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.
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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.
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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.
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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
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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. 2003. Human Adaptive immunity to rotaviruses: A model of intestinal mucosal adaptive immunity. Chapter 11, 307-368. Vol. 9. Viral Gastroenteritis. Elsevier Science
8. Rojas OL, González A.M, Gonzalez R, Perez-Schael I, Greenberg HB, Franco MA, Angel J. 2003. Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells. Virology.314:671-9
9. González A.M, Jaimes MC, Cajiao I, Rojas OL, Cohen J, Pothier P, Kohli E, Butcher EC, Greenberg HB, Angel J, Franco MA. 2003. Rotavirus-specific B cells induced by recent infection in adults and children predominantly express the intestinal homing receptor alpha4beta7. Virology.305:93-105.
10. Jaimes MC, Rojas OL, González A.M, Cajiao I, Charpilienne A, Pothier P, Kohli E, Greenberg HB, Franco MA, Angel J. 2002. Frequencies of virus-specific CD4(+) and CD8(+) T lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults. J Virol. 76:4741-9.
Published Abstracts
1. Saif L.J., Azevedo M.S.P., Gonzalez A.M.,Zhang W.,Wen K.,Lovgren-Begtsson K., Morein B., and Yuan L. Cytokine responses to vaccination with a non- replicating rotavirus like-particle (VLP)-ISCOM vaccine compared to infection with human rotaviruses (HRV) in gnotobiotic pigs. Abstract 5.5. 9th DsRNA Virus Symposium. October 21-26, 2006. Cape Town, South Africa.
2. Yuan L, Gonzalez A.M., Azevedo MSP, Wen K , Zhang W, Lovgren-Bengtsson K., Morein B., and Saif LJ. Distribution of rotavirus-specific effector and proliferating CD4+ and CD8+ T cells induced by human rotavirus infection or vaccination with rotavirus like-particle (VLP)-ISCOM vaccine in gnotobiotic pigs. Abstract 5.10. 9th DsRNA Virus Symposium. October 21-26, 2006. Cape Town, South Africa. viii
3. Zhang W, Azevedo MSP, González AM, Saif LJ, Nguyen TV, Wen K, Yousef AE, Yuan L. Influence of probiotic Lactobacilli colonization on neonatal B cell responses in a gnotobiotic (Gn) pig model of human rotavirus (HRV) infection and disease. Abstract W18-11. 25th Annual Meeting of American Society for Virology. July 15-19, 2006. University of Wisconsin-Madison, Madison,
4. Azevedo MSP, Zhang W, González AM, Saif LJ, Yousef AE, Yuan L. Influence of probiotic lactic acid bacteria (LAB) on cytokine responses of gnotobiotic pigs infected with human rotavirus (HRV). Abstract W18-12. 25th Annual Meeting of American Society for Virology. July 15-19, 2006. University of Wisconsin- Madison, Madison, WI.
5. Yuan L, Azevedo MSP, Zhang W, González AM, Nguyen TV, Wen K, Yousef AE, Saif LJ. Impact of colonization by probiotic Lactobacili on development of T cell responses in neonatal gnotobiotic (Gn) pigs infected with human rotavirus (HRV). Abstract W18-10. 25th Annual Meeting of American Society for Virology. July 15-19, 2006. University of Wisconsin-Madison, Madison, WI.
6. González AM, Nguyen TV, Azevedo MS, Jeong K, Agarib F, Iosef C, Chang K, Lovgren-Bengtsson K, Morein B, Saif LJ. B cell responses to oral priming with rotavirus VP4+ISCOM co-administered with 2/6/7 rotavirus-like particles (VLP) and intranasal (IN) boosting with VP4+ 2/6VLP + ISCOM in gnotobiotic pigs. Abstract and oral presentation. Abstract .25th Annual Meeting of American Society for Virology. July 15-19, 2006. University of Wisconsin-Madison, Madison, WI.
7. Nguyen TV, Yuan L, Azevedo MSP, Jeong K, González AM, Saif LJ. Cytokines transferred from mother to neonates in Swine: Implications for immunomodulation of neonatal immunity by maternal cytokines. Abstract. Proceedings of the 86th Annual Meeting of the Conference of Research Workers in Animal Disease (CRWAD), St. Louis, Missouri. December 4-6, 2005
8. Yuan L, Azevedo MSP, González AM, Nguyen T, Saif LJ. Cytokine and T cell responses to rotavirus infection. Abstract p79. Proceedings of International Symposium on Advanced Virology 2005. Beijing, China. October 10-13, 2005.
ix 9. Jeong K, Azevedo MSP, González AM, Nguyen TV, Yuan L, Saif LJ. Cell- mediated immune responses in gnotobiotic pigs immunized with sequential attenuated human rotavirus (AttHRV) and 2/6-rotavirus-like particle vaccines and challenged with virulent HRV. Abstract W33-5. 24th Annual Meeting of American Society for Virology. Penn State, State College, Pennsylvania. June 18- 22, 2005.
10. Yuan L, Nguyen TV, Azevedo MSP, González AM, Jeong K, Saif LJ. Maternal cytokines in serum and intestinal contents of suckling pigs. Abstract 51459. 12th International congress of mucosal immunology. Abstract .Boston, Massachusetts. June 24-30, 2005.
11. González A.M, Yuan L.J, Azevedo M.P, Nguyen T.V, Jeong K, Lovgren- Bengtsson K*, Morein B*, and Saif L.J. B cell responses elicited by oral/intranasal (IN) immunization of gnotobiotic pigs with a rotavirus-like particle prime/boost vaccine. Abstract. 12th international congress of Mucosal Immunology, June 25-30, 2005, Boston, Massachusetts, USA.
12. Nguyen TV, Yuan L, Azevedo MSP, González AM, Jeong K, Saif LJ. Transfer of cytokines from sows to newborn piglets via colostrum and milk. Abstract IID6. Swine in Biomedical Research Conference. Chicago, January 27-29, 2005.
13. Yuan L, Jeong KI, Nguyen TV, González AM, Azevedo MS, Zhang W, Saif LJ. Effects of maternal antibodies on T cell responses to rotavirus vaccines. Abstract. American Society of Virology 23rd annual meeting, July 2004, Quebec, Canada.
14. Yuan L, Jeong K, Nguyen TV, González AM, Azevedo MSP, Zhang W, Saif LJ. Effects of maternal antibodies on T cell responses to rotavirus vaccines. Abstract W47-6. 23rd Annual Meeting of American Society for Virology. Montreal, Quebec, Canada. July 9-14, 2004.
15. Nguyen TV, Yuan L, Azevedo MS, Jeong K, González AM, Lovgren-Bengtsson K, Morein B, Lewis P, Saif LJ. Effects of maternal antibodies on effector and x memory B cell responses to rotavirus vaccine. Abstract. American Society of Virology 23rd annual meeting, July 2004, Quebec, Canada.
16. Azevedo MS, Pouly S, Yuan L, Jeong K, González AM, Nguyen TV, Saif LJ. Cytokine responses in serum and intestinal contents of gnotobiotic pis after infection with virulent or attenuated rotavirus. Abstract. American Society of Virology 23rd annual meeting, July 2004, Quebec, Canada.
17. Saif LJ, Yuan L, Azevedo MSP, Jeong K, González AM, Iosef C, Nguyen TV, Herrmann JE. Protective immunity induced by live attenuated (Att) human rotavirus (HRV) priming and bovine rotavirus VP6 DNA boosting in a gnotobiotic (Gn) pig model. Abstract 160.4. 90th Anniversary Annual Meeting of American Association of Immunologists. Denver, Colorado. May 6-10, 2003.
18. Yuan L, Azevedo MS, Nguyen TV, González AM, Jeong K, Chang K, Saif LJ. Booster effects of rotavirus 2/6 VLPs vaccine on antibody responses to rotavirus outer-capsid proteins VP4 and VP7 primed by oral attenuated human rotavirus vaccine in gnotobiotic pigs. Abstract. American Society of Virology 22nd annual meeting, Davis, California. July 11-16, 2003.
19. Jeong K, Azevedo MS, Nguyen TV, González AM, Iosef C, Chang K, Yuan L, Hermann JE, Saif LJ. Cellular immune responses and protection in gnotobiotic pigs vaccinated with a VP6 DNA plasmid vaccine regime with and without attenuated human rotavirus. Abstract. American Society of Virology 22nd annual meeting, Davis, California. July 11-16, 2003.
20. Nguyen TV, Azevedo MS, Jeong K, González AM, Iosef C, Lovgren-Bengtsson K, Morein B, Lewis P, Yuan L, Saif LJ. Effect of circulating maternal antibodies on immune responses and protection induced by ISCOM-2/6 VLP vaccine. Abstract. American Society of Virology 22nd annual meeting, Davis, California. July 11-16, 2003.
21. Azevedo MSP, Jeong K, Nguyen TV, González AM, Nielsen P, Lovgren- Bengtsson K, Morein B, Yuan L, Saif LJ. Protective immunity and nasal shedding in gnotobiotic (Gn) pigs after intranasal (IN) or oral priming with attenuated xi human rotavirus (AttHRV) and boosting with 2/6-rotavirus-like-particles (VLPs) and immuno-stimulating complexes (ISCOM). Abstract W47-3. 22nd Annual Meeting of American Society for Virology. Davis, California. July 11-16, 2003.
22. Azevedo MS, Iosef C, Jeong K, González AM, Agarib F, Iosef C, Chang K, Lovgren-Bengtsson K, Morein B, Nguyen TV, Saif LJ. Antibody secreting cell responses and protection in gnotobiotic pigs vaccinated orally with attenuated human rotavirus and intranasally with 2/6 VLPs and immunostimulating complexes. Abstract. American Society of Virology 21st annual meeting, July 2002, Lexington, Kentucky, 11th international congress of Mucosal Immunology, July 2002, Orlando FL and American Society of Virology 22nd annual meeting, July 2003, Davis, California.
23. Rojas OL, González AM, Gonzalez R, Perez-Schael I, Greenberg HB, Franco MA, Angel J. Human rotavirus specific T cells: quantification by ELISPOT and expression of homing receptors on CD4+ T cells. Abstract. 8th International symposium on double-stranded RNA viruses, September 2003, II Ciocco, Castalvecchio Pascoli, Italy and American Society of Virology 22nd annual meeting, July 2003, Davis, California.
24. Yuan L, Azevedo MSP, Jeong K, Nguyen TV, González AM, Iosef C, Herrmann JE, Saif LJ. Evaluation of a live attenuated human rotavirus priming and bovine rotavirus VP6 DNA boosting vaccination strategy in a gnotobiotic pig model. Abstract PR.4. 8th International Symposium on ds-RNA Viruses, Il Ciocco, Lucca, Italy. September 13-18, 2003.
25. Jeong K, González AM, Iosef C , Azevedo MS, Nguyen TV , Agarib F, Chang K, Lovgren-Bengtsson K, Morein B, Saif LJ. Lymphoproliferative responses in gnotobiotic pigs inoculated orally with attenuated human rotavirus and intranasally with 2/6 VLPs and immunostimulating complexes. Abstract. American Society of Virology 21st annual meeting, July 2002, Lexington, Kentucky.
26. Nguyen TV, Azevedo MS, González AM, Jeong K, Agarib F, Iosef C, Chang K, Lovgren-Bengtsson K, Morein B, Saif LJ. Vaccination of gnotobiotic pigs orally with attenuated human rotavirus and intranasally with 2/6 VLP with ISCOM induces similar protection rates but higher antibody titers than attenuated human xii rotavirus alone. Abstract and oral presentation. American Society of Virology 21st annual meeting, July 2002, Lexington, Kentucky.
27. González AM, Nguyen TV, Azevedo MS, Jeong K, Agarib F, Iosef C, Chang K, Lovgren-Bengtsson K, Morein B, Saif LJ. Vaccination of gnotobiotic pigs orally with attenuated human rotavirus and intranasally with 2/6 VLP with ISCOM induces similar protection rates but higher antibody titers than attenuated human rotavirus alone. Abstract and oral presentation. American Society of Virology 21st annual meeting, July 2002, Lexington, Kentucky, USA.
28. Jaimes MC, Rojas OL, González AM, Cajiao I, Charpilienne A, Pothier P, Kohli E, Greenberg HB, Franco MA, Angel J. Frequencies of virus-specific CD4(+) and CD8(+) T lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults. Abstract. 6th International symposium on double- stranded RNA viruses. December 2000, Aruba and 11th international congress of Mucosal Immunology, July 2002, Orlando FL.
29. González AM, Jaimes MC, Cajiao I, Rojas OL, Cohen J, Pothier P, Kohli E, Butcher EC, Greenberg HB, Angel J, Franco MA Rotavirus-specific B cells induced by recent infection in adults and children predominantly express the intestinal homing receptor alpha4beta7. Abstract and oral presentation. 6th International symposium on double-stranded RNA viruses. December 2000, Aruba and 11th international congress of Mucosal Immunology, July 2002, Orlando FL.
FIELD OF STUDY
Major Field: Veterinary Preventive Medicine Studies in Immunology and Virology
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TABLE OF CONTENTS
Page ABSTRACT……………………………………………………………………………....ii DEDICATION…………………………………………………………………………....iv ACKNOWLEDGMENTS..………………………..……………………………………...v VITA…………………………………………………………………………………...... vi LIST OF TABLES………………………………...……………………………………xvii LIST OF FIGURES……………………………………..………………………………xix LIST OF ABBREVIATIONS………………………………………………..………….xxi
CHAPTER 1 HUMAN ROTAVIRUS STRUCTURE,REPLICATION, SPECIFIC IMMUNITY AND VACCINE DEVELOPMENT 1.1 Introduction………………………………………………………………………..1 1.2 Rotavirus Structure………………………………………………………………..4 1.2.1 Overview…………………………………………………………………4 1.2.2 Virion structure…………………………………………………………..5 1.2.3 Physicochemical Properties……………………………………………...6 1.2.4 Genome Structure………………………………………………………..7 1.2.5 Functions of RV proteins………………………………………………...9 1.3 Replication cycle…………………………………………………………………15 1.3.1 Replication overview…………………………………………………...15 1.3.2 Adsorption, penetration and uncoating…………………………………15 1.3.3 Transcription and replication…………………………………………...19 1.3.4 RNA encapsidation and virion assembly……………………………….23 1.3.5 Virus release…………………………………………………………….24 1.4 Rotavirus pathogenesis and pathology…………………………………………...25 1.5 Mucosal Immunity……………………………………………………………….30 1.5.1 Components, inductive and effector sites of mucosal immunity……….30 1.5.2 Intestinal Mucosal Immunity…………………………………………...34
xiv 1.5.3 Structure of the Peyer’s patches (PP)…………………………………...37 1.5.3.1 M cells……………………………………………………………37 1.5.3.2 Subepithelial dome (SED)……………………………………….38 1.5.3.3 Interfollicular region (IFR)………………………………………39 1.5.3.4 The Germinal Center (GC) and B cell follicle…………………...39 1.5.3.5 Role of chemokines in B cell follicle and GC formation………...42 1.5.4 Intestinal B lymphocytes and secretory IgA……………………………43 1.5.4.1 Class switch recombination (CSR)………………………………45 1.5.4.2 Signals for IgA switch…………………………………………...46 1.5.5 Homing mechanisms……………………………………………………48 1.5.5.1 Phenotype and homing of nasally primed lymphocytes…………52 1.5.6 Dendritic cells…………………………………………………………..53 1.5.6.1 Functional characterization of DC subsets………………………56 1.5.6.2 Dendritic cell migration and homing…………………………….57 1.5.6.3 Intestinal dendritic cells………………………………………….58 1.5.6.4 Neonatal dendritic cells…………………………………………..61 1.5.6.5 Porcine dendritic cells……………………………………………62 1.5.7 Intestinal induction of tolerance………………………………………...64 1.5.7.1 T regulatory cells………………………………………………...66 1.6 Immunity to RV in the different animal models and humans……………………72 1.6.1 Mouse model for the study of acquired RV immunity…………………73 1.6.2 Study of RV immunity in knockout mice………………………………76 1.6.3 Mice model for the study of innate immunity after RV infection……...80 1.6.4 Acquired RV-specific immune responses after RV infection of pigs….84 1.6.5 Acquired RV-specific responses after natural infection in children and adults……………………………………………………………………86 1.6.5.1 Humoral antibody responses……………………………………..86 1.6.5.2 B cell responses after natural infection in humans………………90 1.6.5.3 T cell responses after natural infection in humans………………93 1.6.5.4 Innate immunity after RV infection in humans………………….95 1.7 Rotavirus vaccines…………………………………………………………….....98 1.7.1 Replicating and inactivated RV vaccines studied in the mouse model...99 1.7.2 DNA vaccines evaluated in the mouse model………………………...100 1.7.3 Purified capsid proteins as vaccines in the mouse model……………..101 1.7.4 The VLP as candidate vaccines on the mouse model…………………103 1.7.5 Correlates of immunity after RV vaccination…………………………104 1.7.6 Vaccination studies in the gnotobiotic pig…………………………….106 1.7.7 Correlates of protection after RV vaccination of neonatal gnotobiotic pigs ……………………………………………………………………108 1.7.8 Human RV vaccines…………………………………………………..109 1.7.9 New generation of RV vaccines………………………………………112 1.8 References………………………………………………………………114
xv CHAPTER 2 ANTIBODY RESPONSES TO HUMAN ROTAVIRUS (HRV) IN GNOTOBIOTIC PIGSFOLLOWING A NEW PRIME/BOOST VACCINE STRATEGY USING ORAL ATTENUATED HRV PRIMING AND INTRANASAL VP2/6 ROTAVIRUS-LIKE PARTICLES (VLP) BOOSTING WITH ISCOM 2.1 Summary………………………………………………………………………..180 2.2 Introduction……………………………………………………………………..181 2.3 Materials and Methods………………………………………………………….185 2.4 Results…………………………………………………………………………..189 2.5 Discussion………………………………………………………………………193 2.6 Acknowledgments…..…………………………………………………………..199 2.7 References……………………………………………………………………....199
CHAPTER 3 ROTAVIRUS VP4 PLUS IMMUNE STIMMULATING COMPLEXES (ISCOM) CO- ADMINISTERED WITH 2/6/7 VIRUS-LOKE PARTICLES (VLP) FOR PRIMING AND 2/6VLP VACCINES FOR BOOSTING CONFERS PROTECTION AGAINST DIARRHEA IN NEONATAL GNOTOBIOTIC PIGS 3.1 Summary…………………………………………………………………….….217 3.2 Introduction……………………………………………………………….….…218 3.3 Materials and Methods……………………………………………………….…222 3.4 Results…………………………………………………………………………..230 3.5 Discussion……………………………………………………………………....239 3.6 Acknowledgments….……………………….…………………………………..249 3.7 References………………………………………………………………………249
CHAPTER 4 A LOW HUMAN ROTAVIRUS (HRV) DOSE INDUCED HIGHER INTESTINAL IFNα PRODUCING PLASMACYTOID DENDRITIC CELLS (PDC) IN-VIVO AND UPTAKE OR BINDING OF ROTAVIRUS-LIKE PARTICLES (VLPS) IN GNOTOBIOTIC PIGS COMPARED TO A HIGHER HRV DOSE 4.1. Summary………………………………………………………………………..274 4.2. Introduction……………………………………………………………………..275 4.3. Materials and Methods………………………………………………………….279 4.4. Results…………………………………………………………………………..286 4.5. Discussion………………………………………………………………………296 4.6. Acknowledgments.………………….…………………………………………..303 4.7. References………………………………………………………………………303
BIBLIOGRAPHY………………………………………………………………………326
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LIST OF TABLES
Table Page
2.1 Protection rates against virus shedding and diarrhea in gnotobiotic pigs receiving the various vaccination regimes……………...... 205
2.2 Percent of conversion in intestinal contents for IgM, IgA and IgG antibodies in gnotobiotic pigs receiving the various regimes………………………………...206
3.1 Protection rates conferred by the non-replicating VLP vaccines, the combined AttHRV+2/6VLP vaccine regime and controls………………………………...258
3.2 Antibody secreting cell (ASC) responses at pre-challenge determined by ELISPOT induces by the different non-replicating VLP vaccines, the combined AttHRV+2/6VLP vaccine regime and controls………………………………...259
3.3 Virus neutralizing antibody titers at pre-challenge and post-challenge in Gn pigs vaccinated with the different non-replicating VLP vaccines, the combined AttHRV+2/6VLP vaccine regime and controls……………………………..….260
3.4 Memory antibody secreting cell (ASC) responses at pre-challenge determined by ELISPOT induced by the different non-replicating VLP vaccines, the combined AttHRV+2/6VLP vaccine regime and controls………………………..……….261
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4.1 Production of IL-10 by intestinal and splenic pDCs (SWC3low CD4+ CD11b- )…………………………………………………………………..…………...... 313
4.2 Shedding and diarrhea after a low and high HRV doses……………….……....320
4.3 Frequencies of circulating LAP+ CD4+ (CD8- SWC3-) in individual pigs with their correspondent isotype control frequency values………………………….323
xviii
LIST OF FIGURES
Figure Page
1.1 The structure of PP…………………………………………………………...... 36
2.1 Small intestinal contents (SIC) geometric mean antibody titers (GMT, bars) at PID28/PCD0 and PID35/PDC7 of gnotobiotic pigs receiving the various vaccination regimes…………………………………………………………….207
2.2 Serum IgM, IgA and IgG antibody seroconversion at PID0, PID10, PID21, PID28/PCD0 and PID35/PCD7 of gnotobiotic pigs receiving the various vaccinationregimes…………………………………………………….…….....209
2.3 Serum IgM, IgA and IgG geometric mean antibody titers (GMT, bars) at PID28/PCD0 and PID35/PCD7 of gnotobiotic pigs receiving the various vaccination regimes…………………………………………………………….211
2.4 Serum VN geometric mean antibody titers (GMT) at PID0, PID10, PID21, PID28/PCD0 and PID35/PCD7 of gnotobiotic pigs receiving the various vaccination regimes…………………………………………………………….213
2.5 Effect of the dose of 2/6VLP and ISCOM on IgM, IgA and IgG antibody titers to HRV in serum of Att+2/6VLP-ISCOM (250µg/1250 µg or 25 µg/125 µg) and 2/6VLP-ISCOM(250µg/1250µgor 25 µg/125 µg) pigs………………………...215
xix 3.1 Representative dot plots of intestinal MNC from a VP4-2/6/7VLP+VP4-2/6VLP vaccinated and a control gnotobiotic pig……………………………………….262
3.2 Dot plot representing 2/4/6/7VLP and 2/6VLP responses from VP4- 2/6/7VLP+VP4-2/6VLP vaccinated pig (A,B) and a control pig (C,D)………..264
3.3 Correlation coefficient between VP6-specific AEC and RV-specific ASC detected by flow cytometry and ELISPOT, respectively……………………………...…266
3.4 Frequencies (%) of CD21+ VP4-specific AEC…………………………………268
3.5 Frequencies (%) of CD21+ VP7-specific AEC…………………………………270
3.6 Frequencies (%) of CD21+ VP6-specific AEC…………………………………272
4.1 Intestinal MNC from s pig inoculated with low HRV dose at PID2…………...311
4.2 The frequencies of intestinal and splenic activated cytokine producing (IFNα, IL- 12, IL-6 and TNFα) pDCs after a low HRV dose, high HRV dose and controls and PID2………………………………………………………………….…….314
4.3 The frequencies of intestinal and splenic activated cytokine producing (IFNα, IL- 12, IL-6 and TNFα) cDCs after a low HRV dose, high HRV dose and controls and PID2………………………………………………………………………..316
4.4 RV shedding and percentage of pigs with diarrhea for 6 days after inoculation of a high or low dose ……………………………………………………………...... 318
4.5 Intestinal and splenic pDC binding/uptake of 2/4/6/7VLP- GFP…………………………………………………………………………….321
4.6 The frequencies of circulating LAP+CD4+ (SWC3-CD8-) T cells after a low HRV dose, high HRV dose and controls ………………………..……………..324
xx
LIST OF ABBREVIATIONS
Activation-induced deaminase (AID) Antibody secreting cells (ASC) Antigen presenting cell (APC) Attenuated human rotavirus (AttHRV) A proliferation inducing ligand (APRIL) B cell attracting chemokine-1 (BCA-1) B cell receptor (BCR) B-cell lineage specific activator protein (BSAP) B-lymphocyte inducedmaturation protein-1 (Blimp-1) B-lymphocyte stimulator/B-cell-activating factor of the TNF family (BAFF) Bone maroow (BM) Class switch recombination (CSR) Common mucosal immune system (CMIS) Conventional dendritic cell (cDC) Cutaneous lymphocyte antigen (CLA) Cryopatches (CP) Cytokine secreting cells (CSC) Cytomegalovirus (CMV) Dendritic cell (DC) Double-layered particle (DLP) Double stranded RNA (dsRNA) Double stranded RNA dependent protein kinase (PKR) Endoplasmic reticular (ER) Experimental autoimmune encephalitis (EAE) Follicle associated epithelium (FAE) Follicular dendritic cell (FDC) Focus-forming unit (FFU) Gastrointestinal (GI) Genitourinary (GU) Germinal center (GC) Granulocyte-monocyte colony stimulating factor (GM-CSF)
xxi Gut-associated lymphoid tissue (GALT) Heat-shock protein-70 (hsp70) High endothelial venule (HEV) Human rotavirus (HRV) Intranasal (IN) Intramuscular (IM) Isolated lymphoid follicles (iLF) Immunoglobulin (Ig) Immune stimulating complexes (ISCOM) Inflammatory bowel disease (IBD) Intercellular adhesion molecule (ICAM) Intestinal lamina propria (ILP) Interferon regulatory factor-3 (IRF-3) Interfollicular region (IFR) Intraepithelial lymphocytes (IEL) Labile toxin (LT) Lamina propria (LP) Latency associated peptide (LAP) Leukocyte-function associated molecule-1 (LFA-1) Lymph nodes (LN) Lymphotoxin (LT) Maltose binding protein (MBP) Melanoma differentiation associated gene 5 (mda-5) Mesenteric lymph nodes (MLN) Microfold cells (M cells) Monkey Kidney cells (MA104 cells) Mononuclear cells (MNC) Mucosa-associated lymphoid tissue (MALT) Mucosal addressin cellular adhesion molecule-1 (MadCAM-1) Mucosal-associated chemokine (MECK) Myelin basic protein (MBP) Myeloid DC (mDC) Mutant labile toxin (mLT) Nasal-associated lymphoid tissue (NALT) Natural IFNα producing cells (NIPC) Neuraminidase (NA) Open reading frame (ORF) Ovalbumin (OVA) Periarteriolear lymphatic sheaths (PALS) Peripheral lymph nodes (PLN) Peripheral lymph node addressin (PNAd) Peroral (PO) Peyer’s Patches (PP) Plasmacytoid DCs (pDC) Plaque-forming unit (PFU)
xxii Pathogen associated molecular patterns (PAMPs) Pathogen recognition receptors (PRR) Polyacrylamide gel electrophoresis (PAGE) Polymeric immunoglobulin A (pIgA) Polymeric immunoglobulin receptor (pIg) Post-inoculation (PI) Post-inoculation day (PID) Respiratory syncytial virus (RSV) Reticuloendotheliosis viral oncogen related B (RelB) Retinoic acid (RA) Retinoic acid inducible gene-1 (RIG-1) RNA dependent RNA polymerase (RdRp) Rotavirus (RV) Rhesus rotavirus (RRV) Secretory IgA (sIgA) Severe combined immunodeficiency (SCID) Single-stranded RNA (ssRNA) Subcutaneous (SQ) Subepithelial dome (SED) Tobacco mosaic virus (TMV) Thymus-expressed chemokine (TECK) Transmissible gastroenteritis virus (TGEV) T regulatory cells (Tregs) Triple-layered particle (TLP) Toll-like receptor (TLR) Untranslated region (UTR) Vasoactive intestinal peptide (VIP) Virus-like particles (VLP) Virus neutralizing (VN) Wild type (WT)
xxiii
CHAPTER 1
HUMAN ROTAVIRUS STRUCTURE, REPLICATION, SPECIFIC IMMUNITY
AND VACCINE DEVELOPMENT.
1.1 INTRODUCTION
Rotaviruses (RV) are the leading cause worldwide of severe dehydrating diarrhea in children less than 5 years of age (212). Approximately 0.6 million children die from RV infections, mainly in developing countries (420). India alone has a mortality rate of 0.1 million children per year (257). The highest RV mortality of 1 in 200 children occurs in southern Asia and the sub-Saharan African regions (212). Transmission of RV occurs via the fecal-oral route mainly, but respiratory transmission has been also suggested (214,
446). Manifestations of RV disease are severe watery diarrhea, fever and vomiting that usually lead to fluid and electrolyte disequilibrium and other secondary complications
1 (e.g. renal failure) including death (411). Mortality in developed countries is low (20-60
deaths per year), but hospitalizations per year reach 70,000 leading to medical and
societal costs of approximately $1 billion dollars (104, 419). The number of cases per
year in developed countries is 9.1 million and in developing countries is 130 million
(420). Annual doctor visits secondary to moderate and severe diarrhea are 2 million and
25 million in developed and developing countries, respectively (420). For these reasons
both developed and developing countries will greatly benefit from the development of
effective RV vaccines.
The RV are icosahedral, non-enveloped, double-stranded RNA viruses that belong to
the Reoviridae family. The virus capsid is composed of three concentric protein layers. A core composed of VP1, VP2 and VP3, an inner layer composed of VP6 and an outer layer composed of VP4 and VP7. The virus has 11 double stranded RNA (dsRNA) segments, each coding for one protein that can be either structural or non-structural (except gene 11 that encodes two proteins) (177). Four different antigenic specificities define RV
classification in: group, subgroup and serotype, with the latter determined by VP7 and
VP4. Based on antibody specificities to VP6, groups A to G and subgroups I and II have
been described. Serotypes are based on genotype or antigenic specificities related to VP7
and VP4 that induce neutralizing antibodies. The classification of RV by
serotype/genotype G is based on the glycoprotein VP7 and serotype/genotype P is based
on the protease-sensitive viral hemagluttinin VP4 (277).
Group A RV are responsible for most human infections with a minority of cases of
non-group A RV infections described sporadically in adults (mainly groups B and C)
(177). The first licensed RV vaccine, a Rhesus-Human reassortant RV tetravalent vaccine
2 (composed of the four most common human RV G serotypes G1, G2, G3 and G4) was
withdrawn from the market less than a year after its release because of its association
with intussusception (437). A Centers for Disease Control and Prevention case-control
data base from the National Immunization Survey was recently reanalyzed and the results
were consistent with previous reports demonstrating a substantial short term increase of
intussusception among the recipients of the vaccine particularly after the first vaccine
dose. However the incidence of intussusception was only 1 in 11,000 to 16,000 compared
to the previous described incidence of 1 in 5000 to 10,000 (389, 498). In addition the
incidence of RotaShield-associated intussusception was higher in infants >9 months with
a relative risk (RR) of 15.9 compared to a RR of 5-10 in infants from 1-4 months of age
(498). The Global Alliance on Vaccines and Immunization (GAVI) Task Force on
Research and Development considers RV vaccines a priority (437). Similarly, the
Institute of Medicine committee proposed RV vaccines as a priority for developing
countries where RV mortality and morbidity are high (211, 437). New RV vaccines were recently licensed: a bovine-human pentavalent reassortant vaccine that was approved for use in the United States, Canada, Latin American and European countries (543) and an attenuated human monovalent vaccine that was licensed in more than 70 countries (1,
48). The vaccines are highly effective against severe diarrhea; however, testing in developing countries such as India, China and those in Africa where uncommon RV serotypes are more prevalent, has not been performed (207, 211, 480), so the protective efficacy of the vaccines in these countries is unknown. Because both newly licensed vaccines are replicating viruses, induction of intussusception is a risk that can not be ignored. No association has been observed during vaccine trials, but only when millions
3 of children are vaccinated, will the real risk be known. Lately, the FDA issued a warning for potential rise of the intussusception rates after vaccination with Rotateq
(http://www.fda.gov/cber/safety/phnrota021307.htm). For this reason, development of new vaccine strategies remains a priority.
1.2 ROTAVIRUS STRUCTURE
1.2.1 Overview
The RV are composed of three concentric protein layers: the core composed of
VP2, an inner capsid protein composed of VP6, and two outermost proteins, VP4 and
VP7. The inner capsid protein VP6 is the main RV antigen and induces the majority of non-neutralizing antibody responses after natural infection and immunization (103, 512).
The two outermost proteins VP4 and VP7 contain the virus neutralizing antigens that elicit antibodies that block virus cell entry and inhibit virus replication (neutralizing antibodies). There are seven RV groups (A-G) defined by antibody specificities to VP6.
Groups A, B and C RV infect humans whereas groups D, E, F and G infect animals exclusively (277). Humans are mainly infected by group A RV, so this section will focus on this particular group. Group A RV is virus is further classified into serotypes defined by antibody specificities or genotypes defined by genetic differences among VP7 and
VP4. In other words, by the use of neutralization assays (e.g. plaque reduction, neutralization of cytopathic effect assays), RV can be serotyped based on the different neutralizing epitopes of VP4 or VP7 and by sequencing of VP7 and VP4, RV can be genotyped based on genomic differences. With the aid of neutralization assays, 14
4 different VP7 (or G) serotypes have been identified (141). Monoclonal antibodies against
all types of VP4 or hyperimmune anti-serum to all VP4 types are not available making
neutralization assays unsuitable for distinction of all the different VP4 (or P) serotypes
(141). For this reason, sequence analyses are the most useful tool for VP4 classification
(244). By the use of genetic analysis, 21 different VP4 P genotypes (compared to only 11
P serotypes) have been described (244). In current studies, both nomenclatures are widely
used (serotype and genotype), but clearly genotyping is more definitive for VP4
characterization (176).
1.2.2 Virion structure
The RV has three distinctive morphologic forms recognized by electron microscopy: a core (VP1, VP2 and VP3), double (VP1, VP2, VP3 and VP6) and triple layered particles (VP1, VP2, VP3, VP6, VP4 and VP7) (176). Studies of RV particles embedded in vitreous ice provided most of the current information about the structure of the virus. The particles possess icosahedral symmetry with a triangulation number of 13 levo for the two outer layers. The three capsid layers are crossed by 132 channels classified as type I, II and III. Twelve type I channels run down the icosahedral fivefold axes, 60 type II channels surround the fivefold axes in six coordinated positions and 60 type III channels neighbor the icosahedral threefold axes on the six coordinated positions.
All the channels are involved in the transport of nascent RNA transcripts during virus replication. The channels located at the fivefold axes transport mRNAs that interact with the core proteins, VP1 and VP3 (320). The core possesses a triangulation number of 1 and is composed of 120 VP2 molecules organized in dimers. The core is surrounded by
5 780 VP6 molecules organized in trimers at the threefold axes of symmetry. (469). The
outer layer is composed of 60 VP4 spikes and 780 VP7 molecules. Located at the tip of
the type II channels, VP4 protrudes from the surface (444). The viral protein VP4 interacts with 2 molecules of VP7 and 6 molecules of VP6 (176, 444). This interaction suggests that VP4 might play a role in the maintenance of the geometry of the capsid.
Trimers of VP7 molecules compose the majority of the outer layer covering most of the inner capsid. The RV proteins have the capacity of self-assembly suggesting high affinity interactions among the proteins (147).
1.2.3 Physicochemical Properties
The RV infectivity depends on the integrity of the outer capsid which is stabilized by calcium ions (201). For this reason, treatment with EDTA or any other divalent ion- chelating agent leads to destabilization of the outer capsid and loss of infectivity. Calcium has been detected in triple layered particles and potential calcium binding sites have been identified in the structure of VP7 (167, 201). For the generation of core particles, the inner layer can be disrupted by treatment with chaotropic agents such as sodium thiocyanate or high concentrations of calcium chloride (11, 133). Treatment of triple layered particles with chloroform or deoxycholate disrupts VP4 integrity and abrogates infectivity (176, 178). The RV are relatively resistant to inactivation, they are stable from pH 3.0 to pH 9.0 (178) and they can retain infectivity after months of storage at 4°C or even at -20°C with CaCl2 for stabilization of the outer capsid (495). Infectivity and hemagglutination are abolished by repeated freezing and thawing (37, 54). Removal of
VP4 can be accomplished by treatment of the particles with basic solutions, fixatives and 6 ethanol (486). However, among all RV, human RV are somehow more difficult to cultivate and more susceptible to inactivation by decapsidation (176).
1.2.4 Genome Structure
The mRNAs are monocistronic except for gene 11 (363) that encodes for NSP5 and NSP6. The RV are dsRNA viruses with a segmented genome and they have their own RNA dependent RNA polymerase (RdRp) that transcribes each viral RNA segment to its corresponding mRNA (277). The genomic RNA is highly packed within the core and actively interacts with proteins from the core, mainly VP2 (314). Each mRNA has a
5’-methylated guanidine cap followed by several non-coding sequences, an open reading frame and a stop codon. The 3’ portion of each mRNA is composed of non-coding sequences, terminal cytidines and no poly-adenylation sequences.
The NSP2 and NSP5 proteins are involved with packaging of RNA (430). The NSP2 protein has non-sequence specific affinity for nucleotides and single-stranded RNA
(ssRNA) (492). It forms an octamer that undergoes a conformational shift when bound to ssRNA (492) which is a typical feature of packaging proteins. The NSP5 protein undergoes phosphorylation which has been proven indispensable for virus replication.
Also, NSP5 has affinity for ssRNA, dsRNA and VP2 but the functionality of these interactions is unknown (49, 542, 564).
The only known virus infecting mammal species that contains 11 dsRNA segments are
RV. The electrophoretic mobility of the RNA segments from group A RV is characterized by 4 high molecular weight segments, 5 intermediate weight segments grouped in 2 larger and 3 smaller segments and 2 low molecular weight segments.
7 Polyacrylamide gel electrophoresis (PAGE) is a useful tool to classify RV, but other assays like Northern blot and sequencing are important complementary tools for the identification of reassortants that are two different virus strains that interchange one or more RNA segments (560). Reassortants were created for the production of the first licensed RV vaccine (RotaShield). Three different reassortants were generated in-vitro, each with a single-gene substitution of the gene that encodes for VP7 and 10 other genes from the parental strain Rhesus RV (RRV). The reassortant strains were created by coinfecting cells in-vitro with RRV and a human RV strain G1, G2 or G4 serotype and by adding antibodies to RRV to the culture to induce selective pressure. This procedure allowed the incorporation of the 4 most common serotypes (G1, G2 and G4 from human origin and G3 from RRV origin) in a single vaccine to achieve a broader antigenic coverage (277).
Rearrangement is a different type of mutation that occurs within the structure of the genomic viral RNA and also can be detected by PAGE. The most common rearrangement occurs by a head to tail duplication immediately downstream from the open reading frame and usually maintains a normal protein product (176). Typically, missing segments or decreased intensity in one of the segments and appearance of new bands with unusual electrophoretic patterns, can be observed when gene rearrengements are present (176). Rearrangements are commonly encountered in viruses recovered from immunodeficient patients, chronically infected children, asymptomatic immunocompetent children and different animals (pigs, calves and birds) (42, 245, 432,
442, 514). The most common rearrangement occurs in segments 5 (encoding for NSP1) and 11 (encoding for NSP6). Rearrangements in gene 11 have been proven to confer to
8 the virus a selective advantage by improving growth and/or increasing viral stability
(362). Virus rearrangement usually does not impair infectivity and these segments have
the capacity to reassort and replace the normal existing genomic RNA of a non-
rearranged virus strain (10). Analysis of RV with rearranged genome sequences aids in
the study of the function of a gene and determining if a gene is essential for virus
replication. For example, it is known that a zinc finger motif encoded by gene 5 is not
necessary for replication, because rearranged viruses lacking this portion of the gene replicate normally (518). Rearrangement has never been observed in any of the RNAs encoding for VP2, VP6, VP7 or VP4 (176).
1.2.5 Functions of RV proteins
The RV genome encodes six structural proteins that form the capsid of the virus and six nonstructural proteins involved in virus replication and present exclusively in infected cells. Six genome segments code for the six structural proteins (VP1, VP2, VP3,
VP4, VP6 and VP7) and five segments encode the remaining six nonstructural proteins
(NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6). Each core protein (VP1, VP2 and VP3)
has affinity for RNA but only VP1 shows sequence specific viral RNA recognition (132).
Several studies indicate that VP1 acts as the virus polymerase serving for both
transcription and replication (107, 132, 426, 429). The function of VP1 depends on the
core protein VP2 that also binds RNA in a nonspecific manner and interacts with dsRNA
from the inside of the core (72, 314). VP2 is the only structural protein that exhibits self-
assembly into icosahedral core particles suggesting that it directs the assembly of the
capsid (259). Viral protein VP3 interacts with the N terminus of VP2, binds GTP
9 covalently and acts as a guanylyl and methyl transferase generating capped mRNA transcripts (108, 176, 330).
The non-structural proteins are involved in RV replication (NSP2, NSP3, NSP5 and NSP6) and morphogenesis (NSP4). Phylogenetic analyses of NSP1 derived from 12 different RV strains revealed clustering according to species origin except for human and porcine strains that were clustered in one group (171). The NSP1 is not essential for replication; it acts like a virulence factor in mice (but not in rabbits or piglets) (84, 125) and it is the least conserved of the proteins being more variable than VP4 or VP7 (413,
517). Also, NSP1 has been linked to a proteasome-dependent degradation of interferon regulatory factor-3 (IRF-3) (34, 439). The IRF-3 is an important regulator of type I IFNs that are crucial in-vivo antiviral cytokines (395). The in-vivo significance of IRF-3 degradation by NSP1 is not known because soon after infection, RV induces high levels of IFNα in serum (170, 304). The NSP2 is an NTPase, it possesses helix destabilizing properties and it may be involved in RNA encapsidation and virulence. Together with
NSP5, NSP2 has been implicated in viroplasm formation, genome replication and packaging. The NSP5 is an O-glycosylated phosphoprotein that self assembles into dimers and has autokinase activity that is enhanced by NSP2 (5, 17, 202). The gene 11 that encodes NSP5 is frequently rearranged (117, 299) and distinct phosphorylation patterns of NSP5 generate different protein forms (6). The NSP3 recognizes viral mRNAs and facilitates its translation using the cellular machinery. Its N-terminal domain binds viral mRNAs and the C-terminal domain binds the elF4G from the cell, directing the viral mRNAs to the cellular ribosomes for protein synthesis (440, 441). The NSP6
10 interacts with NSP5 and it is suggested that it might regulate the self-assembly of NSP5
(524).
The NSP4 gene 10 has been classified into five groups or genotypes (A-E) among
human and animal RV of different P and G serotypes (126). Human RV derived NSP4
genes belong to genotype A and B only and in 100% of the cases, human NSP4 group A
was associated to VP6 subgroup I and human NSP4 group B was associated with VP6
subgroup II (249). Furthermore, animal NSP4 genotypes A and B clustered
independently from human NSP4 genotypes. For example, porcine and equine NSP4
genotype B, clustered in VP6 subgroup II while human NSP4 genotype B clustered in
VP6 subgroup I. Together with the fact that NSP4 genotypes C-E infect animals only,
interspecies transmission can be tracked by the study of the NSP4 genotypes (249) . On
the other hand, animal NSP4 genotypes clustered according to animal species of origin
(126). In a study of a RV outbreak in children from India, a strain isolated from a patient
(G9P[19]) (538) revealed a high homology with a porcine RV for genes encoding VP6,
VP4, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5. The strain was VP6 subgroup I and had an NSP4 genotype B suggesting a porcine origin of these latter two proteins. The
sequence of the VP7 gene was from human origin suggesting that viral reassortment
might have occurred between human and porcine RV strains and demonstrating human
susceptibility to porcine RV strains.
The NSP4 plays a role in virus morphogenesis and has enterotoxigenic properties
(29). Its C-terminal cytoplasmic domain acts like an intracellular receptor on the
endoplasmic reticular (ER) membrane (273). The NSP4 binds unassembled VP6 and
directs it to the ER for double-layered particle (DLP) formation (21, 334). The VP4 and
11 VP7 form a complex with NSP4 in the ER before the formation of triple-layered particles
(TLPs) (510). The importance of these interactions is unclear because NSP4 is not
essential for DLP or TLP formation. Co-infection of insect cells with recombinant
baculoviruses expressing VP2, VP4, VP6 and VP7 results in the formation of TLPs and
double-layered particles (DLPs) and NSP4 is not required for the self-assembly of these
proteins.
The NSP4 also functions as an age-dependent enterotoxigenic agent in mice (29,
30). Mouse crypt cells and human cell lines in contact with NSP4, mobilize Ca2+ and secrete Cl- which can be a possible mechanism for RV-induced secretory diarrhea in pup mice and, possibly in human neonates (30). Antibodies to NSP4 are induced after infection in different animal models and humans and the NSP4 antibodies reduced diarrhea in mice, but were not protective in pigs (29, 588).
The most antigenic structural RV protein is VP6 followed by VP4 and VP7. The inner capsid protein VP6 plays an important role in maintaining the structure of the capsid by
interacting with VP7, VP4 and VP2. Most of the RV-specific antibodies induced after
natural infection and immunization are directed against VP6 (269, 512); thus, it is
considered the most immunogenic rotavirus protein. Passive immunization of severe
combined immunodeficiency (SCID) mice with non-neutralizing VP6 antibodies results
in reduced virus shedding (90). Furthermore, adult mice and rabbits inoculated with 2/6
virus-like particles (VLP) are protected against RV reinfection. The mechanism of
protection by VP6 induced antibodies is probably related to inhibition of replication by
binding of VP6 antibodies to intracellular core DLPs during enterocyte transcytosis after
surface engagement of the polymeric immunoglobulin (Ig) receptor (pIg) (123, 403, 493).
12 On the other hand, neonatal gnotobiotic pigs inoculated with 2/6VLPs are not protected
from RV infection or disease (586). Additionally, neonatal mice vaccinated with VP6
(with E.Coli labile toxin as adjuvant) or passively immunized with IgA antibodies to VP6
are not protected against virus challenge whereas adult mice get protected with passive
immunization of VP6 antibodies (90, 463, 537). The reason of this discrepancy is
unknown, but it may be related to the immaturity of the neonatal immune system or to the
inability of VP6 antibodies to prevent RV diarrhea in neonatal mice and pigs. Although
high levels of VP6 antibodies are generated after immunization of neonates with VP6 (or
2/6VLPs), the antibodies are apparently insufficient to prevent virus replication. Another
possibility is that the immaturity of innate immunity in neonates facilitates virus
replication whereas the more mature innate immune system of adults controls virus
replication more efficiently. Together with the generation of high titer VP6-specific
antibodies and relatively lower RV doses compared to those inoculated into neonates,
more complete control of RV infection can be achieved in adults (535, 536). The section
on RV induced immunity in various animal species will present a more detailed review of
the information available on RV induced humoral and cellular responses and their role in protection.
The genomic RNA segment 4 encodes VP4, a non-glycosylated protein that forms spikes arranged in trimers in the outermost part of the capsid (168, 176). The VP4 is a hemagglutinin (194) and mediates attachment of the virus to the cell surface (168).
Trypsin cleaves VP4 (arginine 231, 241 and 247) and leads to the formation of VP5* and
VP8* that enhances virus infectivity (19, 148). Recently, the interaction between the cell membrane and two different domains in VP5* has been documented (213). Also, VP5*
13 has been shown to induce the formation of pores in cell membranes (169) but the
mechanism of pore formation has not been defined. However, it has been described that
VP5* adopts dimer and trimer conformations that are highly stable and resemble those of
class-II enveloped virus fusion proteins such as Semliki forest virus and dengue fever
virus envelope glycoproteins, suggesting that VP5* might be directly involved in the
formation of the cell pores (583). Trypsin cleavage does not alter virus binding to the
cellular membrane but it is indispensable for virus entry into the cytoplasm of the cell
probably by exposing VP5* (583) and by uncoating the virus particle (183, 209, 465).
After RV infection, VP4 antibodies that block virus cell entry (neutralizing antibodies)
are induced. Passive immunization of mice with monoclonal neutralizing antibodies to
VP4 confers protection against RV disease after challenge with a virulent strain (463).
Protective immunity was induced in mice after VP4 inoculation (338) and VP4 has been
shown to be immunogenic in children (558).
The outer capsid protein VP7, is a glycoprotein arranged in trimers that depend on
Ca++ for their formation (166). There is a functional interaction between VP7 and VP4 that may cooperate to allow RV entry into the host cells (210). After RV infection, VP7 neutralizing antibodies are induced and VP7 specific CD8+ T lymphocytes are generated in mice (232, 255). Recently, it has been described that VP7 induces nonspecific massive
B cell proliferation in mice (64). Rabbits and mice immunized with 2/6/7VLP are protected from reinfection (136); however, studies in the gnotobiotic pig model and in
humans are lacking.
14
1.3 REPLICATION CYCLE
1.3.1 Replication overview
Replication of RV takes place in mature small intestinal enterocytes in-vivo but most of the replication studies have been performed in-vitro using monkey kidney cells
(MA104 cells) as target cells (176). Early interactions of RV with the cell membrane include viral attachment, entry and virus uncoating (333) which is necessary for subsequent transcription and replication (202). Subsequently mRNAs are generated for the synthesis of viral proteins using ribosomes from the host cell (416). After protein synthesis, virion assembly takes place which also involves the synthesis and packaging of dsRNA using the viral RdRp (110). After the virus particles mature, they bud from the infected cell by using lipid rafts, avoiding direct cell lysis (153) or by direct cell disruption (390). Virus replication reaches a peak at 18 hrs post-inoculation (PI) in
MA104 (128, 365) and 20-24hr PI in polarized human intestinal cells (CaCo-2 cells)
(176, 272).
1.3.2 Adsorption, penetration and uncoating
The initial stages of RV replication have been studied by biochemical techniques and electron microscopy. Triple layered particles containing VP7 and VP4 are the only particles capable of generating productive infections. Virus attachment occurs after cleavage of VP4 and exposure of VP5* and VP8* (594) but cleavage of VP4 is not necessary for virus attachment (147). The RV entry into the cells is a multistep process
15 (333) that might require both VP4 and VP7. Various studies suggest that RV utilizes
multiple cell receptors to gain entry into the cytoplasm of the cell. Studies using RRV
have shown that the virus binds initially a sialic acid (SA) containing molecule (probably a ganglioside) and then to the α2β1 integrin (594). Studies using RRV and a neuroaminidase (NA)-resistant (nar3) mutant isolated from a NA-sensitive RRV strain, described that after α2β1 binding, both viruses sequentially bind αvβ3 and αxβ2 (219).
Antibodies directed to αvβ3 or αxβ2 blocked the entry of RRV, SA11 and Wa RV but not their cell attachment (219). These findings suggest that virus entry is a sequential process where after receptor binding, the virus finally gains access to the cytoplasm of the cell in a sequential and organized manner. It also has been demonstrated that RRV, nar3 and Wa RV bind the heat-shock protein 70 (hsp70) and blocking of the hsp70 prevented infection but not cell attachment (226).
Virus cell entry mechanisms can vary among strains. The RRV strain is SA dependent
whereas the mutant RRV nar3 is an SA-independent strain. Furthermore, RRV infects
through the apical portion of the cell whereas nar3 uses both apical and basolateral cell
membranes (373).
The current model for RV cell entry describes that after VP4 cleavage, VP8* binds the SA containing molecule inducing a conformational change in the structure of
VP4. This conformational change presumably aids in the subsequent binding step of
VP5* to the α2β1 integrin. Interactions are proposed to occur between VP7 and αvβ3 or
αxβ2 and VP5* and the hsp70. It is unknown if this is a sequential or an alternative process (333). Furthermore, it is not completely understood how binding to the different molecules results in virus uncoating and entry into the cytoplasm. 16 Binding of the virus to the receptors on the cell surface is followed by internalization of
the virus particle into the cytoplasm. Virus entry induces permeabilization of the cell
membrane. Using release of 51Cr as a measure of cell entry, only trypsin activated viruses
(RRV) and not anti-VP4 treated particles or double layered virus cores induced 51C
release (276). Based on these findings, it is thought that RV entry occurs by direct viral
penetration. Trypsin cleavage has proven to be indispensable for direct penetration of RV
whereas lack of trypsin leads to receptor-mediated phagocytosis which involves
formation of phagosomes via endocytosis (511). Furthermore, cholesterol depletion
inhibits RV cell entry (153) suggesting that RV uses lipid rafts to gain access to the
cytoplasm. Trypsin activated particles gained access to the cytoplasm in 3 to 5 min
whereas non-activated particles that entered the cell by phagocytosis took 30 to 50 min
and seemed unable to replicate. The process of virus entry into the cell cytoplasm is
usually completed after 60 to 90 min (285)
It has also been described that by bafilomycin inhibition of H+-ATPase bombs
that have a role in the formation of the endocytic compartment, RV (OSU strain) entry
was prevented suggesting that endocytosis, which is an ATP-dependant mechanism, may
be a route used by the virus to enter the target cells (106). Furthermore, virus
internalization is an active cellular process because incubation of the cells at 0° to 4°C inhibits virus entry. Because there is evidence for both direct penetration and endocytosis as mechanisms used by RV to enter target cells, it is possible that the virus exploits both routes to achieve a more efficient infection (467).
Once the virus gains entry into the cytoplasm, decapsidation of the outer-most protein layer is necessary for initiation of transcription of the virus genome (259).
17 Calcium chelation by EDTA treatment induces dissociation of VP7 possibly because
trimerization of VP7 has been shown to depend on [Ca++] concentrations and [Ca++] binding sites have been described within the structure of VP7 (165, 166). Also, solubilization of the whole capsid was achieved by diminishing the concentration of
[Ca++] (335, 466). These observations suggest that VP7 is directly involved in the process of decapsidation. Studies that support endocytosis as a mechanism of virus entry, suggest that the low [Ca++] concentration inside the endosomes will induce the solubilization of
the outer capsid. The decrease in [Ca++] is thought to change the conformation of the capsid (impeding trimerization of VP7) leading to solubilization of the outer capsid but maintaining intact DLPs (465). On the other hand, studies that propose direct penetration as a mechanism of entry contradict the theory of low [Ca++] induced decapsidation
because once the cell is permeabilized, the high extracellular [Ca++] will enter the
cytoplasm preventing decapsidation (467). However, solubilization of the outer capsid
can be independent of the concentration of [Ca++] (151). The hypothesis that endocytosis
and direct permeabilization are both possible mechanisms used by the virus to gain entry
into the cytoplasm is supported by the fact that solubilization of the capsid can be [Ca++] dependent or independent.
1.3.3 Transcription and replication
Once cell penetration is achieved, viral decapsidation will occur and RV transcription will take place in double layered (2/6) subviral core particles (428). It is suggested that TLPs are transcriptionally incompetent possibly because elongation and translocation of the transcripts are inhibited by the outermost capsid (319). Initiation of
18 transcription is dependent on VP6 despite its inherent lack of enzymatic activity (259).
The RV cores composed of VP2 gain transcriptional activity only when VP6 is
incorporated (298). The genome of the virus is packed in close association to VP2
pentamers and VP1, the viral RdRp. The RNA capping protein VP3, a methyltransferase
and guanylyltransferase, is also located in the core (108). Crystallographic and
cryoelectron microscopic analyses have indicated that genomic RNA is highly organized
within the core forming a concentric structure (445). Following virus entry, capsid
solubilization activates the RdRp and capped mRNAs are translocated into the cell
cytoplasm by extrusion through type I channels (445). There are twelve pentamers of
VP2 each associated with an RdRp, and it is thought that each polymerase is in charge of transcribing one RNA segment. Evidence for this observation is supported by the fact that genomic RNAs are produced in equimolar amounts during replication whereas viral mRNAs are not (425). However, this model raises the question as to how the 11 RNA segments are correctly brought together and a possible mechanism suggested is that
RNA-RNA interactions coordinate the packaging process (436) .
The amount of mRNA generated by the polymerase is high, an efficiency
explained by the fact that the polymerase is capable of holding the 5’ and 3’ ends of the
segments, making them operate like circular templates. The role of the core lattice (VP1,
VP2, VP3 and VP6 associated to viral RNAs) in transcription is unknown, but the affinity
of VP2 for RNA and the RdRp suggests that the core might function as a platform that
facilitates dsRNA melting (314, 445).
Once the mRNAs are synthesized, NSP3 specifically recognizes the virus mRNAs
and facilitates their translation by transporting them to the cellular machinery for protein
19 synthesis. The NSP3 is a functional homologue of the cellular poly(A) binding protein
(PABP). The N terminus interacts with the 3’-consensus sequence of the rotaviral
mRNAs and the C terminus interacts with the cellular elF4G to direct the viral mRNAs to
the cellular ribosomes. The binding of NSP3 to the mRNAs has been proposed as a
possible mechanism of transport of the newly synthesized mRNAs to the viroplasm for subsequent replication (436).
Replication, genome packaging and particle assembly occur in a perinuclear, electron-dense structure known as the viroplasm which appears 2-3 hrs PI. It is suggested that the viroplasm is formed by congregations of NSP2 and NSP5, because expression of
NSP2 and NSP5 in uninfected cells leads to the formation of viroplasm-like structures
(179). In vivo studies show that both proteins together with VP1, are the main components of the replication intermediates (519). The NSP2 has NTPase activity, ssRNA binding affinity and helix destabilizing properties and it has been suggested that the energy derived from the NTP hydrolysis will facilitate genome packaging. The NSP5 binds to the same site of recognition of RNA in the NSP2 structure and it has been suggested that NSP5 might regulate the RNA binding to NSP2 during the packaging of the genome (264). Also NSP5 has affinity for ssRNA, dsRNA and VP2 and its phosphorylation is necessary for virus replication (49, 542, 564).
In the infected cells, mRNAs serve as templates for protein synthesis in the ribosomes of the cell and negative-strand RNA synthesis in the viroplasms for the formation of genomic dsRNA. A 3’ consensus sequence, a cis-acting signal recognized by the viral polymerase, has been proven essential for the synthesis of dsRNA (426). A similar sequence is also present in the 5’-untranslated region (UTR) and open reading
20 frame (ORF). From computer analyses it has been proposed that the 3’ and the 5’ ends of
the RNA segments make a complementary base pairing that is stabilized by the critical
regions in the ORF and regulate the synthesis of dsRNA by creating a pan handle
structure that is pivotal in the process of replication. Each of the mRNAs adopt a particular secondary structure that involves the formation of a stem loop that differs among each transcript. The proposed hypothesis is that the secondary structure created by the panhandle is the recognition signal of each of the different transcripts for packaging into the core (109). Additionally, RV mRNA contains 5’ and 3’-UTRs of variable lengths. All viruses from the Reoviridae family have highly conserved sequences located at the 5’ and 3’ terminus of the genomic RNA also known as 5’- and 3’-consensus sequences. These highly conserved sequences differ for each dsRNA segment. Mutations that spare these consensus sequences do not abolish packaging of the RNA segment suggesting that a possible mechanism of packaging operates by the recognition of unique codes in the terminal consensus sequences (162).
Free dsRNA is usually not observed in the cytoplasm and is always associated with core-like structures (427). It is thought that packaging of the templates precedes genomic RNA synthesis and that functional signals for packaging reside in the mRNAs and not in the genomic dsRNA. Both, structural and nonstructural proteins are involved in packaging and mRNA replication. As previously described, the polymerase depends on the core lattice to be fully active (429). Other proteins that compose the replicase complex are the RdRp, small amounts of VP6, large amounts of NSP3 and lesser amounts of NSP1, NSP2 and NSP5 (233). The exact composition of the replicase complex is still unclear because of the difficulty in separating the individual proteins
21 involved in transcription from proteins involved in replication and the inability to avoid contamination. By the use of recombinant baculovirus technology this question will probably be resolved. The exact site of transcription has not been identified but it is believed to be the viroplasm. As previously stated, mRNA synthesis occurs inside the
DLPs in an ATP dependent manner (probably used for initiation and/or elongation of
RNA). Synthesis of RNA has been studied in SA11-infected cells, cell free systems and electrophoretic assays that allow separation of positive and negative RNA strands. Both
RNA species can be detected by 3 hrs PI and by 9 to 12 hrs PI, the rate of synthesis increases considerably reaching a peak for positive RNA synthesis (502). About 20% of the RNA synthesized in-vitro is double-stranded and the rest is single-stranded. The synthesis of dsRNA is an asymmetric process in which positive sense RNA acts as a template for the synthesis of negative strand RNA (427).
1.3.4 RNA encapsidation and virion assembly
Following RNA transcription and protein production, DLP assembly and synthesis of genomic dsRNA take place. The newly formed DLPs are transported to the
ER and the particles mature to TLPs. It is unclear how the 11 different dsRNA segments are selectively introduced inside the virus capsid. The actual working model for RV encapsidation is that RNA packaging occurs at the same time as the capsid is being assembled (435), and each RNA segment is attached to a transcription enzyme complex.
It is unlikely that packaging takes place after assembly of the capsid because organization of the RNA segments and attachment to the core lattice complex are more difficult to achieve if the core is assembled.
22 RNA encapsidation of reoviridae family viruses in general is not understood and there are three current models to explain it (431):
Precore precursor model: In this model, assortment of mRNA occurs before the formation of the core. This model was based on the observation of precore replication intermediates composed of mRNA, RdRp and capping enzymes free of core proteins inside RV infected cells. The polymerase and the capping enzyme could serve as platforms that support the formation of the VP2 core. The association of viral mRNAs to
the core particles increases their degradation by cytosolic RNAases and for this reason it
is thought that the mRNA is not inside the core prior to the onset of the dsRNA synthesis.
Following the formation of the core, the mRNA will be used as a template for the
formation of genomic dsRNA. Then translocation of the dsRNA by the polymerase and
other nonstructural proteins will take place for subsequent packaging and organization of
the dsRNA within the core (431).
RNA-pentamer precursor model. There are 12 pentameric core proteins and each pentamer is associated with an RdRp and a capping enzyme. This organization suggests that there is a functional arrangement of the core. It is proposed that a polymerase, a capping enzyme and an mRNA create a complex that serves as a nucleation site for the congregation of 10 core molecules that will create a pentamer. Specific RNA-RNA interactions will drive the formation of the core. The formation of the core and secondary changes in its structure will activate the RdRp for initiation of the synthesis of the negative RNA strand for the formation of genomic dsRNA.
Empty core precursor model. This model is based on the actual encapsidation mechanism of the φ6 phage. Coexpression of recombinant structural proteins from the
23 phage, lead to the formation of empty cores with their polymerases integrated to the core.
The φ6 phage is composed of three RNAs, a small, a medium and a large segment. In the
φ6 phage model when incubating the empty cores with the three mRNAs, the small
segment is first packaged followed by the medium segment and then the large RNA
segment (450). It is though that initially, the core displays a small RNA specific
attachment site that disappears when the small segment is packaged to allow the
formation of a medium RNA attachment site. After packaging of the medium segment,
the medium attachment site disappears and the large attachment site appears to complete
the packaging of the large RNA segment. Complete packaging of the mRNAs induces
changes in the core that activate the RdRp for synthesis of genomic dsRNA (533). On the
other hand, RV synthesizes dsRNA in the absence of complete mRNA packaging (595)
and there is sufficient evidence that argues against any similarity between φ6 and RV
packaging mechanisms making this model the least similar to the proposed mechanism of
RV RNA packaging.
1.3.5 Virus release
After penetration and replication, RV is released by cell lysis in non-polarized
cells (14). However, other pathways for virus release also have been described. Before cell lysis, RV is released apically in polarized human intestinal Caco-2 cells (272). Also, studies of RV release and assembly demonstrated that VP4 (but not VP6 or VP7) is associated with lipid rafts and can reach the plasma membrane 3 hrs PI (482).
Association of RV with lipid rafts has been characterized in Caco-2 cells infected with
RRV and in mice infected with an homologous RV strain (153). The importance of this 24 finding is that RV uses the machinery of the cell to reach the intestinal lumen without
destroying the infected cell (152). In this way possibly more particles can be synthesized
and released and more epithelial cells can be targeted.
1.4 ROTAVIRUS PATHOGENESIS AND PATHOLOGY
Diarrhea caused by RV induces several pathological changes that are usually extensive in neonatal animals but limited in adult animals. The intestinal epithelium from infected neonatal animals shows shortening and atrophy of intestinal villi, lamina propria
(LP) infiltration with mononuclear cells, distended cisternae of the ER, mitochondrial swelling, denudation of the microvilli and irregular microvilli (277). Virus particles were visualized in phagocytes and goblet cells at the LP; the ER cisternae and lysosomes in epithelial cells. Impaired absorption of d-xylose (364), abnormal gastric motor function
(delayed gastric emptying of a liquid meal) (32) and reduced levels of disacharidases (55,
277) have been also described.
Different animal models have been used to study human RV pathogenesis (mice, rats, rabbits, calves, lambs and gnotobiotic pigs). Neonatal pigs (470), calves (472), rats
(149) and dogs (271) developed diarrhea after infection whereas adults developed infection but not diarrhea (122, 300). For this reason, histopathological studies after RV infection were mainly performed in neonatal animals. Colostrum deprived neonatal gnotobiotic calves and pigs were infected with human RV and intestinal lesions caused by the virus were studied (371, 552). Gnotobiotic calves showed denudation of the upper small intestinal villi and flattening of epithelial cells 30 min PI. Similar lessions were observed in the lower intestine at 7 hrs PI, but at this time, RV antigen was not observed.
25 At 40 hrs after infection the epithelium recovered. Gnotobiotic pigs inoculated with
virulent human RV showed lesions and diarrhea at 13 hrs PI. As in the calves, most
lesions were located in the upper small intestine and no lesions were detected in the ileum
at earlier PI hrs. At PI hr 24, lesions were widespread throughout the small intestine and
lesions were detected up to 96 hrs. Normal morphology was detected at PI day (PID) 7.
Earlier characteristic lesions (24 PI hour) included detachment of epithelial cells, lymphoreticular hyperplasia and cell vacuolation which coincided with the peak of RV antigen detected by immunohistochemistry. Later findings (48-96 PI hrs) were crypt hyperplasia and villous atrophy. Duodenal biopsies of children with RV infection did not always show morphological anomalies (193, 297). From 40 children studied during the acute phase of infection, only two showed morphological anomalies. This finding is not
surprising because in pigs and calves, duodenal villous atrophy only occurs late post-
infection (472, 552). Similar lesions like those observed in gnotobiotic calves and piglets after human RV infection, were also described in children (55). Importantly, malnourished piglets and mice developed more severe diarrhea and mucosal damage.
Similarly, malnourished children had a higher mortality rate after an episode of RV induced diarrhea compared to healthy children (182, 221, 454, 530, 599).
The mechanism of RV diarrhea is probably multifactorial. At early stages of the
disease, where a minimal change in cell morphology is observed, animals and humans
may experience severe diarrhea (336, 552). These early observations suggested that RV
diarrhea was caused by different mechanisms other than enterocyte death and ensuing
malabsorption. In adults, it has been shown that RV diarrhea is associated with
malabsorption due to decreased levels of disaccharidases and loss of absorptive cells after
26 atrophy of the enterocytes (415). Additionally, RV infection stimulates the enteric
nervous system (336) which induces fluid secretion to the intestinal lumen causing
diarrhea. The non-structural protein NSP4 and its synthetic peptide NSP4114-135 have been shown to cause an age dependent watery diarrhea in neonatal mice. The non- structural protein induces Cl- secretion and inhibits the Na+-D glucose (SGLT1) and Na+-
L-leucine symporter activity, increasing the osmolarity at the intestinal lumen and preventing water absorption (by the SGLT1 symporter) which leads to watery diarrhea
(29, 229). It is known that NSP4 has a microtubule binding domain. The ER is composed of microtubules that help in the transport of proteins to the Golgi and then to the extracellular milieu. The NSP4 is capable of binding the ER microtubules, blocking protein transport to the Golgi (578). Researchers suggest that blocking of the extracellular transport will possibly prevent digestive enzymes (e.g disaccharidases, maltases and lactases) from reaching the intestinal brush border, inducing malabsorption and contributing to the enterotoxigenic effect of NSP4 (578).
Recently, systemic invasion after RV infection has been described in different animal models and humans (23, 63, 113, 149, 328). Mice, rabbits, calves, pigs and children with acute RV infection had RV antigen in the serum. From 33 samples of RV positive children, 22 had RV antigen in serum. If a high amount of RV antigen was observed in feces there was a higher possibility of detecting RV in serum. There was an inverse correlation with the amount of RV antigen positive serum samples and the days after initiation of disease. From six RV antigen positive serum samples, 3 also had RV
RNA suggesting the presence of replicating virus (63). Adults and mouse pups infected with homologous or heterologous RV had antigen in serum. Serum from infected mice
27 induced RV infection in naïve recipients suggesting that the circulating RV was
infectious (62). Gnotobiotic pigs developed RV viremia after infection with human RV.
Sera from positive pigs was infectious and intravenous RV inoculation induced intestinal
RV infection suggesting that virus in the blood was able to transit to and invade the intestinal epithelium (23). Further evidence suggesting that circulating RV is able to invade the intestinal epithelium is based on in-vitro and in-vivo studies: (i) Caco-2 cells can be infected via the basolateral membrane (124). (ii) Mice inoculated with RRV by
subcutaneous or intraperitoneal routes developed intestinal infection (386). Recently,
mice and rats infected with heterologous RRV and human RV or homologous RV
showed extraintestinal replication of virus (149, 187). Rat pups infected with RRV
apparently had replicating virus in the liver, kidneys lungs, spleen, pancreas, bladder and
thymus. After isolation of the tissues, the organs were flushed and macerated for ELISA
and fluorescent focus assay for antigen and replicating virus detection, respectively. The
authors described lesions in the parenchyma of the lung and liver suggesting virus
replication. Furthermore, mice infected with homologous RV or heterologous RRV had
viral RNA in the MLN, liver, lungs and kidneys. The RNA and TLPs were found inside
the cells. Mice infected with heterologous or homologous RV had similar quantities of
RNA in the MLN but homologous RV infection generated 1000 times more RNA in the
gut compared to RRV infection suggesting that RRV has a tendency for extraintestinal
rather than intestinal dissemination. Additionally, high virus titers were observed in the
MLN, and NSP4 was detected by confocal microscopy inside DCs, B cells and
macrophages located at the MLN. The fact that NSP4 was observed inside DCs suggested
virus replication or endocytosis of infected epithelial cells by the DCs and subsequent
28 migration to the MLN. It has been suggested that NSP4 is a structural protein meaning that its presence inside cells does not necessarily indicate virus replication (146). Further studies that use different markers for virus replication in DCs, macrophages and B cells and differentiation of virus replication from epithelial cell uptake need to be performed to clarify this finding.
Interestingly, mice infected with RRV developed biliary atresia (303, 337).
Idiopathic biliary atresia causes cirrhosis and is the main cause of liver transplantation in children (337). The cause of idiopathic biliary atresia is unknown but is thought to be secondary to an autoimmune response to the biliary duct induced after a viral infection.
Mice infected with RRV develop biliary atresia and the transfer of the T cells to naive
SCID mice, induces biliary duct inflammation suggesting a role of virus-specific T cells in the generation of the biliary atresia possibly by recognition of biliary duct antigens by cross-reactive RRV-specific T cells.
Infection by RV has been suggested to trigger the development of autoimmune diabetes mellitus type 1a (145, 241, 242). Some studies postulate that due to epitope mimicry, rotavirus infection stimulates the generation of cross-reactive lymphocytes that recognize epitopes expressed by the pancreatic islet cells. Furthermore, there is evidence of RRV replication in primary pancreatic islet cells from non-obese diabetic mice, diabetes-resistant mice, fetal piglets and macaque monkeys (144). However, a direct association between RV infection and diabetes mellitus type 1a has not always been observed (60, 350) and pathogenesis studies characterizing the secondary lesions and physiological abnormalities caused by virus replication in the pancreatic cells are needed.
29 Possibly, other diseases are triggered by RV due to its extraintestinal dissemination and
further studies are needed to explore RV systemic tissue invasion lesions and pathology.
1.5 MUCOSAL IMMUNITY
1.5.1 Components, inductive and effector sites of mucosal immunity
The mucosal immune system mediates the symbiotic relationship between the
host and commensal bacteria and is the first line of defense against invading pathogens.
The innate and acquired immunity components distributed throughout the mucosal surfaces maintain an immunological homeostasis along the epithelial surfaces in the
gastrointestinal (GI), respiratory and genitourinary (GU) tracts (294).
Antigen specific responses are initiated by the interaction of antigens with the
microfold cells (M cells) located in the epithelium overlying the lymphoid follicles of the
mucosa-associated lymphoid tissues (MALT) (397). The MALT contains the
immunological cells required for the generation of antigen-specific immune responses (T,
B lymphocytes and APCs). The Peyer’s patches (PP) in the gut and the nasopharynx-
associated lymphoid tissue (NALT), constitute part of the MALT and mucosal immune
responses are induced in this sites after ingestion or inhalation of antigens, respectively so
they are known as inductive sites. The common mucosal immune system (CMIS),
connects the inductive sites with effector sites where activated antigen-specific T and B
cells reside and actively prevent tissue invasion by pathogens (294). Effector sites are the
LP in the GI tract, the respiratory epithelium in the respiratory tract and glandular tissues
such as the mammary glands, secretory glands in the lower GU tract, respiratory and GI
30 tract. Communication within the CMIS occurs when local lymphocytes induced in mucosal inductive sites, home to mucosal effector sites that may differ from their tissue of origin. This concept will be explained in detail in the section on homing mechanisms.
The T cells (CD4+ T helper lymphocytes) stimulated in the inductive sites mediate the
activation of cytotoxic lymphocytes (CD8+ T lymphocytes) by the secretion of cytokines
such as IFNγ and IL-12 also known as Th1 type immunity or mediate the induction of B
cell responses by cytokines such as IL-4, IL-5 and IL-6, also designated as Th2 type
immunity (80).
The GI tract is initiated within the oral cavity which has a very distinct lymphoid organization. The oral mucosa resembles the epithelial organization of the skin, having a stratified squamous cell epithelium and underlying connective tissue (26). The normal epithelium has tight intercellular junctions that prevent pathogen invasion. Additionally, a number of enzymes present within the epithelium and the saliva serve as a first line of defense (26). There is a especial type of APC, also observed in the skin, known as
Langerhan’s cells. These cells capture antigens, then migrate to the draining lymph nodes and initiate an immune response (26). Deeper into the structure of the GI tract, the small and large intestinal epithelium becomes more complex and there is a frequent association with different types of organized lymphoid structures known as secondary lymphoid tissues that include the PP, isolated lymphoid follicles (iLF), the cryopatches (CP) and the mesenteric lymphoid tissues. The structure of the PP, which are the most studied and essential secondary lymphoid tissues in the gut, will be explained in detail in the following section. The iLF are mononuclear cell aggregates resembling the PP or lymph node (LN) structures located throughout the small intestine and the colon. They contain
31 mainly B cells (70%), CD4+ and C8+ T cells and dendritic cells (231) and are inductive
sites of mucosal immune responses containing M cells and similar structures to the PP.
The CP are loosely organized clusters of cells at the base of the intestinal epithelial crypts
and they are distributed mainly in the small intestine and less abundantly in the colon.
There are no CP in the stomach. They contain stromal cells and different types of DCs,
but the population of mature T or B lymphocytes is scarce (<2%). The most abundant cell
type in the CP is the adult counterpart of a fetal lymphoid tissue inducer cell (lineage–,
IL-7 receptor+, c-kit+) responsible for the generation of signals for the formation of secondary lymphoid tissues (398). Some studies suggest that CP are the site of generation of intraepithelial lymphocytes (IEL) (474). The MLN are the largest LN in the body and are the first to develop during embryogenesis. The lymphocytes and antigen bearing DCs that are primed in the PP migrate via lymphatic vessels to the MLN. A few hrs after oral inoculation, antigen recognition occurs in the MLN and even commensal bacteria can be recovered from the MLN under normal conditions (344). Furthermore, mice that lack
MLN, present bacteremia and commensal bacteria in the spleen, suggesting one of the roles of the MLN is to contain commensals within the gut (345). Also, MLN have an important role in the induction of oral tolerance (explained in the section 1.5.5) and appear to prime for Th2 responses by the generation of IL-4 and IL-10 under non- inflammatory conditions as well as to initiate Th1 responses during intestinal inflammation (13, 58).
The respiratory tract is composed of upper respiratory MALT (or aerodigestive tract) constituted by the palatine tonsils and the nasopharyngeal tonsils or adenoids. The adenoids and tonsils in pigs and humans constitute what is known as the Waldeyer’s ring
32 which plays an important role in the mucosal immunity induced in human and porcine
airways (69). On the other hand, rodents lack tonsils and their NALT is located laterally
to the nasopharyngeal duct and dorsal to the soft palate (311). Children under 2 years of
age present a similar structure in the middle concha in the nasal cavity (160). The lower
respiratory MALT is composed of the bronchus-associated lymphoid tissue (BALT) and
usually develops after birth (526). The NALT consists of follicle-associated epithelium
(FAE), high endothelial venules (HEV), T- B lymphocyte rich areas and DCs. The M
cells present in the PP are also present in the NALT containing all the necessary
components to be an inductive site for the generation of immune responses to airborne
antigens.
The GU tract exhibits features that are unique from other mucosal compartments.
The secretions of the GU tract are mainly composed of IgG whereas the GI , respiratory
tract, colostrum, milk and saliva mainly has IgA of local origin. Weak humoral responses
are induced in the vagina and cervix. These tissues lack secondary lymphoid tissues like
those observed in the gut (e.g. PP), so the lower GU tract relies on the draining LN and
the local innate immune cells to prevent or actively respond to invasive pathogens.
However, ascending infections to the uterus induce strong humoral responses suggesting that active immunity is induced in the upper genital tract (376). The male GU tract
responses have been less studied, but it is known there is a poor induction of humoral responses in the male genitalia. Furthermore, the testis are protected by the blood- testicular barrier at the capillary level making them immune-privileged and a site where tolerogenic responses are the rule; however, local infections can be cleared effectively
(376). The mucosal immune system distribution in the female genital tract is especially
33 controlled by hormones. Hormones control the transport of Ig, the secretion of cytokines
and the cell distribution throughout the GU tract. In addition, the immune cells that reside in the GU tract are influenced by physiological events such as parturition, fertilization, implantation and pregnancy. Also sex hormones influence the commensal flora that colonize the GU tract. The female reproductive tract is rich in cells of the innate immune system (DCs, neutrophils, natural killer cells, epithelial cells) and adaptive immunity is initiated in the regional draining LN (inductor sites) and activated lymphocytes travel to effector sites such as the GU tract epithelium and the secretor glands embedded
throughout the tissue to prevent pathogen invasion (571).
1.5.2 Intestinal Mucosal Immunity
Intestinal mucosal immunity has been studied most extensively in humans and
mice and the most recent and important findings described in the literature will be
summarized in this section. The pig mucosal structures will also be described and
compared to the human structures. Lymphoid aggregates embedded in the small and large
intestinal wall are known as gut-associated lymphoid tissue (GALT). The GALT is
composed of at least 5 to 20 lymphoid follicles (138) and has long been considered a
central lymphoid associated tissue due to its mass, its substantial exposure to antigens
(e.g. commensal flora, food) and its important influence on the responses induced in the
systemic immune compartment. To understand the principles of mucosal immunity, it is
essential to understand the function of the GALT and its interaction with other lymphoid
tissues throughout the body. These lymphoid aggregates located in the anti-mensenteric
side of the small intestinal wall include the PP and the iLF located in the large intestine.
34 The PP in mice are distributed throughout the small intestinal wall, human PP are
aggregated in the terminal ileum and pigs have discrete PP aggregates in the jejunum and ileum (349, 443). The PP and iLF throughout the small intestine (80) are composed M cells which are part of the follicle-associated epithelium (FAE), an underlying subepithelial dome (SED), multiple B cell follicles which contain several germinal centers (GC) and interfollicular regions (IFR) with HEV and efferent lymphatics. No afferent lymphatics are observed in the PP; therefore, all lymphocytes that reside within their structure enter via HEVs and are derived from the blood and not from the lymphatic system. A similar structure composition has been described for PP in the pig (523).
Activated B and T lymphocytes, after being primed in the PP, migrate to the GI effector sites, the LP or the intestinal epithelium in the large and small intestine. The intestinal epithelium is particularly rich in intraepithelial lymphocytes (IEL) that develop independently from the lymphocytes that originated in the PP, are mainly CD8+ and null
T cells (CD4- CD8-) with a distinctive γδ T cell receptor. The LP has T and B lymphocytes, innate immune cells such as macrophages, DCs, neutrophils, mast cells and other granulocytes. The distinction between inductive and effector sites is not absolute since it is known that some effector functions such as the initiation of adaptive immune responses by activated antigen specific lymphocytes also occur in the PP (286).
35 M cell IFR pocket HEV M cell SED Villi
Centrocytes
Centroblast
Germinal center FDC
.
Fig 1.1 Structure of a PP. This is a typical PP equipped with antigen-sampling M cells,
T cell areas (IFR) and B cell follicles (in this case a secondary B cell follicle is shown with a germinal center, immature centrocytes and more mature centroblasts) and DCs
(e.g. FDC). The switch from IgM expressing B cells to IgA occurs in the PP and the
MLN to then migrate to the LP via lymph and blood. The T cells mainly end up in the epithelium whereas IgA+ B cells differentiate to plasma cells in the LP and secrete dimeric IgA + J chain in the intestinal lumen. Activated B cells can also migrate to the PP and the iLF to differentiate mainly to IgA+ B cells but also to IgG+ and IgM+ expressing
B cells or secreting plasma cells.
HEV-High endothelial venule, IFR-interfollicular region, SED-subepithelial dome, FDC- follicular dendritic cell [(80) http://www.geocities.com/artnscience/peripheral-lt.html]
36 1.5.3 Structure of the Peyer’s patches (PP)
1.5.3.1 M cells
Antigens at the intestinal lumen are transported to the PP via M cells that
constitute part of the FAE overlying the PP at the luminal surface (see Fig.1). Contrary to
intestinal epithelial cells, M cells have poorly developed brush borders, reduced
enzymatic activity, a thin glucocalix and a unique cytoskeleton (206). The M cells are
capable of forming large endocytic vesicles facilitating the transport of whole
microorganisms as well as soluble antigens from the intestinal lumen to the SED. Many
different types of microorganisms or antigens can be transported by the M cells.
Nevertheless, the size [e.g. gold nanoparticles (82)], the hydrophobicity [e.g.
hydrophilic (82)] and the expression of surface lectins [e.g. plant or bacterial lectins (97,
551)] appear to be important characteristics for antigens to be captured and transported by the M cells (155, 260).
Specific-IgA receptors recently have been identified on the surface of M cells
(355). It is well known that commensal flora in human feces and saliva are recognized and bind secretory IgA (sIgA) (532) and possibly these IgA coated bacteria can bind the
IgA receptor expressed on the surface of M cells for later transport to the PP. After binding of the receptors, antigens will be transported to the SED to subsequently reach the IFR. Local DCs will capture and present antigens to T lymphocytes abundant in the
IFR to initiate a specific antigen response restricted to the GALT and MLN (346). The PP closely associated with the M cells will prevent systemic invasion by commensal or pathogenic microorganisms. Nonetheless in some cases pathogenic microorganisms are capable of destroying M cells, gaining direct access to the PP (e.g. Salmonella
37 typhimurium) (154), the MLNs and the general circulation to ultimately reach the spleen
and liver. It is known that in the absence of MLN or when their function is compromised
(immunosuppression, infection etc), commensal bacteria can more easily reach the
systemic compartment, causing splenomegaly in mice (343) and suggesting a role for the
MLN in prevention of the dissemination of commensals outside the gut.
The M cells express MHC class II and are able to secrete IL-1 but their role as
antigen presenting cell (APC) is unlikely because of the lack of association with T cells
and the wide variety of underlying professional APC (9). Multiple types of DCs reside
underneath the FAE whereas in the M cell pockets (located at each side of the FAE)
mainly memory B and T cells are located (57, 186, 581). Consequently due to the
abundant memory lymphocyte populations located in the M cell pockets of the PP, they
represent GC extensions where rapid antigen specific recall responses occur (80). The
most likely candidates that mediate T cell activation in the M cell pockets are long-lived plasma cells that are IgM+ CD27+ and that express MHC class II and up-regulate costimulatory molecules upon antigen encounter (581).
1.5.3.2 Subepithelial dome (SED)
The region located between the FAE and the B cell follicle is known as the SED
[(Fig.1, (80)]. Lymphocytes migrate from the HEV located at the parafollicular sites to the SED guided by reticular fibers (412). Many different types of DCs are located in the
SED and they appear more immature than those located in the IFR suggesting that DCs in the SED possibly migrate to the IFR after antigen uptake (289). It has been demonstrated that DCs located in the SED are able to take up and present antigens to T cells for
38 subsequent activation. Also DCs in the SED express the Mucosal Addressin Cellular
Adhesion Molecule-1 (MadCAM-1) which is the ligand of the intestinal homing marker
α4β7 expressed on the surface of T and B lymphocytes activated in the intestine (513).
Expression of MadCAM-1 on the surface of DCs suggests that there is a possible
interaction between DCs and mucosally primed lymphocytes.
1.5.3.3 Interfollicular region (IFR)
The IFR characteristically has abundant T lymphocytes, mature DCs,
macrophages and scarce B cells. Most T cells are CD4+ with αβ-TCRs and usually located around the HEVs. [Fig.1 ,(80)]. In contrast, CD8+ T cells are less abundant and restricted to a central and narrow portion inside the IFR. The B cell follicles and the IFR overlap in certain areas which are proposed to be important for T-B cell interactions necessary for the formation of GCs (341). Additionally atypical macrophages are also found in the IFR close to the efferent lymphatic vessels. It has been proposed that DCs that recently engulfed antigens from the intestinal lumen will migrate to the SED and later will locate at the IFR with a more mature phenotype (289). Thus the IFR is an important site for the generation of antigen-specific T cell responses (see next section )
1.5.3.4 The Germinal Center (GC) and B cell follicle
The intestinal PP are composed of primary B cell follicles with mainly non- stimulated B cells or secondary follicles with activated B cells. Primary lymphoid tissues have naïve B cells (IgD+, IgM+) and follicular DCs (FDC) that play an important role in antigen presentation to T and B lymphocytes. Presentation of antigen by IFR DCs, also 39 known as interdigitating DC (332, 354), will activate antigen-specific CD4+ T cells that stimulate antigen-specific B2 cells which proliferate and turn primary follicles into secondary follicles that contain GCs. The formation of GCs is pivotal in the generation of memory cells, affinity maturation that enhances the avidity of antigen-specific antibodies and Ig isotype switching. The T cell zone is located in the periphery of the follicles and in close contact with follicular B cells allowing T-B cell interactions which are essential for the generation of long-lived antigen-specific B cells and GCs. Antigen-specific B cells once activated, migrate back to the follicle to proliferate and acquire the phenotype of
IgD+IgM+CD38+ B cells also known as GC “founder” B cells (322, 332). Founder cells
secrete low affinity IgM antibodies that when bound to antigen create the immune
complex coated bodies or iccosomes that will be later deposited on the FDC for
prolonged periods of time aiding in the maintenance of B cell memory (8, 174).
The GC is divided into functional compartments (339, 340). Naïve B cells located in the dark zone of the GC undergo exponential proliferation upon antigen stimulation
(79) and become centroblasts. The somatic hypermutation of the Ig expressed by centroblasts will give rise to IgD-IgM+CD38+ centrocytes with an altered B cell receptor
(BCR) (449). Only centrocytes with affinity for antigens presented by FDC will survive; the rest will undergo apoptosis (354). Centrocytes are able to take up the FDC-associated iccosomes serving as alternative APC. Antigen captured by FDC will be presented via
MHC class II for activation of antigen-specific CD4+T cells that will help in B cell and
CD8 T cell activation (573, 574). A close interaction between B cells and T cells is
necessary for GC formation because by blocking CD40-CD40L ligation, GC
development is abolished (157). Moreover the interaction of CD38+CD27+ GC B cells
40 and CD27L on the T cell surface promotes isotype switching and antibody secreting cell
(ASC) or plasma cell formation (254). However, T independent (TI) antigens are able to stimulate GC formation but responses are sporadic, short-lived and induced B cells had few somatic hypermutations (326). These studies suggest that T-B cell interactions are not necessary for GC formation, but are essential for long-lived responses and efficient B cell affinity maturation.
The origin of FDCs is unknown but their development and clustering is dependent on cytokines such as lymphotoxin β and α (LT-β, LT-α)(520). Mice lacking either LT-α or LT-β, can initiate the formation of GCs in peripheral lymph nodes (PLN) but with less efficient B cell affinity maturation. It was later discovered that LT deficient mice lack secondary FDC and their associated iccosomes. The B lymphocytes are a major source of
LT and it promotes B cell proliferation and expression of adhesion molecules that presumably facilitate migration and localization of FDC (527). In addition, mice deficient in B-lymphocyte stimulator/B-cell-activating factor of the TNF family (BAFF) also have poorly developed GC (451). It was later observed that these mice had short-lived secondary FDC. Because BAFF-/- and LT -/- mice developed GCs and both lack normal secondary FDC with their associated iccosomes, it is suggested that FDC-bound antigen is not required for the induction of GC but it is required to maintain a long-lived GC response. It also has been suggested that FDC-bound antigen is longed-lived and remains within the follicle for the generation and maintenance of memory B cell responses (8).
41 1.5.3.5 Role of chemokines in B cell follicle and GC formation
The B cell attracting chemokine (BCA-1) also known as CXCL13, attracts naïve
B cells in-vitro and has been shown to be induced in human lymph node (LN) follicles
(324, 385). In mice this molecule is known as the B lymphocyte chemo-attractant (BLC)
(399). The BLC (and CXCL13/BCA-1) binds the CXCR5 receptor and they are known to
be directly involved in the formation of murine lymphoid tissue and PP (83).
Furthermore, it has been observed that BLC up-regulates the B cell expression of
LTα1β2, that as previously stated, promotes the development of FDC and the production
of additional BLC creating a positive feed back loop important in the development and
homeostasis of follicles (16). The CXCL13/BCA-1 is expressed in human PP and large
intestinal GALT (94) and CXCR5 is expressed in the follicular mantle zones, surface of
+ + naïve B cells (325) and CD4 T cells located at the follicles also known as CD4 FH T
+ cells (489). Follicular CD4 FH T cells differ from Th1 and Th2 cells (121), and because of
the abundant expression of B cell costimulatory molecules such as CD40L, OX40 and B
stimulatory cytokines such as IL-10, efficient B cell activation is achieved (83, 548).
+ + + Furthermore, CD4 CXCR5 CD57 FH T cells located in the GC, are essential for B cell differentiation and antibody production (291). Immunostaining with FDC markers and antibodies to CXCL13/BCA-1 had suggested that FDCs express CXL13/BCA-1 on their peripheral extensions (94), but the main source of CXCL13/BCA-1 are DCs located in the GCs and large cells that appear to have macrophage derivation (95, 224).
42 1.5.4 Intestinal B lymphocytes and secretory IgA
Mucosal surfaces are particularly rich in B cells secreting mainly IgA in the form
of dimers or larger polymers (pIgA). Holding the dimers and polymers together, the IgA
complexes are associated with a joining peptide known as the J-chain (375). The J-chain
is covalently bound to IgA and IgM but not IgG and facilitates a non-covalent interaction
with the pIgA and the pIg receptor (or membrane secretory component) expressed on the
basolateral membrane of intestinal epithelial cells (81). After binding the dimeric or the
polyIgA, the pIg receptor-IgA complex (secretory IgA, sIgA) will be endocytosed and
transported to the intestinal lumen. Immune exclusion or immune clearance of pathogens
or antigens will take place when intestinal sIgA binds luminal antigens preventing their
entry to epithelial cells or facilitating their degradation by innate cells, respectively (74).
Pentameric IgM can also be transported by the pIg receptor in humans but it varies
according to the animal species (402). The free pIg or secretory component bound to a J-
chain-IgA or IgM complex is particularly resistant to intestinal enzyme degradation,
protecting the Igs in the intestinal environment. The vast antigenic load that resides in the
gut is probably the cause of the abundant sIgA present in the intestinal secretions
(40mg/kg body weight) (135). Furthermore, 80% or more of the human plasma cells
reside in the gut and 80-90% of them secrete IgA (402). The amount of IgM secreting
plasma cells is lower in the upper aerodigestive tract and higher in the lower intestinal
tract (78) whereas only 3-4% of IgG plasma cells are observed in the intestine under
normal conditions and rarely are IgD+ plasma cells present in the gut.
Mucosal plasma cells are characterized by the synthesis of J-chain that is a prerequisite for the production of pIgA or pentameric IgM and their interaction with pIg
43 receptor. Mice lacking J-chains produce only IgA monomers and IgM hexamers that can
not interact with the pIg or its free extracellular portion, the secretory component (268,
370). Mucosal IgA+ plasma cells express higher levels of J-chain than systemic IgA+ plasma cells (56). Interestingly, 80-90% of IgG+ plasma cells synthesize J-chain but it is rapidly degraded (56, 73). Downregulation of J-chain in extrafollicular plasma cells appears to be a sign of maturation, terminal differentiation and later apoptosis.
Lymphocytes located in systemic lymph nodes tend to have reduced J-chain levels,
higher IgG isotype expression and this may be secondary to several rounds of division
after stimulation in the GCs (77).
The regulation of J-chain synthesis in intestinally derived B cells is not completely understood and apparently secretion of IL-2, IL-4 and IL-5 cytokines seems to play a role (515). Transcription of J-chain depends on an IL-2-induced chromatin remodeling of the J-chain locus and subsequent interaction of specific transcription factors with the J-chain promoter. The B-cell lineage specific activator protein (BSAP) inhibits J-chain transcription by preventing the binding of transcription factors (USF and
B-MEF-2) to the locus (550). On the other hand, the B lymphocyte-induced maturation protein-1 (Blimp-1) promotes J-chain synthesis by inhibiting BSAP (528).
Two different types of B lymphocytes have been characterized, T dependent or
B2 and T independent (TI) or B1 cells. The B2 cells are capable of somatic hypermutation, affinity maturation, Ig isotype switching and generation of memory. The
B1 cells are long-lived, produce polyspecific antibodies (natural IgM), do not generate memory and do not undergo major somatic hypermutation (44). The human B1 cells are divided into B1a (CD5+) and B1b (CD5-), express IgM and CD43 on their surface and are
44 characteristically CD45low and IgDlow (44). They reside mainly in the peritoneal cavity, but a small percentage of B1 cells have also been described in the spleen (281, 462).
Transfer of B1 cells into SCID mice induces repopulation of IgA secreting cells in the intestinal LP and the MLN suggesting that a high frequency of IgA secreting B cells in the gut derive from B1 cells (302, 342). Typically, TI antigens are large and have repetitive epitopes capable of crosslinking the BCR and inducing B cell proliferation.
However, IgM to IgA isotype switching by B1 cells is dependent on cytokines secreted
by T cells (175) . The TI antigens are divided into TI-1 and TI-2 based on the induction
of antibody responses by TI-1 but not TI-2 in neonatal mice and CBA/N mice (600).
Activation of B2 cells depends on the ligation of CD40L expressed on the T cell surface
and CD40 expressed on the B cell surface (116, 185, 581). Other critical surface
molecules essential for the formation of B2 cell responses are CD28 on the surface of T
cells and B7.1/B7.2 on the surface of activated B cells (204, 222, 485).
1.5.4.1 Class switch recombination (CSR)
The IgM heavy chain gene expressed by naïve B cells is assembled from one of
each of the several variable (V), diversity (D) and joining (J) gene segments. This process
is called VDJ recombination and occurs during B cell maturation. After recombination
takes place, the genes will be transcribed and a heavy IgM chain will be later synthesized.
The heavy chain is associated with a light chain to form a monomeric IgM molecule that
will later be expressed on the B cell surface as the BCR. Class-switch recombination
(CSR) occurs when the B cell is further stimulated by antigen and the IgM heavy chain is replaced by the expression of IgA, IgG or IgE heavy chain genes. The IgM heavy chain
45 gene (Cµ) will be replaced by the expression of one of the downstream genes transcribing
the IgA (constant or Cα gene), IgG (Cγ gene) or IgE (Cε gene) heavy chains. The
enzyme activation-induced cytidine deaminase (AID) is required for this deletion-
recombination reaction. The DNA breaks occur in the switch (S) genes preceding the C
genes. The CSR can be sequential which follows the order of the genes (Cµ→Cγ→Cα) or direct (Cµ→ Cα). The genes located in between Cµ and the next C to be transcribed,
will be excised from the DNA or “looped out” creating a recombinant episomal circle.
The DNA breaks will be rearranged by nuclear repair enzymes. After the DNA
recombination is completed, the Ig heavy chain (e.g. IgA, IgG, IgE) genes will be
transcribed and then when the protein product is finalized, assembly with light chains will
occur (maintaining the same antigen specificity) and surface expression or extracellular
secretion in more mature B cells will take place (80).
1.5.2.2 Signals for IgA switching
The GALT is characteristically rich in IgA and three explanations for the
abundance of IgA switched B cells at intestinal effector sites have been postulated:
1) B cells generated in the GALT preferentially express J-chain and IgA before migrating
to effector sites.
2) Plasmablasts expressing IgA are preferentially attracted to the LP by intestinal homing
molecules.
3) The LP microenvironment favors in situ class switch to IgA.
All three mechanisms have been proven in the mouse model but the in-situ switch to IgA in the LP is restricted to murine B1 cells derived from the peritoneal cavity (71, 181, 46 483). It has been explained earlier that the interaction of CD40-CD40L is essential for
CSR to take place. In addition to the close contact between B and T cells necessary to
induce CSR, certain cytokines secreted by T cells promote CSR to IgA. For example, B
cells from patients with IgA deficiency incubated with IL-10 can become IgA committed
B cells (85) . Furthermore, naïve human B cells activated through CD40 and incubated
with IL-10 and TGFβ can be activated to express IgA (161). After CSR to IgA, IL-2, IL-
5, IL-10 and IFNγ may be important for the expansion and terminal differentiation of
IgA+ B cells (401). However, TGF β has been proven essential for CSR to IgA in human
B cells inducing both IgA1 and IgA2 (423). Autocrine TGF-β directs DNA recombination of the Ig gene S regions to be direct (Sµ→Sα) or sequential (Sµ→Sγ
→Sα) after B-T cell CD40-CD40L engagement (593). However, the mechanism behind the formation of IgA+ B cells in the PP is not completely clear. It was originally thought
that DCs promoted CSR to IgA during antigen presentation and by promoting the
gathering of B and T cells (490). It was later demonstrated that DCs produced IL-6, a
cytokine that has been linked with IgA+ B cell production (483). Additionally DC and
macrophage derived BAFF and a proliferation inducing ligand (APRIL) have been
involved in IgA switching in the murine PP (75). The neuroendocrine factor vasoactive
intestinal peptide (VIP) has also been linked to IgA switching (293). Naïve B cells, after
ligation of CD40 and exposure to VIP, secreted both IgA1 and IgA2.
Many investigators attempted to demonstrate that transfer of IgM+ B1 cells results in the repopulation of the LP with in-situ switched IgA+ B1 cells (180, 181). Evidence that
supports the in-situ IgA switch in the LP is questionable because of the technical
difficulties when performing the experimental procedures. First, it is extremely difficult 47 to isolate the LP lymphocytes without contamination from PP derived lymphocytes.
Second, it is not possible to know if the IgM+B1 cell population was contaminated with
switched IgA+B1 cells; moreover, it is difficult to rule out contamination with IgM+ IgA-
B1 cells that are already switched at the DNA level and will later express the Cα gene to transcribe and synthesize IgA (239). Furthermore, in-situ class switch has only been observed in mice and not in humans. Human IgA+ and IgM+ B cells located in the LP
have highly mutated VH-regions characteristic of B2 cells that only switch in the GCs and
not in the LP (70). From the three explanations above, it seems that only the first two
apply to humans but it is unclear which one is the closest or if both apply to the in-vivo
scenario. The mutation rate of intestinal IgA after an immune response in pigs has not
been determined, so it is unknown if B cells in the porcine PP are from B1 or B2 origin.
However from pig studies, by transferring radiolabeled IgA+ B cells and IgM+ B cells it was observed that IgA+ but not IgM+ B cells, preferentially migrated to the LP. This observation suggests that like in humans, pigs do not have in-situ IgA switch in the LP and that IgA+ B cells preferentially migrate to the LP after CSR.
1.5.5 Homing mechanisms
It was originally described that radiolabeled small lymphocytes were capable of
redistributing to different secondary lymphoid tissues whereas large activated
immunoblasts migrated mainly to tertiary lymphoid tissues (218). After this observation,
many others sought to study lymphocyte migration (246, 424, 461). It was clear that
activated lymphocytes had the tendency to return to the tissue where they were originally
activated and the concept of lymphocyte migration and homing evolved.
48 A naïve lymphocyte enters a secondary lymphoid tissue through HEV (91). Once
the lymphocytes make contact with the endothelium lining the lumen of the HEV, a
series of events aid in halting the cells to later direct them to the stroma of the lymph
node. If a lymphocyte does not find an antigen to which it reacts, the cell will return to
the blood stream to enter other secondary lymphoid tissues. If a naïve lymphocyte
encounters its specific antigen and is subsequently activated, it will migrate to the closest
lymph node to circulate through the lymphatic system until reaching the general
circulation via the thoracic duct. Once in the blood, the activated lymphocyte will
preferentially return to a tertiary lymphoid tissue linked to the site where imprinting took
place via post-capillary venules (479).
The entry of lymphocytes to the lymphoid tissues is a multistep process (91). The
first step involves constitutively active adhesion receptors that aid in the interaction of the
cell with the vascular endothelial cells. Initially, tethering and rolling take place when the
lymphocytes are in close contact with the endothelial cells. Rolling allows the cells to
sample for factors (e.g. chemokines) in the endothelium that could activate a serpentine
G-protein linked chemoattractant receptor on the lymphocyte surface that will initiate an
activation dependent arrest followed by diapedesis through the endothelial cells to the lymphoid tissue stroma (91, 479).
The HEV located at human GALT and MLN but not peripheral LN express the
MadCAM-1 (76). In murine GALT, glycosylated mucin-like domains of the MadCAM-1
molecule promote binding of L-selectin (CD62L), abundant on the surface of naïve and
systemically primed lymphocytes (43). The interaction of L-selectin with the
glycosylated MadCAM-1 promotes tethering of the lymphocytes. Also collaborating in
49 this first interaction of the lymphocytes with the endothelial cells is α4β7, an integrin
expressed at low levels on the surface of naïve lymphocytes that also binds to MadCAM-
1 (91). Naïve cells enter peripheral lymph nodes by the interaction with the peripheral
lymph node addressin (PNAd) and L-selectin (80). Another widely expressed adhesion
molecule, the leukocyte-function-associated molecule-1 (LFA-1) and its ligand the
intercellular adhesion molecule-1 and 2 (ICAM-1, ICAM-2) expressed on the surface of
endothelial cells also aid in the localization of naïve or memory lymphocytes to
secondary lymphoid tissues (91). The G-protein coupled transmembrane receptor that
mediates the activation dependent arrest of the lymphocytes, is activated by chemokines
secreted by the vascular endothelium. For example, the secondary lymphoid tissue
chemokine (SLC, CCL21) and Epstein-Barr virus (EBV)-induced molecule ligand (ELC,
CCL19) preferentially attract CCR7+ T cells to parafollicular zones. In mice, stromal cells from mucosal associated lymphoid tissues (MALT) and LN secrete these chemokines that are then transported apically to reach the surface of HEV (28).
Although, the B cell recruitment to secondary lymphoid tissues is less clear, studies in mice demonstrated that CCR7 with its ligand CCL7-8 and CXCR4 that bind
CXCL12/SDF-1 together with CXCR5 (the receptor of CXCL13/BCA-1 previously acknowledged) are crucial molecules for B cell adhesion to endothelial cells (414).
Interestingly, B cell migration to the follicle appears to occur through HEV-like vessels and not typical HEV and depends on CXCL13 and CXCR5 expression (559).
If lymphocytes encounter their specific antigen in the PP, they will upregulate
α4β7 and downregulate L-selectin to become α4β7high L-selectinlow lymphocytes (91).
Lymphocytes exit the GALT through fenestrated lymphatic vessels and reenter the 50 intestinal LP by binding MadCAM-1. However, the expression of MadCAM-1 in the
human LP has not been demonstrated despite the expression of α4β7high by lymphocytes
after intestinal activation (76). Tethering and rolling of intestinally primed lymphocytes
will occur through interactions of α4β7 and MadCAM-1 whereas activation dependent
arrest will depend on the expression of CCR9 and its interaction with thymus-expressed
chemokine (TECK, CCL25) expressed mainly in the small intestinal mucosa (71, 308).
The mucosa-associated chemokine (MECK, CCL28) that is mainly expressed in the large
intestinal mucosa, by interacting with CCR10, aids in the localization of IgA+ B cells to the large intestinal LP (80, 321); however, it does not direct T cells (80). It was suggested that the differential expression of CCR9 and CCR10 on the surface of intestinally committed lymphocytes facilitated their localization to the small or large intestinal LP, respectively. However, it was recently observed that both chemokines were redundant in their role in attracting B lymphocytes to the small intestinal LP (190). Interestingly, both
TECK and MECK enhanced the α4-mediated adhesion to MadCAM-1 suggesting that
these chemokines not only guide intestinally primed lymphocytes to the gut by CCR9 and
10 interactions, but also promote the interaction with MadCAM-1 (237).
In pigs under non-inflammatory conditions, expression of α4β7 was highest in the
MLN and PP compared to the tonsils (similar to humans); however, it was also observed in the respiratory pharyngeal lymph nodes and the main α4β7 expressing cells were T lymphocytes. Additionally, the expression of α4β1 was high in tonsils, PP and MLN and mainly IgA+ B cells expressed the α4β1 (69). On the other hand, human lymphocytes expressing α4β1 migrate exclusively to BALT and not the GALT (579) For pigs,
51 according to the finding described, it appears that α4β1+ B cells migrate to both BALT
and GALT (MLN) whereas the predominant expression of α4β7 by PP derived T cells is
similar between pigs and humans. A recent publication characterized the distribution of
TECK and MECK mRNAs by real-time PCR in the different mucosal tissues of pigs and
a similar distribution to that of humans was observed. The distribution of TECK was
restricted to the small intestine, and CCR9 expression was particularly high in T
lymphocytes located in the GALT. However in the newborn pig, TECK was initially
observed in the small intestine, large intestine and MLN and after a month, the expression
remained high in the small intestine and diminished in the other tissues. On the other
hand, MECK expression was observed in all tissues tested (small and large intestine,
MLN, mediastinal LN, trachea mammary gland and lungs) but was particularly high in
the trachea, mammary gland and large intestine (377).
1.5.5.1 Phenotype and homing of nasally primed lymphocytes
Primary nasal immunization generates lymphocytes with limited capacity to home
to the intestinal mucosa (534) whereas GALT generated lymphocytes have a wider
dissemination to extraintestinal effector sites (80). The NALT induced lymphocytes
develop a phenotype that is not highly compatible with intestinal effector site localization
(α4β1high, α4β7int/low,CCR9low, CCR10high, CCR7high, L-selectinhigh) but direct their localization to local respiratory or systemic LNs and the BM . Similarly, lymphocytes primed in the tonsils, have the capacity to migrate to respiratory effector sites but not to
the gut (80, 267). The expression of L-selectin and CCR7 on nasally primed lymphocytes
facilitates its homing to systemic LNs (150) and to the genitourinary tract by binding to 52 PNAd, CCL21/SLC and CCL19/ELC expressed on the vascular endothelium of systemic and genitourinary LNs (267). The CCR10 on the surface of respiratory primed lymphocytes probably plays an important role in their localization to a variety of mucosal surfaces because MECK, its ligand, is secreted not only by the large intestinal epithelial cells, but also by the upper aerodigestive tract epithelium (418), lactating mammary glands (570) and BM stromal cells (393). Expression of CCR10 can partially explain the wide distribution of intestinally and upper respiratory primed lymphocytes. As mentioned, a similar distribution of MECK is observed in the pig mucosal tissues.
In the pig, the HEV in the palatine and pharyngeal tonsils express PNAd whereas expression of MadCAM is high in the PP and low in the tonsils. Similarly to humans,
MECK expression is high in the tonsils and low in the PP and the opposite is observed for the expression of TECK (69).
1.5.6 Dendritic cells
The gastrointestinal system is widely colonized by a variety of harmless commensal flora that constantly induce T dependent and independent intestinal IgA responses. Conversely T dependent systemic B cell responses are not usually induced by the commensal microorganisms because the gut microenvironment and the MLN successfully contain the flora, preventing systemic invasion (343). Immune responses in the intestine are tightly controlled and a state of immune hyporesponsiveness, also known as oral tolerance, rules the gut immunity (see section on intestinal induction of tolerance).
Oral tolerance is a state of systemic immunologic unresponsiveness specific to an orally fed protein antigen (566). Others also define oral tolerance as the mucosal immune
53 system unresponsiveness to different antigens present in mucosal tissues which might otherwise induce active immune responses if present in different non-mucosal tissues
(507). Oral tolerance is directly dependent on the type and activation status of local professional and non-professional APC. To understand how intestinal immune responses are generated, it is important to know how intestinal dendritic cells are activated and how they induce tolerogenic or active immune responses. For example, by delivering soluble antigens and the cytokine flt3 that expands DC populations, oral tolerance is induced in mice (569). On the other hand if complex antigens are delivered with adjuvants that can be recognized by DCs such as IL-1 and cholera toxin, an active immune response is more likely to occur because of the induction of DCs to drive T cell activation (547, 569).
In mice and humans two main types of DCs have been described: myeloid DC
(mDC) derived from myeloid precursors (488) and lymphoid or plasmacytoid DCs (pDC) derived from lymphoid precursors (575). The mDCs can be generated in-vitro by incubating monocytes with GM-CSF and IL-4. In-vitro generated mDCs allow cell expansion which is optimal for experimental settings (596). In mice at least nine different subtypes currently exist, all of which possess CD11c, class I major histocompatibility complex (MHC) and costimulatory molecules CD80/86 and CD40.
Three main subsets are located in the spleen (CD11c+ CD11b+ CD4+; CD11c+ CD11b+
CD4- and CD11c+ CD11b+ CD8α+). In the MLN and PP three other major types of DCs have been observed (CD11c+ CD11b- CD8α+; CD11c+ CD11b+ CD4- CD8α- and CD11c+
CD11b- CD4-CD8α-). Another three types of pDCs have been described based on the level of expression of CD11c (287). Other markers such as DEC205 and CD1d, are expressed mainly by pDCs but can be upregulated on mDC after in-vitro culture or by 54 LPS treatment (31, 575). The pDCs are located in the rich T cell areas of the
periarteriolear lymphatic sheaths (PALS) in the spleen and LNs, whereas the mDCs are
located in the marginal zone of the spleen but under inflammatory conditions they can
migrate to the PALS (504). Also, pDCs secrete more IL-12 than mDCs and IL-12 induces
IFNγ secretion by the pDCs but not the mDCs (447). Deficiencies in mDC have been
described in avian reticuloendotheliosis viral oncogene related B (RelB) gene deficient
mice whereas mice carrying an Ikaros mutant gene are deficient in pDCs (89, 576). A
special type of DC is the Langerhan’s cells located in the skin. Some studies of mice
suggest that Langerhan’s cells derive from mDC that migrated from local LNs to the
skin. It is known that TGF-β exposure is critical for Langerhan’s cell differentiation (68).
In humans, mDCs express CD14+ CD11c+ and pDCs lack markers of T cells, B
cells, monocytes and NK cells (also known as lineage-negative or LINneg) and express the
IL3Rα. The LINneg DCs include three non overlapping DC subsets: CD11c+ CD1b/CD1c+
CD16+; CD11c+ BDCA3+ and CD11c- BDCA2+ BDCA4+ CD123+ (287). The mDC give rise to monocytes that can develop into interstitial DCs and tissue macrophages whereas
LINneg are precursors of pDCs. Monocytes express CD11b, CD13, CD14, CD33,
CD45RO,GM-CSFRα and low levels of IL3Rα whereas pDC express only low levels of
GM-CSFRα and high levels of IL3Rα (31). Monocytes have a limited capacity to
produce the innate antiviral cytokine IFNα whereas the main source of IFNα comes from
pDC especially after viral infections (497).
Dendritic cells migrate from primary lymphoid tissues (BM, thymus) to non-
lymphoid tissues where they sample antigens and then migrate to local secondary
lymphoid tissues (LNs, PP and spleen) (452). Their main function is to sample and 55 present antigens to naïve T cells. Under inflammatory conditions, pathogens express ligands that bind and stimulate receptors on the surface of DCs [e.g. toll like receptors
(TLR)]. At the same time cytokines and chemokines are released by immune (e.g. B cells and T cells) and non-immune cells (e.g. epithelial cells) inducing the activation of DCs
by binding their receptors on the surface of the DCs. All these interactions plus tissue
damage byproducts, trigger DC maturation and presentation of antigen to T cells,
inducing their activation and overt immune responses. However, under steady state
conditions, DCs capture self antigens but in the absence of danger signals (tissue damage
byproducts, TLR ligands, cytokines, chemokines etc) the DCs remain immature and are
unable to drive T cell specific responses. The T cells activated by immature DC will
undergo anergy inducing systemic tolerance (see section on intestinal induction of
tolerance) (503).
1.5.6.1 Functional characterization of DC subsets
In mice the expression of CD8α characterizes DCs into two functional groups:
DC1 and DC2. The DC1 cells are CD8+, mainly secrete IL-12 and induce preferentially
Th1 responses after adoptive transfer. The DC2 cells are CD8-, secrete more IL-10 and
preferentially induce Th2 responses. Also DC1 cells are major mediators for the
induction of T-cell tolerance and cross-presentation of self-antigens to CD8+T cells (351,
352). In humans functional differentiation has also been described and the dogma is that mature mDCs exposed to GM-CSF and IL-4 preferentially induce Th1 responses in-vitro
whereas mature pDCs exposed to IL-3 induce Th2 responses. However for both mice and
humans there is evidence of differentiation not being absolute for each subset of DCs. In-
56 vitro manipulation with different types and amount of antigens and cytokines can modify their functional characteristics. For example, it was shown that high doses of antigen biased responses towards Th1 whereas low doses biased towards Th2 responses (67).
Also, pDCs cultivated with CpG that bind the toll-like receptor (TLR) 9 induced Th1 responses whereas LPS that binds TLR4, induced Th2 responses (67). Furthermore, culture of human mDC with IL-3 diminishes its capacity to secrete IL-12 and Th2 responses were induced (173). In mice, when maturing DCs in-vitro with GM-CSF (351) or expanding these cells in-vivo with flt-3 (448), CD11c+CD11b-CD8+ DCs induced Th1 responses whereas CD11c+CD11b+CD8- induced Th2 responses . However when freshly isolated, both subsets of DCs induced a Th1 response (252). Probably the in-vivo decision of Th1 or Th2 induction depends not on the phenotype of the DC presenting the antigen to T cells, but on the environment, the secreted cytokines , the antigen type and the amount of antigen that DCs are exposed to at the time of an ongoing infection.
1.5.4.2 Dendritic cell migration and homing
Under non-inflammatory conditions after the intravenous injection of murine peripheral tissue derived DCs (expanded with flt3), rapid migration to the liver, lung, spleen and BM occurs (66). However, DCs that migrate to the lung rapidly return to the general circulation. Circulating DCs are somehow excluded from other secondary lymph nodes, but a small population gains entry to the PP and peripheral LN by a pertussis toxin-sensitive pathway (a protein G dependent receptor such as CCR receptors (92))
(101). Interestingly, spleen DCs utilize α4β7 to migrate to the PP and by blocking
MadCAM-1 with specific antibodies, DC numbers in the PP greatly diminish (66). After
57 DC injection or under physiological conditions, DCs frequently enter the bone marrow
(BM) and are able to induce proliferation of T cells (101). The DC migration to the BM
and thymus is dependent on expression of E and P-selectin on the vascular endothelium
of the BM as well as the interaction of surface α4β1 with endothelial VCAM-1 (66).
Thus DCs use a variety of mechanisms to enter lymphoid and non-lymphoid tissues.
Murine PP in the steady state, express the chemokines CCL9 (598) and CCL20
(251) that attract immature DCs to the SED. It is known that immature DCs express
CCR6 (receptor of CCL20) and upon DC activation, CCR6 is downregulated and CCR7
is upregulated (100). Upon inflammation TNF-α and IL-1α are produced and CCL20 is
induced and secreted by the intestinal epithelial cells possibly attracting immature DCs
expressing CCR6 (253). Studies of CCR6 knockout mice demonstrated the absence of
DCs in the SED of the PP, preventing mice from initiating specific T cell responses to S.
typhimurium that invades the FAE (478). In-vivo DC activation by soluble antigen
derived from Toxoplasma gondii tachyzoites induces CD11b+ DC migration to the IFR where CCL7 and CCL8 (ligands of CCR7) are constitutively expressed (251). These studies support the hypothesis that upon DC exposure to antigens, DC maturation is induced and upregulation of CCR7 occurs, directing DCs to the IFR which is rich in T cell lymphocytes, favoring the induction of T cell specific responses (287).
1.5.4.3 Intestinal dendritic cells
The PP and iLF throughout the intestinal wall are the main site of induction of
intestinal immune responses and DCs are located within these structures. The SED and
the IFR are particularly rich in CD11c+ DCs. (288). The DCs from the PP induce T 58 independent IgA B cell differentiation and intestinal homing imprinting (384). Production
of retinoic acid (RA) by GALT-derived DCs, upregulated the B cell expression of α4β7
and vitamin A deficiency (RA precursor) significantly reduced the amount of intestinal
IgA B cells. Additionally, DC derived IL-6 and IL-5 cytokines induced B cell IgA
secretion and IL-6 or IL-5 cytokines alone prevented the Ig class switch (384). Thus
intestinal T cells preferentially secrete IL-4 and IL-10 and lower levels of IFNγ compared
to splenic T cells that preferentially secrete IFNγ (250). Furthermore, antibodies to TGF-
β and IL-10 promote the production of IFNγ by T cells isolated from the PP (250). These
studies suggest that DCs in the PP secrete TGF-β and IL-10 and direct Th2 and Th3 T
cell responses (see section on T regulatory cells).
Three types of murine DCs have been described in the PP: CD11chigh CD8α+CD4-
CD11b-; CD11chigh CD8α-CD4-CD11b+ and CD11chigh CD8α-CD4-CD11b-. This latter population is also present in spleen and peripheral LNs but it is especially abundant in the
PP and MLN (252, 287). The PP CD11b+ subpopulation produced high levels of IL-10 compared to the CD11b+ DCs from spleen and peripheral LNs and induced IL-10
secreting T cells (252). Freshly isolated CD11b+ DCs (derived from the SED of the PP) induced higher levels of IgA secretion by naïve B cells compared to splenic DCs (483).
On the other hand, the CD11b -subpopulations (PP derived) induced more IL-12 and
IFNγ secreting T cells and they were mainly described in the IFR which is a T cell rich
area in the PP (251). The CD8α- populations are also found in the SED, B cell follicular
area, the FAE and the M cell pockets (252). The DCs located in the PP have been shown
to express the α4β7 ligand MadCAM-1 suggesting that these cells may preferentially
59 interact with intestinally primed lymphocytes (513). Only DCs from the PP are capable of
inducing the expression of α4β7 and CCR9 on T cells in-vitro that upon transfer to naïve
mice preferentially migrate to mucosal lymphoid tissues (383).
The recently described mouse pDCs, express intermediate levels of CD11c, B220,
Ly6C and CD8 and are located in the IFR of the PP. Depletion of pDC by specific
antibodies abolishes IFNα producing cells (20) suggesting that the major source of IFNα
derives from pDCs. Similar to the CD8+ splenic pDCs the PP pDC, also secrete IL-10
(357).
The DCs are a major population in the intestinal LP in mice and humans. In mice,
DCs are located below the basement membrane and can extend their processes into the intestinal lumen to sample intestinal antigens (382). The mouse small intestine is rich in
LP DCs whereas the colon is rarely colonized by DC except in sites where iLF are located (40). Recently, the same three subsets of CD11chigh DCs and pDCs described in
the mouse PP have been described in the LP (387). Some investigators described the
majority of DCs located in the terminal ileum as CD11b-CD8-, but others have observed a predominant subset of CD11b+ DCs (287). Also, DCs in the LP are associated with bacteria in-vivo, suggesting that under non-inflammatory conditions, LP DCs are constantly sampling commensal flora (287). It has been suggested that LP DCs have an important role in the induction of tolerance (387), but there are no published studies that used pure LP DC populations to address this possibility.
60 1.5.6.4 Neonatal dendritic cells
It has been suggested that the immaturity of the neonatal innate and adaptive
immune responses predisposes the neonates to a variety of bacterial, fungal, parasitic and viral infections (301, 487). Human cord blood monocytes/macrophages have low levels of costimulatory molecules, a reduced capacity to differentiate into DCs in-vitro and an altered IL-12 production as well as a decreased phagocytic activity compared to their adult counterparts (541). Neonatal monocytes and macrophages produce lower levels of pro-inflammatory cytokines (TNFα, IL-1β or IL-12) after stimulation with LPS (TLR4
ligand) (105) and other TLR ligands such as mycoplasma-associated lipopeptide (TLR6
ligand) and triacylated Braun lipoprotein (TLR1/2 ligand) (327) compared to cells
isolated from adults. Additionally, human cord blood DCs express lower amounts of
costimulatory molecules, have an altered response to TLR and CD40 signaling linked to a
reduced production of cytokines and a reduced endocytic activity (541). Human neonatal
pDCs exhibited incomplete upregulation of maturation markers such as CD80, CD86,
CD83 and CD40 after stimulation with CpGs compared to adult pDCs (158). Also,
neonatal pDCs secreted less IFNα in response to CpG stimulation compared to adult
pDCs (158). Similarly incubation of cord blood mDCs with LPS or poly I:C (TLR3
ligand) induced a reduced upregulation of CD40 and CD80 compared to adult mDCs
(159). Total cord blood cells activated with LPS compared to adult peripheral blood cells,
secreted significantly lower amounts of IL-12, IL-15 and IL-18 and contained less
mRNA whith a significantly shorter half life (487). All these findings suggest that
neonatal DCs are immature and that their threshold of activation may be higher than that
61 of adult DCs or that there is may be a weaker interaction between DCs and T cells due to the low expression of costimulatory molecules on neonatal DCs (541).
1.5.6.5 Porcine dendritic cells
Porcine DC populations have been studied from various tissues. Blood derived
DCs are mainly CD11b+CD16intSWC3+CD1intMHCII+ (509), whereas BM derived DCs express CD1,CD14,CD16 and SWC3; the same markers expressed by monocytes (96,
417). In the pig the natural IFNα producing cells (NIPC) or pDCs are characteristically
CD4+SWC3lowCD1int, have IL3 binding capacity and lack lineage markers such as CD21,
CD8, CD3 and CD14 (227, 258) and they also lack CD11b (258). The spleen is particularly rich in pDCs compared to the frequencies observed in MLN, tonsils and peripheral blood. In pigs, pDCs cannot be described as being from lymphoid precursor origin as for mice and humans because interestingly both porcine monocytes and NIPCs express the myeloid lineage marker SWC3. Porcine DCs that differ from pDCs have been designated conventional DCs (cDCs) by some groups and are characteristically SWC3high or lowCD11b+ or -CD4-CD1int. The main difference between pDCs and cDC is the lack of expression of CD4, the presence of CD11b, the lower secretion of IFNα and the predominant secretion of TNFα by cDCs (258). As described for humans, blood derived porcine mononuclear cells incubated with GM-CSF and IL-4 give rise to monocyte- derived DCs (417). It has been described that pDCs but not monocyte-derived DCs secrete IFNα and TNFα after exposure to transmissible gastroenteritis virus (TGEV)
(228). Furthermore, monocyte-derived DCs can be activated and induced to secrete IFNα after transfection with dsRNA (102) and incubation with mutant ∆Npro classical swine 62 fever virus (39). These studies show that DC activation and cytokine secretion depends
not only on the type of activated DC but the antigen encountered and most probably, their
location and the influence of neighboring cells that secrete soluble factors (e.g.
cytokines). To date, there are no studies that describe the progenitor cells that give rise to
pDCs, monocytes or cDCs for swine as have been described for humans and mice.
Four different DC subpopulations have been characterized in the porcine small
intestine (jejunum and ileum): CD11b+SWC3+, CD11b+SWC3-, CD11b-SWC3+, CD11b-
SWC3- with all subsets expressing class II MHC molecules on their surface. The LP is rich in CD11b+SWC3+ eosinophilic granulocytes whereas the majority of DC in SED are
CD11b-SWC3+. The IFR has mainly CD11b-SWC3- DCs and in the MLN,
CD11b+SWC3- DCs predominate (52). Whether CD11b-SWC3+ DCs observed in the
SED are pDCs and other DCs observed in the PP are cDC is unclear. However, after
TGEV infection, the NIPC were located in the gut and the MLN as early as 6 hrs after
inoculation, peaking at 12-18hrs and disappearing after 24hrs. The NIPC were localized
in the epithelial layer, LP and periphery of the PP, but were mainly in the MLNs and did
not express SWC3 (455). Interestingly, by analyzing the phenotype of migrating DCs to the MLN by cannulation of intestinal lymphatics, researchers showed that most migrating
DCs were CD11b+ (52). Further studies of porcine DCs characterizing their multiple surface marker expression in mucosal vs. systemic tissues are needed to extrapolate and directly compare the findings observed in the porcine model with the human and mouse studies.
63 1.5.5 Intestinal induction of tolerance
Induction of intestinal tolerance is directly related to the fact that under non- inflammatory conditions, immature DC constantly sample and present antigens to T cells.
Because of this interaction, T cells become anergic and can directly and indirectly promote tolerance. The current model of intestinal DC uptake of antigens under non- inflammatory conditions (or steady-state conditions) holds that DC precursors enter the
LP, PP and iLF and develop into immature DCs (251, 287). Constitutive expression of
CCL9 and CCL20 in the FAE of the PP guides DC migration via surface chemokine receptors such as CCR1 and CCR6, respectively. Antigens are transported via M cells, epithelial cells or possibly via apoptotic bodies from epithelial cells, facilitating DC antigen uptake and subsequent migration to the SED, IFR or the MLN. Antigen uptake is followed by CCR7 upregulation, and under steady state conditions, only low levels of maturation markers such as costimulatory molecules CD80/86 and cytokines are expressed (287). Antigen loaded immature DCs stimulate specific T cells that differentiate to T regulatory (Tregs) cells that mediate bystander tolerance. The mechanism of action of Tregs is still unclear but to function they require direct cell contact and suppressor cytokine secretion (TGF-β, IL-10 and possibly IL-4). There is evidence that Tregs can be induced by mDCs and pDCs. For example, it has been described that immature mDCs induce Tregs to express foxp3 and secrete IL-10 (134) and in a model of autoimmune diabetes, mDCs prevented the activation of autoreactive T cells in-vivo (516). On the other hand, in a model of cardiac transplantation in mice, pDCs were the main inducers of tolerogenic T cells after cardiac transplantation and this effect was reflected in-vivo as a prolonged allograft survival (408). It was recently
64 described that tolerance induced by oral inoculation of ovalbumin (OVA) is prevented in
CCR7-/- mice and after mesenteric lymphadenoctemy. Because CCR7 guides DC migration to the nearest draining LNs, after oral OVA feeding, WT mice showed robust T cell proliferation in the PP and MLN but not in the PLNs and spleen whereas in CCR7 knockout mice, proliferation was only observed in the PP. Interestingly, after oral OVA administration, mice that had their MLNs removed demonstrated ear swelling after local
OVA injection whereas normal mice did not show signs of local inflammation. This study suggests that antigen-bearing DCs that migrate through the afferent lymphatics to the MLN are key regulators in the generation of systemic tolerance after soluble antigen ingestion (572).
The type and amount of antigen is also central in the induction of active vs. tolerogenic immune responses. High doses of soluble antigen (e.g. OVA) result in systemic anergy-driven tolerance (200, 372) whereas low doses of antigen results in
regulatory T cell driven tolerance (184, 388). It is still unknown how high antigen doses
induce tolerance but it is believed that the excess antigen that gains entry to the systemic
compartment induces anergic T cells or T cell deletion, similar to responses induced by
intravenous injection of antigen (565). Low doses of antigen on the other hand, induce
antigen-specific Tregs following presentation by intestinal APCs that secrete tolerogenic
cytokines such as TGF-β, IL-10 and IL-4 (380). These Tregs then circulate to the systemic compartment inhibiting the generation of effector T cells by the secretion of tolerogenic cytokines (bystander suppression).
In contrast to innocuous antigens, mucosal pathogens induce active immune responses by the recognition of pathogen-associated molecular patterns (PAMPs) by
65 pathogen recognition receptors (PRR) such as toll-like receptors (TLR) and dsRNA
recognition molecules [(retinoic acid inducible gene, I (RIG-1); melanoma
differentiation-associated gene 5 (mda-5) product; and dsRNA dependent protein kinase
(PKR)] (164). Intestinal epithelial cells express surface TLRs and its ligation induces IL-
1, IL-8, IL-6, TNFα, IFNα, CCL5 and CCL20 which induce recruitment of granulocytes and DCs (274). Furthermore, PAMPs induce DC activation and upregulation of class II
MHC molecules, co-stimulatory molecules CD80/86, adhesion molecules and cytokines.
All of these molecules and cytokines aid in the induction of antigen-specific T cells able
to initiate an active immune response against the invading pathogen. In mice, CD11b+ and CD8-CD11b-CD4- DCs localized in the SED of the PP or the LP appear to be the main mediators in the initial response after pathogen invasion (287). On the other hand,
CD8+ DCs may be involved in direct presentation or cross-presentation of antigens
carried to the IFR by DCs or epithelial cell exosomes.
1.5.5.1 T regulatory cells (Tregs)
It is known that self-reactive T cells are present under normal conditions (561) but
are actively controlled by T cells that prevent their activation and expansion (475) and
this T-cell mediated suppression is a key mechanism in the prevention of autoimmunity.
Normal mice deprived of Tregs develop autoimmune diseases and its reconstitution inhibits autoimmunity (476). The Tregs are actively engaged in the control of tumors, organ rejection after transplantation, allergy and microbial immunity (353). These cells are anergic, have suppressive properties and seem to mediate control of other cells by direct contact and by secretion of cytokines. Two types of regulatory T cells have been
66 described: adaptive regulatory T cells and naturally occurring Tregs. Adaptive regulatory
T cells mediate suppression by secretion of tolerogenic cytokines (Th3 and Tr1 T cells)
(184). The naturally occurring Tregs characteristically express CD4, CD25 (IL-2
receptor) (477) and foxp3, a new member of the forkhead/winked-helix family
transcription factors (88). These Tregs are thymus derived and are essential in the
induction of oral tolerance because thymectomized mice developed experimental
autoimmune encephalitis (EAE) after feeding with Myelin Basic Protein (MBP) (501).
The foxp3 gene is a master control gene for the development and function of CD25+CD4+
Tregs. In humans, the immune dysregulation, polyendocrinopathy, enteropathy and X- linked inheritance (IPEX), is characterized by a disregulation of Tregs due to a mutation in the foxp3 gene (27) and is characterized by protracted diarrhea, food allergies, icthyosiform dermatitis, endocrine insufficiency and hemolytic anemia (567).
Other cells involved in tolerance are CD8+ T cells, γδ T cells, NK cells and IFNγ secreting cells. The CD8 T cells can induce tolerance by secretion of TGF-β but CD8 knockout mice have normal oral tolerance indicating that CD8+ T cells are not essential for induction of tolerance (379, 549). Studies have suggested that γδ T cells are important regulators of oral tolerance and secretory IgA production. Oral tolerance can be abrogated by the depletion of these cells and can not be induced in γδ T cell deficient mice (284, 374) . The NK T cells have also been linked with the induction of oral tolerance to different antigens but mice lacking NK T cells develop normal tolerance
(457, 525). Also, Th2 type CD4+ T cells secreting IL-10 and IL-4 cytokines have been implicated in the development of tolerance, mainly by suppressing Th1 type IFNγ secreting T cells (562) and inhibition of IL-2 and IFNγ are widely used parameters to 67 measure the induction of systemic tolerance (184). Nevertheless, Th2 type responses only develop under inflammatory conditions and Th2 type T cells can also cause abnormal inflammatory reactions such as airway inflammation in asthma (468). Furthermore, IL-4 deficient mice develop normal oral tolerance (205), but IL-10 deficient mice suffer from
spontaneous inflammatory bowel disease (IBD) and have an abnormal T cell activation
(305). Interestingly, tolerance can be reversed after depletion of IFNγ secreting cells and cannot be induced in IFNγ deficient mice (313). It has been suggested that IFNγ might be
important for the expression of adhesion molecules necessary for migration of regulatory
T cells (323). However studies of IFNγ receptor deficient mice demonstrated that IFNγ is
not essential for the induction of tolerogenic responses (295).
The Th3 regulatory T cells are characterized by the ability to secrete TGF-β that
has a central role in the induction of tolerance. There are three isoforms of TGF-β: TGF-
β1, TGF-β2 and TGF-β3. The β1 isotype is especially abundant in the intestine and has both immunostimulatory and immunoregulatory properties depending on the cellular environment (120). Intestinal TGF-β1 has a role in epithelial cell differentiation, IgA class switching and lymphocyte suppression. Cloned T cells from orally tolerized mice characteristically express CD4, TGF-β, IL-4 and IL-10 and depend on IL-4 rather than
IL-12 for proliferation (562). These T cells were classified as Th3 and their differentiation can be induced by TGF-β and enhanced by IL-10 or anti-IL-12 antibodies
(184); however, the association between Th3 cells and CD4+CD25+ Tregs is unknown and it is unclear if Th3 cells that are CD4+CD25- later become CD4+CD25+ Tregs or if they represent independent populations. The CD4+CD25+ Tregs secrete high levels of IL-
68 10, TGF-β and express cell membrane bound latency associated peptide (LAP) together with TGF-β and apparently mediate suppression via LAP (184). Anti-TGF-β antibodies and soluble recombinant LAP inhibit suppression mediated by Tregs suggesting that surface LAP-TGF-β possibly mediates a type of cell mediated suppression that might involve direct cell contact (391, 392).
It is known that TGF-β plays a crucial role in the control of immune responses because TGF-β deficient mice die soon after birth due to a widespread inflammatory response and mice expressing truncated TGF-β receptors develop autoimmune diseases and increased susceptibility to allergies (217, 491). However, TGF-β deficient mice can develop oral tolerance after high doses of oral OVA, but not after feeding of lower doses
(33). This result suggests that TGF-β is an important suppressor, but it is not the only cytokine regulating the induction of tolerance.
Recently, an IL-17 cytokine T cell response was characterized resulting in responses independent from the previous Th1, Th2 and Th3 described types of T cells.
The new type of T cell response was characterized as Th17 (51, 316). The Th17 cells depend on IL-23 (from the IL-12 cytokine family) for survival and develop from naive
TGFβ and IL-6 secreting T cells (316). Interestingly, these cells are the main inducers of
EAE and in the same model it was recently described that IL-27 secreting cells (also from the IL-12 cytokine family) were able to control IL-17 secreting T cells and their secondary inflammatory responses describing a new type of regulatory T cell (38, 508).
Only TGFβ together with IL-6 were able to induce Th17 T cells whereas TGFβ alone
69 promotes the generation of Tregs instead. This is an example of how, depending on the
presence of other cytokines, either tolerance or active immune responses can be induced.
Bystander suppression occurs when regulatory cells induced by a fed antigen
suppress responses stimulated by other antigens, as long as the fed antigen is present
(184). Bystander suppression is a promising therapy to control the development of
autoimmune diseases in which the autoimmune antigen that triggers the disease is
unknown. As specific Tregs to fed antigens secrete cytokines, they can suppress
inflammation in their microenvironment or migrate to distant organs and control active
immune responses specific to unrelated antigens elsewhere. It was demonstrated in-vitro
that cells from animals fed with MBP suppressed proliferation of an OVA specific cell
line in a transwell experiment where there was no direct cell contact and the main
suppressing soluble factor was TGF-β (381). Furthermore, OVA-specific Tr1 cells that
secrete IL-10 have also been involved in the control of inflammatory bowel disease, if
OVA is provided to maintain the suppressive effect (225). These studies support that both
TGF-β and IL-10 can induce bystander suppression in models of autoimmune diseases.
However other bystander suppression mechanisms have been proposed like
“deactivation” of APC (12) and contact inhibition through cell surface molecules
expressed on the Tregs (184). It has been observed that IL-4 and IL-10 secreted by a
tolerogenic T cell induce “tolerogenic” DCs that later will activate CD4+ and CD8+ T cells with regulatory phenotype (86, 317). Epitopes that stimulate CD8+ T cells are bound
to class I MHC molecules that usually present intracellular antigens, whereas tolerogenic
antigens are extracellular and usually presented by class II MHC molecules. To stimulate
CD8+ T cells by extracellular tolerogenic antigens, it is necessary that extracellular 70 antigens endocytosed by DCs, exit the endosomes and reach the intracellular millieu to be presented by class I molecules (4). This phenomenon is known as cross presentation. In this way DCs can induce not only CD4+ but also CD8+ T cells specific to tolerogenic
antigens. Because antigen bearing DCs migrate throughout the body, it is postulated that
“tolerogenic” DCs will be able to induce Tregs, in sites distant from the initial
inoculation. This is supported by the fact that inhibition of DC migration in CCR7-/- mice prevents the formation of Tregs and knockout mice but not WT mice exposed to airborne antigen develop allergic airborne diseases (238).
Induction of Th2/Th3 T cells and suppression of Th1 responses is thought to be
the outcome of tolerance. It is known that intraperitoneal or oral administration of IL-4
and oral IL-10 can enhance oral tolerance when co-administered with antigens. But also
oral adjuvants coadministered with tolerogenic antigens can increase oral tolerance. For
example, administration of oral LPS and MBP induces IL-4 in the brain (290). Oral
antigen delivery in multiple emulsions, liposomes or poly lactic-copulic acid also
enhance oral tolerance (292, 359). Antibodies to IL-12 and IFNβ are both promoters of
oral tolerance (356, 396). Also, continuous and serial antigen feeding enhances oral
tolerance by up-regulating TGF-β and IL-10. On the other hand, cholera toxin (CT), one
of the most potent mucosal adjuvants, abrogates oral tolerance when fed with unrelated
antigens as well as large doses of intraperitoneal IFNγ.
Many aspects of mucosal tolerance are still under study or need to be further
investigated. For example, it is unclear how the environment where a cytokine is
produced, antigen presentation and costimulatory molecules, routes of antigen
processing, form of the antigen, IgA and IgE after oral feeding of tolerogenic antigens 71 and γδ T cells affect the induction of oral tolerance. In addition, the amount of oral antigen has to be diminished, perhaps with the use of adjuvants, to be able to feed tolerogenic antigens for the treatment of human autoimmune and other diseases.
1.6 IMMUNITY TO RV IN THE DIFFERENT ANIMAL MODELS AND
HUMANS
Group A RV have a wide host range causing infection in many newborn animals like pigs (45, 521, 522), mice (537), rats (149) and calves(99). Human RV induce diarrhea and/or shedding in many animal models including newborn animals such as gnotobiotic pigs (591), calves (471) lambs (500), newborn cynomolgus monkeys (348), sub-adult macaques (243) puppy dogs (529) and mice (41).
The neonatal gnotobiotic pig model is one of the most useful models to study homologous and heterologous RV [e.g. human RV(HRV)] infection and disease because of its prolonged susceptibility to RV induced diarrhea (8 weeks) and lack of influence of maternal antibodies transferred placentally. Their gnotobiotic status assures an environment free of wild type RV for prolonged periods (473). Furthermore and most importantly, the pigs are optimal as models of HRV infection because of their physiological similarities with the human GI and immune systems and susceptibility to
HRV diarrhea (591).
The mouse model has been widely used to study RV infection. Disadvantages
include that mice have a distinctive immune system compared to that of humans and the
commonly used adult mouse model, is not susceptible to RV diarrhea. The major
72 advantage of the mouse model is the availability of “knockouts” that are genetically
targeted immunodeficient mice that lack a specific population of immune cells. Gene
targeted immunodeficient mice allow studies of the role of RV-specific B and T cells in
protection after primary or secondary RV infection. Also, antibodies to mouse CD
surface molecules are widely available permitting detailed characterization of RV
specific B and T cells as well as innate immune responses.
1.6.1 Mouse model for the study of acquired RV immunity
Adult C57BL/6 mice were studied after primary homologous (EC strain,
G3P[16]) RV infection (584). The IgM ASCs (ASC) to RV were first detected in the PP
and MLN at PID 4. At PID 7, RV-specific IgM ASC were detected in the spleen and the
intestinal LP (ILP). By day 28, RV-specific IgM ASC were detected in the BM. At PID 7
RV-specific IgA and IgG were observed in ILP, PP, MLN, spleen and BM. RV-specific
IgA ASC predominated over IgG ASC in the ILP and RV-specific IgA ASC resided for a
long term (9 months) in the PP. The BM had similarly high amounts of IgA and IgG ASC
at 9 months PI. The RV-specific IgM and IgG ASC responses in the PP, MLN and spleen were low to negative at 9 months PI. Large and small IgD-B220low B cells were first observed in PP and MLN at day 4. These cells are characteristically extrafollicular cells
(class switched non-germinal center B cells) and are the first to appear following immunization (366, 499). Large extrafollicular cells peaked in the PP and MLN at PID 7 and 20% and >50% were α4β7+ in the PP and MLN, respectively. By 14 PI, >50% of the large extrafollicular cells were α4β7+ in the PP and MLN, and by 4 months, these cells diminished to baseline levels in both tissues. Most RV-specific large extrafollicular cells 73 secreted antibodies. However, the kinetics of large extrafollicular cells differ from that of
RV-specific IgA ASC located in the PP. The RV-specific IgA ASC were observed in the
PP at 9 months PI, whereas large extrafollicular cells were absent in PP after 4 months,
suggesting that longed-lived IgA ASC in the PP and the large extrafollicular cells are
different populations. Characteristic GC RV-specific B cells (large IgD-B220high) peaked at PID 14 mainly in the MLN (but were also present in the PP and spleen) and persisted up to 4 months. Memory cells (small IgD-B220high) (568) peaked at PID 14 in PP, MLN and spleen, but they remained high in the PP (up to 9 months) whereas they diminished in
MLN and spleen. The kinetics of memory B cells in the PP corresponded to the kinetics of RV-specific IgA ASC in the PP suggesting that both populations could be overlapping.
Possibly, RV-specific IgA ASC detected in the PP were mainly long-lived “memory” plasma cells. The fact that IgG and IgA ASC were also detected in the BM at 9 months PI supports this possibility since various studies have described that the main location of
long-lived plasma cells is the BM in mice after systemic infection and inoculation (OVA)
(367, 494). Also, at 9 months PI, >60% of RV-specific memory B cells (small, IgD-
B220high) in the PP were α4β7+. In summary, the first RV specific B cells to be activated
after inoculation were IgM ASC and large extrafollicular B220lowα4β7- in the MLN (also observed in lower numbers in the PP and the spleen). These cells and returned to baseline levels at 28 days and 4 months PI, respectively. Large and small GC IgD- B220high B cells peaked at day 14 in the MLN and PP, but large cells disappeared 4 months PI. On the other hand, memory B cells (small GC B cells) and RV-specific IgA ASC (possibly long- lived plasma cells) peaked at PID 14 and 28, respectively and remained elevated in the
74 PP for at least 9 months. This observation suggests that long-lived memory IgA ASC
possibly have a role in protection against RV reinfection.
For the study of T cell immune responses after primary RV infection, C57BL/6
adult mice and pups and BALB/c adult mice. Adult C57BL/6 mice were inoculated with
homologous (EC G3P16) and heterologous (RRV G3P1B[4]) RV strains (255). Both
CD4+ and CD8+ IFNγ secreting T cell responses peaked at 5-7 PID and then declined
rapidly. The frequencies of RV-specific CD8+ IFNγ secreting T cells were considerably higher than those of CD4+ T cells. Furthermore, CD4+ T cell responses were higher after
RRV inoculation compared to the frequencies after homologous EC RV inoculation.
Peptide-specific T cell responses to different fractions of VP6 and VP7 were studied
independently. Responses to VP6 were mainly intestinal, whereas responses to VP7 were
more systemic (mainly liver and spleen). Neonatal C57BL/6 mice inoculated with homologous RV had two peaks of T cell responses at PID 5-7 and at PID 14. In comparison adults had only one peak at PID 5-7.
At PID 5-7 homologous RV elicited mainly responses to VP6 and heterologous responses were mainly against VP7. After homologous infection of adult mice, the highest frequencies of CD8+ T cells were found in the liver, followed by the lung, spleen
and ILP. The phenotype of RV-specific CD8+ T cells was that of an effector memory T cell (CD27int, CD44high, L-selectin-) and independent of the tissue of origin, they expressed α4β7. These observations agree with previous studies that describe CD8+ T cell migration to different nonlymphoid tissues regardless of the original site of activation
(358). In summary, both CD4+ and CD8+ T cells are activated by 5-7 days after RV infection and secrete mainly IFNγ. The peak of T cell frequencies varied mouse pups and 75 adults but generally CD8+ T cell responses were higher than CD4+ T cell responses. The
RV-specific CD8+ T cells were widely distributed throughout the body soon after
infection, were characteristically effector memory T cells and expressed α4β7 suggesting
that they were primed in the gut and not systemic tissues. Studies of knockout mice are
more descriptive in the definition of the role of each lymphocyte subset in protection
against RV reinfection and will be addressed in more detail in the following section.
1.6.2 Study of RV immunity in knockout mice
The role of B and T cells in immunity in protection against RV shedding was
studied in the adult mouse model using JHD (which lack B cells) and β2-microglobulin
(lack CD8 T cells) knockout mice, respectively. The JHD knockout mice developed similar virus shedding patterns to controls after primary infection, but after a secondary challenge, the JHD-/- mice were re-infected whereas wild type (WT) mice were protected
against reinfection. On the other hand, β2-microglobulin knockout mice challenged with
RV, developed a 2 day prolonged virus shedding compared to WT mice. It was also
observed that JHD knockout mice depleted of CD8 T cells by CD8-specific monoclonal
antibodies, developed chronic diarrhea. From these studies it was concluded that B cells
are necessary for complete long-term protection and that CD8+ T cells play a role in RV
short-term protection after primary infection and provide partial long-term protection
(198). Furthermore, it is known that in mice lacking IgA in serum and the intestine (IgA -
/-) and challenged with homologous RV, protection from RV reinfection was achieved
(405). On the other hand, in mice lacking intestinal IgA only (J-chain -/-), when vaccinating with heterologous 2/6VLPs intranasally (IN) and after challenge with 76 homologous RV, a 2 day delay in viral clearance was observed (493). These studies
suggest that possibly intestinal IgA can be substituted by other isotypes (IgM and IgG) or
other immune mechanisms (T cells, innate cells) to confer protection against RV
infection. However, the prolonged shedding observed in J-chain-/- mice might be due to
the combined lack of both intestinal IgM and IgA that are transported by pIg receptor.
These data suggests a partial role of IgM in protection against reinfection. Also, it is
known that protection against RV infection is mediated by T dependent B cells (B2) and
not B1 cells according to studies of mice with severe combined immunodeficiency
(SCID) that develop chronic RV shedding after inoculation and the observation that
adoptive transfer of total B cells, but not purified B1 cells cleared RV shedding (312).
The role of CD4 T cells was determined in wild type adult BALB/c mice (369) and C.B-17 SCID mice (312). With the aid of monoclonal antibodies against CD4, infected mice were depleted of CD4+ T cells and a chronic low level of RV antigen shedding was described. When CD4 depleted mice were further depleted of CD8+ T cells, the amount of RV antigen shed increased. The CD4 depleted mice did not develop RV- specific antibodies providing further evidence of the T cell dependent B2 type cell nature of RV-specific B cell responses. In this study it was concluded that CD4, CD8 and B cells each play a role in protection. Additionally, the individual role of CD4+T cells was
later confirmed in studies of SCID mice transferred with RV-specific CD4+ T cells, which did not produce detectable intestinal RV specific IgA but were able to resolve RV shedding. This study demonstrated the role of independent CD4+T cell responses in the
induction of RV protection against RV infection (312). To further understand the
mechanism for CD8+ T cells to confer complete short-term protection and partial long
77 term protection; perforin, fas, type I IFN receptor, stat1 and IFNγ knockout mice were
infected with RV, but lack of these effector molecules did not affect RV clearance (15,
198, 199, 303, 535).
To determine if intestinally committed B cells are necessary to confer protection
from RV infection, RAG-2 knockout mice (lacking B and T cells) were infected with RV
(568). It is known that lymphocyte trafficking to the gut is controlled by the expression of the integrin α4β7 and its ligand MadCAM-1, the chemokine receptor CCR9 and its
ligand CCL25 (TECK) and the chemokine receptor CCR10 and its ligand CCL28 (MEC)
(309, 310, 418, 568). The chemokine CCL25 (TECK) is widely expressed on crypt
epithelial and endothelial cells of the small intestine (71). On the other hand, the chemokine CCL28 is expressed in the colon, but also in other tissues such as tonsils, salivary glands, mammary glands, trachea, small intestine and appendix (418) Only mice that received passive transfer of α4β7+high IgD- (memory) B cells produced intestinal RV- specific IgA, cleared RV and were protected from RV reinfection. On the other hand, transfer of α4β7- IgD- or α4β7- IgD+ did not protect the mice and even 60 days after infection, virus shedding persisted. Furthermore, blocking of both CCL25 and CCL28
with antibodies prevented RV specific B cells from localizing in the LP (190). Either
CCL25 or CCL28 were blocked, entry of RV specific cells to LP was still observed
suggesting a redundant role of both chemokines for intestinal lymphocyte homing. Also,
RV infected CCR9 knockout mice had significant accumulation of RV-specific IgA+ plasmablasts in the LP and only when CCL28 was also blocked with antibodies, accumulation of IgA+ plasmablasts was prevented. These studies suggest that the localization of B lymphocytes to the LP is a crucial event for the induction of RV 78 immunity and subsequent protection from reinfection and that the phenotype of RV
specific B cells is characterized by the expression of α4β7, CCR9 and CCR10.
Expression of homing markers on the surface of RV specific T cells differs from
that of RV specific B cells. Mice deficient in B7 and devoid of α4β7 expression on the surface of T lymphocytes, clear RV with the same kinetics as wild-type mice but when antibodies to CD8+ T cells are injected, mice developed prolonged shedding. By
transferring B7-/- CD8+ T cells into chronically infected Rag-2 mice, RV infection is efficiently resolved. These studies suggest that the expression of α4β7 on CD8+ T cells is not essential for their role in short and long term protection against RV infection (306).
However, the expression of α4β7 on CD8+ T cells depends on the site of inoculation.
Oral inoculation induced mainly RV specific CD8+α4β7high T cells, but subcutaneous inoculation induced CD8+α4β7high and CD8+α4β7low (306). Primary activated CD8+ T
cells and most memory CD8+ T cells, regardless of the site of antigen priming, can
migrate to many uninfected tissues (358). The mechanism of CD8+ T cell “body patrol” is unclear but it is thought to be an important event in the control and elimination of viral infections. It is known that CD4+ T cells on the other hand, developed a specific
phenotype depending on the tissue of origin. The CD4+ T cells activated in the skin expressed L-selectin and downregulated α4β7, whereas CD4+ T cells activated in the gut, downregulated L-selectin, upregulated α4β7 and were highly reactive to TECK (CCL25), suggesting their expression of CCR9 (93). Small intestinal LP CD4+ T cells expressing
α4β7 were mostly CCR9+ but in studies of CCR9 knockout mice, a population of CD4+ T cells expressing α4β7 was observed in the gut suggesting a CCR9 independent
79 mechanism for CD4 intestinal homing, probably by the expression of CCR10 (190, 505).
In-vitro studies showed that CD4+ α4β7high T cells are much more reactive to RV than
CD4+ α4β7- T cells suggesting an enrichment of RV specific CD4+ T cells in the α4β7high
population. No transfer studies of RV-specific CD4+ T cell expressing α4β7high or α4β7low
have been performed so it is unclear if localization of RV specific CD4+ T cells in the gut is necessary for the induction of protection from RV reinfection. In summary, RV- specific B and CD4+ T cells in adult mice play an important role in complete long-term
protection against RV. Both subsets show selective intestinal compartmentalization by
the expression of α4β7, CCR9 and CCR10. Intestinal B cell localization (not yet studied
for CD4+ T cells) is pivotal for protection from RV reinfection. On the other hand, CD8+
T cells expressing intestinal homing markers are dispensable for complete short-term and
partial long-term protection. Apparently, CD8+ T cell homing is much less selective than that of CD4+ T and B cells.
1.6.3 Mice model for the study of innate immunity after RV infection
Stat-/- knockout adult mice challenged with RV, shed 100 times more virus than
immunocompetent mice from 2-6 days after infection but are able to resolve the infection
with similar kinetics as the infected wild type mice. This observation suggested that the
initial control of virus replication might be achieved by innate immune mechanisms.
Intestinal DCs are probably important players in this initial RV control and their
importance in the generation of RV immunity has been addressed. Using CCR6 knockout
mice, intestinal innate immunity after RV infection was studied. The CCR6 is an
important chemokine receptor that aids in the migration of immature DCs to different 80 non-lymphoid and lymphoid tissues (137). The CCR6-/- mice had significantly less RV
specific IgA antibody in stools compared to wild type mice, whereas systemic RV
specific IgG antibody was similar between knockout and wild type mice. The CCR6
knockout mice lack CD11c+CD11b+ DCs at the SED of the PP (137) suggesting a role of
these particular type of DCs in the induction of intestinal acquired B cell immunity to RV
(and possibly other intestinal pathogens) but likely other DCs might also be involved in
the induction of intestinal responses to RV.
Adult mice inoculated with intramuscular (IM) live RV developed intestinal B cell responses (131). Additionally, the transfer of unfractioned cells treated or untreated with mytomycin C purified from the draining lymph nodes of RV inoculated mice induced intestinal RV specific B cell responses in naïve recipient mice (130). These studies suggested that RV was being transported by local APC that home to the gut and were able to induce intestinal RV-specific responses. However, transfer of a pool of cells depleted of macrophages or DC did induce low quantities of intestinal IgA suggesting that contaminating virus was present in the pool, that not all DC were depleted or that B cells served as DCs and were stimulated to produce antibodies. Recently, NSP4 was detected in B220+ cells (marker of B cells and pDC), CD11c+ cells (a DC marker) and
CD11b+ cells (a marker of macrophages, natural killers, granulocytes and activated T
cells) located in the MLN (187). However, APCs engulfing infected epithelial cells and
then migrating to the MLN has to be ruled out to conclude that RV replicates in DCs and
NSP4 has been suggested to be structural protein thus, is not only expressed when virus is
replicating (146). Further studies need to be performed to prove that RV replicates inside
DCs.
81 After RV infection massive B cell activation takes place (64). This B cell proliferation was detected in the MLN and PP at PID 1 to 6. At PID 3-4 fragmented cultures derived from MLN and PP were positive for RV specific IgM and not IgA antibodies. These findings suggested that B1 cells (T independent B cells) were stimulated soon after RV infection because no other cells (e.g. T cells) were activated at this time. It was later described that RV VP7 induces a polyclonal B cell activation (61).
Although it is known that B1 cells alone can not resolve RV infection (312), RV is able to stimulate B1 cells soon after infection. The in-vivo relevance of the activation of B1 cells after RV infection needs to be studied further.
Mice are particularly susceptible to biliary atresia after heterologous RRV infection (188, 303, 438). Mice pups deficient in type I and type II IFN receptors were infected with RRV and only type I receptor deficient mice were susceptible to biliary atresia (282). Treatment with IFNα prevented the mortality due to cholestasis suggesting that after RV infection, IFNα secreting cells possibly plasmacytoid DC (pDC) are stimulated. However, similar virus titers were observed in the livers and brains derived from wild type and knockout RV infected mice suggesting that type I IFNs are important in the modulation of the induction of RV immune responses but they do not directly control RV replication. The lack of effect of IFN type I was further tested by treatment of mice with oral or intraperitoneal injection IFNα and observing that RV replication or diarrhea in pup mice were not prevented (15). Also, in the mouse model of biliary atresia, treatment with 10,000 IU of IFNα 6 hrs after RV infection resulted in identical RV titers in the liver and brain (303). Type I IFNs exhibit a partial antagonistic effect on Th1 responses by increasing the levels of IL-10, diminishing inflammatory responses by 82 inducing Tregs (408) and playing a critical role in the generation of tolerance (31, 59).
Probably, the biliary atresia induced by the lack of type I IFN is not mediated by a larger
RV load but by the lower induction of RV-specific active immunity. Proof of this concept derives from studies of IFNγ knockout mice after being exposed to RRV. The IFNγ knockout mice developed a suppressed phenotype of biliary atresia, were able to resolve jaundice and had a prolonged long-term survival compared to wild-type mice whereas the
RV titers remain similar between knockout and wild type mice (496). Mice IFNγ-/- treated with IFNγ developed bile duct obstruction after RV infection. A different study tested the development of biliary atresia by adoptive transfer of RRV-specific T cell into naïve syngeneic SCID mice and bile pathology was induced after T lymphocyte transfer in the absence of detectable infective virus (337). These studies suggest that active immunity to RV (IFNγ secreting T cells to RV) mediated the biliary duct inflammation and secondary atresia.
In summary, early in RV infection, CD11c+CD11b+ DCs located at the SED are
responsible for the generation of acquired B and T cell immune responses to RV.
Possibly pDC are also stimulated after RV infection in-vivo, but further studies are
needed for confirmation. The role of type I IFN producing cells may be to modulate
inflammatory immune responses after infection but not to prevent RV infection. Also,
RV VP7 stimulates B1 cells that apparently do not play a crucial role in protection
against RV.
83 1.6.4 Acquired RV-specific immune responses after RV infection of pigs
Immune responses to porcine [OSU G5P7, (112)] and human RV [Wa G1P1A,
(591)] have been studied in neonatal gnotobiotic pigs (470). At PID 3 with homologous virulent porcine RV, virus-specific IgM ASC were first observed in the MLN and peaked at PID 7. Intestinal and splenic IgM ASC were observed at PID 7 and returned to baseline levels at PID 29. RV-specific IgA and IgG ASC were first detected at PID 7 in all tissues
(MLN, spleen, intestine) reaching a peak at PID 14 in the MLN and spleen and at PID 21 in the gut. By PID 29 responses were lower in the systemic compartment than in the gut where mainly IgA ASC B cells were observed (470). After virulent human RV inoculation (591), ASC responses were detected in the gut, MLN, spleen and blood at
PID 8-13 and were mainly of the IgM isotype. At PID 21 similar numbers of IgA and IgG
ASC were detected in all tissues (intestinal LP, MLN, spleen and blood). The numbers of systemic IgA and IgG ASC and intestinal LP RV specific IgA ASC were similar after porcine or human virulent RV inoculation at PID 21 but the human RV strain induced higher IgG ASC in the gut (470). Both viruses induced villous atrophy and diarrhea by
24-72 hrs PI (112) demonstrating that pigs were equally susceptible to both virus strains.
The T cell responses were initially studied using lymphocyte proliferation assays (553).
Lymphocyte proliferation assays are presumably a measure of CD4+ T cell responses because of the help they provide to B and CD8+ T cells; however, T independent B cells are also able to proliferate after antigen exposure and for this reason this assay is not a specific measure of T cell immunity (61, 64). Virulent human RV-inoculated pigs developed RV-induced proliferative responses mainly in the MLN, with lower values observed in the intestinal LP, spleen and blood at PID 21.
84 Recently T cell responses were more accurately measured with the use of an
ELISPOT assay that detects cytokine secreting cells (CSC) (24). After incubating total
mononuclear cells (MNC) with RV antigen for 3 days, memory T cell responses were
determined in systemic and intestinal tissues. After virulent human RV inoculation,
responses were mainly Th1 (IFNγ, IL-12) with lower Th2 (IL-4) and T regulatory (IL-10)
CSC responses. The Th1 CSC responses were highest at PID 28 and were located mainly
in the ileum LP and spleen. Memory IL-12 CSC were detected as early as PID 5 in the
spleen and at PID 21 in the spleen and blood. The T regulatory IL-10 CSC peaked at PID
28 in the ileum LP and at PID 14 in the spleen whereas they remained high in the blood from PID 14 to 28. The Th2 CSC responses were observed mainly in the ileum LP, spleen and blood from PID 5-21. These results show that after virulent human RV inoculation, both Th1 and Th2 CSC responses were elicited. Studies by flow cytometry confirmed that effector memory CD4+ T cells and CD8+ T cells secreted IFNγ, resided mainly in the gut (592) and represent cell mediated immunity responses (CD4+ and
CD8+T cells) whereas the Th2 responses represent CSC that will induce humoral B cell mediated immunity. Both arms of immunity are important for the resolution of RV infection in the mouse model and these results suggest that in pigs these cells also play an important role in protection against RV. However, further studies that characterize CD4+ and CD8+ T cell responses to RV in pigs are needed.
85 1.6.5 Acquired RV-specific responses after natural infection in children and adults
1.6.5.1 Humoral antibody responses
Humoral immune responses are the best characterized after RV infection given the availability of antibody assays and the relative ease in obtaining serum and fecal samples from patients. Primary RV-specific responses in children are characterized by an increase in RV-specific IgM antibody titers in serum, feces and saliva with a described seroconversion rate of 100% and a coproconversion rate of 61% (PI antibody titers increased by 4 times that of the pre-infection titers) (223). Detection of serum IgM antibodies to RV is observed as early as 2 days after diarrhea starts, but more commonly occurs 6 days after symptoms begin. Also children infected with RV developed IgA and
IgG antibodies in serum (68% and 91% seroconversion rates for IgA and IgG antibodies, respectively) as well as IgA antibodies in feces (77%) but no fecal IgG antibodies were detected (18, 223). Fecal RV-specific IgM and IgA antibodies can be detected as early as
3 days after RV induced diarrhea and maximum values appeared at 11-15 days and by 5 weeks after the onset of diarrhea titers faded. Serum IgA and IgG reached a maximum peak at 33.5 days post-diarrhea (1:400 and 1:6400 for IgA and IgG, respectively) and remained elevated for at least 4 months (223). Neutralizing antibodies in stools peaked at
5-8 days post-diarrhea, but only 70% of infected children had detectable fecal antibodies whereas conversion for serum neutralizing antibodies reached 100% (140). Adults after acute symptomatic or asymptomatic infection develop mainly RV-specific IgA antibodies in serum, jejunum, stools and saliva and IgG antibodies in serum with low levels of IgM antibodies characteristic of a secondary RV infection (456, 556).
86 Correlates of immunity were studied in an adult volunteers in which 18
individuals were inoculated orally with RV serotype G1P1A (D strain). Only 5 of the 18
volunteers shed RV in feces and four of the five developed diarrhea. The virus
neutralizing antibody pre-challenge titers ranged from <20 to >5120 pfu. Homotypic and
heterotypic (G3) serum neutralizing antibody titers at >1:20 against an immunogenic VP7 epitope were correlated with resistance against infection and disease; however, not all protected volunteers developed serum neutralizing antibodies. A similar correlation with protection was observed with VP4 epitopes (220). Furthermore, neutralizing antibody titers in intestinal fluids were measured, but a correlation was not observed. Volunteers with >1:100 intestinal virus neutralizing antibody titers tended to develop less diarrhea than volunteers with <1:100 antibody titers but these differences were not statistically significant (280). Other investigators showed that serum and jejunal neutralizing antibody titers correlated with protection, but also described that not all protected volunteers were positive for neutralizing antibodies suggesting that other of immune parameters must also be involved in protection against disease and illness. In addition, serum RV specific IgG antibodies best predicted protection against infection whereas jejunal RV neutralizing antibody titers best predicted protection against illness (555). These observations were later confirmed (554); however, lack of correlation of jejunal and serum neutralizing antibody titers and protection against RV infection and disease have been described as well (556).
Infants from 1-24 months of age infected with RV were studied for serum
neutralizing antibody titers to RV and a correlation with protection against RV diarrhea
was observed. A serum antibody titer of 1:128 or greater against serotype G3 was
87 associated with short-term homotypic protection but the children were constantly
reinfected suggesting that immunity was short-lived (114). These observations were
confirmed in studies conducted in developing and developed countries (407, 458). Upon
primary RV infection children developed predominantly homotypic antibodies and the
subsequent secondary RV infections induced heterotypic responses and a broader protection against disease and infection (406). This finding agrees with the observation that after a primary RV infection, some degree of protection against disease is evident, but after two RV infections complete protection against moderate-severe disease is achieved (539). However, heterotypic and not homotypic neutralizing antibody titers were correlated with protection in a case control study conducted in children from
Bangladesh (557). In conclusion, for both adults and children exposed to RV, the role of
neutralizing antibodies in protection against disease and infection is controversial because
both correlation and lack of correlation have been described and also some adults and
children that were protected against RV reinfection, did not develop neutralizing antibody
responses.
The RV specific IgA and IgG antibody titers in serum and IgA antibodies to RV
in feces of adult volunteers receiving two doses of virulent human RV were examined
and a correlation with protection against disease and illness was observed with all
antibodies except fecal IgA. Many adult volunteers that had RV-specific IgA antibodies
in stools were not protected from reinfection. However, only serum RV-specific IgG
antibodies were significantly correlated with protection (555). Other studies performed in
adults did not find a correlation with protection and serum antibodies (556), so in adults
88 the role of serum and fecal antibodies to RV in protection against RV infection and disease is unclear.
In a cohort study in which children from a developing country were observed from birth up to 2 years of age; serum and stool sampling was performed every month.
Children with serum RV-specific IgA antibody titers >1:800 had a lower risk of RV reinfection and were protected against moderate-severe diarrhea. In addition, serum IgG antibody titers to RV >1:6400 were correlated with protection against infection but not disease. Protective antibody titers were achieved after two consecutive symptomatic or asymptomatic infections (539, 540). These observations were confirmed in a study performed in a developed country (USA) and a titer of serum IgA antibodies of >1:200 and of IgG antibodies of >1:800 to RV were correlated with protection against infection and disease (407). Conversely in a study of Danish children with RV gastroenteritis, higher preexisting serum IgA antibody titers to RV were correlated with milder symptoms (e.g. less vomiting) but serum IgG antibody titers were not correlated (240).
The authors also reported that serum IgA antibody titers were correlated with the presence of serum Ig bound to secretory component suggesting that at least some of the detected serum IgA antibodies to RV was probably of intestinal origin, lasting about 4 months after infection. Other studies had reported lower serum RV-specific IgG but not
IgA, antibodies being correlated with the development of RV-induced diarrhea (129). In addition stool IgA antibodies to RV have been previously correlated with protection against RV infection in children. Two cohort studies demonstrated that fecal virus- specific IgA antibodies correlated with subsequent protection against RV reinfection
(142, 143). The most sensitive method for diagnosis of acute RV infection was RV-
89 specific IgA conversion in stools because 92% of RV infected children coproconverted
(143). Nevertheless a substantial number of children that developed disease had elevated pre-infection IgA antibody titers to RV in stools (142, 360). It is suggested that fecal IgA to RV could be of maternal origin and perhaps the passively acquired IgA antibodies did not prevent virus infection because they lack secretory component and can not mediate virus expulsion (197). Moreover, fecal IgA antibodies are not long lasting. Most children after infection do not maintain fecal IgA antibody titers and only frequent reinfections induce long term RV-specific fecal IgA antibodies (142). For these reasons fecal IgA antibodies are not considered a good long-term marker of protection against RV infection. Because some of the serum RV specific IgA probably derives from the intestine, it lasts for about 4 months after infection and many studies have observed a correlation with protection from RV reinfection, serum IgA antibodies have been suggested to be an adequate marker of protection against RV infection and disease (197).
However others suggested that serum antibodies in general correlate with protection and perhaps, regardless of the isotype, VP4 and VP7 specific intestinal antibodies must be present to prevent RV infection (262).
1.6.5.2 B cell responses after natural RV infection in humans
Characterization of RV-induced B cell responses has been performed in children after acute infection and infected adults or exposed to RV (275). Children with acute RV diarrhea were bled early after onset of symptoms when the patients attended the emergency room and blood MNC were isolated and subjected to an ELISPOT assay for detection of RV-specific ASC. All RV positive children developed circulating IgM ASC
90 to RV (82 RV specific IgM ASC/106 total MNC), 93% developed lower IgG ASC responses (3-4 IgG ASC to RV/106 total MNC) but only 64% of the children developed
low IgA ASC responses to RV (0.02-0.08 IgA ASC to RV/106 total MNC). It was later observed that the presence of circulating IgA ASC to RV correlated with the presence of
RV specific IgA ASC in the small intestinal LP. Interestingly by magnetic isolation of
CD38+ (a marker of ASC) B cells, children apparently not recently infected with RV had persisting CD38+ RV specific ASC in the LP probably representing long-lived memory
plasma cells (87). Other studies supported the observations that circulating ASC to RV after primary infection are mainly of IgM isotype and can be detected as early as 1 day of onset of symptoms (215). Furthermore, RV infected and exposed adults developed 10-
100 times more circulating IgG and IgA ASC to RV than children and 63% and 83% of the adults developed IgG and IgA ASC respectively, reaching a peak at 5-9 days post- infection (215).
Children and adults recently infected with RV mainly had VP6 specific large B
cells (presumably activated) that expressed the intestinal homing marker α4β7 but the
ratio between α4β7+/α4β7- VP6-specific B cells was higher in adults (21.6) than
children (3) suggesting that children might have more systemic responses than adults
possibly secondary to higher viremia due to the lack of intestinal immunity (215). A more
detailed characterization was later performed in children after acute RV infection and it
was described that circulating RV-specific B cells were mainly large CD38+, CD27+
(memory cell marker), CD138+/- (plasmablast marker) , CCR6- (chemokine receptor expressed in naïve and memory cells), α4β7+ (intestinal homing marker), CCR9+ and
CCR10+ (B cell homing chemokine receptor markers), cutaneous lymphocyte antigen 91 (CLA)-(lymphocyte marker for skin migration), L-selectin+/- (peripheral LN homing marker), IgM+ IgA+/- and IgG- likely representing intestinally primed plasma cells and plasmablasts (256). However at convalescence, circulating RV-specific B cells were small and large CD38+/-, CD27+/-, CCR6+, α4β7+/-, CCR9+/-, CCR10- representing possibly both local and systemic B cells with memory phenotype (256). The CD27 was previously described to be a memory marker but more recently it has been described that high expression of CD27 can be observed in ASC (296, 393, 409). Adults demonstrated a phenotype similar to that of children at convalescence suggesting that the observed cells represented mature memory responses and are present in both children after RV infection and RV exposed and infected adults (256). Circulating memory cells to RV in adults and children still need to be correlated with protection to determine if RV-specific memory B cells in blood are responsible for the production of serum IgA antibody that previously correlated with protection against RV infection and disease.
Also it was described that in adults, infants and neonates, similar frequencies of naïve B cells (CD27-, CD19+) recognize RV VP6 but not VP7 (422). This phenomenon
has been described in mice (493) and goats (422) suggesting that this is common among
mammals but its significance in-vivo is unknown. Probably, this will aid in the
generation of stronger VP6 responses and this agrees with the fact that the most
immunogenic RV protein is VP6. Also children with acute RV infection develop B cells
with few somatic hypermutations that are mainly RV-specific CD5+ B cells (marker of
B1a cells) that characteristically secrete natural polyreactive and low affinity antibodies
(563). Between 60-87% of circulating B cells in children were CD5+ compared to 22% in
92 adults. Whether the naïve B cells observed to bind VP6 in adults, neonates and children
express CD5 and represent B1 cells needs to be investigated further.
1.6.5.3 T cell responses after natural RV infection of humans
Cellular immunity to RV has been studied in children after acute RV infection
and in adults after RV exposure and infection. Initially, cellular immunity was studied by
proliferation assays that are presumably a measure of CD4 specific T cell responses and as explained previously are not a specific measure of T cell responses. A study characterizing circulating cellular immunity to RV described that 7 of 8 children with acute RV infection had a positive homotypic and heterotypic proliferative response at convalescence (2-8 weeks, 6 of 8 children) and at late convalescence (3-5 months post- infection, 4 of 4 children) (410). Additionally, cytokine responses have been characterized in the supernatant of circulating MNC in children after acute infection. The supernatants were tested for IFNγ, IL-10, IL-8 and TNFα but only IFNγ was significantly elevated in cell supernatants from RV infected children after 24 hrs of incubation with
RV antigen compared to the cell supernantant levels from uninfected children (25).
The characterization of RV specific T cells has also been performed by intracellular cytokine staining of CD4+ and CD8+ T cells by flow cytometry after acute
RV infection in children (4-84 months of age) and healthy RV exposed or non-exposed and infected adults (255). Circulating MNC were isolated from RV positive and negative children and adults and intracellular IFNγ (Th1) and IL-13 (Th2) were measured in
CD69+ (activation marker) CD4+ or CD8+ T cells. Symptomatic adults had significantly higher activated CD4+ and CD8+ IFNγ secreting T cells than healthy adults and children 93 with acute RV diarrhea. No IL-13 responses were detected in infected or healthy adults.
Children after acute RV infection had low frequencies of CD4+ and CD8+ IFNγ secreting
T cells and, contrary to infected adults, RV infected children developed low frequencies of IL-13 CD4+ T cells. Frequencies of cytomegalovirus (CMV) specific IFNγ secreting
CD4+ and CD8+ T cells in healthy CMV seropositive healthy adults were 20 times higher
compared to RV T cell frequencies in RV seropositive healthy adults suggesting that RV
may be a weak immunogen. Also there was a tendency for slightly higher CD8 than CD4
T cell responses in both children and frequently exposed healthy adults (laboratory
workers) whereas infrequently exposed healthy adults had low and balanced CD4 and
CD8 T cell response. Using an ELISPOT assay for detection of CD4+ and CD8+ IFNγ and IL-4 CSC in blood, mainly IFNγ CSC were stimulated after RV infection whereas
IL-4 CSC were not observed (459). In accordance with the findings observed after intracellular staining detected by flow cytometry (255), after magnetic isolation of CD4 and CD8 T cells and incubation with CD14+ monocyte APCs plus RV antigen, children
demonstrated a slightly higher CD8+ T cell response whereas infrequently exposed
healthy adults had a balanced CD4+ and CD8+ T cell response. However, other investigators reported RV-specific IFNγ CSC by ELISPOT in healthy adults and when depleting CD4+ but not CD8+ T cells, most responses were abolished suggesting that RV-
specific T cell immunity in healthy adults is mediated by CD4+ T cells (283). However, this observation can also be attributed to the lack of CD4 help when depleting CD4+T cells and not to a real predominance of RV-specific CD4+ T cells after RV infection because in the previously acknowledged papers by Jaimes and Rojas et al. and by two different methods (flow cytometry and ELISPOT), CD4+ and CD8+ T cells to RV were 94 present and CD8+T cell frequencies were slightly higher in some cases. However,
Kaufhold et al. noted the importance of measuring RV-specific T cell responses by using
a RV pool that included the main human RV strains (G1-G4) to increase the chances of
detecting positive responses (283).
Additionally, CD4+CD69+IFNγ+ T cells to RV from adult volunteers predominantly expressed the intestinal homing marker α4β7 and were also enriched in the L-selectin- population contrary to CMV-specific CD4+CD69+IFNγ+ that were mainly
L-selectin+α4β7- . These results confirmed that RV induces a mixed Th1-Th2 response in
children whereas in infected adults RV induced mainly a Th1 response and intestinally
committed CD4+ T lymphocytes.
1.6.5.4 Innate immunity after RV infection in humans
Cytokines in serum detected after acute primary RV infection reflect to some extent the innate immune responses induced by the virus. However because RV-specific
T cell responses are detected as early as 1 day after the onset of diarrhea in a primary
infection (255), it is not possible to distinguish which cells are the source of the observed
serum cytokines (innate cells vs. acquired T cells). Some studies in children with acute
RV infection demonstrated that serum IL-12, IFNγ, IL-6 and IL-10 increased
significantly (263, 580) and the type I IFNα reached a peak in serum 2 days after the
onset of symptoms (156, 170). The protein MxA, induced by IFNα, also increased in
children with various acute viral infections [RV, adenovirus, CMV and respiratory
syncytial virus (RSV)] compared to the levels in PBL lysates from children with bacterial
gastroenteritis (115, 230). Type I IFN secretion is probably derived from pDC, but other 95 DCs and epithelial cells (484) secrete these cytokines and the main source of IFNα secretion after RV infection is unclear. The importance of cytokine secretion in the control of viral infections has been widely studied and in-vitro studies of human intestinal cell lines infected with RV demonstrated that the pre-treatment of human colon carcinoma cells (Caco) with IFNγ induced resistance to virus entry whereas IFNα treatment did not prevent virus internalization but diminished lipofected single-shelled
RV replication in a dose dependent manner (36). However, it was recently described that
RV NSP1 protein inhibits the synthesis of type I IFNs by proteosome degradation of the
IRF3 (34). Because after RV infection, IFNα is observed, the in-vivo significance of
IRF3 degradation is unclear.
The epithelial cell responses after RV infection have been mainly studied in-vitro using human intestinal colonic cell lines. Studies of HT29 and Caco cells demonstrated that after RV infection, IL-8, RANTES, the growth related peptide alpha, IP-10 (a C-X-C chemokine), granulocyte-monocyte colony stimulating factor (GM-CSF) and IFNα mRNAs were up-regulated (98, 460). The peak of the epithelial response occurred between 3-16 hrs post-infection and lasted for more than 24hrs. However it was observed that 2/4/6/7VLPs induced IL-8 secretion by HT29 cells whereas incubation with
2/6/7VLPs induced lower quantities of IL-8 production suggesting a role of VP4 in the activation of epithelial cells after infection (460).
The mechanism of generation of cytokine responses by RV infected epithelial cells is unclear, but it was recently shown that RV dsRNA induced TNFα, IFNβ and IL-6 mRNAs as well as apoptosis and a delay in epithelial cell migration to repair the disrupted monolayer caused by RV infected dead cells (484). The TLR3, that specifically 96 recognizes dsRNA, was detected at the mRNA and protein level in various intestinal
epithelial cell lines (IEC-6, HT29 and Caco). The abnormal monolayer repair was in part
TLR3 dependent, because after the addition of TLR3 antibodies, cell migration was
promoted. These observations suggest that TLR3 induction promotes apoptosis and
prevents cell migration by mechanisms still unclear, but probably also aids in
containment of the viral infection. However it is highly possible that these effects are
related to RV pathogenesis. A study of the expression of various TLRs in PBLs from
children with acute RV infection confirmed the up-regulation of TLR3 in-vivo as well as
TLR8 (that recognizes ssRNA together with TLR7) during the first 3 days of illness
(580). It is known that via TLR3 signaling, NF-κβ and the IFNβ promoters are activated
(361) and both mRNAs are known to be up-regulated after RV infection of human cell lines in-vitro (460). However, addition of TLR-3-specific antibodies does not prevent secretion of IL-12, IFNα or IFNβ by immature DCs exposed to RV dsRNA (361)
suggesting that other pathways are also involved in the induction of cytokine secretion by
immature DCs. Other dsRNA recognition molecules that also induce NF-κβ like RIG-1,
mda-5 and PKR might be involved in the induction of cytokine responses by innate
immune cells and epithelial cells after RV infection (164) and RV induced activation of
these molecules and their pathways have not been explored.
Human DC activation after RV exposure has been studied in immature and
mature mDC in-vitro. The DC maturation was not altered after RV exposure and
immature DCs secreted mainly IL-6 whereas mature DCs secreted IL-6 and low amounts
of IL-12 and IL-10. Also, intracellular NSP4 was observed in 4-46% of mature DCs but
rarely in immature DCs suggesting that possibly virus replication occurred to some extent 97 and was promoted only in mature DCs for unknown reasons however with the recent evidence that NSP4 is a structural protein (146), virus replication in DCs needs to be studied further (394). The majority of viruses that replicate in DCs do so in immature rather than mature DCs and it was suggested that because immature DCs have a higher endocytic capacity this facilitates virus cell entry and subsequent replication (318).
However, some viruses like RSV (35) and certain strains of CMV (236) infect mature
DCs more efficiently as suggested for RV.
The contribution of innate immuinity in the initial control of RV replication and in the induction of RV pathogenesis is unclear and requires further characterization. Also, important differences in the innate immune responses to RV in neonates vs. adults might aid in the design of more efficient mucosal vaccines able to induce stronger immune responses by the immature neonatal immune system or overcome the suppressive effects of maternal antibodies.
1.7 ROTAVIRUS VACCINES
As previously stated RV mortality, morbidity and hospitalization costs in developed and developing countries demanded the urgent availability of a RV vaccine for infants worldwide. The licensed RV vaccines (RotaShield®, Rotarix®, Rotateq®) for human use are based on live replicating viruses that do not induce diarrhea. However, as noted previously, delivery of replicating vaccines was associated with a risk of intussusception, transmission to susceptible individuals (such as immunosuppressed
98 patients) and possible mutation to more pathogenic strains. For these reasons, non-
replicating vaccine approaches are still under development. In this section, a summary of
the different replicating and non-replicating RV vaccines (inactive RV, VLP and DNA
based vaccines), the animal models used for their testing and the RV vaccines designed
for human use will be presented.
1.7.1 Replicating and inactivated RV vaccines studied in the mouse model
Sucking mice inoculated with homologous (virus strain that normally infects the
animal species to be tested) or heterologous RV (virus strain that infects animal species
different from the one tested) develop diarrhea in 100% of the cases but shedding varied
from 85-100% (189). Primary inoculation of neonatal mice with high doses of RRV
(G3P[3]) induced diarrhea and shedding and subsequent challenge with an homologous
murine virus (G3P[16]; EC RV) conferred complete protection against reinfection and a correlation with RV-specific fecal and serum IgA but not serum IgG was described (189).
Contrary to neonatal mouse pups, adult mice do not develop diarrhea after virulent virus inoculation and only infection can be assessed in this model. By using a jennerian vaccine approach (using a heterologous live virus as candidate vaccine) adult mice were inoculated with RRV, bovine NCDV (G6P[1]), recombinant DxRRV(G1P[3]),
DS1xRRV(G2P[3]), ST3xRRV(G4P[3]) and protection was achieved after challenge with homologous RV (192) . Moreover, inoculation with inactivated triple or double layered particles together with the saponin adjuvant QS21 intramuscular (IM) induced similar and complete protection rates against shedding (368). Because neonatal mice are susceptible to RV diarrhea only during the first week of life, vaccination and assessment
99 of protection against illness is usually studied in adult mice that no longer are susceptible
to diarrhea. A different approach to measure protection against disease in the mouse is to
vaccinate the dams and challenge the offspring while still newborns to determine the
effect of RV-specific antibodies transferred through the placenta or via nursing.
Following this approach, IM formalin inactivated RRV combined with lipid adjuvants
was administrated to female Balb/c mice. After vaccination was completed, mice were
impregnated and RV challenge was performed in the offspring pups. Protection against
diarrhea was observed in all cases (270). All these studies suggest that neonatal or adult
mice can be easily protected from RV infection after inoculation with replicating or
inactivated vaccines plus adjuvant.
1.7.2 DNA vaccines evaluated in the mouse model
Recombinant DNA genetically engineered to express various RV capsid proteins
have been tested in mice as candidate RV vaccines. DNA expressing a group A murine or bovine RV VP6 was delivered IM and induced high titers of serum IgA and IgG to RV but no fecal IgA. Only partial protection was achieved after challenge with homologous
RV and viral antigen detected at PI day (PID) 2 reached similar amounts to that observed in control mice (582). Interestingly, by immunizing orally or IN with VP6 DNA, intestinal anti-VP6 IgA ASC were induced and reduction in RV shedding was observed for 7 days after challenge compared to controls or mice vaccinated with VP6 DNA IM
(203). These results suggest that mucosal vaccination induced higher protection rates that probably correlated with intestinal IgA ASC responses and systemic vaccination was less protective because it was less effective in the induction of intestinal RV responses.
100 Subcutaneous (SQ) immunization with VP7 and VP4 DNA induced neutralizing
antibodies in serum and after challenge with homotypic RV (a strain with the same G and
P serotype), protection against shedding was achieved and the protection rates were
similar after vaccination with individual VP4, VP7 or VP6 DNA (234). Similarly, oral inoculation with VP4 and VP7 encapsulated microparticles induced intestinal IgA antibodies and protection against homotypic RV challenge (235). Interestingly, fecal IgA antibodies was induced by SQ inoculation and was higher after VP6 DNA injection than
VP4 or VP7 DNA vaccination (111). These results suggest that to achieve protection in adult mice, neutralizing antigens are dispensable and SQ is an optional route for the induction of fecal IgA antibodies to RV.
1.7.3 Purified capsid proteins as vaccines in the mouse model
Mice injected with supernatants of insect cells (Sf9) infected with recombinant insect viruses (baculovirus) expressing VP6, VP4 and VP7 (RRV origin) were used as donors of CD8+ T cells isolated after immunization. Immunodeficient SCID mice
inoculated with murine RV that develop chronic shedding were later injected with CD8+
T cells derived from mice immunized with the individual proteins and virus clearance after transfer was achieved (163). This study showed that VP6, VP4 or VP7 specific
CD8+ T cell mediated immunity induces effective heterotypic viral clearance. The immunogenicity of RV proteins was also assessed in neonatal mice. Spherical particles made of VP6 (BRV) and joined to immunogenic VP7 or VP4 peptides were used to inoculate the dams and the offspring were challenged with homologous or heterologous
RV. It was shown that VP6+VP4 was protective against multiple virus strains (BRV, DS-
101 1, Wa, SA11) and induced slightly higher protection than VP6+VP7 (247). Conversely, immunization of dams with baculovirus expressed VP5* and VP8* was not as effective
as the inoculation with the immunogenic VP4 peptides in the induction of protection
against diarrhea in the pups (172). However VP8* expressed in E.Coli or tobacco mosaic
virus (TMV) systems was shown to induce protection in the offspring of immunized
dams (208, 434) suggesting that perhaps some expression systems might be more
effective for the production of immunogenic peptides and proteins or that some systems
are more efficient than others and generate higher amounts of antigen. Additionally, IN
immunization of dams with VP4 expressed in an adenovirus vector induced higher
protection rates than oral or IM inoculation and IN inoculation induced the highest
antibody titers in serum, intestine and milk (331) suggesting that this vaccine vector may
be better expressed at IN sites compared to the muscle or the gastrointestinal tract or that
the IN route might be more effective for induction of higher intestinal responses and
protection in the mouse.
Also, VP6 administered with maltose binding protein (MBP) or labile toxin (LT)
from E.Coli induces protection against shedding after immunization of adult mice (118,
119) indicating that adult mice develop protective immunity after vaccination with
neutralizing (VP4 and VP7) or non-neutralizing antigens (VP6). However, VP6
administered to mouse pups failed to protect against shedding 10 days after inoculation, whereas in the same study, adult mice inoculated with the same vaccine and challenged also at inoculation day 10, were protected from RV infection (537). Apparently, contrary to mechanisms for induction of immunity against shedding in adult mice, where
102 neutralizing antigens might be indispensable for the generation of protective immunity, in neonates neutralizing antigens may be essential for protection.
1.7.4 The VLP as candidate vaccines in the mouse model
VLPs are produced by self-assembly of the individual RV structural proteins coexpressed by baculovirus recombinants in insect cells. Additionally, VLPs have been shown to be antigenically similar to the native RV by proving that inoculation with
2/6/7VLPs or inactive virus induces similar magnitude of immune responses in the mice
(347). Double-layered (2/6VLPs) and triple layered VLPs (2/6/7VLPs) were tested with cholera toxin (CT) in adult mice by oral and IN routes. Only VLPs inoculated by the IN route induced protection against shedding after homotypic challenge in all cases and inoculation with 2/6 or 2/6/7VLPs induced similar protection rates demonstrating again that IN immunization is the best route to induce protective immunity in the mouse (404).
Heterotypic responses to VLPs were studied by vaccinating mice with G1 VLPs or G3
VLPs (QS21 as adjuvant) and challenging with a G3 or G1 homologous virus, respectively. Vaccination with G1 VLPs induced a partial but high protection (88%) against a G3 RV challenge whereas G3 VLP vaccination induced a complete protection against a G1 RV challenge (261).
It is known that the inbred Balb-c mice are less susceptible to RV infection than the outbred CD-1 mice, so depending on the strain used to test the candidate VLP vaccines, variable protection rates might be obtained (65). When a more susceptible mouse strain (CD-1) is vaccinated with 2/6, 2/6/7 or 2/4/6/7VLPs (with LT as adjuvant) and challenged with high doses of homologous RV, similar intermediate protection rates
103 (54-66%) are achieved with the different VLP preparations compared to the higher protection rates achieved after inactivated RV vaccination (90%). Using the same susceptible strain it was also observed that IN vaccination was significantly more efficient than oral vaccination and an oral/IN combination of regimes did not improve the protection rates achieved. These results suggest that other components in the native particles like dsRNA might be important to induce complete protective immunity.
However, higher protection rates were reported after IN vaccination of CD-1 mice with
2/6VLP+LT (∼80%) but probably, these results are statistically similar to the previously reported protections and reflect just variability among individual studies of mice (50).
The intrarectal (IR) route was recently tested in adult Balb-c mice, which is a more resistant mouse strain to RV infection. After inoculation with 2/6VLP with CT as adjuvant or VP8-2/6/7 (with VP8 inserted in VP2) with CT or LT as adjuvants, and challenge, none of the inoculated mice developed RV shedding (7, 421). However, when inoculating the mice IN with 2/6VLP or IR VP8-2/6/7VLP with CpGs as adjuvant, no protection was achieved. These results demonstrated that the IR route might be an effective route to induce protective immune responses in the adult mouse model of RV shedding.
1.7.5 Correlates of immunity after RV vaccination in the mouse model
The mouse model allows the study of the role of each arm of immunity in protection against many pathogens because of the availability of genetically engineered mice strains. With the aid of immunodeficient mice, it has been determined that B lymphocytes, CD4+ and CD8+ T cells play a role in protection against RV infection;
104 however, they all play a different but overlapping role. It has been shown that B cell
immunity is necessary to achieve robust long-term immunity to RV, whereas CD8+ T cells are needed for long term and partial short term protection against reinfection (198).
Because most RV-specific B cell responses are T cell dependent or of B2 derivation,
CD4+ T cells play an indirect but indispensable role by inducing the activation of B cells and CD8+ T cells (312).
In mice systemic and intestinal IgA and IgG B cell responses are induced after
RV infection; however, only after transfer of intestinally committed B cells (that express
α4β7) does RV clearance occur demonstrating that somehow, intestinal B cell responses
are more protective than systemic responses (568). In most cases in which protection is
achieved, intestinal IgA is induced; however, when mucosal IgA is lacking (e.g. IgA
knockout mice), if RV-specific IgG or IgM are present in the gut, protection can still be
achieved. Moreover, when sufficient serum IgG or IgM to RV are present, even when
intestinal or serum IgA are lacking, protection is accomplished by mechanisms not
completely understood. Perhaps serum transfer of Ig to the intestinal lumen mediates the
observed protection and it is known that IgG can be transfered via Fc receptors on the
intestinal epithelial cells from the serum to the gut (307, 405, 506). Local antibody
mediated protection occurs by two mechanisms: First, virus expulsion may occur during
transcytosis of VP6-specific polymeric IgA or IgM from the basolateral membrane to the
intestinal lumen. Antibodies to VP6 recognizing intracellular viral cores during
transcytosis, can inhibit RV replication (90, 139, 191). The second mechanism is virus
exclusion that is probably mediated by VP7 and VP4 specific antibodies that bind TLPs
105 and prevent virus cell entry or uncoating (197). Furthermore, antibodies against the enterotoxigenic NSP4 have also been shown to be protective in mice (29).
As previously described, VP6 immunization protects adult mice but not neonates.
Furthermore, passive transfer of VP6 specific antibodies induces protection in adult but not sucking mice (90). Only by passive transfer of VP5* or VP8* specific antibodies, are neonates protected from infection (463). These studies demonstrate than VP6 antibodies are only protective for adult mice and for reasons not completely understood, neonates depend on antibodies to neutralizing antigens to achieve protection.
1.7.6 Vaccination studies in the gnotobiotic pig
The neonatal gnotobiotic pig is an animal model susceptible to homologous and heterologous RV (e.g. human RV) infection and unlike any other adult animal model
(e.g. mice, rabbit, monkeys), a model for RV disease (473). Pigs are susceptible to RV induced disease for at least 8 weeks of age allowing sufficient time for inoculation with multiple vaccine doses and subsequent virulent RV challenge. The immune and gastrointestinal systems of neonatal pigs resemble that of human infants making it an optimal model for the study of protective immunity induced by human RV (HRV) vaccines. Additionally, due to their epitheliochorial type of placenta, pigs are born devoid of maternal antibodies facilitating study of the immune responses induced after vaccination (473). Pigs are readily infected with RV as newborns and the gnotobiotic or gnotobiotic status assures that the pigs are RV-free which is ideal for the development of vaccine and challenge studies. In addition, gnotobiotic pigs develop similar diarrhea, shedding and antibody responses to RV regardless of their colonization with lactobacillus
106 species suggesting that lack of commensal flora does not interfere with RV replication, pathogenesis or induction of B cell responses (597). For all these reasons, the neonatal gnotobiotic pig model of HRV disease is optimal for the study and evaluation of human
RV vaccines.
Primary inoculation with virulent HRV induces 88% protection rates against
diarrhea and 100% against virus shedding respectively and after challenge, approximately
100% of the pigs achieve homologous protection against diarrhea and shedding (587).
Vaccinating with two doses of live attenuated (Att) human RV delivered orally induced a protection rate of 31% against diarrhea and 17% against shedding whereas three oral doses increased protection rates to 62% and 67% against diarrhea and shedding, respectively, demonstrating that 3 doses of AttHRV were optimal to induce protective immunity (587, 590). On the other hand, oral or IM vaccination with inactivated virus, although inducing high titers of serum virus neutralizing (VN) antibodies, failed to protect against shedding and only a minimal protection rate against diarrhea was achieved
(590).
Because the live AttHRV vaccine (RotaShield) was associated with intussusception, a non-replicating vaccine approach is desirable and VLPs and DNA vaccines were tested as candidate vaccines in pigs. Three doses of oral 2/6VLPs delivered
with mutant LT (mLT) or immune stimulating complexes (ISCOM) as adjuvants did not
protect against shedding and conferred minimal or no protection against diarrhea (248,
586). Similarly, three IM doses of VP6 DNA did not confer protection against shedding or diarrhea (585). Only a combination of oral AttHRV and 2/6VLPs induced moderate protection rates against diarrhea (44-58%) and shedding (58-75%) with the highest
107 protection rates induced by using ISCOM as adjuvant (248, 589). However, oral AttHRV priming followed by IN boosting with 2/6VLP and ISCOM as adjuvant, induced slightly higher protection rates against diarrhea (71% against shedding and diarrhea) (216). From these studies it was concluded that for the induction of protection against disease in neonatal gnotobiotic pigs, neutralizing antigens are essential, the ISCOM adjuvant was a promising adjuvant because it was safe and induced slightly higher protection rates against diarrhea and the oral/IN immunization routes were effective in the induction of protective immune responses in the gnotobiotic pig model.
1.7.7 Correlates of protection after RV vaccination of neonatal gnotobiotic pigs
Primary inoculation with virulent virus confers the highest protection rates observed after RV challenge compared to any vaccine to date. Virulent virus inoculation also induces the highest RV-specific intestinal IgA ASC compared to responses induced by two or 3 doses of AttHRV (591). Vaccination regimes inducing the highest protection rates also induce the highest intestinal IgA ASC and IgA antibody titers in intestinal contents (22, 248, 400, 585, 589). On the other hand, neutralizing antibodies as correlates of protection are more controversial. Vaccination regimes that induce high neutralizing antibody titers do not always induce protection against reinfection (591); however, complete lack of neutralizing antibodies usually leads to no protection (586). Probably serum neutralizing antibodies do not reflect the presence of intestinal neutralizing antibodies that are usually difficult to test and potentially more correlated with protection.
Perhaps, neutralizing antibodies together with other arms of the immune response might be necessary for the induction of protection against reinfection. Additionally, in the
108 neonatal gnotobiotic pig, it was also showed that NSP4-specific antibodies alone do not
confer high protection rates against shedding or diarrhea (586, 588).
1.7.8 Human RV vaccines
The first time a human RV was cultured was after the passage of a RV-infected
human stool suspension in gnotobiotic pigs followed by passage of the RV+ stool preparation in monkey kidney cell cultures (577). Subsequently, RV could be easily grown in cell culture with the specific aim of developing a vaccine (531). The first human
RV vaccine was a live attenuated Wa (G1P1A[8]) strain administered to adult volunteers in phase I clinical trials for study of safety and antigenicity (279) but further efforts were focused on in the development of a Jennerian approach to vaccines. The most common
RV strains infecting humans are group A RV serotypes G1, G2, G3 and G4 so vaccine formulations were produced to include these four common RV serotypes with a group A
RV VP6 (277).
Around 200 years ago, Edward Jenner established the use of cowpox virus as a vaccine for the prevention of small pox in humans. A common vaccination strategy follows the Jennerian approach in which live virus strains infecting animal hosts are used as attenuated vaccines for human use (277). The Jennerian approach was followed to produce three RV candidate vaccines: two bovine strains (NCDV and WC3) (127) and a
RRV (278). The RRV strain was tested in phase I clinical trials for safety and antigenicity, initially in adults and later in older, younger children and infants less than 6 months (278). The vaccine induced diarrhea in infants over 6 months of age but was safe and immunogenic in infants under 6 months inducing only febrile episodes 3 or 4 days
109 after vaccine inoculation and apparently, maternal antibodies were critical modulators in the control of the secondary effects (544). The Jennerian RRV vaccine was tested in phase II trials and children were vaccinated with 104 or 105 pfu/dose orally (277). The
efficacy of the vaccine varied from 0-85% against severe diarrhea. The highest vaccine
efficacy was detected in Venezuela where the predominant circulating strain shared the
same serotype as the vaccine (G3), suggesting that heterotypic responses were not being
elicited after vaccination (196).
A modified Jennerian approach was adopted to achieve a broader antigenic coverage. Three RRV reassortants were generated by the co-cultivation of RRV plus one of a G1, G2 or G4 human RV exerting selective pressure by adding antibodies to RRV.
The resulting vaccine was a parental RRV strain with a substitution of single gene encoding for VP7 serotype G1, G2 or G4 (277, 378) . The goal was the incorporation of the four most common RV serotypes into a single quadrivalent RV vaccine (G1, G2 and
G4 from human RV origin and G3 from the parental strain). Before combining the three reassortants with the parental strain, each reassortant was tested in phase I clinical trials for safety and immunogenicity and reactions similar to the parental strain were also described (195). In phase II clinical trials, the efficacy of the individual vaccines varied according to the region where the vaccine was tested being moderate in Finland and
Rochester, USA and low in Peru (315, 546).
The reassortant strains were combined for the generation of a quadrivalent vaccine that was evaluated in phase I clinical trials for reactogenicity and immunogenicity. The vaccine combination was safe and only febrile reactions were occasionally observed (195). The vaccine failed to induce neutralizing antibodies and the
110 individual doses of each reassortant strain had to be increased to 105 FFU (195). Phase II trials were performed with the individual reassortants or the tetravalent vaccine formulation in more than 10,000 children worldwide (277) . In a study performed in the
United States during a RV season were a G1 serotype was predominant, the efficacy of the quadrivalent vaccine and one reassortant strain (DxRRV) was 64% and 69% protection against diarrhea, respectively. During a second RV season where only 1/3 of the strains were G1 serotype, the monovalent reassortant vaccine conferred only 7% protection against disease whereas the quadrivalent vaccine conferred a 48% protection rate (47) thus supporting the use of the quadrivalent vaccine formulation. However, when
analyzing the protection rates by diarrhea score, the quadrivalent vaccine conferred 80%
protection against severe diarrhea whereas the monovalent vaccine conferred 69%
protection during the first RV season where the majority of infecting strains where G1
serotype. The protection rates against severe diarrhea during the second season were 77%
and 45% by the quadrivalent vs. the monovalent vaccines, respectively (453). Similar
high protection rates conferred by the higher dose of quadrivalent vaccine were also
observed in Venezuela, Finland and native American populations (265, 266, 433, 481).
The vaccine was later approved by the Food and Drug Administration (FDA) for human
use in June, 1998 and Wyeth Laboratories manufactured and distributed the vaccine
(Rotashield) for immunization of infants with three oral doses at 2,4 and 6 months of age
(277).
In July 1999, the Centers for Disease Control (CDC) reported that between
September 1, 1998 and July 7, 1999, 15 cases of intussusception occurred following the
administration of the rotavirus vaccine to almost 1 million infants (2). Later the CDC and
111 the American Academy of Pediatrics (AAP) postponed the administration of the vaccine
until further data supported the lack of association between vaccination and
intussusception (3). It was later confirmed that the rate of intussusception after RV
vaccination was 1 in 12,274 infants vaccinated and recently a rate up to 1 in 16,000 has
been described (53, 277) with the highest risk at one to two weeks after the first dose of
inoculation of the quadrivalent RV vaccine.
1.7.8.1 New generation RV vaccines
New RV vaccines were recently licensed in many countries with promising
protection rates against all types of diarrhea but particularly severe diarrhea. One vaccine
is a monovalent vaccine consisting of attenuated live HRV (Rotarix) with a G1P1A[8]
serotype strain RIX4414, the most common RV serotype infecting infants worldwide.
Two oral doses at 104.7, 105.2 or 105.8 FFU or placebo were administered to healthy
children concurrently with other routine vaccines (against diphtheria, tetanus, pertussis,
hepatitis B and Haemophilus influenza type b) and two weeks before or after the oral polio vaccine (329). Around 31,000 children were vaccinated in 11 Latin American countries and Finland at approximately two and four months of age (464). The efficacy of
the vaccine against severe RV diarrhea and associated hospitalizations was 85% reaching
100% against the most severe diarrhea cases. Protection against homotypic infections was
91% whereas protection against heterotypic infections was only 45%. Furthermore,
diarrhea due to any cause was significantly reduced by 42% in the vaccine group
suggesting that many of the undiagnosed cases were secondary to RV. No increased rates
of intussusception were detected in the vaccine group compared to the placebo (464). The
112 vaccine is currently licensed in the European Union, 15 Latin American countries and 29
other countries including several in Asia (46).
The other vaccine approved for use in the United States, Canada and several Latin
American countries, is a Jennerian approach pentavalent human-bovine (WC3)
reassortant vaccine (Rotateq) including the most common G and P serotypes circulating
(G1-4 and P1A[8]). Approximately 34,000 children were vaccinated in phase II clinical
trials and the protection rate conferred against severe diarrhea and hospitalization was
98%. Protection against all types of diarrhea was 74%. Intussusception cases did not differ between vaccinated and placebo groups. Heterotypic protection against G9 and
G12 serotypes was 100% (545).
Only when millions of children receive the new RV vaccines, will the real risk of
intussusception be known and until then, different vaccine approaches, like non-
replicating vaccine approaches must be tested and available for human use in case the
live vaccine approaches fail to be safe. Approximately three million doses of Rotateq
have been distributed worldwide since its approval on February 3rd, 2006. Recently, a
notification from the FDA was released to the public to alert for the possible association
of intussusception with the administration of the pentavalent vaccine Rotateq that now is
widely distributed to children all over the U.S, Latin America and Canada
(http://www.fda.gov/medwatch/safety/2007/safety07.htm#RotaTeq). However, the CDC posted a statement to this later alert indicating that the cases observed did not exceed the naturally occurring frequency of intussusception in the population that is 18-43 cases in
100,000. Only 28 children vaccinated with Rotateq developed intussusception with 16 patients requiring surgical intervention and no mortality observed. The CDC replied they
113 were not surprised with the cases reported so far. Nevertheless all health care providers or
any other individual were advised to report any observed case of intussusception after
Rotateq vaccination to the Vaccine Adverse Event Reporting System (VAERS)
(http://www.cdc.gov/od/science/iso/concerns/rotavirus.htm). Rotavirus disease causes an unacceptably high mortality and morbidity worldwide and RV vaccines are necessary to prevent mortality in developing countries and morbidity in developed countries.
1.8 REFERENCES
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3. 1999. Withdrawal of rotavirus vaccine recommendation. MMWR Morb Mortal Wkly Rep 48:1007.
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179
CHAPTER 2
ANTIBODY RESPONSES TO HUMAN 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.
2.1 SUMMARY
Safer and more effective human rotavirus (HRV) vaccines are needed. We
evaluated oral priming with attenuated WaHRV (AttHRV) followed by boosting with 2 intranasal (IN) doses of VP2/6 virus-like particles (2/6VLP) with immunostimulating complexes (ISCOM) to determine if this regime induces protection against diarrhea and viral shedding in the gnotobiotic pig model. IgM, IgA and IgG antibody titers in serum and intestinal contents were quantitated by ELISA and serum neutralizing antibody titers
180 were measured by a virus neutralization (VN) test. Seven groups of neonatal gnotobiotic pigs were vaccinated at post-inoculation days (PID) 0, 10 and 21 and challenged with virulent WaHRV at PID28. The vaccine groups included: 1 & 2) Oral priming with
AttHRV and boosting with 2 IN doses of 2/6VLP-ISCOM (Att+2/6VLP-ISCOM) at VLP concentrations of 250µg or 25µg; 3 & 4) 3 IN doses of 2/6VLP-ISCOM at VLP concentrations of 250µg or 25µg (2/6VLP-ISCOM); 5) 3 oral immunizations with
AttHRV (3xAttHRV); 6) 1 oral immunization with AttHRV (1xAttHRV); 7) Controls
(ISCOM matrix and/or diluent). The pigs that received 3xAttHRV or Att+2/6VLP250-
ISCOM had the highest protection rates against diarrhea upon challenge at PID28 with virulent WaHRV. The IgA antibody titers to HRV in intestinal contents were significantly higher in the Att+2/6VLP250-ISCOM group than in all other groups pre- challenge (PID28). Serum VN antibody titers were statistically similar after the first inoculation among the groups given AttHRV, but at PID28 VN antibody titers were significantly higher for the 3xAttHRV and Att+2/6VLP250-ISCOM groups than for the
1xAttHRV group suggesting that boosting with 2/6VLP also boosted VN antibody responses . In humans, intestinal IgA antibodies have been correlated with protection against symptomatic reinfection. Thus the vaccine regime of one oral dose of AttHRV and two IN doses of 2/6VLP250-ISCOM may be an alternative to multiple-dose live oral vaccines in humans.
2.2 INTRODUCTION
Rotavirus gastroenteritis is responsible for the deaths of 600,000 to 870,000 children worldwide, with the highest impact in developing countries (10). Recently, the
181 first licensed rotavirus vaccine, a tetravalent reassortant rhesus rotavirus, was associated
with an increased risk of intussusception and was withdrawn in Oct, 1999 (22, 36).
The gnotobiotic pig model of human rotavirus (HRV) induced diarrhea has the
advantage of susceptibility of pigs to HRV induced disease, a lack of maternal antibodies
and similarity to infants in development of mucosal immunity (34). We studied a new
prime/boost strategy for rotavirus vaccination using oral priming with attenuated HRV
(Wa strain) followed by boosting with two intranasal (IN) doses of recombinant VP2
(from RF bovine rotavirus)/VP6 (from Wa HRV) virus-like particles (2/6VLP). This same regime induced partial protection and intestinal antibody secreting cell (ASC)
responses in gnotobiotic pigs using 2/6 VLPs with mutant E. coli heat labile-toxin (mLT)
as adjuvant. (58% and 44% protection rates against virus shedding and diarrhea,
respectively) (44). In the same study priming with 2/6 VLP + mLT followed by boosting
with oral AttHRV was also examined, but this vaccine regime induced only low
protection rates, so it was not repeated in the present study. Although we previously
studied ASC responses in systemic and intestinal tissues after oral AttHRV priming and
oral 2/6 VLP boosting (18), neutralizing and isotype antibody responses in serum and
intestinal contents following the use of 2/6VLP vaccines with ISCOM adjuvant
administered IN have not been examined. Analysis of such antibody responses is
important for comparison with the corresponding serum and fecal antibody responses in
human infants given rotavirus vaccines.
Double-shelled VLPs were generated using recombinant baculoviruses expressing
the individual rotavirus proteins VP2 and VP6 (24). The rotavirus inner capsid is
composed of the VP2 core and surrounded by VP6, the major inner capsid protein (5, 14).
182 In the murine model, the generation of non-neutralizing IgA monoclonal antibodies to
VP6 using a back-pack tumor was sufficient to protect adult mice against primary rotavirus infection and induce viral clearance in chronically infected mice (4). In contrast, in sucking mice, only IgA VN antibodies to the VP8 subunit of VP4, but not IgA antibodies to VP6 were protective against diarrhea (32). Because it accounts for more than 50% of the virion mass, VP6 is a dominant antigenic target for HRV specific IgA antibodies detected in fecal specimens (11, 14, 20) but its role in eliciting protective immunity is controversial.
Immune stimulating complex (ISCOM) are cage-like structures composed of
cholesterol and Quillaja saponins (27, 31).They stimulate activation of lymphocytes
through the production of pro-inflammatory cytokines and subsequent leukocyte
migration (1, 2) ISCOM have been previously used as adjuvants and delivery vehicles
with appropriate antigens against a variety of pathogens in different animal models and
humans (13, 18, 31, 38) . Only in our previous studies, have ISCOM been used with
VLPs to elicit intestinal immunity to rotavirus (18).
Intestinal (or fecal), and in some studies, serum rotavirus-specific IgA antibody titers correlate with protection against reinfection in humans and in different animal models. In children with acute rotavirus infection, higher serum titers of rotavirus- specific IgA antibodies were correlated with less severe symptoms (8, 19). Other researchers demonstrated that children with higher serum rotavirus-specific IgA antibody geometric mean titers (GMT) were better protected against reinfection (19, 37). Children
183 that had persisting high titers of rotavirus-specific IgA antibodies in stools, showed lower
rates of reinfection (12).Vaccination studies in pig and mouse models, revealed intestinal rotavirus-specific IgA antibody titers and ASC were also associated with protection after challenge with virulent rotavirus (15, 42-46). Serum neutralizing antibodies have also been correlated with protection against secondary HRV infection in children (6, 7) but there is still controversy about their role in protection (6, 8, 40). Higher serum HRV- specific IgG antibody GMT were also correlated with protection in some studies (19, 37) but not in others (15).
In this study we determined rotavirus-specific IgA, IgM and IgG antibody titers in serum and small and large intestinal contents by ELISA and serum neutralizing antibody titers by a VN assay. Rates of protection induced by oral immunization with AttHRV, IN doses of recombinant (bovine-human) 2/6VLP with ISCOM matrix as adjuvant
(Att+2/6VLP-ISCOM) or the two vaccines combined in a prime/boost strategy were compared. Prior rotavirus vaccines were based on three doses of AttHRV so the responses detected in the Att+2/6VLP-ISCOM group were compared with those obtained from pigs that received three oral doses of AttHRV (3xAttHRV). A group that received three doses of 2/6VLP (2/6VLP-ISCOM), a vaccination regime previously shown to be unprotective in pigs when used with E. coli mLT adjuvant (42), was also studied to determine if ISCOM would stimulate higher and protective immune responses. Finally, to determine the booster effect of the 2/6VLP-ISCOM on the responses elicited by the
Att+2/6VLP-ISCOM vaccine, we vaccinated a group of pigs with AttHRV alone
(1xAttHRV).
184 2.3 MATERIALS AND METHODS
Virulent rotavirus: Virulent Wa HRV (P1A, G1) was pooled from intestinal
contents of gnotobiotic pigs after the 18th pig passage. Pigs were challenged with virulent
6 HRV at a dose of 10 ID50 in order to assure that 100% of the primary mock-inoculated
pigs developed diarrhea after challenge (39).
Attenuated virus: The attenuated cell-culture adapted HRV Wa strain (P1A,G1) was used at the 27th passage level in monkey kidney cells (MA104). It was used for oral
inoculation of gnotobiotic pigs at a dose of 5x107 fluorescent focus-forming units and for the immunoassays (46).
Recombinant 2/6 virus-like particles (VLP): The 2/6VLP containing bovine rotavirus VP2 (RF) and human rotavirus VP6 (Wa) were produced in Spodoptera frugiperda 9 insect cells as previously described (18). The assembled 2/6VLPs were purified by CsCl density gradient ultracentrifugation. Characterization of the VLPs was done by immune electron microscopy (IEM), western blot, protein and endotoxin assays
(18).
VLP-ISCOM preparation: To increase the capacity of the 2/6VLPs to bind to
ISCOM matrix, they were mixed with 2M LiCl for 30 min at room temperature and then incubated overnight at -70° (18). The LiCl treated 2/6VLPs (positively charged by the
Li+ ions) were mixed with lyophilized ISCOM matrix (1mg of 2/6VLP per 5mg of
ISCOM matrix) and then dialyzed in 0.09% NaCl solution for 72 hours. The association of 2/6VLPs with ISCOM matrix was confirmed by IEM.
Gnotobiotic pigs: Near-term pigs were aseptically derived by hysterectomy and kept under sterile conditions in isolation units as previously described (39). The
185 gnotobiotic pigs were assigned to one of the following 7 groups: 1 & 2) 1 oral
immunization with Wa AttHRV and two IN doses of recombinant 2/6VLP at a VLP concentration of 250µg + ISCOM concentration of 1250µg or at a VLP concentration of
25 µg + ISCOM concentration of 125µg per dose (Att+ 2/6VLP-ISCOM); 3 & 4) 3 IN doses of 2/6VLP at a VLP concentration of 250µg + ISCOM concentration of 1250µg or at a VLP concentration of 25 µg + ISCOM concentration of 125µg per dose (2/6VLP-
ISCOM); 5) 3 oral immunizations with AttHRV (3xAttHRV); 6) 1 oral immunization with AttHRV (1xAttHRV); and 7) controls: 3 IN doses of ISCOM matrix or 1 oral immunization with diluent and 2 IN doses of ISCOM matrix. The first inoculation was done at 3-5 days after derivation (post-inoculation day 0, PID0). Subsequent inoculations were at 10 (PID10) and 21 days (PID21). All procedures were conducted in accordance with protocols reviewed by The Ohio State University’s Institutional Laboratory Animal
Care and Use Committee.
Assessment of protection: At PID 28, subsets of pigs from each group were challenged with virulent HRV (post-challenge day 0,PCD 0) and observed for 6 days for signs of diarrhea. Fecal consistency was scored as follows: 0: normal; 1: pasty; 2: semi- liquid; 3: liquid. Fecal scores equal to or greater than 2 were considered diarrheic. The mean cumulative fecal score was calculated as the sum of daily fecal scores from PCD 1 to 6 divided by the total number of pigs in each of the groups. Rectal swabs were collected daily and virus shedding was determined in rectal swabs fluids by antigen capture enzyme-linked immunosorbent assay (ELISA) and cell-culture immunofluorescence assay (CCIF) as previously described (33).
186 Blood and intestinal contents: Blood samples were collected at PID0, 10, 21,
28/PCD0 and PID35/PCD7. Serum was collected and complement inactivated at 56°C for 30 min, then stored at -20°C until tested. Small intestinal contents (SIC) and large intestinal contents (LIC) were collected only at euthanasia at PID28/PCD0 and
PID35/PCD7. The LIC or SIC were diluted 1:2 in diluent containing protease inhibitors
(25ug trypsin inhibitor and 5ug Leupeptin-Sigma, St. Louis, MO). The intestinal contents were stored at -20°C until tested.
ELISA for antibody isotypes: Rotavirus-specific IgA, IgM and IgG antibody titers were determined as previously described (30). Briefly, the reagents were added to
96-well microtiter plates coating initially with guinea pig anti-bovine rotavirus hyperimmune serum (incubated overnight at 4°C or 2 hours at 37°C). Washes were performed 4 times with PBS-Tween 0.5% between each step and most of the incubations, unless otherwise stated, lasted 1 hour at 37°C. The plates were blocked with 2% non-fat dry milk (incubated overnight at 4°C) followed by the addition of a 1:3 dilution in PBS
(pH 7.4) of semi-purified rotavirus or mock infected MA104 supernatants. Serial 4-fold dilutions of serum or intestinal contents in 2% non fat dry milk were followed by the addition of biotin-labeled monoclonal antibodies to porcine IgM (KPL Laboratories, Inc,
Gaithersburg, MD-USA), IgG (clone 3H7D7) or IgA (clone 6A11). Streptavidin- horseradish peroxidase was added (Roche, Indianapolis, IN) and 2-2’-azino-bis(3- ethylbenz-thiazoline-6-sulfonic acid (ABTS- Sigma, St. Louis, MO) was used as a chromagenic substrate. The antibody titers were calculated as the reciprocal of the sample dilution which had a mean absorbance greater than the mean of the negative controls
(three negative controls per plate) plus 3 standard deviations, after subtracting mock 187 coated well absorbances from antigen coated well absorbances for each sample. Serum
samples were considered positive for IgA or IgG if the value was positive at a dilution of
>1:4. Some serum negative samples (controls and PID0 samples) had background
reactions at 1:4 for IgM antibodies; for this reason the initial test dilution was 1:16. For intestinal contents, all three isotypes were considered positive above a 1:4 dilution. Each plate had a positive control with a known antibody titer that was used to estimate reproducibility of the individual assays.
Virus neutralization assay: To determine rotavirus VN antibody titers in serum samples, four-fold dilutions of the sera were mixed with equal volumes of Wa HRV
(initial dilution 1:4) as described previously (33, 46). Briefly after incubation at 37°C for
1 hour, the virus-antibody mixtures were added to MA104 monolayers, incubated 1 hour at 37° and the agar overlay added. The plates were incubated at 37°C and plaques were counted at 4-5 days. Titers were expressed as the reciprocal of the serum dilution that reduced the number of plaques by 80% compared to the control.
Statistical analyses: Antibody responses were compared using one-way analysis of variance followed by Duncan’s multiple-range test (SAS Inc, Cary N.C) using log- transformed data. Percentage of pigs with diarrhea and shedding, intestinal content conversion and seroconversion rates were compared using Fischer’s exact test. A p value of <0.05 was considered significant.
188 2.4 RESULTS
The Att+2/6VLP250-ISCOM and 3xAttHRV groups had the highest
protection rates against diarrhea and shedding after challenge with virulent Wa
HRV. Pigs in the Att+2/6VLP250-ISCOM and 3xAttHRV groups had the highest
protection rates against diarrhea (71% and 44%, respectively) and shedding (71% and
67%, respectively) (Table 2.1), and these rates did not differ statistically for the two
groups. All the pigs from the 2/6VLP250-ISCOM and control groups developed shedding
and diarrhea following challenge. A 2/6 VLP vaccine dose effect with respect to virus
shedding was detected among the Att+2/6VLP250-ISCOM, Att+2/6VLP25-ISCOM and
1xAttHRV groups with 71%, 34% and 14% protection against viral shedding,
respectively, but only shedding in the 1xAttHRV group differed statistically from the
other two groups.
The Att+2/6VLP250-ISCOM group had the highest percent of intestinal content conversion for IgA and IgM antibodies at PID28/PCD0. At pre-challenge
(PID28/PCD0), the Att+2/6VLP250-ISCOM group had the highest percent (100%) pigs
with IgM and IgA antibody titers to HRV in LIC and SIC, but no significant differences
were detected compared to the 3xAttHRV and 1xAttHRV groups in IgA antibody titers
in SIC (Table 2.2). Post-challenge (PID35/PCD7), all pigs had IgA and IgM antibody
titers in LIC and SIC, except for the 3xAttHRV group in which 90% of pigs had IgM
antibody titers to HRV and the control group that failed to develop IgA antibody titers by
PID35/PCD7, but had IgM antibodies consistent with a primary immune response.
At PID28/PCD0, the 2/6VLP250-ISCOM group had the highest IgG conversion
rate in intestinal contents. The percent of pigs with IgG antibody titers to HRV was
189 variable between SIC and LIC; higher percent positives were detected in SIC for the
3xAttHRV and 1xAttHRV groups. At PID35/PCD7, the percent of pigs positive for IgG antibodies to HRV was highest in the Att+2/6VLP250-ISCOM and 2/6VLP250-ISCOM
groups. IgG antibody titers were not detected in the control group by PID35/PCD7.
The Att+2/6VLP250-ISCOM group had the highest intestinal IgA antibody
titers detected pre-challenge in small and large intestinal contents. Antibody titers in
LIC and SIC were measured by ELISA pre-challenge (PID28/PCD0) and post-challenge
(PID35/PCD7). Antibody titers in LIC were analyzed and were similar to SIC antibody titers; for this reason the LIC data was omitted. Prechallenge in SIC, the Att-2/6VLP250-
ISCOM group had the highest rotavirus-specific IgA and IgM antibody titers (Figure
2.1). At post-challenge, all groups had increased IgM and IgA antibody titers (2 to 737-
fold); the 3xAttHRV group had the lowest IgM antibody titer and the control group did
not have IgA antibodies pre or post-challenge. The rotavirus-specific IgG antibody titers
were low pre and post-challenge in the vaccine groups and the Att+2/6VLP250-ISCOM
and 2/6VLP250-ISCOM groups had the highest titers post-challenge. The groups with the
highest protection rates, the 3xAttHRV and Att+2/6VLP250-ISCOM groups, had the
lowest antibody increases (0 to 26-fold) post-challenge compared to pre-challenge titers
(PID28/PCD0) for IgA and IgM (Figure 2.1).
One hundred percent IgA serconversion was first detected in the
Att+2/6VLP250-ISCOM group. All pigs from each of the vaccine groups had IgM
antibody titers to HRV from PID10 until PID35/PCD7. The control group developed IgM
antibody titers only post-challenge at PID35/PCD7 (Figure 2.2). Low titers of IgA
antibodies to HRV in serum were first detected in the Att+2/6VLP250-ISCOM group at
190 PID10, but the percent positive pigs did not differ significantly compared the other groups. At PID21, 100% of the Att+2/6VLP250-ISCOM group had IgA antibody titers; the highest seroconversion rate detected followed by the 3xAttHRV and the 1xAttHRV groups (74% and 72%, respectively). At PID28/PCD0, all of the groups except for the
1xAttHRV (81%) and control groups (0%) had 100% IgA seroconversion and at
PID35/PCD7 all groups except for the control group had 100% IgA seroconversion.
At PID10, the groups that received oral AttHRV had the highest rate of IgG seroconversion to HRV (18% to 51% compared to 0% of the 2/6VLP250-ISCOM and control groups). After PID21 all groups had 100% IgG seroconversion, except for the control group.
Neutralizing antibody seroconversion was evident at PID10 only in the groups that received oral AttHRV and 100% of the pigs in these groups had VN antibody titers at
PID21 through PID35/PCD7 (data not shown). After challenge, 80% of the pigs from the
2/6VLP250-ISCOM group developed neutralizing antibodies.
All groups that received Att-HRV as a first dose had higher serum IgM and
IgG antibody titers at PID28/PCD0 and PID35/PCD7, Antibody titers to the cumulative 3 doses (PID28/PCD0) and 3 doses plus challenge (PID35/PCD7) were measured (Figure 2.3). At PID28/PCD0, IgM and IgG antibody titers to HRV were significantly higher in all the groups that received oral AttHRV. The IgA antibody titers to HRV were higher in the Att+2/6VLP250-ISCOM group, but these were statistically similar to the 2/6VLP250-ISCOM group.
At PID35/PCD7, IgM antibody titers were high, and increased in all vaccine groups (7- to 6,797-fold), but the 3xAttHRV group had the lowest titers and no increase
191 compared to pre-challenge (Figure 2.3). The IgA antibody titers to HRV increased in all groups post-challenge (9- to 79- fold) but the 3xAttHRV group also had the lowest titers and lowest increase (9- fold) and the control group did not show any increase. The IgG antibody titers to HRV were high in all the groups except for the control group. The groups with the highest protection rates (3xAttHRV and Att+2/6VLP250-ISCOM), had the lowest antibody titer increases post-challenge (0- to 15-fold) compared to pre- challenge titers (PID28/PCD0).
Only groups that received AttHRV had neutralizing antibodies to HRV pre- challenge. From PID10 to PID28/PCD0, only the 3 groups that received oral AttHRV as a first dose developed neutralizing antibodies (Figure 2.4). However by PID28/PCD0, both the Att+2/6VLP250-ISCOM and 3xAttHRV groups had significantly higher VN antibody titers than the 1xAttHRV group. At PID35/PCD7, all vaccine groups had VN antibodies, but the ones that received AttHRV had the highest VN antibody titers (Figure
2.4).
Serum antibody titers to HRV were consistently lower in the groups that received 25µg of 2/6VLP compared to the 250µg 2/6VLP dose. Groups that received
Att+2/6VLP250-ISCOM, Att+2/6VLP25-ISCOM, 2/6VLP250-ISCOM and 2/6VLP25-
ISCOM were tested to measure effect of VLP-ISCOM dose on antibody titers (Figure
2.5) and protection rates (Table 2.1). The IgA, IgM and IgG antibody titers to HRV were consistently lower in the groups that received 25µg of 2/6VLP compared to the 250µg 2/6
VLP dose, but statistically lower responses were not detected at all time points (Figure
2.5). Lower protection rates were detected in the Att+2/6VLP25-ISCOM vaccination group (34% and 0% protection against shedding and diarrhea) than in the
192 Att+2/6VLP250-ISCOM group. The 2/6VLP-ISCOM 250µg and 25µg vaccination regimes did not confer any protection.
2.5 DISCUSSION
We studied a new vaccination regime consisting of priming with oral AttHRV and boosting with 2/6VLP IN. Intestinal IgA antibodies were significantly higher at
PID28/PCD0, in the pigs that received this vaccine regime and the protection rates were similar to those pigs that received 3 doses of AttHRV. Intranasal vaccination has been proposed to be more effective than peroral because of the low exposure of the immunogen to the adverse conditions found in the gastrointestinal tract such as proteolytic enzymes, extremes of pH and the abundance of different commensals and non-commensal pathogens (41). Responses in nasal associated lymphoid tissue (NALT) after IN inoculation and gut associated lymphoid tissue (GALT) after oral inoculation were compared in mice inoculated with Streptococcus mutans surface protein AgI/AgII coupled to subunit B of cholera toxin. After IN inoculation, more AgI/AgII specific IgA
ASC were located in NALT compared to the number of IgA ASC located in the Peyer’s
Patches (PP) after oral inoculation with twice the amount of antigen as was given by the nasal dose (1005 vs 6 IgA ASC in NALT and Peyer’s Patches (PP), respectively) (41).
Intranasal inoculation of adult mice with inactivated rotavirus gave better protection rates and higher intestinal and systemic IgA antibody titers to rotavirus than oral inoculation (9). Adult mice inoculated IN with 2/6VLPs and E. coli mLT had higher levels of IL-2 and IL-5 in supernatants of stimulated cells from spleen, mesenteric lymph
193 nodes (MLN), cervical LN and PP compared to the responses generated by the oral route
(17).
In our previous studies of pigs, we used ISCOM as adjuvant and gave oral
AttHRV followed by oral 2/6VLP250-ISCOM, achieving 50% protection against
diarrhea and 75% protection against shedding (18)]. In the current study, oral inoculation
of AttHRV and IN inoculation of 2/6VLP250-ISCOM gave higher protection rates against diarrhea (71%). We further showed that the oral AttHRV priming, IN 2/6VLP boosting regime was better than oral priming and oral boosting. It was highly immunogenic and induced protection rates similar to the actual vaccine regime of three oral doses of AttHRV. In a previous publication from our laboratory, we showed that the converse regime, IN priming with 2/6VLP and oral boosting with AttHRV was much less protective (17% and 25% protection against shedding and diarrhea, respectively) (44)].
The mechanism of protection from an enteric pathogen induced by IN vaccines
has been examined in studies using porcine and murine models (9, 35, 42, 44). In the
gnotobiotic pig model, antibody responses after inoculation of oral Att+2/6VLP250-
ISCOM (28) or IN in the present study were similar except for slightly higher intestinal
IgA antibodies and a significant boosting effect of the 2/6VLP (in the Att+2/6VLP250-
ISCOM compared to the 1xAttHRV) on VN antibodies by IN route in the present report.
The IN inoculation of 2/6VLPs alone induced partial to complete protection (86% to
100%) against rotavirus shedding in adult mice (29). Adult mice knockouts for the polymeric immunoglobulin receptor (pIgR) (with low levels of intestinal IgA and IgM)
and previously inoculated with 2/6VLP IN were not protected against shedding after
challenge with virulent wild-type murine ECw rotavirus, suggesting that transcytosis of
194 IgA and/or IgM is necessary to achieve the protection conferred by IN inoculation with
2/6VLP (35). On the other hand, IN and not oral inoculation of adult mice with inactivated rotavirus induced higher IgA antibody titers in serum and intestinal contents and complete or near-complete protection against rotavirus shedding upon challenge (9).
In order to determine the origin of the antibodies, the authors measured IgA antibodies in
the supernatants of different lymphoid tissue cultures. Interestingly, quantities of IgA
antibodies were higher in the supernatant from RALT (bronchial lymphoid tissue-10.3
ng/ml) than in the supernatant from GALT (PP-1ng/ml) suggesting that the major source
of virus specific IgA came from RALT. However the authors did not determine IgA
antibody titers to rotavirus in intestinal contents at challenge, so it is unknown if IgA was
transudated from serum and by this mechanism, conferred protection against shedding.
Adult mice lacking pIgR and the molecule for intestinal trafficking, α4β7 still had IgA in intestinal contents suggesting that transport of IgA from serum to the intestine may occur but the function of this transudated immunoglobulin remains to be determined (23, 35).
In the study by Fromantin et al. (17), IL-2 and IL-5 responses to IN 2/6VLP were higher than the responses generated by the oral 2/6VLP inoculation suggesting that T cells might play a greater role in the protection generated after IN 2/6 VLP vaccination of mice.
Intestinal IgA has been suggested as a marker of protection against reinfection
after natural rotavirus infection in children and after virus or viral antigen inoculation and
challenge in pigs and sucking mice (12, 15, 25, 39, 42-46). Also in adult mice, IgA
monoclonal antibodies to VP6 secreted by hybridoma cell backpack tumors, conferred
protection against shedding (4), but contrary findings with lack of protection were
reported using a similar approach in infant mice in which only neutralizing IgA
195 monoclonal antibodies to VP8 were protective (32). In the current report, we showed that
the Att+2/6VLP-ISCOM vaccine approach greatly increases IgA antibodies to HRV in large and small intestinal contents, which was associated with higher protection rates detected in this group of pigs. Future studies will assess the levels of protection conferred by a single dose of triple-layered (2/6/4/7) VLPs+ 2 doses of 2/6 VLPs in order to avoid a first oral dose of AttHRV. The primary oral dose of the live rhesus human rotavirus reassortment vaccine was correlated with most of the cases of intussusception after vaccination of infants (3).
Intestinal IgM was also high at PID28/PCD0 in the Att+2/6VLP250-ISCOM group. In previous reports from our lab, gnotobiotic pigs inoculated with Wa HRV oral and Wa 2/6 VLPs IN with mutant E. coli mLT also had the highest rotavirus specific IgM
ASC in duodenum and ileum and intestinal IgM antibody titers to HRV [Azevedo MSP et al 2002, submitted, (44)]. Although IgM has been considered as a primary immunoglobulin, previous studies have shown that human intestinal IgM secreting plasma cells had 8.5% V region gene mutation frequency compared to germ line sequences and that almost all of the intestinal plasma cells had V region genes mutated suggesting that most of them come from germinal centers (16). Human rearranged VH
gene IgM (+) memory B cells from blood, tonsils and spleen showed mutation
frequencies of only 2-6% (3, 21). These data suggest that intestinal IgM responses could be more than simply an expression of the unprimed state of the host and might also be playing an important role in protection against reinfection.
The antibody responses in serum clearly show that the groups that received oral
AttHRV as a first dose had higher HRV specific IgM and VN titers from PID 10 until
196 PID28/PCD0 (also PID35/PCD7 for VN antibody titers) and IgA and IgG titers at PID21
and PID28/PCD0. The VN antibody titers to HRV were statistically higher in the
3xAttHRV and Att+2/6VLP250-ISCOM groups compared to the 1xAttHRV,
2/6VLP250-ISCOM and control groups at PID28/PCD0 suggesting that the second and
third doses of 2/6VLP could boost the antibody responses for the generation of VN
antibodies without necessarily expressing the proteins (VP4 and VP7) containing the
neutralization epitopes and that this strategy was as effective as giving 3 doses of oral
AttHRV. This trend was previously described by our laboratory, and higher, but not
statistically significant responses were detected after the oral AttHRV dose followed by
2/6VLP-E.coli mLT IN (44). No VN antibodies were detected in the 2/6VLP250-ISCOM
or 2/6VLP25-ISCOM groups. The serum IgA antibody titers to HRV were highest in the
Att+2/6VLP250-ISCOM group at PID21 (which also was the group with the highest
percent of pigs that seroconverted to IgA) and at PID28/PCD0, but statistically similar to the 2/6VLP250-ISCOM group at PID28/PCD0. Post-challenge, the groups with the highest protection rates had the lowest antibody titer increases as previously described in mice [46] and in our previous studies of pigs (21, 28)
The ISCOM are cage-like structures composed of cholesterol and Quillaja
saponins (27, 31) with phospholipids added to facilitate incorporation of the antigen.
ISCOM matrix can induce activation of lymphocytes through the production of pro-
inflammatory cytokines and subsequent leukocyte migration (1, 2). Investigators
previously used a variety of viruses including rotavirus, influenza virus and human
immunodeficiency virus with ISCOM adjuvant for vaccine studies in humans and
animals (13, 18, 38). Studies with influenza vaccine had shown that ISCOM can enhance
197 either the production of mucosal (virus specific IgA) or systemic (lymphocyte proliferation and cytotoxic activity) responses measured in vitro (2). Previous studies from our laboratory showed that AttHRV+2/6VLP induced moderate protection rates and intestinal antibody secreting cell (ASC) responses in gnotobiotic pigs using mutant E. coli heat labile-toxin (mLT) as adjuvant. Somewhat lower protection rates against virus shedding (58%) and diarrhea (44%) were noted (44). In the current study we demonstrated that the use of ISCOMs increased protection rates, they did not induce toxicity in the animals and they may be promising for future use in humans (31).
In summary, 2/6 VLP IN increased systemic IgA antibody titers to HRV but only low titers of intestinal IgA antibodies were induced. Three doses of oral AttHRV stimulated intermediate titers of systemic IgA antibodies compared to the
Att+2/6VLP250-ISCOM and 2/6VLP250-ISCOM group responses and lower titers of intestinal IgA antibodies than the Att+2/6VLP250-ISCOM group. Priming with oral
AttHRV followed by boosting with 2/6VLP250 IN increased systemic and intestinal IgA antibody responses. Based on these results, even though Att+2/6VLP250-ISCOM and
3xAttHRV vaccine regimes stimulated the highest protection rates, it seems that each regime may stimulate different types of immune responses since IgA and IgM intestinal antibody responses differed between these groups. There is evidence that adult mice inoculated with live rotavirus depend more on B cell immunity compared to mice inoculated IN with the chimeric protein VP6+ E coli labile-toxin depend more on CD4 T cells, suggesting that successful protection can be achieved by different immune mechanisms (26). It is possible that protection induced by 3xAttHRV is more dependent on VN antibodies than that induced by the Att+2/6VLP250-ISCOM vaccine; however,
198 both groups had similar levels of serum VN antibody titers. The VN antibody responses in intestinal contents are difficult to asses, but a comparison of T cell responses in the
Att+2/6VLP250-ISCOM and 3x-AttHRV groups is feasible to further explore the mechanisms of protection stimulated by each vaccine regime.
2.6 ACKNOWLEDGMENTS
We thank Dr. Juliette Hanson for the clinical care of the gnotobiotic pigs, Paul
Nielsen for technical assistance and Bert Bishop for advice regarding the statistical analysis data. This work was supported by grants from the National Institutes of Health,
NIAID (RO1AI33561 and RO1AI37111).Marli Azevedo is a fellow of National Council of Scientific and technologic development (CNPq),Brazil.
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204 Percent of pigs with Protection rate (%) Protection rate (%) Vaccination Against Against groupa na b c Virus shedding Diarrhea sheddingd diarrhead
Att+2/6VLP250 7 29B 29B 71 71 ISCOM
Att+2/6VLP25 -3 66 B 100 A 34 0 ISCOM
2/6VLP250-I 5 100A 100A 00 ISCOM 3XAttHRV 9 33B 56B 67 44 205
1XAttHRV 7 86A 100A 14 0
Controls 12 100A 100A 00
a Gnotobiotic pigs receiving the various vaccination regimes at PID 0, 10 and 21, were challenged with virulent WaHRV at PID28 Diarrhea scores and virus shedding were determined at PCD 0-6. n= Number of pigs. Proportions in the same column, with different superscript letters differ significantly (Fischer’s exact test). A portion of this protection data was published previously by Iosef C et al, 2002 [6]. bDetermined by ELISA and Cell-Culture Immunofluorescence infectivity assay (CCIF). c Diarrhea determined by fecal scores greater or equal to 2: feces were scored as follows: 0=normal; 1=pasty; 2=semiliquid; 3=liquid. d Protection rate=[1-(%of vaccinated pigs in each group with diarrhea-shedding / % of control pigs with diarrhea-shedding)] x100. TABLE 2.1. Protection rates against virus shedding and diarrhea in gnotobiotic pigs receiving the various vaccination regimes IgM coproconversion IgA coproconversion IgG coproconversion
PID28 PID35 PID28 PID35 PID28 PID35
SICb SICb SICb SICb SICb SICb
Att+2/6VLP250- 100A 100A 100A 100A 16B 71AB
206 ISCOM
2/6VLP250 33.3B 100A 66.6B 100A 66.6A 80A ISCOM
3xAttHRV 66.6B 90A 93B 100A 46A 36B
1xAttHRV 75B 100A 75B 100A 25A 42B
Controls 0C 100A 0C 0A 0C 0C a Gnotobiotic pigs receiving the various vaccination regimes were euthanized at PID28/PCD0 or PID35/PCD7 and LIC and SIC were collected the day of the euthanasia. IgM, IgA and IgG antibody titers to HRV were quantitated by ELISA. bProportions in the same column with different letters differ significantly (Fischer’s exact test)
TABLE 2.2. Percentage of conversion in intestinal contents for IgM, IgA and IgG antibodies in gnotobiotic pigs receiving the various regimens a
Figure 2.1. Small intestinal contents (SIC) geometric mean antibody titers (GMT,bars) at
PID28/PCD0 and PID35/PCD7 of gnotobiotic pigs receiving the various vaccination regimes. Pigs were inoculated with vaccines at PID0,10 and 21 and challenged at
PID28/PCD0. Approximately half of the pigs were euthanized pre-challenge at PID28 and the rest of the group was euthanized post-challenge at PID35/PCD7 and SIC/LIC was collected. Error bars represent standard error of the mean. Antibody titers that differ significantly are marked with different letters (One-way anova and Duncan-multiple range test on log10-transformed titers). Numbers to the right of the bars indicate fold
Increases of the PID35/PCD7 GMT over the PID28/PCD0 GMT.
207
SIC PID28/PCD0 PID35/PCD7
Att+2/6VLP250- Att+2/6VLP250- 645 A 1248 A 2X ISCOM ISCOM 2/6VLP250- 2/6VLP250- 6 BC 1024 A 170X ISCOM ISCOM
3xAttHRV 21 B 3xAttHRV 99 B 5X
1xAttHRV 16 BC 1xAttHRV 689 A 43X
IgM Control 2 C Control 1474 A 737X
0 1 10 100 1000 10000 100000 0 1 10 100 1000 10000 100000 Geometric Mean Titers Geometric Mean Titers
Att+2/6VLP250- 813 A Att+2/6VLP250- ISCOM 2756 A 3X ISCOM 2/6VLP250- BC 2/6VLP250- 6 776 ISCOM ISCOM AB 129X
B 3xAttHRV 38 3xAttHRV 619 B 16X
BC 10 1xAttHRV 1xAttHRV 2261 A 226X
C IgA Control 2 Control 2
0 1 10 100 1000 10000 100000 0 1 10 100 1000 10000 100000 Geometric Mean Titers Geometric Mean Titers
Att+2/6VLP250- Att+2/6VLP250- 2 A 20 ISCOM ISCOM B 10X
2/6VLP250- 2/6VLP250- 5 A 128 A 26X ISCOM ISCOM
A 3xAttHRV 6 3xAttHRV 5 BC
1xAttHRV 2 A 1xAttHRV 5 BC
A C Control 2 Control 2 IgG 0 1 10 100 1000 10000 100000 0 1 10 100 1000 10000 100000 Geometric Mean Titers Geometric Mean Titers
208
Figure 2.2. Serum IgM, IgA and IgG antibody seroconversion at PID 0, PID10,PID21,
PID28/PCD0 and PID35/PCD7 of gnotobiotic pigs receiving the various vaccination regimes. Pigs were inoculated with vaccines at PID0, PID10 and PID21 and challenged at
PID28/PCD0. Approximately half of the pigs were euthanized pre-challenge at PID28 and the rest of the group was euthanized post-challenge at PID35/PCD7 and blood samples were collected at each of the different inoculation days. For each vaccination regime, the number of pigs that were positive for IgM, IgA and IgG antibodies to HRV at each inoculation day were divided by the total number of animals per group and expressed as a percentage. Groups at the same PID with different letters differ significantly (Fischer's exact test).
209
aaa 100 a Att+2/6VLP250- ISCOM on i 80 2/6VLP250-
ers ISCOM 60 3xAttHRV
40 1xAttHRV
20 Controls % seroconv a bb b 0
0 0 1 2 0 7 ID D D P ID1 D C C P PI P 5/P 28/ 3 ID D P PI PID/PCD
a a a 100 b on i 80 b Att+2/6VLP250- ISCOM ers 60 2/6VLP250- ISCOM 40 c 3xAttHRV 20 a 1xAttHRV % seroconv a d c b 0 Controls 0 0 1 1 2 0 ID D D7 P D ID C C PI P P P 28/ 35/ ID ID P P PID/PCD
aaa 100 Att+2/6VLP250-
on ISCOM i 80 2/6VLP250- ers ISCOM 60 a 3xAttHRV 40 a 1xAttHRV 20 % seroconv Controls a b b b c b 0
0 1 0 7 ID0 D D P ID1 ID2 C C P P P P 28/ 35/ ID ID P P PID/PCD
210
Figure 2.3. Serum IgM, IgA and IgG geometric mean antibody titers (GMT,bars) at
PID28/PCD0 and PID35/PCD7 of gnotobiotic pigs receiving the various vaccination regimes. Pigs were inoculated with vaccines at PID0, PID10 and PID21 and challenged at
PID28/PCD0. Approximately half of the pigs were euthanized pre-challenge at PID28 and the rest of the group was euthanized post-challenge at PID35/PCD7and blood samples were collected at each of the different inoculation days. Lines represent standard error of the mean. Antibody titers that differ significantly are marked with different letters (One-way anova and Duncan-multiple range test on log10-transformed titers).
Numbers to the right of the bars indicate fold increases of the PID35/PCD7 GMT over the PID28/PCD0 GMT.
211
SERUM PID28/PCD0 PID35/PCD7
Att+2/6VLP250- Att+2/6VLP250- 920 A 16384 A 7X ISCOM ISCOM
2/6VLP250- A 2/6VLP250- 512 37641 A 124X ISCOM ISCOM
B 3xAttHRV 167 B 3xAttHRV 512
B 1xAttHRV 77 1xAttHRV 16384 A 12X
IgA Control 2 C Control 20393 A 6797X
0 1 10 100 1000 10000 100000 0 1 10 100 1000 10000 100000 Geometric Mean Titers Geometric Mean Titers
Att+2/6VLP250- Att+2/6VLP250- 2403 A 13440 A 15X ISCOM ISCOM
2/6VLP250- 2/6VLP250- 304 B 16384 A 32X ISCOM ISCOM
3xAt t HRV 1024 A 3xAttHRV 1495 B 9X
1xAt t HRV 1318 A 1xAttHRV 6087 AB 79X
IgM Control 3 C Control 2 C
0 1 10 100 1000 10000 100000 0 1 10 100 1000 10000 100000 Geometric Mean Titers Geometric Mean Titers
Att+2/6VLP250- Att+2/6VLP250- 9613 A 44102 ISCOM ISCOM A 4X
2/6VLP250- 2/6VLP250- 4096 65536 ISCOM B ISCOM A16X
3xAttHRV 15533 A 3xAttHRV 44904 A 3X
1xAttHRV 12734 A 1xAttHRV 65536 A 5X
IgG Control 2 C Control 2 B
0 1 10 100 1000 10000 100000 0 1 10 100 1000 10000 100000 Geometric Mean Titers Geometric Mean Titers
212
Figure 2.4. Serum VN geometric mean antibody titers (GMT) at PID0, PID10, PID21,
PID28/PCD0 and PID35/PCD7 of gnotobiotic pigs receiving the various vaccination regimes. Pigs were inoculated with vaccines at PID0, PID10 and PID21 and challenged a
PID28/PCD0. Approximately half of the pigs were euthanized pre-challenge at PID28 and the rest were euthanized post-challenge at PID35/PCD7. Blood samples were collected at each of the different inoculation days. Error bars represent standard error of the mean. VN antibody titers at the same PID with different letters differ significantly
(One-way anova and Duncan-multiple range test on log10-transformed titers)
213
100000
10000 A an Titers e 1000 Att+2/6VLP A 250-ISCOM A B 2/6VLP250- etric M 100 ISCOM m A B 3xAttHRV
Geo 10 1xAttHRV C B B Control
1 PID 0 PID10 PID21 PID28/PCD PID35/PCD 0 7 Days after inoculation
214
Figure 2.5 Effect of dose of 2/6VLP and ISCOM on IgM, IgA and IgG antibody titers to
HRV in serum of Att+2/6VLP-ISCOM (250ug /1250ug or 25ug/125ug) and 2/6VLP-
ISCOM (250ug/1250ug or 25ug/125ug) pigs. Pigs were inoculated with vaccines at
PID0, 10 and 21 and challenged at PID28/PCD0. Approximately half of the pigs were euthanized pre-challenge at PID28 and the rest of the group was euthanized post- challenge at PID35/PCD7 and blood samples were collected at each of the different inoculation days. Error bars represent standard error of the mean. Statistical analysis was performed between groups as follows: Att+2/6VLP250-ISCOM vs. Att+2/6VLP25-
ISCOM and 2/6VLP250-ISCOM vs. 2/6VLP25-ISCOM and significantly lower titers are marked with an asterisk (*) (One-way anova and Duncan-multiple range test on log10 transformed titers).
215
100000 rs
e 10000 * * Tit Att+2/6VLP250-ISCOM n 1000 a * Att+2/6VLP25-ISCOM e 2/6VLP250-ISCOM 100 2/6VLP25-ISCOM etric M 10 om e
G 1 PID0 PID10 PID21 PID28/PCD PID35/PCD 0 7 100000 PID/PCD rs e 10000 Tit n
a Att+2/6VLP250-ISCOM 1000 e Att+2/6VLP25-ISCOM 2/6VLP250-ISCOM 100 * * 2/6VLP25-ISCOM etric M
om 10 e G 1 PID0 PID10 PID21 PID28/PCD PID35/PCD 0 7 100000 PID/PCD s r 10000 te
i *
1000 Att+2/6VLP250-ISCOM Att+2/6VLP25-ISCOM Mean T
c 2/6VLP250-ISCOM i r
t 100 2/6VLP25-ISCOM e m * 10 Geo
1 PID 0 PID 10 PID 21 PID28/PCD PID35/PCD PID/PCD0 7
216
CHAPTER 3
ROTAVIRUS VP4 PLUS IMMUNE STIMULATING COMPLEXES (ISCOM) CO-
ADMINISTERED WITH 2/6/7 VIRUS-LIKE PARTICLES (VLP) FOR PRIMING
AND 2/6VLP VACCINES FOR BOOSTING CONFERS PROTECTION AGAINST
DIARRHEA IN NEONATAL GNOTOBIOTIC PIGS
3.1 SUMMARY
Live rotavirus (RV) vaccines can be transmitted to susceptible individuals, could mutate to become pathogenic and some have been associated with intussusception. Non- replicating RV vaccines were used to prime neonatal gnotobiotic pigs orally with RV
VP4/ISCOM plus 2/6/7VLPs, then boost intranasally with VP4+2/6VLP/ISCOM (VP4-
2/6/7+VP4-2/6VLP). To test VP4 omission, we primed with 2/6/7VLPs, then boosted
217 with 2/6VLPs (2/6/7-2/6VLP). To determine the effect of priming and boosting with VP7 without VP4, we vaccinated with three doses of 2/6/7VLPs (3x2/6/7VLP). Protection rates against diarrhea were 67%, 50% and 33% conferred by the VP4-2/6/7+VP4-
2/6VLP, 3x2/6/7VLP and 2/6/7-2/6VLP vaccines, respectively. The VP4-2/6/7+VP4-
2/6VLP vaccine induced the highest intestinal RV-specific IgA antibody secreting cells,
VP4-specific antibody-expressing cells and efficiently boosted serum neutralizing antibodies 7 days after challenge.
3.2 INTRODUCTION
Rotaviruses (RV) are the major cause of severe diarrhea in children less than 5 years of age. They cause 610,000 deaths, mainly in developing countries (18) but hospitalizations in both developed and developing countries cost billions of dollars annually (18). The first licensed human RV vaccine (RotaShield) was associated with intussusception and was withdrawn from the market (27). Two other live attenuated RV vaccines were recently licensed (Rotarix and RotaTeq) (18, 41, 48). To date no increased association with intussusception has been observed with the new vaccines, but only when millions of children are vaccinated will the real risk be known. These vaccines have not yet been tested in certain developing countries such as India, China and those in Africa where uncommon RV serotypes are more prevalent (16, 17, 45), so their protective efficacy in these countries is unknown. Thus, development of new or second generation
RV vaccine strategies remains a priority.
The neonatal gnotobiotic (Gn) pig remains the only animal model susceptible to human RV-induced diarrhea to test HRV vaccines. After inoculation with homologous
218 RV or heterologous virulent HRV, Gn pigs shed virus and, unlike adult animal models
(e.g. mice, rabbits, monkeys), Gn pigs also develop diarrhea. Neonatal pigs are
susceptible to RV disease for up to at least 8 weeks of age allowing the evaluation of
vaccine efficacy (42, 43). The gastrointestinal tract and mucosal immune system of
neonatal pigs resemble that of human infants (43). Their Gn status assures that they will
not acquire extraneous wild type RV infection during the vaccination and challenge
experiments. Gnotobiotic pigs develop similar diarrhea scores, fecal RV titers and RV-
specific antibody titers after inoculation with HRV regardless of their colonization status with lactobacillus species (57). These findings suggest that their Gn status and lack of commensal flora does not influence RV replication, induction of diarrhea or compromise
RV-specific B cell responses.
The RV capsid is composed of a core (VP2), an inner capsid protein (VP6) and two
outer capsid proteins containing neutralizing epitopes (VP4 and VP7). Non-replicating virus-like particle (VLP) vaccines are a useful tool to understand the role of RV structural proteins in immunity, especially the neutralizing outer capsid proteins (VP4/VP7). We previously determined that three doses of 2/6VLP, conferred no protection against shedding or diarrhea in the neonatal Gn pig model of HRV disease (52). Only by priming
orally with the replicating attenuated HRV (AttHRV) followed by two non-replicating
2/6VLP vaccine booster doses was protection against RV shedding and diarrhea achieved
(21). We concluded that using a non-replicating 2/6VLP vaccine, VP6 alone is not
sufficient to confer protection in this model. We hypothesized that neutralizing antigens
might be necessary to effectively vaccinate neonatal Gn pigs to protect against RV
infection and disease. Therefore, our main objective was to replace the live AttHRV oral
219 vaccine for priming with a non-replicating vaccine that includes VP7 with or without
VP4. Thus we tested 2/6/7VLP and semi-purified recombinant VP4 as components of a
non-replicating RV candidate vaccine. From previous studies, when vaccinating with one
oral dose of AttHRV followed by two oral or intranasal (IN) doses of 2/6VLP with
ISCOM as adjuvant (23), a higher protection rate against diarrhea was achieved with the oral/IN than the oral/oral regime (71% vs. 50%, respectively) (21, 23). These studies suggest that a non-replicating vaccine should target intestinal and nasal tissues for maximum efficacy; therefore, a combination of oral and IN routes was chosen for the current vaccine study.
Protection against RV in humans has been correlated with RV-specific IgA antibodies in serum shortly after infection (14) and intestinal IgA antibodies (10). Serum neutralizing antibody titers correlated with protection in some studies (33, 34), but not in others (49) so their association with protection remains unclear. In Gn pigs, protection has been associated with intestinal effector and memory RV-specific IgA ASC (53, 54) and intestinal RV-specific IgA antibodies (21). In the pig model, the role of RV-specific serum neutralizing antibody titers in protection is also uncertain. High serum neutralizing antibody titers are not necessarily associated with protection (46, 55). However, complete lack of neutralizing antibodies is usually associated with lack of protection (21, 52). To study the correlates of immunity, we measured effector and memory total RV-specific intestinal, systemic (spleen) and circulating IgA, IgM and IgG ASC by ELISPOT, and serum neutralizing antibodies. Also, to study RV protein-specific B cell responses,
CD21+IgA+, CD21+IgM+ and CD21+IgG+, VP6, VP7 and VP4-specific antibody
expressing cells (AEC) were analyzed by flow cytometry.
220 High amounts of double-layered 2/6VLPs can be produced efficiently and stably but
the efficiency of producing triple-layered 2/4/6/7VLPs is compromised by their reduced
stability with loss of high quality intact triple-layered particles after purification. Also, the stability of the triple-layered particles is compromised by the extensive dialysis during incorporation into ISCOM (which is optimal for adjuvanticity). For this reason, we tested semi-purified recombinant VP4 incorporated into ISCOM as adjuvant and mixed with 2/6/7VLPs orally as a priming dose followed by two IN booster doses of semi-purified VP4 plus 2/6VLPs incorporated into ISCOM (VP4-2/6/7+VP4-2/6VLP vaccine). To assess the effect of omission of VP4, we primed with 2/6/7VLP and boosted with 2/6VLPs (2/6/7-2/6VLP group). Alternatively, to determine the effect of priming and boosting with VP7 without VP4, we vaccinated with three doses of 2/6/7VLPs
(3x2/6/7VLP group). Controls were inoculated with ISCOM matrix only. Our results confirmed that to induce protection in neonatal Gn pigs, the neutralizing RV antigens
VP4 and VP7 must be included in the vaccine formulation. Also, semi-purified VP4 with
2/6/7VLPs efficiently induced protective responses and is an alternative approach to the use of 2/4/6/7VLPs. This is the first report of non-replicating RV VLP vaccines with inclusion of neutralizing antigens and RV protein-specific B cell responses assessed by flow cytometry in Gn pigs. To our knowledge, this is the first study reporting the protective immunogenicity induced by semi-purified VP4+2/6/7VLP as an alternative approach to 2/4/6/7VLP vaccination.
221 3.3 MATERIALS AND METHODS
Viruses. The challenge inoculum was the HRV Wa strain (G1,P1A[8]). A pool of
intestinal contents was obtained from the 18th Gn pig passage and diluted in minimum essential media (MEM; Invitrogen, Carlsbad, CA). The pigs received 2% sodium bicarbonate to neutralize gastric acidity followed by 106 fluorescent-focus forming units
(FFU) of virulent HRV orally. The ID50 was previously determined to be 1 FFU (54).
The AttHRV was grown in monkey kidney cells (MA104) and infected cell lysates were collected from the 25-26th passage. Each pig received 2% sodium bicarbonate to neutralize gastric acidity and 5x107 FFU of AttHRV Wa orally as previously reported
(21, 32). The AttHRV pool was also used to infect MA104 cell monolayers in 96-well microtiter plates for the ELISPOT assay, as in-vitro stimulating antigen for the memory
ELISPOT and in the virus neutralization assay.
VP4 and VLP preparation. Recombinant baculoviruses expressing VP2 (RF, bovine RV), VP6 (Wa, G1), VP7 (CJN, G1) and VP4 (Ku, P1A[8]) were constructed as previously described (12, 23). Recombinant baculoviruses expressing VP2-GFP (RF) and
VP2-DsRed (RF) were kindly provided by the late Dr John Cohen †(L’institut national de la recherche agronomique-INRA). For the construction of VLP and VP4, Trichopulsia ni cells (H5 cells) in Express Five serum free media (Invitrogen, Carlsbad, CA) were infected with various combinations of recombinant baculoviruses to generate 2/6, 2/6/7,
2/4/6/7VLPs or VP4 at a total MOI of 5-6. Different fluorescent particles for 2/6VLP-
GFP, 2/6VLP-DsRed, 2/6/7VLP-DsRed and 2/4/6/7VLP-GFP were constructed by using
VP2-GFP or VP2-DsRed recombinant baculoviruses. After 7 days of infection, infected
cell lysates were collected and stored at 4°C. Particles were purified by sucrose-CsCl
222 purification, resuspended in TNC (Tris-HCL 10mM, NaCl 140 mM, CaCl 10mM), pH
7.4 and tested by electron microscopy (EM) for integrity, anaerobic and aerobic culture
media at 37 and 27°C to culture any contaminating bacteria or fungi, western blot for the composition of the VLPs, RV antigen ELISA assay and insect cell (Sf9) protein ELISA for the titer of RV and insect cell protein antigens, respectively. The insect cell protein
ELISA was performed to detect contaminating proteins from insect cell origin by coating
96-well microtiter plates with insect cell (Sf9 or H5) supernatant (baculovirus infected) as a positive control or various dilutions of the VLP preparations. It was developed by adding a rabbit polyclonal anti-Sf9 protein antibody (Research Diagnostics Inc, Flanders,
NJ) that was crossreactive with proteins of H5 cell origin (data not shown), followed by an anti-rabbit HRP (KPL Inc, Gaithersburg, MA) and 2,2-Azino-bis(3-ethylbenzo- thiozoline-6-sulfonic acid) diammonium (Sigma-Aldrich, St Louis, MO) in citrate buffer, pH 4. Protein concentrations were determined by Bradford protein assay (Bio-Rad,
Hercules, CA) and endotoxin levels by Limulus Amebocyte assay (Associates of Capecod
Inc., Woods Hole, MA) as previously described (23). At least 80% or more intact
2/6/7VLP particles as determined by EM were used for inoculation. Furthermore, to assure that most of the inoculated protein was from VLP and not insect cell protein origin, only samples with a ratio of RV antigen/insect cell protein >80 (usually the ratio was higher than 3000) were used for inoculation.
For the semi-purification of VP4, infected H5 cell lysates were collected and centrifuged at 1000xg for 40 minutes. Supernatants were collected and cell pellets were sonicated,
centrifuged at 1000xg for 40 min and the additional supernatants were collected and
pooled. Polyethylene glycol (Sigma-Aldrich, St Louis, MO) and NaCl (Amresco, Solon, 223 OH) were added in solution for a final concentration of 10% and 2.3%, respectively and
incubated overnight at 4°C. The supernatant was centrifuged at 1000xg for 40 min and
pellets were resuspended in TNC buffer, pH 7.4. The VP4 samples were tested with the
same assays as the VLP preparations (except EM and insect cell protein ELISA). Because
the titer of the VP4 samples stored at 4°C diminished after a month, only fresh samples
(less than a month) positive at a 1/1200 dilution or more by ELISA were used for Gn pig inoculation.
Non-replicating VLP vaccine preparation. The incorporation of 2/6VLPs or VP4 to
ISCOM matrix was performed as previously described (23). Briefly, 2/6VLPs and/or
VP4 preparations were incubated with 2M LiCl to positively charge the proteins and
facilitate their incorporation. The mixture was incubated at room temperature for 30 min
and then overnight at -70°C. The VLP samples were mixed with ISCOM (ratio1:5)
injected into a sterile dialysis cassette (Pierce Biotechnology, Inc., Rockford , IL) and
dialyzed for 72hr against 1L of 0.09% NaCl solution (changed every 12hr). The
2/6/7VLPs were mixed with ISCOM alone or with VP4 (after 72hr dialysis) because in
preliminary tests when dialyzed for incorporation, the triple layered structure (2/6/7VLPs
and 2/4/6/7VLPs) was compromised.
Inoculation of Gn pigs. The Gn pigs were derived by hysterectomy and maintained
in isolator units as previously described (56). At 3 days of age and then weekly, rectal
swabs were taken to assess their sterility. At 5 days of age and every 10 days, pigs were
bled and inoculated with a total of three vaccine doses. Pigs were assigned to one of the
following 4 groups: 1) One oral dose of VP4 (500µg) incorporated into ISCOM (2.5mg)
and mixed with 2/6/7VLP (250µg) in 3ml MEM (Invitrogen, Carlsbad, CA) followed by 224 two IN doses of VP4 (250µg) and 2/6VLP (250µg) incorporated into ISCOM (1.25mg) in
500µl of TNC buffer pH 7.4 (VP4-2/6/7+ VP4-2/6VLP vaccine). This vaccine was
designed to test the protective immunity elicited by priming with VP4 and VP7 in VLPs
(2/6/7VLPs) and VP4 boosting without VP7 (VP4-2/6VLP). 2) One oral dose of
2/6/7VLP (250µg) mixed with ISCOM (1.25mg) in 3ml of MEM followed by two IN
doses of 2/6VLP (250µg) incorporated into ISCOM (1.25mg) in 500µl of TNC buffer
(2/6/7-2/6VLP vaccine). This vaccine was designed to test the protective immunity
elicited by priming with VP7 in VLPs (2/6/7VLPs) and boosting with 2/6VLPs in the
absence of VP4. 3) One oral dose of 2/6/7VLP (250µg) mixed with ISCOM in 3ml of
MEM and two IN doses of 2/6/7VLP (250µg) mixed with ISCOM (1.25mg) in 500µl of
TNC buffer (3x2/6/7VLP vaccine). This regime was designed to test the protective
immunity elicited by VP7 incorporated into 2/6/7VLPs for priming and boosting but
without VP4. 4) Controls were inoculated with one oral dose of ISCOM matrix (1.25mg)
diluted in 3ml of MEM followed by two IN doses of 500µl of ISCOM (1.25mg) in TNC
buffer. The AttHRV+2/6VLP vaccine was included to compare protection and immune
responses elicited by oral priming with live HRV to the responses induced by priming
with only non-replicating vaccines. The AttHRV+2/6VLP vaccine was delivered as
previously described (21, 31, 32). A week after the third inoculation (post-inoculation
day (PID) 28), a subset of pigs was challenged with 106 FFU of virulent HRV Wa orally.
Protection assessment. Vaccine efficacy was assessed by scoring fecal consistency and virus shedding for 6 days after challenge as previously described (23). Briefly, fecal consistency was scored as follows: 0= solid; 1=pasty; 2=semi-liquid; 3=liquid. Fecal consistencies with scores of 2 or more were considered diarrheic. Rectal swabs were 225 collected and resuspended in 8ml of MEM and tested by RV antigen ELISA and cell
culture immunofluorescence (CCIF) assays. Virus shedding was considered positive if
the sample was positive by CCIF or ELISA.
Isolation of mononuclear cells (MNC). One week after the last dose of vaccine at
PID28, a subset of pigs was euthanized and blood, small (SIC) and large intestinal
contents (LIC), spleen and ileum-jejunum tissue fragments were collected. Blood, spleen
and intestine were processed for isolation of MNC as previously described (56).
ELISPOT assay for total RV-specific antibody secreting cell (ASC) to detect
effector and memory B cells. After MNC isolation, cell suspensions were prepared in
enriched RPMI (e-RPMI) (Invitrogen, Carlsbad, CA) as previously described (55). For
detection of memory B cells, MNC were incubated in 12-well culture plates using e-
RPMI with semipurified AttHRV as stimulating antigen for 5 days at 37°C, adding fresh media every other day (53). As previously described (56) to detect effector and memory total RV-specific cells, MNC were incubated in 96-well culture plates with acetone-fixed
Wa AttHRV infected MA104 cells. The MNC were incubated for 12hrs at 37°C and then washed 4 times with PBS pH 7.4 with 0.05% tween (PBS-Tw). Biotinylated monoclonal mouse anti-porcine IgA (hybridoma 3D11 and 6D11, 1/500 or 1/5000 respectively), IgM
(hybridoma 5C9, 1/2,000) and IgG (hibridoma 3H7, 1/10,000) (37) were added and plates were incubated overnight at 4°C. Plates were washed 4 times with PBS-Tw and 1/30,000 streptavidin (SA)-peroxidase (Roche, Diagnostics Corp, Indianapolis, IN) in PBS-Tw was added for 2 hrs at room temperature. Tetramethylbenzidine (TMB) with H2O2 peroxidase system (KPL Inc., Gaithersburg, MA) was added for development of the spots. Spots were counted using light microscopy at 10x magnification. 226 Flow cytometry assay for detection of CD21+ RV protein-specific antibody expressing cells (AEC). The following protocol was modified from Gonzalez et al (20).
After isolation of MNC, 106 fresh cells were added to 15ml polypropylene conical tubes
(Becton Dickinson Labware, Franklin Lakes, NJ), washed with PBS-Azide 0.02%
(Sigma, St Louis, MO)-bovine serum albumin 0.5% and centrifuged at 400xg for 5 min.
After discarding the supernatant, 20µl of 1/100 dilution of pig serum (RV negative Gn pig) were added and incubated at 4°C for 10 min. Cells were washed, centrifuged and stained with the following antibodies and fluorescent VLPs: mouse anti-human CD21
(C3d complement receptor co-expressed with CD19 on B cells) allophycocyanin (APC)
(Becton Dickinson, San Jose, CA), mouse monoclonal anti-porcine IgA (Serotec,
Raleigh, NC) biotinylated with a commercial kit-(Amersham-GE Healthcare Biosciences,
Piscataway, NJ), biotinylated mouse monoclonal anti-porcine IgM (KPL Inc,
Gaithersburg, MA) or biotinylated mouse monoclonal anti-porcine IgG (Becton
Dickinson, San Jose, CA). A combination of 2/6VLP-GFP and 2/6/7VLP-DsRed or
2/6VLP-DsRed and 2/4/6/7VLP-GFP were added (0.5ug each) to different tubes. Cells were incubated at 4°C for 10 min in the dark. Cells were washed, centrifuged, labeled with SA-conjugated peridinine chlorophyll protein (PerCP) (Becton Dickinson, San Jose,
CA) and incubated at 4°C for 10 min in the dark. After the incubation, cells were washed, centrifuged and 200µl of 1% paraformaldehyde (Sigma, St Louis, MO) were added and cells were stored at 4°C in the dark until cell acquisition by flow cytometry. Control tubes were stained only with mouse anti-porcine IgA, IgM or IgG and CD21-APC or SA-
PerCP and CD21-APC only. Acquisition of the cells for flow cytometry was performed within a week after staining (usually the next day). Data was analyzed by Cell Quest pro 227 software (Becton Dickinson, San Jose, CA). For the data obtained for the correlation analysis between ELISPOT RV-specific ASC numbers and flow cytometry VP6-specific
AEC frequencies, the AEC were stained differently. To try to mimic the ELISPOT assay, flow cytometric analyses were performed by gating the lymphocyte window in a wide window including large and small cells and no CD21 staining was performed. Only
2/6VLP-GFP, biotinylated mouse anti-porcine IgM (KPL Inc, Gaithersburg, MA) and mouse anti-porcine IgA (Serotec, Raleigh, NC) or mouse anti-porcine IgG (KPL Inc.,
Gaithersburg, MA) were added to different tubes. After incubating at 4°C for 10 min and washing, rat anti-mouse IgG1 phycoerythrin (PE) and SA-PerCP were added. In control tubes, MNC were only stained with anti IgA or IgG and biotinylated IgM and then with
SA-PerCP and rat anti-mouse IgG1 PE or only SA-PerCP and rat anti-mouse IgG1 PE.
Virus Neutralization test. Rotavirus neutralizing (VN) antibody titers were
determined by an immunohistochemistry (IHC) virus infectivity reduction assay. The
MA104 cells were grown at 37°C in a CO2 atmosphere in 96-well plates for three days
using MEM (Invitrogen, Carlsbad, CA) with 10% FBS and 1% antibiotic-antimycotic
(Invitrogen, Carlsbad, CA). Cells were incubated in MEM without FBS for at least 3
hours at 37°C + CO2 before adding the serum dilutions. Serial four-fold serum dilutions
were prepared in MEM and AttHRV was also diluted in MEM. Equal volumes of the
diluted serum were mixed with AttHRV (100 TCID50); 50µl were added to duplicate
wells and incubated for 1 hr at 37°C+ CO2. Trypsin (Sigma-Aldrich, St Louis, MO) was diluted (0.001mg/ml) in MEM and 50µl were added. The plates were incubated for 18 hrs at 37°C+ CO2. Cells were fixed with 80% acetone and air dried. Hyperimmune guinea pig anti-RV serum in PBS, pH 7.4 with 0.05% tween and 2% non-fat dried milk (PBS- 228 Tw-NFDM) was added and incubated for 1hr at 37°C+ CO2. Plates were washed twice
with PBS-Tw and goat anti-guinea pig IgG conjugated to horseradish peroxidase (KPL
Inc, Gaithersburg, MA) in PBS-Tw-NFDM was added and incubated for 1 hr at 37°C+
CO2. Plates were washed twice with PBS-Tw and 3-Amino-9-ethylcarbazole, H2O2 and acetate buffer (Sigma-Aldrich, St Louis, MO) were added according to the manufacturer’s instructions. The VN antibody titers were expressed as the reciprocal of the serum dilution that reduced virus infectivity (FFU) by 100%. To confirm that the
2/6/7VLP preparations induced VN antibodies, a guinea pig was hyperimmunized with 3 doses of 2/6/7VLP (200µg) with complete (first dose only) and incomplete Freund’s adjuvant subcutaneously and a VN titer of >1024 was induced.
Statistical analyses. Statistical analyses were performed using the SAS program (SAS
Institute Inc., Cary, NC). Differences in shedding and diarrhea protection rates among groups were analyzed by Fischer’s exact test. The mean peak fecal titer and the cumulative diarrhea scores were analyzed by one-way ANOVA followed by Duncan’s test. The effector and memory B cells and frequencies of AEC among groups were
compared using Kruskall-Wallis rank sum test. A one-way ANOVA followed by
Duncan’s test was used to compare serum VN antibody titers pre and post-challenge.
Pearson’s correlation coefficient was used to correlate frequencies of VP6-specific AEC
and numbers of RV-specific ASC. Spearman’s correlation coefficient was used to
correlate protection rates with mean intestinal RV-specific IgA, IgG ASC and mean RV-
specific VN antibody titers. A value of p<0.05 was considered significant.
229 3.4 RESULTS
The VP4-2/6/7+VP4-2/VLP vaccine induced the highest protection against diarrhea and shedding compared to the 2/6/7-2/6VLP and 3x2/6/7VLP vaccines
Protection against shedding and diarrhea are summarized in Table 3.1. To compare the non-replicating VLP vaccines with a protective regime, the previously tested
AttHRV+2/6VLP vaccine that induced 71% protection against shedding and diarrhea (21,
32) was included in the analysis. Protection against diarrhea was highest for the VP4-
2/6/7+VP4-2/6VLP vaccine with a 67% protection rate that was similar to the
AttHRV+2/6VLP vaccine (71%). The vaccine without VP4, the 2/6/7-2/6VLP vaccine, induced the lowest protection rate against diarrhea (33%) and the 3x2/6/7VLP vaccine induced an intermediate protection rate (50%). All control pigs had diarrhea. Protection against diarrhea conferred by all vaccines was statistically similar but higher than the protection conferred by ISCOM alone (except for the 2/6/7-2/6VLP vaccine). Mean cumulative diarrhea scores were similar for all vaccines and significantly lower than controls except for the VP4-2/6/7+VP4-2/6VLP vaccine. All non-replicating VLP vaccines and the controls had similar protection rates against HRV shedding. Only the
AttHRV+2/6VLP vaccine conferred significant protection against shedding. The protection rate against shedding induced by the VP4-2/6/7+VP4-2/6VLP vaccine was low, but highest (not significantly higher) among the non-replicating vaccines (33%).
Without VP4, the 2/6/7-2/6VLP vaccine conferred no protection against shedding and the
3x2/6/7VLP vaccine conferred a minimal protection rate (10%). Controls were not protected against shedding. The mean peak RV titer in feces was 100 times lower for the
AttHRV+2/6VLP and the VP4-2/6/7+VP4-2/6VLP vaccine groups than the mean peak
230 fecal titers from the 3x2/6/7VLP and the 2/6/7-2/6VLP vaccine groups and 1000 times
lower than controls (but was statistically similar among vaccines and controls).
The AttHRV+2/6VLP and the VP4-2/6/7+VP4-2/6VLP vaccines induced comparable protection rates against diarrhea and intestinal effector IgA ASC responses pre-challenge. To measure effector B cell responses, we used the ELISPOT assay that detects RV-specific ASC. Effector ASC responses at PID28 (pre-challenge) are summarized in Table 3.2. To compare with responses elicited by a protective regime,
RV-specific ASC induced by the AttHRV+2/6VLP vaccine were included. The RV- specific IgM and IgA ASC responses in ileum were generally higher than in spleen. The blood generally had the lowest mean numbers of ASC. Mean intestinal RV-specific IgA
ASC were associated with protection against shedding (r=0.97; p=0.004) and diarrhea
(r=1.0; p<0.0001) (Table 3.2). Intestinal RV-specific IgA ASC were highest for the
AttHRV+2/6VLP group (ASC, 55±18/5x105 total MNC) with the highest protection rates against shedding and diarrhea (71%). The VP4-2/6/7+VP4-2/6VLP vaccine with the next highest protection rate against diarrhea (67%), had the second highest intestinal RV- specific IgA ASC (19±4 RV-specific ASC/5x105 MNC), but they were statistically similar to the AttHRV+2/6VLP and 3x2/6/7VLP induced responses. The 3x2/6/7VLP vaccine with an intermediate protection rate against diarrhea (50%) also had intermediate intestinal RV-specific IgA ASC (10±2 RV-specific ASC/5x105 MNC) responses; statistically lower than the AttHRV+2/6VLP induced IgA ASC responses and higher than the 2/6/7-2/6VLP induced IgA ASC. Controls and 2/6/7-2/6VLP vaccinated pigs had no or minimal IgA ASC (2±0), no protection against shedding and no or low (33% for the
2/6/7-2/6VLP group) protection against diarrhea. A significant association was also 231 observed between intestinal RV-specific IgG ASC and protection against shedding
(r=0.97, p=0.004) and diarrhea (r=1.0, p<0.0001), but there were no significant differences in IgG ASC numbers among groups (all vaccine groups had higher IgG ASC than controls)(Table 3.2). Responses in spleen and blood were low and were not associated with protection.
The VP4-2/6/7+VP4-2/6VLP and 3x2/6/7VLP vaccine groups had significantly higher VN antibody titers 7 days after challenge. Serum VN antibody titers were determined by the IHC virus infectivity reduction assay and results are summarized in Table 3.3. Pre-challenge, the VP4-2/6/7+VP4-2/6VLP and the
3x2/6/7VLP vaccines induced low but significantly higher RV-specific VN antibody titers than the 2/6/7-2/6VLP vaccine regime and controls. The AttHRV+2/6VLP vaccine induced significantly higher RV-specific VN antibody titers than all other vaccines. At post-challenge day 7, the VP4-2/6/7+VP4-2/6VLP and 3x2/6/7VLP vaccine groups had increased and significantly higher RV-specific VN antibody titers compared to those induced by the 2/6/7-2/6VLP vaccine and controls. The AttHRV-2/6VLP vaccine group had significantly higher RV-specific VN antibody titers post-challenge compared to all other groups. The mean VN antibody titers among vaccine groups was associated with protection rates against diarrhea and shedding pre and post-challenge suggesting their role in protection against RV infection and disease.
The VP4-2/6/7+VP4-2/6VLP vaccine induced the highest intestinal RV- specific memory B cell response and the AttHRV+2/6VLP vaccine induced the highest spleen and circulating RV-specific memory B cell response. Memory RV- specific B cell responses are summarized in Table 3.4. The VP4-2/6/7+VP4-2/6VLP
232 vaccine induced the highest intestinal RV-specific IgA and IgG memory B cell responses
among all groups but they were only significantly higher compared to the responses
induced by the AttHRV+2/6VLP vaccine. On the other hand, the AttHRV+2/6VLP
vaccine induced significantly higher systemic and circulating RV-specific memory B cell
responses compared to all other groups for all isotypes (except for circulating IgG
memory B cells induced by the 2/6/7-2/6VLP vaccine). Memory responses elicited by the
2/6/7-2/6VLP and 3x2/6/7VLP vaccines were frequently higher in intestinal than in
systemic tissues and IgA and IgG memory ASC responses were generally lower (but
statistically similar except for the circulating IgG ASC memory responses induced by the
3x2/6/7VLP regime) to the responses induced by the VP4-2/6/7+VP4-2/6VLP vaccine
regime. Intestinal IgM ASC memory responses induced by 3x2/6/7VLP and 2/6/7-
2/6VLP vaccines were higher but statistically similar to the responses induced by the
VP4-2/6/7+VP4-2/6VLP vaccination.
Flow cytometry analysis of 2/6 (VP6), 2/6/7 (VP7) and 2/4/6/7VLP (VP4)
specific CD21+ AEC responses. The CD21 as a B cell marker. The mouse anti-human
CD21 antibody, which is cross-reactive with porcine CD21 was used to define B cell populations. For all tissues, most CD21+ cells expressed IgM (71-95%). Control pigs had the highest frequency of CD21+IgM+ B cells (93-95%) consistent with their naïve status.
Vaccinated pigs had a tendency for a lower expression of CD21+IgM+ B cells (71-93%).
Expression of both CD21 and IgA (0.38-8.5%) or IgG (0.3-12%) was lower than the
coexpression of CD21 and IgM but the majority of CD21+ B cells expressed IgM, IgA or
IgG (88-100%). Vaccinated pigs commonly had higher frequencies of CD21+IgA+ (1.0-
8.5%) and CD21+IgG+ (0.3-14%) B cells than control pigs (0.3-2.6% IgA+CD21+, 0.3-
233 1.2% IgG+CD21+) consistent with induced B cell maturation after vaccine delivery (data not shown). The CD21+IgA+ B cells in intestinal tissues were higher for the
AttHRV+2/6VLP and the VP4-2/6/7+VP4-2/6VLP vaccines than the other groups although statistically similar for all vaccines (data not shown). The CD21+IgG+ B cells were higher in all vaccine groups compared to controls (similar among vaccine groups, data not shown). Total CD21 frequencies were similar among groups. Some CD21- cells expressed IgM (3-35%), IgA (0.05-6.7%) and IgG (0.18-10%) and further characterization of these cells is needed. In most cases the frequencies of CD21+ Ig+ were higher than the frequencies of CD21-Ig+ for all tissues and groups. Based on these results and because according to the literature CD21 is co-expressed with CD19 (6), CD21 together with surface Ig were considered adequate porcine B cell markers.
RV VP6, VP7 and VP4-specific B cell responses can be studied by using
2/6VLP-GFP, 2/6/7VLP-DsRed and 2/4/6/7VLP-GFP, respectively. To study RV protein-specific B cells, 2/6VLP-GFP, 2/6/7VLP-DsRed and 2/4/6/7VLP-GFP were utilized to detect VP6, VP7 and VP4 specific responses, respectively. A typical dot plot representing intestinal B cell responses of a VP4-2/6/7+VP4-2/6VLP vaccinated pig and a control pig is shown in Figure 3.1. Frequencies of large cells (most RV-specific B cells were large cells), CD21+ expressing IgA, IgM or IgG and binding to the fluorescent
VLPs were analyzed. Double positive cells were usually not detected when staining with
2/6VLP-GFP and 2/6/7VLP-DsRed in the same tube (Fig.3.1.A). Most probably, the VP7 from the 2/6/7VLPs masked the underlying VP6, blocking the binding of VP6 specific B cells. On the other hand, when staining cells with 2/4/6/7VLP-GFP and 2/6/7VLP-DsRed in the same tube, double positive cells were detected (data not shown). It is likely that
234 VP7-specific B cells were able to bind both 2/4/6/7VLP-GFP and 2/6/7VLP-DsRed
because VP7 was not completely covered by the VP4 spikes. In all cases where positive
responses were detected, VP4 responses were several times higher than VP7 responses.
Nevertheless, we deducted VP7 specific AEC frequencies from VP4 specific AEC
frequencies to calculate VP4 specific responses. Also, as previously described for human
and mouse RV-specific B cells characterized by flow cytometry (24), using fluorescent
VLP in three independent experiments measuring IgA+, IgM+ or IgG+ cells, the sum of
the negative population in one experiment (e.g. CD21+IgA-) was higher than the sum of the positive population in the two other experiments (e.g. CD21+IgM+ + CD21+IgG+) and this was true for all tissues and isotypes (data not shown). Possibly fluorescent VLPs bound CD21+Ig- cells that are not B cells (e.g. antigen presenting cells) in a non-specific manner.
Cells binding 2/4/6/7VLP-GFP but not 2/6VLP-GFP or 2/6/7VLP-DsRed
were observed in the large cell population of cells in both controls and vaccinated
pigs. Typical dot plots of cells stained with 2/4/6/7VLP-GFP and 2/6VLP-DsRed are
shown in Figure 3.2. Dot plots on the top represent intestinal MNC from a VP4-
2/6/7+VP4-2/6VLP vaccinated pig and dot plots on the bottom represent a control pig.
Dot plots Fig 3.2.A and C were analyzed for the large lymphocyte population only while
dot plots Fig 3.2.B and D were analyzed for the large CD21+ population of cells. When analyzing only large cells, the vaccinated pig and the control showed a large population of 2/4/6/7VLP+ cells (3.7% and 13.5% for the vaccinated pig and control, respectively).
On the other hand, only the vaccinated pig had significantly higher frequencies of
2/6VLP+ cells (1.8% vs. 0.08% for the vaccinated pig and the control, respectively).
235 When analyzing large CD21+ cells (plots Fig 3.2.B and D), the amounts of 2/4/6/7VLP+ cells diminished for both vaccinated and control pigs (0.4% vs. 0.1% for the vaccinated
and the control pig, respectively), but the vaccinated pig had 4-fold higher frequencies of
2/4/6/7VLP binding cells (See upper left quadrant in Fig.3.2.B and 3.2.D and Fig 3.4.a).
Similarly, when analyzing large CD21+ cells, a significant enrichment of 2/6VLP+ cells was achieved mainly for the vaccinated pig (6.7% vs. 0.2% for the vaccinated and control pig, respectively and see Fig 3.4.c). This is the first study using 2/4/6/7VLP to characterize VP4 specific responses by flow cytometry, so the non-specific binding of
2/4/6/7VLP to control cells has not been explored previously.
The correlations between RV-specific ASC detected by ELISPOT and VP6- specific AEC detected by flow cytometry were low to moderate. To determine if RV- specific AEC detected by flow cytometry were effector RV-specific B cells, a correlation between VP6-specific AEC frequencies detected by flow cytometry and RV-specific
ELISPOT ASC numbers from vaccinated pigs was examined and is summarized in
Figure 3.3. The VP6 (2/6VLP-GFP) specific AEC frequencies were analyzed in a lymphocyte window including small and large cells (unlike the other analyses where only
large cells were analyzed) and no CD21 staining was performed. A wide gating was
performed to mimic the ELISPOT assay where intact MNC populations were added to a
well. Only VP6 responses were analyzed because most of RV specific responses are VP6
specific. The plots in Figure 3.3A-C represent the correlation between RV-specific ASC
and VP6-specific AEC among individual pigs that were vaccinated with 3x2/6/7VLP
(n=7), AttHRV+2/6VLP (n=2), 2/6/7+2/6VLP (n=1) and 3xAttHRV (n=4), for all tissues
at pre-challenge. The VP6-specific IgM+ AEC vs. RV-specific IgM ASC had a low, but
236 significant correlation (r=0.37; p=0.02); the VP6-specific IgA+ AEC vs. RV-specific IgA
ASC (r=0.48; p=0.005) and the VP6-specific IgG+ AEC vs. RV-specific IgG ASC
(r=0.46; p=0.02) had a moderate and significant correlation. These results suggest that most of VP6-specific AEC are not effector RV-specific B cells as reflected by ASC.
The VP4-2/6/7+VP4-2/6VLP vaccine induced similar or higher frequencies of
VP4-specific AEC than the AttHRV+2/6VLP vaccine . The VP4 specific AEC frequencies detected by using 2/4/6/7VLP-GFP are summarized in Figure 3.4.a. Non- specific binding in pigs vaccinated with 3x2/6/7VLP and 2/6/7-2/6VLP was observed.
Background values were calculated as the highest mean frequency detected in
3x2/6/7VLP and 2/6/7-2/6VLP vaccinated pigs for all tissues and isotypes plus or minus
2x SE (usually control pigs had the lowest background value compared to vaccinated pigs). Only frequencies statistically higher than background were considered positive. For most tissues and isotypes, CD21+ VP4-specific AEC responses between the
AttHRV+2/6VLP and VP4-2/6/7+VP4-2/6VLP vaccine groups were statistically similar,
but circulating and spleen CD21+VP4-specific IgM+ AEC were significantly higher for the VP4-2/6/7+VP4-2/6VLP vaccine group, probably because the VP4-2/6/7+VP4-
2/6VLP vaccinated pigs received three doses of VP4 (compared to 1 dose of VP4 in
AttHRV). Frequencies statistically higher than background after AttHRV+2/6VLP vaccination were mainly CD21+VP4-specific IgG+ AEC in ileum and spleen and
CD21+VP4-specific IgA+ AEC in spleen. Frequencies statistically higher than background induced by the VP4-2/6/7+VP4-2/6VLP vaccine regime were CD21+VP4- specific IgA+ and IgG+ AEC in spleen and IgM+ AEC in blood and spleen. Other isotypes
237 in the different tissues were statistically similar to background frequencies observed from
pigs inoculated with vaccines lacking VP4 (3x2/6/7VLP, 2/6/7+2/6VLP) or controls.
The VP7-specific responses were low and similar among vaccine groups
except for the 3x2/6/7VLP vaccine with the highest circulating IgM and IgG VP7-
specific AEC. The VP7-specific AEC frequencies measured using 2/6/7VLP-DsRed
(VP7-CJN-G1) are summarized in Figure 3.4.b. The overall VP7-specific AEC responses
were low and similar among vaccine groups. Non-specific binding observed in cells
derived from control pigs was generally absent or lower than 0.02. Background by tissues
and isotypes was calculated based on the mean frequency from control pigs plus or minus
2xSE . Only frequencies statistically higher than background frequencies were considered positive. The AttHRV-2/6VLP group had no responses in most tissues (statistically similar to background frequencies) and only low frequencies of CD21+ VP7-specific
IgG+ AEC were observed in spleen. The VP4-2/6/7+VP4+2/6VLP vaccine induced
CD21+VP7-specific IgA+ AEC in ileum and IgG+ AEC in spleen. The 3x2/6/7VLP
vaccine induced similar CD21+VP7-specific IgG+ AEC and IgM+ AEC in spleen and circulating IgM+ AEC compared to the other vaccination groups (but statistically higher
than the AttHRV+2/6VLP vaccine for splenic IgM+ AEC) and this group (3x2/6/7VLP) induced the highest circulating CD21+VP7-specific IgG+ AEC because it was the only
vaccine group receiving three doses of VP7.
The VP6-specific ASC responses were highest and similar among vaccine groups.
The VP6-specific AEC frequencies measured using 2/6VLP-GFP are summarized in
Figure 3.4.c. Non-specific binding was minimal or absent. Background was calculated as
for VP7-specific AEC. The VP6-specific AEC responses were generally higher than
238 VP4- and VP7-specific AEC responses. For all isotypes in all tissues, responses were
frequently similar and significantly higher than controls probably because all vaccines
included three doses of VP6. However, the AttHRV-2/6VLP and the VP4-2/6/7+VP4-
2/6VLP vaccines induced significantly higher circulating CD21+VP6-specific IgG+ AEC than the 2/6/7-2/6VLP vaccine and only the AttHRV-2/6VLP vaccine induced statistically higher splenic IgG+ AEC than the 2/6/7-2/6VLP vaccine. Additionally, the
AttHRV-2/6VLP vaccine induced significantly higher splenic IgA+ and IgG+ AEC than the VP4-2/6/7+VP4-2/6VLP regime.
3.5 DISCUSSION
We studied the role of VP7 with and without VP4 in protective immunity induced by rotavirus using the neonatal Gn pig model and various formulations of VLP vaccines.
In previous studies we demonstrated that three doses of 2/6VLP (VP6) alone did not confer protection against diarrhea or shedding (23, 52) . In this study we showed that by adding three doses of VP4 to the 2/6VLP vaccine and priming with VP7 in 2/6/7VLPs
(VP4-2/6/7+VP4-2/6VLP vaccine), we increased protection against diarrhea (33% to
67%) and protection against shedding (0% to 33%). Priming and boosting with VP7 without VP4 (3x2/6/7VLP) induced an intermediate protection rate (50% and 10% protection against diarrhea and shedding, respectively) between the protection rates induced by the VP4-2/6/7+VP4-2/6VLP (67% and 33% protection against diarrhea and shedding, respectively) and 2/6/7-2/6VLP vaccines (33% and 0% protection against diarrhea and shedding, respectively). In the absence of VP4, priming with 2/6/7VLP and boosting without VP7 (2/6/7-2/6VLP vaccine) induced the lowest protection rate against
239 diarrhea (33%) and no protection against shedding. These findings suggest that an
effective non-replicating vaccine should include three doses of both neutralizing antigens
VP4 and VP7 for a higher protection rate against disease and infection. Further
experiments are needed to confirm this possibility.
The lack of protection seen after vaccination with 2/6VLPs in neonatal Gn pigs
was also observed in neonatal mice vaccinated with VP6 alone (using E.coli heat labile
toxin as adjuvant) and challenged with virulent virus 10 days after inoculation (47).
However, of the adults that received the same vaccine, 88% were protected against
shedding, also upon challenge at 10 days after vaccination. Only after inoculating the
pups with virulent heterologous rhesus RV (RRV), was 91% protection against shedding
achieved after challenge at post-inoculation day 10. However neonatal serum IgG and
fecal IgA antibody titers were significantly lower than the adult mice responses.
Furthermore the amount of RV antigen shed by VP6-inoculated neonatal mice was
significantly higher than that of VP6-inoculated adult mice (47), suggesting that in
neonates lower acquired immune responses were induced and that perhaps their immature
innate immune system failed to inhibit or decrease RV replication. Nevertheless, the reason why VP6 protects adult mice and not neonates against RV challenge is unclear. In contrast to neonates, adults have been previously exposed to various pathogens and environmental antigens that promote the maturation of the innate and acquired immune system. Possibly, the adult innate immune responses partially control initial RV replication and a robust acquired immune response is able to eradicate replicating RV.
Also perhaps adults generate a larger and wider repertoire of cross-reactive B and T lymphocytes. Adult vaccination with VP6 might stimulate cross-reactive intestinal
240 lymphocytes broadening the immune responses capable of conferring protection against
often low doses of virulent virus challenge. Conceivably to prevent enteric viral
infections, vaccines delivered to neonatal animals and even to human neonates need to
include higher doses and/or quality components that effectively induce local immune
responses including neutralizing antibodies (e.g. replicating viruses). Because VP6 (or
2/6VLPs) alone does not induce neutralizing antibodies, likely the antibodies induced are
insufficient to provide the degree of protection needed to prevent the massive infection of
neonatal villious epithelial cells caused by RV. To date evidence of VP6 or 2/6VLPs
inducing protection against RV shedding derives from studies of adult mice and rabbits
(1, 9, 11). Vaccination with VP6 (or 2/6VLP) alone has not been performed in naïve adult
pigs or humans so it is unknown if VP6 alone will confer protection. Further evidence of
the dependence on neutralizing antigens for the induction of protection in neonatal mice
was observed in studies using backpack hybridoma transplants. Backpack hybridoma
transplants secreting VP8-specific IgA, prevented diarrhea in neonatal mice. However,
VP6-specific IgA secreting backpack hybridoma transplants did not prevent diarrhea in
the pups (40). Only adult mice were protected from shedding with backpack hybridoma
transplants secreting extremely high titers of non-neutralizing anti-VP6 IgA (7). In addition, calves that received colostrum from 2/4/6/7VLP vaccinated cows were completely protected against diarrhea upon bovine RV challenge compared to colostrum- fed calves from 2/6VLP vaccinated cows that were only partially protected (13).
If the route and type of vaccine induces local protective immunity at the site of entry and initial replication of the virus, then infection will be contained and aborted (3,
4). Recently, we studied nasal and intestinal replication of virulent and AttHRV strains in
241 the Gn pig model (2). Although both viruses replicated in intestinal and nasal tissues, the
AttHRV replicated much less efficiently than the virulent strain in intestinal tissues but
with similar efficiency in nasal tissues. The AttHRV used in the pigs is the same serotype
(P1A[8],G1) as a candidate and licensed HRV vaccine (RIX4414 and Rotarix). Two
doses of RIX4414 (105.8 FFU, PO) or 2-3 doses of Rotarix in humans and AttHRV
(5x107FFU PO) in pigs were at least moderately effective against all types of diarrhea
(63-96% for RIX4414, 70-85% for Rotarix in humans and 63% for pigs) (41, 44, 54). It is
unclear if the tissue culture passaged Wa AttHRV also replicates nasally in humans like
in pigs. In the pig, this is reflected as a relatively low number of intestinal and higher
systemic effector and memory RV specific antibody-secreting cells (ASC) detected after
AttHRV vaccination compared to the virulent Wa HRV (54). Possibly, if a higher amount of AttHRV reaches or is stable in the gut, higher intestinal immune responses and protection rates will be obtained. (21, 23). These studies suggest that a non-replicating vaccine should target intestinal and nasal mucosal tissues for maximum efficacy; therefore, a combination of oral and IN routes such as we tested is probably optimal for candidate rotavirus vaccines.
We studied B cell immunity elicited by the non-replicating VLP vaccines. The B cell responses induced by the AttHRV+2/6VLP vaccine (21, 31, 32), were included to compare responses induced by a known protective regime to the responses induced by the
non-replicating VLP vaccines. The intestinal effector and memory ASC responses
elicited by the AttHRV+2/6VLP vaccine were published recently (31, 32). We
hypothesized that comparable immune responses could be elicited by an optimally
constructed non-replicating VLP vaccine (containing neutralizing antigens) delivered
242 similarly. To measure B cell responses we used an assay that detects RV-specific effector
ASC (ELISPOT) and an AEC assay that measures RV-specific Ig expressing B cells
(flow cytometry) reflecting RV-specific non-effector B cells (RV-specific germinal center B cells, extrafollicular B cells, pre-plasma cells and/or memory B cells) as well as effector B cells. Flow cytometry was used to detect RV-protein specific responses using
detecting antigens in their native conformation as VLPs. We observed that the vaccines inducing the highest protection rates (AttHRV+2/6VLP and VP4-2/6/7+VP4-2/6VLP vaccines), induced the highest intestinal RV-specific IgA ASC. Previous studies from our laboratory described a strong association between intestinal RV-specific IgA ASC or intestinal antibody responses and protection (23, 54). In the current study we also showed a significant association between numbers of intestinal IgA ASC and protection.
However, we did not find an association between protection and VP6, VP7 or VP4- specific AEC measured using specific fluorescent VLPs and detected by flow cytometry.
The correlation coefficient between RV-specific ASC detected by ELISPOT and VP6- specific AEC detected by flow cytometry among individual pigs was moderate to low
(Fig.3.3). This suggested that the majority of VP6-specific AEC were not effector ASC.
Possibly, only effector RV-specific IgA ASC and not VP6-specific AEC (that also include non-effector VP6-specific B cells) correlate with protection. Alternatively, perhaps by flow cytometry we did not detect all VP4 and/or VP7 specific responses explaining the lack of association with protection. In mice the majority of Syndedan-1+ B cells secrete antibodies and thus are ASC. In RV infected mice (PID7) the majority of
Syndecan-1+ B cells have an extrafollicular phenotype (IgD- B220low) and similar frequencies of 2/6VLP specific-Syndecan-1+ IgD- B220low and ASC were observed. On
243 the other hand, only a minority of 2/6VLP specific B cells with germinal center
phenotype secreted antibodies (51) demonstrating that not all AEC are effector ASC. To
determine if RV-protein specific B cells detected by flow cytometry correlate with
protection in pigs, a marker for effector B cells (e.g. CD138, marker of plasma cells (38)
might be needed.
To our knowledge this is the first study to use 2/4/6/7VLP-GFP to assess VP4
specific responses by flow cytometry; 2/6VLP and 2/6/7VLP were previously used for
the characterization of human RV-specific B cells (36). In adults, children and neonates,
a similar large percentage of naïve B cells bound 2/6VLP-GFP while no binding to
2/6/7VLPs was detected. Furthermore, VP7-specific B cell responses were lower than
VP6-specific responses and adults had higher VP7 responses than recently infected
infants. In Gn pigs, VP6 responses were also the highest but no non-specific binding of
2/6VLP was observed (controls did not have VP6 specific responses). In contrast,
2/4/6/7VLP did bind cells from the vaccinated and control pigs, but they were mainly
non-B (CD21-) cells. These cells probably were professional and non-professional antigen presenting cells binding or taking up the VLP particles. Further characterization
of these cells is in progress. Possibly, VP4 is a better target for antigen uptake than VP6
but this needs to be tested further. If this hypothesis is true, this could explain in part the dependence of neonatal Gn pigs on RV neutralizing antigens for induction of protection.
The neonatal immune system is immature and if antigen presenting cells have stronger avidity to antigens containing VP4, then stronger immune responses may be elicited if
VP4 is added to a RV vaccine formulation. The reason why we did not find a significant population of 2/6VLP binding cells in the control pigs may be due to their Gn status. It is
244 known that the gut microflora induces somatic hypermutation in the gut-associated lymphoid tissue, increasing the Ig repertoire in naïve B cells (39). The lack of gut
microflora in the Gn pig may induce a smaller repertoire of naïve B cells diminishing the
chance of detecting naïve B cells bound to 2/6VLPs.
The mouse anti-human CD21 marker was selected to study porcine B cells. This
antibody is the only available antibody to study porcine B cells together with anti-IgA,
anti-IgM and anti-IgG reagents. Cross-reaction of the anti-human CD21 antibody with
porcine CD21 was verified previously (5). The CD21 molecule, also known as the C3d
complement receptor, is expressed later in B cell development (26) and is linked
physically and functionally to CD19 in mice and humans (6, 28, 29). The CD19 B cell
marker has been previously used to study RV B cell immunity in humans (20, 24). This is the first RV study that uses CD21 as a porcine B cell marker. We found that most CD21+
B cells express IgM, IgA or IgG and that CD21- B cells express lower surface Ig with further characterization of these later cells needed.
We measured VN antibody titers in serum and found that the non-replicating vaccines that induced higher protection rates (VP4-2/6/7+VP4-2/6VLP and 3x2/6/7VLP vaccines) induced a significant and similar boost in VN antibody titers by 7 days post challenge. This finding suggests that the 2/6/7VLP ± VP4 regimes were immunogenic
and induced VP4 and/or VP7 specific B cells that were activated at the time of challenge.
The role of serum neutralizing antibodies in protection is conflicting. Studies in humans showed that primary infections induce mainly homotypic neutralizing antibodies and protection is detected only after homotypic infections initially (8, 34). After repeated infections, cross-reactive neutralizing antibodies can protect against heterotypic
245 infections (8, 34). Other studies of humans did not find a correlation between neutralizing
antibodies and protection (50). This discrepancy is also observed in the pig studies. Pigs with high serum VN antibody titers are not always protected from infection or disease
(21, 56). Serum neutralizing antibodies alone may not be sufficient to provide protective immunity or in many cases serum neutralizing antibodies may not adequately reflect the presence of neutralizing antibodies in the intestine which can not be accurately or routinely measured. Also other components of the immune system (T cells, innate responses, cytokines, non-neutralizing antibodies) may be important to confer complete protection. Previous studies from our laboratory showed that VN antibodies together with
intestinal RV-specific IgA ASC might be necessary for induction of protection against disease and infection. Various vaccine regimes delivered to Gn pigs that induced high serum VN antibody titers and intermediate-high intestinal RV-specific IgA ASC induced high protection rates against shedding and diarrhea (21, 23, 30, 54). In the current study, we found that our reference vaccine, AttHRV+2/6VLP that is highly protective, induced high VN antibody titers pre and post-challenge and intermediate-high intestinal RV- specific IgA numbers. The VP4-2/6/7+VP4-2/6VLP vaccine induced similar RV-specific intestinal IgA ASC but low VN antibody titers resulting in a similar (67%) protection rate against diarrhea, but low (33%) protection rate against shedding.
Memory B cell responses were measured as described by Yuan et al. (53). In the present study we found that our reference vaccine, the AttHRV+2/6VLP regime induced significantly lower intestinal memory B cell responses compared to those induced by the
VP4-2/6/7+VP4-2/6VLP vaccine. On the other hand, spleen and circulating memory B cells were highest for our reference vaccine. Because at PID28, intestinal effector IgA
246 ASC were present and a high titer of intestinal IgA antibodies was detected after
AttHRV+2/6VLP vaccination (21), protection conferred by this regime might have been conferred by these effector B cells rather than memory B cell responses. In comparison, the VP4-2/6/7+VP4-2/6VLP vaccine induced significantly higher intestinal memory B cell responses indicating that this non-replicating vaccine regime was effective in inducing intestinal immune responses. The low intestinal and high systemic memory B cell responses induced by the AttHRV+2/6VLP vaccine can be explained based on previous findings from our laboratory. In Gn pigs, AttHRV replicates as extensively as the virulent virus in nasal tissues but at much lower magnitude in intestinal tissues (2). It is also known that IN inoculation induces greater systemic immune responses (19, 22).
Nasal virus replication stimulates lymphocytes that can reach the systemic compartment by expressing the systemic homing markers, CCR7 and L-selectin (25). Also, not only the AttHRV+2/6VLP vaccine, but also the 3xAttHRV vaccine, induced mainly systemic memory B cell responses (53). The 3xAttHRV vaccine induced lower effector ASC (23) and few intestinal memory B cells at PID28 (pre-challenge), but intestinal RV-specific
IgA antibodies (21) were detected and the vaccine induced considerable protection (67% against shedding and diarrhea) (54). The same observation was described for adult mice
(35). Adult mice inoculated with 2/6VLP IN developed fecal IgA antibodies but low intestinal ASC responses and developed protection from RV shedding. The authors concluded that the fecal IgA antibodies may be a consequence of the higher systemic response and that T cells but not intestinal B cells might be important in the induction of protective immunity. From studies of adult knockout mice, both B and CD8+ T cells played an important role in protection against RV shedding. If B or CD8+ T cells were
247 lacking, protective immunity was observed but, only if there were no B or no CD8+ T cells, did mice develop chronic shedding (15). Because the vaccinated pigs with the highest protection rates had intestinal effector B cells at the time of virulent virus
challenge, we can not conclude whether intestinal or systemic memory B cells played a
major role in RV protection.
In conclusion, ours and previous studies showed that protection after RV
vaccination is dependent on the presence of both effector intestinal IgA ASC and
neutralizing antibodies. The AEC detected by flow cytometry did not correlate with
protection because most of these cells might not be effectors (significant moderate to low
correlation between ELISPOT ASC and flow cytometry AEC) and possibly only plasma
B cells that actively secrete antibodies correlate with protection. The importance of
memory B cell responses could not be elucidated from this study because in the vaccine
groups with the highest protection rates, effector intestinal IgA ASC were also present at
the time of challenge. However, the VP4-2/6/7VLP+VP4-2/6VLP vaccine induced high
intestinal memory responses demonstrating that the non-replicating vaccine was
immunogenic even when delivered orally into the intestinal environment for priming. It
also induced effector intestinal IgA ASC, VP4-specific AEC responses and significantly
boosted VN antibody titers 7 days after challenge. In the future, protection against
shedding in the Gn pig model may be improved if pre-challenge VN antibody titers are
increased by delivering more antigen and/or adding other mucosal adjuvants to improve the immunogenicity of the non-replicating vaccines. The semi-purified VP4 was immunogenic and when delivered with 2/6/7VLPs (VP4-2/6/7VLP+VP4-2/6VLP), this vaccine regime conferred protection against diarrhea. Thus this non-replicating VLP
248 vaccine regime appears promising and the difficulty of synthesizing high amounts of
2/4/6/7VLPs and preserving their stability with ISCOM adjuvant was circumvented.
3.6 ACKNOWLEDGMENTS
We thank Dr Juliette Hanson for the clinical care of the Gn pigs, Myung Guk Han
and Guohua Li for assistance with electron microscopy and Rich McCormick, Peggy
Lewis and the summer students from the Agricultural Technical Institute at The Ohio
State University for their technical assistance. We thank Dr Manuel Franco, Juanita
Angel and Maria Cristina Jaimes for helpful comments and Hong Liu for assistance in the
statistical analyses.
This work was supported by a grant from the National Institutes of Health,
National Institutes of Allergy and Infectious Disease (RO1AI033561). Salaries and research support were provided by State and Federal Funds appropriated to the Ohio
Agricultural Research and Development Center at the Ohio State University.
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256 disease., 25th Annual Meeting of American Society of Virology, Madison, Wisconsin.
257 Protection Protection rates (%) rates (%) % Mean peak % Mean against against nb Sheddingc titer shed diarrheae cumulative sheddingg diarrheag Vaccinea in FFU/ml(SEM)d scoref AttHRV +2/6VLP 7 29 6.3x102(1.2X102) 29 6.5(0.4) 71 71 AA ABAA VP4-2/6/7 + VP4-2/6VLP 6 67 2.9X102(1.1X102) 33 8.4(1) 33 67 AB A A AB AB A 3x2/6/7VLP 10 90 4.3X104(8.5X103) 50 6.9(0.5) 10 50 BA ABBA 2/6/7+2/6VLP 6 100 6.6X104(2.8X104) 66 6.3(1.3) 0 33 258 B A AB B B AB Control 15 100 1.7X105(1.3X105) 100 10(0.5) 0 0 BA BABB
a The different non-replicating VLP regimes, the combined AttHRV+2/6VLP vaccine regime and controls are summarized b Number of pigs challenged per group. The AttHRV+2/6VLP results were previously published by Gonzalez et al .[21] and Nguyen et al [32]. c Shedding was determined by cell-culture immuno-fluorescence (CCIF) and enzyme linked immunosorbent assay (ELISA) for rotavirus detection during 6 days after challenge with virulent Wa HRV. The detection limit for CCIF is 250 fluorescent forming units (FFU)/ml. Numbers with different letters differ significantly (Fisher's exact test, p<0.05) d Mean peak rotavirus titers were determined by CCIF and expressed as FFU/ml. Numbers with different letters differ significantly (one-way ANOVA followed by Duncan's test; p<0.05). SEM in parentheses. eFeces were scored for 6 days after challenge with virulent WaHRV as follows: 0=solid; 1=pasty; 2=semi-liquid; 3=liquid. Scores equal or higher than 2 were considered diarrheic. Numbers with different letters differ significantly (Fisher's exact test, p<0.05) f Mean cumulative diarrhea score= [(sum of fecal score for 6 days)/n)]. Numbers with different letters differ significantly (one-way ANOVA followed by Duncan's test, p<0.05). SEM in parenthesis g Protection rate= [1-(%of vaccinated pigs with shedding or diarrhea/% of control pigs with shedding or diarrhea)]x100
Table 3.1. Protection rates conferred by the non-replicating VLP vaccines, the combined AttHRV+2/6VLP vaccine regime and controls RV-specific ASC/ 5x105 total MNC % Protection % Protection Ileum Spleen PBLs against against Vaccinea nb IgM IgA IgG IgM IgA IgG IgM IgA IgG sheddinge diarrheae
AttHRV +2/6VLP 9 10(3)c 55(18) 15(5) 5(2) 9(3) 16(8) 1(0) 3(1) 3(1) 71 71 ABd AA AAA AAA A A
VP4-2/6/7 + 5 1(1) 19(4) 10(5) 0(0) 0(0) 1(1) 0(0) 0(0) 2(1) 33 67 VP4-2/6VLP B AB A DC BC B B BC AB AB A
3x2/6/7VLP 12 13(8) 10(2) 8(2) 4(3) 2(1) 11(10) 1(0) 3(2) 2(1) 10 50 A BAABC B B AB AB B B A
2/6/7+2/6VLP 6 1(1) 2(0) 3(2) 3(1) 0(0) 3(2) 0(0) 0(0) 1(1) 0 33 AB CAAB BC B AB C BC B AB 259 Control 9 0 00 000 000 0 0 C CB DCC BCC B B r=0.97f r=0.97f p=0.004 p=0.004 r=1g r=1g p<0.001 p<0.001 a The different non-replicating VLP regimes, the combined AttHRV+2/6VLP vaccine regime and controls are summarized b Number of pigs per vaccine group cNumbers in parentheses represent the SEM dNumbers with different letters differ significantly (Kruskal-Wallis rank sum test; p<0.05) within an isotype and tissue e Protection against shedding and diarrhea conferred by the different regimes as described in Table 1. f Spearman's correlation coefficient between mean RV-specific IgA and IgG intestinal ASC and protection against shedding. g Spearman's correlation coefficient between mean RV-specific IgA and IgG intestinal ASC and protection against diarrhea.
Table 3.2. Antibody secreting cell (ASC) responses at pre-challenge determined by ELISPOT induced by the different different non- replicating VLP vaccines compared to the combined AttHRV+2/6VLP vaccine regime and controls non-replicating VLP vaccines compared to the combined AttHRV+2/6VLP vaccine regime and controls Vaccinea nb Pre-challengec nb Post-challengec PID28/PCD0 PID35/PCD7
AttHRV +2/6VLP 13 150(110) 8 2896(503) AA
VP4-2/6/7 + 12 7(2) 9 1195(341) VP4-2/6VLP B B
3x2/6/7VLP 11 6(2) 7 1248(439) BB
2/6/7+2/6VLP 10 2(0) 4 362(941) CC
Control 8 2(0) 11 3(0) CD r=0.89d r=0.90d p=0.04 p=0.02 r=91e r=0.87e p=0.03 p=0.053
a The different non-replicating VLP regimes, the combined AttHRV+2/6VLP vaccine regime and controls are summarized b Number of pigs per group. c Serum samples at pre-challenge on post-inoculation day (PID) 28 (PID28/PCD0) and at post-challenge day (PCD) 7 (PID35/PCD7) were tested. Data represents the inverse Geometric Mean Titers (GMT) from 4-13 pigs per group. Numbers in parentheses represent the standard error of the mean. Numbers with different letters underneath differ significantly. (One way ANOVA followed by Duncan's test; P<0.05). For titer calculation, 100% focus forming unit reduction was considered. Titers ≤2 were considered negative. dSpearman's correlation coefficient between diarrhea and mean RV-specific VN antibody titers pre-challenge and post-challenge eSpearman's correlation coefficient between shedding and mean RV-specific VN antibody titers pre-challenge and post-challenge
Table 3.3 Virus neutralizing antibody titers at pre and post-callenge in Gn pigs vaccinated with the different non-replicating VLP vaccines compared to the combined AttHRV+2/6VLP vaccine regime and controls
260 Memory RV-specific ASC/ 5x105 total MNC Ileum Spleen PBL Vaccinea nb IgM IgA IgG IgM IgA IgG IgM IgA IgG
AttHRV +2/6VLP 7 1.4(1)c 1.9(1) 2.1(1) 198(73) 18(9) 1923(877) 64(43) 80(35) 584(246) Ad BCDAAA AAA
VP4-2/6/7 + 9 2(1) 75(48) 143(103) 5(2) 1(0) 100(55) 2(1) 9(7) 28(9) VP4-2/6VLP A A AB B B B B B B
3x 2/6/7VLP 8 3(1) 18(9) 12(7) 1.6(1.4) 1(1) 10(8) 2(2) 1(1) 2(2) 261 AABBCBBB BBCC
2/6/7+2/6VLP 4 12(12) 33(11) 36(10) 0(0) 0(0) 4(3) 2(2) 5(5) 23(14) AB A A BC B B B BC AB
Control 7 0 0 0 0 0 0 0 0 0 BCD CBC CCD a The different non-replicating VLP regimes, the combined AttHRV+2/6VLP vaccine regime and controls are summarized b Number of pigs per vaccine group cNumbers in parentheses represent the SEM dNumbers with different letters differ significantly (Kruskal-Wallis rank sum test; p<0.05)
Table 3.4. Memory antibody secreting cell (ASC) responses at pre-challenge determined by ELISPOT induced by the different non-replicating VLP vaccines compared to the combined AttHRV+2/6VLP vaccine regime and controls AttHRV+2/6VLP regime
Figure 3.1. Representative dot plots of intestinal MNC from a VP4-2/6/7+VP4-2/6VLP vaccinated and a control gnotobiotic pig. Lymphocyte and not the monocyte cell population was gated on side scattered vs. forward scattered plots. Large lymphocytes and CD21+ B cells were analyzed in windows 1 and 2, respectively. Plots of 2/6VLP-
GFP vs 2/6/7VLP-DsRed (A) and 2/4/6/7VLP-GFP vs. 2/6VLP- DsRed (B) on windows
1 and 2 (large CD21+ B cells) were performed. A window along the 2/6VLP or
2/4/6/7VLP-GFP FL1 axis (window 3) and the 2/6/7VLP-DsRed FL2 axis (window 4) was drawn to avoid double positive cells (dead cells). Only frequencies of large
CD21+Ig+VLP+ cells on window 3 (for 2/6VLP or 2/4/6/7VLP analyses) or window 4
(for 2/6/7VLP analysis) were analyzed. A typical dot plot from intestinal IgA responses to VP6 (2/6VLP-GFP), VP7 (2/6/7VLP-DsRed) and VP4 (2/4/6/7VLP-GFP) from a pig vaccinated with VP4-2/6/7+VP4-2/6VLP (C) and a control pig (D) are shown. Quadrants were placed according to the background frequencies in the plots analyzed with IgA, IgM or IgG and CD21 without the VLPs (not shown) and background values were deducted from the plots with fluorescent VLPs.
262 (C) Pig vaccinated (D) Control with VP4-2/6/7+VP4-2/6VLP AM050306.044 AM062906.064
Intestinal MNC at pre-challenge 0.59% 0%
AM062906.064 (A) AM062906.064 (3) 0 1 2 3 4 Large 100 101 102 103 104 10 10 10 10 10 cells (1) 2/6 VLP GFP 2/6VLP GFP R5 R2 2/6VLP-GFP AM062906.064 AM050306.044 R3 R6 (4)
R1 100 101 102 103 104 2/6/7 VLP DsRed 263 0 200 400 600 800 1000 0.01% 0% Forward Scatter
0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 2/6/7 VLP DsRed 2/6/7VLP DsRed AM062906.064 (B)
% CD21+ IgA+ AEC 2/6/7VLP-DsRed AM050306.048 AM062906.067 AM062906.067 (2) 0.14% 0% R4
100 101 102 103 104 100 101 102 103 104 CD21 APC 2/6 VLP DsRed 0 1 2 3 4 10 10 10 10 10 3 4 2/4/6/7 VLP GFP 100 101 102 10 10 2/4/6/7VLP GFP Figure 3.1 2/4/6/7VLP-GFP
Figure 3.2. Dot plots representing 2/4/6/7VLP and 2/6VLP responses from a VP4-
2/6/7+VP4-2/6VLP vaccinated pig (A,B) and a control pig (C,D). Dot plots of only large
(A,C) or large combined with CD21+ B cells (B,D) 2/4/6/7VLP-GFP vs. 2/6VLP-DsRed
binding cells are shown . Both vaccinated and control pigs had large cells that recognized the 2/4/6/7VLP-GFP (A,C- upper left quadrants, respectively) but only the vaccinated pig had significant frequencies of cells recognizing the 2/6VLP-DsRed (A- lower right quadrant). By enclosing CD21+ large cells, the vaccinated pig (B) had higher
2/4/6/7VLP-GFP binding cells (upper left quadrant) and a significant enrichment on
2/6VLP-DsRed binding cells (lower right quadrant) compared to the control pig (D).
264 Large cell Large cell and CD21+ window only windows (A) (B) AM062906.007 AM062906.007 Pig vaccinated 3.7% 1.3% 0.4% 0.1% with VP4-2/6/7VLP+ VP4-2/6VLP 1.8% 6.7% FP G 265 100 101 102 103 104 100 101 102 103 104 2/6 VLP DsRed 2/6 VLP DsRed
(C) AM050306.048 (D) AM050306.048 2/4/6/7VLP-
Control 13.5% 0.3% 0.1% 0.08%
0.08% 0.2%
100 101 102 103 104 100 101 102 103 104 2/6VLP DsRed 2/6VLP DsRed
2/6VLP-DsRed Figure 3.2. Intestinal MNC at pre-challenge
Figure 3.3. Correlation coefficient between VP6-specific AEC and RV-specific ASC detected by flow cytometry and ELISPOT, respectively. (A,B,C)- Plots represent the correlation between ASC and AEC from all tissues among individual vaccinated pigs pre- challenge. (A) VP6-specific IgM+ AEC vs. RV-specific IgM ASC; (B) VP6-specific IgA+
AEC vs. RV-specific IgA ASC; (C) VP6-specific IgG+ AEC vs. RV-specific IgG ASC.
Frequencies of IgA, IgM or IgG+ 2/6VLP-GFP+ total lymphocytes were correlated to numbers of ASC to RV. No CD21 staining was performed.
266 (A) 100
10 r=0.36 p=0.02
RV-specific IgM+ ASC 1 0.01 0.1 1 10
Frequencies of VP6-specific IgM+ AEC (%) (B) 100
10
r=0.48 p=0.001
1 0.01 0.1 1
(C) Frequencies of VP6-specific IgA+ AEC (%) 100
10
r=0.46 p=0.02 1 RV-specific IgG+ ASC RV-specific IgA+ ASC 0.01 0.1 1
Frequencies of VP6-specific IgG+ AEC (%) Figure 3.3 267
Figure 3.4. Frequencies (%) of CD21+ VP4-specific AEC. The CD21+ IgM (top), IgA
(middle), IgG (bottom) VP4 specific AEC frequencies elicited by the different non- replicating VLP vaccines and the AttHRV+2/6VLP regime are summarized. Frequencies of VP7 specific AEC detected by using 2/6/7VLP-DsRed were deducted from the frequencies detected by 2/4/6/7VLP-GFP. Stripped bars represent ileum responses; black bars represent spleen and white bars represent peripheral blood responses (PBL). Within a tissue type or blood and isotype, bars with different letters differ significantly (Kruskal- wallis rank sum test, p≤ 0.05). The gray dotted lines represent background frequency values. Each tissue or blood and isotype has a different background frequency value that was calculated as the highest mean frequency induced in pigs that did not receive VP4 as part of the regime (3x2/6/7VLP and 2/6/7-2/6VLP vaccines or controls) plus or minus 2
SE. Values statistically higher than background frequencies were considered positive.
268 IgM 2.5 2.5 A Ileum Spleen 7.0 PBL 2.0 2.0 A 6.0 A 5.0 1.5 1.5 4.0 1.0 1.0 3.0 B BC BC 0.5 AAB AB B C2.0 0.5 BCBC C 1.0 0.0 0.0 AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls0.0 +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls VP4-2/6VLP VP4-2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP VP4-2/6VLP 1.0 IgA 1.0 2.5 0.8 0.8 269 A 2.0 A 0.6 1.5 0.6 0.4 1.0 0.4 A A 0.2 AB B BC C A AB AB B 0.5 0.2 A A A 0.0 0.0 0.0 AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP VP4-2/6VLP VP4-2/6VLP VP4-2/6VLP 1.0 IgG 1.0 1.0
0.8 0.8 0.8 Frequencies (%) of CD21+ VP4-specific AEC A 0.6 A 0.6 A AB 0.6 0.4 0.4 0.4 A A B B C 0.2 BC B C ABC 0.2 0.2 BC C 0.0 0.0 AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls 0.0 AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls +2/6VLP 2/6/7VLP + 2/6VLP AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls +2/6VLP 2/6/7VLP + 2/6VLP VP4-2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP VP4-2/6VLP Figure 3.4 VP4-2/6VLP
Figure 3.5. Frequencies (%) of CD21+ VP7-specific AEC. The CD21+ IgM (top), IgA
(middle), IgG (bottom) VP7 specific responses elicited by the different non-replicating
VLP vaccines and the AttHRV+2/6VLP regime are summarized. Stripped bars represent ileum responses; black bars represent spleen and white bars represent peripheral blood responses (PBL). Within a tissue type or blood and isotype, bars with different letters
differ significantly (Kruskal-wallis rank sum test, p≤ 0.05). The gray dotted lines
represent background frequency values. Each tissue or blood and isotype has a different
background frequency value that was calculated as the mean frequency induced by
control pigs that did not receive VP7 as part of the regime plus or minus 2 SE. Values
statistically higher than background frequencies were considered positive.
270 1.0 IgM 1.0 Ileum Spleen 1.5 PBL A 0.8 0.8 1.3
1.0 0.6 A 0.6 0.8 0.4 0.4 AC A A B ABD BD0.5 0.2 A 0.2 CD A AB AB A 0.3 AB 0.0 0.0 0.0 AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP VP4-2/6VLP VP4-2/6VLP VP4-2/6VLP 1.0 IgA 1.0 1.0 0.8 0.8 0.8
271 0.6 0.6 A 0.6 0.4 0.4 A AB 0.4 A AB B B A A A A A 0.2 0.2 A A A 0.2
0.0 0.0 0.0 AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP IgG VP4-2/6VLP VP4-2/6VLP VP4-2/6VLP
Frequencies (%) of CD21+ VP7-specific AEC 1.0 1.0 1.5
0.8 0.8 1.3 A 1.0 0.6 A 0.6 0.8 0.4 A 0.4 0.5 A A A B 0.2 0.2 A A A AB B 0.3 BC BC C 0.0 0.0 0.0 AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls +2/6VLP 2/6/7VLP + 2/6VLP AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls +2/6VLP 2/6/7VLP + 2/6VLP VP4-2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP VP4-2/6VLP Figure 3.5 VP4-2/6VLP
Figure 3.6. Frequencies (%) of CD21+ VP6-specific AEC. The CD21+ IgM (top), IgA
(middle), IgG (bottom) VP6 specific responses elicited by the different non-replicating
VLP vaccines and the AttHRV+2/6VLP regime are summarized. Stripped bars represent ileum responses; black bars represent spleen and white bars represent peripheral blood responses (PBL). Within a tissue type or blood and isotype, bars with different letters
differ significantly (Kruskal-wallis rank sum test, p≤ 0.05). Background frequencies were
generally absent in most cases and only CD21+Ig+VP6+AEC spleen cells from control pigs were 0.01 or less. Each tissue or blood and isotype has a different background frequency value that was calculated as the mean frequency induced by control pigs plus or minus 2 SE. The gray dotted lines represent background frequency values.
272 IgM 6.0 Ileum 2.0 6.0 A A Spleen PBL 5.0 5.0 1.5 4.0 A A A A 4.0 A 1.0 A 3.0 A 3.0 A 2.0 A 2.0 0.5 A B B 1.0 B 1.0 0.0 0.0 0.0 AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP VP4-2/6VLP VP4-2/6VLP VP4-2/6VLP 2.0 IgA 2.0 2.0 1.5 1.5 273 A 1.5 A A 1.0 1.0 1.0 A A A BC 0.5 AB AB 0.5 A A B 0.5 C AB
0.0 0.0 0.0 AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP VP4-2/6VLP VP4-2/6VLP VP4-2/6VLP IgG Frequencies (%) of CD21+ VP6-specific AEC 2.0 2.0 2.0 A A A 1.5 A 1.5 1.5 A AB A 1.0 1.0 1.0 A B AB B B 0.5 0.5 B0.5 C C 0.0 0.0 0.0 AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls AttHRV VP4- 3x2/6/7VLP 2/6/7VLP + Controls +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP +2/6VLP 2/6/7VLP + 2/6VLP VP4-2/6VLP VP4-2/6VLP VP4-2/6VLP Figure 3.6
CHAPTER 4
A HIGH HUMAN ROTAVIRUS (HRV) DOSE ASSOCIATED WITH EARLY
DIARRHEA INDUCTION DIMINISHED INTESTINAL AND SPLENIC
PLASMACYTOID DENDRITIC CELL (PDC) RESPONSES COMPARED TO A
LOWER HRV DOSE IN GNOTOBIOTIC PIGS.
4.1 SUMMARY
In-vivo dendritic cell (DC) responses after human rotavirus (HRV) infection are
poorly characterized. To study viral dose effects on DCs ex-vivo and the association with
clinical outcome, we examined DC responses 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 274 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 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.
4.2 INTRODUCTION
Rotavirus (RV) is an enteric pathogen and the leading cause of severe diarrhea in
young children worldwide. Complications due to secondary untreated or refractory
dehydration lead to high mortality rates in developing countries (24). The tissue target is
the small intestinal epithelium; however, RV also causes transient viremia/antigenemia in
humans and different animal models including gnotobiotic pigs (3, 9-11). Rotavirus is an 275 icosahedral, non-enveloped, double-stranded RNA virus composed of three concentric protein layers: a core consisting of VP2, an inner layer composed of VP6 and two outer proteins, VP7 and VP4 that contain the viral neutralizing antigens (33).
The neonatal Gn pig is susceptible to homotypic and heterotypic RV infection
(e.g human RV [HRV]), and unlike any adult animal model (e.g. mice, rats, rabbits, monkeys), it is susceptible to HRV induced diarrhea (50). Furthermore, the gastrointestinal and immune system of neonatal Gn pigs resembles that of infants (50) so responses detected in neonatal pigs may mimic those after natural RV infection of infants. The pigs are maintained in isolator units and are fed sterile infant formula, assuring a pathogen-free status and lack of normal flora, making them an optimal model for the study of in-vivo antigen-induced innate immunity. After inoculation with antigen, intestinal or systemic mononuclear cells (MNC) can be isolated and immediately stained ex-vivo for detection of specific subtypes of dendritic cells (DC). Cytokines in serum or small intestinal contents (SIC) detected on the same day can be correlated with the cytokine producing DCs in the gut and spleen to determine if the DCs were a major source of the serum/SIC cytokines detected, supporting their in-vivo activation.
The types of DCs have been characterized in detail in mice and humans. Two main types are described: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs), but within each type many subtypes exist with varied tissue distribution (2, 53). Depending on the type of DC induced by an antigen and the DC location, immunoregulatory or adaptive immune T cells will be generated leading to tolerance or active immune responses, respectively (35). In mice (43) and humans (23, 34), pDCs prime regulatory
CD4+CD25+ FoxP3+ T cells that induce tolerance. However, pDCs have also been
276 actively involved in the resolution of viral infections. The pDCs are stimulated to produce
type I IFNs and are actively involved in the direct control of viral infections by causing
TNF-related apoptosis inducing-ligand (TRAIL) induced apoptosis (14), indirect resolution of the infection in-vivo by attracting other innate cells such as macrophages and inducing Th1 T cell responses (7, 22). Localization of the pDCs also defines the fate of the immune response: generally if they are located in lymph nodes, tolerance will be induced; if they are located in other non-lymphoid tissues, an active immune response
will be established (37, 43, 58).
Previous characterization of circulating pDCs in pigs (54) has shown them to be
SWC3low CD4+ IL-3 receptor+, negative for lineage markers such as CD8, CD3, CD21,
CD14, express low levels of MHC class II and CD80/86, and secrete IFNα after exposure
to various antigens such as viruses and CpG (26). In pigs, myeloid DCs are known as conventional DCs (cDCs) because both pDCs and cDCs express the myeloid marker
SWC3. The cDCs characteristically are SWC3+and CD11b+ or -, they lack CD4 and
secrete lower amounts of IFNα after antigen stimulation compared to pDCs (31).
Monocytes are SWC3high CD14+ CD16+ and lack CD4 and CD11b and macrophages are
SWC3high and CD14low (19, 38, 54). The regulatory T cell markers in the pig have not been studied. In this study we used surface bound TGFβ1 associated to latency- associated peptide (LAP), described previously on the surface of murine CD4+ T cells
(CD25+ and -) and associated with tolerogenic properties (41, 45). We stained with
antibodies to CD4, CD8, SWC3 and LAP to determine the frequencies of
CD4+LAP+(CD8-SWC3-) T cells after high and low doses of HRV.
277 Type I IFN receptor knockout mice infected with RV cleared the virus with
similar kinetics as infected wild type (WT) mice (1, 55). However, treatment of human
colonic cell lines with IFNα diminished RV infection to a maximum of 90% compared to
untreated cell lines. Cell binding or RV entry into the cells was not affected by IFNα
treatment, but liposomal transfection of RV failed in IFNα treated cells (6). Recently, it
was described that the RV non-structural protein 1 (NSP1) induces interferon regulatory
factor-3 (IRF-3) degradation leading to reduced type I IFN production in-vitro and such
inhibition was mainly in mDC (5, 20). However, after natural infection in humans, serum
IFNα is detected by 2 days after diarrhea onset (21). It is unclear whether RV inhibits
type I IFN secretion in-vivo and the cells responsible for IFNα secretion after RV
infection have not been characterized.
The purpose of this study was to characterize the ex-vivo induction of DCs locally
in the intestine and systemically in the spleen after HRV infection of neonatal gnotobiotic
pigs. We characterized porcine DCs [pDCs and cDC)], their cytokine production profiles
(IFNα, IL-12, IL-10, IL-6, TNFα) and their uptake/binding of HRV 2/4/6/7VLPs.
Because we observed that pDCs were the main DC induced after HRV infection and
pDCs are known to induce regulatory T cells we also measured the frequencies of
regulatory T cells [CD4+LAP+(SWC3-CD8-)] as another parameter of pDC function. We previously observed that a high HRV dose [M strain (G3P1A[8])] of 106 focus-forming units (FFU), caused severe dehydration, with death ensuing if not treated rapidly
(unpublished observations), whereas lower doses (10-1-103 FFU) did not cause severe dehydration and death. For these reasons, we inoculated gnotobiotic pigs with a low dose
(10 FFU) and higher but non-lethal dose (103 FFU) of M strain HRV to determine if there 278 was a dose effect on three different parameters of DC responses (induction of
intracellular cytokines and regulatory T cells and DCs showing antigen (VLP)
uptake/binding) and if the DC responses induced were associated with the HRV shedding
or diarrhea outcome. To our knowledge this is the first study that characterizes the ex-
vivo induced intestinal and systemic DC responses after an enteric viral infection.
4.3 MATHERIALS AND METHODS
Virus. The virus inoculum was the HRV M strain (G3,P1A[8]). A pool of
intestinal contents was obtained from the 11th Gn pig passage and diluted in minimum essential media (MEM; Invitrogen, Carlsbad, CA) to a final concentration of 10 fluorescent-focus forming units (FFU) or 1000 FFU/ml for inoculation and stored at -
70°C until use. The DD50 and ID50 of the HRV M strain in gnotobiotic pigs is < 0.1 FFU.
Animals. The Gn pigs were derived by hysterectomy and maintained in isolator units as previously described (59). At 3 days of age, rectal swabs were collected to assess the pig sterility. At 3-5 days of age pigs were bled and inoculated. The pigs received 2% sodium bicarbonate peroral (PO) to neutralize gastric acidity followed by 10 FFU or 1000
FFU in 3ml MEM (Invitrogen) of virulent M HRV. Controls were inoculated with MEM only. Pigs were bled the day of inoculation and at post-inoculation day (PID) 2 and subsets of pigs were euthanized at PID1, 2 and 4 after the high dose but at PID2 only after a low dose. All procedures were conducted in accordance with protocols reviewed by the Ohio State University's Institutional Laboratory Animal Care and Use Committee
(ILALUC).
279 Assessment of diarrhea and RV shedding. After HRV inoculation, diarrhea was
assessed by scoring of fecal consistency. Virus shedding was assessed by ELISA and cell
culture immunofluorescence (CCIF) after inoculation until the day of euthanasia as
previously described (28). Briefly, fecal consistency was scored as follows: 0= solid;
1=pasty; 2=semi-liquid; 3=liquid, with scores of 2 or more considered diarrheic. Rectal
swabs were collected every day including the day of euthanasia and were resuspended in
8ml of MEM and tested by RV ELISA for assessment of antigen shedding and CCIF for
detection of infectious virus shed as previously described (50). Virus shedding was considered positive if the sample was positive by CCIF or ELISA. Serum samples and
SIC from the day of euthanasia were tested for RV antigen by ELISA and absorbances
(Abs) were analyzed (50)
VLP preparation. Recombinant baculoviruses expressing VP6 (Wa, G1), VP7
(CJN, G1) and VP4 (Ku, P1A[8]) were constructed as previously described (17, 28).
Recombinant baculovirus expressing VP2 (RF, bovine RV) with green fluorescent
protein (GFP) was kindly provided by the late Dr Jean Cohen †(L’institut national de la
recherche agronomique-INRA) (15). For the construction of fluorescent VLPs (using the
VP2-GFP construct) utilized in the uptake/binding assay, Trichopulsia ni cells (H5 cells)
in Express Five serum free media (Invitrogen, Carlsbad, CA) were infected with various
combinations of recombinant baculoviruses to generate 2/4/6/7VLP-GFP at a total MOI
of 5 (15). At PID 7, infected cell lysates were collected and stored at 4°C. Particles were
purified by sucrose-CsCl purification, resuspended in TNC (Tris-HCL 10mM, NaCl 140
mM, CaCl 10mM) buffer, pH 7.4 and tested by electron microscopy (EM) for integrity,
anaerobic and aerobic culture media at 37 and 27°C to culture any contaminating bacteria
280 or fungi, western blot to confirm the composition of the VLPs, RV antigen ELISA assay and insect cell (Sf9) protein ELISA (that crossreacts with H5 cell proteins) for the RV and insect cell protein antigen titers, respectively. Protein concentrations were determined by Bradford protein assay (Bio-Rad, Hercules, CA) and endotoxin levels by Limulus
Amebocyte assay (Associates of Capecod Inc., Woods Hole, MA) as previously described
(28). Preparations with at least 80% or more intact 2/4/6/7VLP-GFP particles as determined by EM and stored less than three months since purification were used in the test. To assure that most of the inoculated protein was from VLP and not insect cell protein origin, only samples with a ratio of RV antigen/insect cell protein >3000 were used.
Isolation of mononuclear cells (MNC). Blood, spleen and ileum-jejunum tissue fragments were collected the day of euthanasia. Anticoagulant (acid citrate D-glucose-
ACD) treated blood, spleen and intestine were processed for isolation of MNC as previously described (59). Isolation of intestinal MNC included diffuse small intestinal peyer's patches, lamina propria and intraepithelial MNC. After isolation the cells were diluted in phenol red free Dulbecco's Eagle's modified MEM (Invitrogen, Carlsbad, CA) supplemented with 10% filtered porcine serum (Sigma Aldrich, St Loius, MO), 1% L- glutamine, sodium pyruvate, non-essential aminoacids and antibiotic-antimycotic
(Invitrogen) (DC media) and kept at room temperature (RT) until testing for VLP uptake/binding assay and flow cytometric staining which were performed the same day immediately after the isolation of all tissue derived cells.
Flow cytometry for detection of cytokine secretion by DCs and lymphocyte expression of membrane bound TGFβ1 (LAP). After isolation of the MNC,
281 approximately 2.4x107 cells were added to 15ml polypropylene conical tubes (Beckton
Dickinson Labware, Franklin Lakes, NJ), cell were diluted in cold filtered (0.1µm filter)
phosphate-buffered saline (PBS)- 0.5% bovine serum albumin (BSA)-0.02% sodium
azide (Sigma Aldrich) or wash media and centrifuged at 400xg for 5 min. After the
supernatant was discarded, 20µl of a 1/100 dilution of uninfected Gn pig control serum
was added and cells were incubated for 10 min at 4°C. Cells were washed, centrifuged
and stained. For staining of cell surface markers, cells were incubated for 10 min at 4°C,
washed with wash buffer and centrifuged at 400xg for 5 min at 4°C. To define DC
subpopulations that secrete IFNα, IL-12, TNFα and IL-6, cells were stained with
monoclonal antibody (mAb) to porcine SWC3 (IgG2b isotype, clone 742215A, Beckton
Dickinson), which is a DC marker (pDCs and cDCs) in pigs, followed by conjugated
fluorescein isothiocyanate (FITC) anti-mouse IgG2b (Beckton Dickinson). Then, biotin
conjugated recombinant human cytotoxic T lymphocyte-associated antigen-4 (hCTLA-4)
(Ancell corp, Bayport, MN) was added. The hCTLA-4 binds to porcine CD80/86 (26)
that are upregulated on activated DCs. The final antibodies added were a conjugated
phycoerythrin (PE) labeled mAb to porcine CD4, (IgG2b isotype, clone 74-12-4, Beckton
Dickinson) and conjugated peridinin chlorophyll (PerCP) streptavidin (SA). To define
cell populations that secrete IL-10, a different set of antibodies to cell surface markers
was used because the only working antibody for flow cytometry that recognized porcine
IL-10 was of IgG2a isotype excluding the use of hCTLA that is a recombinant protein
fused to mouse IgG2a. Instead mAb to porcine CD11R1 (CD11b) (IgG1 isotype clone
MIL4, Serotec, Raleigh, NC) that is a marker of cDCs in pigs was used together with a
mAb to porcine CD4 FITC (IgG2b isotype, clone 74-12-4, Beckton Dickinson) followed 282 by conjugated allophicocyanin (APC) anti-mouse IgG1 (Beckton Dickinson). Then,
biotin conjugated SWC3 (IgG1 isotype, clone 742215, Southern Biotechnologies,
Birmingham, AL) was added followed by SA-PercP. After staining of the cell surface
markers, cytofix-cytoperm (Beckton Dickinson) was added for fixation and
permeabilization of the cells. For intracellular staining (IC), the cells were washed with perm-wash (Beckton Dickinson), the incubation time was 10 min at RT and centrifugation was performed at 400xg for 5 min. After permeabilization, cells were washed and for those cells stained with mAbs to IFNα (IgG1 isotype, clone 27105-1), IL-
12 (IgG1 isotype, clone 116219), TNFα (IgG1 isotype, clone 103314) and IL-6 (IgG1 isotype, clone 77830.11) (R&D systems, Minneapolis, MN), 50µl of uninfected gnotobiotic pig serum diluted 1:100 in perm-wash was added for 5 min at RT to block unspecific antibody binding. Then cells were stained with mAbs to porcine IL-10 (IgG2a isotype, clone 148806), IFNα, IL-12, TNFα and IL-6, and washed twice (except for IL-
10 staining), followed by anti-mouse IgG2a PE (for IL-10 staining) or anti-mouse IgG1
APC (for staining of all other cytokines). All cells were washed twice and diluted in 100-
200µl of perm-wash.
For staining of cell membrane bound TGFβ1 (LAP) a human anti-LAP (IgG1
isotype, clone 27232, R&D systems) was used. This antibody recognizes human and
mouse LAP-TGFβ1 expressed on CD4+CD25+ and CD4+CD25- regulatory T cells (41,
42). Incubation times, washings and centrifugation were the same as for the cell surface marker staining above. Cells were stained initially with mAbs to human LAP that proved to be crossreactive with porcine LAP followed by anti-mouse IgG1 APC (Beckton
Dickinson). Then, mAbs to porcine CD4-PE (IgG2b isotype, clone 74-12-4) porcine 283 CD8-FITC (IgG2a isotype, clone 76-2-11, Beckton Dickenson) and biotin conjugated
mAb to porcine SWC3 (IgG1 isotype, clone 742215A, Southern Biotechnologies) were
added followed by SA PerCP then fixed with 200µl of 1% paraformaldehyde. After
staining, all cells were stored at 4°C in the dark until cell acquisition by flow cytometry.
Two sets of control tubes were stained: one set of controls included all the antibodies to cell surface markers except the cytokine antibodies or LAP and only the secondary antibodies against the cytokine antibody isotype or LAP isotype were added. Another set of controls included all the isotype controls for the cell surface mAbs or the conjugated secondary antibody or SA-PerCP only. Acquisition of 50,000 events was performed by a
FACScalibur flow cytometer (Beckton Dickinson) within a week after staining. Analyses were performed by Cell Quest Pro software (Beckton Dickinson).
DCs Uptake/binding assay: Immediately after isolation, cells were placed in DC media (see isolation of MNC) and kept at RT until the assay was performed on the day of isolation of the cells. Approximately 106 cells were distributed to 5ml sterile polypropylene tubes and resuspended in a maximum volume of 50µl of DC media. Then,
2µg 2/4/6/7VLP-GFP (optimized by prior titration) were added and cells were incubated at 37°C for 20 min. After incubation with VLPs, cells were washed with cold wash media
(PBS- 0.5% BSA-0.02% sodium azide) and centrifuged at 400xg for 5 min at 4°C. The cells were stained with mAb to CD11R1 (CD11b) (clone MIL4, Serotec), followed by anti-mouse IgG1 PE. Then, the biotin-conjugated mAb to porcine SWC3 (clone 742215,
Southern Biotecnologies) and mAb to human CD21-APC (clone B-ly4, Beckton
Dickinson) that crossreacts with porcine CD21 (C3d complement receptor and a porcine
B cell marker) were added followed by SA-PerCP. Incubations for each step were at 4°C 284 for 10 min. The cells were fixed and resuspended in 100-200 µl of 1% paraformaldehyde
and stored at 4°C until cell acquisition by flow cytometry.
ELISA for cytokine detection in serum and SIC. At euthanasia blood samples
without anticoagulant were taken for serum isolation and SIC was collected and treated
with proteinase inhibitors before storage at -20°C until testing. Cytokines in serum and
SIC were detected as previously described (4). Briefly, Nunc Maxisorp 96-well microtiter plates were coated with anti-IL-12, IFNα, IL-10, IL-6 (R&D systems), TNFα (Endogen,
Pierce biotechnologies, Rockford, IL) or active TGFβ1 (Biosource, Camarillo, CA) overnight at RT. The plates were blocked with PBS-0.1% Tween (Tw)-0.5% BSA for 2 hrs at RT. Diluted samples (1:1 in PBS-1% BSA) and cytokine standard dilutions were added to the plates at a total volume of 100µl. Plates were incubated at RT for 2 hrs and then washed five times with PBS-0.1% Tw. Biotinylated mAbs to active TGFβ1
(Biosource, Camarillo, CA), IL-12, IFNα, IL-10 or IL-6 (R&D systems, Minneapolis,
MN) or TNFα (Endogen) were diluted and added for a 2hr incubation at RT. Plates were washed and horseradish peroxidase-conjugated SA (0.1µg/ml) was added followed by the incubation of the plates for 45 min at RT. Tetramethylbenzidine (TMB) with H2O2 peroxidase system (KPL Inc, Gaithersburg, MA) was added for development of the plates. Standard curves for each cytokine were constructed using a computer-generated four parameter curve-fit with the dilutions of the recombinant cytokines used as positive controls. The detection limits of the ELISA assays were 78 pg/ml for IFNα, 15.6 pg/ml
for TGFβ1 and 7.8pg/ml for all the other cytokines.
285 Statistical analyses. Statistical analyses were performed using the SAS program
(SAS Institute Inc., Cary, NC). Differences in shedding titers, mean days to onset of
diarrhea or shedding, mean duration of diarrhea/shedding, diarrhea scores and mean
cumulative diarrhea scores among groups were analyzed by ANOVA followed by
Duncan's test. The percent of pigs that developed diarrhea at each PID were analyzed by
a Fischer's exact test. A p value of <0.05 was considered significant. Frequencies of
cytokine producing DC and cytokines in serum and SIC and CD4+LAP+ (SWC3-CD8-) T cells were analyzed by Kruskall Wallis rank-sum test followed by a Wilcoxon rank-sum test. A p value of ≤0.05 was considered significant. The DC VLP uptake/binding and the frequencies of CD4+LAP+ (SWC3-CD8-) T cells (pairs of experimental values and isotype controls) were analyzed by Wilcoxon rank-sum test. A p value of <0.05 was considered significant. Spearman’s correlation coefficient was used to correlate frequencies of cytokine producing DCs with cytokines in serum and SIC. A p value of
<0.05 was considered significant.
4.4 RESULTS
The low HRV dose induced high frequencies of activated intestinal IFNα
producing pDCs, lower frequencies of IL-12, IL-6, TNFα and no IL-10 producing
pDCs. A representative dot plot of intestinal pDC cytokine responses from pigs receiving a low HRV dose (10 FFU) at PID2 is shown in Fig. 4.4.1. After isolation of the cells from intestine or spleen, staining for DC markers that characterize pDCs (SWC3lowCD4+ cells) or cDC and monocytes/macrophages (SWC3 high or low CD4- cells) and IC staining with mAbs to porcine IFNα, IL-12, IL-10, TNFα and IL-6 cytokines, was performed. After 286 acquisition of the cells, a lymphocyte gate (gate 1) was drawn in a plot of side scatter vs.
forward scatter. Then, gates 2 and 3 were drawn to select CD4+ and CD4- populations
(Fig. 4.4.1B); and gates 4 and 5 were drawn to select hCTLA+ and - (CD80/86, Fig.
4.4.4.1B) all within gate 1. Analyses were performed on CD4+ CD80/86+and CD80/86-
(Fig. 4.4.1) and CD4- CD80/86+ and CD80/86- cells (data not shown) in a plot of IC cytokine vs. SWC3. The isotype controls for the CD4 and the CD80/86 staining are shown. The CD80/86 was expressed at moderate levels on cells which is typical of activated pDCs in pigs and other animal species (43, 54). The majority of cytokine producing DCs were within the activated (CD80/86+) CD4+ SWC3low population that is characteristic of pDCs (Fig. 4.4.1A shows in a plot of IC cytokine vs. SWC3 in gate 1 that the majority of cytokine producing cells are SWC3low). Minimal to absent responses were detected in the CD4- populations. The SWC3high cells that represent monocytes/macrophages, did not co-express CD4, and this population did not contain detectable cytokine producing cells by flow cytometry (data not shown). When analyzing
CD4+ CD80/86+ vs. CD4+CD80/86-, the majority of cytokine producing DCs (except
TNFα producing pDCs) were activated (CD80/86+), but frequencies of intestinal non- activated and activated cytokine producing pDCs for each cytokine for either high or low doses did not differ significantly. The intestinal and splenic pDC production of IL-10 after HRV inoculation was minimal, did not differ between groups and correlated with the very low serum IL-10 concentrations detected at PID2 (see Table 4.1) suggesting IL-
10 was not secreted in-vivo by the pDCs or other cells early after HRV infection.
287 A higher HRV dose induced significantly lower IFNα production by activated intestinal pDCs compared to the lower HRV dose. To determine the dose
effect of HRV on DC cytokine responses, pigs were inoculated with a high or low HRV
dose. Activated pDCs (SWC3low CD4+ CD80/86+) were the major DC type induced after the low dose HRV. No monocytes/macrophages (SWC3high,CD4-) were observed as cytokines producing cells by flow cytometry. The cDC responses were lower and are presented in the next section.
The effect of the low or high HRV dose on the frequencies of intestinal or splenic
IFNα, IL-12, IL-6 and TNFα cytokine producing pDCs at PID2 is shown in Fig. 4.4.2. In
Fig. 4.4.2A, the frequencies of activated intestinal and splenic IFNα producing pDCs are shown. The frequencies of intestinal IFNα producing pDCs were significantly lower after a high HRV dose compared to a low dose and both groups had higher frequencies than controls. On the other hand, although a similar trend to that in the gut was observed in the spleen, the splenic frequencies of IFNα producing pDCs were statistically similar among the three groups and lower than the intestinal frequencies only significantly for the low
dose responses. Within each figure, the serum IFNα concentrations (pg/ml) at PID2 only
from pigs euthanized for the isolation of DCs are shown. Frequencies of intestinal IFNα
producing pDCs correlated highly with the serum IFNα concentrations, whereas the
frequencies in spleen showed a moderate correlation. This suggests that the ex-vivo DC
staining probably reflected the pDC in-vivo activation and cytokine secretion.
Furthermore, primarily intestinal pDCs contributed to the serum IFNα concentrations
detected. The SIC IFNα concentrations were low and similar for high, low dose and
288 control pigs. The IFNα was probably degraded or unstable in the intestine as we
previously showed for most other cytokines but IL-12 and in this study, also TGFβ (4).
The frequencies of intestinal IFNα producing pDCs were low for the high dose inoculated pigs, not only at PID2 (0.53±0.2, Fig. 4.4.2), but also at PID1 (0.78±0.6) and
PID4 (0.53±0.2) compared to the low HRV dose at PID2 (3.29±1, Fig. 4.4.2). The same was observed in the spleen where the frequencies of IFNα producing pDCs after a high dose were absent at PID1 (0±0) and low at PID 2 and 4 (0.13±0.1 at PID2, Fig. 4.4.2;
0.06±0.06 at PID4) compared to low dose pigs at PID2 (0.66±0.5, Fig. 4.4.2).
Non-activated IFNα producing pDCs were induced in the gut but not the spleen
(data not shown). Frequencies were lower (but not significantly) compared to the intestinal activated pDC frequencies and were similar between the two doses, but significantly higher than controls. The non-activated IFNα producing pDCs possibly
represent pDC in the process of activation (see Fig. 4.4.1).
The high HRV dose induced significantly lower frequencies of splenic IL-6
producing pDCs compared to the low dose and lower (but not significantly lower)
intestinal IL-6, intestinal and splenic IL-12 and TNFα. The activated intestinal IL-12,
IL-6 and TNFα producing pDCs are shown in Fig. 4.4.2B. Compared to the frequencies of activated intestinal IFNα producing pDCs, these other cytokine producing pDCs were lower in frequency. For intestinal IL-12, IL-6 and TNFα producing pDCs, a high dose
induced lower (but not significantly lower) frequencies compared to a low HRV dose.
For the low dose, frequencies of activated intestinal IL-12 and IL-6 producing pDCs were significantly higher than controls. Only for activated intestinal IL-6 producing pDCs, did
289 the high dose induce significantly higher frequencies than controls. The frequencies of
activated intestinal TNFα producing pDCs were similar among the three groups. Within
each figure, the SIC (IL-12) or the serum cytokine concentrations (IL-6 and TNFα) are
shown. The IL-12 concentrations in SIC is shown because we previously demonstrated
that IL-12 was a stable cytokine in intestinal contents compared to other cytokines (4).
There was a high and significant correlation between the frequencies of activated
intestinal pDCs and concentrations of IL-12 in SIC but not with serum IL-12 (data not
shown) and a moderate correlation with IL-6 concentrations in serum. The frequencies of
TNFα producing pDCs did not correlate with TNFα concentrations in SIC or serum. This
suggests that the intestinal pDCs were likely activated in-vivo and secreted intestinal IL-
12 and IL-6 in serum contributing to the cytokine concentrations detected. However it is
likely that other IL-6 and IL-12 secreting cells (possibly other DCs) in different systemic
tissues were activated after HRV infection and contributed to their serum concentrations.
The TNFα produced by pDCs after a low dose was not detected in serum or SIC
suggesting those TNFα producing pDCs did not contribute to TNFα cytokine
concentration detected. The TNFα in the serum of controls was slightly higher to that of
low and high dose pigs but did not differ significantly.
In Fig. 4.4.2C, the frequencies of activated splenic IL-12, IL-6 and TNFα
producing pDCs are shown. Frequencies of activated splenic pDCs producing IL-6 were significantly lower after a high HRV dose compared to the low dose and, the high dose and controls had similar frequencies. The high dose induced lower (but not significantly lower) frequencies of activated splenic IL-12 and TNF α producing pDCs compared to a
290 lower dose. The low dose induced significantly higher frequencies of IL-12 and IL-6 (but not TNFα) compared to the controls whereas the high dose induced higher frequencies of only IL-12 (but not IL6 or TNFα) producing pDCs than the controls. Within each figure the cytokine concentrations in serum are shown. For IL-12 in serum only a moderate correlation was observed with the splenic activated pDCs suggesting that probably these pDCs were activated in-vivo, secreted IL-12 contributing to the cytokine concentrations detected but likely other DCs in different systemic tissues were also stimulated to secrete
IL-12 after RV infection. The IL-6 or TNFα in serum did not correlate with the frequencies of activated splenic pDCs suggesting that splenic pDCs did not contribute to the IL-6 or TNFα concentrations in serum .
Low frequencies of cDCs producing IFNα, IL-12 and IL-6 were induced after HRV infection and the high dose induced significantly lower frequencies of activated intestinal IL-12 and splenic IL-6 producing cDCs compared to the low dose. In Fig. 4.4.3A, intestinal and in Fig. 4.4.3B, splenic frequencies of activated cDCs producing IFNα, IL-12 and IL-6 are shown. The frequencies of cytokine producing cDCs were lower than the frequencies of pDCs after HRV infection but were only significantly different for the frequencies of intestinal IFNα producing cDCs (p=0.004, Wilcoxon test). The high dose induced statistically lower frequencies of intestinal IL-12 and splenic
IL-6 producing cDCs and frequencies of intestinal or splenic IFNα and splenic IL-12 were lower but did not differ statistically compared to their frequencies after the low dose. The frequencies of cDCs producing TNFα were very low (data not shown) after
HRV infection. All frequencies of intestinal cytokine producing cDCs had a high to
291 moderate correlation with the concentration of the corresponding cytokine in serum
(IFNα and IL-6) and SIC (IL-12) suggesting that cDCs secreted cytokines in-vivo after
HRV infection. However, correlations were higher for serum IFNα and SIC IL-12 with
intestinal pDCs suggesting that intestinal pDC were major contributors to the serum/SIC
concentrations for these particular cytokines in-vivo. On the other hand, the correlation
between serum IL-6 concentrations and frequencies of intestinal IL-6 producing cDCs
was higher compared to that of the frequencies of intestinal pDCs suggesting cDCs
secreted IL-6 in-vivo whereas pDCs produced IL-6 but did not contribute to its serum
concentration. Frequencies of splenic IL-12 and IL-6 secreting cDCs did not correlate
with the corresponding serum cytokine concentrations suggesting splenic cDCs were not
major contributors to the serum IL-12 or IL-6.
The mean HRV shedding titers did not differ significantly between the two
HRV doses, but diarrhea was observed early only at PID 1 and 2 after a high HRV
dose. The high dose HRV induced significantly lower frequencies of intestinal IFNα and
splenic IL-6 producing pDCs and intestinal IL-12 and splenic IL-6 producing cDCs than
a low HRV dose. To determine if there was an association between the frequencies of
cytokine producing DCs induced after inoculation with the different doses and the
clinical outcome (infectious virus shedding and diarrhea), HRV fecal shedding titers by
CCIF and diarrhea scores were measured on the day the experiments were performed
(PID2) and during 6 days after inoculation of pigs with the high (PID2 n=13, 6 day
follow up, n=5) or low (PID2 n=8, 6 day follow up, n=2) HRV doses (Table 4.2 and Fig.
4.4.4). In Fig. 4.4 we added all pigs tested at the different PIDs and in Table 4.2, except
for the analysis at PID2, only the pigs that were followed for 6 days after inoculation 292 were analyzed. We observed that the peak of HRV replication after each inoculation dose of M strain HRV was PID 2 (Fig. 4.4.4) and titers of infectious virus were statistically
similar during the 6 days that shedding was recorded (Fig. 4.4.4). However, no pigs
developed diarrhea during PID 1 and 2 after a low HRV dose whereas 23% and 69% of
pigs that received a high dose developed diarrhea at PID1 and 2, respectively (Fig. 4.4).
The higher percentage of pigs that developed diarrhea after a high dose was statistically
significant at PID2 (Fischer's exact test, p=0.002). The pigs that received the low dose of
virus developed diarrhea only after PID3. All pigs shed HRV in feces, the onset of
shedding was similar and the average peak HRV titers shed was also similar between the
two groups (Table 4.2). However the mean duration days of viral shedding was
significantly shorter for the lower dose inoculated pigs (2 vs. 4.4 days for the low and
high doses, respectively). The mean fecal HRV titers at PID2, was analyzed by adding all
the pigs studied for the DC responses plus the pigs that were followed for 6 days (low
dose n= 8 and high dose n=13) and no difference in virus shedding titers was observed
between the two doses. Furthermore, if only the pigs analyzed in the DC study were
included in the statistical analysis, infectious HRV titer, HRV antigen in serum and SIC
were similar between doses (data not shown). All HRV inoculated pigs eventually
developed diarrhea, the mean days to onset of diarrhea, the duration of days with diarrhea
and the mean cumulative score were similar between doses. As shown in Fig. 4.4.4, at
PID 2 the low dose did not induce diarrhea and the diarrhea scores were significantly
lower than those after a high dose. In the high dose group however, not all pigs developed
diarrhea at PID 2 (there were two pigs that developed diarrhea at PID5) explaining the
similar mean days to onset of diarrhea between the two groups during the 6 day follow
293 up. In summary, the high and the low HRV doses were similar for most parameters
mentioned except no pigs inoculated with a low dose developed diarrhea at PID1 and 2
and the duration of shedding was shorter for the low dose demonstrating that a higher
dose induced more disease at PID2 and more prolonged shedding.
Uptake of 2/4/6/7VLP-GFP by intestinal and splenic pDCs and splenic cDCs
was reduced after the high HRV dose compared to a low dose. We described that after
a high HRV dose, intestinal and splenic frequencies of cytokine producing pDCs or cDCs
were diminished in most cases and were significantly lower for some cytokine producing
DCs compared to the low dose pigs. To determine if the pDC and cDC response was
effected at the antigen uptake/binding level after a high dose of HRV, we studied the
capacity of freshly isolated DCs diluted in DC media to uptake or bind 2/4/6/7VLP-GFP
(Fig. 4.5). The expression of CD80/86 did not differ between a low or high HRV dose
(intestinal CD80/86+ low dose=7.34%, high dose=10.7%, p=0.5; splenic CD80/86+ low dose=2.3%, high dose=1.7%, p=0.6, Wilcoxon rank-sum test) thus if there was a difference in the uptake/binding of 2/4/6/7VLPs it is not due to an enrichment of activated DC in either group. To characterize pDCs and cDC antigen binding/uptake, markers of pDCs and cDCs (SWC3) and cDCs only (CD11b) were used together with a
B cell marker to exclude IgM+B cells from the analysis and to focus on professional DCs.
No CD4 staining was performed for pDCs because it has been described that pDC do not express CD11b (31) or CD21 (18, 26), so we characterized pDCs in this assay as
SWC3low CD11b- CD21-. The results of the uptake/binding pattern of pDCs are shown in
Fig. 4.5. The intestine or spleen cells were analyzed in a lymphocyte gate to study pDCs
and cDCs uptake/binding capacity after a low or high HRV dose. In Fig. 4.5 frequencies
294 of intestinal or splenic pDCs and cDCs 2/4/6/7VLP-GFP+ are shown. The intestinal and
splenic pDCs after a high HRV dose had significantly lower binding/uptake of
2/4/6/7VLPs compared to pDCs from pigs that received a lower dose. However intestinal
cDCs did not show differences in the uptake/binding of 2/4/6/7VLP after a high or low
dose. In the spleen a higher dose induced lower uptake/binding of 2/4/6/7VLP by cDCs
compared to a low HRV dose. In summary we demonstrated that a high HRV dose
reduced two different parameters of pDC and cDC function: intracellular cytokine
production and antigen uptake/binding.
Inoculation with a high HRV dose induced significantly lower frequencies of
circulating CD4+ (SWC3-CD8-) T cells with surface membrane bound TGFβ (LAP) and lower active TGFβ1 concentrations in SIC than a low dose. After a low HRV dose, frequencies of activated pDCs increased. As a third parameter of pDC response and because pDCs are known to induce regulatory T cells we determined the frequencies of
CD4+ (SWC3-CD8-) T cells expressing the cell membrane bound TGFβ1 (LAP) previously described on the surface of regulatory T cells that actively induce tolerance.
The LAP expression was analyzed in CD4+ (SWC3-CD8-) T cells and shown in Fig. 4.6.
The antibody used was a human anti-LAP mAb that cross-reacts with murine LAP (41,
42). The intestinal, splenic and circulating MNC were stained, but only the frequencies of
circulating LAP+ cells are reported because the background antibody binding in the
spleen and intestinal MNC was high. The values of individual experiments with their
corresponding isotype controls are shown in Table 4.3. The experimental values and the
isotype controls differ statistically only for the low dose inoculated pigs (Wilcoxon rank-
sum test). Expression of LAP on CD4+ (SWC3-CD8-) circulating T cells was significantly 295 higher in peripheral blood cells derived from pigs inoculated with a low dose of HRV
compared to pigs receiving a higher dose or controls. There was also a moderate and
significant correlation between the concentration of active TGFβ1 in SIC and the frequencies of circulating LAP+CD4-(SWC3-CD8-) T cells, but not with serum active
TGFβ1. We previously showed that IL-12 was stable in intestinal contents compared to other cytokines (4); in this study we showed that TGFβ1 was detected in SIC and possibly also stable. Other SWC3low DCs and SWC3high monocytes expressed LAP but their expression did not differ among the three groups (data not shown).
4.5 DISCUSSION
We studied the ex-vivo DC cytokine producing cells, the DC uptake/binding of
2/4/6/7VLPs and circulating LAP+CD4+ (SWC3-CD8-) T regulatory cells induced early
after inoculation with high or low HRV doses. We demonstrated that a low HRV dose
induced primarily intestinal activated IFNα producing pDCs. However after a high HRV
dose frequencies of activated intestinal IFNα pDCs, IL-12 producing cDCs and splenic
IL-6 producing pDCs and cDCs were significantly lower compared to a low HRV dose.
Other cytokine producing pDC and cDC in the gut and spleen were lower (but not
significantly) after a high dose versus low dose. Similarly, the 2/4/6/7VLP
uptake/binding patterns of intestinal and splenic pDCs and splenic cDCs and the
frequencies of LAP+CD4+ (SWC3-CD8-) regulatory T cells were lower after a high than a lower dose of HRV suggesting that a high HRV dose reduced the pDCs responses at the level of cytokine production and antigen uptake/binding and possibly secondarily to this effect, a lower frequency of regulatory T cells were induced. Clinically a high dose 296 induced higher diarrhea scores and higher percentages of pigs with diarrhea at PID2,
when the peak cytokine responses occurred and when DC responses and CD4+LAP+ T cells were studied. Because the pigs given high or low HRV doses developed similar patterns and titers of infectious virus shedding, it is unlikely that RV directly influenced the reduced pDC and cDC responses observed in the gut and spleen after a high dose nor did the IFNα in serum or secreted by pDCs prevent HRV replication. Evidence for the
lack of IFN type I effects on RV replication are also supported by studies of knockout
mice for IFN type I receptor which cleared RV infection similarly to WT mice (1, 55).
Also when neonatal mice were inoculated orally or intravenously with IFNα before and
after inoculation, virus induced diarrhea was not prevented (1).
Four-day-old pigs developed intestinal lesions early (13 hrs PI) after a high (105
FFU) dose of Wa HRV. However, the Wa HRV strain is less pathogenic than the M HRV strain used in this study because after a dose 106 FFU of Wa HRV, pigs were not as dehydrated and mortality was not observed as a consequence of the dehydration like we observed with the same dose of M HRV strain (unpublished observations). Products of
inflammation, associated with enterocyte necrosis after a high HRV dose as suggested by
a higher diarrhea score and percentage of pigs with diarrhea at PID 2, possibly mediated
the reduced cytokine production and VLP uptake/binding by pDC and cDCs. There is
evidence that nitric oxide (NO) that is induced during inflammation, inhibits IFNα, IL-6
and TNFα production by human blood derived pDCs upon stimulation with the TLR9
agonist AAC-30, diminishing the expression of OX40L whereas expression of CD80/86
was unaltered (40). Furthermore the TLR9 agonist, induced strong IFNα, TNFα and IL-6
by pDCs in the absence of NO. It is known that rotavirus non-structural protein NSP4 297 induces NO after infection of humans (49). In the case of HRV, possibly a higher dose
induced an initial NO concentration that was higher than that induced by a lower dose
and together with the exposure of the DCs to RV double stranded RNA that is the
pathogen associated molecular pattern produced by replicating virus, downregulation of
intestinal and splenic pDC responses occurred. We did not detect a difference in the
expression of CD80/86 between the low and high HRV dose as was the case for human
pDCs cultured with AAC-30 and NO. Furthermore, the high mobility group B1
(HMGB1) protein, a damage associated molecular pattern released during necrosis,
suppresses human pDC responses to CpGs diminishing the secretion of IL-12 and IFNα
(46). However inhibition caused by cell damage byproducts remains to be tested for
porcine pDCs as previously shown for human pDCs and the extent of intestinal pathology
needs to be confirmed comparing the high vs. low doses at PID2. It is known that pDCs
regulate cDC activation which this has been demonstrated in pigs (27) and possibly the altered functionality of the pDCs after the high dose affected the activation of cDCs. The low dose pigs developed diarrhea after PID3 and DC responses at later time points need to be confirmed, but possibly will also be lower than at PID2.
In a mouse model of cardiac transplantation, pDCs bearing graft epitopes were
increased, induced regulatory T cells and correlated clinically with the absence of graft
rejection (43). In a different model of mouse airway inflammation, mice that did not
develop airway inflammation had a predominant population of pDCs draining the
pulmonary lymphatics, whereas mice with airway inflammation had myeloid DCs as the
predominant draining population. Furthermore, in a mouse model of biliary atresia after
Rhesus RV (RRV) infection, IFN type I receptor knockout mice were the only ones
298 susceptible to biliary inflammation and secondary atresia, whereas in a different study,
IFNγ-/- mice were protected, suggesting that the IFN type I induction after RRV infection induced an immunoregulatory response that reduced RV-specific Th1 T cell responses resulting in protection against biliary inflammation (36, 52). Our observations show that a low HRV dose induced immunoregulatory cells [(intestinal activated IFNα producing pDCs and LAP+CD4+ (SWC3-CD8-) T cells that may represent regulatory T cells in
pigs)] suggesting that also in the pig, pDCs induce regulatory T cell priming although the
high dose significantly reduced the activation of these cells.
In mice infected with high or low doses of herpes simplex virus (HSV) that
induces chronic infection of the host, observations contrary to those seen for HRV that
causes an acute infection, were described. After a high dose of HSV, frequencies of activated pDC were increased and they secreted high levels of IFNα in-vivo, and when cultured in-vitro, they did not promote T cell proliferation (8). These findings suggest that infection with a chronic virus such as HSV, induces mainly immunoregulatory pDCs that might facilitate long-term survival of the virus in the chronically infected host.
However the outcome in terms of effector T cell responses to RV after a low dose of
HRV remains to be studied, but possibly effector T cell responses after a low dose will be lower than those after a higher dose.
Activated pDCs are involved in the induction of regulatory T cells as well as
adaptive immune responses (13, 43). It has been demonstrated that regulatory T cell
induction by pDCs requires pDCs to be located in lymph nodes (43) and that in the
absence of MLNs, oral tolerance is abolished (58) suggesting that pDCs inducing
adaptive immune responses must be located in tissues apart from lymph nodes. 299 Furthermore, circulating pDCs have been proven to induce potent Th1 polarization after
incubation with influenza virus (13, 22). In a mouse model of genital HSV-2, pDCs were
scarce in the vaginal mucosa in the steady state, but after HSV-2 infection, tissue pDCs
increased greatly and secreted large amounts of IFNα, suppressing local virus replication
and were the main mediators of protection against a lethal dose of HSV-2 after local
treatment with CpGs (37, 51). After a low dose of HRV, a higher frequency of intestinal
IFNα producing pDCs and regulatory T cells were induced and possibly the pDCs
inducing the regulatory T cells were located in the MLN. On the hand, effector T cell
responses to RV possibly can also be induced by intestinal or splenic pDCs although this
was not examined. A high dose, by inducing fewer intestinal pDCs, possibly diminished
the amount of pDCs in the MLN preventing greater regulatory T cell priming. Whether intestinal pDCs are able to induce RV-specific effector T cells that help to clear the infection needs to be explored further .
We used LAP to characterize T regulatory lymphocytes, and because these cells have not been previously examined in pigs, we used T cell and DC markers and studied their LAP expression. It is known that LAP is not exclusive to CD4+CD25+ T regulatory cells and that CD4+CD25-LAP+ cells are able to induce tolerance whereas
CD4+CD25+LAP- cells induced minimal regulatory activity in mice (45). Furthermore,
CD4+LAP+ T cells protected mice from colitis and CD4+LAP- T cells did not (41). These studies suggest that LAP expression is a marker of active tolerance. We observed that a low HRV dose induced high frequencies of IFNα producing pDC were induced together with LAP+CD4+ (SWC3-CD8-) cells and active TGFβ in the gut, possibly produced by local natural regulatory T cells or Th3 lymphocytes but this remains to be determined. 300 Infection of pigs by HRV induced intestinal and splenic mixed Th1/Th2 responses
(4) and children develop circulating Th1/Th2 T cell responses as well (30). In previous
studies in pigs, circulating pDCs produced high amounts of IFNα, IL-12 and TNFα after
CpG stimulation whereas cDCs produced lower amounts of IFNα and TNFα as we also
described (26). Human circulating pDCs incubated in-vitro with CpGs produced IFNα,
IL-6 and TNFα, but no IL-12 suggesting that porcine and human pDCs may differ in the
IL-12 cytokine secretion patterns after in-vitro incubation with the TLR9 agonist CpG
(29). There is evidence of activated pDCs inducing Th1 (13) and Th2 (48) responses, and
possibly in the gut and spleen, pDCs mediate the balanced Th1/Th2 T cell response
observed after RV infection in pigs and infants. Induction of IFNα (13) and IL-12 (12)
have been linked with induction of Th1 responses whereas TGF-β (44), and IL-6 together
with IL-5 can induce mucosal IgA producing B cells (39). Also there is evidence that
type I IFNs together with IL-6 induce plasma cell differentiation (32). An anti-
inflammatory property of IL-6 is the reduced maturation of DCs in humans (47) .We
observed that in the spleen, activated IL-12 and IL-6 producing pDCs were predominant
and IFNα responses were lower than those detected in the gut after a low HRV dose.
Previous studies in mice have demonstrated that the splenic DCs are especially prone to
secrete IL-6 compared to other mucosal DCs like those located in the lung (16).
We studied the binding/uptake of 2/4/6/7VLP by pDCs and cDCs in the spleen
and gut. Most of the total cells isolated after HRV inoculation did not express CD80/86
and immature (CD80/86-) DCs are known to bind antigen more avidly (25) than more mature CD80/86+ DCs (25). Additionally, the expression of CD80/86 was similar
between the high and low dose pigs so the differences observed in the VLP 301 binding/uptake after a high or a low HRV dose are not explained by a differential
expression of CD80/86 by the DCs. We observed that 2/4/6/7VLP uptake/binding by
intestinal and splenic pDCs and splenic cDCs was significantly higher after a low dose of
HRV compared to a higher dose. Uptake of antigen by circulating monocytes, cDCs and
pDCs has been previously described in pigs, and although similar for different antigens
used, it varied depending on the antigen added to the culture. Specifically pDCs and
monocytes were able to uptake more green bovine serum albumin than cDCs but the
difference was not significant (54). We observed that splenic and intestinal cDCs did not
uptake/bind as much 2/4/6/7VLPs as pDCs and this difference may be due to the type of
antigen (2/4/6/7VLP) used in this assay.
Gnotobiotic pigs develop similar diarrhea scores and fecal RV titers after
inoculation with HRV regardless of their colonization status with lactobacillus species
(60). These findings suggest that their gnotobiotic status and lack of commensal flora does not influence RV replication or induction of diarrhea. Furthermore, splenic DCs from both germ-free and pathogen-free mice presented a similar phenotype in terms of expression of CD86, MHC II and stimulation of T cell proliferation and developed similar oral tolerance to OVA (56, 57). Studies of germ-free vs. pathogen-free pigs are lacking but our studies with lactobacillus and the germ-free mice studies suggest that
DCs in germ-free pigs are fully responsive.
To our knowledge this is the first study that describes the ex-vivo DC responses in
the gut and spleen after HRV infection in neonatal gnotobiotic pigs. We show that the
frequencies of intestinal pDCs and cDCs correlated with serum IFNα, IL-6 and SIC IL-
12 cytokine concentrations, highly suggestive of their in-vivo activation and cytokine
302 secretion. How immunoregulatory responses in the gut are balanced when acute enteric
infections occur is unclear. We show that perhaps, depending on the amount of virus that
initiates an acute enteric infection, immunoregulatory mechanisms are induced (low dose) or downregulated (high dose). Possibly, if the initial pathogen load is high, natural immune regulatory mechanisms (natural regulatory T cells) are suppressed by inflammatory products to facilitate the induction of adaptive immunity and control the infection
4.6 ACKNOWLEDGMENTS
We thank Dr Juliette Hanson for the clinical care of the Gn pigs, Myung Guk Han and Guohua Li for assistance with electron microscopy for VLPs and Rich McCormick,
Peggy Lewis and the summer students from the Agricultural Technical Institute at The
Ohio State University for their technical assistance. We thank Drs. Juanita Angel, Manuel
Franco and Ms. Menira Souza for helpful comments and Mr. Hong Liu for assistance in the statistical analyses.
This work was supported by a grant from the National Institutes of Health,
National Institutes of Allergy and Infectious Disease (RO1AI033561). Salaries and research support were provided by State and Federal Funds appropriated to the Ohio
Agricultural Research and Development Center at the Ohio State University.
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310
Figure 4.1 Intestinal MNC from pigs inoculated with a low HRV dose at PID2. After acquisition of the cells by flow cytometry, a gate in the lymphocyte population was drawn
(gate 1) in a plot of side scatter vs. forward scatter, and in a plot in gate 1 of forward scatter vs. CD4, CD4+ (gate 2) and CD4- (gate 3) cells were selected. In a third plot of side scatter vs. hCTLA (binding to porcine CD80/86), hCTLA+ (CD80/86+, gate 4) and hCTLA-
(CD80/86-, gate 5) in gate 1 were selected. After analysis of CD4+ (CD80/86+and CD80/86-) and CD4- (CD80/86+and CD80/86-) in a plot of SWC3 vs. intracellular cytokine (IFNα, IL-
12, IL-6, TNFα) it was observed that the highest frequencies of cytokine producing cells
were in the intestinal SWC3low CD4+ CD80/86+ population, previously characterized as pDC.
In Fig 1A, a plot of SWC3 vs. IFNα analyzed in gate 1 only, shows that the SWC3low cells were the main secretors of IFNα. In Fig.1B, the CD4 and CD80/86 plots are shown with their correspondent isotype controls. The CD80/86+ cells do not express high levels of these
molecules which is characteristic of pDCs. Analysis of CD4+ CD80/86+ (Fig.1C), shows that the frequencies of IFNα, IL-12 and IL-6 were higher than the correspondent frequencies of
CD4+ CD80/86-(which probably represent pDCs in the process of activation), but TNFα was higher in the population of non-activated pDCs (SWC3low CD4+ CD80/86-) in this pig. All
frequencies detected in the control tubes stained with extracellular DC markers and the
intracellular isotype control were deducted from the experimental analyses and the quadrants
were placed according to where the background frequencies were detected. The bars for the
DC markers were placed according to the isotype controls for the DC cell surface markers.
For each experiment, 50,000 events were acquired by the flow cytometer.
311 AM010507.008 C. CD4+CD80/86+ CD4+CD80/86- Lymphocyte AM010506.008 Gate 1 AM010506.008
α 6.9% 3.4%
R1 IFN
0 200 400 600 800 1000 Forward Scatter 100 101 102 103 104 SWC3 FITC 100 101 102 103 104 A. Dot plot in gate 1 SWC3 FITC
AM010506.009 AM010506.009
AM010506.008 0% 0% SWC3low IFNα+
100 101 102 103 104 100 101 102 103 104 SWC3 FITC SWC3 FITC Isotype control
AM010607.007 AM010607.007 0.23% 100 101 102 103 104 0.07% SWC3 FITC IL-12
100 101 102 103 104 100 101 102 103 104 B. Analysis in gate 1: SWC3 FITC SWC3 FITC
AM010607.009 AM010607.009 hCTLA or
α 0.24% 0.51% CD4 CD80/86
AM010507.008 AM010507.008 TNF
0 1 2 3 4 3 100 101 102 103 104 10 10 10 10 10 5.17% SWC3 FITC SWC3 FITC R3 2 4.11% AM010407.010 R2 5 AM010407.010 R7 R8 4 0.25% 0.24% 100 101 102 103 104 100 101 102 103 104 CD4 PE hCTLA PerCP L-6 I Isotype Control
0 1 2 3 4 10 10 10 10 10 0 1 2 3 4 For CD4 and hCTLA staining SWC3FITC 10 10 10 10 10 AM010507.012 SWC3FITC AM010507.012
0.2% AM010407.011 AM010407.011 0.07% 0% 0.5%
100 101 102 103 104 0 1 2 3 4 IgG2bPE 10 10 10 10 10 SA PerCP 100 101 102 103 104 SWC3FITC 0 1 2 3 4
Isotype control 10 10 10 10 10 SWC3FITC
Fig 4.1 Intestinal MNC from low dose SWC3 inoculated pigs at PID2
312 Intestinal pDCa Splenic pDCb 10 FFU c 1000 FFU control 10 FFU c 1000 FFU control pDC IL-10d mean 0.2(0.2) 0.5(0.3) 0.1(0.1) 0(0) 0.13(0.13) 0.02(0.01) (SEM) A A A A A A
IL-10serume mean 18(10) 70(42) 43.8(38) (SEM) A A A r=0.48 r=0.49 p=0.003f p=0.003g a- Intestinal IL-10 producing pDCs isolated at PID2. b- Splenic IL-10 producing pDCs isolated at PID2. c- The different inoculation groups, low dose (10 FFU, n=6) , high dose (1000 FFU, n=5) and controls (n=6). d- The mean frequencies of IL-10 producing pDCs. In parenthesis the standard error of the mean(SEM). Values with a different capital letter are statistically different (Kruskal-wallis rank sum test followed by a Wilcoxon rank sum test). e- The serum cytokine concentration in pg/ml. In parenthesis the SEM. Values with a different capital letters are statistically different (Kruskal-wallis rank sum test) f- Correlation coefficient between intestinal IL-10 producing pDCs and serum IL-10 among all groups (Spearman's correlation coefficient) g-Correlation coefficient between splenic IL-10 producing pDCs and serum IL-10 among all groups (Spearman's correlation coefficient), the p values are shown in parenthesis.
Table 4.1. Production of IL-10 by intestinal and splenic pDCs (SWC3low CD4+ CD11b-)
313
Figure 4.2 The frequencies of intestinal and splenic activated cytokine producing (IFNα,
IL-12, IL-6 and TNFα) pDCs after a low HRV dose, high HRV dose and controls at
PID2 are summarized. Figure 2A, frequencies of intestinal and splenic IFNα producing pDCs. Fig, 2B, intestinal activated IL-12, IL-6 and TNFα producing pDC and Fig.2C,
splenic activated IL-12, IL-6 and TNFα producing pDC. The y axes represent the
frequencies (%) of pDCs by flow cytometry (left) and the cytokine levels (right) detected
by ELISA in pg/ml. The mean frequencies of activated pDCs after a low HRV dose
(white bars), a high HRV dose (gray bars) or control pigs (black bars) are shown as bars.
Inside each plot the value of the mean cytokine level detected in serum (IFNα, IL-16,
TNFα) and SIC for IL-12 are shown (-●-). A correlation coefficient and p values were
placed inside each plot only when significant. The error bars denote the standard error of
the mean (SEM) and bars with different letters differ significantly. Serum or SIC
cytokine values with different numbers of asterisks (*) differ significantly for cytokine
concentrations for the three groups (Kruskall-wallis rank sum test followed by a
Wilcoxon rank sum test, p≤0.05).
314 5.0 12000 (pg/ml) cytokine 5.0 12000 ** IFNa A. A** IFNa 4.0 Spleen 10000 4.0 Intestine 10000 r=0.43 r=0.73 8000 8000 3.0 p=0.01 3.0 p<0.0001 Serum 6000 6000 * * Serum 2.0 IFNa 2.0 IFNa A 4000
Frequency of (%) DC 4000 1.0 1.0 B A * 2000 2000 A C* 0.0 0 0.0 0 10 FFU 1000 FFU control B. 10 FFU 1000 FFU control (pg/ml) cytokine
4.0 IL-6 14 4.0 TNFa 20 4.0 IL-12 70 315 12 Intestine Intestine r=0.64 60 3.0 Intestiner=038 10 3.0 15 3.0 p=<0.0001 50 p=0.03 8 2.0 2.0 10 40 Serum A 2.0 SIC-IL-12 6 30 A IL-6 Serum A** ABAB**** 1.0 4 1.0 5 20 TNFα Frequency of (%) DC 1.0 A 2 A A 10 B B* 0.0 0 0.0 0 0.0 0 C. 10 FFU 1000 FFU control 10 FFU 1000 FFU control 10 FFU 1000 FFU control IL-12
IL-6 TNFa (pg/ml) cytokine 4.0 ** Spleen 400 4.0 A 15 4.0 20 ** r=0.36 Spleen Spleen 3.0 p=0.04 300 3.0 3.0 15 A Serum 10 Serum A 2.0 200 2.0 IL-6 2.0 Serum 10 A**/* IL-12 * 5 TNFα 1.0 A 100 1.0 1.0 5 A Frequency of (%) DC B B A 0.0 0 0.0 B 0 0.0 0 10 FFU 1000 FFU control 10 FFU 1000 FFU control 10 FFU 1000 FFU control n=6n=5 n=5-6 n=3n=5 n=5-6 n=4n=5 n=5-6 M HRV dose (FFU) PID2 Fig.4.2 Intestinal and splenic pDC SWC3lowCD4+ CD80/86+
Figure 4.3 The frequencies of intestinal and splenic activated cytokine producing (IFNα,
IL-12 and IL-6) cDCs after a low HRV dose, high HRV dose and controls at PID2 are summarized. Figure 3A, frequencies of intestinal IFNα , IL-12 and IL-6 producing cDCs.
Fig, 3B, splenic activated IFNα,IL-12 and IL-6 producing cDC. The y axes represent the frequencies (%) of cDCs by flow cytometry (left) and the cytokine levels (right) detected by ELISA in pg/ml. The mean frequencies of activated pDCs after a low HRV dose
(white bars), a high HRV dose (gray bars) or control pigs (black bars) were shown as bars. Inside each plot the value of the mean cytokine level detected in serum (IFNα, IL-
16, IL-12) and SIC (IL-12) are shown (-●-). A correlation coefficient and p values were placed inside each plot only when significant. The error bars denote the standard error of the mean (SEM) and bars with different letters differ significantly. Serum or SIC cytokine values with different numbers of asterisks (*) differ significantly for the cytokine concentrations for the three groups (Kruskall-wallis rank sum test followed by a
Wilcoxon rank sum test, p≤0.05).
316 A.
0.5 12000 IL-12 2.5 IL-6 14 IFNa 1.0 ** 70 ** 12 Ileum r=0.64 10000 Ileum 60 2.0 Ileum 0.4 0.8 0.59 p<0.0001 r=0.47 50 10 8000 p=0.005 p=0.004 Serum 0.6 1.5 8 0.3 SIC-IL-12 40 Serum IFNa 6000 IL-6 6
A * 30 (pg/ml) cytokine 0.2 0.4 1.0 4000 ** 20 4 0.2 A 0.5 0.1 10 2 317 2000 A AB B * B A *B B 0.0 0 0.0 0 0.0 0 10 FFU 1000 FFU control 10 FFU 1000 FFU control 10 FFU 1000 FFU control B. 2.5 IL-6 14 Frequency of (%) DC 0.5 12000 1.0 IL-12 400 A ** IFNa ** Spleen 12 Spleen 350 2.0 0.4 Spleen 10000 0.8 ** 300 10 r=0.46 Serum 8000 250 1.5 Serum 8 0.3 p=0.006 0.6 IL-12 200 IL-6 * Serum 6000 1.0 6 0.2 IFNa 0.4 150 4000 * 4 A 100 * 0.2 0.5 0.1 A 2000 2 AB 50 B B AB B B 0.0 0 0.0 0 0.0 0 10 FFU 1000 FFU control 10 FFU 1000 FFU control 10 FFU 1000 FFU control n=6 n=5 n=5-6 n=6 n=5 n=5-6 n=3 n=5 n=5-6
M HRV dose (FFU) PID2
Fig. 4.3 Intestinal and splenic cDC SWC3lowCD4- CD80/86+
Figure 4.4 RV shedding and percentage of pigs with diarrhea for 6 days after inoculation of a high or low HRV dose. The replicating RV shedding detected by CCIF in rectal
swabs and diarrhea scores from pigs receiving a high (1000 FFU-black squares; % pigs
with diarrhea- ×) and a low (10 FFU- open squares; % of pigs with diarrhea- +) HRV
dose during 6 days after inoculation are shown. The y axis to the left represents the titers of infectious virus in FFU per ml, the y axis to the right, the percentage of pigs that had diarrhea and the x axis represents the post-inoculation days (PID). The number of pigs per group is included in the bottom of the figure. Diarrhea scores from rectal swabs were designated as follows: 0= solid, 1=pasty, 2=semi-liquid; 3=liquid. Fecal consistencies with scores of 2 or more were considered diarrheic. No significant differences were detected for RV fecal shedding at any time point after inoculation (ANOVA followed by
Duncan’s rank sum test, p<0.05). However, at PID1 and 2, only a high dose of HRV induced diarrhea and was significantly higher statistically at PID2 compared to the low dose and is represented by an asterisk (*) (Fischer’s exact test). At no other PID, were significant differences observed for percentage of pigs with diarrhea.
318 9.E+04 100 8.E+04 * 90 7.E+04 80 70
(FFU/ML) 6.E+04
60 diarrhea 5.E+04 50 4.E+04 40 3.E+04 319 30 2.E+04
20 with of pigs % 1.E+04 10 Infectious virus titers titers virus Infectious 0.E+00 0 123456 CCIF Titer 10 0.E+00 6.3E+04 5.0E+03 6.9E+02 2.2E+03 0.0E+00 1000 3.E+03 4.7E+04 4.7E+03 2.9E+03 1.6E+03 1.4E+03 %pigs diarrhea10 0 0 100 100 100 100 with diarrhea diarrhea1000 23 69 40 22 80 80 10FFU n=8 n=8 n=2 n=2 n=2 n=2 1000FFU n=13 n=13 n=10 n=9 n=5 n=5
Fig.4.4 PID Mean Mean Av. Peak Fecal RV Mean Mean Diarrhea Mean Mean Mean HRV Titer shed titer Cumulative score a % days % days duration dose duration f h i h b c d (FFU/ml) g At PID 2 c d dayse Score g At PID2 n Shed. To onset dayse n diarr. To onset n
10 2100 2(0) 2(0)B 10500 8 62510 100 3.5(0.5) 3.5(0.5) 11.9(0.1) 81.6 FFU A A (5500) (1866) A A A A (0.1) A 100%A B
5100 2.2 4.4 10760 13 46857 100 3.2(0.7) 2.8(0.5) 9.8(1) 13 1.95 1000 A (0.2) (0.2) A (3398) (18260)85 A A A A (0.08) FFU A A %A A 320 a- Different HRV doses b-Number of pigs followed for 6 days after inoculation with the different doses c- Percent of pigs that had fecal RV shedding or diarrhea at least one day during the 6 days of follow up d-Days to onset of shedding or diarrhea e-Mean duration days of viral shedding or diarrhea f- The mean peak virus titers in feces detected by cell-culture immuno fluorescence (CCIF) g and h- Number of pigs tested for fecal virus shedding or diarrhea scores at PID2 and the mean fecal HRV titers at PID2 or mean diarrhea score, the percentage represents the number of pigs with shedding/total pigs per group at PID2 i-The mean cumulative score is the sum of the diarrhea scores of each pig per group/number of pigs per group. The consistency of the feces was scored every day for 6 days after inoculation and scores were as follows: 0= solid; 1=pasty; 2= semi-liquid; 3=liquid. Feces were considered diarrheic if the score was equal or higher than 2 For all values, SEM is shown in parenthesis and values with different letters differ significantly (One-way ANOVA p<0.05
Table 4.2 Shedding and diarrhea after low and high doses
Figure 4.5 Intestinal and splenic pDC binding/uptake of 2/4/6/7VLP-GFP. Immediately
after isolation, cells were resuspended in DC media and incubated with 2µg of
2/4/6/7VLP-GFP as described in materials and methods. Then cells were stained with
extracellular markers (SWC3, CD11b, CD21) and cell acquisition was performed by flow
cytometry. Cells were analyzed in a lymphocyte gate in a plot of side scatter vs. forward scatter. Then, in a plot of side scatter vs. SWC3; SWC3 high, SWC3 low and SWC3- cells were selected separately in a gate of CD21- cells. The figure shows the mean frequencies
of pDCs (SWC3low CD11b- CD21-; black bars) and cDC (SWC3low CD11b+ CD21-; white bars) from pigs that received a low HRV dose and a high HRV dose and their binding/uptake of 2/4/6/7VLP. Statistical analyses were performed by a Wilcoxon rank sum test and letters represent significant differences between pDCs (capital letters) and cDCs (small letters). The error bars represent the SEM.
321 322 2/4/6/7VLP-GFP Fig. 4.5 Frequency (%) of DCs 2/4/6/7VLP-GFP+ 10 15 20 25 0 5 IntestinalandsplenicpDC (SWC3 0FU ///VP1000FFU-2/4/6/7VLP 10 FFU-2/4/6/7VLP A = n=5 n=4 aa a pDCs andcDCs Intestine low B CD11b - CD21 - ) cDC(SWC3 10 15 20 25 0 5 low
0FU ///VP1000FFU-2/4/6/7VLP 10 FFU-2/4/6/7VLP CD11b A n=4 + CD21 a pDCs andcDCs - ) anduptake/bindingof Spleen B n=5 b
10 FFU dose a 1000 FFU dosea controlsa pig#b CD4+/SW-/CD8-c isotype controld pig#b CD4+/SW-/CD8-c isotype controld pig#b CD4+/SW-/CD8-c isotype controld 210106 0.53 0 130306 0.04 0 130506 0 0 210206 0.19 0.07 130406 0 0 130606 0 0 210306 0.43 0 201006 0 0 201406 0.01 0.01 210406 0.41 0 200906 0 0 130706 0 0 201106 0 0 130806 0 0 210506 0.03 0.03 210606 0.08 0 p=0.018e p=0.2e p=0.5e a 323 -Inoculated dose: 10 FFU, 1000 FFU, media (controls).
b-Individual pig id.
c- CD4+SW-CD8- staining for individual pigs by groups.
d- isotype control frequencies for each individual experiment.
e-Statistical significance (p value) between experimental and isotype control frequencies (Wilcoxon test). A p value of ≤ 0.05 was considered significant
Table 4.3. Frequencies of circulating LAP+ CD4+ (SWC3-CD8-) in individual pigs with their correspondant isotype control frequency values.
Figure 4.6 The frequencies of circulating LAP+CD4+(SWC3-CD8-) T cells after a low
HRV dose , high HRV dose and controls are summarized. The y axes represent the cell frequency (%) of expression of membrane bound TGFβ (LAP) cells by flow cytometry
(left) and the TGFβ cytokine levels in SIC (right) detected by ELISA in pg/ml. The mean cell frequencies of 4-7 pigs receiving a low HRV dose (white bars) at PID2, a high HRV dose (gray bars) at PID2 and PID4 or control pigs (black bars) were represented as bars.
Inside the plot the value of the mean cytokine concentration in SIC is shown (-●-). A correlation coefficient and p value were placed inside the plot. The error bars denote the standard error of the mean (SEM) and bars with different letters differ significantly
(Kruskall-wallis rank sum test followed by a Wilcoxon rank sum test, p<0.05)
324 + - - n=4 CD4 (SWC3 CD8 ) PBL 0.50 A* r=0.59 60 0.45 p=0.013 50 0.40 SIC
TGFb SIC TGF 0.35 40 0.30
0.25 30 β 325 inpg/ml 0.20 20 0.15 n=7 0.10 n=6 Frequency of LAP+ cells B** 10 0.05 B** 0.00 0 10FFU 1000 FFU control Virulent M strain HRV dose
Fig. 4.6 Cell membrane bound TGFb (LAP) expression on peripheral blood lymphocytes
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