Doctor of Philosophy, The Ohio State University, 2024, Veterinary Biosciences

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to substantial damage to all aspects of our society. Vaccination is the most effective strategy to prevent infectious diseases. Since the pandemic, several vaccine platforms, including inactivated, mRNA, viral vector-based, and protein subunit vaccines have been developed. While these vaccines have been highly effective in reducing severe disease and mortality, they still have several limitations, such as the inability to prevent virus infection and transmission, particularly against emerging variants of concern (VoCs), short duration of protection, failure to induce mucosal IgA antibodies and resident memory T cells in the respiratory tract, and high cost of production or distribution. Therefore, there is an urgent need to develop the next generation of intranasal COVID-19 vaccines that induce durable and broadly protective immunity, both in the airways and systemically. The combined MMR (measles/mumps/rubella) vaccine has been available in the United States since 1971 and is one of the safest and most effective human vaccines. The MMR vaccine contains attenuated strains of measles virus (MeV), mumps virus (MuV), and rubella virus and confers lifelong protection against these three viruses. Both MeV and MuV have been used as delivery platforms for experimental vaccines against highly pathogenic viruses. In this study, we have developed a highly efficacious, intranasally delivered, trivalent measles-mumps-SARS-CoV-2 spike (S) protein (MMS) vaccine candidate that induces robust systemic and mucosal immunity with broad protection. MMS vaccine candidate is based on three components of the MMR vaccine, a measles virus Edmonston, and the two mumps virus strains [Jeryl Lynn 1 (JL1) and JL2], which are known for their high safety and long-lasting immunity. The six proline-stabilized prefusion S protein (preS-6P) genes for ancestral SARS-CoV-2 WA1 and (open full item for complete abstract)

Committee: Jianrong Li (Advisor); Jacob Yount (Committee Member); Zongdi Feng (Committee Member); Mark Peeples (Committee Member) Subjects: Virology

Doctor of Philosophy, The Ohio State University, 2009, Veterinary Preventive Medicine

In this study, several strategies have been explored for the development of live attenuated and killed DIVA influenza vaccines. The NS1 protein of influenza A viruses, a virulence marker, is a good target to attenuate viruses in developing a live attenuated vaccine. Through passage in eggs, we found that several different NS1 truncation variants were generated from a parental non-stable virus strain, A/turkey/Oregon/71-delNS1 (H7N3). Based on the results from both in vitro and in vivo studies, naturally selected NS1 truncation variants are potentially live attenuated influenza vaccine candidates in poultry. With a large deletion in NS genes, those NS1 truncation variants have a low possibility to revert back to virulent viruses. We then checked feasibility of those NS1 truncation variants to be used for in ovo vaccination, and hatchability of vaccinated groups was significantly lower than that of PBS control group, indicating that NS1 truncation variants were not attenuated enough to be used for in ovo vaccination and there was a need for further attenuation. To improve hatchability of NS1 truncation variant vaccinated eggs, temperature sensitive (ts) mutation and HA substitution strategies were utilized. NS1 truncation variants containing ts mutations exhibited ts phenotype in vitro, but were not attenuated enough to improve hatchability of in ovo vaccination. We showed that HA substitution had different effect on hatchability of in ovo vaccination, and the hatchability of eggs inoculated with NS1 truncation variants of different HA subtypes was still significantly lower than PBS control group. Therefore, we explored a strategy of targeting non-conserved parts of non-coding regions (NCR) to attenuate NS1 truncation variant viruses. In vitro studies illustrated that even single nucleotide change in NCR of PA or PB1 segments could affect protein expression. In addition, it was found that mutations in NCR of PA segment alone or PA and PB1 combination affect virus repl (open full item for complete abstract)

Committee: Chang-Won Lee (Committee Chair); Y Saif (Committee Member); Daral Jackwood (Committee Member); Jeffrey LeJeune (Committee Member) Subjects: Virology

Doctor of Philosophy, The Ohio State University, 2022, Comparative and Veterinary Medicine

Porcine epidemic diarrhea virus (PEDV) is an alphacoronavirus. It was initially reported in 1970s in Europe. However, massive porcine epidemic diarrhea (PED) epidemics did not occur in China until late 2010. PEDV was first introduced into the United States (US) and rapidly spread nationwide in 2013. PEDV infection of suckling piglets causes up to 100% mortality and the US pig industry lost 10% of domestic pig population and $0.9 to $1.8 billion during 2013-2014, posing a great threat to the pork industry sustainability. Live attenuated vaccines (LAVs) given orally to pregnant sows/gilts replicate in the intestines and induce lactogenic immunity in sows/gilts. They are the most effective approach to provide passive immunity to neonatal pigs against PED via colostrum and milk. However, safety concerns associated with potential virulence reversion hinder broad application of LAVs. The goal of this dissertation is to identify promising targets in two viral genes and to develop a recombination-resistant platform for the development of effective and safe PEDV LAVs. My first objective was to evaluate whether the exonuclease (ExoN) domain within PEDV nsp14 is a good target for LAV development. Based on the infectious cDNA clone of a highly virulent PEDV strain (icPC22A), eight mutants targeting nsp14 ExoN catalytic sites, zinc finger, or Mg2+-binding site were designed. Only one E191A mutant, carrying the mutation in Mg2+-binding site, was rescued that was characterized by poor growth in Vero or IPEC-DQ cells of the early passages no.1-3 (P1-3) with peak titers of 1.80 ± 0.12 and 1.42 ± 0.16 log10 TCID50/mL, respectively. However, the P4 of E191A was characterized by dramatically improved growth characteristics reaching a high infectious titer (5.55 ± 0.35 log10 TCID50/mL) in Vero cells, like icPC22A. Sequence analysis demonstrated that the introduced mutation site has reverted to wildtype in the P4 virus. To evaluate the pathogenesis of the E191A-P1, 4-5-day-old gnotobi (open full item for complete abstract)

Committee: Qiuhong Wang (Advisor); Zongdi Feng (Committee Member); Linda Saif (Committee Member); Anastasia Vlasova (Committee Member) Subjects: Virology

Doctor of Philosophy, The Ohio State University, 2022, Comparative and Veterinary Medicine

Influenza A virus (IAV) continues to pose an enormous threat to animal and public health owing to the emergence of novel strains with panzootic and pandemic potential. Effective control of emerging IAVs requires new broadly protective vaccines. It is now widely accepted that IAV populations exist as quasispecies (mutant spectra) mainly due to the high rates of spontaneous mutations spawned by their error-prone RNA-dependent RNA-polymerase complex. The IAV quasispecies is an extremely diverse swarm of biologically and genetically heterogeneous particle subpopulations that collectively influence the evolutionary fitness of the virus. Live attenuated influenza vaccines with truncations in nonstructural protein 1 (NS1) have shown broad protective efficacies in birds and mammals, which correlate with the ability to induce elevated interferon-related innate immune responses in the vaccinated hosts. These abilities appear to coincide with the presence of heterogenous population of interferon-inducing and defective-interfering particles in the vaccines. It is therefore of great importance to understand the factors that shape the heterogeneity of IAV populations to facilitate the development of broadly effective preventive and therapeutic measures against emerging IAV strains. In the first part of this study, we asked as to whether we could exploit the extreme diversity of influenza virus populations to improve an NS1-truncated live attenuated influenza vaccine developed for poultry (PC4) by selecting viral subpopulations with enhanced interferon-inducing capacities. We deconstructed a de novo population of PC4 through plaque isolation, created a large library of clones, and assessed their interferon-inducing phenotypes. While most of the clones displayed the parental interferon-inducing phenotype in cell culture, few clones showed enhanced interferon-inducing phenotypes in cell culture and chickens. Through deep sequencing of the whole IAV genome, we showed the enhanced int (open full item for complete abstract)

Committee: Scott Kenney (Advisor); Chang-Won Lee (Committee Member); Qiuhong Wang (Committee Member); Andrew Bowman (Committee Member); Renukaradhya Gourapura (Committee Member) Subjects: Immunology; Virology

Doctor of Philosophy, The Ohio State University, 2022, Microbiology

Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally. Cutaneous leishmaniasis (CL) is the most common form, characterized by chronic skin lesions. Currently, there are no approved vaccines for human use. We have generated centrin knock out Leishmania (L.) mexicana (LmexCen-/-) mutants using CRISPR/Cas9. Centrin is a cytoskeletal protein required only for intracellular amastigote replication in Leishmania. Here, we investigated the safety, immunogenicity, and efficacy of LmexCen-/- parasites in vitro and in vivo. Our data shows that LmexCen-/- amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected macrophages and dendritic cells, compared to LmexWT. Furthermore, LmexCen-/- parasites are safe in susceptible mouse models and efficacious against challenge with LmexWT in genetically different BALB/c and C57BL/6 mice. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens compared to the controls. Overall, we demonstrate that LmexCen-/- parasites are a promising candidate vaccine against CL in pre-clinical models. Next, we explored the metabolic drivers of these vaccine-mediated immunological profiles. Metabolomics are emerging as a useful tool to uncover unknown networks that govern immune regulation and determine functional specialization. We analyzed the metabolic changes occurring after immunization with LmexCen-/- and compared them with LmexWT infection. Our results show enriched aspartate metabolism and pentose phosphate pathway (PPP) in ears immunized with LmexCen-/- parasites. These pathways are both known to promote M1 polarization in macrophages, and PPP in particular induces nitric oxide production in macrophages cultured with LmexCen-/-, suggesting a shift to a pro-inflammatory phenotype following immunization. Furthermore, immunized mice showed enriched t (open full item for complete abstract)

Committee: Abhay Satoskar (Advisor); Pravin Kaumaya (Committee Member); Steve Oghumu (Committee Member); Jesse Kwiek (Committee Member) Subjects: Immunology; Microbiology; Neurosciences; Parasitology

Doctor of Philosophy, The Ohio State University, 2017, Comparative and Veterinary Medicine

Control of avian influenza (AI) has been continuously challenged by the extreme diversity and complicated ecology of the causative agent, avian lineage type A influenza virus. Poultry species serves unique roles in AI ecology as providing an environment for viral evolution and act as an intermediate host to spread novel viral strains to other animal species including humans. Thus, to minimize the socio-economic impact from the emergence of novel strains and zoonotic infection, development of a strong immunity in poultry is desirable as a preventive measure. But currently available influenza vaccines for poultry, inactivated vaccines (IV) or recombinant viral vector vaccines, provide limited protection range confined to a specific target antigen and they require periodic updates to keep pace with the frequent viral mutations. As an alternative, live attenuated influenza vaccine (LAIV) can supplement the current vaccines for providing a broader protective immunity against diverse strains. The long-term aim of this study is to develop a broadly effective LAIV for the use in poultry. Among diverse strategies to produce LAIV candidates, we focused on influenza A virus mutants that encode C-terminally truncated nonstructural protein 1 (NS1-truncated mutants). Nonstructural protein 1 (NS1 protein) is a multifunctional protein which provides the virus a favorable environment, primarily by interfering with host type I interferon (IFN) induction and signaling system. In turn, the NS1-truncated mutants can be a promising live vaccine candidate for their highly attenuated phenotype as well as an induction of a boosted immune response following the unimpeded type I IFN response. Previously, four genetically stable plaque purified NS1 truncated mutants, pc1- to pc4-LAIV, were evaluated for their potential as LAIV candidates in chickens. All the four candidates showed limited replication in chickens, but interestingly, the protective efficacy was not equal among viruses; only the (open full item for complete abstract)

Committee: Chang-Won Lee Dr. (Advisor); Daral J. Jackwood Dr. (Committee Member); Renukaradhya Gourapura Dr. (Committee Member); Andrew S. Bowman Dr. (Committee Member) Subjects: Immunology; Veterinary Services; Virology