Investigating Differential Binding Stabilities of MHC-E Restricted Epitopes in SIV Vaccination

Abstract

HIV continues to pose a global health challenge and has prompted extensive research into exploring effective vaccine strategies. Previous breakthroughs in simian immunodeficiency virus (SIV) vaccine development using a rhesus CMV vector recombinant for SIV antigen (RhCMV 68-1/SIV) showed unprecedented sterile immune clearance against SIV challenge. Subsequent studies sought to determine the underlying mechanism of protection in this promising prophylactic vaccine strategy. An unconventional, MHC-E-restricted subset of CD8+ T cells was identified and shown to be necessary for protection. However, RhCMV 68-1/SIV vaccine-identified SIV epitopes identified by Hansen et al. lack canonical primary anchor residues, present low or negligible binding to human HLA-E, and thus do not support stable HLA-E/B2M complex formation. This inconsistency raises questions about their role and functionality in RhCMV 68-1/SIV vaccination. We therefore performed a comprehensive and direct comparison of SIV peptide binding to human and rhesus MHC-E. This thesis study aims to evaluate the binding characteristics and stability of RhCMV 68-1/SIV vaccine-identified SIV epitopes in complex with a panel of rhesus macaque and human MHC-E orthologs, specifically Mamu-E02:04, Mamu-E02:11, and HLA-E*01:03. The objective is to validate whether these SIV epitopes demonstrate comparable binding to Mamu-E and HLA-E, or if there is enhanced stability for Mamu-E as opposed to HLA-E. If the latter holds true, with Mamu-E exhibiting superior stability in complex with vaccine-identified SIV epitopes, it could explain the mechanism of vaccine efficacy but indicate challenges in translating this vaccine success to human clinical applications. If Mamu-E and HLA-E show similar binding to RhCMV 68-1/SIV vaccine-identified SIV epitopes, then alternative mechanisms of vaccine-mediated protection should be explored. To accomplish these goals, Mamu-E and HLA-E were recombinantly expressed in prokaryotic expression systems and refolded to form MHC-E/B2-microglobulin complexes prior to performing in vitro SIV peptide loading reactions. Thermostability assays using Differential Scanning Fluorimetry were conducted to compare the stability of Mamu-E and HLA-E complexes in the presence of excess SIV peptides. By examining the interactions between the vaccine-identified SIV epitopes and MHC-E molecules, this study aims to clarify the underlying mechanisms of the observed vaccine efficacy thereby facilitating its translation into the human clinic. Our findings reveal that the majority of vaccine-identified SIV epitopes do not stabilize either Mamu-E or HLA-E alleles. Given this weak or insignificant stabilization, it is likely that direct presentation of vaccine-identified SIV epitopes by MHC-E is not the primary mechanism of protection introduced by RhCMV68-1/SIV vaccination.