Doctor of Philosophy, The Ohio State University, 2020, Comparative and Veterinary Medicine

Human respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and young children worldwide for which no vaccine is currently available. In order to understand the pathogenesis of RSV infection and develop efficacious vaccines and/or treatment, researchers must use an appropriate animal model. Cotton rats are the best small animal model for RSV infection, as the virus replicates in both the nose and lungs with physiologic inocula, cotton rats express the same host receptor used for in vivo RSV attachment and infection, and they have been proven to predict the efficacy of prophylactics. In this study, the cotton rat animal model was further characterized to assess its use in maternal vaccination studies and to evaluate the effect of RSV infection, specifically the G attachment protein on inflammation and lung function. I detailed the cotton rat placenta and its Fc receptor expression to better understand maternal antibody transfer in this animal model, established baseline respiratory function parameters and lung mechanics as well as the changes induced after RSV infection and allergen sensitization, and finally determined the inflammatory effects and immunogenicity of the G protein irrespective of viral replication. As stated, there currently is no vaccine or antiviral therapy to prevent or treat RSV infection, but virus neutralizing monoclonal antibodies can be given prophylactically, emphasizing the protective potential of antibodies. One concept of RSV vaccinology is the immunization of mothers to induce high antibody titers, which will lead to the transfer of high levels of maternal antibodies. Studies use the cotton rat to investigate the benefit of maternal immunization for RSV to induce protective passive immunity. In cotton rats, maternal IgG antibodies are transferred via the placenta in utero and postnatally via intestinal absorption from colostrum. In this study, placentas from cotton rats at mid-gestation (~ (open full item for complete abstract)

Committee: Stefan Niewiesk (Advisor) Subjects: Pathology; Veterinary Services; Virology

PhD, University of Cincinnati, 2018, Medicine: Pathobiology and Molecular Medicine

Dendritic cells (DCs) are the most efficient antigen presentation cells and execute a pivotal role in the onset and regulation of innate and adaptive immune responses and the recruitment of a variety of leukocytes to the site of inflammation or injury. To counter the challenge of the non-stop exposure to exogenous pathogens, the lungs developed an intrigue and complex immunology defense orchestra that conducted by DCs. An increasing volume of research on DCs in recent years show that the disruption of DC equilibrium in the lung may result in different diseases. In this study, we used two different disease models to elucidate the importance of DC in disease pathogenesis. Pulmonary Langerhans cell histiocytosis (PLCH) is a rare interstitial lung disease characterized by focal DC accumulation, bronchiolocentric nodule formation, and cystic remodeling of the lung and occurs predominantly in active smokers. Approximately 50% of PLCH patients harbor somatic BRAF-V600E mutations identified mainly within the DC lineage. However, the rare nature of the disease and lack of animal models impedes the study of the pathogenic mechanisms of PLCH. We have established the first mouse model that recapitulates the hallmark characteristics of PLCH. In addition, we show that the BRAF-V600E mutation is associated with increased DC responsiveness towards multiple stimuli including the DC-chemokine CCL20. We provide evidence that DC accumulation in the lung is due to both increased viability and enhanced recruitment. Further evidence indicates that the accumulation of other inflammatory cells in PLCH is a secondary event driven by CCL7 secreted from DCs in a BRAF-V600E-dependent manner. Moreover, we demonstrate that the PLCH-like phenotype in the mouse model can be attenuated following smoking cessation and removal of BRAF-V600E DCs. Furthermore, we show PBMCs isolated from PLCH patients harboring the BRAF-V600E mutation produce CCL7. Collectively, our studies provide the first mechani (open full item for complete abstract)

Committee: Michael Borchers Ph.D. (Committee Chair); Ian Paul Lewkowich Ph.D. (Committee Member); Francis McCormack M.D. (Committee Member); William Miller Ph.D. (Committee Member); Kathryn Wikenheiser-Brokamp M.D. Ph.D. (Committee Member) Subjects: Surgery

Doctor of Philosophy, The Ohio State University, 2018, Biochemistry Program, Ohio State

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization in infants and the second leading cause of respiratory related death in the elderly. A prophylactic monoclonal antibody, palivizumab, is available to prevent infection, but due to cost its use is limited to at risk infants primarily in developed countries. No effective vaccine or small molecule drug is currently available for prevention or treatment of RSV infection. Insight into the entry mechanisms of RSV should aid the development of small molecule drugs and vaccines. The RSV fusion (F) glycoprotein is a trimeric, membrane anchored glycoprotein that mediates fusion of the viral envelope with a target cell membrane to initiate infection, without apparent help from the RSV attachment glycoprotein. The F protein is initially expressed in its active, metastable, and high energy prefusion (pre-F) conformation. To mediate fusion, the F protein refolds into an elongated form, embedding its terminal fusion peptide into the target cell membrane and refolds again into its postfusion (post-F), six helix-bundle conformation. This process brings the viral and cellular membranes together, initiating fusion. The interaction that triggers the F protein to undergo these changes is not known, and is the subject of this work We examined the structural and functional importance of the 12 residues in the apical loop of the RSV F protein by alanine scanning mutagenesis. All alanine mutants were produced and maintained the pre-F confirmation and trimer form. Exposure to low ionic strength buffer, a condition which increased cell-cell fusion caused by the parental F protein, was used to force refolding, thereby bypassing triggering. Only five of the 12 residues were essential for cell-cell fusion: I64, K65, K66, K68, and K75. The positive charge of all four lysines was required for fusion function. K65, K66, and K68 are grouped along the side of the apical loop. Alanine mutatio (open full item for complete abstract)

Committee: Mark Peeples (Advisor); Jianrong Li (Committee Member); Richard Swenson (Committee Member); Thomas Magliery (Committee Member) Subjects: Biochemistry; Virology

Doctor of Philosophy, The Ohio State University, 2017, Molecular, Cellular and Developmental Biology

Respiratory syncytial virus (RSV) is a highly contagious and universally prevalent virus that presents as a respiratory infection, which can range from mild to fatal in severity. An infant's first RSV infection is typically the most severe, but the elderly are also at risk for severe infection. Although life-threatening complications in healthy adults are less common, the burden of illness due to RSV in this population remains substantial. Clinical trials for RSV vaccine candidates began in the 1960's, but to date, there remains no vaccine or therapeutic available. A prophylactic has been developed for RSV, although its use is limited to high-risk pre-term infants during the RSV season. This prophylactic was developed using small animal models of RSV infection. For the work presented herein, RSV pathogenesis and immune response is investigated using the cotton rat. The cotton rat is a less common, but more permissive and predictive model of RSV infection. This document is separated into four chapters. The first chapter is a review of the literature, which provides an overview of RSV, highlights what is known, and indicates areas where there are opportunities to clarify our understanding of the virus, as well as a brief discussion of the strengths and weaknesses of animal models of RSV infection. Chapter two addresses one of the uncertainties in the RSV field, the cellular receptor for the virus. Earlier studies had demonstrated heparan sulfate to be a receptor for RSV, but these studies were performed on immortalized cell lines, which express different proteins on their surface than airway epithelial cells, the target cell for RSV infection. Based on recent work using primary human airway epithelium cells, CX3CR1 was identified as a potential RSV receptor. Adding to that work, chapter two summarizes data that support CX3CR1 as a receptor in vivo using the cotton rat model. Chapter three examines eosinophils in the cotton rat and during RSV infection. This work was (open full item for complete abstract)

Committee: Stefan Niewiesk DVM, PhD (Advisor); Michael Oglesbee DVM, PhD (Committee Member); Mark Peeples PhD (Committee Member); Ian Davis DVM, PhD (Committee Member) Subjects: Immunology; Microbiology; Molecular Biology; Virology

Doctor of Philosophy, The Ohio State University, 2015, Integrated Biomedical Science Graduate Program

Live attenuated respiratory syncytial virus (RSV) vaccine candidates are produced in Vero cells, a cell line that cleaves the attachment (G) glycoprotein. As a result, Vero-derived virus is 5-fold less infectious for primary well-differentiated human airway epithelial (HAE) cultures than virus grown in HeLa. HAE cultures are isolated directly from the human airways, so it is likely that Vero-grown vaccine virus would be similarly inefficient at initiating infection of the nasal epithelium following vaccination, requiring a larger inoculum, thereby raising the cost per dose. Using protease inhibitors with increasing specificity, we identified cathepsin L as the responsible protease and confirmed that virus grown in the presence of protease inhibitors was more infectious for HAE cultures. Our evidence suggests that the G protein interacts with cathepsin L in the late endosome or lysosome via endocytic recycling. While essential for Nipah virus F protein cleavage, endocytic recycling is detrimental to the production of infectious RSV from Vero cells. We found that cathepsin L is able to cleave the G protein in Vero-grown, but not in HeLa-grown virions suggesting a difference in G protein posttranslational modification. Using mutagenesis, we identified a cluster of amino acids that are important for G protein cleavage and they contain a likely cathepsin cleavage site. Virus grown in Vero cells and containing a G protein resistant to cleavage is 5-fold more infectious for HAE than the same virus grown in Vero. Live attenuated RSV vaccine virus containing this mutation would reduce the cost of vaccine production for infants.

Committee: Mark Peeples Ph.D. (Advisor); Douglas McCarty Ph.D. (Committee Chair); Ian Davis DVM/Ph.D. (Committee Member); Stefan Niewiesk DVM/Ph.D. (Committee Member) Subjects: Biology; Biomedical Research; Microbiology; Molecular Biology; Virology

Doctor of Philosophy, The Ohio State University, 2014, Biochemistry Program, Ohio State

The hallmark of a retrovirus is the ability to reverse transcribe single-stranded genomic RNA (gRNA) into double-stranded DNA for subsequent integration into the host cell genome. After integration, the host cell machinery takes over; thus, viral DNA is transcribed into RNA and translated into viral proteins such as Gag. The Gag polyprotein, consisting of domains matrix (MA), capsid (CA), and nucleocapsid (NC), contributes to almost every stage of the retroviral lifecycle, including reverse transcription and gRNA encapsidation. Host cell tRNALys3 and tRNATrp serve as the primers for reverse transcription in human immunodeficiency virus type 1 (HIV-1) and Rous sarcoma virus (RSV), respectively. The NC domain of Gag facilitates tRNA primer annealing to the complementary primer binding site (PBS) in the viral genome, which initiates reverse transcription. Previous in vitro studies showed MA has an inhibitory effect on HIV-1 Gag nucleic acid (NA) chaperone activity and this inhibition can be reversed in the presence of inositol hexaphosphate (IP6). Taken together, these data suggested that IP6 binds to MA and displaces it from nucleic acid, thus stimulating the NA chaperone activity of HIV-1 Gag. In addition, the NC domain of Gag is responsible for binding a packaging signal (psi) in gRNA to facilitate the selective incorporation of gRNA into virions during assembly. A salt-titration approach demonstrated HIV-1 Gag binds psi-containing RNA (psi RNA) with greater specificity and fewer electrostatic interactions when compared to RNA lacking psi (non-psi RNA). Whereas extensive studies have been carried out in HIV-1, little is known about the NA chaperone activity of RSV Gag. In this work, I examine the NA chaperone activity of RSV Gag in vitro. Similar to HIV-1, RSV NC is required for Gag-facilitated tRNA primer annealng to the RSV PBS. In contrast to HIV-1, MA does not inhibit the NA chaperone activity of Gag and I observed no IP6-dependent stimulation in Gag-facilitated (open full item for complete abstract)

Committee: Karin Musier-Forsyth (Advisor); James Hopper (Committee Member); Kathleen Boris-Lawrie (Committee Member); David Bisaro (Committee Member) Subjects: Biochemistry; Virology

MS, University of Cincinnati, 2011, Medicine: Epidemiology (Environmental Health)

Objective: Determine the change in respiratory syncytial virus (RSV) hospitalizations for patients with hemodynamically significant congenital heart disease (hs-CHD) following the 2003 AAP policy revision recommending palivizumab prophylaxis. Methods: Hospital discharge data from the years 2000 and 2006 were obtained from the Kids' Inpatient Database (KID). ICD9-DM codes were used to identify RSV admissions among patients less than or equal to 24 months of age with CHD and hs-CHD. Demographic risk factors were assessed between years and logistic regression was performed to compare odds of RSV admission versus non-RSV admission among patients with CHD and hs-CHD patients. Results: Among CHD and hs-CHD patients, RSV admissions decreased by 26% (2,688 to 2,649) and 37% (635 to 467) respectively from 2000 to 2006 (p<0.01). Patients with hs-CHD were less likely to be admitted for RSV in 2000 than 2006 (OR=0.70, 95% CI: 0.62-0.78). As opposed to patients with private insurance, those with governmental insurance were more likely to be admitted for RSV among patients with hs-CHD (OR=1.55 (95% CI: 1.28-1.89). Conclusions: Nationally representative data demonstrate that RSV admissions among patients with hs-CHD decreased in the palivizumab era. This decrease is temporally related to the release of the 2003 AAP Guidelines and likely represents the clinical impact of prophylaxis. Patients with hs-CHD and governmental insurance remain at higher risk of hospitalization from RSV despite the availability of prophylaxis.

Committee: Erin Nicole Haynes PhD (Committee Chair); James Cnota MD (Committee Member) Subjects: Surgery

Doctor of Philosophy, The Ohio State University, 2011, Comparative and Veterinary Medicine

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, Pneumovirinae subfamily is the most significant respiratory pathogen in infants and second only to influenza virus in the elderly. Despite extensive efforts, no vaccines or small molecule antiviral drugs are available. The RSV fusion (F) glycoprotein has been a major target for vaccine and antiviral drug development because of its importance in the viral replication cycle, its conserved sequence and structure, its exposed position in the virion, and its strong immunogenicity. Like other paramyxoviruses, the RSV F protein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. However, the Pneumovirinae F protein is unique in that it, alone, is sufficient to mediate membrane fusion and virus infection. It is, therefore, the simplest F protein to study. It likely has the ability to attach to target cells from which position it is triggered. Neither the trigger site on the F protein nor the triggering molecule/event has been identified. To begin to study the triggering mechanism of the RSV F protein biochemically, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. This sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopy, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni2+ column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming “hatpin” shaped molecules that aggregated as rosettes, characteristic of the po (open full item for complete abstract)

Committee: Mark Peeples PhD (Advisor); Michael Oglesbee PhD (Committee Member); Stefan Niewiesk PhD (Committee Member); Jianrong Li PhD (Committee Member) Subjects: Virology

Doctor of Philosophy, The Ohio State University, 2011, Integrated Biomedical Science Graduate Program

Respiratory syncytial virus (RSV) infection of most cultured cell lines causes cell-cell fusion and death. Cell fusion is caused by the fusion (F) glycoprotein and is clearly cytopathic, but other aspects of RSV infection may also contribute to cytopathology. To investigate this possibility, we generated an RSV replicon that lacks all three of its glycoprotein genes and so cannot cause cell-cell fusion or virus spread. This replicon includes a green fluorescent protein gene and an antibiotic resistance gene to enable detection and selection of replicon-containing cells. Adaptive mutations in the RSV replicon were not required for replicon maintenance. Cells containing the replicon could be cloned and passaged many times in the absence of antibiotic selection, with 99% or more of the cells retaining the replicon after each cell division. Transient expression of the F and G (attachment) glycoproteins supported the production of virions that could transfer the replicon into most cell lines tested. Since the RSV replicon is not toxic to these cultured cells and does not affect their rate of cell division, none of the 8 internal viral proteins, the viral RNA transcripts, or the host response to these molecules or their activities are cytopathic. However, the level of replicon genome and gene expression is controlled in some manner, well below that of complete virus and, as such, might avoid cytotoxicity. RSV replicons could be useful for cytoplasmic gene expression in vitro and in vivo, and for screening compounds active against the viral polymerase.

Committee: Mark Peeples PhD (Advisor); Douglas McCarty PhD (Other); Michael Oglesbee PhD (Other); Jianrong Li PhD (Other) Subjects: Virology

Doctor of Philosophy, The Ohio State University, 2010, Integrated Biomedical Sciences

Interferons (IFN) are a family of cytokines that are well known for their ability to inhibit virus replication. IFNs are classified as type I (IFN-α/β), type II (IFN-γ), or type III (IFN-λ) according to sequence homology and receptor binding. The type III IFNs are the most recently discovered and, despite evidence of their antiviral effects in vitro, little is known about their relative importance to antiviral immunity in vivo. It has been believed that IFN-α/β represent the most critical component of innate antiviral immune responses based on reports demonstrating heightened sensitivity to viral infection in mouse models lacking the type I IFN receptor (IFNαβR-/-). However, we and others have demonstrated that IFNαβR-/- mice are, in fact, not more susceptible to infection with either respiratory syncytial virus (RSV) or a pneumotropic strain of influenza A virus while STAT1-/- mice, which lack signaling from all three IFN receptors, are more susceptible. The experiments detailed here test the hypothesis that IFN-λ is capable of fully compensating for the loss of IFN-α/β signaling in protecting the murine host against respiratory virus infection. In support of this hypothesis we observed that IFN-λ is the major IFN induced following influenza virus infection, with peak levels exceeding that of IFN-α and IFN-β by ~10-fold in both wild-type and IFN-αβR-/- mice. Importantly, the levels of IFN-λ induced in vivo are more than sufficient to protect respiratory epithelium from virus challenge as demonstrated in an in vitro antiviral assay. Also, mice lacking both type I and type III IFN signaling are highly sensitive to fatal influenza virus infection, a result which supports an important role for IFN-λ in antiviral protection. Surprisingly, we could not detect expression of IFN-λ during RSV infection of IFNαβR-/- mice suggesting that protection, in this case, is coming from a novel signaling pathway. Moreover, protection against RSV infection in the absence of the IFNαβR (open full item for complete abstract)

Committee: Mark Peeples (Advisor); Joan Durbin (Committee Member); Emilio Flano (Committee Member); Yusen Liu (Committee Member); Stefan Niewiesk (Committee Member) Subjects: Immunology