Identification of a superagonist variant of the immunodominant Yellow fever virus epitope NS4b 214-222 by combinatorial peptide library screening

•We identified a 7I superagonist mutant of the NS4b214-22 Yellow Fever virus epitope.•This LLW-7I mutant was found by combinatorial peptide library screening on the NN5048 CD8 T cell clone.•The LLW-7I mutation increases T cell function yet does not impact the monomeric off-rate.•Our modeling suggests that this superior antigen sensitivity rather results from an increased pMHC complex rigidity. The CD8 T cell response to the HLA-A2-restricted epitope LLWNGPMAV (LLW) of the non-structural protein 4b of Yellow Fever Virus (YFV) is remarkably immunodominant, highly prevalent and powerful in YFV-vaccinated humans. Here we used a combinatorial peptide library screening in the context of an A2/LLW-specific CD8 T cell clone to identify a superagonist that features a methionine to isoleucine substitution at position 7. Based on in silico modeling, the functional enhancement of this LLW-7I mutation was associated with alterations in the structural dynamics of the peptide in the major histocompatibility complex (pMHC) binding with the T cell receptor (TCR). While the TCR off-rate of LLW-7I pMHC is comparable to the wild type peptide, the rigidity of the 7I peptide seems to confer less entropy loss upon TCR binding. This LLW-7I superagonist is an example of improved functionality in human CD8 T cells associated with optimized ligand rigidity for TCR binding and not with changes in TCR:pMHC off-rate kinetics.