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Rotavirus Pathogenesis, Innate Immunity and Their Immune Modulation by Probiotics in a Piglet Model and in Vitro

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Rotavirus Pathogenesis, Innate Immunity and Their Immune Modulation by Probiotics in a Piglet Model and in Vitro ROTAVIRUS PATHOGENESIS, INNATE IMMUNITY AND THEIR IMMUNE MODULATION BY PROBIOTICS IN A PIGLET MODEL AND IN VITRO DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Lulu Shao Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2015 Dissertation Committee: Dr. Linda J Saif, Advisor Dr. Anastasia N Vlasova, Co-advisor Dr. Prosper N. Boyaka Dr. Renukaradhya J. Gourapura Dr. Gireesh Rajashekara Copyrighted by Lulu Shao 2015 Abstract Rotavirus (RV) is a major pathogen causing acute gastroenteritis in children under 5 and in young animals. Because no specific antiviral therapy is available, effective RV vaccines are crucial to prevent morbidity and mortality. However, licensed RV vaccines for humans have low efficacy in developing countries, and emerging RV strains (including G9) may decrease vaccine efficacy. Our goal was to improve understanding of RV pathogenesis, cross-protection and correlates of protection and probiotics/commensals, age and tissue-specific effects on innate immunity in a pig model. Our first objective was to assess the pathogenesis of porcine RV (PRV) G9P[13] and evaluate short-term cross-protection between G9P[13] and human RV (HRV) G1P[8] in a gnotobiotic (Gn) pig model. Gn pigs were inoculated with G9P[13] and challenged with G1P[8], or vice versa. G9P[13] induced longer fecal virus shedding than G1P[8] and generated complete short-term cross-protection in pigs challenged with G1P[8] or G9P[13], whereas G1P[8] induced only partial protection against G9P[13] challenge. G9P[13] replicated more extensively in porcine monocyte-derived dendritic cells (DCs) than G1P[8]. Our results suggest that heterologous protection by the current monovalent G1P[8] vaccine against emerging G9 strains should be evaluated in further studies to prevent wider dissemination of G9 strains. ii Our second objective was to compare the influence of age, diet, microbiota and tissue origin on TLR expression in mononuclear cells (MNCs) and DCs isolated from spleen, ileum and mesenteric lymph nodes (MLNs) of germfree (GF) and conventional pigs. TLR mRNA expression profiles were distinct between GF and conventional pigs, and were generally lower in ileal MNCs/DCs which may be due to microbiota-driven immunoregulatory/immunosuppressive mechanisms to avoid overstimulation by dampening TLR expression levels. Comparison of TLR expression profiles in GF and conventional pigs demonstrated that exposure to commensal microbiota may be of more importance than age in TLR expression, further highlighting the critical role of commensal microbiota in the development of systemic and mucosal immune systems of neonates. Our third objective was to examine the differential effects of live probiotic bacteria on splenic, ileal and MLN MNCs from Gn pigs. MNCs were analyzed after treatment with probiotics and/or inactivated HRV (inactHRV) in vitro to determine DC frequencies and cytokine levels. We found that the Gram-positive probiotics Lactobacilus rhamnosus GG (LGG) and Bifidobacterium lactis Bb12 (Bb12) decreased the frequencies of conventional DC (cDC) and activated (MHCII+) cDC in ileal and MLN of inactHRV- exposed or untreated MNCs. In contrast, the Gram-negative probiotic Escherichia coli Nissle 1917 (EcN) increased plasmacytoid DC (pDC) and (MHCII+) pDC frequencies in MLN MNCs. In inactHRV-exposed groups, EcN, LGG and Bb12 reduced cytokine levels of IL-6 (pro-inflammatory), IFN-alpha (innate/anti-viral), IL-12 (Th1), and IL-4 (Th2) in splenic MNCs; whereas, EcN increased IL-10 (T-regulatory) levels. These results iii demonstrate that Gram-positive and Gram-negative probiotic bacteria differentially modulate DC frequencies, surface phenotypes and functions in vitro. Collectively, these findings improve our understanding of RV pathogenesis, correlates of protection, and the development of porcine innate immunity. This knowledge should help to improve RV vaccine efficacy and aid in design of new vaccines. iv This dissertation is dedicated to my parents, Huabin Shao and Lin Zhang, for the love and support. Dedication goes to my husband Zhenyu Li who encourages and supports me to reach my goal. v Acknowledgments I would like to express my sincere appreciation and gratitude to my advisor, Dr. Linda J. Saif, for her guidance and mentorship. Her enthusiasm and attitude to scientific research greatly affected me. Without her support and encouragement, this dissertation would not have been possible. Many thanks to my co-advisor, Dr. Anastasia N. Vlasova, for training me with lab techniques, discussing experimental data analyses, and revising my manuscripts and dissertation. She encouraged me to believe in myself to face challenges and guided me to reach this accomplishment. I would also like to thank all my committee members, Drs. Prosper N. Boyaka, Renukaradhya J. Gourapura and Gireesh Rajashekara, for the guidance and suggestions over the years. My deep appreciation to Dr. Qiuhong Wang, Dr. David Fischer, Dr. Sukumar Kandasamy, Dr. Kuldeep Chattha, Stephanie Neal and Zhongyan Lu with whom I encountered the struggles of research. My gratitude goes to Xiaohong Wang, Marcia Lee, Juliet Chepngeno and Kyle Scheuer, for their technical assistance which helped me to finish my projects. To Dr. Juliette Hanson, Ronna Wood and Jeff Ogg for the help of animal experiments. To Hanna Gehman and Robin Weimer for the support and help. vi Vita 2006 to 2011 ..................................................B. S. Veterinary Medicine, Huazhong Agricultural University 2011 to present ...............................................Graduate student, Department of Veterinary Preventive Medicine, The Ohio State University Publications 1. N. Vlasova, K. Chattha, S. Kandasamy, Z. Liu, M. Esseili, L. Shao, G. Rajashekara, L. J. Saif. (2013) Lactobacilli and Bifidobacteria promote immune homeostasis and modulate innate immune responses to human rotavirus in neonatal gnotobiotic pigs. PLoS ONE 8(10): e76962. doi: 10.1371/journal.pone.0076962 2. L. Shao, D. D. Fischer, S. Kandasamy, A. Rauf, S. N. Langel, D. E. Wentworth, K. M. Stucker, R. A. Halpin, H. C. Lam, D. Marthaler, L. J. Saif, A. N. Vlasova. Comparative in vitro and in vivo studies of porcine rotavirus G9P[13] and human rotavirus Wa G1P[8]. J Virol doi:10.1128/JVI.02401-15 3. S. Kandasamy, A. N. Vlasova, D. D. Fischer, A. Kumar, K. Chattha, A. Rauf, L. Shao, S. N. Langel, G. Rajashekara, L. J. Saif. Differential effects of Escherichia coli vii Nissle and Lactobacillus rhamnosus strain GG on human rotavirus binding, infection, and B cell immunity. J Immunol accepted viii Published Abstracts 1. L. Shao, A.N. Vlasova, K. Chattha, S. Kandasamy, D. Fischer, L.J. Saif. Differential effects of live probiotic bacteria and their metabolites in vitro on conventional and plasmacytoid dendritic cells from gnotobiotic pigs. The American Society for Virology 2013 2. L. Shao, L.J. Saif, D. Fischer, S. Kandasamy, A. Rauf, A. Vlasova. Comparative in vivo and in vitro studies of porcine rotavirus G9P[13] and human rotavirus Wa (G1P[8]) in gnotobiotic pigs. Conference of Research Workers in Animal Diseases 2014 3. L.J. Saif, A.N. Vlasova, S. Kandasamy, L. Shao, A. Rauf, D. Fischer, K. Chattha, A. Kumar, G. Rajashekara. Escherichia coli (Nissle 1917) modulates innate and adaptive immune responses and reduces human rotavirus diarrhea in a neonatal gnotobioitc pig disease model. NIH RO1 Infant Immunity Workshop 2014 4. S. Kandasamy, A. N. Vlasova, K. Chatta, D. Fischer, L. Shao, A. Kumar, G. Rajashekara, L.J. Saif. Escherichia coli Nissle 1917 colonization ameliorates human rotavirus diarrhea and modulates B cell responses in a neonatal gnotobiotic pig disease model. NIH RO1 Infant Immunity Workshop 2014 5. A.N. Vlasova, S. Kandasamy, L. Shao, A. Rauf, D. Fischer, K. Chattha, A. Kumar, G. Rajashekara, L.J. Saif. Escherichia coli Nissle 1917 colonization upregulates innate immunity and partially protects against human rotavirus infection in a neonatal gnotobiotic pig model. The American Association of Immunologists 2014 ix 6. S. Kandasamy, A. Vlasova, K. Chattha, D. Fisher, L. Shao, A. Kumar, G. Rajashekara, L.J. Saif. Escherichia coli Nissle 1917 colonization ameliorates human rotavirus diarrhea and modulates B cell responses in a neonatal gnotobiotic pig disease model. The American Association of Immunologists 2014 7. L.J. Saif, A. N. Vlasova, S. Kandasamy, K. Chattha, G. Rajashekara, A. Kumar, A. Rauf, L. Shao, D. Fischer, H. Huang and S. Neal. Tailoring probiotics as immunomodulators to enhance neonatal mucosal immunity to rotavirus (RV) vaccines or alleviate RV diarrhea: Evaluation in a neonatal gnotobiotic piglet model. Conference of Research Workers in Animal Diseases 2014 8. Y. Wang, A. Vlasova, L.J. Saif, D. Fischer, K. Chattha, S. Kandasamy, L. Shao, Y. Levin, B. Jiang. Immunogenicity and Protective Efficacy of Inactivated Rotavirus Vaccine in Piglets. Presented at the 33rd Annual Meeting, American Society for Virology, Ft. Collins, CO, June 2014. 9. S. Kandasamy, A. Vlasova, K. Chattha, D. Fischer, A. Rauf, L. Shao, S. Neal, A. Kumar, G. Rajashekara, L.J. Saif. Differential effects of Escherichia coli Nissle and Lactobacillus rhamnosus strain GG on human rotavirus infection and B cell responses. Society for Mucosal Immunology, Berlin, Germany, July 2015. Fields of Study Major Field: Comparative and Veterinary Medicine Studies in Virology and Immunology x Table of Contents Abstract ..............................................................................................................................
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