Fig 1.
Localization of study site and design.
(A) The Brazil map in South America showing the localization of the P. vivax malaria outbreak that occurred in small village named Souza, Minas Gerais state (orange), outside of the Amazonian endemic region for malaria in Brazil (grey), and 2000 km away from endemic region (green). (B) Study design of first and brief P. vivax exposure that included individuals with acute malaria infection confirmed by microscopy (Cases, n = 16), and relatives and/ or neighbors without malaria symptoms and negative microscopy diagnosis (non-Case, n = 22). The 12-month follow-up were initiated briefly after the beginning of outbreak (BL, baseline) with three additional and identical cross-sectional surveys carried-out at 3, 6 and 12 months after the first survey. Adapted from U.S. Geological Survey (USGS) (https://www.usgs.gov/).
Table 1.
Demographic, epidemiological, and immunological data of individuals in the P. vivax malaria outbreak area at enrollment.
Fig 2.
IgM and IgG antibodies responses to P. vivax antigens after the first P. vivax exposure.
(A) Frequency and level of IgM antibodies against P. vivax EBP2, DEKnull-2 and MSP1-19 in individuals with acute P. vivax infection (Case, n = 16), and relatives and/ or neighbors without malaria symptoms (non-Case, n = 22). (B) Frequency and level of IgG antibody against P. vivax EBP2, DEKnull-2 and MSP1-19 in Case and non-Case. The IgM and IgG antibody responses were evaluated at the outbreak (BL, baseline), 3, 6 and 12 months after the P. vivax outbreak for Case group, and at BL, 3, 6-month for non-Case group. Sera reactivity was expressed as ELISA Reactivity Index (RI). Percentage (%) of antigen-specific IgM and IgG positive was expressed at the top of the graph. The dashed line represents RI = 1. Samples with RI > 1.0 are considered positive. Differences statistically significant were indicate by asterisk (*P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001).
Fig 3.
Schematic representation of ebp2 structure and amino acid sequence alignment of P. vivax outbreak isolate.
(A) Schematic representation of DBPII and EBP2 domains. The signal peptide (red box), Duffy-binding-like domain (DBL) (blue box), C-terminal cysteine-rich domain (DCR) (yellow box) and transmembrane domain (TM) (green box) are indicated. The polymorphic site in EBP2 is indicated in DBL region (E353K). (B) Amino acid sequence alignment of the DBL domain of P. vivax EBP2 of outbreak isolate (S17 sample). The sequence found at position 353 is highlighted.
Fig 4.
Influence of recurrence on the IgM and IgG responses to distinct P.vivax vaccine candidates.
Individuals who experienced first P. vivax malaria infection (Cases) were grouped into: (i) Recurrence (n = 6)–individuals who experienced one or two additional recurrent P. vivax infection; and (ii) No-recurrence (n = 10)–individuals who did not have additional blood-stage P. vivax infection. IgM (red) and IgG (blue) antibodies responses to EBP2, DEKnull-2 and MSP1-19 were measured by ELISA at baseline (BL), 3, 6, and 12-month after the outbreak. The color gradient indicates the intensity of IgM (red) and IgG (blue) antibody levels categorized by tercile in High (Upper tercile), Medium (Second tercile) and Low (First tercile) for each protein and parasitemia (orange) according to parasitemia range. The time points of follow-up study and recurrent P. vivax infection moment were indicated at the heatmap.
Fig 5.
IgG response to distinct P.vivax DBL vaccine candidates among individuals with different previous malaria episodes.
Frequency and levels of IgG antibody against P. vivax (A) EBP2 and (B) DEKnull-2 among (1) Outbreak (n = 55)—individuals from non-endemic region, including recurrent P. vivax infection (closed circle) and without recurrence (open circle) and, individuals long-term malaria exposure from endemic region with (2) Single previous malaria episode (n = 20) and multiple episodes (n = 49). Sera reactivity was expressed as ELISA Reactivity Index (RI). Samples with RI > 1.0 were considered positive. The percentage (%) of positive IgG antibody response to each protein was expressed at the top of the graph. Differences statistically significant were indicate by asterisk (*P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001).