Host-parasite interactions and immunity to irradiated sporozoites

We compare and contrast the results of immunizing mice with irradiated sporozoites of Plasmodium berghei and Plasmodium yoelii. Host genetic control of protective immunity is different in the two rodent malarias. Few mouse strains are strongly protected by P. yoelii sporozoites, while all are protected by P. berghei sporozoite immunization. The role of CD8+ T cells in the protective immune response to each of these malarias varies with the strain of mouse. Moreover, a single strain will use a CD8+ T cell-dependent mechanism against one malaria, and a CD8+ independent mechanism against the other. Thus, each host-parasite pairing in these rodent malarias engenders a unique set of immune responses. Such variety should be expected in the immune response to the human malarias, and may complicate the development of universally applicable vaccines.     

Intrasplenic immunization with infected hepatocytes: a mouse model for studying protective immunity against malaria pre-erythrocytic stage.

Malaria liver forms are the target of antibody or T-cell-mediated immune mechanisms induced by previous or subsequent developmental stages of the parasite. The potential for vaccine development of antigens expressed exclusively in the liver stages has not been fully explored partly because of the lack of an experimental animal model. Here we show that protective immunity against sporozoite-induced infection with Plasmodium yoelii and P. berghei can be obtained by intrasplenic injection of a small number of liver stages of the parasites. The serum of the protected animals did not contain antibodies against sporozoites, liver or blood stage malaria parasites. Protective immunity was abolished by depletion of either CD4+ or CD8+ T cells from the vaccinated mice before challenge.     

Isolation and characterization of protective cytolytic T cells in a rodent malaria model system.

Protective immunity against malaria is induced by immunization with irradiation-attenuated sporozoites. Here we report the isolation of cytolytic T-cell (CTL) clones from BALB/c (H-2d) mice immunized with either Plasmodium berghei or Plasmodium yoelii sporozoites. The epitopes recognized by these CTL can be mimicked by synthetic peptides corresponding to a homologous region in the CS proteins of both rodent malaria species. Both peptides are recognized by the CTL in the context of the same MHC class I molecule, H-2 Kd. In vivo adoptive transfer of the CTL clones into non-immune syngeneic mice protected them from a lethal challenge of infectious sporozoites.     

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.     

Complete protection against Plasmodium yoelii by adoptive transfer of a CD8+ cytotoxic T-cell clone recognizing sporozoite surface protein 2.

BALB/c mice immunized with irradiated Plasmodium yoelii sporozoites produce antibodies and cytotoxic T lymphocytes against the circumsporozoite protein and against a 140-kDa protein, sporozoite surface protein 2 (PySSP2). Approximately 50% of mice immunized with P815 cells transfected with the gene encoding PySSP2 are protected against malaria, and this protection is reversed by in vivo depletion of CD8+ T cells. To determine if CD8+ T cells against PySSP2 are adequate to protect against malaria in the absence of other malaria-specific immune responses, we produced three CD8+ T-cell clones by stimulating spleen cells from mice immunized with irradiated P. yoelii sporozoites with a mitomycin-treated P815 cell clone transfected with the PySSP2 gene. Adoptive transfer of clone TSLB7 protected 100% of mice against P. yoelii. The second clone protected 58% of mice, and the third clone provided no protection. Clone TSLB7 protected even when administered 3 h after sporozoite inoculation at a time when sporozoites had entered hepatocytes, suggesting that it is recognizing and eliminating infected hepatocytes. These studies demonstrate that cytotoxic T lymphocytes against PySSP2 can protect against P. yoelii sporozoite challenge in the absence of other parasite-specific immune responses.     

Peptide-primed CD4+ cells and malaria sporozoites.

We have mapped a T cell epitope in the circumsporozoite (CS) protein of the murine malaria parasite, Plasmodium yoelii. A 21-mer synthetic peptide corresponding to the amino acid positions 59-79 (referred to as Py1), induced specific proliferation in BALB/c and C57BL/6 mice, and provided help for the production of antibodies to peptides from the repetitive region, (QGPGAP)n, of the same CS protein, when mice were immunized with the Py1 peptide conjugated to the repetitive peptide. Long-term CD3+CD4+CD8-TCR alpha beta+ T cell lines and clones were derived from both strains of mice. These lines and clones, that proliferated in an MHC-restricted fashion, did not recognize peptides from the homologous region of another murine malaria parasite, P. berghei. About 50% of these clones produced detectable amounts of IFN-gamma and IL-2, whereas the remaining produced IL-4, IL-5, and IL-6. In preliminary experiments, some of these clones specifically inhibited P. yoelii sporozoite development in vitro and conferred protection in vivo in passive transfer experiments. These findings show that heterogenous T cell populations are activated in mice upon immunization with a short peptide from the P. yoelii CS protein and that some of these cells could be active in the effector arm of the immune response against malaria sporozoites.     

Specific inflammatory cell infiltration of hepatic schizonts in BALB/c mice immunized with attenuated Plasmodium yoelii sporozoites.

We compared immunization of BALB/c mice with radiation-attenuated versus killed sporozoites of the rodent malaria parasite, Plasmodium yoelii. We employed a suboptimal schedule of only two immunizations, in expectation that some parasites might break through the resultant low level immunity and that it might thus be possible to study the response of the host against these 'breakthrough' schizonts. As a measure of protective immunity, we used histological means to determine the percentages of challenge sporozoites prevented from completing development into hepatic schizonts within the liver. Immunization with attenuated sporozoites led to almost complete protection, whereas immunization with similar dosages of killed sporozoites led to approximately a 75% protection. Fluorescent antibody titers against sporozoites were similar in both sets of immunized animals. However, serum from mice immunized with attenuated sporozoites had a protective effect upon passive transfer into immunologically naive mice subsequently challenged with normal sporozoites; serum from mice immunized with killed sporozoites had no such effect. When mice suboptimally immunized with attenuated sporozoites were challenged, we observed breakthrough schizonts being infiltrated with inflammatory cells, primarily mononuclear cells, and neutrophils; partial depletion of CD4+ or CD8+ cells within these mice prior to challenge prevented the infiltration of breakthrough schizonts. Thus, cellular infiltration of schizonts was apparently secondary to earlier action by lymphocytes. This infiltration was also not observed in mice immunized with killed sporozoites. The more effective protective immunity induced by attenuated sporozoites could be due to their ability to release antigen into the cytoplasm of hepatocytes that they invade or their ability to continue differentiating, thereby presenting new antigens that are not seen after immunization with killed sporozoites.     

GP96NTD-CSP重组DNA疟疾疫苗诱导小鼠产生保护性免疫的研究

目的探讨pFLAG CMV8 gp96NTD-CSP重组DNA疟疾疫苗免疫能否诱导小鼠产生保护性免疫及其效应机制。方法以pFLAG CMV8质粒为载体,构建免疫用重组质粒,按照DNA疫苗免疫方法免疫小鼠;野生子孢子进行攻击后,采用Real-time PCR和吉氏染色观察被攻击小鼠的肝脏虫荷和原虫血症,即免疫小鼠抵御野生子孢子攻击的能力;并通过ELISA和ELISPOT方法探讨免疫小鼠保护性免疫的可能机制。结果核酸疫苗pFLAG CMV8 gp96NTD-CSP免疫小鼠能显著抵御野生子孢子的攻击,并且能诱导小鼠产生较高的抗体水平和较高的CSP特异的CD8+T细胞频率。结论 pFLAG CMV8 gp96NTD-CSP重组DNA疫苗可能通过诱导小鼠CSP特异抗体和CSP特异的CD8+T细胞的产生,一定程度上抵御野生子孢子的攻击。     

The relative contribution of antibodies, CD4+ and CD8+ T cells to sporozoite-induced protection against malaria.

Protective immunity against Plasmodium yoelii, induced by sporozoite immunization, was investigated using a quantitative method based on the measurement of plasmodial ribosomal RNA in the liver of sporozoite-challenged mice. The relative importance of the different immune mechanisms induced by sporozoite immunization was determined by evaluating quantitatively the anti-parasite activity of antibodies, CD4+ and CD8+ T cells. The role of antibodies was determined by passive transfer of immune sera to naive mice. The transfer to mice of sera obtained after a single immunizing dose reduced the liver stages by 47%. The respective contribution of CD4+ and CD8+ T-cell subsets was determined in B10 (H-2b) mice, treated with a monoclonal antibody (mAb) which inhibits B-cell maturation, and subsequently immunized once with irradiated sporozoites. These mice produced low levels of anti-sporozoite antibodies, but were capable of inhibiting the development of liver stages as efficiently as non-manipulated immunized mice. Administration of either anti-CD4 or anti-CD8 mAb to these mice, did not significantly decrease their capacity to inhibit the development of liver stages. We only observed a significant loss of immunity when the mice were depleted in vivo of both CD4+ and CD8+ T cells. In contrast to earlier studies, we found that the induction of protective immunity is not a phenomenon restricted to a few strains of mice having a particular genetic make-up. The apparent non-responsiveness observed in some strains of mice can be overcome by using larger immunizing doses.     

The synthetic, oxidized C-terminal fragment of the Plasmodium berghei circumsporozoite protein elicits a high protective response

A polypeptide of 69 amino acids (PbCS 242-310) encompassing the C-terminal region of the circumsporozoite protein of Plasmodium berghei (PbCS) was generated using solid-phase peptide synthesis. The immunological and protective properties of peptide PbCS 242-310 were studied in BALB/c mice (H-2d). Two subcutaneous injections, in the presence of IFA at the base of the tail, generated (i) high titers of anti-peptide antibodies which also recognized the native P. berghei CS protein, (ii) cytolytic T cells specific for the Kd-restricted peptide PbCS 245-253 and (iii) partial CD8+-dependent protection against sporozoite-induced malaria. The same frequencies of peptide PbCS 245-253 specific CD8+ T cells were found by IFN-gamma ELISPOT in the draining lymph nodes of animals immunized with the short optimal CTL peptide 245-253 or with the polypeptide 242-310, indicating that the longer polypeptide can be processed and presented in vivo in the context of MHC class I as efficiently as the short CTL peptide. Interestingly, higher levels of IFN-gamma producing CD8 T cells and protection were observed when the four cysteine residues present in the C-terminal peptide were fully oxidized. These findings underline the potential importance of the chemical nature of the C-terminal fragment on the activation of the immune system and concomitant protection.     

Cellular mechanisms in immunity to blood stage infection

We studied mechanisms of immunity to blood stage infection in the mouse malarias Plasmodium vinckei and Plasmodium yoelii 17X. Infection with P. vinckei was uniformly lethal, whereas P. yoelii 17X caused a self-limited, nonlethal infection. Transfer of immune CD4+ T cells conferred protection against P. yoelii in nude mice. Previous studies by others had suggested that immunity to P. yoelii may be related to MHC class I expression on reticulocytes and found that CD8+ T cells alone transferred protection in immunodeficient mice. However, in our experiments, immune CD8+ T cells failed to transfer protection. In the P. vinckei system, both B cell-deficient and immunologically intact mice developed immunity to P. vinckei after parasite infection and drug cure. In vivo depletion of CD4+ T cells abrogated immunity in these immune mice. Adoptive transfer of CD4+ T cells failed to protect nude or normal mice from P. vinckei infection, but the transfer of immune CD4+ T cells reconstituted immunity in CD4-depleted immune mice. Splenectomy of immune mice resulted in the complete loss of immunity. Despite the fact that immunity to P. vinckei could be achieved with live parasite infection and drug cure, immunization of mice with killed P. vinckei with various adjuvants failed to protect mice from live challenge. In contrast, immunization with killed P. vinckei antigens in combination with attenuated Salmonella typhimurium SL3235 induced a high degree of protective immunity. These results suggest that induction of immunity against virulent malarias requires both induction of CD4+ T cells and certain splenic alterations caused by parasite infection or S. typhimurium.(ABSTRACT TRUNCATED AT 250 WORDS)     

T helper epitopes enhance the cytotoxic response of mice immunized with MHC class I-restricted malaria peptides.

We have previously derived MHC class I (H-2Kd) restricted cytotoxic T lymphocytes (CTL) from BALB/c mice immunized with irradiated sporozoites from Plasmodium (P.) berghei and P. yoelii. The CTL recognize synthetic peptides corresponding to a region of the circumsporozoite (CS) protein that is homologous in the two species. In the present study, we have attempted to induce CS-specific CTL by immunization with those peptides in incomplete Freund's adjuvant. Only a low level CTL response was detected in BALB/c mice immunized with synthetic peptides corresponding to the Pb or Py CTL epitopes. In contrast, CS-specific CTL responses could be readily detected in mice injected with mixtures of peptides that combined the P. berghei or P. yoelii CTL epitopes with previously defined T helper epitopes. Several different T helper epitopes were shown to enhance the response when injected as separate peptides in a mixture, or when covalently linked to a CTL epitope. These results may have general implications for the elicitation of CTL responses to defined CTL epitopes and for the design of peptide-based synthetic vaccines.     

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.      

CD8+ cytolytic T cell clones derived against the Plasmodium yoelii circumsporozoite protein protect against malaria.

Immunization of BALB/c mice with radiation-attenuated Plasmodium yoelii sporozoites induces cytotoxic T lymphocytes (CTL) specific for an epitope located within the amino acid sequence 277-288 of the P. yoelii circumsporozoite (CS) protein. Several CD8+ CTL clones were derived from the spleen cells of sporozoite-immunized mice, all displaying an apparently identical epitope specificity. All the clones induced high levels of cytolysis in vitro upon exposure to peptide-incubated MHC-compatible target cells. The adoptive transfer of two of these clones conferred complete protection against sporozoite challenge to naive mice. This protection is species and stage specific. Using P. yoelii specific ribosomal RNA probes to monitor the in vivo effects of the CTL clones, we found that their target was the intrahepatocytic stage of the parasite. The protective clones completely inhibited the development of the liver stages of P. yoelii. Some CTL clones were only partially inhibitory in vivo, while others failed completely to alter liver stage development and to confer any detectable degree of protection. The elucidation of the effector mechanism of this CTL mediated protection against rodent malaria should facilitate the design of an effective malaria vaccine. From a broader perspective this model may provide further insight into the mechanism(s) of CTL mediated killing of intracellular non-viral pathogens in general.     

Dynamic production of tumour necrosis factor-alpha (TNF-alpha) messenger RNA, intracellular and extracellular TNF-alpha by murine macrophages and possible association with protein tyrosine phosphorylation of STAT1 alpha and ERK2 as an early signal.

Cytotoxic T lymphocytes (CTL) recognize and lyse target cells through the interaction of the T-cell receptor complex with the class I or class II major histocompatibility complex (MHC). The production of class I-restricted CTL has been shown to be critical to the elimination of specific pathogens including Listeria monocytogenes. However, the function of class II-restricted CTL in the clearance of intracellular pathogens is poorly understood. H-2b beta 2-microglobulin-deficient mice (beta 2M-/-) are not able to produce CD8+ CTL in response to infection with L. monocytogenes. We used this model to evaluate the efficacy of class II-restricted CTL, in the absence of a class I-restricted response, during a primary infection with L. monocytogenes. We demonstrate that, despite their effectiveness in adoptive transfer of protection, Listeria-specific CD4+ class II-restricted cytotoxic lymphocytes are ineffective in decreasing titres of L. monocytogenes in the spleen was found established infection. In beta 2M-/- mice, persistence of L. monocytogenes in the spleen was found preferentially in class II-negative cells. Surprisingly, class I-restricted CTL from C57BL/6 mice were capable of decreasing bacterial titres during an established infection even in the absence of detectable class I on the surface of cells from beta 2M-/- mice. These data strongly suggest that, in the absence of a class I-restricted response, pathogens that elicit a class II-restricted cytotoxic response may escape prompt eradication by the immune system.     

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.      

Memory phenotype CD8(+) T cells persist in livers of mice protected against malaria by immunization with attenuated Plasmodium berghei sporozoites

Natural exposure to Plasmodium parasites induces short-lived protective immunity. In contrast, exposure to radiation-attenuated sporozoites (gamma spz) promotes long-lasting protection that is in part mediated by CD8(+) T cells that target exoerythrocytic stage antigens. The mechanisms underlying the maintenance of long-lasting protection are currently unclear. The liver is a repository of Plasmodium antigens and may support the development and / or homing of memory T cells. While activated CD8(+) T cells are presumed to die in the liver, the fate of anti-Plasmodium CD8(+) T cells remains unknown. We propose that inflammatory conditions in the liver caused by Plasmodium parasites may allow some effector CD8(+) T cells to survive and develop into memory cells. To support this hypothesis, in this initial study we demonstrate that liver mononuclear cells from P. berghei gamma spz-immune mice transferred protection to naive recipients and moreover, that CD4(+) and CD8(+) T cells responded to Plasmodium antigens by up-regulating activation / memory markers. While CD4(+) T cells under went a transient activation following immunization with gamma spz, CD8(+) T cells expanded robustly after spz challenge and exhibited stable expression of CD44(hi) and CD45RB(lo) during protracted protection. These results establish a key role for intrahepatic T cells in long-lasting protection against malaria.     

Persistence of protective immunity to malaria induced by DNA priming and poxvirus boosting: characterization of effector and memory CD8(+)-T-cell populations

The persistence of immunity to malaria induced in mice by a heterologous DNA priming and poxvirus boosting regimen was characterized. Mice were immunized by priming with DNA vaccine plasmids encoding the Plasmodium yoelii circumsporozoite protein (PyCSP) and murine granulocyte-macrophage colony-stimulating factor and boosting with recombinant vaccinia encoding PyCSP. BALB/c mice immunized with either high-dose (100 microg of p PyCSP plus 30 microg of pGM-CSF) or low-dose (1 microg of p PyCSP plus 1 microg of pGM-CSF DNA) priming were protected against challenge with 50 P. yoelii sporozoites. Protection 2 weeks after immunization was 70 to 100%, persisted at this level for at least 20 weeks, and declined to 30 to 40% by 28 weeks. Eight of eight mice protected at 20 weeks were still protected when rechallenged at 40 weeks. The antigen (Ag)-specific effector CD8(+)-T-cell population present 2 weeks after boosting had ex vivo Ag-specific cytolytic activity, expressed both gamma interferon (IFN-gamma) and tumor necrosis factor alpha, and constituted 12 to 20% of splenic CD8(+) T cells. In contrast, the memory CD8(+)-Ag-specific-cell population at 28 weeks lacked cytolytic activity and constituted only 6% of splenic CD8(+) T cells, but at the single-cell level it produced significantly higher levels of IFN-gamma than the effectors. High levels of Ag- or parasite-specific antibodies present 2 weeks after boosting had declined three- to sevenfold by 28 weeks. Low-dose priming was similarly immunogenic and as protective as high-dose priming against a 50-, but not a 250-, sporozoite challenge. These results demonstrate that a heterologous priming and boosting vaccination can provide lasting protection against malaria in this model system.     

Immunization with a circumsporozoite epitope fused to Bordetella pertussis adenylate cyclase in conjunction with cytotoxic T-lymphocyte-associated antigen 4 blockade confers protection against Plasmodium berghei liver-stage malaria

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8+ T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8+ T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-gamma) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.     

Acute malaria prolongs susceptibility of mice to Plasmodium berghei sporozoite infection.

The fate of immune response against sporozoite stage in malaria infection was investigated. Two groups (A and B) of mice were inoculated twice with infective sporozoites of Plasmodium berghei. The mice in group A were maintained on chloroquine prophylaxis to prevent the sporozoite infection from causing malaria. Group B animals on the other hand were allowed to develop acute malaria from the infection which was subsequently cured with chloroquine. Upon examination for stage specific immune responses, it was found that the animals in group A produced high antibody titres against sporozoites and none against erythrocytic stages. The mice in group B produced little anti-sporozoite antibodies but had high antibody titres against blood forms. Challenge infection with P. berghei sporozoites showed that group A animals had become resistant against sporozoite-induced parasitaemia, whereas the mice in group B remained susceptible. The possible significance of suppression of protective immunity by malaria in host-parasite relationship is discussed.