SARS-CoV-2 and influenza viruses remain major global public health threats, particularly for individuals with underlying health conditions. A crucial study in this thesis was focused on developing a bivalent replication-incompetent single-cycle pseudotyped vesicular stomatitis virus (VSV) vaccine incorporating a prefusion-stabilized SARS-CoV-2 spike protein lacking a furin cleavage site and a full-length influenza A virus neuraminidase (NA) protein. Vaccination of K18-hACE2 and C57BL/6J mouse models generated durable neutralizing antibody responses, T cell immunity, and protection from morbidity and mortality upon challenge with either virus. Notably, the vaccine provided heterologous protection against a different influenza strain, highlighting the advantage of using NA to enhance cross-strain immunity. Given that no bivalent vaccine is currently approved for use against both SARS-CoV-2 and influenza, these findings support the potential of the VSV platform in developing safe and efficient multivalent vaccines.

Beyond seasonal respiratory viruses, emerging coronaviruses such as MERS-CoV and SARS-CoV-1 continue to pose significant pandemic threats, with mortality rates of 30% and 10%, respectively. Despite being classified as priority pathogens, no approved vaccines exist for human use. The COVID-19 pandemic has further emphasized the need for broad-spectrum coronavirus protection due to the ongoing emergence of variants with diverse transmission and virulence profiles. This thesis also covers the design and in vivo efficacy testing of a replication-defective VSV-based multivalent vaccine expressing prefusion-stabilized spike proteins of SARS-CoV-2, SARS-CoV-1, and MERS-CoV. Using a prime-boost regimen in KIKO mice (human ACE2 knock-in, mouse ACE2 knockout), the vaccine elicited strong antibody responses against all three coronaviruses and conferred protection against ancestral SARS-CoV-2 infection four months post-vaccination, significantly reducing viral load and lung pathology. These findings demonstrate the potential of this multivalent VSV platform to induce cross-reactive immunity and serve as a viable approach for developing broad-spectrum coronavirus vaccines to strengthen pandemic preparedness.

In addition to vaccine development, this thesis addresses critical limitations in existing Long COVID mouse models. Current models rely on mouse-adapted viral strains that fail to fully represent human disease, hACE2 transgenic mice with non-physiological ACE2 expression, and humanized models that suffer from limitations in hematopoietic stem and progenitor cell (HSPC) engraftment. Moreover, existing models primarily focus on lung fibrosis and immune responses, overlooking other key manifestations of Long COVID as a multi-organ disease. In addition, current studies using murine models for Long COVID only extends across 30 days.

The interplay between persistent viral reservoirs, immune dysregulation, and various risk factors underscores the urgent need for improved models to study disease mechanisms and evaluate potential therapeutic interventions. This study introduces a novel murine model that better recapitulates the complexities of Long COVID, offering a more accurate platform for understanding disease progression, testing therapeutic strategies, and advancing countermeasure development.

Together, these findings contribute to pandemic preparedness, vaccine innovation, and the development of effective countermeasures against emerging infectious diseases. The results support the use of VSV-based platforms for bivalent and multivalent vaccine development and provide a much-needed framework for studying Long COVID, addressing major gaps in current research and therapeutic evaluation.