Genetically engineered defective interfering particles of influenza A virus for antiviral treatment and vaccination

Abstract

The Influenza A virus (IAV) is a major human respiratory pathogen causing seasonal outbreaks and occasional pandemics. Owing to its continuous evolution, particularly of the surface proteins of IAV strains, annual updates to vaccine formulations are necessary. This process has several limitations including lengthy screening periods for identifying and generating strains for annual update of vaccines. Additionally, the emergence of antiviral-resistant IAV strains has challenged the efficacy of current treatments, necessitating the exploration of alternative therapeutic strategies. This dissertation investigates defective interfering particles (DIPs) of IAV as a promising antiviral approach. DIPs are naturally occurring viral mutants that produce particles typically with deletions in one of their eight viral RNA (vRNA) segments. Among these, DI244 is a well- characterized DIP with a deletion in segment 1 (seg 1), which encodes polymerase basic protein 2 (PB2). In addition, a new type of DIP “OP7” was identified by our research group that has been extensively studied. OP7 carries multiple point mutations in seg 7, which encodes matrix proteins. In a normal scenario, DIPs require co-infection with a standard virus (STV) for their replication, as the DIP is unable to generate a functional protein. However, during co-infection, DIPs can inhibit STV replication (both in vitro and in vivo), indicating their potential as natural antivirals. Additionally, the development of mutations that lead to resistance to DIPs is highly unlikely. In previous collaborations, a modified IAV reverse genetics system was developed that utilizes PB2-expressing cells to generate clonal DI244-DIPs without STV contamination in the final virus harvest. This approach involved eight plasmids encoding seven full-length vRNA segments along with a seg 1 DI vRNA encoding plasmid. After transfection, PB2 protein expression from the host cell facilitated DIP propagation. Subsequently, genetically engineered suspension Madin-Darby canine kidney (MDCK) cells were used to optimize DIP production achieving high titer DIP preparations for antiviral applications. This innovative method formed the basis for this PhD project, which aimed to develop improved DIP constructs for use as antivirals or vaccines. Notably, the concept of using seg 1 DIPs as vaccine constructs has not been described previously. In the first part of this thesis, evolutionary studies (performed by a colleague) are described in which novel deletion junctions with presumably better interference capabilities compared to the previously described prototypic and well- characterized DIP “DI244” were identified by next generation sequencing (NGS). Besides the emergence of diverse DIPs, differences in their propagation and accumulation were observed. It was hypothesized that DI vRNA displaying strong growth properties may also demonstrate high antiviral activity. In the context of this PhD thesis, for experimental validation, the aforementioned reverse genetics system was utilized to construct and reconstitute these newly identified seg 1 DIPs in a clonal population devoid of any infectious STV. Subsequent in vitro co-infection studies confirmed that rapidly propagating DIPs indeed exhibit higher antiviral activity compared to the slower growing DIPs, including DI244. Therefore, these newly identified seg 1 DIPs are promising candidates for antiviral therapy. In the second part of this thesis, options for the generation of DIPs for use as live vaccines were explored. The primary objective was to harness the potential of DIPs to additionally induce adaptive immune responses against seasonal infections. Specifically, the surface glycoproteins of the DIPs should be replaced by those of seasonal vaccine strains. Such a live vaccine would be administered by a nasal spray via the mucous membranes. This strategy was designed to elicit mucosal immunity at the primary site of infection, thereby promoting a comprehensive immune response that includes cellular, humoral, and systemic adaptive immunity. Although these initial attempts were not successful, potential alternative experimental approaches to allow for the reconstitution of these constructs evolved and various promising strategies are currently under investigation. OP7 has exhibited superior antiviral activity against STV replication compared to conventional DIPs like DI244 in various studies. In the last part of this thesis, the challenge of reconstituting OP7 DIPs free from infectious STV was addressed. Here, the reverse genetics approach for generating seg 1 DIPs was refined by introducing a ninth plasmid encoding seg 7-OP7. This change resulted in a population of DIPs that included OP7 chimera (with deleted seg 1 and mutated seg 7-OP7) alongside seg 1-DIP (with wild-type seg 7). Due to the deletions in seg 1, both DIPs were restricted to growth in PB2-expressing adherent MDCK cells and did not require any inactivation steps for further use. The seed virus obtained was subsequently passaged in suspension MDCK cells in bioreactors and optimized (by our team) for high-yield production. In a next step, conducted by a collaborator (Dunja Bruder, Helmholtz Centre for Infection Research, Braunschweig, Germany), OP7 preparations were tested intranasally in mice. At high doses, they showed no toxicity and provided complete protection against a fatal STV challenge. This demonstrated the remarkable potential of OP7 chimera DIP preparations for use as an antiviral. In the future, OP7 chimera DIPs will be used to establish GMP-compliant production processes advancing clinical development and enhancing pandemic preparedness with this new class of broad-spectrum antivirals.