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. 2026 Jan 28:16:1710869.
doi: 10.3389/fimmu.2025.1710869. eCollection 2025.

B-cell epitope mapping and characterization of antibody responses to recombinant PvRipr in malaria-exposed individuals

Isabela Ferreira Soares  1 , Cinthia Magalhães Rodolphi  1 , Ana Luiza Carneiro Alencar  1 , Ada da Silva Matos  1 , Rodrigo Nunes Rodrigues-da-Silva  2 , Barbara de Oliveira Baptista  3 , Rodrigo Medeiros Martorano  4 , Hugo Amorim Dos Santos de Souza  3 , Evelyn Kety Pratt Riccio  3 , Jenifer Peixoto de Barros  3 , Paulo Renato Rivas Totino  3 , Cláudio Tadeu Daniel-Ribeiro  3   5 , Lilian Rose Pratt-Riccio  3 , Josué da Costa Lima-Junior  1
Affiliations

B-cell epitope mapping and characterization of antibody responses to recombinant PvRipr in malaria-exposed individuals

Isabela Ferreira Soares et al. Front Immunol. .

Abstract

Introduction: Malaria caused by P. vivax continues to be a serious public health problem, especially in countries like Brazil where P. vivax accounts for more than 80% of diagnosed cases. Since this plasmodial species is characterized as one of the most difficult to eliminate, the development of a specific vaccine for P. vivax may be an essential tool for effective control of the disease. The protein Ripr has been described in P. falciparum as an essential part of the erythrocyte invasion complex. Given the limited number of P. vivax vaccine antigens currently under investigation, this study aimed to characterize, the naturally acquired humoral immune response to Ripr protein of P. vivax.

Methods: ELISA assays were performed using plasma samples from individuals naturally exposed to malaria in the Brazilian Amazon in order to determine levels of IgM, IgG and IgG subclasses against PvRipr. In addition, linear B-cell epitopes within the protein were identified.

Results and discussion: Our results demonstrated that PvRipr is naturally immunogenic, as more than 60% of the individuals presented IgM or IgG antibodies against recombinant PvRipr. The profile of IgG subclasses was also investigated and higher frequencies of seropositive individuals for IgG1 and IgG2 were observed. After in silico prediction, a total of four linear B cell epitopes were identified in PvRipr, from these sequences, B-PvRipr(879-888) and B-PvRipr(923-958) had higher frequencies of seropositive individuals and reactivity indexes in comparison to the other tested epitopes. Moreover, levels of IgG antibodies specific for these two epitopes were strongly correlated with the levels of IgG antibodies against recombinant PvRipr and especially with IgG3 antibodies, a cytophilic subclass widely cited in the protective immune response against malaria.

Keywords: B-cell epitopes; P. vivax; Ripr; malaria; vaccine candidate.

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Conflict of interest statement

The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
PvRipr expression and purification results. (A) Western blot result. Anti-His detection and ECL revelation. M: Protein Marker and 3: induced sample. (B) SDS-PAGE result of affinity chromatography showing the first results of expression and purification. M: Protein Marker; 1: Precipitate; 2: Supernatant; 3: Flow through; 4-5: Wash; 6: First Elution. (C) Final product: SDS-PAGE of the final protein expression after subsequent elutions and new dialysis step. M: Protein Marker; S: Final Sample.
Figure 2
Figure 2
Frequencies of IgM and IgG seropositive individuals for recombinant PvRipr. The p values are <0.0001 for **** and 0.0049 for *. SN = seronegative individuals. (B) RIs of IgM and IgG antibodies against recombinant PvRipr. Triangles represent RIs of each individual for IgM antibodies and circles for IgG antibodies. The p value is <0.0001 (****). Asterisks above bars that are not connected by capped lines indicate that the p value applies to all groups.
Figure 3
Figure 3
(A) Frequencies of seropositive individuals for IgG subclasses against recombinant PvRipr. The p values are IgG1 vs IgG3 – p=0.0245 (*), IgG2 vs IgG3 – p=0.0175 (*), p=0.0037 (**) and p<0.0001 (****) (B) RIs of IgG subclasses against recombinant PvRipr. The p values are p=0.0403 (*), p=0.0036 (**) and p<0.0001 (****). Asterisks above bars that are not connected by capped lines indicate that the p value applies to all groups.
Figure 4
Figure 4
Location of predicted B cell epitopes in PvRipr 3D structure. The protein chain is indicated by a gray and transparent (40%) surface. Epitopes B-PvRipr(698-725), B-PvRipr(751-771), B-PvRipr(879-888), and B-PvRipr(923-958) are highlighted in yellow, green, pink and blue, respectively. (A) and (B) show different rotations of the protein.
Figure 5
Figure 5
(A) Frequencies of seropositive individuals for IgG antibodies specific for B cell epitopes of PvRipr. The p values are p=0.0004 (***) and p<0.0001 (****), (B) RIs of IgG antibodies against B cell epitopes of PvRipr. The p values are p=0.0046 (**), p=0.0002 (***) and p<0.0001 (****) and (C) Different epitope response combinations identified in the studied population. Colored circles under n values indicate that the number of samples described is capable of recognizing epitopes of each respective color, as demonstrated in the colored rectangles.
Figure 6
Figure 6
Correlation matrix of the humoral immune response against recombinant PvRipr and its B cell epitopes. Inside each box is the r value referring to the correlation between the antibodies that form the intersection. Asterisks were placed in boxes with significant p-values (* = p<0.05 / ** = p < 0.01 / *** = p < 0.001 and **** = p < 0.0001.
Figure 7
Figure 7
RIs of IgG antibodies against recombinant PvRipr pre and post-depletion of epitope-specific antibodies. Each grey circle represents the RI of an individual sample tested against recombinant PvRipr (A) RIs of 70 individuals seropositive for both PvRipr and B-PvRipr(879-888). (B) RIs of 37 individuals seropositive for both PvRipr and B-PvRipr(923-958). In both graphs **** indicates p < 0.0001.
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