Functional study of IFIRM in influenza A virus infection and the development of COVID-19 vaccine and diagnostic

Abstract

Seasonal influenza and the recent COVID-19 have caused global public health burdens and seriously affect immunocompromised individuals. These infectious diseases are caused by respiratory tract infections by influenza viruses and SARS-CoV-2 respectively. To fight against these highly transmissible respiratory viruses, it is imperative to have deep understanding and adequate knowledge of host immune responses and host-virus interactions. Here in this two-part thesis, the immunology of the defence against respiratory viruses was studied. The first part of this thesis focused on innate antiviral responses during influenza A virus infection. A novel functional role of the interferon-stimulated gene IFIRM against influenza A virus infection was revealed and mechanistic investigations demonstrated that IFIRM effectively restricts influenza A virus replication in a m6A-dependent manner. Using immunoprecipitation-liquid chromatography with tandem mass spectrometry, m6A-related proteins such as YTHDF and IGF2BP families were identified as the novel interacting partners of IFIRM. Antiviral function of IFIRM was abolished in loss-of-function experiments utilizing METTL3 knockout cells and m6A-deficient mutant virus, further demonstrated that IFIRM’s antiviral function is dependent on both host and viral m6A. More importantly, induction of IFIRM suppressed the infection of several strains of influenza A viruses, including H1N1 and H3N2 subtypes, suggesting that the m6A-dependent antiviral mechanism of IFIRM is general but not strain specific. The second part of this thesis focused on the recently emerged SARS-CoV-2. Prophylactics and diagnostics for COVID-19 were explored. Specifically, a novel concept regarding nasal vaccine booster and a novel target for diagnostics were tested. An unadjuvanted, protein-only nasal vaccine booster, namely N-RBD, was rationally designed and tested in mice model. The protein vaccine booster was administered intranasally to primed mice that were pre-immunized with two doses of COVID-19 mRNA vaccine, mimicking human primary vaccination. The results showed that a single booster dose was sufficient to enhance antibody neutralization of live SARS-CoV-2 viruses and induce mucosal immunity. On the other hand, orf8-based ELISA was developed as a novel COVID-19 diagnostic tool. Orf8-specific antibodies were detected in patient sera and was found to be more sensitive for detecting early infections. The results demonstrated the potential use of N-RBD nasal booster and orf8-ELISA in real world applications, aiming to help control disease spread. Lastly, the study of virus evolutions and mutations of SARS-CoV-2 variants along the COVID-19 pandemic were carried out. Two unique SARS-CoV-2 mutations, namely the loss of orf3b and D119/F120 double deletions of orf8, were investigated. Phylogenetic and sequence analysis showed that the Q57H substitution in orf3a contributed to an early stop codon in orf3b, resulting in loss of function. Double amino acid deletions in Delta variant orf8 resulted in loss of dimerization and secretion of the protein. Both mutations resulted in molecular changes that affect protein phenotypes, which warrant further studies to understand their impact on virus pathogenesis. Taken all together, from mechanistic study of interferon-stimulated gene’s antiviral function to the development of vaccines and diagnostics, this study provided a comprehensive view of the broad aspects of immune protection against respiratory infectious disease.