Jenefer M Blackwell, Michaela Fakiola, Joyce Oommen, Toolika Singh, Noel H. Smith, Shyam Sundar
In a previous (Nature Genetics, 2013, 45:208-213) genome-wide association study (GWAS) we identified HLA DRB1 allele groups tagged by ancestral haplotypes that confer disease risk (DRB1*11/*13/*14 allele groups), or protect (DRB1*15/*16/*01 allele groups) from, human visceral leishmaniasis in India and Brazil (Combined P=2.76x10-17, OR=1.41, 95%CI 1.30-1.52). One mechanism to account for this association is that the top single nucleotide polymorphisms (SNPs) associated with visceral leishmaniasis tag variants that determine functional differences at the amino acid level which directly influence epitope selection and antigen presentation. To understand how amino acid sequence differences in these genetic risk factors could influence binding of leishmanial epitopes for presentation to the immune system, we successfully captured leishmanial epitopes from antigen presenting cells. We eluted peptides from HLA class II molecules purified from 4-5x106 dendritic cells and characterized the amino acid sequences of peptides eluted from risk versus protective DRB1 molecules. Based on 9-mer cores for the 20-mers with strongest DRB1 allele-specific binding affinity there was, as we had also demonstrated in a purely in silico analysis of epitopes from 43 candidate vaccine antigens, a greater number and complexity of amino acid residues making up the 9-mer cores of peptides eluted from the DRB1*1301 or *1404 risk alleles than for the DRB1*1501 protective allele, suggesting greater promiscuity in binding of Leishmania epitopes to risk compared to protective DRB1 alleles. In addition, there was a bias towards hydrophobic and polar AAs in DRB1*1501-specific peptides at 4 and 6, pointing to the potential importance of these anchor residues interacting with the DRB1 residues at position 11 and 13, that are the core positions determining the association with visceral leishmaniasis.
Overall this epitope capture experiment demonstrated greater promiscuity of amino acid usage across the 9-mer core epitopes for risk DRB1 alleles (DRB1*1404 and DRB1*1301) compared to more restricted amino acid usage in the protective allele group (DRB1*1501). This appears to be different to viral infections where HLA-DRB1 variants linked with low HIV viremia promiscuously present a larger breadth of peptides with lower functional avidity when compared to DRB1 variants linked with high HIV viremia, or Hepatitis C where dominant and highly promiscuous epitopes characterize the CD4+ T helper cell response of spontaneously controlled infection. For VL it could be an indication that an over-enthusiastic pro-inflammatory CD4+ T cell response associated with DRB1 risk alleles early in infection could be counter-protective, a hypothesis consistent with high TNF in clinical VL that will be testable in humanized HLA_DRB1*1404 and HLA_DRB1*1501 transgenic mice which we are now using to pursue this research.
Plain language summary: Visceral leishmaniasis is a parasitic disease found in resource poor regions of tropical countries and is life-threatening in susceptible individuals. Drugs are toxic and expensive, with drug resistance a growing problem. No vaccines are available to protect against this disease, and there is a particular need to ensure that next generation defined vaccines will be effective even in genetically susceptible individuals. We have therefore employed modern genomics to identify the major genetic risk factor for visceral leishmaniasis, knowledge of which is helping us to engineer vaccines that will specifically aid in protecting susceptible individuals.
Funder: NIH Tropical Medicine Research Centre (Leader S Sundar, India; Project leaders J M Blackwell and M Fakiola)
External collaborators:
- Professor Shyam Sundar, Institute for Medical Science, Banaras Hindu University, Varanasi, India
- Ms Toolika Singh, Institute for Medical Science, Banaras Hindu University, Varanasi, India
- Dr Michaela Fakiola, Institute of Molecular Genetics, Milan, Italy
- Professor Mary Wilson, University of Iowa, Iowa, USA
- Dr Noel Smith, Lonza Biologics plc, Cambridge, UK