Fig 1.
Seropositive responses and inhibitory function of anti-PvDBPII antibodies.
(A) Total IgG levels against PvDBPII in plasma from acutely infected subjects (PV; n = 40) were determined and compared with healthy controls (HCs; n = 21). The total IgG reactivity index against PvDBPII is shown. The total IgG levels of seropositive responders were classified into high responders (HRs; ≥ cut-off + 2SD of the OD of HCs), low responders (LRs; > cut-off + 2SD of the OD of HCs). The seronegative samples were defined as non-responders (NRs; < cut-off of the OD of HCs). (B) Seropositive IgG P. vivax samples (n = 23) and HCs (n = 17) were used to assess IgG subclass (IgG1-4) responses against PvDBPII. The dashed line represents cut-off value of seropositivity (RI = 1) calculated from the mean of RI + 2SD of HC samples. Statistical testing was performed by Mann-Whitney Rank Test (for comparing two non-parametric groups; *p-value < 0.05, **p-value < 0.005, ****p-value < 0.0001. (C) Top: Representative images of PvDBPII binding to Duffy-positive human erythrocytes. Bright field (BF), GFP fluorescence, and merged pictures are shown. Bottom: Functional inhibition of plasma from acutely infected subjects seropositive for total IgG (n = 23) against PvDBPII–human erythrocyte binding. High-responder (HR) samples exhibiting > 80% inhibition are indicated by red stripes, HR samples with < 80% inhibition by black stripes, and low-responder (LR) samples by blue. Data are presented as mean ± SD of percentage inhibition.
Fig 2.
Frequency of cTfh2 cells in seropositive anti-PvDBPII antibody responses.
PBMCs from acutely P. vivax infected (PV; n = 22) and healthy controls (HC; n = 14) were phenotyped by flow cytometry. (A) The frequencies of cTfh cells (CXCR5+ PD-1+) and cTfh subsets based on the expression of CXCR3 and CCR6 (cTfh1; CXCR3+CCR6-, cTfh2; CXCR3-CCR6-, cTfh17; CXCR3-CCR6+, and cTfh1/17; CXCR3+CCR6+). (B) The frequency of cTfh2 cells, comparing HR and LR subjects. (C) The MFI expression of ICOS molecules on the surface of cTfh2 cells was shown. (D) The frequency of IL-4- or IL-10-producing cTfh2 cells in P. vivax (n = 11) and healthy controls (HCs; n = 6). Each bar represents median, and error bar represents interquartile range (IQR). High-responder (HR) samples are shown in red, with > 80% inhibition indicated by red stripes, and low-responder (LR) samples in blue. Statistical testing was performed by Mann-Whitney Rank Test for comparing two non-parametric groups and Wilcoxon matched-pairs signed rank test; *p-value < 0.05; **p-value < 0.005; ***p-value < 0.0005; ****p-value < 0.0001; ns, non-significant.
Fig 3.
Activation of cTfh2 cells upon PvDBPII stimulation.
PBMCs from acutely P. vivax infected subjects (AC; n = 7), recovered subjects (RC; n = 7), and healthy controls (HC; n = 6) were stimulated in vitro with recombinant PvDBPII (rPvDBPII) for 6 days. (A). Representative gating strategy for cTfh subsets between stimulated with rPvDBPII and un-stimulated (unstim). (B) The frequency of cTfh cells (CXC5+ PD-1+) and the Stimulation Index (SI) of cTfh cells after PvDBPII stimulation. (C) The SI of cTfh2 cells in culture of AC, RC, and HC subjects. (D) The representative of histogram and MFI ICOS expression on the surface of cTfh2 cells after in vitro culture with PvDBPII antigen. Each bar represents median, and error bar represents interquartile range (IQR). Statistical testing was performed by Wilcoxon matched-pairs signed rank test; *p-value < 0.05; ns, non-significant.
Fig 4.
Proliferation and activation of the cTfh2-like cells after co-culture stimulation.
(A). Experimental design: naïve CD4+T cells were sorted from P. vivax subjects (n = 6) and HCs (n = 5), labeled with or without CFSE, and then stimulated for 5 days to drive differentiation of cTfh2-like cells. The cTfh2-like cells were then co-cultured with autologous MBCs and the following stimuli: rPvDBPII antigen, R848 or IFN-γ. After 5 days, the cells were analyzed by flow cytometry to detect the proliferation and activation of cTfh2-like cells, along with the presence of ASCs. The culture supernatant was collected to detect total IgG and anti-PvDBPII antibody levels. The representative gating strategy for cTfh2 was based on CXCR3-CCR6-cTfh-like cells. (B) Representative proliferation of cTfh2-like cells under different conditions: PvDBPII stimulation, non-stimulation, or PHA stimulation. Data analysis was performed using FlowJo software, and the proliferation index was measured. (C) The frequency of ICOS+cTfh2-like cells in culture conditions: stimulation 1 (S1) rPvDBPII; S2) rPvDBPII and R848 stimulation; S3) rPvDBPII and IFN-γ; S4) No antigen stimulation (negative control). Positive control was PHA stimulation. Each bar represents the median, and the error bars represent the interquartile range (IQR). Statistical testing was performed by one-way ANOVA followed by Dunn’s multiple comparison test. Significance levels are as follows: *p-value < 0.05; **p-value < 0.005; ***p-value < 0.0005; ****p-value < 0.0001. The image was created in BioRender. Salsabila, Z. (2026) https://BioRender.com/np0qrx7.
Fig 5.
ASC differentiation and anti-PvDBPII antibody secretion after cTfh2-MBC co-cultures.
(A) Representative gating of ASC differentiation based on CD19+CD27+CD38+ cells in cTfh2-like cell-MBC co-cultures with various stimulation conditions: S1) rPvDBPII stimulation, S2) R848 and rPvDBPII stimulation, S3) rPvDBPII and IFN-γ, S4) no antigen stimulation (negative control). (B) The frequency of ASCs after co-culture. (C) Total IgG and anti-PvDBPII antibody levels were measured in culture supernatants. Each bar represents median, and error bar represents interquartile range (IQR). Statistical testing was performed by one-way ANOVA analysis and multiple-comparison by Dunn’s multiple comparison; *p-value < 0.05; **p-value < 0.005; ***p-value < 0.0005; ****p-value < 0.0001.