William P. Weidanz

Production of interleukin 10 during malaria caused by lethal and nonlethal variants of Plasmodium yoelii yoelii

We investigated the induction of T-helper cell subsets during the course of lethal or nonlethal blood-stage Plasmodium yoelii 17X infection in C57BL/6 mice, which are relatively susceptible to these intraerythrocytic parasites. C57BL/6 mice infected with the nonlethal variant (PyNL) showed a moderate level of parasitemia and resolution of primary acute infection by week 4. Mice infected with the lethal variant (PyL) developed fulminating parasitemia and ultimately died. T-helper subset function was assessed during infection by determining the kinetics of in vitro production of the Thl-derived cytokine interferon-γ (IFN-γ) and the Th2-derived cytokine interleukin 10 (IL-10) by means of bioassay and enzyme-linked immunosorbent assay (ELISA), respectively. Spleen cells obtained from mice infected with PyL within the 1st week of infection produced high levels of IL-10 and IFN-γ in response to malaria antigen. IL-10 also appeared in sera from PyL-infected mice at the same time at which the in vitro IL-10 response peaked. In contrast, spleen cells from mice infected with PyNL failed to produce IL-10 during the course of infection. CD4+ T-lymphocytes from mice infected with the lethal variant were a major source of IL-10, although non T-cells were also involved in the production of IL-10 during this malaria infection. In addition, the initial burst of IL-10 in response to malaria antigens was seen concomitantly with the production of IFN-γ within the 1st week of infection. These results indicate that both Thl and Th2 subsets of T-helper lymphocytes are activated during infection with the lethal variant of P. yoelii and support the contention of other investigators that a strong Th2 response early in infection is associated with the lethal outcome of malaria.

Protection against Plasmodium chabaudi Malaria Induced by Immunization with Apical Membrane Antigen 1 and Merozoite Surface Protein 1 in the Absence of Gamma Interferon or Interleukin-4

ABSTRACT Strategies to optimize formulations of multisubunit malaria vaccines require a basic knowledge of underlying protective immune mechanisms induced by each vaccine component. In the present study, we evaluated the contribution of antibody-mediated and cell-mediated immune mechanisms to the protection induced by immunization with two blood-stage malaria vaccine candidate antigens, apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-1). Immunologically intact or selected immunologic knockout mice were immunized with purified recombinant Plasmodium chabaudi AMA-1 (PcAMA-1) and/or the 42-kDa C-terminal processing fragment of P. chabaudi MSP-1 (MSP-142). The efficacy of immunization in each animal model was measured as protection against blood-stage P. chabaudi malaria. Immunization of B-cell-deficient JH−/− mice indicated that PcAMA-1 vaccine-induced immunity is largely antibody dependent. In contrast, JH−/− mice immunized with PcMSP-142 were partially protected against P. chabaudi malaria, indicating a role for protective antibody-dependent and antibody-independent mechanisms of immunity. The involvement of γδ T cells in vaccine-induced PcAMA-1 and/or PcMSP-142 protection was minor. Analysis of the isotypic profile of antigen-specific antibodies induced by immunization of immunologically intact mice revealed a dominant IgG1 response. However, neither interleukin-4 and the production of IgG1 antibodies nor gamma interferon and the production of IgG2a/c antibodies were essential for PcAMA-1 and/or PcMSP-142 vaccine-induced protection. Therefore, for protective antibody-mediated immunity, vaccine adjuvants and delivery systems for AMA-1- and MSP-1-based vaccines can be selected for their ability to maximize responses irrespective of IgG isotype or any Th1 versus Th2 bias in the CD4+-T-cell response.

Immunization against Plasmodium chabaudi malaria using combined formulations of apical membrane antigen-1 and merozoite surface protein-1

The control of Plasmodium falciparum malaria by vaccination will require immunization with multiple parasite antigens effectively formulated in combination. In this regard, proteins expressed on the surface of blood-stage merozoites are attractive as vaccine targets given their functional importance in the invasion of erythrocytes and accessibility to serum antibodies. We have utilized a Plasmodium chabaudi vaccine model to begin to evaluate the efficacy of immunization with combined formulations of apical membrane antigen-1 (AMA-1) and merozoite surface protein-1 (MSP-1). Using a pET/T7 RNA polymerase bacterial expression system, we have expressed, purified and refolded recombinant antigens representing the 54 kDa ectodomain of Pc AMA-1 and the 42 kDa C-terminus of Pc MSP-1. Immunization with recombinant Pc AMA-1+Pc MSP-1(42) induced a high level of protection against P. chabaudi malaria with protective efficacy varying with antigen dose, choice of adjuvant, and immunization protocol. Based on the reduction of P. chabaudi parasitemia, Alum proved effective for use with the combination of Pc AMA-1 and Pc MSP-1(42). The use of Quil A was similarly effective with single or combined antigen immunizations, particularly with low antigen dose. In general, serological analysis of prechallenge sera indicated a dominant IgG1 response. For a given formulation, immunization with the combination of Pc AMA-1 and Pc MSP-1(42) elicited IgG responses comparable to those observed following immunization with each antigen alone. However, prechallenge antibody titers alone were not predictive of protective efficacy. While Pc AMA-1 and Pc MSP-1(42) can be effectively formulated in combination, further study is needed to define measurable parameters of protective T cell and B cell responses induced by Pc AMA-1+Pc MSP-1(42) that are predictive of vaccine efficacy.

Immunity to blood-stage murine malarial parasites is MHC class II dependent

To determine whether MHC class II antigen presentation is essential for the induction of protective immunity against blood-stage malarial parasites, we used gene-targeted knockout (KO) mice to follow the time-course of nonlethal Plasmodium yoelii and Plasmodium chabaudi infections in two models of MHC class II deficiency. Infection of MHC class II KO (A(-/-)) mice with either parasite species resulted in an unremitting hyperparasitemia, whereas MHC-intact control mice resolved their parasitemia. In contrast, invariant chain KO (Ii(-/-)) mice, which present antigen via recycled but not nascent MHC class II molecules, eventually cured their infections when infected with P. yoelii. P. chabaudi parasitemia declined to subpatent levels in most Ii(-/-) mice but then recrudesced. Immunity to blood-stage malaria may be achieved by cell-mediated and antibody-mediated mechanisms of immunity, as such, the findings in A(-/-) mice indicate an essential role for MHC class II presentation of malarial antigens. Moreover, they suggest that protective immune responses to malarial antigens capable of eliminating blood-stage parasites are T cell dependent and can be induced with antigens processed in early and late endosomes.

Expansion of the gammadelta T cell subset in vivo during bloodstage malaria in B cell-deficient mice.

Mice rendered B cell‐deficient either by chronic anti‐μ treatment initiated at birth or by gene knockout (JHD and μ‐MT mice) suppressed acute Plasmodium chabaudi infections with a time course similar to intact control mice. Moreover, both kinds of B cell‐deficient mice showed a 50‐ to 100‐fold increase in splenic γδ T cell number after suppression of parasitemia compared with uninfected B cell‐deficient controls; the magnitude of this increase resulted in significantly (P < 0.05) greater numbers of splenic γδ T cells in the B cell‐deficient mice than in infected B cell‐intact controls (about 10‐fold). In contrast, the number of splenic CD4+ αβ T cells was only slightly elevated (< 2‐fold) in both kinds of B cell‐deficient mice compared with their intact controls. The number of splenic γδ T cells following suppression of P. vinckei parasitemia was approximately ninefold greater in JHD mice than in C57BL/6 controls, whereas similar numbers of splenic CD4+ αβ T cells were detected. Maximal numbers of γδ T cells were in cell‐cycle in both JHD and C57BL/6 mice during descending P. chabaudi parasitemia, but the number of γδ T cells in cell‐cycle was greater in B cell‐deficient mice than in intact controls. Interleukin‐10 (IL‐10), a potent TH1 cell‐suppressive molecule, does not appear to down‐regulate the γδ T cell response during malaria in B cell‐intact mice because the magnitude of the γδ T cell response was not significantly greater in IL‐10 knockout mice compared with heterozygote controls. These findings collectively indicate that a markedly enhanced expansion of the γδ T cell population occurs in the absence of B cells, and this expansion occurs predominantly during acute malaria when parasite burdens are similar in B cell‐deficient animals and intact controls. J. Leukoc. Biol. 60: 221–229; 1996.

The effects of interleukin-15 on human γδ T cell responses to Plasmodium falciparum in vitro

We observed that the gammadelta T cell subset expands when human peripheral blood mononuclear cells (PBMC) from malaria-naive donors are cultured with Plasmodium falciparum lysate in the presence of IL-2 or IL-15, cytokines that utilize two common IL-2 receptor subunits. IL-15 induced the expansion of the gammadelta T cell subset at all levels tested, whereas IL-2 was not stimulatory at high levels. Flow cytometric analysis of apoptosis using the TUNEL assay indicated that the percentage and absolute number of gammadelta T cells undergoing apoptosis were greater in cultures stimulated with antigen and IL-2 than in cultures stimulated with either antigen and IL-15 or control erythrocyte lysate and IL-2. The ability of IL-15 to enhance gammadelta T cell function was also assessed; the results suggest that IL-15 can function with IL-2 to enhance the capacity of gammadelta T cells to inhibit parasite replication. Together these data indicate that IL-2 and IL-15, which both bind to IL-2Rbeta and IL-2R(gamma)c, enhance gammadelta T cell function, but they appear to have different effects on proliferation and survival.

Analysis of antigen‐specific antibodies and their isotypes in experimental malaria

Measuring antibody production in response to antigen exposure or vaccination is key to disease prevention and treatment. Our understanding of the mechanisms involved in the antibody response is limited by a lack of sensitive analysis methods. We address this limitation using multiplexed microsphere arrays for the semi ‐quantitative analysis of antibody production in response to malaria infection.

Signalling through the IL-2 receptor γ(c) peptide (CD132) is essential for the expression of immunity to Plasmodium chabaudi adami blood-stage malaria.

A genetic dissection approach was employed to determine whether the IL‐2 receptor complex (IL‐2R) comprised of α, β and γ chains is required for the suppression of Plasmodium chabaudi adami parasitemia. Blood‐stage infections in IL‐2Rγc−/y mice failed to cure with parasitemia remaining elevated for >50u2003days indicating the IL‐2Rγc through which all members of the γc family of cytokines signal has an essential role in protective immunity against blood‐stage malarial parasites. In contrast, the curing of parasitemia in IL‐2/15Rβ−/− mice, deficient in both IL‐2 and IL‐15 signalling was significantly delayed but did occur, indicating that neither cytokine plays an essential role in parasite clearance. Moreover, the observation that the time course of parasitemia in IL‐15−/− mice was nearly identical to that seen in controls suggests that the parasitemia‐suppressing role of stimulating through the IL‐2/15Rβ chain is owing to IL‐2 signalling and not a redundant function of IL‐15.