Quantifying memory CD8 T cell-mediated immune pressure on influenza A virus infection in vivo

The conservation of T cell epitopes in human influenza A virus has prompted the development of T cell-inducing influenza vaccines. However, the selection pressure mediated by memory CD8 T cells upon influenza virus has not been directly measured. Using a droplet digital PCR technique to distinguish wild-type and an epitope-mutant PR8 influenza viruses in vivo, this study quantifies the viral replicative fitness of a CD8 T cell-escaping mutation in the immunodominant influenza NP366-374 epitope in C57BL/6 (B6) mice under different settings of cellular immunity. Although this mutation does not result in a viral fitness defect in vitro or during the early stages of in vivo infection in naive B6 mice, it does confer a moderate but consistent advantage to the mutant virus following heterosubtypic challenge of HKx31-immunized mice. In addition, this advantage was maintained under increased MHC diversity but became more substantial when the breadth of epitope recognition is limited. Finally, we showed that lung-resident, but not circulating, memory CD8 T cells are the primary source of cellular immune pressure early during infection, prior to the induction of a secondary effector T cell response. Integrating the data with an established modeling framework, we show that the relatively modest immune pressure mediated by memory CD8 T cells is one of the important factors responsible for the conservation of CD8 T cell epitopes in influenza A viruses that circulate among humans. Thus, a T cell-inducing vaccine that generates lung-resident memory CD8 T cells covering a sufficient breadth of epitopes may transiently protect against severe pathology without driving the virus to rapidly evolve and escape. Author SummarySince the historic Spanish flu in 1918, influenza has caused several pandemics and become an important public health concern. The inactivated vaccines routinely used attempt to boost antibodies, which may not be as effective when antigenic mismatch happens and could drive the virus to evolve and escape due to their high immune pressure. In contrast, the ability of influenza-specific T cells to reduce pathology and the conservation of T-cell epitopes across subtypes have shed light on the development of universal vaccines. In this study, we assessed the CD8 T cell-mediated selection pressure on influenza virus in mouse using a digital PCR technique. Within mice that have influenza-specific systemic and lung-resident memory CD8 T cells established, we found the advantage conferred by an escaping mutation in one of the immunodominant epitopes is around 25%. This advantage becomes much greater when the cellular immunity focuses on the focal epitope, while it is delayed when only systemic cellular immunity is established. Combining the data with our previous modeling work, we conclude that the small selection pressure imposed by CD8 T cells can explain the overall conservation of CD8 T cell epitopes of influenza A virus in addition to functional constraint.