A hybrid multistage protein vaccine induces protective immunity against murine malaria

Abstract

We have previously reported the design and expression of chimeric recombinant proteins as an effective platform to deliver malaria vaccines. The erythrocytic and exoerythrocytic protein chimeras described included autologous T helper epitopes genetically linked to defined B cell epitopes. Proof-of-principle studies using vaccine constructs based on the Plasmodium yoelii circumsporozoite protein (CSP) and P. yoelii merozoite surface protein-1 (MSP-1) showed encouraging results when tested individually in this mouse malaria model. To evaluate the potential synergistic or additive effect of combining these chimeric antigens, we constructed a synthetic gene encoding a hybrid protein that combined both polypeptides in a single immunogen. The multistage vaccine was expressed in soluble form in Escherichia coli at high yield. Here we report that the multistage protein induced robust immune responses to individual components, with no evidence of vaccine interference. Passive immunization using purified IgG from rabbits immunized with the hybrid protein conferred more robust protection against the experimental challenge with P. yoelii sporozoites than passive immunization with purified IgG from rabbits immunized with the individual proteins. High antibody titers and high frequencies of CD4(+)- and CD8(+)-specific cytokine-secreting T cells were elicited by vaccination. T cells were multifunctional and able to simultaneously produce interleukin-2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α). The mechanism of vaccine-induced protection involved neutralizing antibodies and effector CD4(+) T cells and resulted in the control of hyperparasitemia and protection against malarial anemia. These data support our strategy of using an array of autologous T helper epitopes to maximize the response to multistage malaria vaccines.

Fig 1

Expression and antigenic characterization of the P. yoelii LPC/RMC recombinant hybrid protein. (A) Sequence of the PyLPC/RMC protein. The amino acid sequence is shown in single-letter code. The broken underlined sequence shows the preerythrocytic PyLPC. The underlined sequence indicates the erythrocytic PyRMC. Sequences enclosed in open boxes are four putative promiscuous CD4⁺ T cell epitopes included in the original PyRMC (PyT8, PyT53, I₁₆₂₀-S₁₆₃₁, and I₁₆₄₂-L₁₆₅₅) (27). Sequences enclosed in dotted boxes are the P. falciparum and P. berghei tag sequences originally included at the C-terminal end of individual proteins as an alternative for protein purification. A carboxyl-terminal 6×His tag was included for protein purification. (B) SDS-PAGE (left) and Western blot (right) analysis of the purified PyLPC/RMC protein expressed in E. coli BL21(DE3). Left, Coomassie stain after SDS-PAGE separation of the purified PyLPC/RMC (lane 1), PyLPC (lane 2), and PyRMC (lane 3) proteins. Molecular weight markers (Bio-Rad) are indicated to the left. Right, Western blot analysis of the purified PyLPC/RMC protein incubated with serum samples from mice immunized with PyLPC (lane 4), monoclonal antibody 2A10 that recognizes the P. falciparum tag sequence (16) (lane 5), serum samples from mice immunized with a synthetic peptide representing the promiscuous T cell epitope PyT8 (lane 6), serum samples from mice immunized with PyT53 (lane 7), serum samples from mice immunized with PyMSP-1₁₉ (27) (lane 8), monoclonal antibody 3D11 that recognizes the P. berghei tag sequence (33) (lane 9), or an anti-6×His tag monoclonal antibody (lane 10). (C) Antigenicity of the P. yoelii chimeric protein determined by ELISA. Recombinant chimeras were tested using anti-PyLPC/RMC (▲), anti-PyLPC (□), anti-PyRMC (♢), 2A10 (), 3D11 (▽), or anti-6×His tag (●). Data are presented as the geometric mean OD obtained at different concentrations of the corresponding monoclonal antibodies (10 μg/ml to 0.00047 ng/ml) or the reciprocal of the serum dilution obtained from mice immunized with P. yoelii PyLPC/RMC, PyLPC, or PyRMC (dilutions 1:1,000 to 1:209,715,200). Numbers on the x axis indicate the dilutions tested (dilution number 1 was 10 μg/ml for monoclonal antibodies or 1:1,000 for polyclonal antibodies). Antigen specificity was confirmed using preimmune serum samples as a control (data not shown). (D) Immunofluorescence images of P. yoelii parasites (sporozoites and schizonts) obtained by incubation with anti-PyLPC/RMC antibodies showing the characteristic staining patterns of CSP and MSP-1.

Fig 2

(A) Top, kinetics of the antibody response to PyLPC/RMC in CAF1/J mice immunized with the hybrid chimeric protein (●) or the mixture of PyLPC and PyRMC (♢), determined by ELISA. Antibody titers were measured 20 days after each immunization. Horizontal bars represent arithmetic mean values for each group. Bottom, comparative antibody titers to individual chimeric proteins measured 20 days after the third immunization. Reciprocal endpoint ELISA titers were determined using the specified recombinant proteins and serum samples from mice immunized with PyLPC/RMC or the mixture of PyLPC and PyRMC and expressed as arithmetic mean values ± standard deviations (SD). (B) Effects of immunization with the hybrid PyLPC/RMC protein (●), the combination of PyLPC and PyRMC (♢), or placebo (▲) on the course of infection after experimental challenge with P. yoelii sporozoites (n = 10 mice). Top, summary of the course of parasitemias expressed as percentage of parasitemia (average ± SD). Bottom, impact of immunization on the hemoglobin concentration after challenge, expressed as percentage of reduction in hemoglobin concentration relative to baseline levels (average ± SD). *, P = 0.02 by t test; ****, P < 0.0001 by one-way ANOVA of AUC analysis with Bonferroni's multiple comparison posttest.

Fig 3

(A) Kinetics of the antibody response to PyLPC/RMC in CAF1/J mice immunized with different doses of the hybrid chimeric protein, determined by ELISA. Groups of 10 mice were immunized three times with 0.2 μg to 20 μg of PyLPC/RMC emulsified in Montanide ISA 51. Antibody titers were measured 20 days after each immunization and expressed as reciprocal geometric mean antibody titers ± standard deviations for mice immunized with 0.2 μg (●), 1 μg (♢), 10 μg (▲), or 20 μg (○). (B) Antibody avidity is expressed as avidity index measured as the concentration of ammonium thiocyanate required to dissociate 50% of bound antiprotein antibodies. Boxes of box-and-whisker plots summarize the medians and 25th and 75th percentiles, and whiskers indicate the upper and lower adjacent values from 10 mice. (C and D) Effects of immunization with different doses of PyLPC/RMC on the course of infection after experimental challenge with P. yoelii sporozoites. Summary of kinetics of parasitemia (C) and reduction in hemoglobin levels (D) in mice immunized with 0.2 μg (●), 1 μg (♢), 10 μg (▲), 20 μg (○), or placebo (■). Data are presented as the mean values expressed as percentage of parasitemia or the mean change in hemoglobin concentration per day determined by comparison with baseline concentrations and expressed as percentage of reduction in hemoglobin levels (n = 6). ***, P < 0.0001 by t test in comparison with results for 1, 10, or 20 μg/dose; *, P < 0.01 by Wilcoxon matched-pairs signed rank test. For panels C and D, the P values were calculated by one-way ANOVA of AUC analysis with Bonferroni's multiple comparison posttest: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 4

Effects of passive immunization on the course of parasitemia (A) or hemoglobin levels (B) after experimental challenge with P. yoelii sporozoites. Results are shown for groups of mice immunized as follows: purified IgG derived from rabbits immunized with PyLPC (♦); purified IgG derived from rabbits immunized with PyRMC (▽); purified IgG derived from rabbits immunized with PyLPC/RMC (●); mixture of IgG antibodies derived from PyLPC- or PyRMC-immunized rabbits (○); and purified IgG derived from normal rabbits (△). Data are the summary of the results of two independent experiments (n = 11 mice). Error bars show standard deviations. The arrows indicate days of passive immunization with purified rabbit antibodies. †, animals that were euthanized due to severe anemia; *, P < 0.05, **, P < 0.01, and ***, P < 0.001 by one-way ANOVA of AUC analysis with Bonferroni's multiple comparison posttest in comparison to mice that received purified IgG from rabbits immunized with PyLPC/RMC.

Fig 5

T cell responses following immunization with the hybrid PyLPC/RMC protein. Antigen-specific cytokine production was assessed at different time points after the final immunization. Groups of nine CAF1/J mice were immunized twice with 20 μg of PyLPC/RMC emulsified in Montanide ISA 51. The magnitudes and quality of PyLPC/RMC-specific CD4⁺ or CD8⁺ T cell responses were determined using flow cytometry multiparametric analysis. (A) Kinetics of the frequencies of CD4⁺ (left) or CD8⁺ (right) T cells producing IL-2, IFN-γ, or TNF-α upon stimulation with pools of synthetic peptides: PyLPC (closed bars), PyRMC pool 1 (hatched bars), and PyRMC pool 2 (open bars). Bars represent mean responses ± standard deviations for three mice per group. (B) To determine the proportions of multifunctional CD4⁺ or CD8⁺ T cells, Boolean gate analysis was used to identify and quantify the fraction of the total response of cells that produced three, two, or one cytokine in response to the corresponding antigen. The pie charts summarize the fractions of single, double, or triple producers for indicated groups in the experiment described for panel A.

Fig 6

Effects of CD4⁺ or CD8⁺ T cell depletion in CAF1/J mice immunized with PyLPC/RMC formulated in Montanide ISA 51. Mice were treated the day before challenge and again on day 2 using 500 μg of GK1.5 anti-CD4⁺ monoclonal antibody (♦), YTS169.4 anti-CD8⁺ monoclonal antibody (□), or a combination of both (○). Control mice were treated with normal rat IgG (▲). As a control for infection, placebo-immunized mice were also included (■). (A) Summary of the course of parasitemias expressed as percentage of parasitemia (average ± standard deviation [SD]). (B) Impact of immunization on the hemoglobin concentration after challenge, expressed as percentage of reduction in hemoglobin concentration relative to baseline levels (average ± SD). Data are representative of two independent experiments (n = 6 mice). The arrows indicate days of antibody treatment with specified monoclonal antibodies. †, animals that were euthanized due to severe anemia; **, P < 0.01, and *, P < 0.05 by one-way ANOVA of AUC analysis with Bonferroni's multiple comparison posttest in comparison to mice that received normal rat IgG.