Characterization of T and B cell epitopes in PvCyRPA by studying the naturally acquired immune response in Brazilian Amazon communities

Abstract

Plasmodium vivax, a challenging species to eliminate, causes millions of malaria cases globally annually. Developing an effective vaccine is crucial in the fight against vivax malaria, but considering the limited number of studies focusing on the identification and development of P. vivax-specific vaccine candidates, exploring new antigens is an urgent need. The merozoite protein CyRPA is essential for P. falciparum growth and erythrocyte invasion and corresponds to a promising candidate antigen. In P. vivax, a single study with multiple vaccine candidates indicates PvCyRPA with strong association with protection, outperforming classic malaria vaccine candidates. However, little is known about the specific naturally acquired response in the Americas, as well as the antigen epitope mapping. For this reason, we aimed to investigate the cellular and humoral immune response elicited against PvCyRPA in Brazilian endemic areas to identify the existence of immunodominant regions and the potential of this protein as a single or even a multi-stage specific malaria vaccine candidate for P. vivax. The results demonstrated that PvCyRPA is naturally immunogenic in Brazilian Amazon individuals previously exposed to malaria, which presented anti-PvCyRPA cytophilic antibodies. Moreover, our data show that the protein also possesses important immunogenic regions with an overlap of B and T cell epitopes. These data reinforce the possibility of including PvCyRPA in vaccine formulations for P. vivax.

Fig. 1

(a) Frequency of IgM and IgG antibodies responders. Bars indicate the percentage of IgM responders (light gray bar), IgG responders (dark gray bar), and both combined (black bar). **** = p < 0.0001. (b) Reactivity of IgM and IgG antibodies. RI’s of the entire population for both IgM and IgG are demonstrated in this graph. The red dotted line separates responders from non-responders, values of RI’s above 1 are considered IgM (light gray) and/or IgG (dark gray) responders. (p < 0.0001 = ****). (c) Frequency of IgM responders per group. Bars indicate the percentage of IgM responders in each group: P. vivax (pink bar), cohabitants (blue bar) and exposed (orange bar). P values are 0.0015 for ** and 0.0004 for ***. (d) Reactivity of IgM antibodies per group. RI’s of each group for IgM antibodies against PvCyRPA is demonstrated in this graph. P vivax group is represented with a pink bottle, the cohabitants group with a blue bottle, and the exposed one with an orange bottle. P values are 0.0010 for *** - P. vivax x exposed and 0.0001 for *** - P. vivax x cohabitants. (e) Frequency of IgG responders per group. Bars indicate the percentage of IgG responders to in each group: P. vivax (pink bar), cohabitants (blue bar) and exposed (orange bar). (f) Reactivity of IgG antibodies per group. RI’s of each group for IgM antibodies against PvCyRPA is demonstrated in this graph. P vivax group is represented with a pink bottle, the cohabitants group with a blue bottle, and the exposed one with an orange bottle. The red dotted line separates responders from non-responders, values of RI’s above 1 are considered responders. RI’s analyzes were done by Kruskal-Wallis test and Dunn’s multiple comparison test, while frequencies were compared through Fisher’s exact test.

Fig. 2

Reactivity of IgG subclasses. RI’s of 90 malaria-exposed individuals for IgG subclasses IgG1, IgG2, IgG3, and IgG4 are shown in this graph. Each dot represents the RI of a patient. The red dotted line separates responders from non-responders, values of RI’s above 1 are considered responders to each of the subclasses. P < 0.0001=****. RI’s analyzes were done by Kruskal-Wallis test and Dunn’s multiple comparison test.

Fig. 3

Location of predicted epitopes in PvCyRPA 3D structure. The protein chain is indicated by a gray and transparent (40%) surface. The locations of epitopes B-PvCyRPA_(81−91), B-PvCyRPA_(119−129), B-PvCyRPA_(134−151), B-PvCyRPA_(181−192), B-PvCyRPA_(241−257), B-PvCyRPA_(257−272), and B-PvCyRPA_(312−330) were indicated by licorice sticks green, blue, red, cyan, magenta, orange, and yellow, respectively. Different rotations of the protein are shown in (A) and (B).

Fig. 4

(a) Frequency of IgG responders to different PvCyRPA B cell epitopes. The percentage of IgG antibody responders to each one of the 7 predicted; (b) Reactivity of IgG antibodies against PvCyRPA B cell epitopes. RI’s of 51 individuals that respond with IgG antibodies are demonstrated in this graph for each of the 7 predicted B cell epitopes. The red dotted line separates responders from non-responders, values of RI’s above 1 are considered responders to each one of the B cell epitopes. B1 x B2 (p = 0.0084**), B1 x B3 (p < 0.0001****), B1 x B6 (0.0004***), B7 x B2 (p = 0.0248*), B7 x B3 (p = 0.0001***), B7 x B6 (p = 0.0115) and B4 x B3 (p = 0.0115*). RI’s analyzes were done by Kruskal-Wallis test and Dunn’s multiple comparison test, while frequencies were compared through Fisher’s exact test.

Fig. 5

Heat map with RI’s of IgG antibodies against B cell epitopes. RI’s of 51 individuals that respond to IgG antibodies are demonstrated in this heat map for each of the 7 predicted B cell epitopes. Each line from 1 to 51 represents one of the tested individuals and different epitopes that were recognized by them. The gradient color bar indicates different values of RI’s.

Fig. 6

Numbers of Spots forming units of IFN-γ in the population and per group. The graph demonstrates the numbers of adjusted SFU of IFN-γ for each of the 6 tested pools using the entire population and each studied group. The dotted red line represents the mark of 20 SFU, values above this line indicate individuals capable of producing IFN-γ against the respective pool. The infected group is represented in pink, the exposed with previous malaria infections in blue, and the exposed without previous malaria infections in orange. The red dotted line separates responders from non-responders and represents the mark of adjusted 20 SFU. Analyses using the number o SFU were done by Kruskal-Wallis test and Dunn’s multiple comparison test.

Fig. 7

Correlation matrix of cellular and humoral immune response against PvCyRPA. Results were obtained by Spearman correlation test. Numbers correspond to r value of each correlation. P ≤ 0,05 = * and p ≤ 0,01= **.