STUDIES OF HUMAN ROTAVIRUS CANDIDATE NON-REPLICATING VACCINES AND INNATE IMMUNITY IN A GNOTOBIOTIC PIG MODEL OF HUMAN ROTAVIRUS DISEASE DISSERTATION Presented as Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ana María González, M.D * * * * The Ohio State University 2007 Dissertation Committee Approved by Distinguished University Professor Linda J. Saif, Adviser ________________________ Adviser Adjunct Assistant Professor Lijuan Yuan Graduate Program of Veterinary Preventive Associate Professor Kenneth W. Theil Medicine Professor Caroline Whitacre ABSTRACT Rotavirus is the major cause of severe dehydrating diarrhea in children and young infants worldwide. The mortality rates reach 600,000 annually, mainly in developing countries and vaccination is an important preventive measure. The first two objectives of my PhD research were to produce and test a combination of replicating and non-replicating human rotavirus (HRV) vaccines or non-replicating HRV vaccines in the gnotobiotic pig model to minimize or avoid the use of more reactogenic live HRV vaccines. The third objective was to assess the mucosal and systemic dendritic cell responses after RV infection because these responses are largely uncharacterized but are important in understanding immunity induced after infection and for design of vaccines. The neonatal gnotobiotic pig is susceptible to HRV for more than 8 weeks and their gnotobiotic status assures that wild type rotavirus infection does not occur during vaccination. Additionally gnotobiotic pigs are optimal for the study of innate immune responses to HRV in-vivo by excluding any confounding factors (e.g. commensal flora. other pathogens etc). For the first objective, gnotobiotic pigs were vaccinated priming with a peroral (PO) live attenuated human rotavirus (AttHRV) and boosting (2x) with a non-replicating 2/6 virus-like particles (VLPs) intranasally (IN) using ISCOM as adjuvant. High protection rates against diarrhea and shedding (71%) were induced which coincided with higher IgA antibody titers in small intestinal contents and serum virus neutralizing (VN) antibody responses. In contrast, vaccination with 2/6VLP alone conferred no protection against diarrhea or shedding suggesting that neutralizing antigens, VP4 and VP7 were needed as part of a non-replicating vaccine formulation to induce protective immune responses in neonatal pigs. Consequently the second objective was to test a non-replicating vaccine that included RV neutralizing antigens. A combination of semipurified VP4 and 2/6/7VLP PO followed by VP4+2/6VLP IN using ISCOM as adjuvant was tested. A 67% protection rate against diarrhea and 33% protection rate against shedding were elicited with high to moderate numbers of IgA antibody secreting cell responses in the gut but low VN antibody titers in serum. Vaccination with 2/6/7VLP PO and 2/6VLP IN (that lacked the semipurified VP4) conferred low protection rates (33% against diarrhea and no protection against shedding) suggesting that VP4 was an essential component of a non-replicating ii rotavirus vaccine. This study confirmed that RV neutralizing antigens are needed for a non-replicating HRV vaccine formulation to induce protective immune responses in neonatal pigs. Additionally, to improve protection rates against shedding, VN titers likely need to be enhanced by giving more potent adjuvants and/or a higher vaccine concentrations. For the third objective, viral dose effects on DCs ex-vivo and the association with clinical outcome were examined in gnotobiotic pigs after a high or low dose of HRV. We assessed intestinal and splenic activated (CD80/86+) IFNα, IL-12, IL- 10, IL-6 and TNFα producing DCs and their uptake/binding of fluorescent 2/4/6/7VLPs by flow cytometry and serum/intestinal cytokines by ELISA. Because infection with HRV induced mainly intestinal plasmacytoid DCs (pDCs), we studied membrane bound TGFβ1-latency associated peptide (LAP) CD4+ regulatory T cells known to be induced by pDCs. At post-inoculation day (PID) 2, a high HRV dose induced significantly lower frequencies of intestinal activated IFNα+ pDCs (and lower IL-12, IL-6 and TNFα+ pDCs) than a low dose. The frequencies of intestinal IFNα+ pDCs correlated with serum IFNα concentrations (r=0.73 p<0.01) suggesting that the pDCs were activated in-vivo. Furthermore a high HRV dose induced lower uptake/binding of 2/4/6/7VLP-GFP by intestinal and splenic pDCs and lower frequencies of circulating membrane bound TGFβ LAP+ CD4+(SWC3-CD8-), T cells compared to a lower dose suggesting that pDC responses (intracellular cytokine production, antigen engagement and regulatory T cell induction) were diminished by a high dose. At PID2, 69% of high dose pigs developed diarrhea compared to none of the low dose pigs, whereas titers of infectious virus shed were similar. Cell-damage byproducts are known to inhibit pDCs. Higher early rates of diarrhea, possibly associated with enterocyte damage byproducts may decrease pDC function thereby preventing induction of regulatory T cells and facilitating adaptive immune responses to HRV. These results will improve our knowledge of HRV immununopathogenesis and the mechanisms to maintain a balance between tolerance and adaptive immunity suggesting that perhaps, depending on the size of the inocula, immune regulatory responses and adaptive immune responses are regulated distinctively in the gut. iii Dedicated to my parents, my brothers, my sister "in-law", Laura, Sofia and all my family. With their support and love my life was always so much better. To Colombia for all its greatness. iv ACKNOWLEDGMENTS I deeply thank my advisor, Dr Linda J. Saif who believed in me and supported my ideas during these years. I really admire her perseverance, patience and effort to reach perfection in all her actions. I also thank the members of my graduate committee, Dr. Lijuan Yuan, Dr. Kenneth Theil and Dr. Caroline Whitacre for their guidance. I appreciate the help and daily support of my laboratory colleagues: Marli Azevedo, Wei Zhang, Ke Wen, Myung Guk Han, Guohua Li, Trang Van Nguyen, Kwang-il Jeong and Cristiana Iosef. With their daily help, assistance and enthusiasm, work was always easier. I especially thank Peggy Lewis because with her attitude and appreciation for all members of the laboratory she always made us feel special. I also appreciate the support of my colleagues: Menira Souza and Veronica Costantini, for always being there for me with their friendship and good company. I also thank all the staff members, graduate students, postdoctoral fellows and visiting scientists for their help and for making the laboratory a pleasant place to be. I especially thank Robin Weimer, Hannah Gehman, Juliette Hanson, Rich McCormick, Janet McCormick. Paul Nielsen, Mary Decker and Robert Dearth. I am sincerely grateful to my advisors at the Pontificia Universidad Javeriana, Dr Juanita Angel and Manuel Franco, who always taught me accurate science, provided me with an environment that was just and truthful and guided me all the way. I do not have words to express the admiration and appreciation I feel for them. To my colleagues from Colombia and dear friends, Maria Cristina Jaimes, Olga Rojas and Irma Pelaez. The experiences we all lived together are still in my heart and will be forever. Thank you for your friendship, company, support and ideas. I also thank my mother for all her support, dedication and example of kindness, Alvaro for his prayers and good advice; my brother and Pilar for being always close to support me. I thank my father for his unconditional admiration and for supporting my dreams. v VITA September 21, 1976 …………………………………………….Born, Bogotá, Colombia 1994-1998 ………………………………………………..1st-4th year medical school Instituto de Ciencias de la salud CES 1998-2000 …………………………………….Diploma. Medical Doctor and surgeon Pontificia Universidad Javeriana 1999-2001 ………………………………………………………….Research Assistant Human Genetics Institute Pontificia Universidad Javeriana 2001-present………………………………………………….Graduate Research Assistant Food Animal Health Research Program Department of Veterinary Preventive Medicine OARDC, The Ohio State University vi PUBLICATIONS 1. Nguyen TV, Yuan L, Azevedo MS, Jeong KI, González A.M, Iosef C, Lovgren- Bengtsson K, Morein B, Lewis P, Saif LJ.2006. High titers of circulating maternal antibodies suppress effector and memory B-cell responses induced by an attenuated rotavirus priming and rotavirus-like particle-immunostimulating complex boosting vaccine regimen. Clin Vaccine Immunol.13:475-85. 2. Nguyen T.V., L. Yuan, M.S.P. Azevedo, K.I. Jeong, González A.M, C. Iosef, K. Lovgren-Bengtsson, B. Morein, P. Lewis and L.J. Saif. 2006. Low titer maternal antibodies can both enhance and suppress B cell responses to a combined live attenuated human rotavirus and VLP-ISCOM vaccine. Vaccine 24:2302-2316. 3. Azevedo, M. S. P., L. Yuan, S. Pouly , González A.M, KI. Jeong, T. V. Nguyen, and L. J. Saif. 2006. Cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus (HRV). J. Virol. 80:372-382. 4. Azevedo, M. S. P., L. Yuan, K. I. Jeong, González A.M, T. V. Nguyen, Pouly S., M. Gochnauer, W. Zhang, A. Azevedo, and L. J. Saif. 2005. Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus .J Virol. 79:5428-36. 5. Yuan, L., Azevedo, M., González A.M, Jeong, K., Nguyen, T., Iosef, C., Lewis, P., Herrmann, J., and Saif, L. J. 2005. Mucosal and systemic antibody responses and protection induced by a prime/boost rotavirus-DNA vaccine in a gnotobiotic pig model. Vaccine. 23:3925-36. 6. González A.M, Nguyen TV, Azevedo MS, Jeong K, Agarib F, Iosef C, Chang K, Lovgren-Bengtsson K, Morein B, Saif LJ. 2004. Antibody responses to human vii rotavirus (HRV) in gnotobiotic pigs following a new prime/boost vaccine strategy using oral attenuated HRV priming and intranasal VP2/6 rotavirus-like particle (VLP) boosting with ISCOM. Clin Vaccine Immunol. 135:361-72. 7. González A.M, Jaimes MC, Rojas OL, Angel J, Greenberg H, Franco M.