Antibody-Dependent, Gamma Interferon-Independent Sterilizing Immunity Induced by a Subunit Malaria Vaccine

Abstract

The development of effective malaria vaccines is hampered by incomplete understanding of the immunological correlates of protective immunity. Recently, the moderate clinical efficacy of the Plasmodium falciparum circumsporozoite protein (CSP)-based RTS,S/AS01_E vaccine in phase 3 studies highlighted the urgency to design and test more efficacious next-generation malaria vaccines. In this study, we report that immunization with recombinant CSP from Plasmodium yoelii (rPyCSP), when delivered in Montanide ISA 51, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. This immunity was antibody dependent, as evidenced by the complete loss of immunity in B-cell-knockout (KO) mice and by the ability of immune sera to neutralize sporozoite infectivity in mice. Th2-type isotype IgG1 antibody levels were associated with protective immunity. The fact that immunized gamma interferon (IFN-γ)-KO mice and wild-type (WT) mice have similar levels of protective immunity and the absence of IFN-γ-producing CD4⁺ and CD8⁺ T cells in protected mice, as shown by flow cytometry, indicate that the immunity is IFN-γ independent. Protection against sporozoite challenge correlated with higher frequencies of CD4⁺ T cells that express interleukin-2 (IL-2), IL-4, and tumor necrosis factor alpha (TNF-α). In the RTS,S study, clinical immunity was associated with higher IgG levels and frequencies of IL-2- and TNF-α-producing CD4⁺ T cells. The other hallmarks of immunity in our study included an increased number of follicular B cells but a loss in follicular T helper cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants and vaccine dosage and determine the correlates of immunity in the search for superior malaria vaccine candidates.

Keywords: antibodies; malaria; protection; vaccine.

FIG 1

SDS-PAGE and Western blot analysis of purified recombinant circumsporozoite protein (CSP) from Plasmodium yoelii (rPyCSP). Lane 1, molecular weight marker; lane 2, purified rPyCSP; lane 3, ECL-Western blot of rPyCSP using anti-PyCSP MAb NYS1 followed by goat anti-mouse alkaline phosphatase (AP)-conjugated antibody as described in Materials and Methods. rPyCSP was run in duplicate on the same gel and sliced into two portions for use in Coomassie blue staining or Western blot analysis.

FIG 2

IgG responses in C57BL/6 and genetic knockout (KO) mice immunized with rPyCSP in CpG ODN 1826 and Montanide ISA 51 as determined by ELISA. (A) Total IgG responses in wild-type (WT), unimmunized WT (WT control), CD4-KO, CD8-KO, IFN-γ-KO, and B-cell-KO mice (n = 10). The data were statistically tested by one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test (P = 0.01). (B) IgG responses categorized as values for protected mice versus nonprotected mice in WT, CD4-KO, and CD8-KO groups (n = 10) as determined by ELISA. (C) IgG isotype responses in WT, unimmunized WT (WT control), CD4-KO, CD8-KO, IFN-γ-KO, and B-cell-KO mice (n = 10) as determined by ELISA. The titers were log transformed and plotted as geometric means of log-transformed titers. The data were statistically tested by one-way ANOVA followed by Tukey’s multiple-comparison test. (D) IgG isotypes characterized as protected versus nonprotected mice in WT, CD4-KO and CD8-KO mouse groups as determined by ELISA. The titers were log transformed and plotted as geometric means of log-transformed titers. The ELISA values shown are calculated values determined as interpolated titers at an optical density at 405 nm of 0.5. All error bars show standard errors of the geometric mean values. Dots show the values for individual mice. *, P < 0.01; **, P < 0.001; ***, P < 0.0001; ****, 0.00001; ns, not significant.

FIG 3

Analysis of CD4⁺ and CD8⁺ T cells and their secreted cytokines in C57BL/6 mouse groups by flow cytometry. Groups (n = 6) comprised rPyCSP + adjuvant (prechallenge), naive (unimmunized), adjuvant alone, and protected mice. Protected mice were immunized with rPyCSP and challenged with Py17XNL sporozoites and did not develop blood stage infection. Splenocytes were isolated 14 days after sporozoite challenge, and the expression of IL-2, IFN-γ, IL-4, and TNF-α was measured in CD4⁺ TCRαβ⁺ cells (A) and CD8⁺ TCRαβ⁺ cells (B). Representative dot plots are shown. All error bars show standard deviations of the mean values. *, P < 0.01; **, P < 0.001; ***, P < 0.0001; ns, not significant by Student’s t test.

FIG 3

Analysis of CD4⁺ and CD8⁺ T cells and their secreted cytokines in C57BL/6 mouse groups by flow cytometry. Groups (n = 6) comprised rPyCSP + adjuvant (prechallenge), naive (unimmunized), adjuvant alone, and protected mice. Protected mice were immunized with rPyCSP and challenged with Py17XNL sporozoites and did not develop blood stage infection. Splenocytes were isolated 14 days after sporozoite challenge, and the expression of IL-2, IFN-γ, IL-4, and TNF-α was measured in CD4⁺ TCRαβ⁺ cells (A) and CD8⁺ TCRαβ⁺ cells (B). Representative dot plots are shown. All error bars show standard deviations of the mean values. *, P < 0.01; **, P < 0.001; ***, P < 0.0001; ns, not significant by Student’s t test.

FIG 4

Comparison of B cells and follicular B and follicular T-helper cells in C57BL/6 mouse groups by flow cytometry. Groups (n = 6) comprised rPyCSP + adjuvant (prechallenge), naive (unimmunized), adjuvant-alone, and protected mice. Protected mice were immunized with rPyCSP and challenged with Py17XNL sporozoites and did not develop blood stage infection. Splenocytes were isolated 14 days after sporozoite challenge. (A) Top, B cells were quantitated in live single splenocytes by gating on the CD19⁺ cell marker. Bottom, CD19⁺ B cells were gated on IgD and IgM to get IgM^lo IgD^hi, IgM^hi IgD^hi, and IgM^hi IgD^lo subpopulations. The IgM^lo IgD^hi population was further gated on CD38 and CD23 to quantitate follicular B cells in mice. (B) CD4⁺ T cells were gated on CXCR5 and PD-1 to quantitate follicular T-helper cells in mice. Representative dot plots are shown. All error bars show standard deviations of the mean values. *, P < 0.01; **, P < 0.001; ***, P < 0.0001 by Student’s t test.