The in Vitro Antigenicity of Plasmodium vivax Rhoptry Neck Protein 2 (Pv RON2) B- and T-Epitopes Selected by HLA-DRB1 Binding Profile

Abstract

Malaria caused by Plasmodium vivax is a neglected disease which is responsible for the highest morbidity in both Americas and Asia. Despite continuous public health efforts to prevent malarial infection, an effective antimalarial vaccine is still urgently needed. P. vivax vaccine development involves analyzing naturally-infected patients' immune response to the specific proteins involved in red blood cell invasion. The P. vivax rhoptry neck protein 2 (PvRON2) is a highly conserved protein which is expressed in late schizont rhoptries; it interacts directly with AMA-1 and might be involved in moving-junction formation. Bioinformatics approaches were used here to select B- and T-cell epitopes. Eleven high-affinity binding peptides were selected using the NetMHCIIpan-3.0 in silico prediction tool; their in vitro binding to HLA-DRB1^*0401, HLA-DRB1^*0701, HLA-DRB1^*1101 or HLA-DRB1^*1302 was experimentally assessed. Four peptides (39152 (HLA-DRB1^*04 and 11), 39047 (HLA-DRB1^*07), 39154 (HLADRB1^*13) and universal peptide 39153) evoked a naturally-acquired T-cell immune response in P. vivax-exposed individuals from two endemic areas in Colombia. All four peptides had an SI greater than 2 in proliferation assays; however, only peptides 39154 and 39153 had significant differences compared to the control group. Peptide 39047 was able to significantly stimulate TNF and IL-10 production while 39154 stimulated TNF production. Allele-specific peptides (but not the universal one) were able to stimulate IL-6 production; however, none induced IFN-γ production. The Bepipred 1.0 tool was used for selecting four B-cell epitopes in silico regarding humoral response. Peptide 39041 was the only one recognized by P. vivax-exposed individuals' sera and had significant differences concerning IgG subclasses; an IgG2 > IgG4 profile was observed for this peptide, agreeing with a protection-inducing role against P. falciparum and P. vivax as previously described for antigens such as RESA and MSP2. The bioinformatics results and in vitro evaluation reported here highlighted two T-cell epitopes (39047 and 39154) being recognized by memory cells and a B-cell epitope (39041) identified by P. vivax-exposed individuals' sera which could be used as potential candidates when designing a subunit-based vaccine.

Keywords: HLA-DRB1 typing; Plasmodium vivax; PvRON2; antigenicity; cellular and humoral response; epitope; synthetic peptide.

Figure 1

PvRON2 peptides in vitro binding to purified HLA-DRB1* molecules. A cut-off line is shown at 50% binding, used for selecting high-binding peptides for further evaluation of IC50 value. Each plot shows percentage epitope binding to HLA-DRB1* in this study and that for their control peptide.

Figure 2

In vitro assays for calculating a PvRON2 peptide's IC50 value. Different epitope concentrations were evaluated for calculating the value at which control peptide was displaced by 50% (using a second order exponential decay function). Each point under the curve represents evaluated epitope concentration-dependent control peptide (μM) binding.

Figure 3

Gating strategy for the proliferation assays and PBMC proliferative response to PvRON2 epitopes from individuals exposed to P. vivax infection compared to control group. (A) P. vivax-exposed individuals' PBMC stimulated with universal peptide (39153). (B) Non-stimulated P. vivax exposed individuals' PBMCs. (A,B) Upper left plot, selected lymphocyte population (SSC-A vs. FSC-A), upper right plot selection of single cells from lymphocyte population (FSC-H vs. FSC-A). The lower left-hand plot shows gated CFSE label lymphocytes (Comp-FITC-A) for analyzing CD4+ T-cells (Comp-Pacific Blue-A). Lower right-hand plot shows CD4+ lymphocyte proliferation analyzed by FlowJo software (v7.6.5, Ashland, Oregon, USA) using 7 peaks or cell generations. (C) Mann-Whitney and Student's t-tests were used for assessing statistically significant differences between exposed individuals and control group. Universal peptide 39153 (n = 29), DRB1*04 and DRB1*11 peptide 39152 (n = 15), DRB1*07-specific peptide 39047 (n = 8), DRB1*13-specific peptide 39154 (n = 6), low-binding control peptide 39115 (n = 29) and P. vivax lysate (n = 29) responses are shown. The CD4+ cells were labeled with Pacific Blue mouse anti-human CD4 (RPA-T4 clone) antibody. Statistically significant differences (p ≤ 0.05) are shown and data represents the means ± SEM for all values. *p < 0.05 and **p < 0.005.

Figure 4

Exposed individuals' supernatant culture in vitro cytokine production. Individual data shows the mean value of non-stimulated and PBMCs stimulated with universal epitope (39153), specific epitopes 39047, 39152, and 39154, and P. vivax lysate. IFN-γ, TNF, IL-10, and IL-6 levels were measured by CBA kit; cytokine concentration is expressed in pg/mL. Statistically significant differences (p ≤ 0.05) are shown and data represents the means ± SEM for all values. *p < 0.05, **p < 0.005 and ***p < 0.0005.

Figure 5

Exposed individuals and control group supernatant culture in vitro cytokine production. Individual cytokine values from PBMCs stimulated with universal peptide (39153), specific epitopes 39047, 39152, and 39154, and P. vivax lysate. TNF and IL-6 levels were measured by CBA kit and cytokine concentration is expressed in pg/mL. Statistically significant differences (p ≤ 0.05) are shown and data is the means ± SEM for all values. *p < 0.05 and **p < 0.005.

Figure 6

IgG antibody response against four PvRON2 B-cell epitopes (n = 30). Seropositive samples were those above the cut-off point (0.218, dotted line), calculated as control group's mean plus two standard deviations. The Kruskal-Wallis test was used for analyzing differences between each B-epitopes response in P. vivax-exposed individuals' samples. *p < 0.05.

Figure 7

IgG antibody response to PvRON2 B epitopes by endemic area. Significant differences (calculated by Mann-Whitney test) between samples from Colombia's Chocó (n = 13) and Córdoba (n = 17) departments are shown. The dashed line indicates the cut-off point for seropositive samples. *p < 0.05 and ***p < 0.0005.

Figure 8

Evaluating IgG subclass response to the 39041 epitope (n = 6). The Kruskal-Wallis test was used for analyzing differences between each IgG subclass response in P. vivax-exposed individuals' samples. **p < 0.005 and ***p < 0.0005.