Designing immunogens that confer simultaneous protection against Influenza and SARS-CoV-2.

Abstract

Embargo up to 6/4/2027

Respiratory viruses remain a persistent global health challenge due to their high transmissibility, rapid mutational dynamics, and seasonal resurgence. Among them, Influenza A virus (IAV) and SARS-CoV-2 represent two major pathogens responsible for significant morbidity and mortality worldwide. While seasonal influenza causes approximately 650,000 deaths annually, SARS-CoV-2 has resulted in over 777 million reported infections and 7 million deaths since its emergence. Both viruses exhibit antigenic drift and immune escape mechanisms that limit long-term vaccine effectiveness. Current vaccine platforms, including mRNA, viral vector, and inactivated formulations, provide variable protection and require frequent updates due to waning immunity and viral evolution. These limitations underscore the need for rationally designed, broadly protective, and cost-effective next-generation vaccines. This thesis presents a structure-guided approach toward the development of a chimeric subunit vaccine candidate targeting both Influenza A virus and SARS-CoV-2. The work focuses on two immunodominant neutralizing regions: the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the hemagglutinin (HA) head domain of H1N1 Influenza A virus. The central objective was to engineer a bivalent antigen capable of eliciting focused and potent humoral responses against both viruses while improving antigen stability and manufacturability. Structure-guided immunogen design principles were applied to optimize epitope exposure and enhance conformational stability. Recombinant constructs were expressed in mammalian and yeast systems and purified using affinity and size-exclusion chromatography. Stability enhancement was achieved through saturation suppressor mutagenesis, particularly on destabilized HA backgrounds. High-throughput yeast surface display screening enabled identification of stabilizing mutations that improved folding and antibody recognition. Deep sequencing of sorted libraries provided residue-level maps of stabilizing substitutions, generating a valuable dataset for future vaccine design. A chimeric trimeric immunogen was successfully engineered by genetically fusing the HA domain of H1N1 with the SARS-CoV-2 RBD. The construct retained structural integrity and demonstrated robust antigenicity, establishing proof-of-concept for a dual-targeting subunit vaccine that could potentially replace seasonal influenza vaccination without requiring new manufacturing infrastructure. Further, HA stabilization studies identified multiple suppressor mutations that decreased thermal stability by 3°C. Oligomerization strategies using Foldon and synthetic scaffolds demonstrated that multimeric display enhances immunogenicity and protective efficacy in murine models, underscoring the importance of antigen architecture in B cell activation. Genetically engineered HA ectodomain–head conjugates were developed to overcome the poor immunogenicity of isolated head domains. These constructs maintained proper folding and stability and represent promising candidates for broader and more cost-effective influenza vaccines. Additionally, cysteine-scanning mutagenesis provided insights into HA conformational flexibility and residue accessibility, contributing to a deeper understanding of membrane fusion mechanisms and informing future stabilization strategies. Collectively, this work demonstrates the power of structure-guided immunogen engineering in overcoming challenges of antigen instability, immune escape, and limited breadth. By integrating protein engineering, high-throughput screening, structural validation, and preliminary immunological assessment, this thesis provides a rational framework for the development of next-generation subunit vaccines with enhanced stability, breadth, and translational potential against evolving respiratory viruses.