High affinity antigen-specific T cells play a critical role during protective immune responses. Epitope enhancement can elicit more potent T cell responses and can subsequently lead to a stronger memory pool; however, the molecular basis of such enhancement is unclear. We used the consensus peptide-binding motif for the Major Histocompatibility Complex molecule H-2K b to design a heteroclitic version of the mouse hepatitis virus-specific subdominant S598 determinant. We demonstrate that a single amino acid substitution at a secondary anchor residue (Q to Y at position 3) increased the stability of the engineered determinant in complex with H-2K b . The structural basis for this enhanced stability was associated with local alterations in the pMHC conformation as a result of the Q to Y substitution. Recombinant viruses encoding this engineered determinant primed CTL responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second CTL determinant and consequent disease progression in persistently infected mice. Collectively, our findings provide a basis for the enhanced immunogenicity of an engineered determinant that will serve as a template for guiding the development of heteroclitic T cell determinants with applications in prevention of CTL escape in chronic viral infections as well as in tumor immunity. Author Summary Enhancing the immune responses to pathogens is a chief goal of vaccine development. Here, we describe the development of an engineered CD8+ T cell epitope that elicits an immune response to the native epitope that is more potent than the one that occurs during the natural infection. We showed that this “improved” (heteroclitic) epitope protects against clinical disease and against cytotoxic T cell escape that frequently occurs in the immunodominant epitope expressed by the virus. We also performed structural analyses and showed that enhanced immunogenicity was associated with changes in the conformations of both the peptide and the region of the MHC class I molecule that is in close association with the peptide. These studies provide a model for designing T cell epitopes with enhanced immunogenicity that will be useful in vaccine development, with particular emphasis on diseases, such as HIV and hepatitis C, in which epitope mutation and escape is common.
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