Rapid parasite multiplication rate, rather than immunosuppression, causes the death of mice infected with virulent Plasmodium yoelii.

Protective immunity against a lethal malaria challenge infection was passively transferred to naive recipient mice with spleen cells from donor mice bearing a lethal infection with the virulent YM strain of Plasmodium yoelii. Successful transfer of protection was contingent upon the elimination of residual, viable parasites from donor spleen cell suspensions prior to the infusion of cells. Passive transfer experiments failed to detect suppressor cells in the spleens of lethally infected mice because unfractionated spleen cells or T-cell-enriched spleen cells from mice infected with P. yoelii YM did not enhance parasitemias upon infusion into mice infected with cross-reactive nonvirulent P. yoelii 17X. We concluded that a form of protective immunity was generated during the course of virulent infection but that its expression was inconsequential because parasite growth apparently exceeded the capacity of the immune system to clear the infection.     

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.     

Antibody recognition and isoelectrofocusing of antigens of the malaria parasite Plasmodium yoelii.

Inbred BALB/c mice were either immunized with Triton X-100-extracted antigens of blood-stage Plasmodium yoelii or infected with P. yoelii and cured in three successive schedules. Whereas the immunized BALB/c became only partially protected from subsequent challenge infection with blood-stage P. yoelii, the convalescent mice acquired total immunity. When total P. yoelii antigen extract was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with anti-P. yoelii serum, five major protein bands of 150, 84, 40, 19, and 16 kDa were recognized by the sera of fully protected convalescent mice but not by the sera of partially protected mice. The utility of comparing reactivities of sera from fully protected and partially protected malaria hosts and the possibility that antigens uniquely recognized by the convalescent mouse sera may contribute to immunity against P. yoelii infection are discussed. Although previously reported to be an effective adjuvant for immunization against P. yoelii infection in (BALB/c x C57BL)F1 hybrid mice, saponin did not promote protection any better than did Freund adjuvant in BALB/c mice immunized with detergent-extracted P. yoelii antigen. Most of the P. yoelii proteins (14 to 250 kDa) found in Triton X-100 extracts of P. yoelii-parasitized erythrocytes isoelectrofocused as a single peak in the pH region 4.4 to 4.6, suggesting a rationale for previous findings that the most anti-P. yoelii protective and T-helper activities are induced by antigens isoelectrically focused in a fraction of similar pH.     

Activation of liver macrophages in murine malaria is enhanced by vaccination.

During blood-stage infection of mice with a lethal variant of Plasmodium yoelii, cells in both spleen and liver became activated to reach a peak at day 5. In mice protected by vaccination, activation was accelerated after infection. The most striking difference observed was in the 10-fold greater yield of infiltrating cells, including macrophages, obtained from the liver just before the mice recovered. Their capacity to give an oxidative burst and their cytotoxic activity against tumour cells was also more than 10 times normal. This suggests that the recruitment of inflammatory cells to the liver plays an important role in the protection of vaccinated mice against malaria.     

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.     

Host defenses in murine malaria: evaluation of the mechanisms of immunity to Plasmodium yoelii infection.

The immune response of random-bred mice to infection with a relatively avirulent strain of Plasmodium yoelii was measured in terms of parasitemia, splenomegaly, immediate and delayed hypersensitivity to a P. yoelii antigen preparation, resistance to challenge with a virulent variant of P. yoelii, and nonspecific resistance to L. monocytogenes. Avirulent P. yoelii produced a self limiting infection which resolved in 21 days. Peak parasitemia and splenomegaly were observed at 14 days, and infected mice were resistant to challenge with virulent P. yoelii from 7 days through at least 126 days. Mice infected with avirulent P. yoelii developed humoral immunity as judged by immediate hypersensitivity reactions and the capacity of their serum to passively protect normal mice against virulent P. yoelii. At no time did mice infected with the avirulent P. yoelii display evidence of cell-mediated immunity, as expressed by delayed-type hypersensitivity and increased resistance to L. monocytogenes. In fact, at the height of avirulent P. yoelii infection there was decreased resistance to L. monocytogenes in both liver and spleen, and the macrophages of the undisturbed peritoneal cavity were similarly defective. It was concluded that the defense mechanism of mice against P. yoelii is mediated by humoral factors in the absence of demonstrable cell-mediated immunity.     

Variation in expression of antibody-dependent cell-mediated cytotoxicity in rodents with malaria.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against mouse erythrocytes sensitized with immunoglobulin G was studied in mice with malaria. Spleen cells from mice had enhanced cytotoxic activity early in Plasmodium berghei infection but not later in the disease. Sera from infected animals and partially purified malarial immune complexes inhibited ADCC. In addition, ADCC was diminished in spleen cells from mice infected with the lethal variant of P. yoelii 17x compared with that in mice infected with the nonlethal variant. P. berghei-infected erythrocytes did not release 51Cr when incubated with effector cells unless the erythrocytes were sensitized with antibodies against normal mouse erythrocytes.     

Mycobacterium-induced potentiation of type 1 immune responses and protection against malaria are host specific

Malaria and tuberculosis are endemic in many regions of the world, and coinfection with the two pathogens is common. In this study, we examined the effects of long- and short-term infection with Mycobacterium tuberculosis on the course of a lethal form of murine malaria in resistant (C57BL/6) and susceptible (BALB/c) mice. C57BL/6 mice coinfected with M. tuberculosis CDC1551 and Plasmodium yoelii 17XL had a lower peak parasitemia and increased survival compared to mice infected with P. yoelii 17XL alone. Splenic microarray analysis demonstrated potentiation of type 1 immune responses in coinfected C57BL/6 mice, which was especially prominent 5 days after infection with P. yoelii 17XL. Splenocytes from coinfected C57BL/6 mice produced higher levels of gamma interferon (IFN-gamma) and tumor necrosis factor alpha than splenocytes from mice infected with either pathogen alone. Interestingly, mycobacterium-induced protection against lethal P. yoelii is mouse strain specific. BALB/c mice were significantly more susceptible than C57BL/6 mice to infection with P. yoelii 17XL and were not protected against lethal malaria by coinfection with M. tuberculosis. In addition, M. tuberculosis did not augment IFN-gamma responses in BALB/c mice subsequently infected with P. yoelii 17XL. These data indicate that M. tuberculosis-induced potentiation of type 1 immune responses is associated with protection against lethal murine malaria.     

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.     

Plasmodium yoelii blood-stage antigens newly identified by immunoaffinity using purified IgG antibodies from malaria-resistant mice

As the search for an effective human malaria vaccine continues, understanding immune responses to Plasmodium in rodent models is perhaps the key to unlocking new vaccine strategies. The recruitment of parasite-specific antibodies is an important component of natural immunity against infection in blood-stage malaria. Here, we describe the use of sera from naturally surviving ICR mice after infection with lethal doses of Plasmodium yoelii yoelii 17XL to identify highly immunogenic blood-stage antigens. Immobilized protein A/G was used for the affinity-chromatography purification of the IgGs present in pooled sera from surviving mice. These protective IgGs, covalently immobilized on agarose columns, were then used to isolate reactive antigens from whole P. yoelii yoelii 17XL protein extracts obtained from the blood-stage malaria infection. Through proteomics analysis of the recovered parasite antigens, we were able to identify two endoplasmic reticulum lumen proteins: protein disulfide isomerase and a member of the heat shock protein 70 family. Also identified were the digestive protease plasmepsin and the 39 kDa-subunit of eukaryotic translation initiation factor 3, a ribosome associated protein. Of these four proteins, three have not been previously identified as antigenic during blood-stage malaria infection. This procedure of isolating and identifying parasite antigens using serum IgGs from malaria-protected individuals could be a novel strategy for the development of multi-antigen-based vaccine therapies.     

Antibody-independent immunity to reinfection malaria in B-cell-deficient mice.

Immunity to "reinfection malaria" or "premunition" was studied in B-cell-deficient mice which had previously experienced acute malaria caused by the avirulent plasmodia Plasmodium yoelii or P. chabaudi or by the lethal P. vinckei. Such mice resisted challenge infection with large numbers of homologous parasites but differed in their capacity to resist challenge with heterologous species. Mice immune to P. yoelii resisted infection with P. chabaudi but developed acute-type, albeit nonlethal, infections when challenged with P. vinckei. Whereas mice immune to P. chabaudi resisted challenge with P. vinckei and vice versa, they developed fulminating malaria and died when infected with P. yoelii. The data suggest that immunity to reinfection malaria in B-cell-deficient mice, although antibody independent, is mediated by different mechanisms of resistance depending upon the plasmodial species used to initiate acute infection. Additional evidence supporting this concept was gained from preliminary experiments in which immunity to reinfection was measured by the ability of chronically infected mice to control endogenous parasites at low levels. B-cell-deficient mouse strains showed genotypic differences in their ability to develop immunity to reinfection with P. yoelii. In contrast, the same mouse strains uniformly developed immunity to reinfection with P. chabaudi. These findings suggest that different genetic loci control resistance to reinfection malaria caused by different species of plasmodia. Finally, B-cell-deficient mice acutely infected with lethal plasmodia, P. vinckei or P. berghei, died at the same time or earlier than similarly infected immunologically intact mice, indicating that "early death" in virulent malarial infections is an antibody-independent phenomenon.     

Experimental cutaneous leishmaniasis. V. Protective immunity in subclinical and self-healing infection in the mouse.

This study shows how infection of CBA mice with L. tropica can be manipulated so as to mimic the principal features of both subclinical and self-healing cutaneous leishmaniasis in man. CBA mice were infected with graded inocula of L. tropica promastigotes. The pattern of primary infection was found to be dependent on dose of infecting organisms: mice given low dose inocula (10(2), 10(3)) developed subclinical infections; those given high dose inocula (10(4), 10(5), 10(6)) developed overt, clinical lesions. Size and duration of lesions, and antibody production were directly proportional to dose; delayed hypersensitivity responses were inversely proportional to dose. Protective immunity to challenge infection was induced by both subclinical and clinical infection; and was manifest both during and after the healing stages of primary lesions. Protective immunity was also induced by artificial immunization with sonicated promastigotes in adjuvants but was only manifest if the challenge dose was not too large. The course of challenge infections differed depending on the method of immunization, i.e. whether by infection or artificial immunization. Lymphoid cells from immune CBA mice conferred protection on recipient syngeneic CBA mice against challenge infection; serum from immune mice did not, but suspension of immune peritoneal cells in immune serum enhanced their protective capacity. The experimental induction of protective immunity by low-dose infection, without a clinical allergic response at the site of inoculation, is of importance in designing an immunoprophylactic approach to human leishmaniasis.     

Protective immunity against Plasmodium yoelii malaria induced by immunization with particulate blood-stage antigens.

The Plasmodium yoelii murine model was used to test several combinations of blood-stage antigens and adjuvants for the ability to induce immunity to blood-stage malaria. Upon fractionation of whole blood-stage antigen into soluble and insoluble components, only the particulate antigens (pAg) induced protective immune responses. Of a number of adjuvants tested, Quil A was the most effective. Immunization with pAg plus Quil A induced solid protection against nonlethal and lethal P. yoelii challenge infection. Analysis of cytokine production revealed mRNA for Th1-type cytokines (interleukin 2 [IL-2] and gamma interferon) as well as Th2-type cytokines (IL-4 and IL-10) in the spleens of both protected and susceptible animals. The data suggested that the protective pAg response was associated with the earlier production of cytokine mRNA with a Th2 phenotype somewhat favored. Immunization of B-cell-deficient JHD mice indicated that the protection against P. yoelii induced by pAg immunization was B cell dependent. Although immunization with pAg plus Quil A increased the levels of antigen-specific antibodies of all four immunoglobulin G (IgG) isotypes, protection correlated most closely with the presence of IgG1 and IgG2b antibodies. Sera from pAg-plus-Quil A-immunized animals recognized only a limited subset of six to eight distinct P. yoelii antigens, primarily associated with the pAg fraction. These results provide the basis for the identification and characterization of potential vaccine antigens, selected solely for their ability to immunize against blood-stage malaria.     

T-cell-mediated immune response in murine malaria: differential effects of antigen-specific Lyt T-cell subsets in recovery from Plasmodium yoelii infection in normal and T-cell-deficient mice.

For analysis of the role of immune T cells in protective immunity against murine malaria, Plasmodium yoelii-immune Lyt T-cell subsets were functionally characterized in vitro and in vivo. Selected Lyt2- and Lyt2+ T cells from P. yoelii-immune C57BL/10 mice differed in their capability to proliferate in response to P. yoelii antigen in vitro. Only the Lyt2- T-cell population produced T-cell growth factor upon restimulation, and none of the selected T-cell subsets produced detectable amounts of macrophage activating factor. Lyt2- but not Lyt2+ lymphocytes were capable of transferring protection to normal C57BL/10 mice. When transferred into T-cell-deficient C57BL/6-nu/nu mice, adoptive resistance to P. yoelii by Lyt2- lymphocytes was only demonstrable after prior reconstitution of recipients with normal T cells. These results suggest an interaction between P. yoelii-immune Lyt2- T cells and normal T lymphocytes via T-cell growth factor in the development of protective immunity to malaria.     

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)     

Macrophages expressing heat-shock protein 65 play an essential role in protection of mice infected with Plasmodium yoelii

C57BL/6 (B6) mice are resistant to infection with the non-lethal (NL) strain of Plasmodium yoelii 17X, while being susceptible to that with the lethal (L) strain. The 65 000 MW heat-shock protein (hsp 65) was strongly expressed in splenic adherent cells of B6 mice 10 days after infection with the NL strain of P. yoelii but only slightly in those from mice infected with the L strain. Mice which had survived infection with the NL strain were resistant to challenge with the L strain and hsp 65 was strongly expressed in splenic adherent cells of these mice. Severe combined immunodeficient mice and nude mice were susceptible to malaria infection even with the NL strain and did not express hsp 65 after infection, suggesting that T cells are required for the expression of hsp 65 as well as for protective immunity. B6 mice treated intraperitoneally with carrageenan, which impairs the macrophage function, became susceptible to NL strain infection, indicating that macrophages play an important role as the final effectors in protective immunity. These results demonstrate that the hsp 65 expressed by macrophages correlates closely with protection against P. yoelii infection.     

Mechanisms of resistance to infection with Coccidioides immitis in mice.

Serum from vaccinated mice was ineffective in neutralizing the infectivity of arthrospores of Coccidioides immitis for recipient mice. However, a T-cell-enriched lymphocyte population was effective in preventing a lethal infection. Spleen cells from immune mice were passaged through nylon wool columns resulting in a T-cell enriched, B-cell-depleted population as shown by the susceptibility of the cell population to anti-theta serum and the inability of the cells to transfer adoptively an immune response to ovalbumin. Whereas transfer of 5 x 10(7) unfractionated immune spleen cells was required to protect 100% of the recipients against a lethal infection with C. immitis, 7 x 10(6) T-cell-enriched immune spleen cells were sufficient for the same level of protection. Thus, transfer of resistance to infection was achieved with fewer cells after the removal of B cells from the transferred spleen cells. The results confirm that T cells are crucial in transferring resistance against infection with C. immitis in mice.     

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.     

Role of T lymphocytes in recovery from murine cytomegalovirus infection.

Congenitally athymic nude (Nu/Nu) mice inoculated intraperitoneally with murine cytomegalovirus (MCMV), in doses as low as 1.3 X 10(1) plaque-forming units succumbed to the infection. In contrast, the mean lethal dose for heteroxygous euthymic (Nu/+) littermates was 4 X 10(3) plaque-forming units. Though histopathological changes consistent with MCMV infection were found in the spleen, lungs, and adrenals of nude mice, there were only small focal areas of involvement in the liver. In contrast, Nu/+ mice dying from infection had pathological evidence of severe hepatitis. Spleen cells from immune and control BALB/c mice were injected intravenously into syngeneic mice that had been inoculated previously with lethal doses of MCMV intraperitoneally. Mice receiving 1 X 10(7) or more immune spleen cells were protected against the infection, whereas mice receiving 1 X 10(8) control spleen cells or immune serum were not. Treatment of immune spleen cells with anti-theta serum and complement significantly reduced their protective effect. Immune mechanisms associated with T lymphocytes appear to be critical for recovery from MCMV infection.     

Roles of CD4+ T Cells and Gamma Interferon in Protective Immunity against Babesia microti Infection in Mice

Babesia microti produces a self-limiting infection in mice, and recovered mice are resistant to reinfection. In the present study, the role of T cells in protective immunity against challenge infection was examined. BALB/c mice which recovered from primary infection showed strong protective immunity against challenge infection. In contrast, nude mice which failed to control the primary infection and were cured with an antibabesial drug did not show protection against challenge infection. Treatment of immune mice with anti-CD4 monoclonal antibody (MAb) diminished the protective immunity against challenge infection, but treatment with anti-CD8 MAb had no effect on the protection. Transfer of CD4(+) T-cell-depleted spleen cells resulted in higher parasitemia than transfer of CD8(+) T-cell-depleted spleen cells. A high level of gamma interferon (IFN-gamma), which was produced by CD4(+) T cells, was observed for the culture supernatant of spleen cells from immune mice, and treatment of immune mice with anti-IFN-gamma MAb partially reduced the protection. Moreover, no protection against challenge infection was found in IFN-gamma-deficient mice. On the other hand, treatment of immune mice with MAbs against interleukin-2 (IL-2), IL-4, or tumor necrosis factor alpha did not affect protective immunity. These results suggest essential requirements for CD4(+) T cells and IFN-gamma in protective immunity against challenge infection with B. microti.