https://orcid.org/0000-0003-4415-8646
Figures
Abstract
Background
Clinical immunity to malaria has been associated with humoral immune responses targeting asexual-stage parasite proteins. However, the variability in antibody-driven responses may be influenced by genetic factors in the human host. This study aimed to evaluate the frequency of SNPs in the TLR9 gene and their association with IgG antibody responses against PvCSP variants (VK247, VK210, and V-like) and PvMSP-119 among individuals presenting with symptomatic Plasmodium vivax infections in a malaria-endemic region of Venezuela.
Methodology/Principal findings
A cross-sectional study was conducted in Bolívar state, Venezuela, involving 210 individuals infected with P. vivax. IgG reactivity against P. vivax recombinant antigens was assessed by ELISA, and three TLR9 gene SNPs (rs5743836, rs352140, and rs187084) were genotyped by PCR. The median age of individuals was 29 years, with the majority being male miners with a prior history of malaria. Over 90% of individuals exhibited IgG antibodies against the three PvCSP variants and PvMSP-119. High responders to the PvCSP variants reported fewer symptoms compared to medium (p < 0.001) and low (p < 0.001) responders. Among the analyzed SNPs, heterozygous genotypes were the most prevalent. Using an overdominant inheritance model, carrying the heterozygous genotype (T/C) for TLR9 gene SNP rs5743836 was associated with lower IgG antibody response against PvCSP VK247 (aOR = 0.26, 95% CI = 0.09 to 0.73, p = 0.0094) and PvCSP V-like (aOR = 0.37, 95% CI = 0.14 to 0.99, p = 0.047). No significant associations between inheritance models for the three SNPs and parasitemia were observed.
Conclusions/Significance
These findings suggest that TLR9 gene variability, particularly the heterozygous genotype for SNP rs5743836, may influence the IgG antibody response against PvCSP VK247 and V-like. Identifying genetic traits that impact immune response development could be valuable for malaria vaccine design and implementation.
Author summary
In this study, we investigated how human genetic differences might affect the immune responses to malaria. Specifically, we looked at how certain genetic variations in the TLR9 gene, which is part of the immune system, might affect the production of antibodies against malaria proteins in 210 Venezuelan individuals infected with Plasmodium vivax, one of the parasites that causes malaria. Most of these individuals were male miners likely infected in a highly endemic area. Our findings suggest that people with a specific genetic variation, known as a heterozygous genotype in the TLR9 gene, had different levels of antibodies against two malaria proteins. Interestingly, those with higher antibody levels had fewer symptoms, suggesting that a stronger immune response may protect against malaria symptoms. Understanding these genetic influences on immune response could be valuable in designing vaccines and treatments that work more effectively in different populations.
Citation: Carrión-Nessi FS, Salazar-Alcalá E, Forero-Peña DA, Curiel KA, Lopez-Perez M, Fernández-Mestre M (2025) TLR9 gene polymorphism -1237T/C (rs5743836) is associated with low IgG antibody response against PvCSP variants in symptomatic P. vivax infections in Venezuela. PLoS Negl Trop Dis 19(6): e0013262. https://doi.org/10.1371/journal.pntd.0013262
Editor: Kevin Shyong-Wei Tan, National University of Singapore, SINGAPORE
Received: November 28, 2024; Accepted: June 18, 2025; Published: June 30, 2025
Copyright: © 2025 Carrión-Nessi 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: All relevant data are within the manuscript and its Supporting information files.
Funding: The authors received no funding for this research.
Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: Mary Lopez-Perez is an Editorial Board Member of the journal PLOS Neglected Tropical Diseases. The rest of the authors have declared that no competing interests exist.
Introduction
Plasmodium vivax remains a leading public health problem in America, particularly in rural areas of Venezuela, Brazil, and Colombia [1]. Since 2016, Venezuela has reported the highest number of malaria cases and deaths in the region [1], with the southeastern part of the country, bordering Brazil and Guyana, remaining at high risk for malaria transmission [2]. There, Bolívar state accounts for 60–88% of the country’s malaria cases and has become the hottest malaria hotspot in America [3].
The host immune response to P. vivax is triggered by various parasitic components, resulting in a synergistic interaction between innate and adaptive immunity to control and eliminate the parasite [4]. This response begins with the bite of an infected female Anopheles mosquito, which inoculates sporozoites into the host’s skin, and develops through both hepatic and blood schizogony processes. The parasite’s blood stages contain pathogen-associated molecular patterns, such as glycosylphosphatidylinositol anchors, hemozoin, and parasite nucleic acids, which trigger an innate immune response via diverse pattern recognition receptors, including Toll-like receptors (TLRs) [5]. To date, 10 TLRs have been identified in humans, located either on the cell surface (TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10) or on endosomes (TLR3, TLR7, TLR8, and TLR9) [6].
TLR9 participates in microbial DNA recognition, specifically unmethylated CpG motifs, and is expressed on professional antigen-presenting cells, such as dendritic cells and B cells [6]. Additionally, TLR9 regulates B cell viability, apoptosis, and antibody and IL-10 production by upregulating the Fc receptor-like 3 [7]. The activation of memory B cells by TLR9 leads to cell expansion and immunoglobulin production, which may be critical for memory B cell homeostasis and sustained antibody production [8]. In malaria, TLR9 recognizes free or hemozoin-bound parasite DNA [5,9]. Indeed, the high content of CpG motifs in P. vivax is likely responsible for the species’ high fever-inducing capacity [10].
In malaria-endemic areas, particularly where transmission rates are high, individuals become less susceptible to symptomatic malaria episodes with increasing age and exposure [11]. The role of antibodies in malaria protection is well documented [12], and the host’s genetic makeup is known to significantly influence the development of an effective immune response against the parasite [13]. Compared to several studies in P. falciparum [14], only two studies have reported TLR9 gene polymorphisms in P. vivax [15,16]. Moreover, only a few studies have documented the role of immune-related gene polymorphisms on antibody production against P. vivax proteins [17–21]. To date, no study has evaluated the effect of single nucleotide polymorphisms (SNPs) of the TLR9 gene on the IgG antibody response against circumsporozoite protein (PvCSP), and only one study has done so against merozoite surface protein 1 (PvMSP-119) [16].
Here, we evaluated the frequency of TLR9 gene SNPs rs5743836 (-1237T/C), rs352140 (1635A/G), and rs187084 (-1486C/T) and their associations with IgG antibody response against PvCSP variants (VK247, VK210, and V-like) and PvMSP-119 among individuals with symptomatic P. vivax infections in Bolívar state, Venezuela.
Methods
Ethical approval and consent to participate
This study adhered to the ethical guidelines established by national and institutional committees overseeing human experimentation following the ethical principles of the Declaration of Helsinki. The study protocol was reviewed and approved by the Independent Bioethics Committee for Research of the Bioethical National Center of Venezuela (Approval No.: CIBI-CENABI-03/2022). All patients received a comprehensive explanation of the study design and the data and sample collection methodology. Only those who voluntarily agreed to participate and provided signed consent were included in the study.
Study area
This study was conducted in the Laboratory of Malariology of the “Ruiz y Páez” University Hospital Complex (Bolívar state), which serves as the main reference center for diagnosis and treatment of malaria patients in the state. Bolívar state, situated in the southeastern part of the country, is Venezuela’s largest political and administrative region, with an estimated population of over 1,947,403 [22]. Most of the population resides in the northern and eastern areas of the state, where primary economic activities include mining, steel and aluminum manufacturing, hydroelectric power generation, commerce and services, forestry, livestock rearing, and agricultural development [3].
According to the most recent official publication on the national epidemiological situation of malaria in Venezuela (Week No. 41, 2022) [23], a total of 60,329 accumulated cases had been reported in Bolívar state, representing 59% of malaria cases in Venezuela. P. vivax (78%) was the dominant parasite species, followed by P. falciparum (16%) and mixed (P. vivax/P. falciparum) infection (6%) [23].
Study design and population
A cross-sectional study was conducted between June 2022 and January 2023 involving unrelated Venezuelan individuals infected with P. vivax, either primo-infected or with previous infections, who presented with malaria-compatible symptoms at the Laboratory of Malariology of the “Ruiz y Páez” University Hospital Complex. All individuals included in the study were classified as uncomplicated malaria cases according to the Venezuelan criteria [24]. The diagnosis of Plasmodium spp. was performed by thick and thin blood smears stained with the Giemsa method according to the WHO guidelines [25]. For quality assurance, each slide was independently examined by two experienced microscopists using 100X oil immersion magnification. Both microscopists identified Plasmodium spp. and quantified parasitemia from the thick blood smear [26]. This diagnosis was subsequently confirmed by nested PCR [27] at the Laboratory of Pathophysiology (Immunogenetics Section) of the Instituto Venezolano de Investigaciones Científicas. Individuals with mixed (P. vivax/P. falciparum or other species) infection, those undergoing antimalarial treatment at the time of blood sample collection, immunocompromised individuals (e.g., transplant recipients or chemotherapy patients), and those with other self-reported infectious, autoimmune, or hematologic diseases (e.g., sickle cell disease), were not enrolled.
Additionally, 40 healthy individuals without previous malaria exposure (negative control samples) from non-endemic areas and no history of blood transfusion were included as controls. This control group was used in the ELISA assays to establish the cut-off point necessary to determine the antibody reactivity index (RI).
Blood sample collection and genomic DNA extraction
After applying a clinical-epidemiological data collection form, two blood samples were collected from each individual by peripheral venipuncture. One sample was collected in a tube without anticoagulant to obtain serum and a second with EDTA for molecular analysis. The genomic DNA was extracted from whole blood using the protocol described by Bunce [28]. The concentration and purity of the genomic DNA were quantified by ultraviolet spectrophotometry using the NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The quality of the genomic DNA was verified by agarose gel electrophoresis.
Recombinant antigens
Recombinant PvMSP-119 (amino acids 1616–1704, Belém strain) was expressed in Escherichia coli with a polyhistidine affinity tag (6xHis tag), as previously described [29], at the Department of Molecular Biology and Immunology of the Fundación Instituto de Inmunología de Colombia. Peptides corresponding to the three variants (VK247, VK210, and V-like) of the PvCSP repetitive region were synthesized in Fmoc solid-phase [30] at the Instituto de Medicina Tropical (Laboratory of Peptide Synthesis) of the Universidad Central de Venezuela. Sequences are shown in S1 Table.
Evaluation of IgG antibody response against the three PvCSP variants and PvMSP-119
IgG reactivity against P. vivax recombinant antigens was determined by ELISA. All steps were performed at room temperature in a humidity chamber. Briefly, 96-well flat-bottom plates (Nunc MaxiSorp ELISA, Thermo Fisher Scientific, Waltham, MA, USA) were coated for 2 hours with PvCSP variants (5 µg/mL) or PvMSP-119 (10 µg/mL) diluted in carbonate/bicarbonate pH 9.6. After washing three times with 0.05% Tween 20 in phosphate-buffered saline (PBS-T), the plates were blocked for 30 minutes with 1% bovine serum albumin (BSA) in PBS. Then, 100 µL of diluted serum (1:200 for PvCSP variants and 1:100 for PvMSP-119 in 0.5% BSA in PBS-T) were added to triplicate wells and incubated for 1 hour. After washing, 1:10,000 horseradish peroxidase-conjugated anti-human IgG (Santa Cruz Biotechnology, Dallas, TX, USA) was added to each well and incubated for 1 hour. Bound antibodies were detected by adding TMB (Sigma-Aldrich, Burlington, MA, USA) and the reaction was stopped with 0.1 M sulfuric acid. The absorbance was read on an Infinite M200 ELISA plate reader (Tecan, Männedorf, Switzerland) at 405 nm.
IgG antibody response against P. vivax recombinant antigens was expressed as a RI, which was determined by dividing the optical density (OD) value of the test sample by the cut-off point OD value. The cut-off values were calculated by averaging the OD values plus three standard deviations obtained with negative control samples. Samples from individuals with an RI ≥ 1 (also known as responders) were considered positive.
Genotyping of TLR9 gene SNPs
The SNP rs5743836 was determined by bi-directional PCR amplification of specific alleles, using previously described primers [31]. For this, 1 µL of DNA was mixed with 1X Tonbo VerityMAX DNA polymerase master mix, 0.4 µM primer P, 0.4 µM primer Q, 0.05 µM primer W, and 0.1 µM primer M. Amplified products were separated by electrophoresis in a 1.5% agarose gel and visualized using a ChemiDoc MP imaging system (Bio-Rad Laboratories, Hercules, CA, USA). Depending on the genotype, two or three overlapping fragments were produced. PQ primers served as the internal control (644 bp), PW primers amplified the T allele (395 bp), and MQ primers amplified the C allele (275 bp) (S2 Table).
SNPs rs352140 and rs187084 were determined by PCR-restriction fragment length polymorphism, using previously described primers and protocols [32]. Here, 1 µL of DNA was mixed with 1X Tonbo VerityMAX DNA polymerase master mix, 1 pM forward and 1 pM reverse primers. Amplified products were digested with restriction enzymes (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions, separated by electrophoresis in a 2% agarose gel, and visualized using a ChemiDoc MP imaging system. Genotypes were assigned based on the number and size of fragments resulting from the digestion of the amplified product (S2 Table).
Statistical analysis
Data were analyzed using SPSS version 27 (IBM Corp., Armonk, NY, USA) and plotted using GraphPad Prism version 10.1.2 (GraphPad Software, Boston, MA, USA). Individual data were summarized using descriptive statistics. A two-stage cluster analysis was performed to identify natural groupings (or clusters) of parasitemia and RI against the three PvCSP variants and PvMSP-119. The method used a Log-likelihood distance measure and involved an initial pre-clustering step creating a Cluster Feature tree, followed by an agglomerative hierarchical clustering stage on the subclusters. The optimal number of clusters was determined automatically by comparing potential solutions using the Schwarz Bayesian Information Criterion or the Akaike Information Criterion. The resulting clusters were examined based on their mean RI profiles and subsequently designated as “low”, “medium”, and “high” responder groups. The distribution of numerical variables was assessed using the Kolmogorov-Smirnov test, and one-way ANOVA or Kruskal-Wallis tests were applied as appropriate. Pearson’s chi-square and Fisher’s exact tests were used for categorical variables. Genetic analyses were conducted using the SNPStats program [33]. Allele, genotype, and haplotype frequencies, Hardy-Weinberg equilibrium, linkage disequilibrium, and inheritance models were analyzed. Multiple regression models adjusted for age, sex, mining occupation, probable area of infection, and history of malaria were performed to evaluate the effect of potential confounders on the association between IgG antibody response level and parasitemia, and TLR9 gene SNPs. A p value < 0.05 was considered statistically significant.
Results
Clinical-epidemiological characteristics of the study population
A total of 210 unrelated Venezuelan individuals with symptomatic malaria by P. vivax were included in the analysis (Fig 1). The median age of the individuals was 29 (IQR 21–43) years, most were male (60.5%, n = 127), and practiced illegal gold mining (56.2%, n = 118). The probable area of infection for all individuals was Bolívar state, primarily in Sifontes (38.1%, n = 80), Sucre (26.7%, n = 56), and Angostura del Orinoco (22.4%, n = 47) municipalities. More than 60% of individuals had low parasitemia (≤ 4800 parasites/μL of blood), and over 80% reported a history of malaria, with a median of 5 (IQR 2–12) episodes (Table 1).
Individuals who self-identified mainly as Caucasian (n = 7, primary European ancestry) or Amerindian (n = 2, Venezuelan Indigenous ethnic group) were excluded to minimize confounding factors related to population stratification in the genetic association study of TLR9 gene SNPs. This focused the analysis on the general admixed population (“criollo”/“mestizo”) characteristic of the study area in Bolívar state.
All participants reported at least one symptom, with a median number of 7 (IQR 5–10). The most common symptoms were fever (94.3%, n = 198), headache (92.4%, n = 194), chills (76.7%, n = 161), low back pain (71.9%, n = 151), and arthralgia (68.6%, n = 144).
High seroprevalence of PvCSP and PvMSP-119-specific IgG
More than 90% of individuals had IgG antibodies (RI ≥ 1) against the three PvCSP variants and PvMSP-119 (Fig 2). Using a two-stage cluster analysis, we grouped individuals into low, medium, and high responders according to their IgG antibody response levels. This classification was used for following analyses. No statistically significant differences were observed between the clinical-epidemiological characteristics of the individuals and their IgG antibody response levels against the three PvCSP variants (S3–S5 Tables). However, a higher proportion of previous malaria was observed in high responders against PvMSP-119 compared to low responders (85.7% vs. 68.1%, p = 0.013) (S6 Table).
(A) Specific IgG levels against recombinant PvCSP and PvMSP-119 in serum from adults (n = 210) determined by ELISA. Values are expressed as reactivity index (RI). Medians and p values are shown using the Kruskal-Wallis test followed by Dunn’s multiple comparison test. Negative cut-off values (RI = 1) are shown as dotted lines and were used to calculate seroprevalence. (B) Seroprevalence of PvCSP- and PvMSP-119-specific IgG.
High responders against the three PvCSP variants had a lower number of symptoms compared to medium (p < 0.001) and low (p < 0.001) responders (Fig 3).
Number of symptoms among low, medium, and high responders against the VK247 (n = 198), VK210 (n = 200), V-like (n = 204), and PvMSP-119 (n = 208). Responders were defined by a two-stage cluster analysis using their reactivity index (RI) for each tested antigen. Median, IQR, and whiskers (1.5 times the IQR) are shown. P values using the Kruskal-Wallis test followed by Dunn’s multiple comparison test are also shown.
TLR9 gene SNP rs5743836 (-1237T/C) is associated with low IgG antibody response against PvCSP VK247 and V-like
Genotypic and allelic frequencies of TLR9 gene SNPs are summarized in Fig 4. Heterozygous genotypes T/C (66.7%, n = 140), A/G (57.1%, n = 120), and C/T (81.4%, n = 171) for SNPs rs5743836, rs352140, and rs187084, respectively, were the most frequent among 210 analyzed individuals. For SNP rs187084, the homozygous genotype for the minor allele (T/T) was not observed. None of the analyzed TLR9 gene SNPs conformed to Hardy-Weinberg equilibrium (p < 0.05). Moderate linkage disequilibrium (D’ = 0.512, r = 0.47) was observed between SNPs rs5743836 and rs187084 (p < 0.001), and strong linkage disequilibrium (D’ = 0.753, r = 0.64) was observed between SNPs rs352140 and rs187084 (p < 0.001).
Proportion of individuals infected with P. vivax (n = 210) carrying a specific genotype (A) or allele (B) for SNPs rs5743836, rs352140, and rs187084 of the TLR9 gene.
Then, we evaluated the effects of TLR9 gene SNPs on the IgG antibody response, comparing low and high responders. According to the best-fit inheritance model (overdominant), carrying the heterozygous genotype (T/C) for the SNP rs5743836 of the TLR9 gene was associated with low IgG antibody response against PvCSP VK247 (aOR = 0.26, 95% CI = 0.09 to 0.73, p = 0.0094) (Table 2) and PvCSP V-like (aOR = 0.37, 95% CI = 0.14 to 0.99, p = 0.047) (Table 3). No other significant differences were observed between SNP rs5743836 inheritance models and IgG antibody response level against the PvCSP VK210 (S7 Table) or PvMSP-119 (S8 Table). Likewise, no differences were observed between SNP rs352140 inheritance models and IgG antibody response level against the three PvCSP variants (Tables 2, 3 and S7) and or against PvMSP-119 (S8 Table).
Haplotype analysis revealed that the combination of major alleles of the SNPs rs5743836, rs352140, and rs187084 of the TLR9 gene (TAC) was the most common among the individuals. Other haplotypes were observed with variable distribution (S9 Table). CAT and CGC haplotypes were not observed in high responders against PvCSP VK247, and PvCSP VK210 and V-like, respectively. Despite some trends, no significant differences in haplotypic frequencies were observed between low and high responders (S9 Table).
No significant differences between inheritance models for the SNPs rs5743836 and rs352140 and parasitemia were observed (Table 4). Due to the absence of the homozygous genotype for the minor allele (T/T), it was not possible to establish inheritance models for the SNP rs187084.
Discussion
Innate and adaptive mechanisms of the immune system have evolved to adapt and control the pathogenesis of infectious diseases and to repair tissues [34]. The pathogen-host interaction in P. vivax malaria is biologically complex and influenced by environmental factors, parasitemia, immune status, and the genetic makeup of the human host [35]. Whereas most of the studies have focused on P. falciparum, we explored here the effects of TLR9 gene polymorphisms on the IgG antibody response against two malaria antigens, PvCSP and PvMSP-119.
As previously reported in Bolívar state [36,37], most individuals were young adult males engaged in illegal gold mining. Over 80% of individuals reported previous malaria episodes, with a median of five episodes, highlighting the importance of the state as a hotspot in Latin America [3]. In this study, the parasitemia distribution reflects the reported in Colombia [38], but appears constrained compared to broader ranges reported in the Asia-Pacific region [39,40]. This difference is probably due to the symptomatic status of all participants and a single-timepoint sampling, potentially missing wider density fluctuations [41,42]. In this study, over 90% of individuals had IgG antibodies (RI ≥ 1) against PvCSP or PvMSP-119, similar to other studies conducted in Latin America [21,43,44]. Although no significant differences were observed between the clinical-epidemiological characteristics of individuals according to their specific-IgG response, male miners were commonly high responders. This agrees with previous studies showing an association between mining activity and high malaria transmission in this area [3,45].
High responders against PvCSP variants exhibited fewer malaria symptoms than medium and low responders. Specific-PvCSP antibodies block the invasion and development of sporozoites in human hepatocytes [46], which may prevent the development of blood stages and, in turn, clinical malaria. A higher proportion of previous malaria was observed in high responders against PvMSP-119 compared to low responders, confirming a potentiating effect on the antibody response against this protein in individuals previously exposed to malaria [21], and suggesting an increased number of antibody-producing short-lived plasma cells, likely due to the presence of a memory B cell response, which rapidly proliferates and differentiates upon reinfection [47].
TLRs play a critical role in immune protection and have been subjected to selection in response to immune challenges, as observed in studies associating TLRs and their SNPs with disease [6]. To ensure the representativeness of our findings, SNPs were selected based on available data from genomic databases, particularly those focused on Latin American populations. Additionally, the chosen SNPs were selected based on prior research demonstrating significant associations with the diseases under investigation, with known functional effects. In this study, heterozygous genotypes of SNPs rs5743836, rs352140, and rs187084 of the TLR9 gene were most frequent among evaluated individuals, as reported in African individuals [48] and among three United States ethnic groups (African American, European, and Hispanic) [49]. High heterozygosity has also been reported at SNPs rs352140 and rs187084 [15,50,51] or only rs5743836 [52]. These genetic differences across populations, as a result of balanced selection [53], complicate comparisons between studies conducted in different locations [14].
After controlling for potential biases that could influence the IgG antibody response, we found that the heterozygous genotype (T/C) for SNP rs5743836 of the TLR9 gene was associated with low IgG antibody response against PvCSP VK247 and V-like in the evaluated individuals. Findings on the promoter activity for SNP rs5743836 of the TLR9 gene are contradictory. Studies have reported higher promoter activity in the homozygous genotype for the major allele (T/T) [54], but also for the minor allele (C/C) [55,56], and the heterozygous genotype (T/C) [57,58]. These discrepancies could be explained by differences in the etiologies of the models studied, the timing of transcriptional evaluation, and/or the presence of other functional SNPs that interact with rs5743836, regulating TLR9 expression [55,56]. Therefore, it may be deduced that in individuals carrying the heterozygous genotype for SNP rs5743836, TLR9 gene transcription and B cell activation are dysregulated, affecting the IgG antibody response against PvCSP. Notably, the overall clinical presentation and the specific IgG response in P. vivax malaria are shaped by a complex interplay of such host factors. Beyond TLRs, these encompass gene polymorphisms in cytokine [59–61], B-cell co-stimulatory molecules [21], human leukocyte antigens molecules [19,62], and Fc gamma receptors [63], alongside factors like active malaria infection status [64], prior exposure, and inherent PvCSP immunogenicity [65–67]. However, current scientific evidence on the precise contributions and interactions of these factors is limited and often inconsistent, highlighting the need for comprehensive studies across diverse geographic regions and genetic background [14].
We did not observe an association between parasitemia and the evaluated SNPs of the TLR9 gene, similar to those reported in the Brazil-French Guiana border [16]. However, the literature on this association varies according to the specific SNP analyzed. Regarding SNP rs5743836, a study conducted in Brazil reported an association between the homozygous genotype for the minor allele (C/C) and high parasitemia [15]. Other studies reported an association between the homozygous genotype for the major allele (T/T) and low parasitemia [48,68]. These differences could be attributed to the genetic background of the population and the high genetic diversity of Plasmodium, which has multiple haplotypes and varied genetic patterns worldwide [14,16]. However, it has been shown that the homozygous genotype for the major allele (T/T) has higher promoter activity than the homozygous genotype for the minor allele (C/C) [54], potentially resulting in higher production of proinflammatory cytokines during malarial infection, favoring parasite control and elimination [48].
Several studies did not observe any association between SNP rs187084 and parasitemia [15,16,48]. However, one study in Brazil reported an association between the homozygous genotype for the major allele (C/C) and high parasitemia [68]. Peripheral parasitemia is only one indicator of disease state, but may not fully represent the total parasite burden, particularly in P. vivax infections where significant parasite biomass may be sequestered in organs such as the spleen and bone marrow [69]. It is plausible that this total biomass acts as a stronger driver influencing both the host’s symptoms and the magnitude of the IgG response. Consequently, discrepancies in associations with peripheral parasitemia are perhaps expected. Moreover, numerous factors influence the clinical profile of the disease beyond parasite density, such as endemicity levels, multiplicity of infection [70], and the broader genetic composition of the human host [14,16]. These characteristics could act as confounding factors contributing to the variable findings observed between this and other studies on endemic malaria and should be considered when analyzing and interpreting the results [16]. No studies on the association between SNP rs352140 and parasitemia were found, suggesting that future research should investigate this.
We acknowledge limitations regarding our findings. Using symptom counts rather than severity grading (such as hemoglobin levels or splenomegaly quantification) impacts clinical interpretability. Financial and practical constraints prevented the collection of detailed hematological data (e.g., hemoglobin, platelets) and certain key symptoms (e.g., gastrointestinal distress, palpable splenomegaly), hindering a robust assessment of genotype-phenotype relationships. Viral co-infections present at the time of malaria infection are frequent in the study region [37], but were not assessed in the recruited individuals. Such co-infections could influence malaria symptomatology and immune responses, potentially confounding the relationships observed in our study. We also recognized that sample size and the focus on uncomplicated patients within a point-of-care setting limit generalizability. Future hospital- and community-based studies with larger, more diverse cohorts (including asymptomatic, mild, and severe cases) and comprehensive clinical/paraclinical assessments, including screening for relevant co-infections, are necessary to confirm our findings.
Conclusions
Our findings suggest that genetic variation within the TLR9 gene, specifically the rs5743836 (-1237T/C) heterozygous genotype, may modulate IgG antibody response against key P. vivax antigens: PvCSP VK247 and V-like. Elucidating how host genetic factors shape pathogen-specific immunity is pertinent to the rational design of effective malaria vaccines. Future research should incorporate functional assays to determine the biological significance of the observed association between rs5743836 and IgG antibody response. Such studies should also measure total parasite burden and include asymptomatic and severe cases to assess the variant’s impact across the P. vivax clinical spectrum. Longitudinal studies on populations with diverse malaria exposure and carrying the TLR9-1237T/C polymorphism may also reveal the risk of recurrent parasitemia and anemia, and the durability of specific IgG responses. Integrating standardized clinical evaluations within such longitudinal frameworks will be instrumental for establishing the contribution of TLR9 gene polymorphisms to protective immunity against P. vivax malaria.
Supporting information
S1 Table. Peptide sequences corresponding to the three variants (VK247, VK210, and V-like) of the PvCSP repetitive region.
https://doi.org/10.1371/journal.pntd.0013262.s001
(DOCX)
S2 Table. Description of polymorphisms, primer sequences, PCR amplification program, restriction enzymes, and fragments resulting from the genotyping of TLR9 gene SNPs.
https://doi.org/10.1371/journal.pntd.0013262.s002
(DOCX)
S3 Table. Clinical-epidemiological characteristics of individuals infected with P. vivax by their IgG antibody response level against the VK247 PvCSP variant.
https://doi.org/10.1371/journal.pntd.0013262.s003
(DOCX)
S4 Table. Clinical-epidemiological characteristics of individuals infected with P. vivax by their IgG antibody response level against the VK210 PvCSP variant.
https://doi.org/10.1371/journal.pntd.0013262.s004
(DOCX)
S5 Table. Clinical-epidemiological characteristics of individuals infected with P. vivax by their IgG antibody response level against the V-like PvCSP variant.
https://doi.org/10.1371/journal.pntd.0013262.s005
(DOCX)
S6 Table. Clinical-epidemiological characteristics of individuals infected with P. vivax by their IgG antibody response level against PvMSP-119.
https://doi.org/10.1371/journal.pntd.0013262.s006
(DOCX)
S7 Table. Association of genotypic frequencies of TLR9 gene SNPs with IgG antibody response level against the VK210 PvCSP variant.
https://doi.org/10.1371/journal.pntd.0013262.s007
(DOCX)
S8 Table. Association of genotypic frequencies of TLR9 gene SNPs with IgG antibody response level against PvMSP-119.
https://doi.org/10.1371/journal.pntd.0013262.s008
(DOCX)
S9 Table. Association of haplotypic frequencies of TLR9 gene SNPs with IgG antibody response level against the three PvCSP variants and PvMSP-119.
https://doi.org/10.1371/journal.pntd.0013262.s009
(DOCX)
Acknowledgments
We thank all volunteers who participated in this study and the technical staff involved in the samples collection.
References
- 1.
WHO. World malaria report 2024. Geneva: World Health Organization; 2024.
- 2. Moreno JE, Rubio-Palis Y, Martínez ÁR, Acevedo P. Evolución espacial y temporal de la malaria en el municipio Sifontes del estado Bolívar, Venezuela. 1980-2013. Bol Malariol y Sal Amb. 2014;54:236–49.
- 3. Grillet ME, Moreno JE, Hernández-Villena JV, Vincenti-González MF, Noya O, Tami A, et al. Malaria in Southern Venezuela: the hottest hotspot in Latin America. PLoS Negl Trop Dis. 2021;15(1):e0008211. pmid:33493212
- 4. Chua CLL, Brown G, Hamilton JA, Rogerson S, Boeuf P. Monocytes and macrophages in malaria: protection or pathology?. Trends Parasitol. 2013;29(1):26–34. pmid:23142189
- 5. Gowda DC, Wu X. Parasite recognition and signaling mechanisms in innate immune responses to malaria. Front Immunol. 2018;9:3006. pmid:30619355
- 6. Kawai T, Ikegawa M, Ori D, Akira S. Decoding toll-like receptors: recent insights and perspectives in innate immunity. Immunity. 2024;57(4):649–73. pmid:38599164
- 7. Liu Q-B, Zhou R-H, Liu C-M. TLR9/FCRL3 regulates B cell viability, apoptosis, and antibody and IL-10 production through ERK1/2, p38, and STAT3 signaling pathways. In Vitro Cell Dev Biol Anim. 2022;58(8):702–11. pmid:36121575
- 8. Bernasconi NL, Traggiai E, Lanzavecchia A. Maintenance of serological memory by polyclonal activation of human memory B cells. Science. 2002;298(5601):2199–202. pmid:12481138
- 9. Parroche P, Lauw FN, Goutagny N, Latz E, Monks BG, Visintin A, et al. Malaria hemozoin is immunologically inert but radically enhances innate responses by presenting malaria DNA to Toll-like receptor 9. Proc Natl Acad Sci U S A. 2007;104(6):1919–24. pmid:17261807
- 10. Anstey NM, Douglas NM, Poespoprodjo JR, Price RN. Plasmodium vivax: clinical spectrum, risk factors and pathogenesis. Adv Parasitol. 2012;80:151–201. pmid:23199488
- 11. Doolan DL, Dobaño C, Baird JK. Acquired immunity to malaria. Clin Microbiol Rev. 2009;22(1):13–36. pmid:19136431
- 12. Cohen S, McGregor IA, Carrington S. Gamma-globulin and acquired immunity to human malaria. Nature. 1961;192:733–7. pmid:13880318
- 13. Duah NO, Weiss HA, Jepson A, Tetteh KKA, Whittle HC, Conway DJ. Heritability of antibody isotype and subclass responses to Plasmodium falciparum antigens. PLoS One. 2009;4(10):e7381. pmid:19812685
- 14. Ramirez Ramirez AD, de Jesus MCS, Rossit J, Reis NF, Santos-Filho MC, Sudré AP, et al. Association of toll-like receptors in malaria susceptibility and immunopathogenesis: a meta-analysis. Heliyon. 2022;8(4):e09318. pmid:35520620
- 15. Costa AG, Ramasawmy R, Ibiapina HNS, Sampaio VS, Xábregas LA, Brasil LW, et al. Association of TLR variants with susceptibility to Plasmodium vivax malaria and parasitemia in the Amazon region of Brazil. PLoS One. 2017;12(8):e0183840. pmid:28850598
- 16. Ramírez ADR, de Jesus MCS, Menezes RAO, Santos-Filho MC, Gomes MSM, Pimenta TS, et al. Polymorphisms in Toll-Like receptors genes and their associations with immunological parameters in Plasmodium vivax malaria in the Brazil-French Guiana Border. Cytokine. 2023;169:156278. pmid:37356261
- 17. Pandey JP, Morais CG, Fontes CJ, Braga EM. Immunoglobulin GM 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. pmid:20696056
- 18. Dewasurendra RL, Suriyaphol P, Fernando SD, Carter R, Rockett K, Corran P, et al. Genetic polymorphisms associated with anti-malarial antibody levels in a low and unstable malaria transmission area in southern Sri Lanka. Malar J. 2012;11:281. pmid:22905743
- 19. 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(5):e36419. pmid:22649493
- 20. Storti-Melo LM, da Costa DR, Souza-Neiras WC, Cassiano GC, Couto VSCD, 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(2):152–5. pmid:22107686
- 21. Cassiano GC, Furini AAC, Capobianco MP, Storti-Melo LM, Cunha MG, Kano FS, et al. Polymorphisms in B cell co-stimulatory genes are associated with IgG antibody responses against blood-stage proteins of Plasmodium vivax. PLoS One. 2016;11(2):e0149581. pmid:26901523
- 22.
INE. Proyecciones de población con base al censo 2011. Instituto Nacional de Estadística; 2011. 2023 November 11. http://www.ine.gob.ve/index.php?option=com_content&view=category&id=95&Itemid=9
- 23.
MPPS. Boletín epidemiológico. Semana epidemiológica n°41. 09 de octubre al 15 de octubre del 2022. Año de edición LXIII. Caracas, D.C.: Ministerio del Poder Popular para la Salud; 2022.
- 24.
MPPS. Programa nacional de eliminación de malaria: pautas de tratamiento en casos de malaria. Caracas: Ministerio del Poder Popular para la Salud; 2017.
- 25.
WHO. Microscopy examination of thick and thin blood films for identification of malaria parasites. World Health Organization; 2016.
- 26.
Alger JJRMH. Densidad parasitaria en malaria: métodos de determinación y su interpretación. 2001;69:118–20.
- 27. Usluca S, Celebi B. Comparison of different 18S rRNA primers with conventional PCR methods in determination of Plasmodium spp. and evaluation of nested PCR method. J Basic Clin Health Sci. 2020.
- 28.
Bunce M. PCR-SSP typing. In: Histocompatibility testing. World Scientific; 2000. 149–86.
- 29. Cunha MG, Rodrigues MM, Soares IS. Comparison of the immunogenic properties of recombinant proteins representing the Plasmodium vivax vaccine candidate MSP1(19) expressed in distinct bacterial vectors. Vaccine. 2001;20(3–4):385–96. pmid:11672901
- 30. Hansen PR, Oddo A. Fmoc solid-phase peptide synthesis. Methods Mol Biol. 2015;1348:33–50. pmid:26424261
- 31. Carvalho A, Marques A, Maciel P, Rodrigues F. Study of disease-relevant polymorphisms in the TLR4 and TLR9 genes: a novel method applied to the analysis of the Portuguese population. Mol Cell Probes. 2007;21(4):316–20. pmid:17482427
- 32. Joshi A, Punke EB, Mehmetoglu-Gurbuz T, Peralta DP, Garg H. TLR9 polymorphism correlates with immune activation, CD4 decline and plasma IP10 levels in HIV patients. BMC Infect Dis. 2019;19(1):56. pmid:30651082
- 33. Solé X, Guinó E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics. 2006;22(15):1928–9. pmid:16720584
- 34. Medzhitov R. Recognition of microorganisms and activation of the immune response. Nature. 2007;449(7164):819–26. pmid:17943118
- 35. López C, Yepes-Pérez Y, Hincapié-Escobar N, Díaz-Arévalo D, Patarroyo MA. What is known about the immune response induced by Plasmodium vivax malaria vaccine candidates?. Front Immunol. 2017;8:126. pmid:28243235
- 36. Forero-Peña DA, Carrión-Nessi FS, Chavero M, Gamardo Á, Figuera L, Camejo-Ávila NA, et al. The clinical-epidemiological profile of malaria patients from Southern Venezuela, a critical hotspot in Latin America. Malar J. 2021;20(1):375. pmid:34544438
- 37. Forero-Peña DA, Carrión-Nessi FS, Lopez-Perez M, Sandoval-de Mora M, Amaya ID, Gamardo ÁF, et al. Seroprevalence of viral and bacterial pathogens among malaria patients in an endemic area of southern Venezuela. Infect Dis Poverty. 2023;12(1):33. pmid:37038195
- 38. Arévalo-Herrera M, Lopez-Perez M, Medina L, Moreno A, Gutierrez JB, Herrera S. Clinical profile of Plasmodium falciparum and Plasmodium vivax infections in low and unstable malaria transmission settings of Colombia. Malar J. 2015;14:154. pmid:25889074
- 39. Groves ES, Simpson JA, Edler P, Daher A, Pasaribu AP, Pereira DB, et al. Parasitaemia and fever in uncomplicated Plasmodium vivax malaria: a systematic review and individual patient data meta-analysis. PLoS Negl Trop Dis. 2025;19(3):e0012951. pmid:40153391
- 40. Markwalter CF, Petersen JEV, Zeno EE, Sumner KM, Freedman E, Mangeni JN, et al. Symptomatic malaria enhances protection from reinfection with homologous Plasmodium falciparum parasites. PLoS Pathog. 2023;19(6):e1011442. pmid:37307293
- 41. Mehlotra RK, Kasehagen LJ, Baisor M, Lorry K, Kazura JW, Bockarie MJ, et al. Malaria infections are randomly distributed in diverse holoendemic areas of Papua New Guinea. Am J Trop Med Hyg. 2002;67(6):555–62. pmid:12518843
- 42. Kalilani L, Mofolo I, Chaponda M, Rogerson SJ, Meshnick SR. The effect of timing and frequency of Plasmodium falciparum infection during pregnancy on the risk of low birth weight and maternal anemia. Trans R Soc Trop Med Hyg. 2010;104(6):416–22. pmid:20207387
- 43. Furini AA, Capobianco MP, Storti-Melo LM, Cunha MG, Cassiano GC, Machado RLD. Cytokine gene polymorphisms are not associated with anti-PvDBP, anti-PvAMA-1 or anti-PvMSP-119 IgG antibody levels in a malaria-endemic area of the Brazilian Amazon. Malar J. 2016;15(1):374. pmid:27435973
- 44. Ventocilla JA, Tapia LL, Ponce R, Franco A, Leelawong M, Aguiar JC, et al. Evaluation of naturally acquired immune responses against novel pre-erythrocytic Plasmodium vivax proteins in a low endemic malaria population located in the Peruvian Amazon Basin. Malar J. 2024;23(1):163. pmid:38783317
- 45. Fletcher IK, Grillet ME, Moreno JE, Drakeley C, Hernández-Villena J, Jones KE, et al. Synergies between environmental degradation and climate variation on malaria re-emergence in southern Venezuela: a spatiotemporal modelling study. Lancet Planet Health. 2022;6(9):e739–48. pmid:36087604
- 46. Julien J-P, Wardemann H. Antibodies against Plasmodium falciparum malaria at the molecular level. Nat Rev Immunol. 2019;19(12):761–75. pmid:31462718
- 47. Yman V, White MT, Asghar M, Sundling C, Sondén K, Draper SJ, et al. Antibody responses to merozoite antigens after natural Plasmodium falciparum infection: kinetics and longevity in absence of re-exposure. BMC Med. 2019;17(1):22. pmid:30696449
- 48. Omar AH, Yasunami M, Yamazaki A, Shibata H, Ofori MF, Akanmori BD, et al. Toll-like receptor 9 (TLR9) polymorphism associated with symptomatic malaria: a cohort study. Malar J. 2012;11:168. pmid:22594374
- 49. Lazarus R, Klimecki WT, Raby BA, Vercelli D, Palmer LJ, Kwiatkowski DJ, et al. Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium, and haplotypes in three U.S. ethnic groups and exploratory case-control disease association studies. Genomics. 2003;81(1):85–91. pmid:12573264
- 50. Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, Korbel JO, et al. A global reference for human genetic variation. Nature. 2015;526(7571):68–74. pmid:26432245
- 51. Costa AG, Ramasawmy R, Val FFA, Ibiapina HNS, Oliveira AC, Tarragô AM, et al. Polymorphisms in TLRs influence circulating cytokines production in Plasmodium vivax malaria: TLR polymorphisms influence cytokine productions in malaria-vivax. Cytokine. 2018;110:374–80. pmid:29656958
- 52. Sam-Agudu NA, Greene JA, Opoka RO, Kazura JW, Boivin MJ, Zimmerman PA, et al. TLR9 polymorphisms are associated with altered IFN-gamma levels in children with cerebral malaria. Am J Trop Med Hyg. 2010;82(4):548–55. pmid:20348497
- 53. Minias P, Vinkler M. Selection balancing at innate immune genes: adaptive polymorphism maintenance in toll-like receptors. Mol Biol Evol. 2022;39(5):msac102. pmid:35574644
- 54. Novak N, Yu C-F, Bussmann C, Maintz L, Peng W-M, Hart J, et al. Putative association of a TLR9 promoter polymorphism with atopic eczema. Allergy. 2007;62(7):766–72. pmid:17573724
- 55. Ng MTH, Van’t Hof R, Crockett JC, Hope ME, Berry S, Thomson J, et al. Increase in NF-kappaB binding affinity of the variant C allele of the toll-like receptor 9 -1237T/C polymorphism is associated with Helicobacter pylori-induced gastric disease. Infect Immun. 2010;78(3):1345–52. pmid:20038537
- 56. Lange NE, Zhou X, Lasky-Su J, Himes BE, Lazarus R, Soto-Quirós M, et al. Comprehensive genetic assessment of a functional TLR9 promoter polymorphism: no replicable association with asthma or asthma-related phenotypes. BMC Med Genet. 2011;12:26. pmid:21324137
- 57. Carvalho A, Osório NS, Saraiva M, Cunha C, Almeida AJ, Teixeira-Coelho M, et al. The C allele of rs5743836 polymorphism in the human TLR9 promoter links IL-6 and TLR9 up-regulation and confers increased B-cell proliferation. PLoS One. 2011;6(11):e28256. pmid:22132241
- 58. Carvalho A, Cunha C, Almeida AJ, Osório NS, Saraiva M, Teixeira-Coelho M, et al. The rs5743836 polymorphism in TLR9 confers a population-based increased risk of non-Hodgkin lymphoma. Genes Immun. 2012;13(2):197–201. pmid:21866115
- 59. 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 isotype profiles in complicated and uncomplicated malaria. Malar J. 2009;8:286. pmid:20003246
- 60. 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. pmid:23668806
- 61. Segbefia SP, Asandem DA, Amoah LE, Kusi KA. Cytokine gene polymorphisms implicated in the pathogenesis of Plasmodium falciparum infection outcome. Front Immunol. 2024;15:1285411. pmid:38404582
- 62. 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(1):53–8. pmid:23745572
- 63. Adu B, Jepsen MPG, Gerds TA, Kyei-Baafour E, Christiansen M, Dodoo D, et al. Fc gamma receptor 3B (FCGR3B-c.233C>A-rs5030738) polymorphism modifies the protective effect of malaria specific antibodies in Ghanaian children. J Infect Dis. 2014;209(2):285–9. pmid:23935200
- 64. Keitany GJ, Kim KS, Krishnamurty AT, Hondowicz BD, Hahn WO, Dambrauskas N, et al. Blood stage malaria disrupts humoral immunity to the pre-erythrocytic stage circumsporozoite protein. Cell Rep. 2016;17(12):3193–205. pmid:28009289
- 65. Soares IF, López-Camacho C, Rodrigues-da-Silva RN, da Silva Matos A, de Oliveira Baptista B, Totino PRR, et al. Recombinant Plasmodium vivax circumsporozoite surface protein allelic variants: antibody recognition by individuals from three communities in the Brazilian Amazon. Sci Rep. 2020;10(1):14020. pmid:32820195
- 66. Storti-Melo LM, de Souza-Neiras WC, Cassiano GC, Joazeiro ACP, Fontes CJ, Bonini-Domingos CR, et al. Plasmodium vivax circumsporozoite variants and Duffy blood group genotypes in the Brazilian Amazon region. Trans R Soc Trop Med Hyg. 2009;103(7):672–8. pmid:18804827
- 67. González JM, Hurtado S, Arévalo-Herrera M, Herrera S. Variants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Colombian isolates. Mem Inst Oswaldo Cruz. 2001;96(5):709–12. pmid:11500776
- 68. Leoratti FMS, Farias L, Alves FP, Suarez-Mútis MC, Coura JR, Kalil J, et al. Variants in the toll-like receptor signaling pathway and clinical outcomes of malaria. J Infect Dis. 2008;198(5):772–80. pmid:18662133
- 69. Barber BE, William T, Grigg MJ, Parameswaran U, Piera KA, Price RN, et al. Parasite biomass-related inflammation, endothelial activation, microvascular dysfunction and disease severity in vivax malaria. PLoS Pathog. 2015;11(1):e1004558. pmid:25569250
- 70. Pacheco MA, Lopez-Perez M, Vallejo AF, Herrera S, Arévalo-Herrera M, Escalante AA. Multiplicity of infection and disease severity in Plasmodium vivax. PLoS Negl Trop Dis. 2016;10(1):e0004355. pmid:26751811