Diversity analysis of MSP1 identifies conserved epitope organization in block 2 amidst high sequence variability in Indian Plasmodium falciparum isolates

Abstract Background Despite its immunogenicity, the polymorphic nature of merozoite surface protein 1, an important vaccine candidate for Plasmodium falciparum malaria, remains a concern. This study analyses the impact of genetic variability and parasite population structure on epitope organization of different MSP1 segments. Methods Altogether 98 blood samples collected from P. falciparum infected mild and severe malaria patients of Chhattisgarh and West Bengal were used to sequence regions encoding block 2 and MSP1-19 of msp1. Sequences were analysed using MEGA7, DnaSPv5, Arlequin3.5 and BepiPred. Results All three major MSP1 block 2 allele families namely K1, MAD20 and RO33 were detected in the samples and they together resulted in 41 indel variants. Chhattisgarh samples displayed an average MOI of 2.07 ± 1.59 which was higher in mild malaria and in age group < 18 years. Ultra-structure of block 2 alleles revealed that mutation and repeat expansion were two major mechanisms responsible for allelic variability of K1 and MAD20. Regions flanking block 2 were highly variable in Chhattisgarh with average mismatch differences (k) ranging from 1.198 to 5.156 for three families. In contrast, region encompassing MSP1-19 exhibited limited heterogeneity (kChhattisgarh = 1.45, kWest Bengal = 1.363). Of the 50 possible B cell linear epitopes predicted from block 2 variants, 94.9% (131 of 138) of the parasites could be represented by three conserved antigens. Conclusions Present data indicates that natural selection and transmission intensity jointly play a role in controlling allelic diversity of MSP1 in Indian parasite isolates. Despite remarkable genetic variability, a limited number of predominant and conserved epitopes are present in Indian parasite isolates reinstating the importance of MSP1 as a promising malaria vaccine candidate

Gene-Gene Interaction and Functional Impact of Polymorphisms on Innate Immune Genes in Controlling <em>Plasmodium falciparum</em> Blood Infection Level

<div><p>Genetic variations in toll-like receptors and cytokine genes of the innate immune pathways have been implicated in controlling parasite growth and the pathogenesis of <em>Plasmodium falciparum</em> mediated malaria. We previously published genetic association of <em>TLR4</em> non-synonymous and <em>TNF-α</em> promoter polymorphisms with <em>P.falciparum</em> blood infection level and here we extend the study considerably by (i) investigating genetic dependence of parasite-load on interleukin-12B polymorphisms, (ii) reconstructing gene-gene interactions among candidate <em>TLR</em>s and cytokine loci, (iii) exploring genetic and functional impact of epistatic models and (iv) providing mechanistic insights into functionality of disease-associated regulatory polymorphisms. Our data revealed that carriage of AA (P = 0.0001) and AC (P = 0.01) genotypes of <em>IL12B</em> 3′UTR polymorphism was associated with a significant increase of mean log-parasitemia relative to rare homozygous genotype CC. Presence of <em>IL12B+1188</em> polymorphism in five of six multifactor models reinforced its strong genetic impact on malaria phenotype. Elevation of genetic risk in two-component models compared to the corresponding single locus and reduction of <em>IL12B</em> (2.2 fold) and lymphotoxin-α (1.7 fold) expressions in patients'peripheral-blood-mononuclear-cells under <em>TLR4Thr399Ile</em> risk genotype background substantiated the role of Multifactor Dimensionality Reduction derived models. Marked reduction of promoter activity of <em>TNF-α</em> risk haplotype (C-C-G-G) compared to wild-type haplotype (T-C-G-G) with (84%) and without (78%) LPS stimulation and the loss of binding of transcription factors detected <em>in-silico</em> supported a causal role of <em>TNF-1031</em>. Significantly lower expression of <em>IL12B+1188</em> AA (5 fold) and AC (9 fold) genotypes compared to CC and under-representation (P = 0.0048) of allele A in transcripts of patients' PBMCs suggested an Allele-Expression-Imbalance. Allele (<em>A+1188C</em>) dependent differential stability (2 fold) of <em>IL12B</em>-transcripts upon actinomycin-D treatment and observed structural modulation (P = 0.013) of RNA-ensemble were the plausible explanations for AEI. In conclusion, our data provides functional support to the hypothesis that de-regulated receptor-cytokine axis of innate immune pathway influences blood infection level in <em>P. falciparum</em> malaria.</p> </div

Best predictive gene-gene interaction models identified by multifactor dimensionality reduction analysis.

a<p> <i>The best model was selected as the one with the minimum prediction error and maximum CVC.</i></p>b<p> <i>TBA corresponds to the testing balanced accuracy defined as the prediction error (PE) = 1-TBA.</i></p>c<p> <i>CVC corresponds to cross validation consistency.</i></p>*<p> <i>P values for gene-gene interaction models were calculated after 10,000 permutations in MDRpt software.</i></p

<i>IL12B</i> mRNA-microRNA interaction assay.

<p>(A) Schematic representation of reporter gene constructs for <i>IL12B</i> 3′UTR A and C alleles used for transfection assays. (B) Entire 3′UTR region was mapped for putative microRNA binding sites. The highlighted, boldfaced and underlined segments within <i>IL12B</i> sequence were the seed sequences for the miRNAs. (C) Schematic representation of the score and seed position of four miRNAs on target IL12B. (D & E) Normalized luciferase relative light units (RLU) in HepG2 cells were measured for <i>IL12B+1188</i> A and C allele containing pSiCHECK2 constructs with (+) and without (−) the effect of miRNAs. Co-transfection of the empty pRNAU6.1 (+) vector with pSiCHECK2-IL12B+1188 construct was set as 100% and reductions in luciferase expression in presence of four miRNAs were measured in relation to this. hsa-miR-23a and hsa-miR-23b resulted in significant reduction in luciferase activities for both pSiCHECK2-IL12B+1188A and pSiCHECK2-IL12B+1188C constructs. Statistical significance was measured with t-test. (*) indicates the P values and percentage reduction to be statistically significant.</p

Genetic association of <i>IL12B</i> promoter and 3′UTR polymorphisms with parasitemia at genotypic level.

a<p> <i>MAF denotes minor allele frequency and SD denotes standard deviation.</i></p>*<p> <i>P-Value<0.05.</i></p

Genetic and functional association of multifactor models obtained by Multifactorial Dimensionality Reduction analysis.

<p>(A) Forest plots presented the comparison of risk estimates in terms of odds ratio (OR) and 95% confidence interval (CI) for significant gene-gene interaction models and component single loci. The risk corresponding to each single and two factor models was denoted by a dot and the horizontal lines represented odds ratio and 95% CI respectively. The model IDs (A–J) and respective ORs (95% CI) were given at the left and right side of each dot in the forest plot. (B) <i>IL12B</i> and (C) <i>LTA</i> gene expression in patients' PBMCs classified according to <i>TLR4Thr399Ile</i> genotype status by real time quantitative PCR. Distribution of ΔC_t was plotted and compared between the genotypic groups. Statistical significance was determined by Mann Whitney U test. P values and corresponding fold changes obtained for each pairwise comparison were shown in the box plots. (*) indicates these differences to be statistically significant.</p

<i>IL12B</i> 3′UTR polymorphism based RNA ensemble structures.

<p>(A–D) display the SNPfold derived partition function heat maps generated for 101 nucleotide sequences harboring A (risk), G, U and C (non-risk) alleles respectively at the 51^st position. The partition function matrix illustrates the base-pairing probabilities represented by dots. We estimated the pairwise Pearson correlation coefficient with respect to wild-type A allele and P values to quantify the overall modulation in the RNA structural ensemble caused by a mutation. (*) indicates the P value to be statistically significant. (E & F) show the mountain plot diagrams for <i>IL12+1188</i>A and C allele for 101 bases using RNAfold. The upper panel demonstrates the height vs position graph in which the red, green and blue lines depict the minimum free energy structure, the partition function of all possible RNA secondary structures and ensemble centroid structure respectively. The lower panel represents the entropy vs position profile and arrowhead denotes the 51^st position, the location of <i>A+1188C</i>. (G & H) shows allele specific minimum free energy (MFE) conformations generated from RNA MFOLD. Numbers indicate the base position while the arrow directs the position of the polymorphic site.</p