Human astrovirus is an important cause of viral gastroenteritis worldwide. Young children, the elderly, and the immunocompromised are especially at risk. Few studies have been conducted examining human astrovirus incidence and seroprevalence to properly assess disease burden and significance. Furthermore, no vaccines or antiviral therapies exist to address human astrovirus infection, but several lines of evidence suggest that a protective antibody response mounted against a childhood infection can prevent future reinfection in adulthood. Therefore, we expect that vaccines eliciting such an antibody response will protect vulnerable individuals from disease. However, successful design of vaccine antigens requires knowledge of how neutralizing antibodies target sites of importance. In this thesis, I discuss my work on the antigenicity and conformational epitopes of the human astrovirus capsid. I begin with our seroprevalence evaluation of all eight classical human astrovirus serotypes in the United States, followed by a review of our simplified workflow for monoclonal antibody sequencing. Finally, I present a structural and mechanistic characterization of two neutralizing monoclonal antibodies which target the human astrovirus capsid spike at two separate, non-overlapping epitopes and both block virus attachment to host cells. These studies provide a basis for the development of therapies to prevent and treat human astrovirus disease.

Respiratory syncytial virus (RSV) is a top cause of severe lower respiratory tract disease and mortality in young children and the elderly. The viral envelope G glycoprotein contributes to pathogenesis through its roles in host cell attachment and modulation of host immunity. Although the G glycoprotein is a target of protective RSV-neutralizing antibodies, its development as a vaccine antigen has been hindered by its heterogeneous glycosylation and sequence variability outside a conserved central domain (CCD). We describe the cocrystal structures of three high-affinity broadly neutralizing human monoclonal antibodies bound to the RSV G CCD. All three antibodies bind to conformational epitopes that span a highly conserved surface, illuminating an important region of vulnerability. We further show that isolated RSV G CCD activates the chemokine receptor CX3CR1 and that antibodies block this activity. These studies provide a template for rational vaccine design targeting this key contributor to RSV disease.

This research focuses on three primary topics: (1) the development of biolayer interferometry serology tests to detect viral antigen reactivity with microliter scale human serum samples; (2) characterization of targets and epitopes for antibody-based cancer therapeutics with an emphasis on immune checkpoint regulation of natural killer cells via KIR receptors; and (3) preliminary investigation of the human astrovirus 1 RNA-dependent-RNA-polymerase.

BLI-ISA: The biolayer interferometry based antibody test (BLI-ISA) represents a novel, high throughput platform with numerous advantages over conventional enzyme-linked immunosorbent assays (ELISA) in terms of sample requirements, labor, and overall assay time while maintaining high accuracy. Compared to the COV2T antibody test for SARS-CoV-2, BLI-ISA had 94\% positive percent agreement and >98\% negative percent agreement in detection of serum IgG reactivity to viral antigen. The BLI-ISA antibody test is readily adaptable to new antigens, and was quickly deployed to assess reactivity to a panel of eight human astrovirus serotypes with good agreement to studies using more laborious methods.

Antibody therapeutics for cancer: a comparison of bioinformatic methods for the identification of tumor surface markers using RNAseq reveals statistical methods like DESeq outperform some dimensionality reduction techniques like PCA. Then, the epitope of an immune checkpoint inhibitor antibody is characterized by X-ray crystallography in the context of KIR receptor variants, highlighting the importance of population-wide polymorphism analysis.

HAstV RdRp: expression methods for this under-studied polymerase were developed to aid future structural work and activity assays.

Human astrovirus (HAstV) is a leading cause of viral diarrhea in young children. HAstV is also associated with chronic diarrhea and even systemic infection in immunocompromised patients. Currently, no vaccines or antiviral therapies exist for HAstV infections. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against future HAstV infection. Thus, we hypothesize that a vaccine that elicits protective antibodies will protect children from HAstV disease. A fundamental gap in our knowledge is an understanding of the binding sites and mechanisms of action of neutralizing antibodies that block HAstV infection. Our studies demonstrate that a single domain in the HAstV capsid protein is involved in binding to a potent neutralizing antibody. Furthermore, we have exciting structural and mechanistic studies on both the HAstV capsid domain and the neutralizing antibody. The overall studies have uncovered a point of vulnerability on the HAstV virus capsid surface and can lead the way for the development of a safe and effective prophylactic vaccine.

Respiratory syncytial virus (RSV) is the top cause of severe lower respiratory disease in infants and children and causes over 100,000 deaths in children worldwide each year. RSV is also a leading cause of respiratory disease in immunocompromisedand elderly populations. However, no FDA approved vaccine exists to protect against RSV infection. RSV contains two immunogenic proteins on its envelope, RSV F and RSV G, that have been the targets of vaccine developmental strategies. Most current efforts to create a vaccine against RSV focus on RSV F, and several candidates are in phase III clinical trials. The only current prophylaxis available for RSV is a monoclonal antibody against RSV F, yet it does not prevent infection. RSV G as a vaccine immunogen is linked to vaccine enhanced disease and is poorly immunogenic, however anti-RSV G antibodies have been shown to be protective in animal models of RSV disease and correlate with protection in humans. In this thesis, I present the history of an inactivated RSV vaccine that caused vaccine enhanced disease in children, a summary of studies using RSV G as a vaccine antigen, and an overview of the RSV G vaccine antigens currently in clinical trials. I present data showing that the structure-based design of an RSV G immunogen with a mutation in the central conserved domain is a promising vaccine candidate. Finally, I present studies to investigate RSV G binding and activation of the human chemokine receptor CX3CR1 on mammalian cells. Overall, these studies provide a foundation for the further development of the RSV G immunogen as a vaccine candidate to protect against RSV disease.

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia worldwide especially in infants, young children, immunocompromised individuals, and the elderly. In the United States alone, the economic burden to treat or prevent severe respiratory disease caused by RSV estimated 6.6 billion dollars in a study conducted with financial reports from 2021. Since its discovery in the late 1950’s, the question of how to elicit a protective immunogenic response against RSV remained unclear, that is, until 2023 when the first FDA approved vaccines became commercially available. These vaccines serve to prevent severe RSV-associated respiratory disease with endpoints involving major economic and quality of life burdens like hospital visits. However, studies have shown that correlates of protection involve antibody responses against two major surface glycoproteins that are required for efficient viral entry: RSV F and RSV G. Currently, all commercially available vaccines and prophylactic monoclonal antibodies use RSV F as their antigenic target. To investigate RSV G as an antigenic target for protective immune responses, commercially unavailable correlates of protection, I develop structure guided RSV G based vaccine designs and determine novel RSV G conformational epitopes recognized by broadly reactive monoclonal antibodies. I begin by discussing our work to address the issue of RSV G’s poor immunogenicity and its association to the apparent immune modulating activity seen in natural infection and vaccination using a nanoparticle vaccine platform where my role was the design, production, and characterization of the vaccine constructs. Following this, I describe a synthetically produced microparticle vaccine design based on layer-by-layer technology where my role was to determine its reactivity with an antibody that relies heavily on correct CX3C folding to recognize the CCD. Finally, I present our structural studies involving five broadly reactive antibodies in complex with the CCD where we characterize three novel conformational epitopes and reveal two non-competing antigenic sites. This work serves as a blueprint for the generation of structure guided mutagenesis, structure guided vaccine design, and antibody therapeutic or prophylactic strategies to protect from and prevent RSV-associated respiratory disease.

Diarrheal diseases remain a leading cause of death for young children worldwide. However, the reduction of total child deaths from viral sources of diarrhea may be possible through preventative vaccination. Human astroviruses (HAstVs) are a significant cause of viral diarrhea, with young children and immunocompromised populations most at risk. Some evidence indicates that antibodies induced by childhood infection leads to protection in adulthood, suggesting that vaccination against human astrovirus may be possible, but no vaccine or approved therapeutics currently exist. To develop an effective vaccine, it is important to understand how antibodies target critical regions of the virus. However, few studies have explored what regions of the HAstV capsid are responsible for inducing neutralizing antibodies or how these antibodies neutralize HAstV infection. In this thesis research, I have investigated the vulnerable antigenic regions on the HAstV capsid spike by structurally defining the epitopes of three neutralizing monoclonal antibodies, 3B4, 3H4, and 4B6 using X-Ray crystallography or cryogenic electron microscopy. I have discovered three new neutralizing antibody epitopes, with the epitope of antibody 3H4 being an entirely novel and highly conserved antigenic location near the base of the spike. Additionally, all three of these antibodies block virus attachment to cells, and I show using biolayer interferometry that they either fully or partially block HAstV spike binding to the newly discovered HAstV receptor human neonatal Fc receptor (FcRn). Finally, I establish a proof-of-concept system to test a spike-based protein vaccine using the murine astrovirus (MuAstV) infection model in mice. I find that recombinant MuAstV spike folds into a native dimeric structure, and its use as a vaccine immunogen induces anti-spike IgG antibodies in mice, paving the way for future MuAstV challenge and vaccine protection studies. Taken together, my thesis research lays a foundation for HAstV vaccine design and monoclonal antibody therapies to prevent and treat HAstV infection and disease.