Dynamics of IgM and IgG Antibody Response Profile against Linear B-Cell Epitopes from Exoerythrocytic (CelTOS and TRAP) and Erythrocytic (CyRPA) Phases of Plasmodium vivax: Follow-Up Study

Abstract

Malaria is a serious health problem worldwide affecting mainly children and socially vulnerable people. The biological particularities of P. vivax, such as the ability to generate dormant liver stages, the rapid maturation of gametocytes, and the emergence of drug resistance, have contributed to difficulties in disease control. In this context, developing an effective vaccine has been considered a fundamental tool for the efficient control and/or elimination of vivax malaria. Although recombinant proteins have been the main strategy used in designing vaccine prototypes, synthetic immunogenic peptides have emerged as a viable alternative for this purpose. Considering, therefore, that in the Brazilian endemic population, little is known about the profile of the humoral immune response directed to synthetic peptides that represent different P. vivax proteins, the present work aimed to map the epitope-specific antibodies' profiles to synthetic peptides representing the linear portions of the ookinete and sporozoite cell passage protein (CelTOS), thrombospondin-related adhesive protein (TRAP), and cysteine-rich protective antigen (CyRPA) proteins in the acute (AC) and convalescent phases (Conv30 and Conv180 after infection) of vivax malaria. The results showed that the studied subjects responded to all proteins for at least six months following infection. For IgM, a few individuals (3-21%) were positive during the acute phase of the disease; the highest frequencies were observed for IgG (28-57%). Regarding the subclasses, IgG2 and IgG3 stood out as the most prevalent for all peptides. During the follow-up, the stability of IgG was observed for all peptides. Only one significant positive correlation was observed between IgM and exposure time. We conclude that for all the peptides, the immunodominant epitopes are recognized in the exposed population, with similar frequency and magnitude. However, if the antibodies detected in this study are potential protectors, this needs to be investigated.

Keywords: PvCelTOS; PvCyRPA; PvTRAP; humoral immune response; malaria.

Figure A1

Primary infected individuals and more than 1 previous malaria individuals of (A) PvCelTOS^(I133-G147), (B) PvTRAP^(P344-G374), and (C) PvCyRPA^(T289-G307) in the acute phase of P. vivax for IgM. The dashed line represents the cut-off that separates responders from non-responders. The horizontal line in the scatter plots represents the median RI value. Each sample was tested in duplicate by ELISA. Statistical significance of the RI observed at different points for each antigen was assessed using the Kruskal Wallis test with Mann-Whitney post-test for multiple comparisons (p < 0.05). The bars represent statistical differences between the groups. Ac: n = 56.

Figure 1

Profile of the antibody response (frequency of responder and reactivity index) against (A) PvCelTOS^(I133-G147), (B) PvTRAP^(P344-G374), and (C) PvCyRPA^(T289-G307) in the acute phase (AC) of P. vivax infection and cohabitant (CH) individuals. The dashed line represents the cut-off that separates responders from non-responders. The horizontal line in the scatter plots represents the median reactivity index (RI) value. Each sample was tested in duplicate by ELISA. Statistical significance of the frequency of responders (blue bars) at different points for each antigen was assessed using the chi-square test with Fischer’s post-test for multiple comparisons. Statistical significance of the RI (black bars) observed at different points for each antigen was assessed using the Kruskal–Wallis test with Mann–Whitney post-test for multiple comparisons (p < 0.05). Asterisks represent p value summary, * p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0001. AC (acute), CH (cohabitants). AC: IgG and IgM: n = 56, CH: IgG and IgM n = 23.

Figure 2

Profile of the antibody response against (A) PvCelTOS^(I133-G147), (B) PvTRAP^(P344-G374), and (C) PvCyRPA^(T289-G307) during the convalescent phase (30- and 180-days post-infection) of P. vivax infection. The dashed line represents the cut-off that separates responders from non-responders. The horizontal line in the scatter plots represents the median reactivity index (RI) value. Each sample was tested in duplicate by ELISA. Statistical significance of the frequency of responders (blue bars) at different points for each antigen was assessed using the chi-square test with Fischer’s post-test for multiple comparisons. Statistical significance of the RI (black bars) observed at different points for each antigen was assessed using the Kruskal–Wallis test with Mann–Whitney post-test for multiple comparisons (p < 0.05). Asterisks represent p value summary, * p < 0.05; ** p < 0.005; **** p < 0.0001. Conv 30 (convalescent 30 days), Conv 180 (convalescent 180 days). Conv 30: n = 46, Conv. 180: n = 24.

Figure 3

Response profile of IgG antibody subclasses (IgG1, IgG2, and IgG3) against PvCelTOS^(I133-G147), PvTRAP^(P344-G374) and PvCyRPA^(T289-G307) in the acute (AC) and convalescent phases of P. vivax (Conv 30 and Conv 180). The dashed line represents the cut-off that separates responders from non-responders. The horizontal line in the scatter plots represents the median reactivity index (RI) value. Each sample was tested in duplicate by ELISA. Statistical significance of the frequency of responders (blue bars) at different points for each antigen was assessed using the chi-square test with Fischer’s post-test for multiple comparisons. Statistical significance of the RI (black bars) observed at different points for each antigen was assessed using the Kruskal–Wallis test with Mann–Whitney post-test for multiple comparisons (p < 0.05). Asterisks represent p value summary, * p < 0.05; ** p < 0.005; *** p < 0.0005. 30 (convalescent 30 days), 180 (convalescent 180 days). (A) PvCelTOS^(I133-G147); acute: n = 27, Conv 30: n = 21, Conv 180: n = 12, CH: n = 12. (B) PvTRAP^(P344-G374); acute: n = 31, Conv 30: n = 26, Conv 180: n = 11, CH: n = 11. (C) PvCyRPA^(T289-G307); acute: n = 16, Conv 30: n = 12, Conv 180: n = 5, CH: n =7.

Figure 4

Dynamics of IgG response during acute (AC) and convalescent phase (Conv 30 dpi) of vivax malaria for (A) PvCelTOS^(I133-G147), (B) PvTRAP^(P344-G374) and (C) PvCyRPA^(T289-G307). The dashed line represents the cut-off that separates responders from non-responders. Each line with spheres at the ends represents an individual (n = 38). All samples were tested in duplicate by ELISA. Statistical significances were tested using the Wilcoxon test (p < 0.05).

Figure 5

Dynamics of IgG response during acute (AC)and convalescent phase (Conv 180 dpi) of vivax malaria for (A) PvCelTOS^(I133-G147), (B) PvTRAP^(P344-G374) and (C) PvCyRPA^(T289-G307). The dashed line represents the cut-off that separates responders from non-responders. Each line with spheres at the ends represents an individual (n = 19). All samples were tested in duplicate by ELISA. Statistical significances were tested using the Wilcoxon test (p < 0.05).

Figure 6

Dynamics of IgG response during acute (AC) and convalescent phase (Conv 30 and Conv 180 dpi) of vivax malaria for (A) PvCelTOS^(I133-G147), (B) PvTRAP^(P344-G374) and (C) PvCyRPA^(T289-G307). The dashed line represents the cut-off that separates responders from non-responders. Each line with spheres at the ends represents an individual (n = 11). All samples were tested in duplicate by ELISA. Statistical significances were tested using the Wilcoxon test.

Figure 7

Correlation between the RIs of IgM and IgG during (A) acute (AC) and convalescent phases ((B) Conv 30 and (C) Conv 180) phases of infection for PvCelTOS^(I133-G147), PvTRAP^(P344-G374) and PvCyRPA^(T289-G307). All samples were tested in duplicate by ELISA. Statistical significances were tested by the Spearman correlation.