Development and implementation of a direct detection, quantitation and validation system for class I MHC self-peptide epitopes

Gene and protein expression studies demonstrate that viral-infected and malignant cells undergo a complex series of transcriptional and translational changes. As class I MHC molecules reflect the proteome (and changes therein) by presenting intracellular peptide epitopes, the development of a direct discovery and validation technology for the identification of these epitopes is needed. We developed our technology using HIV-1-infected cells as a model. A combination of hollow fiber class I HLA protein production and mass spectrometric epitope analysis indicated a 3-fold increase in the host-peptide VLMTEDIKL720–728, [eIF4G(720)] presented by the HLA-A⁎0201 of HIV-1-infected cells. This peptide is derived from the host-protein translation of eukaryotic initiation factor 4-gamma (eIF4G) that plays a pivotal role in cellular protein synthesis. Direct confirmation of expression of this self-encoded antigen was performed through development of a T cell receptor mimic (TCRm) monoclonal antibody (mAb). The resulting 4F7 TCRm demonstrated specific recognition of the eIF4G(720)–A⁎0201 complex. Staining of normal PBMCs with 4F7 showed only low levels of endogenous eIF4G(720) presentation by HLA-A⁎0201, while 4F7 staining of HIV-1-infected PBMCs revealed a ∼ 3-fold increase in eIF4G(720)–A⁎0201. The MHC–peptide complex was initially detectable by 4F7 at 3 days post-infection, with a steady increase through day 8. We therefore demonstrate the successful development and implementation of an integrated discovery and validation technology system for direct identification and confirmation of class I MHC–peptide epitopes on cells.