Abstract
HLA-DRB1 is the major genetic risk factor for visceral leishmaniasis (VL). We used SNP2HLA to impute HLA-DRB1 alleles and SNPTEST to carry out association analyses in 889 human cases and 977 controls from India. NetMHCIIpan 2.1 was used to map epitopes and binding affinities across 49 Leishmania vaccine candidates, as well as across peptide epitopes captured from dendritic cells treated with crude Leishmania Ag and identified using mass spectrometry and alignment to amino acid sequences of a reference Leishmania genome. Cytokines were measured in peptide-stimulated whole blood from 26 cured VL cases and eight endemic healthy controls. HLA-DRB1*1501 and DRB1*1404/DRB1*1301 were the most significant protective and risk alleles, respectively, with specific residues at aa positions 11 and 13 unique to protective alleles. We observed greater peptide promiscuity in sequence motifs for 9-mer core epitopes predicted to bind to risk (*1404/*1301) compared with protective (*1501) DRB1 alleles. There was a higher frequency of basic amino acids in DRB1*1404/*1301-specific epitopes compared with hydrophobic and polar amino acids in DRB1*1501-specific epitopes at anchor residues pocket 4 and pocket 6, which interact with residues at DRB1 positions 11 and 13. Cured VL patients made variable, but robust, IFN-γ, TNF, and IL-10 responses to 20-mer peptides based on captured epitopes, with peptides based on DRB1*1501-captured epitopes resulting in a higher proportion (odds ratio 2.23, 95% confidence interval 1.17-4.25, p = 0.017) of patients with IFN-γ/IL-10 ratios > 2-fold compared with peptides based on DRB1*1301-captured epitopes. Our data provide insight into the molecular mechanisms underpinning the association of HLA-DRB1 alleles with risk versus protection in VL in humans.