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-1(42)). 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-1(42) were partially protected against P. chabaudi malaria, indicating a role for protective antibody-dependent and antibody-independent mechanisms of immunity. The involvement of gammadelta T cells in vaccine-induced PcAMA-1 and/or PcMSP-1(42) 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-1(42) 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.

FIG. 1.

Isotypic profile of antigen-specific IgG antibodies induced by protective immunization with recombinant PcAMA-1 and/or PcMSP-1. (A) Groups of C57BL/6 mice (n = 5) were immunized with recombinant PcAMA-1 (A), PcMSP-1₄₂ (M), or the combination of PcAMA-1 and PcMSP-1₄₂ (A+M) with Quil A as adjuvant. The concentrations of IgG1, IgG2a/c, IgG2b, and IgG3 antibodies specific for PcAMA-1 (white bars) or PcMSP-1₄₂ (black bars) present in prechallenge sera were determined by ELISA. Background values obtained when adjuvant control sera (n = 5) were used have been subtracted. (B) Groups of C57BL/6 mice (n = 5) immunized with purified recombinant PcAMA-1 (♦), PcMSP-1 (•), or PcAMA-1 plus PcMSP-1 (★) with Quil A as adjuvant were challenged with 10⁶ P. chabaudi-parasitized erythrocytes. Mice immunized with Quil A alone (□) or phosphate-buffered saline (PBS) alone (▿) served as controls. Resulting parasitemias were monitored by enumerating parasitized erythrocytes in thin tail blood smears stained with Giemsa stain. The results obtained are similar to previously reported data (7).

FIG. 2.

Protective AMI and CMI induced by PcAMA-1 and PcMSP-1₄₂ immunization. (A) Groups of TCR-δ^−/− mice (n = 5) were immunized with recombinant PcAMA-1 (A), PcMSP-1₄₂ (M), or the combination of PcAMA-1 and PcMSP-1₄₂ (A+M) with Quil A as adjuvant. The concentrations of IgG1, IgG2a/c, IgG2b, and IgG3 antibodies specific for PcAMA-1 (white bars) or PcMSP-1₄₂ (black bars) present in prechallenge sera were determined by ELISA. Background values obtained by using adjuvant control sera (n = 5) have been subtracted. (B and C) Groups of TCR-δ^−/− mice (n = 5) (B) and J_H^−/− (JHD) mice (n = 5) (C) immunized with purified recombinant PcAMA-1 (♦), PcMSP-1 (•), or PcAMA-1 plus PcMSP-1 (★) with Quil A as adjuvant were challenged with 10⁶ P. chabaudi-parasitized erythrocytes. Mice immunized with Quil A alone (□) or PBS alone (▿) served as controls. Resulting parasitemias were monitored by enumerating parasitized erythrocytes in thin tail blood smears stained with Giemsa stain. Similar protection data were obtained in studies of C57BL/6, TCR-δ^−/−, and J_H^−/− mice immunized with PcAMA-1 and/or PcMSP-1₄₂ formulated with Freund's adjuvant and recombinant IL-12 in place of Quil A.

FIG. 3.

IFN-γ is not required for PcAMA-1 and PcMSP-1₄₂ vaccine-induced protection. (A) Groups of IFN-γ^−/− mice (n = 5) were immunized with recombinant PcAMA-1 (A), PcMSP-1₄₂ (M), or the combination of PcAMA-1 and PcMSP-1₄₂ (A+M) with Quil A as adjuvant. The concentration of IgG1, IgG2a/c, IgG2b, and IgG3 antibodies specific for PcAMA-1 (white bars) or PcMSP-1₄₂ (black bars) present in prechallenge sera were determined by ELISA. Background values obtained by using adjuvant control sera (n = 5) have been subtracted. (B) Groups of IFN-γ^−/− mice (n = 5) immunized with purified recombinant PcAMA-1 (♦), PcMSP-1 (•), or PcAMA-1 plus PcMSP-1 (★) with Quil A as adjuvant were challenged with 10⁶ P. chabaudi-parasitized erythrocytes. Mice immunized with Quil A alone (□) or PBS alone (▿) served as controls. Resulting parasitemias were monitored by enumerating parasitized erythrocytes in thin tail blood smears stained with Giemsa stain. Mean peak parasitemia in IFN-γ^−/− mice immunized with Quil A or saline is significantly higher than that observed in immunologically intact C57BL/6 controls (P < 0.01). This finding is consistent with previously reported data (3, 49).

FIG. 4.

IL-4 is not required for PcAMA-1 and PcMSP-1₄₂ vaccine-induced protection. (A) Groups of IL-4^−/− mice (n = 5) were immunized with recombinant PcAMA-1 (A), PcMSP-1₄₂ (M), or the combination of PcAMA-1 and PcMSP-1₄₂ (A+M) with Quil A as adjuvant. The concentration of IgG1, IgG2a/c, IgG2b, and IgG3 antibodies specific for PcAMA-1 (white bars) or PcMSP-1₄₂ (black bars) present in prechallenge sera were determined by ELISA. Background values obtained by using adjuvant control sera (n = 5) have been subtracted. (B) Groups of IL-4^−/− mice (n = 5) immunized with purified recombinant PcAMA-1 (♦), PcMSP-1 (•), or PcAMA-1 plus PcMSP-1 (★) with Quil A as adjuvant were challenged with 10⁶ P. chabaudi-parasitized erythrocytes. Mice immunized with Quil A alone (□) or PBS alone (▿) served as controls. Resulting parasitemias were monitored by enumerating parasitized erythrocytes in thin tail blood smears stained with Giemsa stain.