The CD8+ T cell response to virus infection is dependent upon recognition of peptide epitopes complexed to MHC molecules and presented on the surface of infected cells. Whilst the mechanisms underlying this processing pathway are known, there is a paucity of quantitative information regarding the dynamics of presentation and how this may impact upon the magnitude of the T cell response. Here we have used targeted mass spectrometry to investigate the display of epitopes during vaccinia virus infection of three different cell types; namely in vitro fibroblasts and dendritic cells, and ex vivo bone marrow-derived dendritic cells. LC-MRM was used to simultaneously track the kinetics of 47 vaccinia-derived epitopes presented to the immune system, incorporating stable isotope-labeled peptides in order to provide measures of absolute abundance, whilst data-independent SWATH-MS was used to track antigen levels and profile virus proteome expression. The CD8+ response to each epitope was measured in parallel from infected mice in order to determine the immunodominance hierarchy each peptide elicits.

We reveal that the nature of vaccinia epitope display is highly diverse during infection, with peptide levels spanning many orders of magnitude. Further, whilst the relative kinetic of each epitope was remarkably similar across each cell type, abundance levels were found to vary considerably, with notably diminished levels on fibroblasts. Antigen expression and epitope kinetics were tightly coupled, as is predicted in order to engender a rapid T cell response to infection. However, correlations between epitope abundance and CD8+ response magnitudes remain difficult to predict, despite here using ex vivo dendritic cells as a model of in vivo infection. This study represents an advancement in our understanding of virus infection in the context of the immune system and will be necessary to drive further investigation into factors that can tailor immunity to pathogens for therapeutic benefit.