RESEARCH ARTICLE Polymorphisms in B Cell Co-Stimulatory Genes Are Associated with IgG Antibody Responses against Blood–Stage Proteins of Plasmodium vivax Gustavo C. Cassiano1*, Adriana A. C. Furini2, Marcela P. Capobianco1, Luciane M. Storti- Melo3, Maristela G. Cunha4, Flora S. Kano5, Luzia H. Carvalho5, Irene S. Soares6, Sidney E. Santos7, Marinete M. Póvoa8, Ricardo L. D. Machado1,8 1 Department of Biology, São Paulo State University (Universidade Estadual Paulista - UNESP), São José do Rio Preto, state of São Paulo (SP), Brazil, 2 Department of Dermatologic, Infectious, and Parasitic Diseases, College of Medicine of São José do Rio Preto, São José do Rio Preto, SP, Brazil, 3 Department of Biology, Federal University of Sergipe, Aracaju, Sergipe, Brazil, 4 Laboratório of Microbiology and Immunology, Institute of Biological Sciences, Federal University of Pará (Universidade Federal do Pará - UFPA), Belém, state of Pará (PA), Brazil, 5 Laboratory of Malaria, René Rachou Research Center, Oswaldo Cruz Foundation, Belo Horizonte, state of Minas Gerais (MG), Brazil, 6 Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of São Paulo (Universidade de São Paulo - USP), São Paulo, SP, Brazil, 7 Laboratory of Human and Medical Genetics, Federal University of Pará, Belém, PA, Brazil, 8 Laboratory of Basic Research in Malaria, Section of Parasitology, Evandro Chagas Institute, Belém, PA, Brazil * gcapatti@hotmail.com Abstract The development of an effective immune response can help decrease mortality from malaria and its clinical symptoms. However, this mechanism is complex and has significant inter-individual variation, most likely owing to the genetic contribution of the human host. Therefore, this study aimed to investigate the influence of polymorphisms in genes involved in the costimulation of B-lymphocytes in the naturally acquired humoral immune response against proteins of the asexual stage of Plasmodium vivax. A total of 319 individuals living in an area of malaria transmission in the Brazilian Amazon were genotyped for four SNPs in the genes CD40, CD40L, BLYS and CD86. In addition, IgG antibodies against P. vivax api- cal membrane antigen 1 (PvAMA–1), Duffy binding protein (PvDBP) and merozoite surface protein 1 (PvMSP–119) were detected by ELISA. The SNP BLYS –871C>T was associated with the frequency of IgG responders to PvAMA–1 and PvMSP–119. The SNP CD40 –1C>T was associated with the IgG response against PvDBP, whereas IgG antibody titers against PvMSP–119 were influenced by the polymorphism CD86 +1057G>A. These data may help to elucidate the immunological aspects of vivax malaria and consequently assist in the design of malaria vaccines. PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 1 / 15 OPEN ACCESS Citation: Cassiano GC, Furini AAC, Capobianco MP, Storti-Melo LM, Cunha MG, Kano FS, et al. (2016) Polymorphisms in B Cell Co-Stimulatory Genes Are Associated with IgG Antibody Responses against Blood–Stage Proteins of Plasmodium vivax. PLoS ONE 11(2): e0149581. doi:10.1371/journal. pone.0149581 Editor: Laurent Rénia, Agency for Science, Technology and Research - Singapore Immunology Network, SINGAPORE Received: September 28, 2015 Accepted: February 1, 2016 Published: February 22, 2016 Copyright: © 2016 Cassiano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its supporting information files. Funding: Conselho Nacional de Desenvolvimento Científico e Tecnológico, No 471605/2011-5, http:// www.cnpq.br/, RLDM, and Fundação Amazônia de Amparo a Estudos e Pesquisas do Pará, No ICAAF- 005/2011, http://www.fapespa.pa.gov.br/, MMP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. http://crossmark.crossref.org/dialog/?doi=10.1371/journal.pone.0149581&domain=pdf http://creativecommons.org/licenses/by/4.0/ http://www.cnpq.br/ http://www.cnpq.br/ http://www.fapespa.pa.gov.br/ Introduction Plasmodium vivax is the most prevalent species outside Africa, and P. vivax infections are responsible for the high morbidity observed in affected populations despite the lower lethality compared with infections caused by P. falciparum [1]. In endemic areas for malaria, particu- larly where the transmission rate is high, as age and exposure increase, subjects tend to become less susceptible to malaria episodes due to the development of an effective immune response against the parasite [2]. The role of antibodies in protection against malaria is well docu- mented, and the passive transfer of antibodies from the serum of immune individuals to patients infected with P. falciparum effectively controls blood-stage parasites and reduces the clinical signs of the disease [3, 4]. Therefore, the development of a vaccine capable of providing protection against the blood stages of the malaria parasite will greatly decrease the clinical and economic burden of the disease. The blood stage proteins of Plasmodium considered to be the main candidate targets for vaccine development include merozoite surface protein 1 (MSP–1), Duffy binding protein (DBP), and apical membrane antigen 1 (AMA–1). After two successive cleavages, only a 19 kDa C-terminal portion of MSP–1 (MSP–119) remains anchored to the surface of merozoites during erythrocyte invasion, and it is believed that MSP–119 is involved in the initial adhesion of merozoites to erythrocytes [5]. AMA–1 is an integral membrane protein that is essential for the reorientation of merozoites prior to erythrocyte invasion [5]. Furthermore, the binding of AMA–1 to rhoptry neck protein (RON2) is an important step in the formation of the junction complex during invasion [6]. In P. vivax, the binding of DBP to its receptor Duffy antigen receptor for chemokines (DARC) plays an important role in the binding of merozoites of this species to host reticulocytes [7]. Antibodies directed against these proteins have been shown to inhibit the binding of these proteins and prevent the invasion of erythrocytes by merozoites [8–11]. In addition, some longitudinal studies have associated AMA–1 and MSP–119 antibod- ies with a decreased risk of malaria [12,13]. B cells require two types of signals to become activated and produce antibodies. The first sig- nal is provided by antigen binding to the B cell receptor (BCR). Activated T cells generally pro- vide the second signal for B cell activation through a variety of proteins. The CD40 protein is a member of the tumor necrosis factor (TNF) receptor family, which are expressed on the surface of a wide variety of cells, including B cells. The binding of CD40 to its ligand CD40L expressed on the surface of activated T cells provides the major costimulatory signal for B cells to mount a humoral response [14]. The interaction mediated by this signaling pathway is responsible for B cell proliferation and differentiation, immunoglobulin isotype switching, and antibody secretion [15,16]. Upon B cell activation, the expression of the CD86 molecule increases. In addition to the important role of this molecule in T cell activation, the binding of CD86 to its receptor, CD28, provides bidirectional signals that appear to be important for IgG production in B cells [17]. B-lymphocyte stimulator (BLyS) is a member of the TNF family present on the surface of many cells, including monocytes, macrophages, and activated T cells, or it can occur in a soluble form. Its main function is to provide signals for B cell survival and proliferation [18]. It is known that the genetic component of the host plays an important role in the develop- ment of an immune response against malaria [19]. The role of gene polymorphisms in the immune system in the production of naturally acquired antibodies has been documented in P. falciparum [20–27]. However, few studies have assessed the genetic mechanisms involved in the production of antibodies against P. vivax proteins [28–31]. Thus, this study aimed to evalu- ate the effects of single nucleotide polymorphisms (SNPs) in the genes CD40, CD40L, CD86 and BLYS on the production of IgG antibodies against candidate vaccine proteins from P. vivax in a naturally exposed population in the Brazilian Amazon. B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 2 / 15 Competing Interests: Luzia H. Carvalho is a PLOS ONE Editorial Board member. Materials and Methods Study area and population sample The study was conducted in the municipality of Goianésia do Pará (03°50'33 "S, 49°05'49" W), approximately 300 km from the city of Belém, capital of the state of Pará, in the Brazilian Ama- zon region. The climate is tropical semi-humid, with an average annual temperature of 26.3°C and average annual rainfall of approximately 2,000 mm3. In this municipality, despite the seasonal rainfall pattern characterized by a dry season between June and November and a rainy season between December and May, malaria trans- mission is unstable and occurs throughout the year. The annual parasite incidence rates in 2011 and 2012 were 99 and 39 per 1,000 inhabitants, respectively. More than 80% of malaria cases are due to P. vivax, and the main vector in the region is Anopheles darlingi (Primo, unpublished data). Samples were collected at the municipal health center between February 2011 to August 2012, and 223 individuals infected with P. vivax with classic symptoms of malaria who sought the malaria diagnostic service were recruited. In addition, 96 uninfected individuals who sought medical care offered during the study were invited to participate in the study. These participants had no close kinship and, therefore, were genetically unrelated, which was evi- denced by a demographic questionnaire. Samples from 40 malaria-naive individuals residing in a non-endemic area (São José do Rio Preto, Brazil) and who never visited malaria transmis- sion areas were used as controls. Blood sample collection and malaria diagnosis After applying a questionnaire to assess demographic and epidemiological data, blood was col- lected in EDTA-containing test tubes, after which plasma samples were separated by centrifu- gation and stored at –20°C. Malaria was diagnosed using thick smears stained with Giemsa according to the malaria diagnosis guidelines of the Brazilian Ministry of Health. Subsequently, all participants (including the non-infected) had their diagnoses confirmed by nested–poly- merase chain reaction (PCR) [32]. All participants or their guardians signed an informed con- sent form. The project was approved by the health authorities of Goianésia do Pará and by the Research Ethics Committee of the College of Medicine of São José do Rio Preto (CEP/FAMERP No. 4599/2011). Genotyping of the genes CD86, CD40L, CD40, and BLYS DNA was extracted from peripheral blood samples using the Easy-DNA™ extraction kit (Invi- trogen, California, USA). The following SNPs were identified using PCR–restriction fragment length polymorphism (RFLP): +1057G>A in CD86 (rs1129055), –726T>C in CD40L (rs3092945), –1C>T in CD40 (rs1883832), and –871C>T in BLYS (rs9514828). To amplify the polymorphisms in CD40 and CD40L, the protocol described by Malheiros and Petz-Erler [33] was used with modifications, and amplification of the polymorphisms in CD86 and BLYS fol- lowed the protocol described by Cassiano et al. [34]. Briefly, all PCR reactions were performed in a final volume of 25 μL containing 1× Buffer (20 mM Tris-HCl, 50 mM KCl, pH 8.4), 1.5 mMMgCl2, 0.2 mM of each dNTP, 0.4 pmol of each primer, and 0.5 U of Platinum Taq DNA Polymerase (Invitrogen, São Paulo, Brazil). Amplification was performed under the following reaction conditions: an initial step of 5 min at 94°C, 35 cycles of 30 s at 94°C, 30 s at 56°C (except for the SNP in gene BLYS, where the annealing temperature was 50°C) followed by 1 min at 72°C, and a final step of 10 min at 72°C. The amplification products were digested using restriction enzymes (Fermentas, Vilnius, Lithuania) according to the manufacturer's B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 3 / 15 recommendations. Primer sequences and their restriction enzymes, and the restriction frag- ments obtained after digestion of each polymorphism are presented in S1 Table. Antigens Three recombinant P. vivax proteins were used in this study. PvMSP–119, corresponding to amino acids 1616–1704 of MSP–1 protein from the Belém strain, was expressed in Escherichia coli with a polyhistidine affinity tag (6xHis tag) [35]. A gene coding for a recombinant protein corresponding to amino acids 43–487 of the ectodomain of PvAMA–1 was synthesized by GenScript USA Inc. (Piscataway, NJ) and expressed in Pichia pastoris [11]. Region II of DBP of P. vivax strain Sal1 (PvDBP), which includes amino acids 243–573, was expressed in E. coli as a 6xHis fusion protein [36]. Antibody assays The assessment of IgG antibodies against P. vivax recombinant proteins was performed as described previously [11, 35, 36]. Briefly, the concentrations used for PvMSP–119, PvAMA–1, and PvDBP were 2 μg/mL, 2 μg/mL, and 3 μg/mL, respectively. All plasma samples were diluted at 1:100 and added in duplicate. Monoclonal antibody binding was detected using per- oxidase conjugated anti-human immunoglobulin (Sigma, St Louis, USA). The results for total IgG are expressed as reactivity index (RI), which was calculated by dividing the optical density (OD) of the sample by the cut-off value, which in turn was calculated by averaging the OD val- ues of the 40 plasma samples from the control subjects residing in the non-endemic area plus three standard deviations. Individuals with RI> 1 (also known as responders) were considered positive. Estimates of interethnic admixture The population of northern Brazil is highly mixed and formed mainly by crosses between Europeans, Africans, and Native Americans. To avoid spurious interpretations resulting from population substructure, we used a panel of 48 ancestry informative markers (AIMs) to esti- mate the proportion of individual interethnic admixture in our sample, following a previously described protocol [37]. The Structure software version 2.3.4 was used, and three parental pop- ulations (European, African, and Native Americans) were assumed as described by Santos et al. [37]. These estimates were used as covariates in the multivariate analyses to adjust for popula- tion stratification. Statistical analysis Statistical analysis was performed using the SNPassoc R package (R software version 3.1.1) [38]. Genotypic deviations from Hardy-Weinberg equilibrium were assessed using the exact test described by Wigginton et al. [39]. For univariate analysis, differences in proportions were assessed using the chi-square test, and differences between means were assessed using Student's t-test or Mann-Whitney U-test, depending on whether the data were parametric. The correla- tion between IgG antibody titers against PvAMA–1, PvDBP, and PvMSP-119 was assessed using the Spearman correlation coefficient. The analysis of association between the SNPs and the antibody responses frequency used a logistic regression model, and the factors significantly associated with the antibody responses in the univariate analysis (gender, previous history of malaria infection, and current infection) were included as covariates. Similarly, generalized lin- ear regression was used to assess associations between the SNPs and the magnitude of the IgG antibody responses. In all the multivariate analyses, SNPs were included following different B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 4 / 15 genetic models: dominant (11 vs 12 + 22), recessive (11 + 12 vs 22), and log-additive (0, 1, 2 alleles). Values of p<0.05 were considered significant. Results Population profile The profile of the study population is summarized in Table 1. The age of the study population varied between 14 and 68 years (median of 30 years) and the male/female sex ratio was 1.47. The period of residence in the study area varied between 0 (newly arrived migrants) to 37 years (median of 7 years). The exact period of time for which individuals had been continuously exposed to malaria could not be reliably determined because of the high rates of migration characteristic of the Brazilian Amazon population. As previously reported by Cassiano et al. [34], the population of Goianésia do Pará is highly mixed, showing a higher proportion of European genetic ancestry (44%) and significant contributions of African (31.4%) and Native American ancestry (24.6%). Most participants (78.7%) reported having had previous malaria infections, and at the time of blood collection, 69.9% (223/319) were infected with P. vivax (diagnosed by thick smear). Further analysis by nested PCR indicated that 13 of the 223 infected individuals (5.8%) were infected with both P. falciparum and P. vivax, and no individ- ual diagnosed as negative by thick smear was positive by nested PCR. Naturally acquired IgG antibodies against blood-stage proteins of P. vivax Of the 319 participants, 296 (92.8%) had their plasma samples evaluated for IgG against PvAMA–1, 284 (89.0%) were evaluated for IgG against PvDBP, and 291 (91.2%) were evalu- ated for IgG against PvMSP–119, which reflects the differences shown in Table 1 regarding the total number of individuals. In addition, 69.1% (202/291) of the participants had antibodies (RI> 1) against PvMSP–119, 63.4% (180/284) had antibodies against PvDBP, and 55.4% (164/296) had antibodies against PvAMA–1. Among the subjects evaluated for the three pro- teins, 80% (223/279) had antibodies against at least one protein and 46.2% (129/279) showed responses against all three proteins. Significant positive correlations were observed between the IgG antibody titers against PvAMA–1 and PvMSP–119, between PvAMA-1 and PvDBP, and between PvDBP and PvMSP–119 (r = 0.72, 0.68, and 0.51, respectively, using Spearman correla- tion, p< 0.0001). We assessed whether the frequency of individuals with antibodies against the proteins studied was correlated with any demographic or epidemiological variables (Table 1). The genetic ances- try proportions did not differ between subjects with or without antibodies (all p values> 0.34). Furthermore, no association was observed between the period of residence in the study area and the antibody response. However, a higher frequency of responders to all the proteins evaluated was observed among male subjects (p< 0.001). A higher proportion of individuals who had never contracted malaria was observed among those without antibodies (p< 0.0001) regardless of the protein evaluated. Furthermore, as expected, individuals who were infected at the time of blood collection showed a higher frequency of responses to all three proteins evaluated (p< 0.0001). Associations of B-cell co-stimulatory gene polymorphisms with the frequency of IgG responses against PvAMA-1, PvDBP, and PvMSP–119 Polymorphisms in the genes studied were successfully genotyped in all samples, except for SNP rs1883832 in the CD40 gene, which was not identified in two samples. No significant deviation B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 5 / 15 from Hardy-Weinberg equilibrium was observed for any polymorphism (all p values> 0.06). The minor allele frequencies (MAF) were as follows: 0.249 for SNP rs9514828 in the BLYS gene (allele T), 0.216 for SNP rs1129055 in the CD86 gene (allele A), 0.155 for SNP rs1883832 in the CD40 gene (allele T), and 0.112 for SNP rs3092945 in the CD40L gene (allele C) (S2 Table). These allele frequencies were similar to those found in a previously analyzed subset of these samples [34]. The effects of polymorphisms in the BLYS, CD86, CD40, and CD40L genes on the IgG anti- body responses against the proteins PvAMA–1, PvDBP, and PvMSP–119 are shown in Table 2 and S3 Table. The additive, recessive, and dominant genetic models were tested for each SNP. With regard to PvAMA–1, the IgG antibody response was positively correlated with the pres- ence of the T allele for SNP rs9514828 in the BLYS gene based on an additive model (OR = 1.59, 95% CI: 1.05–2.40; p = 0.03). The T allele for SNP rs1883832 in the CD40 gene also followed an additive model and was negatively correlated with the IgG antibody response against PvDBP (OR = 0.57; 95% CI: 0.35–0.92, p = 0.02). Considering a dominant model, there was a greater likelihood for individuals harboring the T allele in genotypes TT and TC of SNP rs9514828 in the BLYS gene to have antibodies against PvMSP–119 compared with individuals who harbored the CC genotype (OR = 2.01; CI: 1.12–3.61, p = 0.01). An analysis of interactions between the polymorphisms produced no additional information beyond the information obtained by the individual analysis of the polymorphisms (data not shown). In addition, we evaluated the distribution of genotypes/alleles according to the number of proteins for which individuals had antibodies, i.e., whether they responded against one, two, three, or no proteins Table 1. Summary of the epidemiological data and seropositivity of the study population. PvAMA-1 PvDBP PvMSP-119 Characteristics All individuals (319)a Positive (164)a Negative (132)a pe Positive (180)a Negative (104)a pe Positive (202)a Negative (89)a pe Gender, male (%) 59.6 68.3 47.7 0.0004 64.8 47.1 0.0004 66.8 40.4 <0.0001 Age, median years (range) 30 (14–68) 30.5 (14–68) 29 (14–66) 0.52 29 (14–68) 30.5 (14–66) 0.63 30 (14–68) 29 (15–65) 0.37 Time of residenceb, median years (range) 7 (0.1–37) 6.0 (0.1–37) 8.5 (0.1–37) 0.06 6.5 (0.1–37) 8.0 (0.1–37) 0.16 6.5 (0.1–37) 8.5 (0.1–37) 0.15 Genetic ancestryc, mean ± SD (%) African 31.4 ± 11.0 31.3 ± 11.6 31.9 ± 10.3 0.66 31.6 ± 11.1 31.8 ± 11.3 0.92 31.6 ± 11.4 30.6 ± 9.7 0.55 European 44.0 ± 11.9 44.0 ± 11.9 43.8 ± 12.6 0.91 44.1 ± 11.8 43.6 ± 13.2 0.76 43.6 ± 12.4 45.3 ± 11.6 0.34 Native American 24.6 ± 9.4 24.6 ± 9.7 24.2 ± 9.1 0.70 24.3 ± 9.5 24.6 ± 9.5 0.77 24.8 ± 9.4 24.1 ± 9.5 0.60 Previous malaria infectiond (%) 78.7 95.0 55.9 <0.0001 92.0 52.2 <0.0001 90.1 49.4 <0.0001 Individuals infected with P. vivax (%) 69.9 81.7 50.0 <0.0001 78.9 44.2 <0.0001 80.2 37.1 <0.0001 aNumber of individuals. The differences in the total number of individuals evaluated for each protein corresponding to samples that lacked plasma. bTime of residence in Goianésia do Pará. cData of genetic ancestry obtained from 273 individuals. dProportion of individuals who contracted malaria in the past. eP-values were calculated from a chi-squared test for qualitative variables, the Mann-Whitney test for nonparametric continuous variables and Student’s t- test for parametric continuous variables. doi:10.1371/journal.pone.0149581.t001 B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 6 / 15 (Fig 1). It was observed that the frequency of carriers of the T allele of SNP rs1883832 in the CD40 gene progressively decreased as the antibody response increased (χ2 = 9.01; p = 0.002, Chi-squared for trends). In addition, the presence of allele T from SNP rs9514828 in gene BLYS was higher among the individuals who responded against all proteins tested (χ2 = 25.30; p< 0.0001, Chi-square for trends). Associations of polymorphisms with IgG antibody titers against PvAMA– 1, PvDBP, and PvMSP–119 We evaluated the effects of the polymorphism on IgG antibody titers against the three P. vivax proteins. The variables that affected antibody titers, corresponding to the same variables associ- ated with response frequency (gender, past history of malaria infection, and current infection) (S4 Table), were included in the multivariate analysis together with the polymorphisms. There was no significant effect of any genotype/allele investigated on the antibody titers (Fig 2). How- ever, when individuals with or without a P. vivax infection at the time of blood collection were analyzed separately, a significant increase in IgG antibody titers against PvMSP–119 was observed among infected individuals harboring genotype AA of the CD86 polymorphism com- pared to those harboring the GG and GA genotypes (median [Q1–Q3]: 7.87 [4.74–8.40] vs. 5.05 [1.56–7.60]; p = 0.03). Table 2. Associations between Polymorphisms and Antibody Responses against Blood-Stage Proteins of P. vivax. PvAMA-1 PvDBP PvMSP-119 Gene SNP Model Genotype OR (95%CI)a pb OR (95%CI)a pb OR (95%CI)a pb BLYS rs9514828 Dominant C/C 1.00 0.04 1.00 0.39 1.00 0.01 C/T–T/T 1.68 (1.01–2.79) 1.26 (0.74–2.14) 2.01 (1.12–3.61) Recessive C/C–C/T 1.00 0.22 1.00 0.80 1.00 0.83 T/T 1.91 (0.65–5.57) 0.87 (0.30–2.51) 0.89 (0.29–2.74) Log-Additive 0,1,2 1.59 (1.05–2.40) 0.03 1.14 (0.74–1.74) 0.56 1.47 (0.91–2.37) 0.11 CD86 rs1129055 Dominant G/G 1.00 0.85 1.00 0.45 1.00 0.22 G/A–A/A 0.95 (0.57–1.58) 1.23 (0.72–2.11) 0.70 (0.39–1.24) Recessive G/G–G/A 1.00 0.61 1.00 0.17 1.00 0.31 A/A 1.32 (0.45–3.85) 2.19 (0.69–6.96) 1.84 (0.56–6.07) Log-Additive 0,1,2 1.01 (0.67–1.52) 0.97 1.28 (0.83–1.97) 0.26 0.88 (0.56–1.38) 0.58 CD40 rs1883832 Dominant C/C 1.00 0.36 1.00 0.03 1.00 0.31 C/T–T/T 0.77 (0.44–1.35) 0.53 (0.30–0.94) 0.73 (0.39–1.35) Recessive C/C–C/T 1.00 0.66 1.00 0.21 1.00 0.92 T/T 1.35 (0.35–5.27) 0.39 (0.09–1.66) 1.09 (0.23–5.13) Log-Additive 0,1,2 0.87 (0.55–1.37) 0.54 0.57 (0.35–0.92) 0.02 0.81 (0.48–1.35) 0.42 CD40Lc rs3092945 Dominant T/T 1.00 0.63 1.00 0.36 1.00 0.70 T/C–C/C 1.24 (0.51–3.06) 1.54 (0.61–3.88) 0.83 (0.31–2.18) Recessive T/T–T/C 1.00 0.70 1.00 0.60 1.00 0.92 C/C 1.46 (0.21–10.39) 0.59 (0.09–4.03) 0.89 (0.08–9.86) Log-Additive 0,1,2 1.21 (0.59–2.48) 0.60 1.24 (0.58–2.64) 0.58 0.86 (0.38–1.94) 0.72 aOR stands for odd ratio and CI stands for confidence intervals. bp values based on fitting logistic regression models adjusted for gender and current malaria infection. P values < 0.05 are in bold. cGenotypes available only for women because the CD40L gene is located on chromosome X. doi:10.1371/journal.pone.0149581.t002 B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 7 / 15 Fig 1. Positive antibody response and carrier frequency of mutant alleles. Frequency of carriers of mutant alleles of SNPs in genesCD40, BLYS, CD86, andCD40L according to the number of proteins for which subjects were responders. Individuals with antibodies against the three proteins (n = 279) were classified according to their reaction against zero (n = 57), one (n = 55), two (n = 38), or three (n = 129) proteins of blood-stage P. vivax. doi:10.1371/journal.pone.0149581.g001 B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 8 / 15 Fig 2. BLYS,CD40,CD86, andCD40L genotypes in relation to antibody titers against the merozoite proteins. Antibody titers were expressed as log- transformed reactivity indices (RI). For the SNP in the geneCD40L, men and women harboring the C allele were grouped and compared with individuals who did not possess this allele given that CD40L is located on chromosome X. Multivariate logistic regression analysis found no significant differences in the antibody titers between the different genotypes. doi:10.1371/journal.pone.0149581.g002 B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 9 / 15 Discussion The present study aimed to evaluate the effects of polymorphisms in co-stimulatory genes of B cells on antibody responses against recombinant proteins of P. vivax in a population sample from the Brazilian Amazon. Although the number of studies aimed at identifying genetic mechanisms involved in regulating the production of antimalarial antibodies has increased in recent years [20–31], the development of an effective vaccine will most likely require a thor- ough understanding of the host–parasite relationship. Therefore, studies such as the one reported herein will help to elucidate the responses of individuals to certain vaccine protein candidates. Importantly, in this study, potential biases due to population substructure were considered. The frequencies of the alleles CD86 +1057A and CD40L –726T are higher among individuals with higher proportions of European ancestry [34], and consequently, if individuals with anti- bodies exhibited more European ancestry than non-responders, a false association between these alleles and antibody production could be found. However, the fact that no differences were observed in the proportion of genetic ancestry between the responders and non-respond- ers for the three proteins studied and our inclusion of individual ancestry values as covariates in the multivariate analyses precluded the possibility of false associations in our results. Differences were observed in the frequency of responders against the three proteins evalu- ated, and the proportion of responders against PvMSP–119 was the highest (69.1%), followed by responders against PvDBP (63.4%), and those against PvAMA–1 (55.4%). The higher pro- portion of responders against PvMSP–119 was an expected result considering that previous studies have shown that this protein is highly immunogenic [40, 41], most likely owing to the low degree of polymorphism found in the MSP–1 region [42], and also because MSP–119 is car- ried into the infected cell, persists until the end of the intracellular cycle, accumulates in the digestive vacuole, and is discarded together with digestion products, possibly increasing its exposure to the immune system [43]. Both the frequency of individuals with antibodies and the magnitude of the IgG response were significantly higher in individuals who had had previ- ous episodes of malaria and/or who were infected at the time of blood collection, suggesting the occurrence of a boosting effect in the antibody responses directed against these proteins compared with individuals who never contracted malaria. In addition, it was noted that the IgG response against the three proteins was higher in men than women. Although the reasons for this result remain unclear [29], men are most likely more exposed to malaria transmission from working for longer periods in the field compared to women. The main result found in our study was that polymorphisms in the genes CD40 and BLYS seem to influence the IgG antibody responses against PvAMA–1, PvDBP, and PvMSP–119 of P. vivax in the population studied. Considering that the binding of CD40 to its ligand CD40L is critical for the production of antibodies in B cells [14], it is possible that this molecule is involved in the immune response against malaria. Indeed, this co-stimulatory pathway is important for the production of IgG antibodies against P. falciparum proteins because PBMCs from individuals living in holo- or meso-endemic malaria areas were found to produce more antibodies in vitro when CD40L costimulation was provided [44]. In our study, the presence of the T allele of SNP rs1883832 in the CD40 gene was negatively correlated with the production of IgG antibodies against PvDBP. This polymorphism is located at position –1 of the start codon and affects the Kozak sequence, which is crucial to the initiation of the protein transla- tion process [45], and it is believed that the presence of the T allele can decrease gene expres- sion by 15%–30% [46]. Whether the correlation between the SNP in the CD40 gene and the production of IgG antibodies against PvDBP is associated with clinical immunity requires fur- ther investigation. However, it is noteworthy that the frequency of the T allele is significantly B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 10 / 15 lower in African populations than in European populations [47], and this frequency may sug- gest a selective advantage of the C allele due to pressure exerted by malaria. However, in a case- control study conducted in the city of Macapá in the Brazilian Amazon, no association was found between polymorphism in the CD40 gene and susceptibility to vivax malaria [48]. This same study evaluated the influence of BLYS, CD40, and CD40L polymorphisms on the anti- body response against recombinant proteins of P. vivax in a subgroup whose serological results were available, and the results indicated no correlation with antibody response. However, it is of note that this subgroup consisted of approximately 50 individuals. Moreover, it remains pos- sible that the conditions of malaria transmission and the genetic background of the populations differ between the municipalities of Macapá and Goianésia do Pará, which could explain the differences observed [48]. BLyS is a critical cytokine for B cell survival and differentiation; its serum levels were found to be higher among children with acute malaria and were positively correlated with the levels of IL-10 and IFN-γ [49]. A study conducted by Liu et al. [50] demonstrated that the production of memory B cells in response to vaccination with MSP–119 from P. yoelli in mice was depen- dent on the production of BLyS by dendritic cells (DCs). In our study, the presence of the T allele of SNP rs9514828 in the BLYS gene was positively correlated with the production of IgG antibodies against PvAMA–1 and PvMSP–119. This SNP is located in the promoter region of the gene at position -871 relative to the start codon, corresponding to a binding site for the transcription factor MZF1, and it can therefore modulate gene expression [51]. However, the role of this SNP in gene expression has not been elucidated—some studies have associated the T allele with higher levels of BLYSmRNA [51,52], whereas others have not established this association [53]. It is of interest that the observed associations involving these two SNPs could not be extrap- olated to the antibody response against all proteins tested, i.e., although SNP BLYS rs9514828 was associated with the response of IgG antibodies against PvAMA-1 and PvMSP-119, no asso- ciation involving this SNP and PvDBP was found. Similarly, SNP CD40 rs1883832 was associ- ated with PvDBP but not with PvAMA-1 and PvMSP-119. Although the reasons for these results are unknown, they are likely to reflect intrinsic differences among these proteins, including the degree of polymorphism, exposure to the immune system, and antigen presenta- tion via HLA, among others. However, as shown in Fig 1, there was a higher frequency of allele T carriers of SNP BLYS rs9514828 among those individuals who had antibodies against all three proteins evaluated, whereas the frequency of allele T carriers of SNP CD40 rs1883832 was lower among the responders of all proteins. In addition, we found a correlation between SNP rs1129055 in the CD86 gene and the mag- nitude of the IgG response against PvMSP-119, but only among individuals infected with P. vivax. In murine models of malaria, the CD86 molecule seems to be involved in the differentia- tion of the Th2-type response [54]. Furthermore, for gene CD28, which is the receptor of ligand CD86, was observed a lower production of IgG antibodies against AMA-1 and MSP-1 when CD28 knockout mice were infected with P. chabaudi [55]. In our study, we found that individu- als infected with P. vivax harboring the AA genotype had the highest antibody titers against PvMSP-119. The SNP rs1129055 is located in exon 8 of the gene and causes a non-silent substi- tution of alanine for threonine at amino acid position 304 of the protein, introducing a poten- tial phosphorylation site in the cytoplasmic region of the molecule [56]. Although the functional implications of this polymorphism are not yet elucidated, we speculate that individ- uals with the AA genotype infected with P. vivaxmay have a response directed more towards the Th2-type, with increased production of antibodies, particularly against PvMSP-119. In conclusion, we found evidence for the role of co-stimulatory B cell molecules in the genetic control of the immune response against P. vivax. The identification of individual B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 11 / 15 genetic traits that influence the development of an immune response may be important in the development of vaccines against malaria. However, further investigations involving these genes are necessary to confirm whether their effect on antibody production is associated with the control of P. vivax infection. Supporting Information S1 Table. Reaction conditions for the amplification and enzymatic digestion of polymor- phisms in the genes CD40, CD40L, BLYS, and CD86. (DOCX) S2 Table. Minor Allele Frequencies of Polymorphisms in Genes CD40, CD40L, BLYS, and CD86. (DOCX) S3 Table. Polymorphism Distribution between the Groups with and without Antibodies against Blood-Stage Proteins of P. vivax. (DOCX) S4 Table. Antibody levels (RI) for PvAMA-1, PvDBP, and PvMSP-119 according to gender, current infection status, and previous episode of malaria. (DOCX) Acknowledgments We are grateful to all the residents of Goianésia do Pará who made this study possible, espe- cially Darci Rodrigues, who assisted in sample collection. We are also grateful to Luciana Moran, Valéria Conceição, Katia Françoso, Michaelis Tang, and Marcos Amador for assistance with laboratory testing. Author Contributions Conceived and designed the experiments: GCC RLDM. Performed the experiments: GCC ACF MPC LMS MGC FSK LHC ISS SES. Analyzed the data: GCCMMP RLDM. Contributed reagents/materials/analysis tools: MGC FSK LHC ISS SES MMP RLDM. Wrote the paper: GCC LMS MGC FSK LHC ISS SES RLDM. Revised the manuscript critically: ACF MPC LMS. References 1. World Health Organization. World Malaria Report 2013. Geneva, Switzerland: World Health Organiza- tion; 2013. 2. Doolan DL, Dobaño C, Baird JK. Acquired immunity to malaria. Clin Microbiol Rev. 2009; 22: 13–36. doi: 10.1128/CMR.00025-08 PMID: 19136431 3. Cohen S, McGregor IA, Carrington S. Gamma-globulin and acquired immunity to human malaria. Nature. 1961; 192: 733–737. doi: 10.1038/192733a0 PMID: 13880318 4. Sabchareon A, Burnouf T, Ouattara D, Attanath P, Bouharoun-Tayoun H, Chantavanich P, et al. Para- sitologic and clinical human response to immunoglobulin administration in falciparummalaria. Am J Trop Med Hyg. 1991; 45: 297–308. PMID: 1928564 5. Li X, Chen H, Oo TH, Daly TM, Bergman LW, Liu SC, et al. A co-ligand complex anchors Plasmodium falciparummerozoites to the erythrocyte invasion receptor band 3. J Biol Chem. 2004; 279: 5765– 5771. PMID: 14630931 6. Lamarque M, Besteiro S, Papoin J, Roques M, Vulliez-Le Normand B, Morlon-Guyot J, et al. The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan para- sites. PLoS Pathog. 2011; 7: e1001276. doi: 10.1371/journal.ppat.1001276 PMID: 21347343 B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 12 / 15 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0149581.s001 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0149581.s002 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0149581.s003 http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0149581.s004 http://dx.doi.org/10.1128/CMR.00025-08 http://www.ncbi.nlm.nih.gov/pubmed/19136431 http://dx.doi.org/10.1038/192733a0 http://www.ncbi.nlm.nih.gov/pubmed/13880318 http://www.ncbi.nlm.nih.gov/pubmed/1928564 http://www.ncbi.nlm.nih.gov/pubmed/14630931 http://dx.doi.org/10.1371/journal.ppat.1001276 http://www.ncbi.nlm.nih.gov/pubmed/21347343 7. Singh SK, Hora R, Belrhali H, Chitnis CE, Sharma A. Structural basis for Duffy recognition by the malaria parasite Duffy-binding-like domain. Nature. 2006; 439: 741–744. doi: 10.1038/nature04443 PMID: 16372020 8. Egan AF, Burghaus P, Druilhe P, Holder AA, Riley EM. Human antibodies to the 19kDa C-terminal frag- ment of Plasmodium falciparummerozoite surface protein 1 inhibit parasite growth in vitro. Parasite Immunol. 1999; 21: 133–139. doi: 10.1046/j.1365-3024.1999.00209.x PMID: 10205793 9. Kocken CH, Withers-Martinez C, Dubbeld MA, van der Wel A, Hackett F, Valderrama A, et al. High- level expression of the malaria blood-stage vaccine candidate Plasmodium falciparum apical mem- brane antigen 1 and induction of antibodies that inhibit erythrocyte invasion. Infect Immun. 2002; 70: 4471–4476. doi: 10.1128/IAI.70.8.4471-4476.2002 PMID: 12117958 10. Souza-Silva FA, da Silva-Nunes M, Sanchez BA, Ceravolo IP, Malafronte RS, Brito CF, et al. Naturally acquired antibodies to Plasmodium vivax Duffy binding protein (DBP) in Brazilian Amazon. Am J Trop Med Hyg. 2010; 82: 185–193. doi: 10.4269/ajtmh.2010.08-0580 PMID: 20133990 11. Vicentin EC, Françoso KS, Rocha MV, Iourtov D, Dos Santos FL, Kubrusly FS, et al. Invasion-inhibitory antibodies elicited by immunization with Plasmodium vivax apical membrane antigen-1 expressed in Pichia pastoris yeast. Infect Immun. 2014; 82: 1296–1307. doi: 10.1128/IAI.01169-13 PMID: 24379279 12. Stanisic DI, Richards JS, McCallum FJ, Michon P, King CL, Schoepflin S, et al. Immunoglobulin G sub- class-specific responses against Plasmodium falciparummerozoite antigens are associated with con- trol of parasitemia and protection from symptomatic illness. Infect Immun. 2009; 77: 1165–1174. doi: 10.1128/IAI.01129-08 PMID: 19139189 13. Fowkes FJ, Richards JS, Simpson JA, Beeson JG. The relationship between anti-merozoite antibodies and incidence of Plasmodium falciparummalaria: A systematic review and meta-analysis. PLoSMed. 2010; 7: e1000218. doi: 10.1371/journal.pmed.1000218 PMID: 20098724 14. Chatzigeorgiou A, Lyberi M, Chatzilymperis G, Nezos A, Kamper E. CD40/CD40L signaling and its implication in health and disease. Biofactors. 2009; 35: 474–483. doi: 10.1002/biof.62 PMID: 19904719 15. Noelle RJ, Ledbetter JA, Aruffo A. CD40 and its ligand, an essential ligand-receptor pair for thymus- dependent B-cell activation. Immunol Today. 1992; 13: 431–433. doi: 10.1016/0167-5699(92)90068-I PMID: 1282319 16. Clark EA. A short history of the B-Cell-Associated surface molecule CD40. Front Immunol. 2014; 5: 472. doi: 10.3389/fimmu.2014.00472 PMID: 25324844 17. Rau FC, Dieter J, Luo Z, Priest SO, Baumgarth N. B7-1/2 (CD80/CD86) direct signaling to B cells enhances IgG secretion. J Immunol. 2009; 183: 7661–7671. doi: 10.4049/jimmunol.0803783 PMID: 19933871 18. Stadanlick JE, Cancro MP. BAFF and the plasticity of peripheral B cell tolerance. Curr Opin Immunol. 2008; 20: 158–161. doi: 10.1016/j.coi.2008.03.015 PMID: 18456486 19. Duah NO, Weiss HA, Jepson A, Tetteh KK, Whittle HC, Conway DJ. Heritability of antibody isotype and subclass responses to Plasmodium falciparum antigens. PLoS One. 2009; 4: e7381. doi: 10.1371/ journal.pone.0007381 PMID: 19812685 20. Carpenter D, Abushama H, Bereczky S, Färnert A, Rooth I, Troye-Blomberg M, et al. Immunogenetic control of antibody responsiveness in a malaria endemic area. Hum Immunol. 2007; 68: 165–169. doi: 10.1016/j.humimm.2006.12.002 PMID: 17349871 21. Tangteerawatana P, Perlmann H, Hayano M, Kalambaheti T, Troye-Blomberg M, Khusmith S. IL4 gene polymorphism and previous malaria experiences manipulate anti-Plasmodium falciparum antibody iso- type profiles in complicated and uncomplicated malaria. Malar J. 2009; 8: 286. doi: 10.1186/1475- 2875-8-286 PMID: 20003246 22. Kajeguka D, Mwanziva C, Daou M, Ndaro A, Matondo S, Mbugi E, et al. CD36 c.1264 T>G null muta- tion impairs acquisition of IgG antibodies to Plasmodium falciparumMSP119 antigen and is associated with higher malaria incidences in Tanzanian children. Scand J Immunol. 2012; 75: 355–360. doi: 10. 1111/j.1365-3083.2011.02661.x PMID: 22050542 23. Afridi S, Atkinson A, Garnier S, Fumoux F, Rihet P. Malaria resistance genes are associated with the levels of IgG subclasses directed against Plasmodium falciparum blood-stage antigens in Burkina Faso. Malar J. 2012; 11: 308. doi: 10.1186/1475-2875-11-308 PMID: 22947458 24. Lokossou AG, Dechavanne C, Bouraïma A, Courtin D, Le Port A, Ladékpo R, et al. Association of IL-4 and IL-10 maternal haplotypes with immune responses to P. falciparum in mothers and newborns. BMC Infect Dis. 2013; 13: 215. doi: 10.1186/1471-2334-13-215 PMID: 23668806 25. Maiga B, Dolo A, Touré O, Dara V, Tapily A, Campino S, et al. Human candidate polymorphisms in sympatric ethnic groups differing in malaria susceptibility in Mali. PLoS One. 2013; 8: e75675. doi: 10. 1371/journal.pone.0075675 PMID: 24098393 B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 13 / 15 http://dx.doi.org/10.1038/nature04443 http://www.ncbi.nlm.nih.gov/pubmed/16372020 http://dx.doi.org/10.1046/j.1365-3024.1999.00209.x http://www.ncbi.nlm.nih.gov/pubmed/10205793 http://dx.doi.org/10.1128/IAI.70.8.4471-4476.2002 http://www.ncbi.nlm.nih.gov/pubmed/12117958 http://dx.doi.org/10.4269/ajtmh.2010.08-0580 http://www.ncbi.nlm.nih.gov/pubmed/20133990 http://dx.doi.org/10.1128/IAI.01169-13 http://www.ncbi.nlm.nih.gov/pubmed/24379279 http://dx.doi.org/10.1128/IAI.01129-08 http://www.ncbi.nlm.nih.gov/pubmed/19139189 http://dx.doi.org/10.1371/journal.pmed.1000218 http://www.ncbi.nlm.nih.gov/pubmed/20098724 http://dx.doi.org/10.1002/biof.62 http://www.ncbi.nlm.nih.gov/pubmed/19904719 http://dx.doi.org/10.1016/0167-5699(92)90068-I http://www.ncbi.nlm.nih.gov/pubmed/1282319 http://dx.doi.org/10.3389/fimmu.2014.00472 http://www.ncbi.nlm.nih.gov/pubmed/25324844 http://dx.doi.org/10.4049/jimmunol.0803783 http://www.ncbi.nlm.nih.gov/pubmed/19933871 http://dx.doi.org/10.1016/j.coi.2008.03.015 http://www.ncbi.nlm.nih.gov/pubmed/18456486 http://dx.doi.org/10.1371/journal.pone.0007381 http://dx.doi.org/10.1371/journal.pone.0007381 http://www.ncbi.nlm.nih.gov/pubmed/19812685 http://dx.doi.org/10.1016/j.humimm.2006.12.002 http://www.ncbi.nlm.nih.gov/pubmed/17349871 http://dx.doi.org/10.1186/1475-2875-8-286 http://dx.doi.org/10.1186/1475-2875-8-286 http://www.ncbi.nlm.nih.gov/pubmed/20003246 http://dx.doi.org/10.1111/j.1365-3083.2011.02661.x http://dx.doi.org/10.1111/j.1365-3083.2011.02661.x http://www.ncbi.nlm.nih.gov/pubmed/22050542 http://dx.doi.org/10.1186/1475-2875-11-308 http://www.ncbi.nlm.nih.gov/pubmed/22947458 http://dx.doi.org/10.1186/1471-2334-13-215 http://www.ncbi.nlm.nih.gov/pubmed/23668806 http://dx.doi.org/10.1371/journal.pone.0075675 http://dx.doi.org/10.1371/journal.pone.0075675 http://www.ncbi.nlm.nih.gov/pubmed/24098393 26. Sabbagh A, Courtin D, Milet J, Massaro JD, Castelli EC, Migot-Nabias F, et al. Association of HLA-G 3' untranslated region polymorphisms with antibody response against Plasmodium falciparum antigens: preliminary results. Tissue Antigens. 2013; 82: 53–58. doi: 10.1111/tan.12140 PMID: 23745572 27. Adu B, Jepsen MP, Gerds TA, Kyei-Baafour E, Christiansen M, Dodoo D, et al. Fc gamma receptor 3B (FCGR3B-c.233C&GtA-rs5030738) polymorphismmodifies the protective effect of malaria specific antibodies in Ghanaian children. J Infect Dis. 2014; 209: 285–289. doi: 10.1093/infdis/jit422 PMID: 23935200 28. Pandey JP, Morais CG, Fontes CJ, Braga EM. Immunoglobulin GM; 2010. p. 3 23 5,13,14 phenotype is strongly associated with IgG1 antibody responses to Plasmodium vivax vaccine candidate antigens PvMSP1-19 and PvAMA-1. Malar J. 2010; 9: 229. doi: 10.1186/1475-2875-9-229 PMID: 20696056 29. Dewasurendra RL, Suriyaphol P, Fernando SD, Carter R, Rockett K, Corran P, et al. Genetic polymor- phisms associated with anti-malarial antibody levels in a low and unstable malaria transmission area in southern Sri Lanka. Malar J. 2012; 11: 281. doi: 10.1186/1475-2875-11-281 PMID: 22905743 30. Lima-Junior JC, Rodrigues-da-Silva RN, Banic DM, Jiang J, Singh B, Fabrício-Silva GM, et al. Influence of HLA-DRB1 and HLA-DQB1 alleles on IgG antibody response to the P. vivax MSP-1, MSP-3α and MSP-9 in individuals from Brazilian endemic area. PLoS One. 2012; 7: e36419. doi: 10.1371/journal. pone.0036419 PMID: 22649493 31. Storti-Melo LM, da Costa DR, Souza-NeirasWC, Cassiano GC, Couto VS, Póvoa MM, et al. Influence of HLA-DRB-1 alleles on the production of antibody against CSP, MSP-1, AMA-1, and DBP in Brazilian individuals naturally infected with Plasmodium vivax. Acta Trop. 2012; 121: 152–155. doi: 10.1016/j. actatropica.2011.10.009 PMID: 22107686 32. Snounou G, Viriyakosol S, Zhu XP, Jarra W, Pinheiro L, do Rosario VE, et al. High sensitivity of detec- tion of human malaria parasites by the use of nested polymerase chain reaction. Mol Biochem Parasi- tol. 1993; 61: 315–320. doi: 10.1016/0166-6851(93)90077-B PMID: 8264734 33. Malheiros D, Petzl-Erler ML. Individual and epistatic effects of genetic polymorphisms of B-cell co-stim- ulatory molecules on susceptibility to pemphigus foliaceus. Genes Immun. 2009; 10: 547–558. doi: 10. 1038/gene.2009.36 PMID: 19421221 34. Cassiano GC, Santos EJ, Maia MH, Furini AdC, Storti-Melo LM, Tomaz FM, et al. Impact of population admixture on the distribution of immune response co-stimulatory genes polymorphisms in a Brazilian population. Hum Immunol. 2015; 76: 836–842. doi: 10.1016/j.humimm.2015.09.045 PMID: 26429313 35. Cunha MG, Rodrigues MM, Soares IS. Comparison of the immunogenic properties of recombinant pro- teins representing the Plasmodium vivax vaccine candidate MSP1(19) expressed in distinct bacterial vectors. Vaccine. 2001; 20: 385–396. doi: 10.1016/S0264-410X(01)00359-0 PMID: 11672901 36. Ntumngia FB, Schloegel J, Barnes SJ, McHenry AM, Singh S, King CL, et al. Conserved and variant epitopes of Plasmodium vivax Duffy binding protein as targets of inhibitory monoclonal antibodies. Infect Immun. 2012; 80: 1203–1208. doi: 10.1128/IAI.05924-11 PMID: 22215740 37. Santos NP, Ribeiro-Rodrigues EM, Ribeiro-Dos-Santos AK, Pereira R, Gusmão L, Amorim A, et al. Assessing individual interethnic admixture and population substructure using a 48-insertion-deletion (INSEL) ancestry-informative marker (AIM) panel. HumMutat. 2010; 31: 184–190. doi: 10.1002/humu. 21159 PMID: 19953531 38. González JR, Armengol L, Solé X, Guinó E, Mercader JM, Estivill X, et al. SNPassoc: an R package to perform whole genome association studies. BioInformatics. 2007; 23: 644–645. doi: 10.1093/ bioinformatics/btm025 PMID: 17267436 39. Wigginton JE, Cutler DJ, Abecasis GR. A note on exact tests of Hardy-Weinberg equilibrium. Am J HumGenet. 2005; 76: 887–893. doi: 10.1086/429864 PMID: 15789306 40. Soares IS, Levitus G, Souza JM, Del Portillo HA, Rodrigues MM. Acquired immune responses to the N- and C-terminal regions of Plasmodium vivaxmerozoite surface protein 1 in individuals exposed to malaria. Infect Immun. 1997; 65: 1606–1614. PMID: 9125537 41. Barbedo MB, Ricci R, Jimenez MC, Cunha MG, Yazdani SS, Chitnis CE, et al. Comparative recognition by human IgG antibodies of recombinant proteins representing three asexual erythrocytic stage vac- cine candidates of Plasmodium vivax. Mem Inst Oswaldo Cruz. 2007; 102: 335–339. doi: 10.1590/ S0074-02762007005000040 PMID: 17568939 42. Dias S, Longacre S, Escalante AA, Udagama-Randeniya PV. Genetic diversity and recombination at the C-terminal fragment of the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) in Sri Lanka. Infect Genet Evol. 2011; 11: 145–156. doi: 10.1016/j.meegid.2010.09.007 PMID: 20933611 43. Moss DK, Remarque EJ, Faber BW, Cavanagh DR, Arnot DE, Thomas AW, et al. Plasmodium falcipa- rum 19-kilodalton merozoite surface protein 1 (MSP1)-specific antibodies that interfere with parasite growth in vitro can inhibit MSP1 processing, merozoite invasion, and intracellular parasite develop- ment. Infect Immun. 2012; 80: 1280–1287. doi: 10.1128/IAI.05887-11 PMID: 22202121 B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 14 / 15 http://dx.doi.org/10.1111/tan.12140 http://www.ncbi.nlm.nih.gov/pubmed/23745572 http://dx.doi.org/10.1093/infdis/jit422 http://www.ncbi.nlm.nih.gov/pubmed/23935200 http://dx.doi.org/10.1186/1475-2875-9-229 http://www.ncbi.nlm.nih.gov/pubmed/20696056 http://dx.doi.org/10.1186/1475-2875-11-281 http://www.ncbi.nlm.nih.gov/pubmed/22905743 http://dx.doi.org/10.1371/journal.pone.0036419 http://dx.doi.org/10.1371/journal.pone.0036419 http://www.ncbi.nlm.nih.gov/pubmed/22649493 http://dx.doi.org/10.1016/j.actatropica.2011.10.009 http://dx.doi.org/10.1016/j.actatropica.2011.10.009 http://www.ncbi.nlm.nih.gov/pubmed/22107686 http://dx.doi.org/10.1016/0166-6851(93)90077-B http://www.ncbi.nlm.nih.gov/pubmed/8264734 http://dx.doi.org/10.1038/gene.2009.36 http://dx.doi.org/10.1038/gene.2009.36 http://www.ncbi.nlm.nih.gov/pubmed/19421221 http://dx.doi.org/10.1016/j.humimm.2015.09.045 http://www.ncbi.nlm.nih.gov/pubmed/26429313 http://dx.doi.org/10.1016/S0264-410X(01)00359-0 http://www.ncbi.nlm.nih.gov/pubmed/11672901 http://dx.doi.org/10.1128/IAI.05924-11 http://www.ncbi.nlm.nih.gov/pubmed/22215740 http://dx.doi.org/10.1002/humu.21159 http://dx.doi.org/10.1002/humu.21159 http://www.ncbi.nlm.nih.gov/pubmed/19953531 http://dx.doi.org/10.1093/bioinformatics/btm025 http://dx.doi.org/10.1093/bioinformatics/btm025 http://www.ncbi.nlm.nih.gov/pubmed/17267436 http://dx.doi.org/10.1086/429864 http://www.ncbi.nlm.nih.gov/pubmed/15789306 http://www.ncbi.nlm.nih.gov/pubmed/9125537 http://dx.doi.org/10.1590/S0074-02762007005000040 http://dx.doi.org/10.1590/S0074-02762007005000040 http://www.ncbi.nlm.nih.gov/pubmed/17568939 http://dx.doi.org/10.1016/j.meegid.2010.09.007 http://www.ncbi.nlm.nih.gov/pubmed/20933611 http://dx.doi.org/10.1128/IAI.05887-11 http://www.ncbi.nlm.nih.gov/pubmed/22202121 44. Garraud O, Perraut R, Diouf A, Nambei WS, Tall A, Spiegel A, et al. Regulation of antigen-specific immunoglobulin G subclasses in response to conserved and polymorphic Plasmodium falciparum anti- gens in an in vitro model. Infect Immun. 2002; 70: 2820–2827. doi: 10.1128/IAI.70.6.2820-2827.2002 PMID: 12010968 45. Tomer Y, Concepcion E, Greenberg DA. A C/T single-nucleotide polymorphism in the region of the CD40 gene is associated with Graves' disease. Thyroid. 2002; 12: 1129–1135. doi: 10.1089/ 105072502321085234 PMID: 12593727 46. Jacobson EM, Concepcion E, Oashi T, Tomer Y. A Graves' disease-associated Kozak sequence sin- gle-nucleotide polymorphism enhances the efficiency of CD40 gene translation: a case for translational pathophysiology. Endocrinology. 2005; 146: 2684–2691. doi: 10.1210/en.2004-1617 PMID: 15731360 47. 1000 Genomes Project Consortium, Abecasis GR, Auton A, Brooks LD, DePristo MA, Durbin RM, et al. An integrated map of genetic variation from 1,092 human genomes. Nature. 2012; 491: 56–65. doi: 10. 1038/nature11632 PMID: 23128226 48. Capobianco MP, Cassiano GC, Furini AA, Storti-Melo LM, Pavarino EC, Galbiatti AL, et al. No evidence for association of the CD40, CD40L and BLYS polymorphisms, B-cell co-stimulatory molecules, with Brazilian endemic Plasmodium vivaxmalaria. Trans R Soc Trop Med Hyg. 2013; 107: 377–383. doi: 10. 1093/trstmh/trt031 PMID: 23604864 49. Nduati E, Gwela A, Karanja H, Mugyenyi C, Langhorne J, Marsh K, et al. The plasma concentration of the B cell activating factor is increased in children with acute malaria. J Infect Dis. 2011; 204: 962–970. doi: 10.1093/infdis/jir438 PMID: 21849293 50. Liu XQ, Stacey KJ, Horne-Debets JM, Cridland JA, Fischer K, Narum D, et al. Malaria infection alters the expression of B-cell activating factor resulting in diminished memory antibody responses and sur- vival. Eur J Immunol. 2012; 42: 3291–3301. doi: 10.1002/eji.201242689 PMID: 22936176 51. Kawasaki A, Tsuchiya N, Fukazawa T, Hashimoto H, Tokunaga K. Analysis on the association of human BLYS (BAFF, TNFSF13B) polymorphisms with systemic lupus erythematosus and rheumatoid arthritis. Genes Immun. 2002; 3: 424–429. doi: 10.1038/sj.gene.6363923 PMID: 12424625 52. Novak AJ, Grote DM, Ziesmer SC, Kline MP, Manske MK, Slager S, et al. Elevated serum B-lympho- cyte stimulator levels in patients with familial lymphoproliferative disorders. J Clin Oncol. 2006; 24: 983–987. doi: 10.1200/JCO.2005.02.7938 PMID: 16432079 53. de Almeida ER, Petzl-Erler ML. Expression of genes involved in susceptibility to multifactorial autoim- mune diseases: estimating genotype effects. Int J Immunogenet. 2013; 40: 178–185. doi: 10.1111/j. 1744-313X.2012.01152.x PMID: 22928528 54. Taylor-Robinson AW, Smith EC. Modulation of experimental blood stage malaria through blockade of the B7/CD28 T-cell costimulatory pathway. Immunology. 1999; 96: 498–504. doi: 10.1046/j.1365-2567. 1999.00718.x PMID: 10233733 55. Rummel T, Batchelder J, Flaherty P, LaFleur G, Nanavati P, Burns JM, et al. CD28 costimulation is required for the expression of T-cell-dependent cell-mediated immunity against blood-stage plasmo- dium chabaudimalaria parasites. Infect Immun. 2004; 72: 5768–5774. doi: 10.1128/IAI.72.10.5768- 5774.2004 PMID: 15385476 56. Delneste Y, Bosotti R, Magistrelli G, Bonnefoy J, Gauchat J. Detection of a polymorphism in exon 8 of the human CD86 gene. Immunogenetics. 2000; 51: 762–763. doi: 10.1007/s002510000203 PMID: 10941851 B Cell Co-Stimulatory Polymorphisms and Malaria PLOS ONE | DOI:10.1371/journal.pone.0149581 February 22, 2016 15 / 15 http://dx.doi.org/10.1128/IAI.70.6.2820-2827.2002 http://www.ncbi.nlm.nih.gov/pubmed/12010968 http://dx.doi.org/10.1089/105072502321085234 http://dx.doi.org/10.1089/105072502321085234 http://www.ncbi.nlm.nih.gov/pubmed/12593727 http://dx.doi.org/10.1210/en.2004-1617 http://www.ncbi.nlm.nih.gov/pubmed/15731360 http://dx.doi.org/10.1038/nature11632 http://dx.doi.org/10.1038/nature11632 http://www.ncbi.nlm.nih.gov/pubmed/23128226 http://dx.doi.org/10.1093/trstmh/trt031 http://dx.doi.org/10.1093/trstmh/trt031 http://www.ncbi.nlm.nih.gov/pubmed/23604864 http://dx.doi.org/10.1093/infdis/jir438 http://www.ncbi.nlm.nih.gov/pubmed/21849293 http://dx.doi.org/10.1002/eji.201242689 http://www.ncbi.nlm.nih.gov/pubmed/22936176 http://dx.doi.org/10.1038/sj.gene.6363923 http://www.ncbi.nlm.nih.gov/pubmed/12424625 http://dx.doi.org/10.1200/JCO.2005.02.7938 http://www.ncbi.nlm.nih.gov/pubmed/16432079 http://dx.doi.org/10.1111/j.1744-313X.2012.01152.x http://dx.doi.org/10.1111/j.1744-313X.2012.01152.x http://www.ncbi.nlm.nih.gov/pubmed/22928528 http://dx.doi.org/10.1046/j.1365-2567.1999.00718.x http://dx.doi.org/10.1046/j.1365-2567.1999.00718.x http://www.ncbi.nlm.nih.gov/pubmed/10233733 http://dx.doi.org/10.1128/IAI.72.10.5768-5774.2004 http://dx.doi.org/10.1128/IAI.72.10.5768-5774.2004 http://www.ncbi.nlm.nih.gov/pubmed/15385476 http://dx.doi.org/10.1007/s002510000203 http://www.ncbi.nlm.nih.gov/pubmed/10941851