Evaluation of Plasmodium vivax Cell-Traversal Protein for Ookinetes and Sporozoites as a Preerythrocytic P. vivax Vaccine

Abstract

Four different vaccine platforms, each targeting the human malaria parasite Plasmodium vivax cell-traversal protein for ookinetes and sporozoites (PvCelTOS), were generated and assessed for protective efficacy. These platforms consisted of a recombinant chimpanzee adenoviral vector 63 (ChAd63) expressing PvCelTOS (Ad), a recombinant modified vaccinia virus Ankara expressing PvCelTOS (MVA), PvCelTOS conjugated to bacteriophage Qβ virus-like particles (VLPs), and a recombinant PvCelTOS protein expressed in eukaryotic HEK293T cells (protein). Inbred BALB/c mice and outbred CD-1 mice were immunized using the following prime-boost regimens: Ad-MVA, Ad-VLPs, and Ad-protein. Protective efficacy against sporozoite challenge was assessed after immunization using a novel chimeric rodent Plasmodium berghei parasite (Pb-PvCelTOS). This chimeric parasite expresses P. vivax CelTOS in place of the endogenous P. berghei CelTOS and produces fully infectious sporozoites. A single Ad immunization in BALB/c and CD-1 mice induced anti-PvCelTOS antibodies which were boosted efficiently using MVA, VLP, or protein immunization. PvCelTOS-specific gamma interferon- and tumor necrosis factor alpha-producing CD8⁺ T cells were induced at high frequencies by all prime-boost regimens in BALB/c mice but not in CD-1 mice; in CD-1 mice, they were only marginally increased after boosting with MVA. Despite the induction of anti-PvCelTOS antibodies and PvCelTOS-specific CD8⁺ T-cell responses, only low levels of protective efficacy against challenge with Pb-PvCelTOS sporozoites were obtained using any immunization strategy. In BALB/c mice, no immunization regimens provided significant protection against a Pb-PvCelTOS chimeric sporozoite challenge. In CD-1 mice, modest protective efficacy against challenge with chimeric P. berghei sporozoites expressing either PvCelTOS or P. falciparum CelTOS was observed using the Ad-protein vaccination regimen.

Keywords: CelTOS; Plasmodium; malaria; preerythrocytic; vaccine; vivax.

FIG 1

Vaccination regimens and induction of antibody responses against P. vivax CelTOS in outbred CD-1 and inbred BALB/c mice. (A) Flowchart of the vaccination regimens used in this study. Three groups of 6 mice each were primed with the viral ChAd63 vector (Ad) expressing PvCelTOS (ChAd63-PvCelTOS). These groups were subsequently boosted with (i) the MVA viral vector expressing PvCelTOS (MVA-PvCelTOS), (ii) the PvCelTOS protein expressed in eukaryotic HEK293T cells (PvCelTOS), or (iii) the PvCelTOS protein conjugated to bacteriophage Qβ VLPs (VLP-PvCelTOS). Blood samples were collected at 14 days after the Ad prime and at day 63 after the boost. (B) Endpoint titer ELISA showing the total IgG antibody response against recombinant PvCelTOS protein in CD-1 mice after priming with Ad (day 14) or after boosting with MVA, protein, or VLPs (day 63), as shown in panel A. Means with standard errors of the means (SEMs) are shown. P values were determined by Tukey's multiple-comparison test. *, P < 0.05; ****, P < 0.0001. (C) Endpoint titer ELISA showing the total IgG antibody response against recombinant PvCelTOS protein in BALB/c mice after priming with Ad (day 14) or after boosting with MVA, protein, or VLPs (day 63), as shown in panel A. Means with SEMs are shown. P values were determined by Tukey's multiple-comparison test. **, P < 0.01; ****, P < 0.0001.

FIG 2

(A to F) Ex vivo production of IL-2, TNF-α, and IFN-γ by CD3⁺/CD8⁺ cells upon PvCelTOS stimulation of PBMCs obtained from immunized CD-1 and BALB/c mice. Samples were collected 1 week after boosting with MVA, protein, and VLPs, as shown in Fig. 1A, and PBMCs were analyzed with (+) or without (−) stimulation with a peptide pool of PvCelTOS (n = 3 for naive mice, n = 6 for the other groups). (A to C) Frequencies of CD3⁺/CD8⁺ cells in CD-1 mice producing IL-2 (A), TNF-α (B), and IFN-γ (C); (D to F) frequencies of CD3⁺/CD8⁺ cells in BALB/c mice producing IL-2 (D), TNF-α (E), and IFN-γ (F). MVA, group boosted with MVA-PvCelTOS; Protein, group boosted with PvCelTOS protein plus the Matrix-M adjuvant; VLP, group boosted with PvCelTOS coupled to VLPs plus the Matrix-M adjuvant. Means with SEMs are represented. P values were determined by one-way ANOVA followed by Tukey's multiple-comparison test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (G) Representative dot plots showing the production of the three cytokines in the gated CD3⁺/CD8⁺ population obtained from one representative BALB/c mouse vaccinated with a ChAd63 prime and a boost with MVA expressing PvCelTOS. PerCP, peridinin chlorophyll protein; Comp, compensated; A, area.

FIG 3

Ex vivo production of IL-2, TNF-α, and IFN-γ by CD3⁺/CD4⁺ cells upon PvCelTOS stimulation of PBMCs obtained from vaccinated CD-1 and BALB/c mice. Samples were collected 1 week after boosting with MVA, protein, and VLPs, as shown in Fig. 1A, and PBMCs were analyzed with (+) or without (−) stimulation with a peptide pool of PvCelTOS (n = 3 for naive mice, n = 6 for the other groups). (A to C) Frequencies of CD3⁺/CD4⁺ cells in CD-1 mice producing IL-2 (A), TNF-α (B), and IFN-γ (C); (D to F) frequencies of CD3⁺/CD8⁺ cells in BALB/c mice producing IL-2 (D), TNF-α (E), and IFN-γ (F). MVA, group boosted with MVA-PvCelTOS; Protein, group boosted with PvCelTOS protein plus Matrix-M; VLP, group boosted with PvCelTOS coupled to VLPs plus the Matrix-M adjuvant. Means with SEMs are represented. P values were determined by one-way ANOVA followed by Tukey's multiple-comparison test. *, P < 0.05.

FIG 4

Protective efficacy in BALB/c and CD-1 mice immunized with the different vaccine platforms and challenged with chimeric and wild-type P. berghei sporozoites. Three groups of 6 BALB/c mice each and nine groups of 10 CD-1 mice each were primed with the viral vector ChAd63 (Ad) expressing PvCelTOS (ChAd63-PvCelTOS). The groups were subsequently boosted with (i) the MVA viral vector expressing PvCelTOS (MVA-PvCelTOS), (ii) the PvCelTOS protein expressed in eukaryotic HEK293T cells (PvCelTOS), and (iii) the PvCelTOS protein conjugated to bacteriophage Qβ VLPs (VLP-PvCelTOS) (see the flowchart in Fig. 1A). All mice were challenged 66 days after the boost either with wild-type (WT) P. berghei sporozoites or with chimeric sporozoites expressing PvCelTOS or PfCelTOS (29). (A) Protective efficacy after challenge of immunized CD-1 mice with 1,000 sporozoites of the chimeric parasite line expressing P. vivax CelTOS. Protective efficacy was significantly higher for mice immunized with Ad-protein (P = 0.0002) and Ad-VLPs (P = 0.0001) than for the naive group (log-rank Mantel-Cox test for comparison of survival curves). (B) Protective efficacy after challenge of immunized BALB/c mice with 1,000 sporozoites of the chimeric parasite line expressing P. vivax CelTOS. (C) Protective efficacy after challenge of immunized CD-1 mice with 1,000 sporozoites of the chimeric parasite line expressing P. falciparum CelTOS (29). Protective efficacy was significantly higher for mice immunized with Ad-protein (P = 0.0488) than for the naive group (log-rank Mantel-Cox test for comparison of survival curves). (D) Protective efficacy after challenge of immunized CD-1 mice with 1,000 sporozoites of wild-type P. berghei.