Antigenicity and immunogenicity of Plasmodium vivax merozoite surface protein-3

Abstract

A recent clinical trial in African children demonstrated the potential utility of merozoite surface protein (MSP)-3 as a vaccine against Plasmodium falciparum malaria. The present study evaluated the use of Plasmodium vivax MSP-3 (PvMSP-3) as a target antigen in vaccine formulations against malaria caused by P. vivax. Recombinant proteins representing MSP-3α and MSP-3β of P. vivax were expressed as soluble histidine-tagged bacterial fusions. Antigenicity during natural infection was evaluated by detecting specific antibodies using sera from individuals living in endemic areas of Brazil. A large proportion of infected individuals presented IgG antibodies to PvMSP-3α (68.2%) and at least 1 recombinant protein representing PvMSP-3β (79.1%). In spite of the large responder frequency, reactivity to both antigens was significantly lower than was observed for the immunodominant epitope present on the 19-kDa C-terminal region of PvMSP-1. Immunogenicity of the recombinant proteins was studied in mice in the absence or presence of different adjuvant formulations. PvMSP-3β, but not PvMSP-3α, induced a TLR4-independent humoral immune response in the absence of any adjuvant formulation. The immunogenicity of the recombinant antigens were also tested in formulations containing different adjuvants (Alum, Salmonella enterica flagellin, CpG, Quil A,TiterMax® and incomplete Freunds adjuvant) and combinations of two adjuvants (Alum plus flagellin, and CpG plus flagellin). Recombinant PvMSP-3α and PvMSP-3β elicited higher antibody titers capable of recognizing P. vivax-infected erythrocytes harvested from malaria patients. Our results confirm that P. vivax MSP-3 antigens are immunogenic during natural infection, and the corresponding recombinant proteins may be useful in elucidating their vaccine potential.

Figure 1. Human antibody response to recombinant PvMSP-3α, PvMSP-3β, and PvMSP1₁₉ proteins during patent P. vivax infection.

A) The bars express the percent response for each of the analyzed proteins. Sera from 220 individuals were analyzed for the presence of specific IgG antibodies by ELISA and tested at a 1∶100 dilution in duplicate. The cutoff proteins obtained from the PvMSP-3α, PvMSP-3β (FP-1), PvMSP-3β (FP-2), PvMSP-3β (FP-3), and PvMSP1₁₉ were 0.179, 0.715, 0.195, 0.396, and 0.185, respectively. B) Comparison of individual IR IgG antibodies to MSPs in sera of individuals infected by P. vivax. The line indicates the limit of positivity (IR = 1). IR: Index of reactivity (mean absorbance of test serum/cutoff). C) Comparative analysis of the IgG antibody response against MSP proteins and the frequency of previous episodes of vivax malaria. We analyzed 213 serum samples from individuals who reported the number of previous episodes of malaria for the presence of specific IgG antibodies by ELISA. All sera were tested in duplicate at 1∶100 dilution. *: the percentage of responders with statistically significant correlation to the frequency of previous malaria episodes.

Figure 2. Reactivity against recombinant PvMSP-3 and PvMSP1₁₉ proteins in 220 sera from individuals with patent P. vivax malaria infection.

Each panel represents the reactivity index of serum samples against the indicated recombinant proteins. The serum samples were tested at a 1∶100 dilution, as described in Figure 1B. Symbols represent the IR IgG antibodies against recombinant MSP proteins in the sera of P. vivax-infected individuals. The values of the Spearman correlation coefficient (r) and p values are shown in each panel.

Figure 3. IgG antibody response in C57BL/6 wild-type (WT) and TLR4 KO mice after immunization with MSP-3 in the absence of adjuvant.

Groups of 5 mice were immunized 3 times (s.c.) with 10 µg of PvMSP-3α, PvMSP-3β (FP-1), PvMSP-3β (FP-2), or PvMSP-3β (FP-3) and antibody titers to homologous PvMSP-3 were determined after each dose. Results are expressed as the means of antibody titers (log₁₀) ± SEM and were compared by one-way ANOVA followed by Tukey’s test for multiple comparisons. After the third dose, non-significant differences between groups of immunized mice (C57BL/6 WT vs. TKR4 KO) are denoted on the graph as “n.s.” Significant difference between 2 groups of mice immunized with 3 doses of PvMSP-3β (FP-2) are denoted on the graph (*p<0.05).

Figure 4. IgG anti-PvMSP-3α in mice immunized with various adjuvant formulations.

Groups of 6 female BALB/c mice were immunized 3 times (s.c.) with 10 µg of protein in the following adjuvant formulations: Alum, FliC, CpG ODN 1826, Quil A, TiterMax, or IFA. Anti-PvMSP-3α in the sera of immunized mice was analyzed by ELISA 2 weeks after the first (A), second (B), and third (C) immunizing dose. Results are expressed as mean IgG antibody titers (log₁₀) ± SEM and were compared by one-way ANOVA followed by Tukey’s test for multiple comparisons. Significant differences are noted on the graph: *p<0.05; **p<0.01; ***p<0.001. Non-significant (n.s.) differences are indicated (p>0.05). Data representative of 2 independent experiments.

Figure 5. IgG anti-PvMSP-3β and IgG subclass profiles in mice immunized in the presence of adjuvant.

Groups of 6 females BALB/c were immunized 3 times (s.c.) with 10 µg of protein in the presence of the following adjuvant formulations: Alum, FliC, CpG ODN 1826, Quil A, TiterMax, or IFA. The adjuvants Alum, FliC, and CpG ODN 1826 were also tested in combination (Alum plus CpG ODN 1826 and FliC+CpG ODN 1826). Anti-PvMSP-3β in the sera of immunized mice was analyzed by ELISA 2 weeks after the first (A), second (B), and third (C) doses. Results are expressed as mean IgG antibody titers (log₁₀) ± SEM and were compared by one-way ANOVA followed by Tukey’s test for multiple comparisons. Significant differences are noted on the graph: *p<0.05; **p<0.01; ***p<0.001. Non-significant (n.s.) differences are indicated (p>0.05). Data representative of 2 independent experiments.

Figure 6. Serum IgG isotype responses in mice after immunization with PvMSP-3β in the presence of adjuvant.

BALB/c mice were immunized with recombinant PvMSP-3β in the presence of adjuvant as described in Figure 5. PvMSP-3β-specific IgG1 and IgG2a antibody titers in the sera of immunized mice were analyzed by ELISA 2 weeks after the third dose. Results are the means of IgG1/IgG2a ± SEM for 6 mice per group. All groups were compared by one-way ANOVA and Tukey’s test for multiple comparisons.

Figure 7. Immunofluorescence of P. vivax parasites and anti-MSP-3 antibodies.

Immunofluorescence patterns in the sera of mice immunized with recombinant PvMSP-3α and PvMSP-3β (FP-3) on acetone-fixed P. vivax-infected erythrocytes (Pv-iE). The smears were incubated with pooled antisera (1∶100) from mice immunized with: (A) PBS emulsified in adjuvant, (B) PvMSP-3α, or (C) PvMSP-3β in Freund’s Adjuvant. Antibody binding was detected with secondary Alexa 568-labeled antibody (red) and nuclei were visualized by DAPI staining (blue). BF, bright field.