Vaccine Containing the Three Allelic Variants of the Plasmodium vivax Circumsporozoite Antigen Induces Protection in Mice after Challenge with a Transgenic Rodent Malaria Parasite

Abstract

Plasmodium vivax is the most common species that cause malaria outside of the African continent. The development of an efficacious vaccine would contribute greatly to control malaria. Recently, using bacterial and adenoviral recombinant proteins based on the P. vivax circumsporozoite protein (CSP), we demonstrated the possibility of eliciting strong antibody-mediated immune responses to each of the three allelic forms of P. vivax CSP (PvCSP). In the present study, recombinant proteins representing the PvCSP alleles (VK210, VK247, and P. vivax-like), as well as a hybrid polypeptide, named PvCSP-All epitopes, were generated. This hybrid containing the conserved C-terminal of the PvCSP and the three variant repeat domains in tandem were successfully produced in the yeast Pichia pastoris. After purification and biochemical characterization, they were used for the experimental immunization of C57BL/6 mice in a vaccine formulation containing the adjuvant Poly(I:C). Immunization with a recombinant protein expressing all three different allelic forms in fusion elicited high IgG antibody titers reacting with all three different allelic variants of PvCSP. The antibodies targeted both the C-terminal and repeat domains of PvCSP and recognized the native protein on the surface of P. vivax sporozoites. More importantly, mice that received the vaccine formulation were protected after challenge with chimeric Plasmodium berghei sporozoites expressing CSP repeats of P. vivax sporozoites (Pb/PvVK210). Our results suggest that it is possible to elicit protective immunity against one of the most common PvCSP alleles using soluble recombinant proteins expressed by P. pastoris. These recombinant proteins are promising candidates for clinical trials aiming to develop a multiallele vaccine against P. vivax malaria.

Keywords: Plasmodium vivax; circumsporozoite protein; malaria; prime-boost regimens; recombinant vaccine.

Figure 1

Schematic representation of the Plasmodium vivax circumsporozoite protein (CSP) recombinant antigens. The individual recombinant proteins have the specific individual repeats [yPvCSP-VK210 (n = 19), yPvCSP-VK247 (n = 19), or yPvCSP-P. vivax-like (n = 16)] and the C-terminal (CT) region. For each repeat sequence, the amino acids positions and the histidine tag are indicated. The classic repeat sequence VK210 is represented in yellow, VK247 in green, and P. vivax-like in blue. The yPvCSP-All epitopes merges the repeats of all three allelic variants describe above (n = 6/6/5 repeat sequences of VK210/P. vivax-like/VK247, respectively) and the CT region.

Figure 2

Purification and biochemical characterization of the Plasmodium vivax circumsporozoite protein (CSP) recombinant proteins. (A) 13% SDS-PAGE under reduced conditions stained with Coomassie blue. Proteins migrate between 50 and 55 kDa. Fractions were 2 µg of: (1) yPvCSP-VK210, (2) yPvCSP-VK247, (3) yPvCSP-P. vivax-like, and (4) yPvCSP-All epitopes. (B) Western blot using 1 µg of the same fractions of proteins described above. Antibodies used were monoclonal antibody (MAb) anti-His, MAb VK210 (2F2), and MAb VK247 (2E10.E9). The secondary antibody used was anti-mouse IgG HRP-labeled, and detection was performed by ECL assay. (C) The purity of proteins, after the combination of chromatographic methods, was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), in which the gradient elution was developed combining 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 90% acetonitrile, 24°C, 1 mL/min for 40 min, in a C18 column.

Figure 3

Specific antibody response in mice immunized with Plasmodium vivax circumsporozoite protein (PvCSP) recombinants. (A) C57BL/6 mice were immunized s.c. with three doses 28 days apart with the purified proteins in the presence of the adjuvant Poly(I:C), whereby the antibody response was analyzed according to the timeline described. (B) The IgG antibody serum titers against recombinant proteins representing each allelic form of PvCSP were analyzed by ELISA. The results are expressed as the means ± SD (n = 6). Mouse groups were immunized with: (1) Poly(I:C); (2) Poly(I:C) plus yPvCSP-VK210 (1 μg/dose/mouse); (3) Poly(I:C) plus yPvCSP-VK247 (1 μg/dose/mouse); (4) Poly(I:C) plus yPvCSP-P. vivax-like (1 μg/dose/mouse); (5) Poly(I:C) plus a protein mix [yPvCSP-VK210 (1 μg/dose/mouse) + yPvCSP-VK247 (1 μg/dose/mouse) + yPvCSP-P. vivax-like (1 μg/dose/mouse)]; or (6) Poly(I:C) plus yPvCSP-All epitopes (3 μg/dose/mouse). (C) IgG isotypes were determined by ELISA in sera of mice from groups 5 and 6 described above. Asterisk denotes statistical differences (P < 0.01) when comparing IgG1 isotype with IgG2b, IgG2c and IgG3.

Figure 4

Specificity of IgG antibody in mice immunized with Plasmodium vivax circumsporozoite protein (CSP) recombinants. (A) Schematic representation of recombinant proteins used as a substrate bound to the plates in the ELISA assay. (B) C57BL/6 mice were immunized s.c. with the purified proteins in the presence of the adjuvant Poly(I:C) according to the timeline described in Figure 3A. The specificity of IgG antibody serum titers toward rFliC, FliC-PvCSP-repeats and No repeats proteins were analyzed by ELISA in sera of mice from groups 2 to 6 described in Figure 3B. The results are expressed as mean ± SD (n = 6 animals/group). P values are indicated. Differences were considered statistically significant when P < 0.05.

Figure 5

Recognition of native protein in Plasmodium vivax isolates. Indirect immunofluorescence analysis was performed using pool of sera (diluted 1:100) from C57BL/6 mice immunized with (A) yPvCSP-All epitopes, (B) protein mixture or (C) phosphate-buffered saline (PBS) in adjuvant, as a negative control. (D) As a control positive anti-PvCSP-VK210 (MAb 2F2) was used. Microscope slides containing wild-type P. vivax were obtained from Thailand isolates. Antibody binding was detected with secondary Alexa 568-labeled antibody (red) and nuclei were visualized by DAPI staining.

Figure 6

Interferon gamma (IFN-γ)-producing cells in mice immunized with recombinant proteins. (A) C57BL/6 mice were immunized following the scheme with yPvCSP-All epitopes (3 μg/mouse) or a mix of yPvCSP-VK210 + yPvCSP-VK247 + yPvCSP-P. vivax-like (1 μg/each protein/mouse), in the presence of the adjuvant Poly(I:C). (B) To measure the number of cells secreting IFN-γ, splenocytes collected from immunized C57BL/6 mice on day 40 after priming were used for enzyme-linked immunospot (ELISPOT) assays. The recombinant proteins yPvCSP-VK210 (yVK210), yPvCSP-VK247 (yVK247), yPvCSP-P. vivax-like (yVL), and yPvCS-All epitopes (yAll epitopes) were used as stimuli (10 µg/mL). SFC, spot forming cells. Asterisk denotes statistical differences (P < 0.05).

Figure 7

CD4⁺ and CD8⁺ T cell-mediated immunity of mice immunized with recombinant proteins. C57BL/6 mice were immunized s.c. with the recombinant proteins following the scheme described in Figure 6A. To determine the intracellular expression of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), intracellular cytokine staining (ICS) was performed following the in vitro culture of splenocytes in the presence or absence of the antigenic stimuli yPvCSP-VK210 (yVK210), yPvCSP-VK247 (yVK247), and yPvCSP-P. vivax-like (yVL). (A) Frequency of CD4⁺ cytokine-producing cells. (B) Frequency of CD8⁺ cytokine-producing cells. Asterisks denote statistically significant (P < 0.05) higher frequencies of cells from mice immunized with the recombinant proteins when compared to cells from mice injected with adjuvant only. Media backgrounds of all samples were subtracted before plotting.

Figure 8

Protection against experimental challenge in mice immunized with a prime-boost regimen. (A) C57BL/6 mice were immunized i.p. with Poly(I:C) or a mixture of Poly(I:C) plus 5 µg of yPvCSP-All epitopes following the scheme described. On day 67 after priming, mice were challenged with 4,000 Pb/PvVK210 transgenic sporozoites. (B) Parasitemia was analyzed by Giemsa-stained blood smears on the following 10 days. Percentage of non-infected mice in each experimental group are shown. (C) Log of parasitemia at day 5 (D5) postchallenge measured in mice immunized with Poly(I:C) (n = 4) or a mixture of Poly(I:C) plus 5 µg of yPvCSP-All epitopes (n = 6). (**) P = 0.0095. (D) The liver load was evaluated by RT-PCR. The infection level in liver in mice immunized with a mixture of Poly(I:C) plus 5 µg of yPvCSP-All epitopes (n = 5) is compared with control group that received Poly(I:C) alone (n = 4). (*) P = 0.0159. C-terminal (CT) differences (delta CT) between the mouse HPRT and Pb 18S CT values are shown.