Immunogenicity of a prime-boost vaccine containing the circumsporozoite proteins of Plasmodium vivax in rodents

Abstract

Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. In the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I·C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund's adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus-protein) vaccine regimens. The antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. The vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine.

FIG 1

Production and purification of P. vivax CSP recombinant proteins. (A) Schematic representation of the P. vivax CSP antigen and the sequences of the repeat regions present in each allelic form (PvCSP-VK210, PvCSP-VK247, or PvCSP-Vivax-like). (B) The predicted amino acid sequence of each recombinant protein. (C) Recombinant proteins were purified by affinity purification followed by anion-exchange chromatography and separated by SDS-PAGE under reducing conditions. Lanes: a, His₆-PvCSP-VK210; b, His₆-PvCSP-VK247; c, His₆-PvCSP-Vivax-like; d, His₆-PvCSP-All-CSP-epitopes. Each lane was loaded with 1 μg of protein.

FIG 2

Antibody recognition of P. vivax CSP recombinant proteins. ELISAs were performed with each of the four PvCSP recombinant (Rec.) proteins bound to the plates to determine whether the polypeptides retained the epitopes recognized by specific MAbs generated against radiation-attenuated P. vivax sporozoites. (A) MAb against the His tag. (B) MAb 2F2 specific to the allelic form PvCSP-VK210. (C) MAb 2E10.E9 specific to the allelic form PvCSP-VK247. (D) Polyclonal antibodies to the PvCSP-Vivax-like repeat region.

FIG 3

Induction of specific antibody responses in mice immunized with recombinant P. vivax CSP antigens. (A) Mice were immunized with three doses 21 days apart, and the antibody titers were analyzed according to the timeline shown. The serum IgG responses to P. vivax CS antigens were determined in C57BL/6 mice immunized s.c. with the purified proteins in a formulation containing the adjuvant poly(I·C) (B to D) or emulsified in CFA or IFA (E to G). Mice were immunized with His₆-PvCSP-VK210 (10 μg/dose/mouse), His₆-PvCSP-VK247 (10 μg/dose/mouse), His₆-PvCSP-Vivax-like (10 μg/dose/mouse), a protein mixture (His₆-PvCSP-VK210, His₆-PvCSP-VK247, and His₆-PvCSP-Vivax-like, 30 μg/dose/mouse), or His₆-PvCSP-All-CS-epitopes (30 μg/dose/mouse). Serum IgG titers of antibodies against recombinant proteins representing each allelic form of PvCSP were measured as indicated. The results are expressed as means ± SD (n = 6). Statistical comparisons by one-way ANOVA of the antibody titers from mice immunized with formulations containing the adjuvant poly(I·C) (B to D) show significantly higher antibody levels in animals immunized with the protein mixture (P < 0.01, asterisks). Comparisons of the antibody titers of mice immunized with formulations containing the adjuvant CFA-IFA and recombinant proteins did not show statistically significant differences.

FIG 4

Induction of specific antibody responses in mice immunized with recombinant P. vivax CSP antigens. C57BL/6 mice were immunized at 30 or 3 μg/dose/mouse with a formulation containing a protein mixture (His₆-PvCSP-VK210, His₆-PvCSP-VK247, and His₆-PvCSP-Vivax-like) in the presence of the adjuvant poly(I·C). The immunizations were s.c. with three doses given 21 days apart according to the timeline shown in Fig. 3A. Serum IgG titers of antibodies against recombinant proteins representing each allelic form of PvCSP were measured as indicated. Mice immunized with 30 (inverted triangles) and 3 (squares) μg/dose/mouse of the protein mixture responded equally well according to statistical comparisons (one-way ANOVA, no statistically significant difference). The results as expressed as means ± SD (n = 6).

FIG 5

Induction of specific antibody responses in tlr4^−/− mice immunized with recombinant P. vivax CS antigens. Mice were immunized with three doses 21 days apart according to the protocol and timeline shown in Fig. 3A. The serum IgG responses against P. vivax CSP recombinant proteins were determined in tlr4^+/+ and tlr4^−/− mice immunized with formulations containing poly(I·C) and a PvCSP mixture (His₆-PvCSP-VK210, His₆-PvCSP-VK247, and His₆-PvCSP-Vivax-like, 3 μg/dose/mouse) or control recombinant FliC (10 μg/dose/mouse). tlr4^+/+ (inverted triangles) and tlr4^−/− (diamonds) mice immunized with the PvCSP protein mixture responded well according to statistical comparisons (one-way ANOVA, no statistically significant difference). The results are expressed as means ± SD (n = 6).

FIG 6

Induction of specific antibody responses in mice immunized with recombinant adenoviruses expressing P. vivax CSP as part of a heterologous prime-boost regimen of vaccination. (A and B) Schematic representations and deduced amino acid (aa) sequences of the PvCSP-All-CSP-epitopes expressed by recombinant replication-defective simian or human adenovirus. (C) C57BL/6 mice were immunized i.m. at 2 × 10⁷ PFU/mouse with AdHu5-βgal (control), AdC68-PvCSP, or AdHu5-PvCSP. Serum titers of IgG against recombinant proteins of each allelic form of PvCSP were determined 21 days later as indicated. Mice immunized with AdC68-PvCS and AdHu5-PvCS responded equally well according to statistical comparisons (one-way ANOVA, no statistically significant difference [NS]). The results are expressed as means ± SD (n = 5). (D) Mice were boosted s.c. 42 days after being primed with a formulation containing a protein mixture (His₆-PvCSP-VK210, His₆-PvCSP-VK247, and His₆-PvCSP-Vivax-like, 3 μg/dose/mouse) in the presence of poly(I·C). The titers of specific antibodies to the different allelic forms of PvCSP were measured at day 63. According to statistical comparisons (one-way ANOVA, Tukey's HSD test), mice immunized with AdC68-PvCS and AdHu5-PvCS and boosted with a protein mixture (G5 and G6) had serum IgG antibody titers higher than those of the other groups, as shown.

FIG 7

Induction of specific antibody responses in mice immunized with a heterologous (adenovirus-protein) or a homologous (protein-protein) vaccination regimen. (A) C57BL/6 mice were immunized with three doses of the indicated immunogens, and the antibody titers were analyzed according to the timeline shown. The first dose consisted of 2 × 10⁷ PFU/mouse of AdHu5-βgal (control), AdC68-PvCSP, or AdHu5-PvCSP administered i.m. The first and second boosts consisted of a formulation containing a protein mixture (His₆-PvCSP-VK210, His₆-PvCSP-VK247, and His₆-PvCSP-Vivax-like, 3 μg/dose/mouse) in the presence of poly(I·C). (B) Serum IgG titers against His₆-PvCSP-VK210. (C) Serum IgG titers against His₆-PvCSP-VK247. (D) Serum IgG titers against His₆-PvCSP-Vivax-like. Mice immunized with AdHu5-βgal, AdC68-PvCS, and AdHu5-PvCS and boosted twice with a formulation containing a protein mixture in the presence of poly(I·C) (G4, G5, and G6) responded equally well according to statistical comparisons (one-way ANOVA, Tukey's HSD test, no statistically significant difference). The results are expressed as means ± SD (n = 5).

FIG 8

Specificity of the antibodies elicited by a homologous (protein-protein) or a heterologous (adenovirus-protein) immunization regimen. (A) Schematic representation of each recombinant protein used as a substrate bound to the plates in the ELISA. AA, amino acid; term, terminus. (B) Serum IgG antibodies from mice immunized with the homologous [protein-protein with poly(I·C)] or the heterologous (AdC68-PvCSP/protein mixture) regimen. The detailed immunization protocol is described in the legends to Fig. 3 (homologous) and 7 (heterologous). Sera were collected 2 weeks after the third dose was given. The results are expressed as means ± S.D (n = 5).

FIG 9

IIA for the recognition of the native protein PvCSP. Pools of sera from mice immunized with the homologous [protein-protein with poly(I·C)] and heterologous (AdC68-PvCSP/protein mixture) regimens were used. The detailed immunization protocol is described in the legends to Fig. 3 (homologous) and 7 (heterologous).

FIG 10

Cell-mediated immunity of mice immunized with recombinant P. vivax CSP antigens. (A) C57BL/6 mice were immunized with three doses given 14 days apart, and IC expression assays were performed according to the timeline shown. (B) Twenty-six overlapping peptides spanning the entire sequence of the N or C terminus of PvCSP were used to stimulate splenocytes. AA, amino acid. (C and D) G1 mice (control) were primed i.m. at 2 × 10⁸ PFU/mouse with AdHu5-b-gal and boosted s.c. twice with poly(I·C). G2 mice were primed i.m. at 2 × 10⁸ PFU/mouse with AdC68-PvCSP and boosted twice with a protein mixture (His₆-PvCSP-VK210, His₆-PvCSP-VK247, and His₆-PvCSP-Vivax-like, 30 μg/dose/mouse) in the presence of poly(I·C). G3 mice were primed and boosted with a protein mixture (His₆-PvCSP-VK210, His₆-PvCSP-VK247, and His₆-PvCSP-Vivax-like, 30 μg/dose/mouse). Fourteen days later, splenic cells of these mice were cultured in the presence of anti-CD28 antibody, monensin, and brefeldin A with or without the peptides or recombinant proteins or ConA as indicated. The PvCS-VK210, PvCS-VK247, and PvCS-Vivax-like peptides represent the repeat regions of the allelic variants. After 12 h, cells were stained with anti-CD4, anti-CD8, anti-IL-2, anti-IFN-γ, and anti-TNF-α antibodies. The results are expressed as the total frequency (%) of CD4⁺ or CD8⁺ cells stained for each molecule. Results were obtained from pooled cells from five mice.

FIG 11

Cell-mediated immunity of mice immunized with recombinant P. vivax CSP antigens. C57BL/6 mice were immunized as described in the legend to Fig. 10. Splenic cells of these mice were cultured in the presence or absence of the peptides or ConA, as indicated. The PvCS-VK210, PvCS-VK247, and PvCS-Vivax-like peptides represent the repeat regions of the allelic variants. After 48 h, IFN-γ-secreting cells (spot-forming cells [SFC]) were estimated by ELISPOT assay. The results are expressed as the frequency of SFC per 10⁶ spleen cells obtained from pooled cells of five mice. The asterisk denotes a statistically significant difference (P < 0.01).