TLR9 polymorphisms in African populations: no association with severe malaria, but evidence of cis-variants acting on gene expression

BACKGROUND: During malaria infection the Toll-like receptor 9 (TLR9) is activated through induction with plasmodium DNA or another malaria motif not yet identified. Although TLR9 activation by malaria parasites is well reported, the implication to the susceptibility to severe malaria is not clear. The aim of this study was to assess the contribution of genetic variation at TLR9 to severe malaria. METHODS: This study explores the contribution of TLR9 genetic variants to severe malaria using two approaches. First, an association study of four common single nucleotide polymorphisms was performed on both family- and population-based studies from Malawian and Gambian populations (n>6000 individual). Subsequently, it was assessed whether TLR9 expression is affected by cis-acting variants and if these variants could be mapped. For this work, an allele specific expression (ASE) assay on a panel of HapMap cell lines was carried out. RESULTS: No convincing association was found with polymorphisms in TLR9 for malaria severity, in either Gambian or Malawian populations, using both case-control and family based study designs. Using an allele specific expression assay it was observed that TLR9 expression is affected by cis-acting variants, these results were replicated in a second experiment using biological replicates. CONCLUSION: By using the largest cohorts analysed to date, as well as a standardized phenotype definition and study design, no association of TLR9 genetic variants with severe malaria was found. This analysis considered all common variants in the region, but it is remains possible that there are rare variants with association signals. This report also shows that TLR9 expression is potentially modulated through cis-regulatory variants, which may lead to differential inflammatory responses to infection between individuals

Investigation of Host Candidate Malaria-Associated Risk/Protective SNPs in a Brazilian Amazonian Population

The Brazilian Amazon is a hypo-endemic malaria region with nearly 300,000 cases each year. A variety of genetic polymorphisms, particularly in erythrocyte receptors and immune response related genes, have been described to be associated with susceptibility and resistance to malaria. In order to identify polymorphisms that might be associated with malaria clinical outcomes in a Brazilian Amazonian population, sixty-four human single nucleotide polymorphisms in 37 genes were analyzed using a Sequenom massARRAY iPLEX platform. A total of 648 individuals from two malaria endemic areas were studied, including 535 malaria cases (113 individuals with clinical mild malaria, 122 individuals with asymptomatic infection and 300 individuals with history of previous mild malaria) and 113 health controls with no history of malaria. The data revealed significant associations (p<0.003) between one SNP in the IL10 gene (rs1800896) and one SNP in the TLR4 gene (rs4986790) with reduced risk for clinical malaria, one SNP in the IRF1 gene (rs2706384) with increased risk for clinical malaria, one SNP in the LTA gene (rs909253) with protection from clinical malaria and one SNP in the TNF gene (RS1800750) associated with susceptibility to clinical malaria. Also, a new association was found between a SNP in the CTL4 gene (rs2242665), located at the major histocompatibility complex III region, and reduced risk for clinical malaria. This study represents the first association study from an Amazonian population involving a large number of host genetic polymorphisms with susceptibility or resistance to Plasmodium infection and malaria outcomes. Further studies should include a larger number of individuals, refined parameters and a fine-scale map obtained through DNA sequencing to increase the knowledge of the Amazonian population genetic diversity

Antibody responses to <i>P. falciparum</i> blood stage antigens and incidence of clinical malaria in children living in endemic area in Burkina Faso

Abstract Background High parasite-specific antibody levels are generally associated with low susceptibility to Plasmodium falciparum malaria. This has been supported by several studies in which clinical malaria cases of P. falciparum malaria were reported to be associated with low antibody avidities. This study was conducted to evaluate the role of age, malaria transmission intensity and incidence of clinical malaria in the induction of protective humoral immune response against P. falciparum malaria in children living in Burkina Faso. Methods We combined levels of IgG and IgG subclasses responses to P. falciparum antigens: Merozoite Surface Protein 3 (MSP3), Merozoite Surface Protein 2a (MSP2a), Merozoite Surface Protein 2b (MSP2b), Glutamate Rich Protein R0 (GLURP R0) and Glutamate Rich Protein R2 (GLURP R2) in plasma samples from 325 children under five (05) years with age, malaria transmission season and malaria incidence. Results We notice higher prevalence of P. falciparum infection in low transmission season compared to high malaria transmission season. While, parasite density was lower in low transmission than high transmission season. IgG against all antigens investigated increased with age. High levels of IgG and IgG subclasses to all tested antigens except for GLURP R2 were associated with the intensity of malaria transmission. IgG to MSP3, MSP2b, GLURP R2 and GLURP R0 were associated with low incidence of malaria. All IgG subclasses were associated with low incidence of P. falciparum malaria, but these associations were stronger for cytophilic IgGs. Conclusions On the basis of the data presented in this study, we conclude that the induction of humoral immune response to tested malaria antigens is related to age, transmission season level and incidence of clinical malaria

Heritability of antibody isotype and subclass responses to Plasmodium falciparum antigens.

BACKGROUND: It is important to understand the extent to which genetic factors regulate acquired immunity to common infections. A classical twin study design is useful to estimate the heritable component of variation in measurable immune parameters. METHODOLOGY/PRINCIPAL FINDINGS: This study assessed the relative heritability of different plasma antibody isotypes and subclasses (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE) naturally acquired to P. falciparum blood stage antigens AMA1, MSP1-19, MSP2 (two allelic types) and MSP3 (two allelic types). Separate analyses were performed on plasma from 213 pairs of Gambian adult twins, 199 child twin pairs sampled in a dry season when there was little malaria transmission, and another set of 107 child twin pairs sampled at the end of the annual wet season when malaria was common. There were significantly positive heritability (h(2)) estimates for 48% (20/42) of the specific antibody assays (for the seven isotypes and subclasses to the six antigens tested) among the adults, 48% (20/42) among the children in the dry season and 31% (13/42) among the children in the wet season. In children, there were significant heritability estimates for IgG4 reactivity against each of the antigens, and this subclass had higher heritability than the other subclasses and isotypes. In adults, 75% (15/20) of the significantly heritable antigen-specific isotype responses were attributable to non-HLA class II genetic variation, whereas none showed a significant HLA contribution. SIGNIFICANCE: Genome-wide approaches are now warranted to map the major genetic determinants of variable antibody isotype and subclass responses to malaria, alongside evaluation of their impact on infection and disease. Although plasma levels of IgG4 to malaria antigens are generally low, the exceptionally high heritability of levels of this subclass in children deserves particular investigation

IgG1 as a potential biomarker of post-chemotherapeutic relapse in visceral leishmaniasis, and adaptation to a rapid diagnostic test.

BACKGROUND: Visceral leishmaniasis (VL), caused by protozoa of the Leishmania donovani complex, is a widespread parasitic disease of great public health importance; without effective chemotherapy symptomatic VL is usually fatal. Distinction of asymptomatic carriage from progressive disease and the prediction of relapse following treatment are hampered by the lack of prognostic biomarkers for use at point of care. METHODOLOGY/PRINCIPAL FINDINGS: All IgG subclass and IgG isotype antibody levels were determined using unpaired serum samples from Indian and Sudanese patients with differing clinical status of VL, which included pre-treatment active VL, post-treatment cured, post-treatment relapsed, and post kala-azar dermal leishmaniasis (PKDL), as well as seropositive (DAT and/or rK39) endemic healthy controls (EHCs) and seronegative EHCs. L. donovani antigen-specific IgG1 levels were significantly elevated in relapsed versus cured VL patients (p<0.0001). Using paired Indian VL sera, consistent with the known IgG1 half-life, IgG1 levels had not decreased significantly at day 30 after the start of treatment (p = 0.8304), but were dramatically decreased by 6 months compared to day 0 (p = 0.0032) or day 15 (p<0.0001) after start of treatment. Similarly, Sudanese sera taken soon after treatment did not show a significant change in the IgG1 levels (p = 0.3939). Two prototype lateral flow immunochromatographic rapid diagnostic tests (RDTs) were developed to detect IgG1 levels following VL treatment: more than 80% of the relapsed VL patients were IgG1 positive; at least 80% of the cured VL patients were IgG1 negative (p<0.0001). CONCLUSIONS/SIGNIFICANCE: Six months after treatment of active VL, elevated levels of specific IgG1 were associated with treatment failure and relapse, whereas no IgG1 or low levels were detected in cured VL patients. A lateral flow RDT was successfully developed to detect anti-Leishmania IgG1 as a potential biomarker of post-chemotherapeutic relapse