficient mice, compared with control mice. These results identify divergent roles for TNF and LTalpha in the regulation of host immune responses during P. chabaudi chabaudi AS infection.     

Relationships between sequestration, antigenic variation and chronic parasitism in Plasmodium chabaudi chabaudi– a rodent malaria model

We describe here a rodent malaria model using cloned lines of Plasmodium chabaudi chabaudi in inbred CBA/Ca mice that exhibits both clonal antigenic variation in late stage-specific surface antigens, and deep vascular schizogony in the liver. We show that both these features are modulated by the spleen, and that surface antigen expression is crucially involved in the sequestering phenotype. Surface antigens are variant in chronic infection, and host protective immune responses can distinguish between these variants. Splenectomy abolishes this difference. The acute infection with non-sequestering cloned lines is kinetically indistinguishable from sequestering clones, but parasites unable to express variant sequestration-associated antigen do not form a chronic recrudescing infection. Another clone, able to re-express this antigen in the presence of the spleen, undergoes typical chronic recrudescence. In this model, the biological significance of sequestration-associated variant antigen seems to enable the establishment of chronic infection in the presence of a primed spleen.     

Antigenic variation in Plasmodium chabaudi: analysis of parent and variant populations by cloning

Nineteen of 22 recrudescent populations of Plasmodium chabaudi AS strain were found to be significantly less sensitive to the protective activity of pools of immune serum, than the parent population from which they were derived. The immune sera were collected from donor mice which had been infected with the parent population and had just reduced the patent primary parasitaemia to subpatent levels. Clones prepared from the parent population (which had previously been cloned) and recrudescent variant populations were tested for their sensitivity to the immune sera. It was found that all the clones from the parent population were sensitive to the immune sera but some were more sensitive than others and that a recrudescent variant population could include both sensitive and insensitive parasites. Two insensitive clones of the recrudescent population were found to be different from each other.     

Antigenic variants of Plasmodium chabaudi chabaudi AS and the effects of mosquito transmission

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.