The Proteomic Landscape during Influenza Infection

Influenza A virus (IAV) is a highly contagious respiratory pathogen causing seasonal epidemics that result in around one billion infections and up to 650,000 deaths annually. IAV poses a major zoonotic threat, as transmission between animals and humans can generate novel strains capable of causing pandemics. Through genetic mutation or reassortment, IAV infects both humans and multiple animal species. Historical pandemics, including the 1918 H1N1 Spanish flu and the 2009 swineorigin H1N1 outbreak, demonstrate IAV’s global impact. Although swine have similar respiratory receptors to humans, the viral and host factors enabling cross-species transmission remain unclear, representing key knowledge gaps essential to predict zoonotic potential and prevent future influenza pandemics. When IAV infects respiratory epithelial cells, innate immune sensors trigger antiviral responses, including cytokine and interferon production, which stimulate antiviral molecules in infected and neighbouring cells. Meanwhile, the virus hijacks host machinery to replicate, manipulating signalling pathways via post-translational modifications and evading immune defences to ensure successful replication.

In Manuscript I, this study gained insight into how hostadaptation of IAV shapes the innate immune response by infecting pigs with swine- and human-adapted strains and analysing upper and lower tracheal tissues using multiomics. A classical antiviral innate immune response was induced in both regions following infection, characterised by upregulation of genes and increased abundance of proteins linked to viral recognition and infection. This was accompanied by a significant induction of interferon-stimulated genes and proteins. The swine-adapted strain induced a stronger response in the lower trachea, likely reflecting higher viral load and inflammation.

Manuscript II examined host adaptation of swine- and human-adapted H1N1 strains using a longitudinal air-liquid interface culture of swine nasal epithelial cells. Samples collected 1–48 hours postinfection allowed analysis of early host responses via global proteome and phosphoproteome profiling. The human-adapted strain triggered rapid antiviral signalling within one hour, while the swine-adapted strain induced weaker, delayed responses. Transcriptomic profiling showed broader antiviral gene induction by the human-adapted strain, and phosphoproteomics revealed strain-specific regulation of immune and Hippo signalling, with early YAP/TAZ inactivation for the human-adapted strain but delayed modifications for the swine-adapted strain. These results highlight distinct temporal host responses shaped by viral adaptation and the reverse zoonotic potential of human-adapted strain.

In Manuscript III, we mapped the protease networks across three respiratory tissues after infection with a dualhost IAV H1N1 strain using comprehensive proteomics and degradomics approaches. Protease abundance and activity were highly tissue-specific, the nasal mucosa showed strong antiviral responses along with proteases that elicited a broad specificity, the trachea exhibited moderate modulation, and the lung maintained high protease activity despite lower viral loads, associated with severe tissue damage. Distinct cleavage preferences linked proteolytic activity to antiviral defence, antigen processing, and tissue remodelling, defining an organised proteolytic landscape that shapes tissue-specific host responses and H1N1 pathogenesis.

Overall, this thesis highlights that IAV host adaptation fundamentally shapes immune and proteolytic responses across tissues and species. Human- and swine-adapted H1N1 strains elicited different temporal, transcriptional, and proteomic responses, while dual-host strains triggered tissue-specific protease networks linked to antiviral defence, antigen processing, and lung pathology. These findings highlight how viral adaptation and host factors govern infection dynamics and cross-species transmission, demonstrating the power of integrated multiomics approaches to understand influenza pathogenesis and predict zoonotic or pandemic strains.