Modulation of mucosal immunity and tissue resident memory formation by NS1-deficient influenza A virus

Abstract

<p><strong>Background</strong>: Recurrent influenza A virus (IAV) epidemics and occasional pandemics as a result of viral genome reassortment and human adaptation pose significant health burdens globally. The non-structural protein 1 (NS1) mediates attenuation of host response and facilitates viral replication in the nucleus. While intranasal vaccination using NS1-deleted live attenuated virus has been shown to induce pulmonary mucosal immunity in animal models, the immunological details have not been fully studied.</p><p><strong>Methods</strong>: The model WSN H1N1 virus (WSN/WT), together with the NS1-deleted version (WSN/DelNS1) were used for intranasal inoculation of Balb/c mice. During acute and memory phases, lungs and mediastinal lymph nodes (mLN) were harvested for flow cytometry. Immunized and recovered mice were rechallenged with H3N2 to assess heterosubtypic CD8+ T cell response. To evaluate how CD8+ T cells were differentially primed in WSN/WT and WSN/DelNS1 infections, dendritic cells from mLN and influenza-specific CD8+ T cells were sorted during priming, acute, memory and reactivated phases for single cell RNA-seq (scRNA-seq) analysis. </p><p><strong>Results</strong>: As expected, WSN/DelNS1 induced massive type I interferon response in murine lungs with minimal pulmonary inflammation. Skewed migration of CD103+ dendritic cells (DC) to mediastinal lymph nodes upon WSN/DelNS1 infection was observed with enhanced formation and persistence of tissue resident influenza-specific memory CD8 T cells (TRM). After rechallenge, while influenza-specific TRM in both groups expanded comparably, more polyfunctionality was seen in TRM from WSN/DelNS1 immunized mice than WSN/WT recovered mice. Single cell RNA-seq of migratory DC in initial immunization showed differential gene programs associated with WSN/DelNS1 infection.</p><p><strong>Conclusions:</strong> The data altogether stipulate that the nuclear localization of NS1 determines virulence and host suppression at the transcriptional level. Robust innate and adaptive responses were evoked when NS1 was sequestered in the cytoplasm. Ongoing work is undertaken to identify the changes in transcriptional profile and chromatin landscape by disrupting the nuclear distribution of NS1. We hope our work can provide additional knowledge on the role of nuclear NS1 and unravel how IAV utilizes NS1 to manipulate the functional consequence of an infection through transcriptional and epigenetic regulations. </p>