Emerging and reemerging viruses remain a significant threat to human health. Multiple viral outbreaks have occurred since the start of the twenty-first century. Particularly concerning is the emergence of novel pandemic viruses such as the 2009 H1N1 influenza virus and SARS-CoV-2 coronavirus. Adaptive immune responses to these viruses provide a major mode of protection against virus infection and disease. Antibodies against the hemagglutinin protein of influenza viruses and the Spike protein of SARS-CoV-2 play an essential role in providing protection, since they can block virus entry into host cells and neutralize virus infection. However, these responses are often suboptimal. This dissertation examines how different modifications to viral proteins effects anti-viral immune responses. We tested the effect of nitration of influenza virus proteins on virus infectivity and immunogenicity. In vitro nitration of influenza virus proteins was found to reduce infectivity of multiple strains of influenza viruses. Additionally, chemical in vitro nitration of influenza HA protein led to reduced antibody responses against unmodified HA protein. However, nitration of influenza HA protein in vivo was not observed. While overall nitration of the influenza HA protein reduced antibody responses to the HA protein, it was hypothesized that modifications of specific amino acids of the HA protein could increase antibody responses to HA. Mutants of HA incorporating the unnatural amino acid p-nitrophenylalanine were generated and used for vaccination. However, increased antibody responses to the HA protein were not observed in unnatural amino acid mutants. Finally, we examined the ability of pre-existing immunity to influenza viruses to boost antibody responses to a novel vaccine antigen: the SARS-CoV-2 Spike protein. We found that a fusion protein vaccine made of the receptor-binding domain of Spike and the influenza NP protein generated accelerated antibody responses against Spike in mice with pre-existing immunity to influenza virus. The knowledge gained in these studies will help guide the optimization of vaccine strategies for better immune responses against novel emerging viral pathogens.

The Flavivirus genus consists of over 70 vector-borne, single-stranded RNAcontaining

viruses, including dengue virus (DENV) and Zika virus (ZIKV), which cause

major epidemics among humans and pose a serious threat to global public health. No

vaccines or antivirals exist to prevent or treat infections caused by DENV and ZIKV. To

establish infection, it is vital for flaviviruses to overcome the antiviral state induced by

type 1 interferon. ZIKV and DENV NS5 proteins can bind to human signal transducer

and activator of transcription 2 (hSTAT2) protein, a key signaling intermediary for

interferon (IFN) responses, which triggers the subsequent proteasome-dependent

degradation of hSTAT2. NS5 does not have a host counterpart; thus, NS5 or the NS5-

hSTAT2 interaction are logical antiviral targets. However, the underlying mechanism of

the Flavivirus NS5-hSTAT2 interaction remains elusive. Here, we have elucidated the

structure of the ZIKV NS5 protein and re-purposed a small molecule inhibitor against

DENV for the inhibition of ZIKV RdRp activity. We have also delineated the biochemical and structural basis of the DENV and ZIKV NS5-hSTAT2 interactions and used the knowledge gained from those studies to generate hSTAT2 and NS5 mutants

deficient in interaction. We discovered that the mechanism of hSTAT2 interaction is

conserved among the DENV2 and ZIKV NS5 proteins. In addition to hSTAT2

degradation, we found that ZIKV NS5 also competes with IRF9 for hSTAT2 binding,

resulting in a two-pronged ISGF3 suppression mechanism. Finally, the hSTAT2-

interaction deficient NS5 mutants were used as tools to interrogate the functional

consequences of the hSTAT2-NS5 interaction during ZIKV infection. Disruption of the

ZIKV NS5-hSTAT2 interaction resulted in loss of NS5-mediated hSTAT2 degradation

and IFN suppression. Importantly, during infection, this interaction is critical for efficient

ZIKV propagation. The work described here provides critical insights into DENV2 and

ZIKV NS5-mediated hSTAT2 degradation and suppression of the IFN response and will

facilitate the development of novel vaccines and inhibitors of Flavivirus infections.

Objectives

Results

Influenza B virus (IBV), in conjunction with Influenza A virus (IAV), results in yearly epidemics. While IAV is a well-studied pathogen, IBV lags in understanding of its pathology, replication mechanics, and vaccination use strategies. For example, smoking is a known risk factor for IAV, but little is known regarding its effects on IBV infections and pathology. Second, the IBV nucleoprotein (BNP) fulfills a critical role during replication, but its associated host factors remain largely unclear. Last, live virus vaccinations for IAV during pandemics are considered a potential risk due to the possibility the vaccine virus will reassort with circulating IAV, restoring virulence and exposing a naïve population to a pathogenic virus unintentionally. To fill these gaps, we set out to understand: a) what are the impacts of cigarette smoke on IBV infections b) what host factors are interacting with BNP and how are they involved in replication and c) whether we could attenuate recombinant IBVs (rIBVs) expressing the IAV HA by progressive mutations to the IAV ectodomain. To these ends, we developed an animal model to study the impact of cigarette smoke extract on IBV infections in mice not previously available. We found that low concentration of CSE increased IFN- production in spleenocyte of infected mice, and high concentrations of CSE resulted in lower humoral responses to infection and lower survival rates. Additionally, we confirmed that BNP interacts with the host protein IMPα4, a nuclear import adaptor protein, through yeast-two-hybrid and Co-IP analysis. Further, knockout of IMPα4 expression reduced IBV replication. Finally, progressive mutation of IAV ectodomain using IBV coding sequence did result in attenuation of replication ex vivo, and these candidates could elicit significant humoral and neutralizing responses that protected mice from lethal challenge with homologous strains of IAVs. Together, each of these studies represent a necessary step forward in understanding how lifestyle factors impact IBV infections, the potential specific host-viral protein interactions that could be drug therapeutic targets of the future, and necessary development of innately safe and efficacious live vaccines for use in pandemic scenarios.

Influenza A Viruses (IAV’s) pose a significant threat to public health. On average, Influenza infections account for 25,000 deaths and 250,000 hospitalizations every year in the United States. Despite having been discovered in the 1930’s, and despite decades of study influenza viruses continue to cause significant morbidity and mortality. Furthermore, with the emergence of Highly Pathogenic Avian Influenza Viruses (HPAIV) in humans, IAV’s pose a greater threat than ever before. The innate immune response is critical to controlling these viral infections, but despite over half a century passing since the discovery of the first innate immune proteins, the full scope of innate immunity has yet to be elucidated. The Interferon-Induced Transmembrane (IFITM) family of proteins has been shown to strongly restrict a broad range of viruses, including IAV5,6,7. However, the exact mechanism of this inhibition as well as the mechanism of IFITM regulation and metabolism is poorly understood. Secretory Membrane Protein 3(SCAMP3) was first identified in 1997 and has since been implicated in EGFR recycling and Salmonella pathogenesis. Here we demonstrate a new function of SCAMP3 in the innate immune response. We show that SCAMP3 can function to stabilize IFITM3 by decreasing its lysosomal degradation. We show that SCAMP3 can alter the trafficking of IFITM, resulting in the protein being recycled from early endosomes back to the Golgi apparatus. Upon SCAMP3 depletion, this recycling is ablated and IFITM3 undergoes increased lysosomal degradation. We also show that SCAMP3 can directly restrict Influenza A Viruses. We show that SCAMP3 and interfere with cleavage of Highly Pathogenic Avian Influenza (HPAIV) Hemagglutinin (HA) cleavage, resulting in viruses that are deficient in their ability to enter target cells. We also show that SCAMP3 expression, phosphorylation and localization is altered upon viral infection or stimulation with various innate immune proteins, suggesting that SCAMP3 is an innate immune effector. This research deepens our understanding of the innate immune response and presents a potential therapeutic strategy for treatment or prophylaxis of viral disease.