Master of Science, The Ohio State University, 2024, Animal Sciences

Salmonellosis continues to be one of the major public health concerns worldwide causing a gastrointestinal disease. Poultry meat and eggs are recognised as one of the major sources of Salmonella food poisoning in humans. Our study evaluated the protective efficacy of mannose-conjugated chitosan-nanoparticle (mChitosan-NP) based oral subunit vaccine consisting of outer membrane proteins and flagella of Salmonella Enteritidis against Salmonella colonization in the intestines of broilers by incorporating two known mucosal adjuvants, c-di-GMP (stimulator of interferon gene agonist) and whole cell lysate (WCL) of Mycobacterium smegmatis. We try to identify the optimal dose of c-di-GMP and WCL adjuvants by using three different amounts (2.5µg, 10µg and 50µg/dose) in vivo to potentiate the efficacy of Salmonella subunit vaccine formulation. In vitro analysis revealed that mChitosan-NP Salmonella vaccine and mChitosan-NP adjuvant formulations were carrying high positive charge (Zeta potential +20-25mV), size 235-260nm, and polydispersity index 0.35-0.52, conducive for in vivo studies. Subsequently, the vaccine-adjuvant formulations were evaluated for efficacy in vivo in broiler chickens by challenging with Salmonella Enteritidis. Our data showed that mChitosan (OMP+FLA)/FLA-NP WCL 10µg/dose formulation reduced over 0.5 log10 reduction of challenge bacterial load comparable to a commercial live vaccine at both day post challenge 4 and 10. The systemic and mucosal antibody responses were found superior in adjuvanted mChitosan-NP Salmonella vaccine groups. Additionally, most of the vaccine groups had an increased frequency of B cells compared to mock group at day post-challenge 4, associated with upregulation of TGF-β mRNA at day post-challenge 10. Overall, mChitosan (OMP+FLA)/FLA-NP WCL 10 µg/dose and mChitosan (OMP+FLA)/FLA-NP GMP 50µg/dose performed well in inducing immune responses and efficacy.

Committee: Renukaradhya Gourapura (Advisor); Liesa Bielke (Committee Member); Gireesh Rajashekara (Committee Member) Subjects: Animal Sciences

Doctor of Philosophy, Case Western Reserve University, 2024, Molecular Medicine

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus causing hemorrhagic fever with mortality rate ranging up to 30%. However, licensed vaccine or targeted therapy against SFTSV is yet to be developed. In this study, we developed a protein subunit vaccine employing 24-mer self-assembling ferritin (FT) nanoparticles as a platform to present the head region of the SFTSV Gn (GnH). The purified GnH-FT nanoparticles preserved structural integrity and induced robust humoral and cellular immunity against SFTSV Gn. Furthermore, aged ferrets that received immunization with GnH-FT nanoparticles exhibited complete protection from lethal SFTSV challenge and symptoms of body weight loss, viremia, fever, thrombocytopenia, leukopenia, and mortality. We also developed mRNA vaccines encoding GnH and GnH-FT, which potently induce immunity against SFTSV and provides full protection against lethal SFTSV infection in mouse model. Furthermore, we repurposed the GnH-FT nanoparticle to develop therapeutic antibody against SFTSV. We identified five unique antibodies specifically binding SFTSV, including two candidates with sub-nanomolar KD. Our study suggests two vaccine candidates and potential therapeutic antibodies against SFTSV.

Committee: Jae Jung (Advisor); Feixiong Cheng (Committee Chair); Xinghong Dai (Committee Member); Christine O'Connor (Committee Member); Jun Qin (Committee Member); Frank Esper (Committee Member) Subjects: Immunology; Microbiology; Virology

Master of Science, The Ohio State University, 2023, Comparative Biomedical Sciences

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic human retrovirus which causes a lifelong infection. An estimated 5-10 million persons are infected worldwide. However, the true amount of global HTLV-1 carriers is likely much higher given recent reports from newly endemic areas. HTLV-1 is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL), an aggressive and fatal CD4+ T-cell malignancy. The projected lifetime risk of developing ATL is ~5% in HTLV-1-infected individuals and 25% in perinatal HTLV-1 carriers. Despite the exceptional oncogenicity of this virus, no preventative vaccine has been tested in clinical trials to date. However, several vaccine studies have shown protection against HTLV-1 challenge in animals, demonstrating that developing vaccine is feasible. The COVID-19 pandemic and the success of SARS-Cov-2 spike protein mRNA vaccines spurred the popularity of the mRNA vaccine platform. Immunization with encapsulated mRNA offers numerous benefits over conventional vaccines, including improved safety through the delivery of a non-infectious agent and ability to regulate in vivo half-life, as well as enhanced efficacy through modulation of stability and translation. The use for mRNA vaccines in HTLV-1 research warrants investigation. In this study, we developed an mRNA lipid nanoparticle (mRNA-LNP) vaccine encoding for a codon optimized HTLV-1 envelope (Env) and evaluated its efficacy as a vaccine candidate in New Zealand white rabbits. Rabbits (n=6) were vaccinated with two doses of either Env mRNA-LNP or control GFP mRNA-LNP, then challenged with lethally irradiated HTLV-1-infected cells. All rabbits were rechallenged fifteen weeks later to evaluate the durability of vaccine-induced immune responses in protected rabbits. Anti-Env antibody responses were detected by western blot and quantitatively measured using infected cell binding assays in all Env mRNA-LNP vaccinated rabbits after two doses. Three (out of six) Env mRNA-LN (open full item for complete abstract)

Committee: Amanda Panfil (Advisor); Kai Xu (Committee Member); Stefan Niewiesk (Committee Member) Subjects: Biology; Biomedical Research; Immunology

Doctor of Philosophy, The Ohio State University, 2020, Animal Sciences

Salmonellosis causes around one million illness every year in the Unites States. The symptoms of the disease in human include diarrhea, fever and vomiting. Animal-origin foods especially Salmonella Enteritidis contaminated poultry-related produce, remain the most common source of Salmonella outbreaks, which is responsible for half of the total cases of food poisoning. With the huge consumer demand of chicken meat in the U.S., reducing Salmonella colonization in broilers can largely decrease the risk of foodborne salmonellosis. Current Salmonella vaccines in broiler have failed to provide protection and reduce food contamination. Therefore, our goal is to develop an innovative Salmonella oral vaccine which elicits robust immunity and reduces bacterial colonization and shedding in broilers. Our preliminary data showed gastric pH resistant biodegradable and mucoadhesive chitosan (CS) nanoparticles based vaccine containing entrapped immunogenic outer membrane proteins (OMP) and flagellin (FLA) of Salmonella Enteritidis (SE) with surface labeled flagella [CS(OMP+FLA)-F] was found targeted to intestinal immune sites and induced the expression of important Toll-like receptors in cecal tonsils of birds. In this study, we first prepared CS(OMP+FLA)-F and the temporal dynamic immune responses of the immunized broilers were examined after 1st dose to 3-day-old broilers and 2nd dose inoculated 2 weeks later. We observed increased mRNA expression level of Toll-like receptors (TLRs) 1, 4, 7 and 7 in the cecal tonsils of birds received CS(OMP+FLA)-F, especially at 7 days post 1st dose of vaccine delivery. The antigen specific splenocytes proliferation response was enhanced after 7 days post 1st and 2nd dose of vaccine. This study showed our candidate vaccine can induce both innate and adaptive immune responses. To determine the efficacy of candidate vaccine and investigate the ideal vaccination schedule for maximum protection, dose- and age-dependent study was carried out followed (open full item for complete abstract)

Committee: Renukaradhya Gourapura (Advisor); Ramesh Selvaraj (Advisor); Michael Lilburn (Committee Member); Sheila Jacobi (Committee Member); Thaddeus Ezeji (Committee Member) Subjects: Animal Sciences

Doctor of Philosophy, The Ohio State University, 2015, Comparative and Veterinary Medicine

Porcine reproductive and respiratory syndrome (PRRS) is responsible for greater than $664 million direct annual loss to the US pork industry. The causative agent, PRRS virus (PRRSV), is an enveloped positive sense, single stranded RNA virus belongs to the family Arteriviridae. There are two genotypes, European (type I) and the North American (type II), with varying inter- and intra-genotypic genetic and antigenic diversity, signifying extreme mutagenic nature of the PRRSV. Though, since early 1990s both PRRS modified live virus (PRRS-MLV) and inactivated PRRSV vaccines are in use, control of PRRS remains unsuccessful. Moreover, PRRS-MLV has been implicated in the spread of vaccine origin variant virus to susceptible pigs. Available inactivated vaccines are safe but poorly immunogenic. Considering its safety advantage, development of a potent inactivated PRRSV vaccine is warranted. Intranasal delivery of vaccine is non-invasive and has the potential to stimulate a protective immune response not only in the respiratory tract, but also in the genital, gastric tracts. Additionally, efficient Th1 response inducing adjuvants are necessary in subunit/inactivated vaccines. Recently, we identified potent adjuvanticity of Mycobacterium tuberculosis whole cell lysate (M. tb WCL) to PRRS-MLV. FDA approved and biodegradable PLGA (poly [lactide-co-glycolide]) based micro/nanoparticles are widely used for targeted/sustained delivery of drugs and vaccines. In this doctoral dissertation, we report two novel approaches of production of inactivated/multivalent subunit vaccines and their protective efficacy assessment in pigs. Recently, we have shown that single dose of PLGA nanoparticle (NPs) based inactivated PRRSV (NP-KAg) vaccine delivered intranasally to pigs, elicited immune response with partial viral clearance. Our hypothesis was that co-administration of NP-KAg vaccine twice with nanoparticle-entrapped/unentrapped M. tb WCL, intranasally, elicits robust anti-PRRSV cross-pr (open full item for complete abstract)

Committee: Renukaradhya Gourapura (Committee Chair) Subjects: Immunology; Microbiology; Nanotechnology; Veterinary Services; Virology