Background: A small fraction of HIV-infected patients known as “elite controllers” maintain very low levels of plasma HIV and near normal levels of CD4+T-cells without antiretroviral therapy. The majority of these long-term non-progressors possess HLA alleles such as HLA-B*57:01 or -B*27:05 that are thought to offer superior T-cell mediated immunity. The majority of peptide epitopes presented by these protective haplotypes have been identified using synthetic peptide libraries and no systematic study has been performed to identify the T-cell epitopes naturally presented by these apparently protective alleles. The use of synthetic peptides may fail to identify post-translationally modified epitopes, epitopes of non-canonical lengths and those arising from translation of alternative reading frames. We have used a mass spectrometry-based immunopeptidomics approach to identify the peptides naturally presented by protective alleles.
Methodology: HLA class I molecules were immunoaffinity purified from cells transfected with plasmids expressing HLA B*57:01 and HIV Env gp140, Rev and Vpu. HLA- bound peptide cargo was separated using reversed-phase chromatography and analyzed on a high resolution AB SCIEX 5600plusTripleTOF mass spectrometer. The data obtained was searched using ProteinPilot software. Peptides assignments were rigorously curated and validated with isotopically labeled HIV peptides.
Results: Over 2900 B*57:01-restricted human peptides were identified and we confirmed ten B*57:01 restricted, naturally presented HIV-gp140 peptides using synthetic peptides. Six of these peptides form part of a larger immunogenic region that was assigned on the LANL-HIV Epitope database. One of the four peptides is part of the Env signal peptide and has a post-translationally modified tryptophan residue. Additionally, one Rev peptide was identified and there were no peptides representing Vpu.
Conclusions: Using a cutting-edge immunopeptidomics approach we have identified novel epitopes that are non-conventional or post-translationally modified. Our methodology has enabled the identification of one of the largest sets of naturally presented human B*57:01 epitopes reported to date along with several novel B*57:01-restricted HIV peptides, setting the stage for elucidation of epitopes from other HIV proteins and analysis of T-cell responses.