The in Vitro Antigenicity of Plasmodium vivax Rhoptry Neck Protein 2 (PvRON2) B- and T-Epitopes Selected by HLA-DRB1 Binding Profile

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.

More than 50 proteins have been described to date as being involved in malarial parasite's red blood cell (RBC) invasion; most have been identified at molecular level and characterized immunologically in P. falciparum (Bozdech et al., 2003;Cowman and Crabb, 2006). Conversely, studying P. vivax proteins involved in host invasion has been difficult, mainly due to technical restrictions such as the lack of a continuous in vitro parasite culture, leading to inadequate study of parasite biology (Udomsangpetch et al., 2008;Mueller et al., 2009).
The development of bioinformatics tools during the last few decades has enabled predicting vaccine candidates based on peptide binding affinity for major histocompatibility complex (MHC) class I or class II (Sturniolo et al., 1999;Nielsen and Lund, 2009;Wang et al., 2010;Zhang et al., 2012;Andreatta et al., 2015). The immune system's function is to recognize and differentiate between self and non-self-antigens so as to trigger cellular and/or humoral immune responses. MHC class II proteins (HLA-II in humans) are expressed on antigen presenting cells' (APC) surface (i.e., macrophages, dendritic cells and B-lymphocytes). These recognize extracellular antigens and can bind 13-to 18-aa-long peptides. One of the main difficulties in designing a vaccine is the high HLA polymorphism, especially from HLA-DRB1, this being the most polymorphic locus. Antigen binding capability varies from one allele to another, increasing or reducing affinity and driving immune responses. Selecting peptides as good vaccine candidates relies on their ability to be recognized by HLA-DRB1 alleles to ensure a protection-inducing immune response (Stern and Calvo-Calle, 2009). T-cells can trigger stronger immune responses after their APC recognition, depending on the peptides bound to MHC receptors (Blum et al., 2013).
Antigen-antibody interaction plays an essential role in humoral immune responses against pathogens. Bioinformatics tools are extremely useful for identifying antigenic determinants or B-cell epitopes when designing vaccines (Bergmann-Leitner et al., 2013;Panda and Mahapatra, 2017). Predicting linear B-cell epitopes (contiguous aa in a protein sequence) is based on several methods for determining aa physicochemical properties, such as solvent accessibility, hydrophilicity and flexibility (El-Manzalawy et al., 2017;Solihah et al., 2017). This paper describes naturally-acquired T-cell and antibody immune responses to PvRON2 in P. vivax-exposed individuals from two of Colombia's endemic areas (Córdoba and Chocó), in the search for vaccine candidates. Eleven high-affinity epitopes were selected by NetMHCIIpan-3.0 (Andreatta et al., 2015) in silico prediction and their in vitro binding to at least one of HLA-DRB1 * 0401, HLA-DRB1 * 0701, HLA-DRB1 * 1101 and HLA-DRB1 * 1302 was assessed by competition assays. The Bepripred 1.0 tool was used for selecting four B-cell epitopes in silico. A good immune response was observed against two T-cell and one B-cell epitopes; further studies aimed at testing these peptides as components of a subunit vaccine against P. vivax are thus recommended.
Bepipred 1.0 (Larsen et al., 2006) and Antheprot 2000 V6.0 (Deléage et al., 2001) were used for predicting B-cell epitopes. Four epitopes were chosen as they agreed with average high Parker antigenicity, hydrophilicity and solvent accessibility values obtained with Antheprot software, and the high values obtained with the Bepipred tool (0.35 default threshold and 75% specificity); such peptides were further used for analyzing humoral responses in vitro ( Table 2).

Synthetic Peptides
Peptides selected in silico were purchased from Twenty First Century Biochemicals Inc. (260 Cedar Hill Street Marlboro, MA 01752 USA) and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The biotinylated peptides used as control for HLA peptide binding in competition assays were synthesized using sulfo-NHS-LC-Biotin (Pierce Chemical, Rockford).

In Vitro Peptide-Binding Assays and IC50 Values
Peptide binding competition assays were performed to test PvRON2 high-affinity binding peptides selected by in silico analysis using NetMHCIIpan 3.0 software. Selected unlabeled peptides competed with biotinylated control peptide in binding to HLA-DRB1 * . The biotinylated peptides used were haemagglutinin antigen HA 306−318 (PKYVKQNTLKLAT) for HLA-DRB1 * 04 and HLA-DRB1 * 11 (Hammer et al., 1994;Saravia et al., 2008) and tetanus toxoid (TT) (QYIKANSKFIGITE) for HLA-DRB1 * 07 and HLA-DRB1 * 13 (Doolan et al., 2000).  IgG absorbance readings for individuals exposed to natural P. vivax infection from endemic areas of Colombia. B-cell epitope code, aa sequences, control protein and average response (OD) are shown. SE, standard error.
HLA-DR molecules (0.1 µM) were incubated for 24 h with 5 µM biotinylated HA or TT peptides and a 50-fold excess of unlabeled peptide (250 µM). The mix was incubated for 2 h in Maxisorb NUNC-immune modules (Thermo Scientific) covered with anti-DR. The complex was incubated with alkaline phosphatase streptavidin (Vector Labs) and as substrate alkaline phosphatase yellow (pNPP) liquid substrate (Sigma-Aldrich). Optical density (OD) was determined at 405 nm using a Multiskan GO (Thermo Scientific, Waltham, Massachusetts, USA) ELISA reader. Inhibition was calculated as a percentage, by using the following formula: 100 * 1 − OD in the presence of competitor OD in the absence of competitor IC50 values (50% concentration inhibition) were determined for peptides able to inhibit high-affinity control peptide binding to a particular HLA-DR by more than 50% (Saravia et al., 2008). The peptides and control peptides were tested in 5-250 µM serial dilutions for the competition assays; Mathematica (version 10.1) software (Wolfram Research, Inc., Mathematica, Champaign, IL 2015) was used for calculating IC50 values, using two-phase exponential decay. IC50 values were calculated as a relative value using the following formula: 1 − OD in the presence of competitor OD in the absence of competitor

Study Population
Peripheral blood was obtained from 79 people living in the Colombian departments of Chocó and Córdoba (known P. vivax malaria endemic areas, having the highest case incidence) who had suffered previous episodes of malaria. Inclusion criteria consisted of being over 18 years-old, residing in a P. vivaxendemic area, having had 1 or more episodes of P. vivax malaria (the last one 6 months beforehand) and having received suitable treatment for the disease. Although a stronger immune response would have been expected in acutely-infected P. vivax individuals, the construction of study groups required a prior HLA typing and thus, a second sample had to be taken from individuals matching the alleles of interest to assess antigenicity. Taking this into account, the antigenicity sample was taken from people that had suffered P. vivax malaria at least 6 months earlier. A control group of 50 individuals was selected; this consisted of healthy adults residing in Bogotá, Colombia, who had never lived in malaria-endemic areas and who had never experienced malarial infection. This study was performed according to the legal framework for research in Colombia and Ministry of Health's Resolution 8430 of 1993. The patients had the least risk, all data were kept confidential and were rigorously protected. The samples were collected after all individuals signed an informed consent form; all procedures were evaluated and approved by FIDIC's ethics committee.

HLA-DRB1 Typing
Genomic DNA (gDNA) from 300 µL peripheral blood samples was extracted using a Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, USA), following the manufacturer's instructions. gDNA was used for high resolution HLA-DRB1 typing by Histogenetics (Ossining, NY, USA) through Next Generation Sequencing (NGS) technology using Illumina MiSeq.

PBMC Isolation
Twenty-nine people carrying HLA-DRB1 typing for HLA-DRB1 * 04, HLA-DRB1 * 07, HLA-DRB1 * 11 and HLA-DRB1 * 13 alleles were selected from P. vivax endemic areas of Colombia's Córdoba and Chocó departments. Eight people carrying the same HLA-DRB1 * alleles from a non-endemic area formed the control group. About 40 mL peripheral blood was collected in citrate phosphate dextrose (CPD) tubes and 6 mL peripheral blood in BD vacutainer serum collection tubes (BD Vacutainer Oakville, ON). Thick blood smears were used for confirming samples negative for malaria. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Paque PLUS (GE Healthcare) gradient centrifugation. Briefly, the buffy coat was resuspended in RPMI 1640 (Gibco) and separated by Ficoll, spinning at 1,000 g for 30 min at room temperature (RT). Mononuclear cells were collected, washed and spun at 800 g for 10 min, twice. Cell viability was evaluated by trypan blue exclusion test and cells were counted in a Neubauer chamber.
The CD4-Pacific Blue-stained cell stimulation index was calculated by proliferative cells' relative percentage loss of carboxyfluorescein succinimidyl ester (CFSE) in the presence of antigen, divided by percentage relative CFSE loss for proliferative cells without antigen (Racanelli et al., 2011). Data was averaged for each antigen and for both exposed individuals and control groups. SI ≥ 2 was taken as antigen-specific positive proliferation. A Pacific Blue-labeled mouse anti-human CD4 (RPA-T4 clone) antibody (BD Biosciences), was used for CFSE-cell cluster measure. The samples were then read on a FACS Canto II flow cytometer; FlowJo software (v7.6.5, Ashland, Oregon, USA) was used for analyzing the results.

Cytokine Secretion
IFN-γ, TNF, IL-10, and IL-6 levels in lymphocyte culture supernatant were determined with a BD CBA Human Th1/Th2 Cytokine Kit II (San Jose, CA, USA), following the manufacturer's instructions. Supernatants were read on a FACS Canto II flow cytometer; FCAP Array software (v3.0.1) was used for analyzing the results. Results were expressed in pg/mL for each cytokine; data were compared between unstimulated and stimulated PBMC supernatant culture. Two standard deviations higher than that for control group were taken as positive antigen-specific production.
Indirect Immunofluorescence Assays (IFA) IFA followed that previously described by Moreno-Pérez et al. (2013) with some modifications. Briefly, blood samples from individuals having active P. vivax infection were spun at 1,750 g for 12 min at RT. Both plasma and buffy coat were recovered and parasite red blood cells (pRBC) were washed with saline solution. pRBC were passed through a 60% Percoll gradient and spun at 1,750 g for 20 min. P. vivax-pRBC were diluted until 5-7 schizonts per field and confirmed by acridine orange. Twenty µL of diluted pRBC were placed into multitest microscope slide wells (Tekdon Incorporated) and incubated for 30 min. The supernatant was then removed, and microscopic slides left overnight (ON) at room temperature (RT) to air dry. The slides were then blocked for 30 min at RT with tris-buffered saline (TBS) 1% bovine serum albumin (TBSA) solution and washed three times. The serum samples from 30 exposed individuals and 8 sera from the control group were diluted in TBSA at 1:50 dilution and incubated for 1 h in a humid chamber. Reactivity was observed by fluorescence microscopy using anti-human IgG-FITC antibody (Sigma-Aldrich) diluted 1:50 in TBSA for 45 min in a humid chamber. The parasite nuclei were stained with 4 ′ , 6-diamidino-2-phenylinodole dihydrochloride (DAPI) (0.25 µg/mL) for 5 min at RT and washed twice with 0.05% TBS-Tween 20 and three washes with TBS to remove excess reagent. The slides were visualized on an Olympus BX51 fluorescence microscope, using 100X oil immersion objective; DP2-BSW software (v2.2 Olympus Corporation) was then used to take images and ImageJ 1.51n software (National Institutes of Health, USA) for merging images.

Enzyme-Linked Immunosorbent (ELISA) and Subclass IgG Assays
Total IgG antibodies were measured in serum from exposed individuals and control group. Maxisorb NUNC-immune modules (Thermo Scientific) were coated with 1 µg of each epitope and of rPvGAMA (10 µg/mL) (Baquero et al., 2017) in phosphate-buffered saline (PBS), pH 7.2, and incubated ON at 4 • C. The immune modules were washed three times with PBS-0.05% Tween 20 solution (PBST) the next day and then blocked with 2.5% (wt/vol) non-fat powdered milk in PBST solution for 1 h at RT. Serum at 1:100 dilution was incubated for 2 h (100 µL per well) in duplicate. Secondary antibody horseradish peroxidase-conjugated goat anti-human IgG (Vector labs) was added at 1:10,000 dilution in blocked solution and incubated for 1 h at RT. TMB 2-Component Microwell Peroxidase Substrate (Sera-Care) was added at 100 µL/well to detect monoclonal antibody binding. The reaction was stopped by adding an equal volume of 1M phosphoric acid (H 3 PO 4 ); OD was measured at 450 nm using a Multiskan GO (Thermo Scientific, Waltham, Massachusetts, USA) ELISA reader. Cut-off value was determined as negative control serum samples' mean plus two standard deviations; IgG subclasses were determined for positive total IgG serum.

Statistical Analysis
GraphPad Prism software (version 5.0, San Diego, CA, USA) was used for analysis and constructing graphs. A Mann-Whitney test was used for comparing two groups regarding non-parametric data and Kruskal-Wallis test (with Dunn's multiple comparison post-test) for comparing more than two groups. Student's t-test was used for comparing two groups of data having a normal distribution. A 95% confidence interval was used. p ≤ 0.05 was considered significant. Significance level has been highlighted on all graphs by asterisks, as follows: * p < 0.05; * * p < 0.005, and * * * p < 0.0005.

T-Epitope Selection According in Vitro
Binding Profile Table 1 gives PvRON2 antigenic epitope prediction results. Eleven epitopes were selected in silico for HLA-DRB1 * 04, * 07, * 11, and * 13 alleles (3 epitopes for each allele and 2 epitopes for the * 07 allele). These epitopes were evaluated in vitro for their ability to bind all alleles of interest; those having greater than 50% binding were carefully chosen as high-binding peptides (Figure 1).
The IC50 value was calculated for all high-binding peptides to select the ones displaying higher affinity. Different peptide concentrations (µM) were used and the point at which 50% of the control peptide was displaced was thus calculated. IC50 µM value was calculated using a second order exponential decay function (Figure 2). IC50 assays demonstrated that epitope 39152 had the lowest IC50 ratio for HLA-DRB1 * 04 (0.15) and HLA-DRB1 * 11 (0.4), so it was thus selected as a good epitope for both alleles. Epitope 39047 (0.26) was selected as specific epitope for HLA-DRB1 * 07 and epitope 39154 (0.88) for HLA-DRB1 * 13. Of the four universal epitopes, peptide 39153 had the lowest IC50 mean value (Table 1, Figure 2). The selected epitopes were screened for antigenicity according to their HLA-DRB1 * binding profile in previously typed patients.

Evaluating T-Cell Response Against Selected Epitopes
The people in the study had to have resided in the area for at least the last 5 years (average 29 years) and have had 1 or multiple episodes of P. vivax malaria, the last episodes dated between 2011 and 2015. It is well known that a naturally-acquired response requires a long period of time and multiple exposures to the parasite (Wipasa et al., 2002). HLA-DRB1 * allele distribution for the 79 people here typed is shown in the Supplementary Table 1.
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.
Among the alleles of interest, HLA-DRB1 * 04 had a frequency of 12.66%, HLA-DRB1 * 07 a frequency of 12.03%, HLA-DRB1 * 11 a frequency of 8.23% and HLA-DRB1 * 13 a frequency of 9.49%. PBMC from individuals exposed to P. vivax infection (and control group) were stimulated with 10 µg/mL PvRON2 peptides and incubated for 5 days to evaluate proliferative response. Both universal epitope 39153 and DRB1 * allele-specific peptides induced proliferation (≥2 Stimulation Index) in individuals from endemic areas, whereas there was no proliferation regarding specific peptides for each allele or parasite lysate in the control group (Table 3 and Supplementary Table 2). There were statistically significant differences for universal epitope 39153 (p = 0.0075), DRB1 * 13-specific peptide 39154 (p = 0.0387) and DRB1 * 07-specific peptide 39047 (p = 0.0260) between the group of exposed individuals and the control group. This response to the different antigens used in the lymphoproliferation assay, was compared with a Kruskal-Wallis test (with a Dunn's multiple comparison post-test), where no statistically significant differences were found in response to the different peptides.
Low-binding control peptide 39115 showed the lowest proliferative response (SI = 2.018) and was significantly different regarding the control group (p = 0.0289). P. vivax lysate also induced a greater proliferative response in exposed individuals, despite no statistically significant differences were observed when compared to the control group (Figure 3). However, peptide 39115 induced T cell proliferation in 7 of the 29 exposed individuals, despite no binding to HLA-DRB1 * was either predicted or observed in vitro. Considering that this peptide is a T epitope from the Pv12 protein as shown by the lymphoproliferation assays, further analyses to assess whether it is being presented by other class II molecules such as HLA-DP or HLA-DQ are worth carrying out.
P. vivax lysate also induced a greater proliferative response in exposed individuals; however, no statistically significant differences were observed when compared to control group (Figure 3). The mean SI = 12.47 ± 1.636 SE is for exposed individuals and mean SI = 5.45 ± 1.138 SE for the control group. Despite the SI values of PMBCs stimulated with PHA were significantly higher between exposed individuals and control group (p = 0.0103), 96.6% of exposed individuals and the 100% of control group responded to PHA (data not shown).
compared with the Kruskal-Wallis test (with a Dunn's multiple comparison post-test) between 39115 and all other peptides in exposed individuals, a significantly higher TNF and IL-6 production was observed for peptide 39154 (p < 0.0001).

This suggests that naïve T-cells and/or other innate cells, such as macrophages, natural killer (NK), natural killer T-cells (NKT) and non-cytotoxic innate lymphoid cells recognized peptides and lysate and produced cytokines (Artis and Spits, 2015) (Supplementary
Cytokine levels were compared between exposed individuals and control group, significant differences being found regarding TNF production for epitope 39153 (p = 0.0127). Significant differences were found for epitope 39047 (p = 0.005) and 39152 epitope (p = 0.010) IL-6 production ( Figure 5).

Detecting Antibodies Against P. vivax-Infected RBC by IFA
Naturally-acquired anti-malarial antibodies were detected using Multitest slides (MP Biomedicals) coated with parasitized RBC. The thirty exposed patients' sera reacted against P. vivax but the control group's sera did not. Supplementary Figure 2 shows the fluorescence pattern obtained with these sera.

An Analysis of PvRON2 B-Epitope Humoral Immune Response in P. vivax-Exposed Individuals
Antibody response against four in silico selected PvRON2 Bepitopes was evaluated in sera from 30 individuals exposed to natural P. vivax infection (Figure 6, Table 2). The highest number of seropositive samples was for the 39041-epitope (6/30, 20% of samples), followed by 39042 and 39043 (3/30, 10% of samples) and 39044 (2/30, 6.6% of samples). Peptides 39041 and 39043 showed the highest mean response (0.1598 and 0.1422, respectively), and significant differences were observed regarding 39044 which had the lowest mean response (0.0949) out of all four peptides. The Kruskal-Wallis test (with Dunn's multiple comparison post-test) was used for statistical analysis, there were significant differences regarding epitope response (p = 0.003). Although there were no statistically significant differences between exposed individuals and control group, there was a tendency for a greater response in the first group (data not shown). rPvGAMA was used as positive control, 70% of samples being seropositive (21/30 samples); significant differences with control group were observed (p = 0.0004) (Supplementary Figure 3).
The ELISA results were analyzed by endemic area, showing an evident tendency for a greater response in the samples from the Chocó department compared to the Córdoba department (Figure 7). The Mann-Whitney test gave a significantly higher response for peptides 39041 (p = 0.0135), 39042 (p = 0.0171), 39043 (p = 0.0007) and 39044 (p = 0.0492) in P. vivax-exposed individuals. Samples from Colombia's Chocó (n = 13) and Córdoba (n = 17) departments. From these samples, six reacted positively to at least one PvRON2 B-epitope, where 83% (n = 5) of the samples were from Chocó's department. Positive sera reacted to the four peptides, 83% were from samples from the Chocó department. Although 39041, 39042, and 39044 peptides had no significant differences between exposed individuals from the Chocó and control group, 39043 had a significantly higher response (p = 0.0186). Differences between endemic areas were only observed in response to PvRON2 B-cell epitopes since there were no significant differences between both areas regarding PvGAMA (p = 0.4265), as it was expected to occur when a whole recombinant protein is used as antigen (several epitopes present within it, could be differentially recognized by exposed individuals, and a similar overall response was thus detected). Seropositive samples were selected for evaluating IgG subclasses.

The Prevalence of IgG Subclass Response
Against PvRON2 B-Epitopes in Individuals Exposed to Natural P. vivax Infection Seropositive samples from exposed individuals recognizing Bepitopes were selected for IgG subclass evaluation. Of the four peptides evaluated, only 39041 had significant differences between IgG subclasses (p = 0.0004) while there were no 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)

DISCUSSION
Developing countries desperately need strategies aimed at preventing malaria (especially that caused by P. vivax), such as approaches for developing specific drugs and protective vaccines which are currently unavailable. Although there are P. vivax vaccines in phases I and IIa (López et al., 2017), they have not induced sterile protection (Bennett et al., 2016). Developing a P. vivax vaccine requires studies analyzing naturally-infected patients' immune response regarding proteins involved in erythrocyte invasion. Several P. vivax vaccine candidates' binding regions have been characterized to date, such as reticulocyte binding proteins (RBPs) , the Duffy binding protein (DBP) , the P. vivax GPI-anchored micronemal antigen (PvGAMA) (Baquero et al., 2017), some 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.  -Pinzón et al., 2011). RONs have been strongly associated with MJ formation, thereby helping parasite entry into erythrocytes; RON2 is a vaccine candidate since anti-AMA1 and anti-RON2 antibodies can block erythrocyte invasion (Arévalo-Pinzón et al., 2011;Lamarque et al., 2011;Srinivasan et al., 2011;Tyler et al., 2011;Vulliez-Le Normand et al., 2017).
The data presented here has described naturally-acquired Tcell immune responses to PvRON2 high-affinity binding-MHC-II DRB1 * peptides in P. vivax-exposed individuals from two endemic areas of Colombia. Certain requirements are involved in protection-inducing vaccine design; for example, the proper antigen presentation by MHC-II and MHC-I molecules to Tcell receptors, so as to induce strong immune responses. MHC class II and I molecules are critical in host-pathogen interactions as they determine host immune response quality. High-affinity peptide-MHCII-TCR binding time or peptide-MHCII complex amount is critical for mounting protective T-cell responses (Blum et al., 2013;Tubo et al., 2013). Post-genomic era bioinformatics tools and reverse vaccinology approaches have drawn scientists' attention, since an individual antigen can be screened from one or several microorganisms and the highest affinity epitopes be determined from these might induce protective immune responses (Sette and Rappuoli, 2010).
This study has analyzed the PvRON2 sequence aa to determine high-binding HLA-DRB1 * T-cell epitopes in silico. NetMHCIIpan 3.0 (Andreatta et al., 2015) predicted eleven high-affinity HLA-DRB1 * epitopes where at least one epitope bound to one HLADRB1 * molecule; this was confirmed by in vitro competition assays using biotin-control peptides. However, some predicted epitopes have not bound to HLA-DRB1 * , according to other studies (Bergmann-Leitner et al., 2013), while HLA-DRB1 * 04 bound to 10/11 peptides here. A previous study has shown a strong naturally-acquired humoral response in HLA-DRB1 * 04 people living in the Brazilian Amazon against 5/9 recombinant P. vivax proteins (Lima-Junior et al., 2012). HLA-DRB1 * 04 is one of the most frequently occurring alleles in Colombian Amerindian groups, accompanied by DR2 (DRB1 * 1602), DR6 (DRB1 * 1402) and DR8 (DRBl * 0802). The patients' blood samples used in this study were mostly taken from Amerindians from Córdoba and Chocó. Tule (5 HLA-DRB1 * 04 alleles) is the main Amerindian population in Córdoba and the Waunana (4 HLA-DRB1 * 04 alleles) in the Chocó region, having more diverse DRBl alleles than other groups (Trachtenberg et al., 1996).
Previous studies had suggested that some alleles' over dominance in a population could be due to the spread of advantageous alleles (positive selection) after pathogen-driven selection. This has been shown by Hill et al., in a study of West African alleles (HLA-DRB1 * 1302-DQB1 * 0501) which were not 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. present in other racial groups and were associated with protection against severe malaria, i.e., directional selection (Hill et al., 1991).
Several factors (e.g., parasite evasion mechanisms and immune molecule polymorphism) can affect protection-inducing immune responses to malarial parasites and, consequently, malaria vaccine development. Parasite evasion mechanisms include immunodominant antigen polymorphism, antigenic variation and diversion, epitope masking and the smoke-screen strategy. P. vivax and P. ovale have additional escape mechanisms which are mediated by long-lasting hypnozoites, as well as using different erythrocyte invasion pathways (Rénia and Goh, 2016). MHC Class I and II have enormous allele polymorphism and aa sequence variation in the peptide binding region, thereby enabling peptides to bind to different alleles (Blum et al., 2013). Such variability hampers a single epitope-based vaccine against Plasmodium parasites being developed; however, in silico analysis of T-and B-cell epitopes could be useful for identifying vaccine candidates represented by different epitopes which could provide coverage of the whole target population. The exposed patients studied here were grouped according to their HLA-DRB1 * to cover the most frequently occurring alleles in the endemic population worldwide, such as HLA-DRB1 * 04, HLA-DRB1 * 07, HLA-DRB1 * 11, and HLA-DRB1 * 13. Cell-mediated immune 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. responses were investigated based on MHC class II peptide binding specificity and humoral immune responses by detecting antibody levels against linear B-cell epitopes.
Antibodies against P. vivax blood-stage proteins are important elements for blocking RBC invasion (Wipasa et al., 2002); such antibodies thus play an important role in identifying and validating P. vivax vaccine candidates (Soares et al., 1999;Lima-Junior et al., 2008;Storti-Melo et al., 2012;Changrob et al., 2017;Rodrigues-Da-Silva et al., 2017). We confirmed the presence of naturally-acquired humoral responses against four PvRON2 Bepitopes which were recognized by IgG antibodies and subclasses. Low individual PvRON2 B-epitope responder frequency was observed (20% 39041, 10% 39042, 6.6% 39043 and 39044); such low responses have previously been reported for other bloodstage antigens such as PvMSP8. A loss of mean response to a target protein has been observed as time has elapsed when average response has been about ten times greater to recombinant protein than linear epitopes in acute-infection patients (Cheng et al., 2017). This has been associated with short-lived antibodies, due to short-lasting memory responses or parasite-induced Bcell dysregulation (Rénia and Goh, 2016) and parasite genetic variations or in exposed populations. Antibody response differences against PvRON2 B-cell epitopes between exposed individuals from Chocó (83%) and Córdoba (17%), could be attributed to several factors, including the level of parasitemia and the number of episodes (Druilhe and Pérignon, 1994). The last 3 SIVIGILA reports (2015-2017), showed a higher incidence of P. vivax infection in Chocó regarding Córdoba (Instituto-Nacional-De-Salud, 2017). Other intrinsic factors from the responders such as their HLA, sex, age, psychological stress, nutrition and other infectious diseases could also be involved in such differences (Van Loveren et al., 2001). Two of the selected B-epitopes (39042 and 39044) were located on an α-helical coiled motif protein and other studies have shown that selected in silico peptides related to these motifs have been recognized by naturally-acquired antibodies and have been immunogenic in mice (Villard et al., 2007;Arévalo-Pinzón et al., 2011); however, 39042 (10%) and 39044 (6.6%) epitopes had the lowest recognition values in our study. 39041 had significant differences in the IgG subclasses analyzed; IgG2 predominated while low IgG4 and IgG1 levels were observed. A predominant IgG2 response and low IgG4 reactivity in previous studies has been associated with P. falciparum infection resistance and clearance, IgG2/IgG4 relationship being associated with a protective role (Aucan et al., 2000). Similar results have been found in PvMSP8 studies recording IgG2 noncytophilic antibody predominance which has been associated with resistance to P. vivax malaria (Cheng et al., 2017). 39041 has been seen to be immunogenic in mice (Arévalo-Pinzón et al., 2011) and its potentially protective role makes this peptide a pivotal PvRON2 epitope for inclusion in a subunit-based vaccine.
It has been thought that antibodies would be enough to protect against malaria and that T-cells do not play an important role during the erythrocyte stage. Advances in immunology-related knowledge have demonstrated that B-cells must be activated by CD4+ T-helper cells to prompt good humoral responses, thereby inducing cytokine, memory cell and antibody production (Batista and Harwood, 2009;Tubo et al., 2013;Yuseff et al., 2013). The role of exposed patients' T-cell response against PvRON2 high-affinity binding peptides was studied in cytokine proliferation and production assays. Only exposed individuals' PBMC cultures showed proliferation induced by universal and specific binding peptides, suggesting that PvRON2 induced memory T-cells against high-affinity peptides. However, Th1 and Th2 cytokine responses were low, except for IL-6. Low cytokine responses/production have been observed in other studies; Silva-Flannery et al., found that immunization with monomeric peptide did not result in peptide-specific IFN-γ-secreting cell expansion and was not protective. They also reported that the monomeric peptide was less taken up by antigen-presenting cells and was not going through the phagolysosome (Silva-Flannery et al., 2009).
Cytokine production by unexposed individuals' PBMCs against PvRON2 synthetic peptides may have been due to dendritic or macrophages cells priming naïve T-cells and inducing effector T-cell cytokine production; nevertheless, secretion was very low for some of them.
Unlike the other cytokines tested here, IL6 was highly secreted by exposed patients' PBMCs; this was not surprising, since one of IL6's multi-functions is to stimulate hybridoma and plasmacytoma cell growth and help antibody production (Matsuda et al., 1988). IL6, together with IL12 and VDR, have been associated with reduced parasitemia, its severity and 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. gametocytemia clearance in P. vivax-exposed individuals (Sortica et al., 2014). P. vivax lysate-induced cytokine responses in unexposed individuals' PBMCs could be explained by innate immune cell cytokine production, i.e., macrophages, dendritic cells, NK, NKT and naïve T-cells which become effector cells (Stevenson and Riley, 2004). High cytokine induction in healthy individuals compared to P. vivax-exposed individuals might be related to a parasite evasion mechanism for inhibiting effective immune responses able to eliminate the parasite (Rénia and Goh, 2016). Nonetheless, the healthy individuals had not been exposed to P. vivax since immunofluorescence assays did not show their antibodies' reactivity to the parasite.
Taken together, the in silico T-cell and B-cell epitope selection results highlighted two T-cell epitopes (39047 and 39154) and one B-cell epitope (39041) as promising vaccine candidates. Despite the significant differences observed in immune responses evoked in the exposed individuals compared to the control group, overall the responses were relatively low. All selected peptides were conserved among the 11 P. vivax strains which may also explain such low immune responses. This has been demonstrated in P. falciparum studies where conserved high activity binding peptides (HABPs) were poorly antigenic and poorly immunogenic (Patiño et al., 1997;Lougovskoi et al., 1999;Ocampo et al., 2000;Parra et al., 2000;Hensmann et al., 2004). It should be stressed that caution must be taken, since using these 3 promising peptides in a multi-epitope vaccine in their unmodified state would probably mean that they could induce low immunogenicity and not provide longlasting protection; however, proven approaches have shown that modifying their critical residues should induce a strong and longlasting protection-inducing immune response (Patarroyo et al., 2010).
HABPs have been seen to be P. falciparum vaccine candidates during the last two decades ; however, they must be modified to make them antigenic and protectioninducing by replacing critical aa with others having the same mass but different polarity Patarroyo et al., 2011). Such HABPs can only be used in a tailor-made vaccine targeting a specific HLA-DRB1 * endemic population; however, a universal protection-inducing vaccine will require studying other peptides which can bind to other HLA-DRB1 * alleles. Future studies should be carried out using modified peptides aimed at assessing immunogenicity and protectioninducing ability in the Aotus experimental model to confirm their suitability as P. vivax vaccine candidates. Likewise, additional peptides should be included to cover all parasite stages, aiming at a 100% protection-inducing, multistage, multi-epitope, minimal subunit-based vaccine.

AUTHOR CONTRIBUTIONS
CL: designed and performed the experiments, analyzed the data, drafted the manuscript. YY-P: designed and performed the experiments, analyzed the data, drafted the manuscript, designed the figures. DD-A: drafted the manuscript. MEP: critical suggestions regarding the manuscript. MAP: conceiving the work and drafting all versions of the manuscript. All authors have revised the manuscript and approved the version to be submitted.

FUNDING
This work was financed by the Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) through grant RC # 0309-2013. CL received support from Colciencias within the framework of the Convocatoria Nacional para Estudios de Doctorado en Colombia (call for candidates # 6172). and 39154) and P. vivax lysate. IFN-γ, TNF, IL-10, 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 of all values.
Supplementary Figure 2 | Immunofluorescence patterns for exposed individuals from Colombia's P. vivax-endemic areas and the control group. The upper panels show one Bahía Solano's exposed individual serum recognition of pRBC. Nuclei were stained with DAPI, the parasite with anti-parasite FITC and then, both were merged. The bottom panels show serum from one non-exposed individual.
Supplementary Figure 3 | IgG antibody response against PvGAMA control recombinant protein. Significant differences (calculated by Mann-Whitney test) are shown between samples from exposed individuals and control. The dashed line indicates the cut-off point for seropositive samples.