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Home , Birnaviridae, HBsAg, Listeria, Neonatal Fc receptor, Polyomaviridae, Rotavirus

ROTAVIRUS

AND IMPACT OF MATERNAL AND

ON NEONATAL IMMUNE RESPONSES IN SWINE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree of Doctor of Philosophy in

the Graduate School of The Ohio State University

By

Trang Van Nguyen, B.Sc.

*****

The Ohio State University 2005

Dissertation Committee:

Distinguished University Professor Dr. Linda J. Saif, Adviser

Professor John H. Hughes Approved by

Assistant Professor Dr. Jeffrey T. Lejeune

Adjunct Assistant Professor Dr. Lijuan Yuan ______Adviser Graduate Program in Veterinary Preventive Medicine

ABSTRACT

Group A (RV) is the most important cause of viral in

young children and worldwide and the need for effective vaccines against RV is

urgent. Because occur in and nursing pigs, the should be

delivered as early as the first few days or weeks of life, which poses problems because of

the impact of maternal antibodies (MatAb) on vaccine responses. The transfer of cytokines from /milk to neonates which influences the development of neonatal has not been studied in or animals. The main goals of this thesis were:

(1) to study the efficacy, immunogenicity and correlates of protective immunity after oral with -like particles (VLP) with or without attenuated rotavirus

(AttHRV) in a prime/boost strategy; (2) and (3) to understand the impact of high and low

titer circulating MatAb to RV on protective immunity and B responses induced by

RV vaccines (replicating vs. non-replicating); and (4) to examine the transfer of

cytokines from mothers to neonates and the implications of such transfer for immunomodulation of neonatal immunity.

ii We evaluated responses and protection induced by a replicating vaccine

consisting of an oral of AttHRV for priming and two intranasal doses of a

2/6VLP(100 or 250ug)/ISCOM vaccine for boosting (AttHRV/VLP) or an intranasal

VLP/ISCOM prime/boost vaccine (VLP/ISCOM3x) in gnotobiotic (Gn) pigs and compared them with the 3 doses AttHRV (AttHRV3x) oral vaccine. The AttHRV-

VLP250µg/ISCOM and AttHRV3x groups had significantly higher serum IgA and IgG and intestinal IgA antibody titers to HRV pre-challenge than the 3-dose-

VLP100µg/ISCOM group (VLP/ISCOM3x) and controls (diluent/ISCOMmatrix). The pre-challenge serum virus neutralizing and IgA antibodies correlated moderately with protection against challenge with virulent HRV. Protection rates against and were highest in the AttHRV-VLP250µg/ISCOM and AttHRV3x groups, lower in the AttHRV-VLP100µg/ISCOM group, and with no protection in the

VLP/ISCOM3x group and controls. Thus the VLP/ISCOM vaccine boosted antibody titers and protection after priming with the AttHRV vaccine, but not after priming with

VLP/ISCOM.

We next investigated effects of high titer MatAb on protection and immune responses induced by the above AttHRV/VLP and VLP/ISCOM3x vaccines. Passive circulating MatAb (hyperimmune sow serum) injected into Gn pigs contributed to partial protection against virulent HRV challenge; however, MatAb interference led to no or low intestinal IgM, IgA and IgG antibody titers and significantly reduced intestinal IgA and

IgG antibody secreting cell (ASC) and memory responses in the AttHRV/VLP pigs pre- and post-challenge. The MatAb suppression was not alleviated by extending the vaccination/challenge interval from 28 to 42 days. The suppression by high titer MatAb iii was also observed in the non-replicating 3xVLP/ISCOM vaccinated pigs, as indicated by

a reduction in intestinal IgG ASC responses, serum and intestinal IgA antibody titers and

the pre-challenge memory B cell responses. Consequently, alternative strategies are needed to overcome the high titer MatAb suppression of immune responses to vaccines in neonates.

We then investigated the effect of low titer circulating MatAb (LoMatAb) on protection and immune responses induced by these two vaccine regimens. Protection rates in the AttHRV/VLP groups with and without LoMatAb were similar against viral shedding and diarrhea when challenged with virulent HRV. The LoMatAb had both enhancing and suppressive effects on B cell responses, depending on antibody isotype, tissue and vaccine. For the AttHRV/VLP (replicating) vaccine, LoMatAb enhanced intestinal IgM and IgA-ASC numbers pre- or post-challenge, but it suppressed systemic

(spleen and blood) IgA-ASC numbers and intestinal IgA antibody responses pre- and post- challenge. For the VLP (non-replicating) vaccine, LoMatAb also enhanced intestinal and splenic IgM-ASC numbers post-challenge, but it suppressed IgG-ASC numbers pre-challenge in all tissues. Pre-challenge IgA memory B cells in ileum and spleen were unaffected, but they were suppressed in blood of both vaccine groups. The differential effects of LoMatAb on the IgA responses suggests that LoMatAb did not suppress the induction of IgA-ASC and memory B cells at the induction site (ileum) but it impaired the homing of activated B cells to secondary lymphoid or effector tissues, reducing IgA-ASC and antibodies at these sites. Thus, even LoMatAb exerts different effects on B cell responses to replicating versus non-replicating vaccines.

iv In addition to MatAb, other important immune regulators such as cytokines may also play significant roles in the development of the neonatal and the characteristics of the reduced responsiveness and Th2 biased immune responses in neonates. The effects of maternally-acquired cytokines on development of neonatal immunity are undefined. We investigated IL-6 and TNF-α (pro-inflammatory), IFN-γ and IL-12, (Th1), IL-10 and IL-4 (Th2) and TGF-β1 (Th3) concentrations in sow serum and colostrum/milk and in serum and intestinal contents of their suckling piglets at 0-13 post-partum days (PPD) or post-weaning. All cytokines except for TNF-α were detected in sow colostrum/milk. No IL-6, TNF-α, IFN-γ, IL-4 and IL-10 were detected in piglet sera at PPD0 documenting absence of transplacental transfer, whereas

IL-12 and TGF-β1, present at birth, may be constitutively produced or maternally- derived. The peak mean cytokine concentrations in piglet sera were detected at PPD1-2

(IL-4>TGF-β1>IL-6>IL-12>IFN-γ>IL-10) with high concentrations of IL-4 and TGF-β1 likely contributing to the neonatal Th2 bias. Increased concentrations of intestinal IL-6,

IL-12 and TNF-α in suckling piglets and of serum IL-6 and IL-12 in weaned piglets were potentially induced by intestinal colonization with commensal . Low concentrations of IFN-γ in suckling and weaned piglets may be due to its down- regulation by TGF-β. In summary, we comprehensively documented the transfer of maternal cytokines from colostrum/milk to neonates and have provided new evidence for their potential role in the Th2 bias of neonatal immune responses.

These findings have improved our understanding of the differential effects of circulating MatAb on different RV vaccines and protection and should facilitate the

v design of new rotavirus vaccines to overcome such interference. Our research also provides new information about the cytokines in maternal milk, their transfer and persistence in neonates and their potential role in the development of neonatal immune responses.

vi

DEDICATION

I dedicate this dissertation to my parents, Nguyen Ngoc Thien and Tran Thi Binh An,

whose love and devotion always motivated me

To my husband, Hoang, whose love supported and encouraged me

from thousands miles away

vii

ACKNOWLEDGMENTS

I am in debt to my adviser, Dr Linda J. Saif for giving me the opportunity to work in her lab. I am grateful for her guidance, support and patience during my study, the preparation of the manuscripts and this dissertation and during my unexpected delay in

Vietnam. I learned a lot from her critical thinking and original ideas. Her knowledge and dedication to research was an inspiration during my study.

My deep gratitude goes to my committee member Dr. Lijuan Yuan for innovative ideas and extensive revision of my manuscripts and dissertation. I thank her for the constant encouragement from the first day I started this PhD program.

I sincerely thank Dr Jeoffrey LeJeune for helpful comments on the manuscripts and my dissertation, in special his help with the statistical analysis. I thank Dr. John

Hughes for valuable comments on my dissertation.

Many thanks to Dr. Mo Saif, Dr. Kenneth Theil, Dr. Joy Pate and Dr. Srinand

Sreevatsan for reviewing my manuscripts.

To my colleagues, graduate students and post-doctors: Dr. Marli Azevedo, Dr.

Kwang-il Jeong, Dr. Cristiana Iosef and Ana Gonzalez with whom I shared the hard work in research in the pig model. Thank you for the friendship in special to

viii Dr. Quihong Wang, Menira Souza, Veronica Costantini, Sonia Cheetham, Peggy Lewis,

Marcela Azevedo, Severin Pouly, Stacie Shafer and Ke Wei. Many thanks go to Hong

Liu for his valuable help and suggestions with statistical analyses.

To Dr. Juliette Hanson and Greg Myers for their technical assistance and efforts to make my work with animals possible. To Terry Meek, Todd Root and Richard

McCormick for their technical assistance.

To Hannah Gehman and Robin Weimer, the secretaries of the Department for their indispensable support.

Thanks to the Health Research Program-OARDC for giving me the fellowship for my PhD (from September 2002 to August 2005). Thanks to the OARDC for granting me the Charles Thorne fellowship for the year 2003-2004.

My thanks to the National Institute of Hygiene and Epidemiology, Hanoi,

Vietnam in special to Dr Vu Tan Trao for her support and encouragement.

My sincere appreciation to Samar Al Maalouf for her precious friendship and support during stressful times. I also thank her for her invaluable comments on my dissertation.

My appreciation once again to my husband, Hoang, for his patience, love and understanding that helped me to pass through this great challenge.

ix

VITA

July 20, 1976………………. Born- Hanoi, Vietnam

1994-1997………………….. Bachelor of Science

The University of Western Australia, Perth, Australia

1998-2000………………….. Research Scientist

National Institute of Hygiene and Epidemiology, Hanoi,

Vietnam

2000-2005 …………………. Graduate Student

Food Animal Health Research Department

Department of Veterinary Preventive Medicine,

OARDC, The Ohio State University

PUBLICATIONS

1. Azevedo, M. S., Yuan, L., Jeong, K. I., Gonzalez, A., Nguyen, T. V., Pouly, S., Gochnauer, M., Zhang, W., Azevedo, A., and Saif, L. J. (2005). Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus. J Virol 79(9), 5428-36.

x 2. Yuan, L., Azevedo, M. S., Gonzalez, A. M., Jeong, K. I., Van Nguyen, T., Lewis, P., Iosef, C., Herrmann, J. E., 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(30), 3925-36.

3. Azevedo, M. S., Yuan, L., Iosef, C., Chang, K. O., Kim, Y., Nguyen, T. V., and Saif, L. J. (2004). Magnitude of serum and intestinal antibody responses induced by sequential replicating and nonreplicating rotavirus vaccines in gnotobiotic pigs and correlation with protection. Clin Diagn Lab Immunol 11(1), 12-20.

4. Gonzalez, A. M., Nguyen, T. V., Azevedo, M. S., Jeong, K., Agarib, F., Iosef, C., Chang, K., Lovgren-Bengtsson, K., Morein, B., and Saif, L. J. (2004). 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. Clin Exp Immunol 135(3), 361-72.

5. Nguyen, T. V., Iosef, C., Jeong, K., Kim, Y., Chang, K. O., Lovgren- Bengtsson, K., Morein, B., Azevedo, M. S., Lewis, P., Nielsen, P., Yuan, L., and Saif, L. J. (2003). Protection and antibody responses to oral priming by attenuated human rotavirus followed by oral boosting with 2/6-rotavirus-like particles with immunostimulating complexes in gnotobiotic pigs. Vaccine 21(25-26), 4059-70.

6. Iosef, C., Van Nguyen, T., Jeong, K., Bengtsson, K., Morein, B., Kim, Y., Chang, K. O., Azevedo, M. S., Yuan, L., Nielsen, P., and Saif, L. J. (2002). Systemic and intestinal antibody secreting cell responses and protection in gnotobiotic pigs immunized orally with attenuated Wa human rotavirus and Wa 2/6-rotavirus-like- particles associated with immunostimulating complexes. Vaccine 20(13-14), 1741-53.

7. Nguyen, V. M., Hoang, T. N., Huynh, T. P., Nguyen, T. V., Nguyen, K. G., Nguyen, M. L., Nguyen, T. T., Dunia, I., Cohen, J., and Benedetti, E. L. (2001a). Immunocytochemical characterization of and antigenic macromolecules in viral vaccines. C R Acad Sci III 324(9), 815-27.

8. Nguyen, V. M., Nguyen, V. T., Huynh, P. L., Dang, D. T., Nguyen, T. H., Phan, V. T., Nguyen, T. L., Le, T. L., Ivanoff, B., Gentsch, J. R., and Glass, R. I. (2001b). The epidemiology and disease burden of rotavirus in Vietnam: sentinel surveillance at 6 hospitals. J Infect Dis 183(12), 1707-12.

9. Yuan, L., Iosef, C., Azevedo, M. S., Kim, Y., Qian, Y., Geyer, A., Nguyen, T. V., Chang, K. O., and Saif, L. J. (2001). Protective immunity and antibody-secreting cell responses elicited by combined oral attenuated Wa human rotavirus and intranasal Wa 2/6-VLPs with mutant heat-labile in gnotobiotic pigs. J Virol 75(19), 9229-38.

xi PUBLISHED ABSTRACTS

1. Yuan, L.J., Nguyen, T.V., Azevedo, M.S.P., Gonzalez, A.M., Jeong, K-I and Saif, L.J. (2005). Maternal cytokines in serum and intestinal contents of suckling pigs. The 12th International Congress of Mucosal Immunology, Boston, Massachusetts, June 26-30, 2005. 2. Gonzalez, A.M., Yuan, L., Azevedo, M.S.P., Nguyen, T.V., Jeong, K-I, Lovgren-Bengtsson, K., Morein, B. and Saif, L.J. (2005). B cell responses elicited by oral/intranasal (IN) of gnotobiotic pigs with a rotavirus-like particle prime/boost vaccine. The 12th International Congress of Mucosal Immunology, Boston, Massachusetts, June 26-30, 2005. 3. Nguyen, T., Yuan, L., Azevedo, M.S.P., Gonzalez, A.M., K.-I Jeong and Saif, L.J. (2005). Transfer of cytokines from sows to newborn piglets via colostrum and milk. Swine in Biomedical Research Conference, Chicago, IL, January, 27-29. 4. Jeong, K-I, Azevedo, M., Gonzalez, A.M., Nguyen, T., Yuan, L. and Saif, L. Cell-mediated immune responses in Gnotobiotic pigs immunized with sequential attenuated human rotavirus (AttHRV) and 2/6-rotavirus-like-particles vaccines and challenge with virulent HRV, 24th American Society for , Pensylvania, June 18- 22, 2005 5. Nguyen, T.V., Yuan, L., Azevedo, M.S.P., Jeong, K., Gonzalez, A.M., Lovgren-Bengtsson, K., Morein, B and Saif, L.J. Effects of maternal antibodies on effector and memory B cell responses to rotavirus vaccines. 23rd American Society for Virology, Monstreal, Canada, July 10-14, 2004. 6. Yuan, L., Jeong, K., Nguyen, T. V., Gonzalez, A.M., Azevedo, M.S.P., Zhang, W. and Saif, L.J. Effects of maternal antibodies on responses to rotavirus vaccine. 23rd American Society for Virology, Montreal, Canada, July 10-14, 2004 7. Saif, L.J., Yuan, L., Azevedo, M.S.P., Jeong, K., Gonzalez, A.M., Nguyen, T. V. and Herrmann, J.E. Protective immunity induced by live attenuated (Att) human rotavirus (HRV) priming and bovine rotavirus VP6 DNA boosting in a gnotobiotic (Gn) pig model. 22nd American Society for Immunology, Denver, CO, May 6-10, 2003. 8. Yuan, L., Azevedo, M.S.P., Nguyen, T. V., Gonzalez, A.M., Jeong, K., Chang, K. and Saif, L. J. Booster effects of rotavirus 2/6 virus-like particle (VLP) vaccine on antibody responses to rotavirus outer- VP4 and VP7 primed by oral attenuated human rotavirus (Att HRV) vaccine in gnotobiotic pigs. 22nd American Society for Virology, Davis, CA, July 12-16, 2003. 9. Jeong, K-I, Azevedo, M., Nguyen, T., Gonzalez, A., Iosef, C., Chang, K., Yuan, L., Herrmann, J.E. and Saif, L. Cellular immune responses and protection in gnotobiotic pigs vaccinated with a VP6 DNA vaccine regimen with or without attenuated human rotavirus (AttHRV). 22nd American Society for Virology, Davis, CA, July 12-16, 2003.

xii 10. Nguyen, T.V., Yuan, L., Azevedo, M.S.P., Jeong, K., Gonzalez, A.M., Lovgren-Bengtsson, K., Morein, B. and Saif, L.J. Effects of maternal antibodies on immune responses and protection induced by immunostimulating complexes (ISCOM)- VP2/6 rotavirus-like-particles (VLP) vaccine. 22nd American Society for Virology, Davis, CA, July 12-16, 2003. 11. Azevedo, M.S.P., Jeong, K., Nguyen, T.V., Gonzalez, A.M., Nielsen, P., Lewis, P., Lovgren-Bengtsson, K., Morein, B., Yuan, L. and Saif, L. J. Protective immunity in gnotobiotic (Gn) pigs after intranasal (IN) or oral priming with attenuated human rotavirus (AttHRV) and boosting with 2/6-rotavirus-like-particles (VLPs) and immuno- stimulating complexes (ISCOM), and detection of nasal shedding of AttHRV. 22nd American Society for Virology, Davis, CA, July 12-16, 2003. 12. Saif, L.J., Azevedo, M.S.P., Yuan, L., Jeong, K. I., Gonzalez, A., Nguyen, T. V., Pouly, S. and Gochnauer, M. 2005. Nasal and rectal shedding and viraemia in gnotobiotic pigs after oral or intranasal inoculation with Wa human rotavirus. Abstract W6.3. 8th International Symposium on ds-RNA viruses, II Ciocco, Lucca, Italy. September 13-18, 2003. 13. Yuan, L., Azevedo, M. S., Gonzalez, A.M., Jeong, K. I., Van Nguyen, T., Lewis, P., Iosef, C., Herrmann, J.E., and Saif, L.J. (2005). Evaluation of a live attenuated human rotavirus priming and bovine VP6 DNA boosting vaccination strategy in a gnotobiotic pig model. Abstract PR.4. 8th International Symposium on ds-RNA viruses, II Ciocco, Lucca, Italy. September 13-18, 2003. 14. Azevedo, M., Iosef, C., K. Jeong, Nguyen, T.V., Gonzalez, A.M., Kim, Y., Agarib, F. and Saif, L.J. Rotavirus and rotavirus-like particle (VLP) vaccines with immunostimulating complexes (ISCOM) as a prime/boost strategy induce intestinal IgA antibody secreting cells and protective immunity to human rotavirus in a gnotobiotic pig disease model. 11th International Congress Mucosal Immunology, Orlando, FL, June 16- 20, 2002. 15. Azevedo, M., Iosef, C., K. Jeong, Nguyen, T.V., Gonzalez, A.M., Agarib, F., Chang, K., Lovgren-Bengtsson, K., Morein, B., Nielsen, P., Nguyen, T.V. and Saif, L. J. Antibody secreting cell responses and protection in gnotobiotic pigs vaccinated orally with attenuated human rotavirus and intranasally (IN) with 2/6-rotavirus-like particles (VLPs) and immunostimulating complexes (ISCOM). 21st American Society for Virology, Lexington, KY, July 20-24, 2002. 16. Gonzalez, A. M., Nguyen, T. V., Azevedo, M. S., Jeong, K., Agarib, F., Iosef, C., Chang, K., Lewis, P., Lovgren-Bengtsson, K., Morein, B. and Saif, L.J. Vaccination of gnotobiotic pigs orally with attenuated human rotavirus (AttHRV) and intranasal (IN) VP2/6 rotavirus-like particles (2/6VLP) with ISCOM induces similar protection rates but higher antibody titers than AttHRV alone. 21st American Society for Virology, Lexington, KY, July 20-24, 2002. 17. Jeong, K-I, Gonzalez, A.M., Iosef, C., Azevedo, M., Nguyen, T., Chang, K., Agarib, F., Lovgren-Bengtsson, K., Morein, B. and Saif, L. Lymphoproliferative responses in gnotobiotic pigs inoculated orally with attenuated human rotavirus

xiii (AttHRV) and intranasally (IN) with 2/6 rotavirus-like particles (VLP) with immunostimulating complexes (ISCOM). 21st American Society for Virology, Lexington, KY, July 20-24, 2002. 18. Iosef, C., Nguyen, T. V., Jeong, K., Kim, Y., Lovgren-Bengtsson, K., Morein, B., Nielsen, P., Azevedo, M.S.P. and Saif, L.J. Analysis of antibody secreting cell responses and protection to Wa human rotavirus (HRV) in gnotobiotic pigs immunized with attenuated Wa HRV (AttHRV) and 2/6 rotavirus-like particles (VLP) administered with immunostimulating complexes (ISCOM). 20th American Society for Virology, Madison, WI, July 22-25, 2001 19. Nguyen, T. V., Iosef, C., Jeong, K., Kim, Y., Lovgren-Bengtsson, K., Morein, B., Nielsen, P., Azevedo, M. S.P. and Saif, L.J. Antibody responses to oral rotavirus- like particles (VLP) administered with immunostimulating complexes (ISCOM) and attenuated rotavirus vaccines in gnotobiotic pigs 20th American Society for Virology, Madison, WI, July 22-25, 2001 20. Saif, L. J., Yuan, L., Iosef, C., Azevedo, M.S.P., Nguyen, T.V., Jeong, K., Kim, Y. Vaccine strategies to induce intestinal antibody secreting cells (ASC) and memory B-cells to rotavirus and protection in gnotobiotic pigs. Proceedings to Sixth International Veterinary Immunology Symposium, Uppsala, Sweden, July 15-20, 2001 21. Saif, L. J., Yuan, L., Iosef, C., Azevedo, M.S.P., Nguyen, T.V., Jeong, K., Kim, Y. Vaccination stratergies to induce protective immunity to human rotavirus in a neonatal gnotobiotic pigs disease model. Proceedings to Vaccines for Enteric Diseases, Tampere, Finland, September 12-14, 2001 22. Saif, L. J., Yuan, L., Iosef, C., Azevedo, M.S.P., Nguyen, T.V., Jeong, K., Kim, Y. Evaluation of vaccines to induce intestinal antibody secreting cell (ASC) and memory B-cells to rotavirus and protection in a gnotobiotic pigs model of human rotavirus diarrhea. Brazillian Society for Virology, Caldas Novas, Goias, Brazil, Nov. 25- 28, 2001.

FIELDS OF STUDY

Major Field: Veterinary Preventive Medicine

Studies in Virology and Immunology

xiv

TABLE OF CONTENT

Page

Abstract……………………………………………………..………………………...... ii

Dedications……………………………………………………………………………...vii

Acknowledgment..……………………………………..……………………………….viii

VITA……………………………………………………………………………………...x

List of tables……………………………..………………………………………………xx

List of figures……………………..……………………………………………………..xxi

List of abbreviation………….…………………………………………………………xxiii

CHAPTER I: LITERATURE REVIEW

ROTAVIRUS VACCINES AND INFLUENCE OF MATERNAL ANTIBODIES AND CYTOKINES ON NEONATAL IMMUNE RESPONSES...... 1

1.1 Rotavirus and rotavirus vaccine...... 1 1.1.1 Impact of rotavirus diseases- diarrhea morbidity and mortality in children worldwide ...... 1 1.1.2 Rotavirus structure and ...... 3 1.1.3 Rotavirus classification...... 4 1.1.4 Rotavirus disease...... 6 1.1.5 Rotavirus replication...... 8 1.1.6 Rotavirus pathogenesis and mechanisms of diarrhea ...... 10 1.1.7 Cross- infections...... 17 1.1.8 The viral NSP4 ...... 19 1.1.8.1 Structure and functions of NSP4...... 19 1.1.8.2 Genotypes of NSP4...... 21 1.1.8.3 Mechanisms of diarrhea caused by NSP4...... 21 1.1.8.4 Diarrhea induction by NSP4 ...... 23

xv 1.1.8.5 Immune responses to NSP4...... 24 1.1.8.6 Homotypic and heterotypic NSP4 specific antibodies and protection...... 25 1.1.8.7 Passive immunity conferred by NSP4 antibodies...... 27 1.1.9 Immunity to RV ...... 27 1.1.9.1 Role of innate immunity ...... 27 1.1.9.1.1 Natural killer cells...... 29 1.1.9.2 Adaptive immunity to RV: Studies of different animal models and humans…………………………………………………………………………29 1.1.9.2.1 Mouse model...... 30 1.1.9.2.2 Rabbit model...... 33 1.1.9.2.3 Pig model...... 33 1.1.9.2.4 Non-human primate models ...... 37 1.1.9.2.5 Immunity to RV-human studies: The immune determinants of protection against RV ...... 38 1.1.9.2.5.1 Serum and fecal IgA antibodies...... 38 1.1.9.2.5.2 Markers on B and T cells and the association with RV and protection………………………………………………………………….40 1.1.9.2.5.3 Role of cytokines in RV infections...... 42 1.1.9.2.6 Rotavirus specific antibody levels in children worldwide...... 44 1.1.10 Interference by MatAb with RV infections ...... 44 1.1.10.1 RV antibodies in milk and passive protection ...... 44 1.1.10.2 Circulating antibody and protection against RV...... 46 1.1.10.3 Maternal immunization...... 47 1.1.10.4 Protection against RV by other milk components ...... 50 1.1.10.5 Interference with active immune responses to RV in infants and in animal models ……………………………………………………………………………50 1.1.11 Rotavirus vaccines...... 52 1.1.11.1 Jennerian and modified Jennerian approaches to RV vaccines ...... 52 1.1.11.2 Non Jennerian approach to RV vaccines ...... 54 1.1.11.3 Rotavirus-like particles as vaccines in different animal models...... 55 1.1.11.4 Individual RV proteins as potential vaccines...... 59

1.2 The mucosal immune system...... 62 1.2.1 General structure...... 62 1.2.2 Innate immunity...... 63 1.2.2.1 Mucosal DC...... 64 1.2.2.2 Toll-like receptors...... 64 1.2.2.2.1 Roles of TLR in the recognition of microbial components ...... 65 1.2.2.2.2 Distribution of TLRs in different tissues of humans and animals ...... 67 1.2.2.2.3 Role of TLRs in the regulation of adaptive immunity...... 69 1.2.3 Adaptive immunity ...... 70 1.2.3.1 CD4+ T cells...... 70 1.2.3.2 CD8+ T cells...... 72 1.2.3.3 B cells...... 73 1.2.3.4 Biological roles of IgA antibody...... 74 1.2.3.4.1 Inhibition of adherence ...... 74 xvi 1.2.3.4.2 Neutralization of viruses, and ...... 74 1.2.3.4.3 Inhibition of presentation...... 75 1.2.4 Leukocyte trafficking in the mucosal immune system ...... 75

1.3 Neonatal immune responses ...... 77 1.3.1 Overview...... 77 1.3.2 Innate immunity...... 78 1.3.2.1 Dendritic cells...... 78 1.3.2.2 Monocytes...... 80 1.3.2.3 Natural killer (NK) cells ...... 81 1.3.3 Adaptive immunity ...... 81 1.3.3.1 T effector and memory cell responses ...... 81 1.3.3.2 Neonatal B cell repertoire ...... 84 1.3.3.2.1 Activation of neonatal B cells- in vitro system ...... 86 1.3.3.3 Mucosal immune system in neonatal pigs ...... 88 1.3.3.3.1 Gnotobiotic pig model for human enteric viruses and mucosal immunity 89

1.4 Maternal interference with neonatal active immune responses: sources and mechanisms of interference ...... 91 1.4.1 Sources of maternal antibodies (MatAb) ...... 91 1.4.1.1 Maternal antibodies from milk...... 92 1.4.1.2 Transfer of sIgA antibodies into mucosal ...... 93 1.4.1.2.1 Structure of the pIgR ...... 93 1.4.1.2.2 Functions of pIgR ...... 94 1.4.1.3 Transfer of IgG across the and from milk to neonates ...... 96 1.4.1.3.1 Structure of the FcRn...... 96 1.4.1.3.2 Functions of FcRn...... 97 1.4.1.3.3 Mechanisms of FcRn mediated IgG homeostasis...... 102 1.4.2 Cells in passive immunity...... 104 1.4.2.1 Origin of plasma cells in the mammary gland...... 104 1.4.2.2 Transfer of functional immune cells in milk to neonates ...... 105 1.4.3 ...... 106 1.4.4 Cytokines ...... 107 1.4.4.1 Transfer of cytokines via the placenta ...... 109 1.4.5 CD14...... 110 1.4.6 Levels of MatAb transferred and factors influencing the transfer...... 110 1.4.7 Interference of neonatal responses by MatAb ...... 112 1.4.7.1 Inhibition by Mat Ab ...... 112 1.4.7.2 Enhancement of the immune responses by MatAb...... 114 1.4.7.3 Mechanisms of MatAb interference...... 115 1.4.7.3.1 Neutralization of live viral vaccines ...... 115 1.4.7.3.2 Interference of passive antibodies via the Fc ...... 116 1.4.7.3.3 Interference via idiotypic interaction...... 118

xvii 1.5 Vaccine strategies to overcome the immaturity of neonatal immunity and interference by MatAb ...... 120 1.5.1 Type of vaccines ...... 121 1.5.1.1 Virus-like particles (VLPs)...... 121 1.5.1.2 DNA vaccines ...... 122 1.5.2 Antigen delivery system ...... 127 1.5.2.1 Aluminum ...... 127 1.5.2.2 Liposomes, virosomes and Archaeosomes ...... 127 1.5.2.3 Microspheres...... 129 1.5.2.4 Vectored vaccines ...... 129 1.5.2.5 Monophosphoryl lipid A (MPL)...... 130 1.5.2.6 Saponins and derivatives...... 131 1.5.2.7 Muramyl dipeptide derivatives (MDP)...... 133 1.5.2.8 Hormones...... 133 1.5.2.9 Cytokines ...... 134 1.5.2.10 CpG Oligodeoxynucleotides...... 135

1.6 REFERENCES ...... 136

CHAPTER 2 ...... 213 PROTECTION AND ANTIBODY RESPONSES TO ORAL PRIMING BY ATTENUATED HUMAN ROTAVIRUS FOLLOWED BY ORAL BOOSTING WITH 2/6-ROTARUS-LIKE-PARTICLES WITH IMMUNOSTIMULATING COMPLEXES IN GNOTOBIOTIC PIGS ...... 213 2.1. Summary...... 213 2.2. Introduction...... 214 2.3. Materials and methods ...... 216 2.4. Results...... 220 2.5. Discussion...... 225 2.6. Acknowledgments...... 231 2.7 References...... 232

CHAPTER 3 …………………………………………………………………………..246 HIGH TITER SERUM MATERNAL ANTIBODIES IMPACT PROTECTIVE IMMUNITY AND B CELL RESPONSES INDUCED BY ATTENUATED ROTAVIRUS PRIMING AND A ROTAVIRUS-LIKE PARTICLE –ISCOM BOOSTING VACCINE REGIMEN …………………………………………………..246 3.1. Summary …………………………………………………………………………..246 3.2. Introduction ………………………………………………………………………..247 3.3. Materials and Methods ...…………………………………………………………..250 3.4. Results ....…………………………………………………………………………..256 3.5. Discussion……...…………………………………………………………………..272 3.6. Acknowledgments……...…………………………………………………………..279 3.7. References ……..…………………………………………………………………..280

xviii CHAPTER 4……………………………………………………………………………298 LOW TITER MATERNAL ANTIBODIES CAN BOTH ENHANCE AND SUPPRESS B CELL RESPONSES TO A COMBINED LIVE ATTENUATED HUMAN ROTAVIRUS AND IMMUNOSTIMULATING COMPLEX-BASED ROTAVIRUS- LIKE PARTICLE VACCINE…………………………………………………………..298 4.1. Summary …………………………………………………………………………..298 4.2. Introduction ………………………………………………………………………..299 4.3. Materials and Methods ...…………………………………………………………..301 4.4. Results ……………………………………………………………………………..306 4.5. Discussion ……..…………………………………………………………………..315 5.6. Acknowledgments ……..…………………………………………………………..322 4.7. References ……..…………………………………………………………………..323

CHAPTER 5 ...... 336 CYTOKINES TRANSFERRED FROM MOTHER TO NEONATES IN SWINE: IMPLICATIONS FOR IMMUNOMODULATION OF NEONATAL IMMUNITY BY MATERNAL CYTOKINES...... 336 5.1 Summary...... 336 5.2. Introduction...... 337 5.3. Materials and Methods...... 339 5.4. Results...... 343 5.5. Discussion...... 353 5.6. Acknowledgements...... 361 5.7. References...... 362

BIBLIOGRAPHY………………………………………………………………………376

xix

LIST OF TABLES

Table Page Table 1.1: Level of circulating rotavirus specific antibody of infants in some developing countries...... 204 Table 1.2: Level of circulating rotavirus specific antibody of mothers and infants in some developed countries ...... 205 Table 1.3: Routes of of MatAb across taxa ...... 206 Table 1.4: Expected loss (days) of MatAb from the newborn in different species ...... 206 Table 1.5: Levels of cytokines in human colostrum and milk (pg/ml)...... 207 Table 1.6: Virus-like particles for various virus families ...... 208 Table 2.1: Summary of protection rates against virus shedding and diarrhea in gnotobiotic pigs receiving different vaccine treatments…………………………...... 238 Table 2.2: Isotype antibody responses to Wa HRV in serum of pigs at PID 28/PCD0 and the correlation between antibody titers and protection against virus shedding and diarrhea at this time point………………………………………………………………239 Table 3.1: Rotavirus shedding and diarrhea in gnotobiotic pigs after challenge with Vir HRV………………………………………………………………………………...284 Table 3.2: Comparison of memory B cell responses in pigs given AttHRV/VLP vaccines in the presence or absence of MatAb and between vaccine groups in Exp.I and II…………………………………………………………………………..286 Table 4.1: Rotavirus shedding and diarrhea in gnotobiotic pigs after challenge with VirHRV ………………………………………………………………………………...328 Table 5.1: Concentrations (pg/ml) of IL-12 and TGF-β1 in serum and small intestinal contents (SIC) of neonatal piglets derived by hysterectomy or natural birth…………..365

xx

LIST OF FIGURES

Figure Page

Figure 1.1: Structure of rotavirus……………………………………………………….209

Figure 1.2: Structure of FcRn and pIgR………………………………………………...210

Figure 1.3: Expected influence of maternal antibodies to neonatal immune responses …………………………………………………………….211

Figure 1.4: Association of RV 2/6-VLP with immunostimulating complexes………....212

Figure 2.1: Immunoblot of protein components in 2/6VLP and double-layered inactivated HRV (dl-inact-HRV) …………………………………………………………………...240

Figure 2.2: Geometric mean virus neutralizing (VN) antibody titers in sera of pigs that received different vaccine regimens and their correlation with the protection rates against viral shedding and diarrhea….………………………………………………….241

Figure 2.3: GMT of isotype-specific antibody to Wa HRV in serum of gnotobiotic pigs at various time-points from each vaccine treatment group….…………………………….242

Figure 2.4: The IgG subclass responses to Wa HRV in serum samples at PID28/PCD0 from different vaccine groups…………………………………………………………..244

Figure 2.5: GMT of isotype-specific antibodies to Wa HRV in small (SIC) and large (LIC) intestinal contents of gnotobiotic pigs euthanised at various time-points from each vaccine group (1-7)………………………………………………………………..245

Figure 3.1: Virus neutralizing (VN) and isotype specific geometric mean antibody titer of pigs vaccinated and challenged in the presence or absence of MatAb…………………………………………………………………………………...288

Figure 3.2: Isotype specific geometric mean antibody titers in small intestinal contents of pigs following vaccination and challenged in the presence or absence of MatAb…..290

Figure 3.3: IgM ASC responses to Wa HRV in pigs vaccinated in the presence or absence of MatAb………………………………………………………………………292

xxi Figure 3.4: IgA ASC responses to Wa HRV in pigs vaccinated in the presence or absence of MatAb…………………………………………………………………….....294

Figure 3.5: IgG ASC responses to Wa HRV in pigs vaccinated in the presence or absence of MatAb……………………………………………………………………….296

Figure 4.1: Virus neutralizing (VN) and isotype specific geometric mean antibody titers in serum of pigs vaccinated and challenged in the presence or absence of LoMatAb……………………………………………………………………329

Figure 4.2: Isotype specific geometric mean antibody titers in small intestinal contents of pigs following vaccination and challenged in the presence or absence of LoMatAb……………………………………………………………………330

Figure 4.3: IgM ASC responses to Wa HRV in pigs vaccinated in the presence or absence of LoMatAb ………………………………………………………………...331

Figure 4.4: IgA ASC responses to Wa HRV in pigs vaccinated in the presence or absence of LoMatAb ...………………………………………………………………333

Figure 4.5: IgA ASC responses to Wa HRV in pigs vaccinated in the presence or absence of LoMatAb …...……………………………………………………………334

Figure 4.6: Memory B cell responses in pigs vaccinated with AttHRV/VLP, VLP vaccine regimens or ISCOM control in the presence and absence of LoMatAb at PID28/PCD0 ……………………………………………………………...335

Figure 5.1: Cytokine concentrations in colostrum/milk and the relationship with the cytokine concentrations in sows‘serum samples ……………………………………….366

Figure 5.2: Different forms of TGF-β1 in sows’colostrum/milk ………………………368

Figure 5.3: Cytokines in sera of suckling piglets and correlations with the concentrations in sows’ colostrum/milk ………………………………………………………………..369

Figure 5.4: Comparison of cytokine concentrations in sera of weaned and suckling piglets…………………………………………………………………………………....371

Figure 5.5: The concentrations of IL-6 and TNF-α (pro-inflammatory cytokines) and TGF-β (Th3 cytokine) in the intestinal contents and serum of suckling piglets………..372

Figure 5.6: The concentrations of Th1 cytokines (IFN-γ and IL-12) in the intestinal contents and serum of suckling piglets………………………………………………….374

Figure 5.7: The concentrations of Th2 cytokines (IL-10 and IL-4) in the intestinal contents and serum of suckling piglets………………………………………………….375 xxii

LIST OF ABBREVIATIONS

Aluminium hydroxide (AlOH) Antibody secreting cells (ASC) Bacille Calmette-Guerin (BCG) Bovine rotavirus (BRV) Bovine (BSA) immunofluorescence (CCIF) Chemokine (C-C motif) ligand 25 CCL25 Chemokine (C-C motif) receptor (CCR) Chemokine (CXC motif) receptor 5 (CXCR5) Cholera toxin (CT) Cluster differentiation (CD) (human) colonic carcinomas cell line (Caco-2) Cytotoxic T cells (Tc) Dendritic cells (DC) 2,4 dinitrophenyl (DNP) (ER) -linked immunosorbent assay (ELISA) Enzyme-linked immunospot assay (ELISPOT) Fluorescence forming unit (FFU) Gnotobiotic (Gn) pig Granulocyte- colony-stimulating factor (GM-CSF) Gut associated lymphoid tissue (GALT) Hen egg white lyzozyme (HEL ) B surface antigen (HBsAg) High endothelial venules (HEV) Human virus (HIV) Human leukocyte antigen (HLA) Human rotavirus (HRV) Human synsytial virus (HSV) Idiotope (Id) Immunoglobulin (Ig) Immunostimulatory complexes (ISCOMTM) (IFN) Interferon regulatory factor (IRF) Interleukin (IL) Intraepithelial lymphocytes (IEL) Intramammary route (IMm) xxiii Intramuscular route (IM) Intranasal route (IN) Keyhole limpet hemocyanin (KLH) Lamina propia (LP) LCMV (lymphocytic choriomeningitis virus) Lymphocyte proliferation assays (LPA) Major histocompatibility complex (MHC) Maternal antibodies (MatAb) Mesenteric lymph node (MLN) Microfold (M) cell Monoclonal antibodies (Mab) Mononuclear cells (MNC) Mucosal adhesion cell adhesion molecule (MadCAM-1) Mucosal associate lymphoid tissues (MALT) Mutant Escherichia coli heat labile enterotoxin (mLT) Nasal associate lymphoid tissues (NALT) Natural Killer (NK) Neonatal (FcRn) Non-structural protein (NSP) associated molecular pattern (PAMP) Pattern recognition receptor (PRR) Peyer’s Patches (PP) Phosphate buffered saline (PBS) PLG (Poly DL-lactide-co-glycolide) Polymeric Ig receptor (pIgR) Porcine respiratory and reproductive virus (PRRV) Post challenge day (PCD) Post inoculation day (PID) Protection receptor of serum IgG (FcRp) Quillaja saponina 21 (QS21) Recombinase-activating (RAG) Regulated upon Activation, Normal T-cell Expressed and Secreted (RANTES) Rhesus monkey kidney cells (MA104) Rhesus rotavirus strain (RRV) RRV rhesus rotavirus RRV-TV rhesus rotavirus based tetravalent vaccine Secretory IgA (sIgA) Severe combined immune deficient mice (SCID) Spodoptera frugiperda (Sf-9 cells) T helper cells (Th) Toll-like receptor (TLR) Transfroming growth factor (TGF) Tulane University and Cincinnati Children’s hospital (TICH) Tumor necrosy factor (TNF) Tetravalent –rhesus rotavirus vaccine (TV-RRV)

xxiv Vascular cell adhesion molecule (VCAM-1) (VP) Virus neutralization (VN) Virus-like-particle (VLP)

xxv

CHAPTER 1: LITERATURE REVIEW

ROTAVIRUS VACCINES AND INFLUENCE OF MATERNAL ANTIBODIES

AND CYTOKINES ON NEONATAL IMMUNE RESPONSES

1.1 Rotavirus and rotavirus vaccine

1.1.1 Impact of rotavirus diseases- diarrhea morbidity and mortality in children

worldwide

Rotaviruses (RV) are the most common cause of gastroenteritis in children

worldwide. They are responsible for about 608,000 deaths annually (74). In a ten year

study, from 1990-2000, in 20 countries, the estimated incidence of diarrhea was 3.8

episodes per child per year for children less than 11 months old, and 2.1 episodes per

child per year for children between the age of 1-4 years (374). Based on these numbers,

it is estimated that 1.4 billion diarrhea episodes per year occur in children less than 5

years of age (520). More importantly, the incidence rate of RV disease is similar in

children from developing and developed countries, suggesting that the virus infection

does not discriminate between economic status. Nevertheless, children from developing

countries showed a higher rate of diarrhea related deaths due to poor medical care

systems and malnutrition. In Mexico, RV causes 2148 deaths a year, whereas in the US

1 the numbers were 102 (23). It is estimated that 1205 children die from rotavirus disease each day in developing countries with 82% of deaths occurring in the poorest countries.

Rotavirus infection occurs year-round in developing countries with tropical climates, whereas the infection peaks in the winter season and decreases in the summer in developed countries with temperate climates (157). This difference in seasonality of RV infection explains the younger median age of illness in children from developing compared to developed countries (6-8 months vs.14-18 months, respectively) (86). As a consequence, it has been suggested that RV vaccine be delivered to children in developing countries at an earlier age, even as early as when they receive the

Calmette-Guerin (BCG) vaccine (from birth to 3 months of age) (494).

Because of an increasing awareness of the disease, the estimated annual mortality rate from RV has decreased over the past 2 decades from 873,000 deaths (1985) to

608,000 (2004) (74). Yet a study in Mexico showed that although the overall mortality rates decreased, the decline in the winter season was not evident (709). The economic burden caused by RV is high in industrialized countries with the estimated hospitalization rate among children of 445 per 100,000 children (520). In the United States, it is estimated that RV cause 24,000-110,000 hospitalizations and 75-125 deaths in young children annually. The costs associated with RV disease in the US were estimated at

$100-400 million annually.

Thus the incidence of RV can not be eliminated just by improvement of living standards, i.e. improving hygiene and and water supplies: a vaccine is

2 necessary to prevent and reduce the disease mortality. The use of a RV vaccine could reduce hospitalizations by 40-60% and could decrease RV associated deaths by 10-20%

(1, 173)

1.1.2 Rotavirus structure and proteins

Rotavirus is a non-enveloped double stranded (ds) RNA virus, in the family (197). The RV is composed of 11 segments of dsRNA ranging from 660-

3300 bp. The genome encodes 12 proteins, six structural proteins (VP) 1, 2, 3, 4, 6 and 7 and six non-structural proteins (NSP) 1, 2, 3, 4, 5 and 6. The gene segment 11 encodes both NSP 5 and 6 (196). The inner shell of the virion is composed of 60 dimers of VP2, which surrounds the viral genome and 12 copies each of VP1 (RNA polymerase) and

VP3 (guanylyl-transferase and methylase). Addition of 780 molecules of VP6 on the VP2 produces double-layered particles. The outer layer of the virion is composed of 780 molecules of VP7 forming a smooth surface, organized as trimers plus sixty dimers of

VP4 forming spikes extending outward [Figure 1.1] (324). The VP4 plays an important role in virus replication including receptor binding and cell penetration (25). The VP7 interacts with the cell surface molecules after the attachment of the virus via the VP4

(256). Detailed steps of the virus replication will be discussed subsequently.

The non-structural proteins. The NSP1, encoded by gene segment 5, has been implicated as a factor in mice but not in pigs or rabbits (87, 91, 142). A recent study suggested that NSP1 subverts the innate immune response by inducing degradation of interferon regulatory factor (IRF)3 (46). The NSP2, encoded by gene segment 8, is an oligomeric NTPase, possessing helix destabilizing activity and may be involved in RNA

3 encapsidation and virulence (119, 349, 669, 704). The NSP3, encoded by gene segment

7, binds to eIF4G and inhibits cellular protein synthesis as as enhances of viral mRNA (536, 703). The exact functions of NSP5 and NSP6 are not yet known; due to their RNA binding capacity, it is postulated that they may function as a transporter of other viral proteins from the sites of synthesis into the or they may facilitate

RNA encapsidation or they may be involved in the movement of viral particles from the viroplasms into the ER membrane (523). The NSP4, encoded by gene segment 10, plays a role in viral morphogenesis, acting as the receptor for double layer particles, mediating the budding of these particles into the ER lumen (42). It is also an important enterotoxin, involved in the pathogenesis of RV. The detailed functions of the NSP4 will be discussed later.

1.1.3 Rotavirus classification

Rotavirus classification is based on VP4 and VP7 proteins corresponding to P and

G types, respectively. There are currently 15 G /genotypes identified. Of 22 different P genotypes reported, only 13 P serotypes have been identified (301, 303, 412,

433, 506). Common serotypes of RV infected humans include P[8]G1, P[4]G2, P[8]G3 and P[8]G4 and smaller percentages of G5, G8 and G9. Rotavirus G9 strain, initially detected in the US, is becoming more common in Asian countries such as Australia,

China (up to 18.1%) and Japan (71.4%) (114, 216, 413, 436, 594, 778).

Animal RV has been suggested to cross the species barrier and cause infection in humans. The RV G5 strain which is common in swine has also been found in Brazilian children with an incidence ranging from 5.6% to 57% (406, 435). Rotavirus G8 strains,

4 which are found in cows, are also present in children with increasing frequency, from few

cases in India (320) to as high frequency as 27.7% and 34.1% of RV strains in Nigeria

and Malawi, respectively (4, 166). Rotavirus G6 strains, commonly found in cows, sheep

and were isolated from children with acute diarrhea in Italy, US, Australia, Belgium

and Hungary in combination with P[9] or P[14] strains or in India with unknown P

specificity (594). Sporadic cases of RV-G12 strains were found in Thailand, Korea, India,

Philippines and the US (594). A single case with P[14]G3 lapine-like RV was found in a

6-year old Belgium child with severe diarrhea (171). Rotavirus strain P[11]G10

commonly found in cattle was reported to cause both and symptomatic

infections in human neonates and infants in India (317). These potential cross species

infections can be traced to the ability of RV to undergo genomic rearrangement and

between homologous and heterologous strains, due to the distinctive

segmented RV genome. Molecular characterization of a human RV (HRV) strain

RMC321 from an outbreak of RV diarrhea in India revealed porcine characteristics in

most of the including VP4, VP6 and NSP1-5 (95-99% identity) (699).

This study provided strong evidence that porcine RV can cross the species barrier and

cause severe gastroenteritis in humans.

Apart from the extensive genome rearrangements and genetic ,

evidence of point mutations and accumulations have been reported (594). Monotypes

within G1, G2, G3, G4 and G5 RV strains have been proposed due to the

existence of distinct VP7 gene phylogenetic lineages. The antigenic differences found

between the vaccine G1 strain in the reassortant rhesus vaccine and the circulating G1

strain might be the reason for vaccine failure (331). Antisera to one lineage of the G9

5 strains failed to neutralize another lineage of the G9 strains efficiently (302). Likewise

genetic diversity between VP4 genes of the same P type was identified (594). Thus the

pool of RV infecting humans has the potential to increase, with different combinations of

P and G serotypes and different monotypes within serotypes, complicating the

development of a globally effective vaccine for RV.

However, heterotypic protection has been induced by a single RV serotype against other serotypes, thus it might not be necessary to include all the RV serotypes in vaccine. A monovalent G3 Rhesus RV (RRV) vaccine induced a 58.5% heterotypic protection rate against the circulating G1 serotype, compared to the 72.8% homotypic protection rate induced by the G1 vaccine (427). Interestingly, a study in Peru showed that the G3 RRV monovalent vaccine had a 29% heterotypic protection rate against the circulating G1 or G2 RV strains, whereas neither the G1 nor G2 RRV reassortant vaccines induced homotypic protection against G1 or G2 RV (393). The P6[1]G6 vaccine

(RIT 4237) also showed high heterotypic protection against RV G1, G2 and G3 strains in several preliminary studies (531, 707). A RV vaccine of the G1 strain (RIX4414 vaccine) induced protection against severe diarrhea caused by both G1 and non-G1 RV strains

(172). Thus it is expected that inclusion of at least the most common RV strains may increase the vaccine efficacy by induction of both homotypic and heterotypic protection.

1.1.4 Rotavirus disease

Rotaviruses are transmitted via the oral-fecal route. The symptoms of RV disease include diarrhea, , , , anorexia, cramping and malaise. The symptoms can be either mild or severe; in the latter case resulting in severe and death.

6 Rotavirus infections occur in children as early as 3 months and up to 5 years of age;

however 90% of children in both developing and developed countries encounter RV

infection before the age of 3. Severe symptoms often occur in young children from 6-24

months of age. Rotavirus infections also occur in neonates, but are usually asymptomatic

(263). For this reason, the neonatal strains have been evaluated as candidate RV vaccines

in some settings. Rotavirus infection also occurs in adults; the symptoms however are

usually mild (128, 201, 268)

Rotavirus was first observed in the intestine of mice (EDIM) and monkeys

(SA11) by electron microscopy in 1963 (5, 430). After this discovery, RVs were

associated with diarrhea in calves (461), humans (62) and pigs (71, 583). Human and

animal rotaviruses belong to 7 groups (A-G) based on the common group antigen (VP6).

Rotavirus groups A-C are associated with human infections of which Group A

rotaviruses account for the majority of cases. However, group B-RV which causes adult

diarrhea and group C-RV caused large outbreaks in China and Japan (296, 308, 330, 438,

577, 585).

Porcine RVs are recognized as an important pathogen associated with diarrhea in

both nursing and weaned pigs (71, 323, 746). Rotaviruses in swine are also classified into

several antigenically distinct groups: A, B, C and E (585). Group A RV causes diarrhea

in pigs and belongs to G types 1-6 and 8-11 and P types [5]-[8], [13], [19] and [23] (432).

Two G serotypes of Group C porcine RV have been identified (684). Porcine RVs cause

both clinical infections, manifested by diarrhea, anorexia, and depressed growth rates, and subclinical infections (50, 69, 224).

7 1.1.5 Rotavirus replication

Rotavirus replication consists of the following steps: attachment, penetration,

uncoating, replication of the genome and translation of viral proteins, assembly and

release.

Attachment. Many attempts were made to identify the cellular receptors for RV.

Some animal rotaviruses require (SA) on the cell surface for efficient binding and infection whereas the human viruses do not (140). Thus the SA binding is not an essential step for effective infection. Neuraminidase (NA) resistant strains, which do not require SA to infect cells, have been isolated (419). Ciarlet et al.(143) further classified the need for SA according to P genotypes of the RV strains: RV of P genotypes [1], [2],

[3] and [7] require SA residues for infection whereas RV strains with other P genotypes are SA independent. Gangliosides GM1 and GM3 were also suggested as receptors for

RV (262, 564). The α2β1 binds to the VP4 via the DGE tripeptide motif, whereas

α4β1 and αxβ2 bind to the VP7 via distinct motifs. The heat shock protein

(HSP) 70 and the β3 integrin have also been identified as post-attachment receptors (260,

261). The removal of these proteins by the non-ionic detergent octyl-β-glucoside under non-lytic conditions renders the cell resistant to the virus binding. Antibodies against

αvβ3 reduce the infectivity of RV RRV, but do not block attachment to the cells. The

HSP70 binds specifically to triple, but not to double layered particles and this interaction can be blocked by antibodies against the VP4 and VP7. Antibodies against α2β1 and

αvβ3 combined also suppressed the virus infection, suggesting that the two integrins may

be involved in different steps of virus entry. All of the above mentioned receptors must

be organized in a lipid microdomain of the , called lipid rafts to maintain 8 the ability to bind to RV. The search for RV cellular receptors is ongoing. However, the

available information suggests that there are at least 3 receptors involved in the

attachment and post-attachment stages of .

Penetration. Upon infection, RV VP4 acts as a receptor, binding and allowing the virus to penetrate the cell (26). The cleavage of VP4 by into VP8 and VP5 is

associated with the virus entry. The mechanism of activation by trypsin is not yet known;

however, it is postulated that VP4 cleavage leads to either a conformational change or a

new terminal region in VP4 molecules which favors penetration. Evidence supporting

both and direct cell penetration as the routes of viral entry are available but

still do not give a definite conclusion of the mode of entry of RV.

Uncoating. Once inside the cell, the viral transcriptase is activated to transcribe

the viral genome. This process is triggered by the uncoating and removal of VP4 and VP7

proteins from the triple-layered virus particles. The removal of VP4 and VP7 proteins can

be achieved by removing Ca2+ ions from the medium. The low intracellular Ca2+ concentration is not essential for initiating because increasing the levels in the cell did not affect viral protein synthesis (165).

Replication of the viral genome and translation of the viral proteins. Once the uncoating process is completed, the RNA polymerase activity in the VP1-VP3 enzyme complex (which is latent in the triple-layered particles) is activated in the double-layered virus particles due to the removal of the outer capsid, resulting in genome and extrusion of the eleven viral mRNAs from such particles. The mRNAs direct both

9 protein synthesis and minus-strand synthesis to yield dsRNAs. It was shown that the positive and negative stranded can be detected 3h post infection (PI) (648). The level of transcription peaks between 9-12h.

Viral assembly. The synthesis of dsRNAs, which are still associated with subviral particles, is an event that occurs following packaging of viral mRNAs into core- like assembly intermediates with the viral RNA-dependent RNA polymerases and the (524). Most of RV structural and non-structural proteins are produced on the free . The VP7 and NSP4 are synthesized on ribosomes associated with the

ER membranes and then cotranslationally inserted into the ER membranes. The subviral particles are assembled in the , and then bud through the ER membranes which is modified with VP7 and NSP4 to form transient enveloped particles. The transient envelope is lost as the particles move into the ER and VP4 and VP7 assemble to form the outer capsid while NSP4 is excluded from the particles (415). The membrane destabilizing activities of NSP4 and/or VP4 and the high Ca2+ concentration in the ER lumen are implicated in the removal of the transient lipid envelope (176, 679).

1.1.6 Rotavirus pathogenesis and mechanisms of diarrhea

The common recognized mechanism for RV-induced diarrhea in humans and other animals is the caused by loss of the absorptive villous epithelial cells in the intestine (346). Diarrhea is also accompanied by loss of digestive enzymes, loss of sodium, potassium, bicarbonate and water and osmotic loss of fluid due to undigested (37, 170, 211, 311, 468, 745) Other mechanisms of diarrhea induced by RV infection have been demonstrated only in the mouse model. In neonatal mice,

10 NSP4 functions as an enterotoxin that mediates a secretory diarrhea (44). Shaw et al

(615) and Ball et al showed (44) that RV associated diarrhea also occurs independently of viral replication in mice. Diarrhea can be induced upon inoculation of mice with a large number of virus particles genetically inactivated using psoralen or long-wave light which cross-links the viral RNA. It was hypothesized that morphological changes in murine intestinal cells and diarrhea are associated with attachment of virus to the cells.

Thus the mechanism of RV-induced diarrhea is similar to a toxin effect, which is associated with the function of the NSP4 (44), as discussed subsequently. Another mechanism is the intestinal inflammatory response which causes disruption of the intestinal mucosa and leaky membranes that allows toxins or bacteria to penetrate though the intestinal epithelial cell barrier (66).

In mice, RV infection also stimulates the (ENS), leading to intestinal secretion (422). Drugs that inhibit the ENS could attenuate the intestinal secretory response induced by RRV in mice. Therefore the involvement of ENS was proposed to be another mechanism of RV diarrhea.

In pigs, RV infection is initiated in the upper after oral inoculation, causing cytolytic infections of the villous resulting in shortening of the villi

(460, 715). The virus replicates in the nondividing mature near the tips of the villi leading to the extensive destruction of these cells (100, 674). The villi become covered with immature, non-differentiated cuboidal epithelium. Extensive destruction of enterocytes causes an impaired digestive-absorptive process, leading to an acute and malabsorptive diarrhea. Rotavirus also infects a few cecal and colonic epithelial cells without colonic lesions (675). The villous tips are denuded and mononuclear cells

11 migrate into the underlying lamina propria (LP). The infection starts at the proximal end of the small intestine and advances distally (88). After infection of gnotobiotic (Gn) pigs with HRV Wa strain, moderate to severe villous atrophy occurred in the small intestine by 48-96h PI (715). The lesion was most severe in the caudal region of the intestine, whereas lesion in the proximal part was less extensive and persisted for shorter time (24-

48h PI) (584). The low pathogenic strains tend to infect the proximal part of the small intestine, whereas the highly virulent strains infect the mid and distal small intestine (88).

In neonatal Gn and colostrum-deprived calves infected with HRV-D strain, diarrhea occurred within half an hour of inoculation with denuding of the villi in the upper small intestine, whereas the lower small intestine remained intact (462).

Morphological changes in the lower small intestine occurred later (7h PI), yet RV were not detectable by immunofluorescence.

In pigs, orally inoculated with 105-106 focus-forming units (FFU) of virulent (Vir)

Wa HRV, porcine OSU or SB1A, diarrhea was induced within 12-18h PI, in parallel with the presence of RV antigen within villous epithelial cells (125, 305, 306, 715). Villous atrophy was observed at 24-72h PI, coincident with the peak of virus replication and was prominent in the caudal small intestine. Destruction of the cells on the villous tips causes a malabsorptive and osmotic diarrhea in pigs. Hyperplasia occurs in lymphoid tissues such as Peyer’s patches (PP) and mesenteric lymph nodes (MLN) (715). The duration of diarrhea and viral shedding for VirHRV in pigs is between 4-7 days PI. Pigs inoculated with the attenuated (Att) Wa HRV strain showed no signs of diarrhea and no villous atrophy; instead, the villous epithelial cells remained attached to the basement membrane with only minor morphological alterations. The pathophysiological changes contributing

12 to the maladsorptive diarrhea include impaired glucose-coupled sodium transport (170), decreased activity (255, 781) and increased thymidine kinase activity

(170).

The degree of villous atrophy and the distribution of these damaged villi vary according to RV strains (152), serogroups (580) and the age of the pigs (577, 614).

Rotavirus groups A-C generally replicate and cause villous atrophy in the lower intestine, but not the colon. The groups A and C RV cause infection of cells forming scattered foci throughout the intestine, but limited to the tip or sides of the villi (72, 460, 676). Group

B RV produced scattered foci of infection in the distal small intestine and generated mild but highly acute diarrhea (585). Younger piglets exhibit more severe villous atrophy than older pigs. Group B RV caused an acute, transient but non-fatal diarrhea in pigs less than

6 days of age whereas Groups A and C RV infections led to dehydration and death in most Gn pigs less than 6 day old (577).

Infections with non-Group A RV have been reported in chickens (Group D), Gn

piglets (Cowden porcine Group C and several Group B strains (577)) and calves (bovine

Ohio Group B) resulting in villous atrophy and diarrhea similar to that induced by Group

A RV [review in Saif et al (577)]. Infections of Gn pigs and Calves with group B or C

RV lead to a more rapid onset of diarrhea, with fewer shedding virus for shorter period of time, compared to Group A RV (577).

In children, similarly to pigs, RV replicates in the nondividing mature enterocytes near the tips of the villi (169). Histopathologic changes vary from broadening of the villi

to complete villous atrophy and from mild to heavy mononuclear cell filtration (373,

13 584).This variation in histopathologic changes may represent the spectrum of illness or may indicate the limited information obtained from the small size of biopsy duodenal or jejunal specimens. Diarrhea is due to sugar maladsorption including lactose and D-xylose

(211, 442) and loss (345). Human intestinal biopsies showed mononuclear cell infiltration, mitochodria swelling and denudation of the microvilli (169, 294).

Diarrhea induced by RV in children has an of 24-48h, and lasts up to 7 days. Symptomatic infections with RV are restricted to the young infants, between 6-24 months of age (258, 346). Asymptomatic infections also occur in premature and newborn babies, which may be due to the attenuated nature of the virus or the presence of maternal antibodies (14, 258).

In mice, RV associated diarrhea occurs only during the first 2 weeks of life (514).

In neonatal mice, murine RV causes diarrhea with villous ischemia (514) but with little or no villous atrophy and inflammation (5). In adult mice, RV infection occurs without causing disease or lesions. Acute inflammatory cell recruitment and mucosal ulceration observed in calves and piglets are not seen in the mucosa of mice upon heterologous RV infection (503, 649). Homologous RV infection in mice (EDIM) is associated with shortening of the villi, disordered microvilli and epithelial vacuolation, whereas heterologous RV infection results in little change in villous structure (346, 503). In addition, malabsorption is not the major diarrhea mechanism on the murine RV disease model (151). Two phases of villous morphological change were observed after infection of neonatal mice with murine RV (514). In the first phase (18-48 h PI), ischemia and transient shortening of the villi occurred throughout the small intestine. The villi recovered to their normal height at 72h PI due to a slow rate of cell division, yet initial

14 signs of hyperemic microcirculation were observed in the villi. The decreased rate of cell

division failed to replace the lost cells leading to hypersecretion. The second phase

occurred around 120-144 h PI with few villous shortening and the changes were

restricted to the upper and middle regions of the intestine with hyperemic

microcirculation in the villi. The increased number of red blood cells in the villi led to

perturbance in the countercurrent system and decreases in the osmolarity in the villous

tips thus leading to impaired water absorption and more prolonged diarrhea. Recovery from diarrhea occurred at 168h PI, coincident with the recovery of the villous

microcirculation. Thus the villous atrophy upon RV infection in neonatal mice is not as

pronounced as observed of humans, pigs or calves.

Rotaviruses infect mainly enterocytes of the tip of the small intestinal villi

although not exclusively. Blutt et al. (67) recently demonstrated antigenemia and viremia

associated with of different RV strains infecting adult humans, mice and rabbits and

neonatal humans, rats and calves. Furthermore, these viruses from serum were shown to

be infectious in neonatal and adult mice. A study of neonatal Gn pigs (3-5 days of age)

demonstrated viral antigen in serum between post infection day (PID) 1 to PID 7 after

inoculation with VirHRV (34). This study also showed that serum from pigs infected

with VirHRV Wa strain was infectious for pigs, providing further proof for viremia

associated with HRV infection of pigs.

Rotavirus antigens and RNA have been observed in the central nervous system,

liver and kidney of immunodeficient children (244, 409, 496), and in the blood, spleen,

liver, kidneys, lungs and mesenteric lymph nodes (MLN) of RV inoculated mice (93,

15 177). Viral RNA has also been detected in the blood of 64% of RV infected

immunocompetent children, suggesting that viremia is a common phenomenon (126).

The RV also spreads to mucosal tissues other than the intestine. Oral or gavage

inoculation of pigs with Wa VirHRV also caused nasal virus shedding from PID 1 (oral)

or PID2 (gavage) to PID 7, longer than the duration of rectal virus shedding (34).

Similarly, RV antigen has also been found in nasopharyngeal of children with

respiratory symptoms (218).

Virus induced hepatitis has been described in a neonatal mouse model (1-3 day-

old) inoculated with RRV (687). Liver spread of RRV and hepatitis were demonstrated in both BALB/c mice and the severe combined immunodeficiency (SCID) mice. Not all strains of RV can spread to the liver. The RRV, but not the SA11-Cl4 strain can infect the liver following oral inoculation of CD-1 neonatal mice. The gene segment 7 encoding

NSP3 has been correlated with the spread of RV to the liver (477). Rotavirus infection occurs via the oral-fecal route, yet bypassing of the intestinal barrier by an intraperitoneal

(IP) injection allows the infection and replication of RV in the liver regardless of whether the strain is liver prone (RRV) or not (SA11-Cl4) (477).

One of the mechanisms used by RV to escape the intestine and enter the circulation is the transepithelial transport of the virus through M cells of the Peyer’s patches (PP), which act as an intermediate among the immune, lymphatic and circulatory systems (100). Rotavirus has been demonstrated to enter the lymphatic system (476).

Infection of neonatal mice with RRV or the spread-competent clone SA11-CI4 led to the

progression of virus particles from the intestine to the MLN and then to the peripheral

tissues. Gene segment 7 (encodes NSP3) was identified as the primary determinant of the

16 spread of virus to the MLN and this spread via a lymphatic pathway could be modified by

gene segment 6 (encodes VP6) (476). Rotavirus can also be taken up by

which travel in the blood circulation and cause infections in other tissues (93). Evidence for the spread of RV via the circulation to cause infection in other tissues can be found in

a study by Azevedo et al (34). Pigs inoculated intravenously with serum or intestinal

contents from the viremic VirHRV-inoculated pigs developed diarrhea, rectal and nasal

virus shedding, and viremia, similar to orally inoculated pigs.

1.1.7 Cross-species infections

Although many RV strains are species specific, cross species infections are not

uncommon as mentioned earlier for animal viruses infecting humans. In addition,

infection of adult mice with the non-human primate strains RRV and SA11, has been

widely studied for pathogenesis and immunological responses in the mouse model.

Infection of mice with these heterologous viruses does not induce the typical RV virus

related symptom, i.e. diarrhea. On the other hand, experimental infection with the PP-1

strain of bovine RV, Wa or M strain of HRV caused diarrhea in pigs (266, 589).

The mechanism of cross species infection is still under investigation. It has been

hypothesized that the VP4 phenotype is the determinant of cross species specificity and

pathogenicity (186). This hypothesis was supported by the finding that the PP-1 strain,

which was isolated from calves in which it was asymptomatic, caused diarrhea in pigs.

This strain has 96-97% homology in VP4 amino acid sequence with the porcine P[7]

strain but only 62-79% homology in VP4 amino acid sequence with other bovine strains

(186). Similarly, Gentsch et al (235) showed that the 116E strain HRV which causes

17 asymptomatic infections in human infants, has high homology in VP4 nucleotide and amino acid sequences with the bovine B223 strain. In addition, RV may exist as a

population of reassortants from which a new RV species appears under special

conditions, i.e. a new species emerges during infection of pigs with the bovine PP-1 strain (254). From an original fecal sample containing P[5] and P[7] gene profiles, the

bovine pathogenic P[5] and porcine pathogenic P[7] strains were isolated after serial passage in Gn calves and pigs, respectively. Naturally occurring “assumingly” reassortants have been reported between HRV with VP4 from porcine or bovine strains; all of which induce asymptomatic infections in human infants (168, 251). The NSP4, although it plays a crucial role in RV diarrhea induction in mice, hasn’t been clearly associated with RV species specific virulence due to contradictory reports (186).

The VP7 gene might also contribute to inter-species transmission of RV.

Homologies in amino acid sequences (90%) were identified between the human G6 strain

(PA151 and PA169) and the bovine G6 strain (UK and NCDV) and monoclonal antibodies against the bovine G6 strain cross–reacted with the human G6 strains (239,

240, 312). Human RV G10 isolated in Thailand may have originated from a bovine strain (688). A bovine RV G1 strain (T449) isolated in Argentina had 90% homology in the VP7 amino acid sequence with a human G1 serotype (Wa strain) (65). An outbreak of gastroenteritis in infants in Manipur, India identified a new HRV strain, RMC321 in which the genes encoding VP4, VP6, VP7, NSP 1-5 showed identity to porcine RV but not to HRV (699). As previously mentioned, reassortment is a common event due to the

18 segmented genome of rotavirus, which allows gene segment(s) of one origin to reassort

with rotavirus segments of other origins including between animal and human strains.

1.1.8 The viral enterotoxin NSP4

The NSP4, encoded by gene segment 10, serves as an intracellular receptor during subviral particle morphogenesis (30). It was also identified as a viral enterotoxin and was recognized as the diarrhea inducing mechanism before morphological damages of intestinal cells occur in neonatal mice (44). The NSP4 induces mobilization of intracellular Ca2+ in the intestinal epithelial cells, leading to an efflux of Cl- across the

plasma membrane, thus initiating secretory diarrhea independently of cAMP. Other ion

channels are also activated by the increase in Ca2+ concentrations which increase fluid secretion and reduce absorption (474).

1.1.8.1 Structure and functions of NSP4

Rotavirus NSP4 was the first viral enterotoxin identified (42, 44). There are only two other which were identified following the discovery of RV NSP4: the surface unit of immunodeficiency virus (SIV) and those of equine infectious anemia virus (43, 657). They are called enterotoxins because they stimulate intestinal secretion without altering the tissue morphology (42). Other enterotoxins are found in bacteria such as (cholera toxin), E.coli (heat-labile and stable toxins) and other cholera-like enterotoxins in A.hydrophila, ,

Salmonella spp, Bacillus sp, Clostridioum difficile and Yersinia enterocolitica.

The NSP4, a 28kD acts as an intracellular receptor for VP6. Because

NSP4 resides in the ER, the binding of NSP4 with VP6 is crucial to the budding of

19 double-layered particles through the ER membrane, which facilitates the addition of VP7 and VP4 and a transient ER membrane to form the mature particles. The NSP4 contains a transmembrane domain (residues 24-44) with the C terminus spanning the ER bilayer, the

N terminus remaining in the lumen of the ER and residues 45-175 extending in the cytoplasmic domain. The NSP4 doesn’t contain the classical ER retention signal; thus it was unclear how NSP4 retains at this site. The double-layered RV receptor activity of

NSP4 is localized at the C terminus of the protein in which the methionine residue is essential for ligand binding (671). The NSP4 also binds to VP4 and VP7 and facilitates the addition of the outer coat proteins to the double layer to form a triple layer. The binding site of NSP4 to VP4 is localized to residues 112-148 (672).

A functional study of NSP4 was achieved using RNA interference (RNAi), which is the small interference RNA (siRNA) triggered event in which any RNA with 100% sequence match with the siRNA is degraded (245). Silencing of NSP4 by RNAi led to

75% reduction in RV replication, whereas the silencing was removed when there were 4 nucleotide mismatches in the sequence of siRNA compared to NSP4 (415). The NSP4 silencing led to a reduction in the number of double-layered and triple-layered particles and redistribution of other RV proteins. In the presence of NSP4 silencing, the VP6 forms filaments at the periphery of the cell whereas VP2 scatters throughout the cytoplasm instead of in the . The VP4 is found in the cytoplasm instead of the nucleus and the VP7 diffuses in the perinuclear area instead of into the ER (250, 490). Thus NSP4 is the crucial protein for the later stages of RV morphogenesis.

20 1.1.8.2 Genotypes of NSP4

The NSP4 genes of mammalian RV are classified into 4 groups (genotypes A, B,

C and D) based on the nucleotide and amino acid sequences. Genotypes A, B and C are detected in human viruses and show 87-99% identity between genotypes (142, 297, 298,

368). Murine RV-NSP4 is classified into genotype D due to only 60-65% sequence identity to other human genotypes. Avian RV NSP4 is classified into 2 genotypes (E and

F) that are distinct from the mammalian RV NSP4s because of low identities (31-37%) between them (473). Turkey and pigeon RV NSP4 belong to the same genotypes, whereas the chicken RV NSP4 is of a separate genotype.

1.1.8.3 Mechanisms of diarrhea caused by NSP4

Diarrhea occurs early in RV infection before the initiation of any inflammatory response with little infiltration of mononuclear cells and limited vacuolization of some enterocytes at the tips of the villi (44). Thus the mucosal destruction is not the cause of diarrhea at this early stage. In mice, diarrhea occurs 4-8h post IP inoculation of the purified NSP4 and persists for 24h. Diarrhea induced by NSP4 is age dependent in that 6-

7 days old mice developed diarrhea after IP injection of the protein whereas mice of up to 9 days old developed diarrhea when infected intraileally with NSP4. The region responsible for Ca2+ mobilization encompasses the 114-135 peptide segment, which is localized in the cytoplasmic domain of the molecule, folded as an amphipathic helix. The crystal structure of the NSP4 95-137 peptide segment showed a homotetrameric pore which could span the ER membrane and act as a Ca2+ channel.

21 Multiple pathways may be involved in diarrhea induction by NSP4 (42). The

NSP4 114-135 induces chloride secretory currents across the mouse intestinal mucosa

that potentiate the cAMP induced effects (44). The NSP4 also induces an iodide influx

into mouse crypt cells. This influx only occurs in neonatal mice and requires Ca2+ (475).

The NSP4 of RV group A-C mobilizes Ca2+ in all regions of crypt cells. Thus NSP4- induced diarrhea may occur via activation of the anionic halide permeability pathway, which is dependent on the age of the mice and Ca2+ (475). The NSP4 is also responsible

for inhibition of sodium-dependent glucose transporter (SGLT1) and sodium leucine

symporter activity in the intestinal border of young rabbits which leads to fluid

accumulation in the intestinal lumen, because the SGLT functions in water reabsorption

(265). The NSP4 is hypothesized to function as an enteric nerve secretagogue, i.e. NSP4

may activate the secretory reflexes of the ENS to induce fluid loss and this process is also

Ca2+ dependent (422).

Because of the association of NSP4 with diarrhea induction, it was hypothesized

that NSP4 may play a role in virulence. This hypothesis was supported by sequence

comparisons and site-directed mutational study of NSP4 sequences of the virulent and

attenuated strains of both OSU and Gottfried RVs (774). However the comparison of the

NSP4 nucleotide sequence between the virulent unpassaged HRV 89-12 strain and the

attenuated strains resulting after 33 passages of this virus failed to document any

difference (720). It is possible that a mutation in the NSP4 sequence is not the only

mechanism for loss of virulence (774).

22 1.1.8.4 Diarrhea induction by NSP4

Residues 114-135 in the NSP4 protein are responsible for the enterotoxigenic activity of this protein. Synthetic peptides encompassing these residues induced diarrhea in mice in a dose-dependent and age-dependent manner (44). Administration of purified

NSP4 IP (≥1µmol) or intraileally (IL) (0.5nmol) caused diarrhea in mice within 1-4 hours PI; the diarrhea could last up to 8h PI. The rate of diarrhea was the highest in 6-7 days old mice (IP route) or 8-9 day-old (IL route), but lower in older animals and no diarrhea occurred in 17-18-day-old mice.

The route of administration is important in the diarrhea induction by NSP4.

Intramuscular inoculation of NSP4 of the same dose as for the IP route did not induce diarrhea in CD1 mice. The administration of the synthetic NSP4 corresponding to residues 114-135 led to a lower rate of diarrhea (60 and 71% by IP and IL, respectively) in 6-7-day-old mice, whereas IP or IL inoculation of older mice (11 days or more) led to low or no diarrhea even when the dose was increased 2-4 fold. The sensitivity to NSP4 induced diarrhea increased when the protein was delivered directly into the intestinal lumen. The effective dose of synthetic NSP4 was higher than the purified full length

NSP4.

Diarrhea in neonatal mice can be induced by NSP4 from different virus strains: the full-length SA11-NSP4 and the peptide, the RV EW-NSP4 in CD1 mice, or by the group C-NSP4 (44, 298, 597). The ability of NSP4 to induce diarrhea was also demonstrated in vitro. The addition of rNSP4 to the intestinal cell culture induced enterotoxigenic signaling activity, i.e. an increase in Ca2+ concentrations (775). It is 23 speculated that the concentrations of the receptor for NSP4 in the intestine were higher in infant mice than in adult mice, resulting in reduction of the disease caused by NSP4 in older animals.

Avian RV NSP4 also induced diarrhea in suckling mice despite the low homology

(only 31-37% identity) between avian and mammalian RV NSP4. The enterotoxin domain in avian RV NSP4 lies in a similar region (residues 109-135) as in SA11 NSP4

(472).

1.1.8.5 Immune responses to NSP4

The NSP4, although a non-structural protein, is able to stimulate humoral and cellular immune responses in humans (both children and adults) and other animals, e.g. rabbits, pigs, etc (154, 313, 315, 332, 766, 768). In human infants, the serum IgG antibody responses to recombinant NSP4 (rNSP4) and native NSP4 114-134 segments were observed in all children naturally infected or in 90% of vaccinated infants (332). In that study, following vaccination with reassortant tetravalent vaccine, none of the children had detectable serum IgA antibody responses to rNSP4 or NSP4 114-134. In naturally infected infants, NSP4 specific IgG and IgA antibodies could be measured in serum samples and no differences in these responses between different RV strains of different NSP4 genotypes were observed (544). However, Yuan et al (768) showed that broad heterotypic responses to NSP4 occurred after natural RV infections, with 50%-70% seroconversion to IgA and IgG serotype specific antibodies to NSP4 from RV groups A,

24 B or C. Both homotypic and heterotypic NSP4 antibody responses also were detected in patients given various live oral RV vaccines, including the reassortant tetravalent vaccine.

In humans, the cellular immune responses to NSP4 (indicated by the production of IL-2 and IFN-γ or by the increase of T cell proliferation) can be detected after injection with NSP4, which suggests that Th1-like responses and cytotoxic T cell responses occur

(332). However the secretion of IL-2 and IFN-γ were demonstrated in RV infected adults only, whereas T cell proliferation responses were observed in less than 50% of RV infected infants. Thus NSP4 is a poor inducer of T cell responses in infants.

Similarly, in animal models, such as Gn pigs, infection with a Vir or Att HRV led to 100% seroconversion to the homotypic NSP4 by 28 days post-inoculation (PID) (313).

However, VirHRV induced 5-6-fold higher NSP4-specific antibody titers than the

AttHRV. Virulent HRV also induced NSP4-IgA antibody secreting cells (ASC) in the intestine of the pigs and vaccination and challenge with the VirHRV induced a high number of NSP4-IgG ASC in the MLN. The presence of NSP4-ASC may be related to the extracellular release of NSP4 from infected cells (120). In mice, however, no measurable amounts of antibody against NSP4 were found after homologous and heterologous infections (315), which was due to the low homology between genotypes D and C of NSP4s used in the study.

1.1.8.6 Homotypic and heterotypic NSP4 specific antibodies and protection.

Evidence for the role of NSP4 in diarrhea induction in mice and immunogenicity of NSP4 in mice, humans, calves and pigs suggest the possible role of NSP4 in protection

25 against RV diarrhea by the integration of NSP4 into a RV vaccine containing VP4, VP6

and VP7. Potatoes expressing the of NSP4 with cholera toxin-subunit B

(CTB) were fed to mouse pups, which showed a significant decrease in diarrhea when challenged with RV (24, 761). Another NSP4 fusion protein was expressed in potatoes using the HIV-Tat transduction domain, which allowed direct entry of the protein into the cytosol of mammalian cells (205). The CTB-NSP4 86-175 induced higher serum IgG antibody titers than CTB-NSP4 114-134, Tat-NSP4 86-175 or NSP4 86-175 in orally immunized mice. Mice immunized with a combination of CTB-NSP4 86-175 and Tat-

NSP4 86-175 showed lower IgG antibody titers but higher IgG2 antibody titers than

CTB-NSP4 86-175 alone, indicating an increase of Th1 responses in the presence of Tat

fusion protein (364). The Tat fusion protein was shown to induce Th1 responses leading

to cytotoxic T lymphocyte (CTL) responses, but not mucosal IgA antibodies, whereas a

CTB fusion protein generated high secretory IgA (sIgA) antibodies. Unlike the fecal and

serum IgA antibody responses to the whole virus which correlates with protection against

RV infection and disease, the serum NSP4-IgA may not play any role in protection against RV disease and it is not yet clear whether mucosal NSP4-IgA serves any role.

Antibodies against NSP4 might just represent the number of exposures to RV. It was

observed that Nicaraguan adults showed higher, but not statistically significant, NSP4-

IgG antibody titers than those of Swedish adults (550).

The NSP4 antibody responses and protection against RV disease conferred by

NSP4 antibodies are species specific but not genotype specific. Following primary

infection and challenge with Vir RV in Gn calves and pigs, higher antibody titers to

homologous host homotypic NSP4s than to heterologous host homotypic or heterologous

26 host heterotypic NSP4s were induced (766). In piglets, antibodies to NSP4 induced by previous oral infection failed to confer protection against challenge from a porcine RV bearing serotypically different VP4 and VP7 but essentially identical NSP4 to the porcine

RV in primary infection.

1.1.8.7 Passive immunity conferred by NSP4 antibodies

All of the passive immunity studies using NSP4 specific antibodies were

conducted in the mouse model. The NSP4 antibodies can block induction of disease in

mice. The administration of antiserum to NSP4 peptide 114-135, 5 min before IP delivery

of 50-100 nmol of NSP4 peptide caused a 90% reduction in diarrhea in mice (44). In the

absence of the NSP4 antibody, 67% of mice showed diarrhea upon IP injection with

NSP4. In addition, pups born to dams immunized with NSP4 peptide showed significant

reduction in severity and duration of diarrhea when challenged with SA11 virulent RV.

Pups infected with SA11 RV and fed with NSP4 serum every 4-6h for 60h also showed a significantly reduced diarrhea compared to infected pups fed with control serum. Thus in the mouse model, the presence of NSP4 antibodies in the circulation and in the intestinal tract provides protection against NSP4 and RV induced diarrhea, respectively.

1.1.9 Immunity to RV

1.1.9.1 Role of innate immunity

The role of innate immunity has been explored for RV infection only recently, although RV, being a dsRNA virus, can induce the production of interferon type I from macrophages and fibroblasts (440). The involvement of dendritic cell (DC) in the

27 recognition of RV antigen via the toll-like receptor (TLR) might play a very important

role in antigen presentation to cells of the adaptive immune response (440). Unknown innate immune factors have been suggested as the mechanisms for RV clearance in SCID

mice (C57BL/6 background) (219). The involvement of natural killer (NK) cells in

immunity against RV has also been investigated (471, 486).

Rotavirus antigens are found in monocytes/macrophages during infection and the

presence of RV in these cells was suggested to cause spread of RV to tissues other than

the intestine (93). Microcapsules containing RV were observed to bind specifically to

antigen presenting cells (APCs), i.e. B cells, macrophages and DC in vitro which might

explain the enhanced immune response when microencapsulated RV antigens are used

for immunization (96). The role of DC is further supported by a study in which mice

lacking chemokine receptor (CCR) 6 showed a deficiency in DCs expressing the DC and

monocyte/macrophage markers CD11b and CD11c from the subepithelial dome of the PP

and impaired humoral responses to orally inoculated RV antigens (156). The CCR6

mediates the migration of DCs and lymphocytes during immune responses. In addition,

human immature DCs produced IL-12p70, IFN-γ and IFN-β after stimulation with

dsRNA from RV (440). Of note, only professional APCs produce the p40 component of

the biologically active IL-12p70, whereas other cells only synthesize the p35 subunits of

IL-12 (2). Further understanding of the role of DCs in RV infection requires knowledge

of the interaction between the RV and the TLRs.

However RV also exhibits mechanism(s) to escape the recognition by innate

immune surveillance. In knock–out mice lacking signal transducers and activators of

transcription-1 [Stat1(-/-)] leading to lack of IFN type I and II responses, oral infection of

28 suckling Stat1(-/-) and immunocompetent mice with RV induced diarrhea and virus

shedding of similar intensity (697). A later study identified the RV-NSP1 (encoded by gene segment 5) which causes rapid degradation of the interferon regulatory factor

(IRF)3 during the replication cycle, thus antagonizing the IFN-signaling pathway of the innate immune response (46). Of note, IRF3, a constitutively expressed protein in the cytoplasm, is an important effector of the innate immune responses.

1.1.9.1.1 Natural killer cells

Apart from the cytotoxic effect conferred by CD8 T cells, the cytotoxic activity of

NK cells also contributes to innate immunity and clearance of RV. Prolonged shedding of

RV has been observed in elderly patients and impaired NK cell cytotoxic activity has

been suggested to be the possible cause (471). Cytotoxicity by NK cells was identified in

intestinal intraepithelial cells of chickens infected with RV. This activity was not MHC

restricted (486) and it was not restricted to cells from RV infected chickens.

Upregulation of NK cytotoxicity via IL-15 was observed in an in vitro reovirus infection

(204). Further studies are needed to expand an understanding of the role of NK cells in

RV immunity.

1.1.9.2 Adaptive immunity to RV: Studies of different animal models and humans

In response to RV infection, both arms of the adaptive immune system are involved, i.e. B and T cells (CD4 and CD8 T cells). However, to what extent each

29 component contributes to protective immunity against RV varies considerably in studies

of humans and different animal models. The roles of all components are summarized in

regard to humans and each model in the following sections.

1.1.9.2.1 Mouse model

In this model, both adult and neonatal mice have been used which yield different

interpretations of the determinants of protection against RV.

In the adult mouse model, protection against live virus doesn’t correlate with the

serum or intestinal neutralizing antibodies, but does correlate with the serum and stool

RV IgA antibodies and IgA ASC responses (207, 511). Long-term protection also

depends on antibody production as B cell deficient mice shed virus for a prolonged time

(220). Rotavirus IgG antibodies also play an important role in protection in the adult mice

model, especially in gene knockout mice that do not produce IgA antibodies (511). The

defense mechanism of intestinal IgA antibodies in this model operates via surface

exclusion and intracellular neutralization of the viruses.

In the adult mouse model, the VP6 which does not induce virus neutralizing

(VN) antibodies induces a protective response (208). Non-neutralizing IgA monoclonal

antibodies against the VP6 protein can confer both homologous and heterologous

protection (572). It is postulated that the transcytosis of the dimeric IgA antibody with the pIgR blocks crucial steps in the viral replication cycle (102, 572, 607). Protection induced by VP6 proteins in the adult mouse model also depends on the route of vaccination and 30 adjuvants (134, 136). Intranasal (IN) immunization of adult mice with EDIM VP6 with

E.coli mutant heat labile toxin mLT (LT-R192G) adjuvant induced more than 99% protection against viral shedding after homologous virus challenge. Less protection was induced by IN administration of VP6 with CTA1-DD, Adjumer and CpG- oligodeoxynucleotide (CpG ODN) adjuvants (95, 80 and 74%, respectively). Of note,

CTA1-DD is a gene fusion protein which combine the active subunit A1 of cholera toxin

(CT) with a B cell targeting peptide, D, derived from protein A of S.aureus (424). The use of VP6 with QS21 adjuvant (purified form of Quil A) via the IN route induced only

43% protection compared to a 16% protection rate when the VP6 was administered alone.

The oral route was less effective in inducing protection with all of the above adjuvants.

In the adult mouse model, B cells, CD8 and CD4 T cells were all identified as effectors that play different roles in protection and resolution of the viral infection. The resolution of RV shedding and protection against subsequent infection was associated with RV-specific CD8 T cells. In B cell deficient mice, the depletion of CD8 cells prevented the resolution of infection (221). In immunocompetent mice, CD8 T cell depletion only delayed the resolution of shedding. The CD8 T cells therefore appear to play an effector role in short term protection, whereas long term protection is mediated by RV antibodies. The CD4 T cells are essential for the complete resolution of viral shedding as they provide help for B cell development and antibody production. A study by McNeal et al (459) indicates that CD4 T cells are the only lymphocytes required for protection, as B cell deficient mice, depleted of CD8 T cells, were completely protected against reinfection whereas the removal of CD4 T cells led to a loss of protection.

31 In the neonatal mouse model, unlike the adult mouse model, the role of VN

antibodies was emphasized in protection against RV. The neutralizing IgA antibodies

against VP4, but not against VP6, in a backpack implanted hybridoma protected against

RV induced diarrhea in neonatal mice (572). Infant mice receiving milk from dams

immunized IN with VP2/VP6 protein expressed by recombinant S.typhi (virus-like

particles (VLP) were not formed in this system) were not protected against diarrhea

(158). Similarly, pups born to mice immunized with recombinant viruses

expressing RV VP7 but not VP6 were protected against diarrhea (21). The protection against diarrhea in the newborn mice was mediated by VN antibodies present in milk, but not by serum antibodies because mice born to unimmunized dams but raised by dams immunized with VP8 were protected against diarrhea upon challenge with homologous virus (243). Thus in this model, local but not serum IgA antibodies against neutralizing is suggested to play a role in protection against diarrhea. However, in this model, the protection against viral shedding was not assessed. On the contrary, in the adult mouse model, the protection against virus shedding, but not diarrhea has been used throughout as adult mice do not develop diarrhea after RV challenge.

Despite the ease of studies using mice, this model is far from being a true representative of the human system. The human newborn immune system at birth is more developed than that of the mouse: only 7-day old mice are similar to human newborns

(11). In addition, neonatal mice can quickly loose the bias for Th2 response by 5-6 days after birth whereas human infants remain still under the influence of Th2 cytokines for a longer time (10).

32 1.1.9.2.2 Rabbit model

In the rabbit model, Ciarlet et al reported that VP4 and VP7 were the perquisites

for protection against infection after challenge with a lapine RV (G3 ALA) (139).

Parenteral injection of SA11 derived 2/6/7, 2/4/6 or 2/4/6/7 VLP into rabbits induced

fecal IgG and not IgA antibodies which were also associated with protection or partial

protection against ALA virus (155). Similar to mice, induction of diarrhea in rabbits by

RV is age dependent (141). Only rabbits less than 2 weeks old develop diarrhea and shed virus when infected with lapine RV. According to this study, the rabbits up to 11 months of age, although not having diarrhea, still exhibit histological changes in intestinal villi, typical of diarrhea induced symptoms. The rabbit model has only been used in very limited studies of rotavirus immunity.

1.1.9.2.3 Pig model

Compared to the mouse (both neonates and adults) and rabbit models, the neonatal pig model is a more accurate representation of what happens in human neonates.

Pigs are the only animals in which diarrhea can be induced by HRV strains, which make

them more relevant for the study of RV vaccines for use in human infants. The window

of susceptibility of pigs is longer (up to 8 weeks or more after birth), which allows

studies of the immune responses and the evaluation of vaccine efficacy with booster

doses (589, 770). In mice, on the other hand, diarrhea can be induced only within the

first two week, in comparison to the 14-24 week lifetime of mice.

Within the pig model, there are currently two approaches for the study of RV

induced protection and immune responses: conventional pigs (raised under normal

33 management conditions) and the Gn pigs (raised in germ-free conditions). The former has been used by many investigators, but pigs varied in age, the presence or absence of

MatAb (colostrum fed or deprived pigs) and the confounding effects of extraneous RV infectious. Some conventional pigs raised by dams with previous exposure to RV still shed RV (25% of pigs shed during nursing and 70% during the post-nursing period).

Naturally occurring RV-associated diarrhea is reported in 1- to 41-day-old suckling pigs

(29, 71, 174, 558, 656, 752) or within 7 days following weaning (71, 403, 686, 746).

Uncomplicated RV diarrhea in suckling pigs usually resolves in 2–3 days.

Morbidity is usually less than 20% and mortality due to dehydration is typically less than

15% in diarrheic pigs. Mortality is highest in young pigs. Neonatal pigs remain susceptible to RV infection and exhibit pathological signs until 12 weeks of age (233).

The MatAb usually provides protection against disease and infection only until 2 weeks of ages (225). Natural infection of pigs with RV occurs when MatAb levels in these pigs drops to antibody titers of 1600 (ELISA) (225). High persisting levels of passive IgG RV antibodies transudated from serum back to the gut were also protective (714).

Post-weaning and neonatal pigs have currently been used to study diarrhea pathophysiology and the immune responses and the determinants of protection against

RV. Enterotoxigenic E.coli (ETEC) or RV were detected in the of weaned pigs with diarrhea (487). Colostrum-deprived pigs and 3-week-old newly weaned pigs were exposed to RV before oral treatment with natural human IFN-α (402) . The IFN-α treatment was successful in reducing virus secretion and mortality rates in colostrum- deprived pigs, but not in weaned pigs. However the use of conventional pigs only allows

34 limited interpretation of the development of active neonatal immune responses to RV due

to the presence of other , extraneous RV infection and interference by MatAb.

Rotavirus has been detected in stools of weaned pigs without diarrhea (487). One of the

approaches to avoid such interference was to infect fetal pigs in utero, 15-36 days before

birth and orally challenge the newborn pigs at 2-4h after derivation in germ-free

conditions to assess protection against diarrhea and shedding (683). In this model, diarrhea was prevented in 12 of 14 pigs and reduced in the other two pigs, but fecal viral shedding was still detected in 12 out of 14 pigs.

Neonatal Gn pigs have been used extensively to study immune responses to RV,

RV vaccines and the correlates of protective immunity. In this model, intestinal villous atrophy was induced by infection with the VirHRV Wa strain, but not by the attenuated strain (589, 715). Studies were conducted of the magnitude and kinetics of IgM, IgA and

IgG ASC in pigs inoculated with porcine RV SB1A and Gottfrield strains or HRV Wa strain using an ELISPOT assay (125, 771). The IgM ASC appears as early as 3 days after infection and peak at 7 PID in MLN and spleen and later in lamina propria (LP) of the intestine. The IgA and IgG ASC numbers peak later at PID 14-21 and the IgA ASC responses are found mostly in intestinal lymphoid tissues. The magnitude of the antibody responses are highest in the tissues close to the site of viral replication, i.e. the intestine.

In the Gn pig model, a correlation of protection against infection and disease has been associated with intestinal IgA ASC, whereas the IgA ASC and antibodies in blood can serve as indicator of the IgA intestinal response, and hence protection (493, 681,

771). Unlike the mouse model, in Gn pigs, protection against diarrhea requires the presence of antibodies to the neutralizing antigens VP4 and/or VP7. The 2/6-VLP with 35 mLT, ISCOM or a DNA plasmid vaccine encoding VP6 did not induce protection

against viral shedding and diarrhea in Gn pigs (249, 314, 493, 763, 765). Repeated inoculations with an inactivated vaccine via either the oral or the intramuscular (IM) route (with incomplete Freund’s adjuvant for the IM vaccine) induced low levels of protection, and were less effective in inducing intestinal IgA ASC (769). In contrast with

the mouse model, when RV was inoculated via the IM route, the high IgG ASC and

antibody responses induced in systemic tissues did not confer protection (769). However,

a combination of priming with Wa AttHRV followed by two booster doses of VP6-DNA

vaccine IM induced moderate to high levels of protection (30-70%) against diarrhea and

virus shedding, respectively (763). Of interest, the subsequent boosting with 2/6VLP

(contains no neutralizing epitopes) following the initial priming with Wa AttHRV

enhanced the responses to other neutralizing epitopes on VP4 and VP7, as indicated by

the enhanced levels of neutralizing antibodies (763). This cross induction and

enhancement between epitopes of the same virus or different closely related viruses has

also been observed for vaccinia virus, LCMV etc. (610).

The route of vaccine delivery has also been investigated intensively in the Gn

model. The administration via mucosal routes has shown clear advantages over the

systemic route in inducing mucosal antibody responses as well as in the ease of delivery

of the vaccine. The oral administration of 3 doses of Wa AttHRV induces higher

protection against RV than the intranasal (IN) route (Azevedo and Saif, unpublished).

Boosting of the Wa HRV orally primed pigs with 250µg VLP IN resulted in higher

protection against shedding and diarrhea than boosting with 2/6VLP/ISCOM orally (249,

314). The order of the routes used for priming and boosting also determines the level of 36 protection in pigs. Oral priming with attenuated HRV followed by IN boosting with

2/6VLP or IM boosting with VP6-DNA vaccine, but not the reverse, induced high protection against viral infection and disease (763, 767).

1.1.9.2.4 Non-human primate models

Recently pigtailed have been used as host for simian RV infections

(731). Infection of seronegative macaques aged 11-16 months with YK-1 simian RV led

to high shedding of RV antigen in stools for 2-10 days after challenge, but without signs

of diarrhea. Infection of the seropositive animals also led to shedding of RV antigen in

stools but with shorter duration. The infection induced RV specific antibody responses

and conferred protection of the macaques against challenge virus after 28 days. This

model also demonstrated the role of existing RV IgG antibodies in reduction of virus

shedding when macaques were infected with YK-1 virus. However the macaques, similar

to the adult mice and rabbit models, do not exhibit RV-related diarrhea. Even the very

young macaques (4-6 months old) did not develop symptoms upon infection.

In another non-human primate model for RV, a new strain of simian RV namely

TUCH (Tulane University and Cincinnati Children’s hospital), characterized as a new

P[23]G3 strain, was used to infect juvenile rhesus macaques (458, 611). The T cell

immune responses were induced by this infection, as demonstrated by increased IL-6 and

IL-12 in secretions from the peripheral T lymphocyte cultures. In this model, the CD4+ and CD8+ T cells exhibiting intracellular IL-6 and IFN-γ and the HLA-DR+ differentiated

monocyte-derived DCs were detected after virus infection. The baboons and the velvet 37 monkeys were also investigated as animal infection models for HRV. Shedding of RV in

the feces of 5 of 5 velvet monkeys and 1 of 2 baboons was observed with increased IgG

and VN antibody titers (122). These non-human primates could be useful to study

candidate vaccines for human use. Yet like other infection models, the lack of diarrhea

related symptoms remains a disadvantage for these models.

1.1.9.2.5 Immunity to RV-human studies: The immune determinants of protection

against RV

1.1.9.2.5.1.1 Serum and fecal IgA antibodies

The association of fecal IgA antibodies with protection against RV infection is

indicated by human studies. Fecal IgA antibody responses are considered a good marker for protection (161, 437). A study of RV outbreaks in children in day care centers in the

US showed that the geometric mean RV fecal IgA antibody titers before exposure were highest in children who remained free of infection, lower in children with asymptomatic infections and the lowest in children with diarrhea (437). Thus a RV-fecal IgA antibody titer ≥ 80 correlates with protection against infection, whereas titers ≥ 20 correlate with

protection against diarrhea. Intestinal IgA antibodies are an even better correlate of

protection (92, 288). A study of Danish children with acute gastroenteritis showed an

increase in IgA antibodies in duodenal fluids (intestinal IgA antibody) 10 days after

infection and increased fecal IgA antibody titers at 5-7 months after infection (288).

Furthermore, the presence of IgA ASC in the small intestine of children was correlated

with serum IgA antibody titers (92). Furthermore, salivary IgA antibody titers reflected

38 mucosal IgA RV antibodies and could be a substitute measure for intestinal rotavirus antibody, as suggested by a RV (CJN strain) challenge study of adult volunteers (722).

The correlation between serum antibody and protection is still controversial as contradictory conclusions were drawn from studies of adults and infants with natural infection or following RV vaccination.

Studies of adult volunteers inoculated with HRV-D strain showed a correlation between serum antibody titers and protection against diarrhea. Pre-challenge VP7 antibody titers ≥ 20 correlated with protection against diarrhea or shedding of the virus upon challenge at 19 months after inoculation (257, 347). The presence of the strain- specific or cross-reactive VP4 antibodies also correlated with protection in this study.

Ward et al (717) also found that serum IgG and VN antibodies in jejunal fluid (obtained endoscopically using a jejunal catheter) correlated with protection of adult volunteers administered 2 doses of CJN (P[8]G1)-HRV. However this correlation was not consistently found in another study by the same investigators (718). It is postulated that the existing antibodies in adults due to previous exposure to RV were the reason for the inconsistency of these studies (328).

Studies of young children with natural infection also showed contradictory results regarding the role of serum antibody in protection against RV. A study of Danish children suggested that serum IgA but not IgG antibodies correlated with a decreased severity of symptoms (287). But a study in Bangladesh found that serum IgG antibodies correlated with protection against severe illnesses (147). Serum RV specific IgA, IgA1,

IgG, IgG1 and IgG3 antibodies are recognized as markers for RV infection in

Bangladeshi children with RV (36). A study of US children, who previously developed

39 gastroenteritis, showed that both an IgA antibody titer of >200 and an IgG antibody titer

of >800 obtained after the first RV season correlated with protection in these children in

the second RV season (512). Similarly a study in Mexico demonstrated that both IgA

and IgG RV antibodies were protective, yet the antibody titers required to achieve

protection were higher than those in the US study (702). A serum VN antibody of ≥ 128

was protective in Japanese children against subsequent illness due to G1-RV (127).

Homotypic antibodies acquired via the placenta were associated with protection against dehydration in infants 1-6 months of age during RV induced diarrhea (561). Some vaccine trials of young children also showed a correlation of serum VN and IgA RV antibody responses with protection (708, 719).

In contrast, Ward et al (721) found that protection against RV diarrhea after natural infection did not depend on homologous serum VN antibodies. Similarly, a lack of correlation between serum antibodies and protection were reported in vaccine trials

with RRV-TV, RIT 4237, WC3, Wa, RRV and DxRRV (328). Jiang et al (328) postulated that these discrepancies may result from differences in sample size, vaccine type and dose, demographics and laboratory diagnostics used.

1.1.9.2.5.2 Markers on B and T cells and the association with RV infection and

protection

The use of a flow cytometry assay allows the study of circulating B and T cells

and their subsets using surface markers and the association with RV infection and

protection. The presence of large IgD- B cells expressing RV specific surface

immunoglobulin (sIg) correlates with RV-specific ASC during acute but not during 40 convalescent RV infection (248). Lymphocytes detected during acute infection were RV

ASC whereas those detected during convalescence were memory B cells. The human

cells expressing RV-sIg also express the integrin α4β7, an intestinal homing receptor,

which indicates that RV-sIg B cells are primed to traffic back to the intestine. Therefore the flow cytometric assay of blood lymphocytes bearing α4β7 is considered as an indirect measure of intestinal RV ASC which are responsible for protection against RV infection

(321). Chemokines TECK/CCL25 (thymus-expressed chemokine/chemokine (C-C motif) ligand 25), MEC/CCL28 (mucosa associated epithelial cytokines) and the receptors CCR9 and CCR10 respectively, play an important role in the process of B cell homing to the gut (388, 397, 772). The CCL25 is exclusively expressed in the small intestine and attracts B cells that express CCR9 and IgA. The CCL25 and CCR9 may serve to compartmentalize the small intestine immune response because RV-IgA ASC migrated preferentially to tissues expressing CCL25. In addition, B cells expressing

CCR9 are mainly found in the small intestine whereas they are rarely found in the colon and absent in other epithelial tissue (79, 385). The roles of CCR9 and CC10 were confirmed in that RV specific IgM and IgA ASC, predominantly large lymphocytes, also express CCR9 and CCR10 in response to acute RV infection, which likely targets these cells to the gut (321). During convalescence, the B cell population consists of both small and large cells expressing low or no CCR9 and CCR10, most likely representing RV specific memory B cells with both gut and systemic trafficking profiles. The CCR6 which is expressed by most B cells, subsets of CD4 and CD8 memory T cells and subsets of DC helps positioning leukocytes at mucosal locations (739). The RV memory cells express

CCR6, which allows their recruitment upon inflammation. 41

1.1.9.2.5.3 Role of cytokines in RV infections

A number of cytokines have been implied in disease manifestations and

protection against RV infection in humans. In a study of Bangladeshi children with RV

diarrhea, IFN-γ was higher in children with RV diarrhea than those with diarrhea unassociated with any enteric pathogen (36). In children with persistent diarrhea, plasma

IFN-γ levels were higher than in those with acute diarrhea, indicating that plasma IFN-γ levels may be associated with subsequent development of persisting RV infections (35).

In this study, TNF-α correlates with the acute phase of the diarrhea, related to the pro- inflammatory effect of this cytokine. In another study of US children with RV diarrhea, it was shown that serum IL-6 was correlated with fever, whereas serum IL-6, IL-10 and

IFN-γ correlated with acute RV infection. In regard to the other symptoms of RV infection, the presence of TNF-α was associated with fever and multiple diarrhea episodes whereas IFN-γ levels were an indicator of vomiting (329).

Cytokines in RV infections in Gn pigs have been studied (32). The study was conducted using Gn pigs inoculated with either VirHRV or AttHRV. Generally, higher cytokine levels occurred early after infection with VirHRV compared to AttHRV. The pro-inflammatory cytokine TNF-α levels peaked and remained elevated in serum of the

VirHRV inoculated pigs early in the infection (PID 3) and later (PID 21) in the AttHRV inoculated pigs. In serum, IL-6 was significantly elevated at PID 1 in the VirHRV group and at PID 3 in both HRV groups. For Th1 cytokines, only low and transient IFN-γ responses (PID 3-5) occurred in serum and intestinal contents of the AttHRV-infected

42 pigs, compared to significantly higher and prolonged IFN-γ responses (PID 3-28) in the

VirHRV-infected pigs which correlated with viremia and diarrhea induced by VirHRV but not by AttHRV. The role for IL-12 in the induction of immune responses to rotavirus infection was confirmed in both groups. Early in the infection, there were higher levels of the TH2 cytokine IL-10 in the serum of the VirHRV group compared to the AttHRV group. A delayed initiation of Th2 responses occurred after AttHRV infection of pigs as indicated by higher IL-10 cytokine secreting cell numbers in ileum and spleen of the

AttHRV group compared to the VirHRV group at later times, PID 14 and 28.

Similarly to pigs, in mice, a mixed pattern of Th1 and Th2 cytokines were induced upon infection of mice either with heterologous (SA11) or homologous (EHPw)

RV (223). In mice IL-6 does not play a significant role in protection against RV nor in

Th1 and IgA development (696). Unlike in pigs, type I and II IFN did not play a role in protection of neonatal mice against RV shedding (22, 697). In knock–out mice lacking signal transducers and activators of transcription-1 [Stat1(-/-)], oral infection of suckling

Stat1(-/-) and immunocompetent mice with RV induced diarrhea and virus shedding of similar intensity. Clearance of RV from stools of adult Stat1(-/-) mice occurred at the same time as in wild-type mice. However, adult Stat1(-/-) mice shed up to 100-fold more rotavirus antigen in stools than did immunocompetent mice after infection. Type I IFN receptor -/- suckling mice and IFN-γ -/- suckling mice developed diarrhea with similar duration and had comparable quantities of viral antigen in their intestines as did immunocompetent mice (22). Intestinal epithelial cells also produce chemokines, IFNs and GM-CSF in RV infections, yet the roles in these cytokines and chemokines in protection of mice against RV are unknown (563).

43

1.1.9.2.6 Rotavirus specific antibody levels in children worldwide

A number of studies in the 1980s and 1990s have focused on the antibody levels specific to RV in infants, children and their mothers. The results of these studies are summarized in Tables 1.1 and 1.2. Our studies of the influence of MatAb were based on these antibodies levels (Chapters 3 and 4). Due to the variety of techniques used to measure the antibody titers, it is not possible to unify all of the studies to get the full spectrum of antibody levels in children and mothers in many countries. In addition, the obvious challenge is to identify which are the RV antibodies transferred from the mother to the infants and which antibodies are due to previous encounter with RV and active immune responses in infants.

1.1.10 Interference by MatAb with RV infections

1.1.10.1 RV antibodies in milk and passive protection

The roles of milk antibodies in passive protection against RV have been suggested in humans and demonstrated in animals. In humans, it is suggested that breast milk IgA antibodies confer partial protection against RV infection. A study of Nicaraguan infants from birth until 2 years of age showed a positive correlation between RV IgA antibodies in colostrum and the time of onset of viral shedding (195). In particular, colostral IgA antibody titers were significantly lower in children first demonstrated RV excretion before 6 months of age than in children first demonstrated excretion of the virus after 6 months of age. This study also suggested a relationship between the duration of breast

44 feeding and asymptomatic infections (195). Breast-fed infants who did not excrete RV over the 5-day period received milk with significantly higher RV sIgA antibody titers than breast-fed infants who were infected with RV (456). Infants fed daily with colostrum from cows hyperimmunized with RV were protected from RV associated diarrhea whereas 70% of infants not receiving the bovine colostrum developed diarrhea

(184).

In pigs, MatAb were detected in the feces of suckling pigs up to 18 days of age.

Natural RV infection of these pigs occurred when the geometric mean ELISA titers of

MatAb in their sera declined to 1600, suggesting that MatAb is protective against RV infection in pigs but only for the first one or two weeks (225). The passive antibody doses which conferred protection against RV were quantitated in Gn pigs fed with colostrum/milk antibody from cows immunized with different strains of HRV. Dose levels of 15.8 x 106 and 19.5 x 106 VN antibody units were required to achieve a 50% reduction of diarrhea and viral shedding, respectively (599). However, in nursing piglets,

Saif et al (578) showed that maternal vaccination IMm with live attenuated porcine RV serotypes I and II did not prevent natural infection with OSU and Gottfried RV of suckling pigs, but it did decrease the duration and delay the onset of RV shedding and diarrhea.

In mice, the milk antibodies specific to VP4 but not VP2 or VP6 were associated with protection against RV induced diarrhea. After nasal administration live recombinant

Salmonella expressing RV VP2 and VP6 to female mice, high antibody responses specific to both VP2 and VP6 were induced in serum and milk, but these antibodies failed to protect pups against diarrhea after challenge with bovine RV (BRV) (RF strain) (158,

45 159). However VP4 antibodies in milk provided homotypic protection in the mouse model. Pups born to dams immunized with SA11 VP8* were protected from diarrhea when orally challenge with the SA11 strain. In addition, pups born to naive dams but nursed by VP8*-immunized dams were also protected against diarrhea after challenged with the SA11 strain, whereas pups born to VP8*-immunized dams but nursed by naïve dams were not. Thus neutralizing antibodies in the milk rather than serum antibodies transferred through the placenta provided protection against RV induced diarrhea (243).

The RV-specific IgY antibodies from egg yolk provided complete homotypic protection against RV MO strain-challenge in BALB/c mice.

Rotavirus specific colostrum from cows also protected mice against infection with four different RV G types (184). Colostrum from cows immunized with the adjuvanted modified live Ohio Agricultural Research and Development Center (OARDC) RV vaccine provided complete protection to calves against virulent bovine RV when given as a 1% dietary supplement and partial protection when given as 0.1% supplement (586).

Thus passive protection by RV milk antibodies is dose dependent. Shortly after cessation

of colostrum feeding, two of three calves shed virus at 14 days post exposure,

highlighting the importance of continued presence of passive antibody in colostrum/milk

(579).

1.1.10.2 Circulating antibody and protection against RV

Circulating antibodies specific to RV were also implicated in protection. A study

of children from New Delhi, India showed that neonates infected with the neonatal RV

strain 116E-like had significantly lower levels of cord blood neutralizing antibodies to

46 116E than the neonates who did not become infected, suggesting that MatAb acquired transplacentally provided passive protection against neonatal RV (543). The effect of circulating RV specific IgG antibodies was investigated in the pigtailed model

(730). Upon oral challenge with YK-1 virus, the animals which did not receive the antibody containing serum, shed virus starting at 1-3 days after challenge and the shedding lasted for 6-8 days. On the contrary, in animals passively given IV the immune or control serum (RV specific antibody titers of 10,000 or 300, respectively) did not shed virus or showed delayed shedding at low titers for a limited time. The Gn pigs injected IP with high antibody titer serum from RV hyperimmunized sows exhibited 76 and 36% protection against diarrhea and shedding, respectively when orally challenged with virulent HRV 5 days after IP injection (289). In contrast, the low antibody titer serum derived from naturally infected sows did not provide any protection. Oral feeding with colostrum/milk from RV hyperimmune sows in addition to IP injection of the hyperimmune serum did not improve the protection against virus shedding and diarrhea but significantly delayed the onset of virus shedding (289).

1.1.10.3 Maternal immunization

Maternal immunization may provide protection against RV in newborns. The stability of RV antibody in milk was assessed to evaluate the duration of milk antibody to provide protection against RV. Significantly higher concentrations of antibody to RV in milk persisted for 4 months in postpartum women who received a RRV monovalent reassortant vaccine or the tetravalent vaccine (535). When hyperimmune bovine colostrum containing different levels of RV antibody was administered to children, 3

47 times a day, for a period of 6 days, the antibody activity was detected as early as 8 hours

after ingestion of hyperimmune colostrum and up to 72 hours after consumption had

ceased. These results showed that anti-RV activity survived passage through the gut;

therefore, passive immunotherapy may be used to prevent or treat infectious diseases that

affect the (517). Vaccination of pregnant baboons intramuscularly

with a RRV vaccine with ISCOM repeatedly at 1-2 and 14 weeks after delivery caused

significant increases in RV-specific maternal serum IgG and VN antibodies and in milk

IgA, IgG and VN antibodies (639). The combined IND/2292B VLP vaccine specific for

two different serotypes of BRV induced comparable VN responses to each BRV serotype

in serum, colostrum and milk compared to the responses induced by the individual IND

or 2292B VLP vaccines (366).

Maternal immunization can also be enhanced by feeding bacteria to

mothers, which translated to better protection against RV diarrhea in the mouse model.

Mouse pups born to and nursed by dams fed with Bifidobacterium breve YIT4064 and immunized orally with the simian RV SA11 were more highly protected against SA11- induced diarrhea than those born to and nursed by dams immunized with RV only (757).

In addition, the titers of RV IgA antibody in milk and feces of dams fed B. breve

YIT4064 and immunized orally with RV were higher or significantly higher than those of

dams immunized with RV only.

Passive protection to heterologous RV depends on the route and the type of

antigen. Parenteral immunization of dams with RV, homotypic or heterotypic to the

challenge virus protected suckling mice against diarrhea, whereas oral immunization with

homotypic, but not heterotypic RV strains to the challenge virus conferred protection

48 (502). Colostrum from cows immunized with the simian RV SA11-VLP but not

inactivated SA11 afforded a high rate of protection to colostrum-fed unsuckled calves against challenge with heterologous BRV (209, 210). The highest titers of RV antibodies in mammary secretions of pregnant cows were induced by IM immunization at ~9 week

pre-partum and IMm inoculation at 2 weeks pre-partum with the OARDC modified live

NCDV bovine RV, but not after IM immunization with a commercial inactivated Rota-

Coronavirus vaccine (587). Feeding colostrum from IM plus IMm immunized cows to

newborn calves challenged by RV prevented diarrhea and shedding of RV (587). The

antibody titer induced by this vaccine via these routes remained significantly elevated in

milk for at least 30 days post-partum. The rationale for this vaccination scheme was that

parenteral stimulation near involution resulted in seeding of sensitized plasmablasts to the

mammary gland with boosting by IMm RV injection. The antigen dose in the vaccine

plays a significant role in RV maternal vaccination. Saif and Smith (587) also showed

that the antigen dose of the live OARDC RV vaccine was 1x108 plaque forming unit (pfu)

/ml, whereas that of the commercial live RV vaccine was 1x104 pfu/ml, which likely

influenced the RV antibody titers in milk. The authors also indicated that the efficacy of

inactivated vaccine could be influenced by the inactivating reagent. Binary ethylenimine

(BEI) inactivated RV induced 10-fold greater antibody titers in cow’s milk compared to

β-propiolactone as inactivating agent. The use of adjuvants is important in enhancing antibody titers in milk. Enhanced colostral RV antibody titers were achieved by incorporating Freund’s incomplete adjuvant with the commercial RV- vaccine, compared to the same vaccine without adjuvant or with AlOH adjuvant (587).

49 1.1.10.4 Protection against RV by other milk components

Human but not bovine lactadherin in milk has been shown to inhibit Wa HRV infection in vitro (in RV-infected MA104 and Caco-2 cell lines) (390). Milk-fat globule membrane protein MUC1 in bovine milk inhibits the neuraminidase-sensitive RV RRV strain efficiently but not the Wa strain which is NA resistant. Therefore it is possible that milk feeding can interfere with a live RV vaccine based on the RRV data (e.g. RRV-TV).

A study also suggested an association of trypsin inhibitors in human milk with protection of neonates against RV infection in the first 5 days of life (457). Breast-fed infants were significantly less likely to become infected with RV and showed significantly lower stool tryptic activity or higher trypsin-inhibitory capacity than did bottle-fed infants. Human milk mucin can bind to RV and inhibit viral replication in vitro and in vivo, also providing protection (759).

1.1.10.5 Interference with active immune responses to RV in infants and in animal

models

Interference by circulating MatAb with the development of active immune responses after RV infection or vaccination has only been studied in animal models, whereas interference by milk antibodies has been widely studied in both humans and animal models. In a comparison of milk from New York and Venezuelan mothers, both milk and infants' serum pre-immunization RRV VN antibody titers had a negative effect on seroconversion after RRV vaccination (P = .008 and .02, respectively). Infants fed milk containing RRV VN antibody titers ≥ 160 showed a lower RRV seroconversion rate compared to infants fed milk containing RRV VN antibody titers ≤ 160 after vaccination

50 with RRV. In addition, VP4-specific milk antibodies may interfere with RRV

seroconversion (553). A study of milk from women in Caracas, Venezuela and in

Rochester, New York, USA, also indicated the role of VP4 antibodies, but not VP7

antibodies in the reduction of vaccine responses to RRV in children (416). Seventy-five

percent of RRV vaccinated infants who were breast-fed with milk containing antibodies

against VP4 did not exhibit a seroresponse whereas 45% of infants who consumed breast

milk with VP7 antibodies did not respond to the vaccine. On the contrary, among those

children fed with breast milk negative for VP4 antibodies and positive for VP7

antibodies, 71% experienced a seroresponse. Interference by RV MatAb, although not

proven, was suggested as the reason for the failure of vaccine trials such as RIT4237,

WC3, or RRV-TV (54, 110, 371, 416).

In the Gn pig model, various titers of RV specific MatAb at vaccination have

been shown to influence the outcome of active immunity. Hodgins et al (289)

demonstrated that in Gn pigs, induction of ASC responses was suppressed after Wa

VirHRV primary infection and challenge in the presence of circulating high titer MatAb.

The RV antibodies from milk enhanced suppression of the intestinal IgA ASC responses

before and after challenge with VirHRV. In addition, the serum and intestinal IgA

antibody responses were also suppressed in pigs receiving high titer MatAb in circulation

and/or in milk diet (522). At low titer MatAb, only IgG ASC, but not IgA ASC numbers

in and MLN were significantly reduced after HRV challenge (289). A passive antibody study of calves showed that calves fed with colostrum from normal cows

actively produced serum IgG1 antibodies at 14 days post exposure with the virus,

compared to 7 days in calves without colostrum feeding, whereas calves fed with

51 colostrum from immunize cows did not show any increase in serum IgG responses (579).

Similarly, calves fed with colostrum before inoculation with BRV showed an inverse relationship between the IgG1 titers in colostrum and the ASC responses (521). In particular, fewer IgG ASC numbers in response to BRV inoculation were observed in calves fed with colostrum from naturally infected cows (low titers of RV antibody, or control colostrum, CC), whereas significantly lower ASC responses of all isotypes occurred in calves receiving BRV hyperimmune colostrum (IC). In addition, calves fed with IC only developed fecal IgM antibody upon inoculation with BRV. Calves fed with

CC showed significantly lower serum and fecal IgA and IgG1 antibody titers compared to colostrum-deprived calves after BRV inoculation. Thus passive RV antibodies interfere with neonatal active immune responses in a dose-dependent manner, similar to observations for virus in neonatal mice (624).

1.1.11 Rotavirus vaccines

1.1.11.1 Jennerian and modified Jennerian approaches to RV vaccines

Initially, several RV vaccines using animal viruses were based on the Jennerian approach to vaccination. Of the four vaccines developed in this way, 3 were discontinued before licensure (RIT4237, bovine P[1]G6; WC3, bovine P[5]G6 and RRV, rhesus

P[3]G3) due to variable efficacy between the different countries where the vaccines were tested. Only the RV lamb strain LLR vaccine (P[12]G10) is still used in China (85).

Vaccination of infants with WC and RIT 4237 bovine RV gave good protection in

Finland, but not in other developing countries (706). Similarly the bovine WC3 RV was effective in Philadelphia, but not in Cincinnati where only 9% of children produced

52 antibodies and no difference in the numbers with illness and the severity of diarrhea were

observed between vaccinated and placebo groups (54, 144). The RRV was also tested in

Venezuelan and Swedish children (217, 252) but gave inconsistent results.

Because of the failure of animal RV vaccines, the subsequent vaccine approaches

attempted to include HRV components (VP7 or VP4) to the animal viruses (Modified

Jennerian approach). Reassortants were created, either monovalent (Wa x UK, P[8]G6;

Wa x DS-1xUK, P[8]G2) or tetravalent (Rotashield and UK-based reassortants) or

pentavalent (RotaTeq, P[5]G1-4 and P[8]G6), which cover the four most common human

G1-G4 serotypes in the background of rhesus(P[3]) and bovine P[5]RV. The first vaccine

for RV approved by the US Food and Drug Administration (FDA) in 1998 was

RotaShield, a tetravalent vaccine of simian/human reassortants, created by Dr.

Kapikian’s group at the National Institutes of Health and licensed for production to

Wyeth-Ayerst Laboratories for use in infants at 2, 4 and 6 months of age. However due to

its association with a high rate of intussusception among vaccinees, it was withdrawn one

year after appearing in the market. During a phase III trial of this vaccine in Caracas,

Venezuela, the vaccine induced 88% protection against severe diarrhea (531, 635). The tetravalent vaccine, licensed as Rotashield in 1998, is now licensed to a biotech company

BioVirX for global marketing. Merck Incorporation produces RotaTeq®, composed of five bovine x human reassortant rotaviruses of the most prevalent human serotypes (G1,

G2, G3, G4 and P1A[8]), which is in Phase III trials. In a clinical study of a G1 reassortant strain WI79-9 (G1 serotype VP7 on a BRV WC3 strain background), a component of the pentavalent vaccine RotaTeq®, the vaccine induced a 95% response rate in vaccinees against the WC3 VP4 (P7) and significant antibody responses to VP7 53 were achieved in more than 70% of infants after three oral doses (146). A combination

G1 and G2 human-bovine reassortant RV vaccine has been tried without any adverse

effects (145). A lower rate of seroconversion to WC3 (45-59%) was observed after 3

doses. However, the IgA coproconversion rate was intermediate (~80%) among

vaccinees. It is of great importance to know whether vaccination with 5 strains of

recombinant viruses can improve the immunogenicity of the vaccine.

Other RV vaccines are also in consideration. Because these rare strains of RV are

becoming more common each year, as mentioned previously, the need for a vaccine that carries the full spectrum of HRV serotypes becomes more apparent. A subunit vaccine

based on RV VP4 recombinant protein is being considered because the need for the cold-

chain is eliminated making it ideal for use in developing countries (304). Hoshino et al

(300) generated pentavalent or hexavalent RV vaccines containing the existing reassortants of the RRV tetravalent vaccine with the addition of VP4 gene substitution reassortants of human P1A[8] or P1B[4] in the RRV background.

1.1.11.2 Non Jennerian approach to RV vaccines

The non-Jennerian approach to RV vaccination includes the use of monovalent human neonatal strains based on the observation that these strains are asymptomatic in neonates but immunogenic in both neonates and older children. These vaccines include

M37 (P[6]G1), 116E (P[11]G9 and I321 (P[11]G10) which are in phase I vaccine trials.

A naturally attenuated human neonatal strain RV3 (G3P2A[6]) was tested as a RV vaccine in a limited phase II trial (45). The virus was propagated on African green monkey kidney (AGMK) cells and given at 6.5x105 FFU to children at 3, 5 and 7 months

54 of age. With this strategy, only 46% of vaccinated infants developed any immune

response (serum neutralizing antibodies, serum IgG, IgA, IgM or copro IgA antibodies)

and only 2 of 39 infants developed both seroconversion and coproconversion. Of those

that developed immune responses, 76% were protected against RV infection and diarrhea

following the first epidemic season, compared to the 55% in those with no immune

response or those who received placebo. However the severity of the disease was not

reduced compared to the non-vaccinated group. This low rate of immune response and

protection rates can be attributed to the presence of MatAb, the lack of heterotypic

protective immunity induced by the P[2]G3 strain against the more common P[8]G1,

P[8]G2 or P[6]G2 strains, or the low vaccine dose used (594)

Another non-Jennerian monovalent live oral vaccine is based on the human 89-12

strain (P[8]G1) called Rotarix. GlaxoSmithKline PLC (GSK) is responsible for the

development of the Rotarix® vaccine which was licensed in Mexico in 2004. Phase II

and III trials of Rotarix were conducted in Finland and 11 Latin American countries with

a total of 65,000 subjects enrolled (557). This vaccine induced a high rate of

seroresponses and intestinal IgA responses, yet only 7 out of 20 subjects experienced

more than a 4-fold increase in VN antibody titers (55). The protection against severe

diarrhea due to G1 or G9 strains was both 77% for each (531), indicating cross protection

of the vaccine G1 strain against the other heterotypic strains.

1.1.11.3 Rotavirus-like particles as vaccines in different animal models

Virus-like particles (VLP) offer attractive approaches for neonatal RV vaccines

due to their potential safety (non-replicating so lack of infectivity) compared to live

55 vaccines. The viral VP2 and VP6 proteins spontaneously form a double-shelled structure

and the addition of VP7 or both VP7 and VP4 results in triple layered particles. The

VLPs were constructed from simian, bovine and human strains or a hybrid structure

between RV proteins from different species e.g. bovine and human, bovine and simian or

proteins from different RV groups, e.g. hybrid VLP from group A VP2 with group C

Shintoku VP6 (153, 365, 765).

The synthesis of VLPs can be achieved in an insect cell expression system using

Spodoptera frugiperda 9 (Sf9) or High Five insect cells (326). In the insect cell system,

recombinant baculoviruses expressing different RV proteins were coinfected into insect

cells. The majority of VLPs are found in the culture medium. A higher yield of VLPs per

cell (5-fold) was achieved using the High five cultures compared to that of the Sf9

cultures (326).

The immunogenicity and efficacy of VLPs as a vaccine were assessed in different

animal models including mice, rabbits, cows and Gn pigs. In adult mice, 2/6VLPs

containing bovine RF VP2 and simian SA11 VP6 administered IN with cholera toxin

(CT), E.coli heat labile toxin (LT) or mutant LT with an amino acid substitution (LT-

R192G) induced a high level of protection (91-100%) against challenge with live murine

RV strains including ECwt (509, 510). Even in the absence of an adjuvant 100ug of

2/6VLP administered IN, also resulted in a 38% reduction in virus shedding (510).

Similarly, adjuvant was not necessary for 2/6VLP (0-100µg) to induce systemic and mucosal immune responses when administered orally or IP, suggesting that in mice the

VLPs themselves can act as adjuvant (619). The inclusion of VP7 in the particles as

2/6/7 VLP did not improve the immunogenicity or protective efficacy against challenge 56 with wild type murine RV. A high level of protection was found using 2/6VLP produced

from a dual recombinant baculovirus vector expressing both VP2 and VP6 of simian RV

origin (SA11), especially when administered by IM or IN routes (57). In addition, the

2/6-VLP primed for heterotypic (non-G3) immune responses, but the induction of

heterotypic neutralizing antibodies required replication of the challenge virus. Thus VP6-

T helper cells can provide cognate help to B cells specific for neutralizing epitopes on the

VP7 and VP4 of the challenge virus. Similarly, in the Gn pig model, a vaccination

scheme consisting of an oral dose of Wa AttHRV followed by 2 IN doses of 2/6VLP with

either mLT or ISCOM induced significantly higher VN antibody responses and serum

IgA and IgG antibody titers to VP4 and VP7 compared to one oral dose of Wa AttHRV

alone at PID28 (764). Similar results were found in studies using the rabbit model of RV

infection (without disease). In this model, 2/6-VLP from bovine RF-VP2 and simian

SA11 VP6 in Freund’s adjuvant administered parenterally resulted in a 41% reduction

rate in virus antigen shedding (139). A lower protection rate (5-26%) was achieved by

using 2/6-VLP with QS-21 adjuvant. A high protection rate against viral shedding (65.8-

97.9% reduction in virus shedding) was induced when the 2/6VLP particles were

combined with CT-E29H, the detoxified version of CT adjuvant, and oral or IN

administration (620). In both mouse and rabbit models, the presence of VP7 in the VLP

did not improve protection against virus shedding. Nevertheless, in the rabbit model, the

role of VP4 in protection was realized when 2/4/6/7 VLP conferred higher mean rates of

protection than 2/6/7VLP.

Contradicting results have been observed in neonatal mouse, Gn pig and calf

models. Only suckling newborn mice, born to mothers immunized with 2/6/7-VLP but 57 not 2/6-VLP were protected against RV diarrhea when challenged with RF RV 4-days

after parturition (159). In Gn pigs, 2/6 VLP, composed of RF BRV-VP2 and Wa HRV

VP6 alone did not induce any protection against virus shedding and diarrhea when pigs were challenged with Wa VirHRV. No protection was induced when 2/6-VLP were administered alone or with mLT or ISCOM adjuvants via IN or oral routes (33, 249, 314,

493, 765). In the calf model, only conventional colostrum-deprived calves fed with colostrum and milk from cows immunized with VLP 2/4/6/7 were protected against diarrhea, and 60% of these calves were protected against viral shedding (209). All the calves fed with 2/6VLP immunize colostrum shed virus and one calf out of 5 (20%) had diarrhea when challenged with virulent IND BRV. Differences in the ability of 2/6VLP to induce protective immunity in different animal models may be attributed to use of the adult vs. neonatal of animals and the species origin of the VLPs. Bovine VLPs of different serotypes P[5]G6 and P[11]G10 have been used to coimmunize and they induced lactogenic antibody responses in cow colostrum and milk against both serotypes, confirming the efficacy of these VLPs for passive immunization of cows (366).

The VLPs also have potential for the induction of heterotypic protection. The G1-

2/6/7 VLPs in which VP7 was derived from the human G1 Houston strain 8697 strain and the VP2 and VP6 from the bovine RF and C486 strains, respectively, induced partial protection (88%) in adult mice against oral challenge with the murine EC RV (G3) when given IM in 2 doses with QS21 adjuvant (327). However, this heterotypic protection was highly dependent on type of adjuvant because the G1-VLP in AlOH adjuvant did not protect mice from G3 RV challenge. Similarly in rabbits, two IM doses G1-2/6/7 VLP or

G1-2/4/6/7 VLP or alternative G1 then G3-VLP doses in QS-21 adjuvant induced 58 protection against G3 ALA virus similar to the homotypic protection induced by G3-VLP

(162). Furthermore, the inclusion of VP4 in the VLPs was not critical for the induction of

heterotypic neutralizing antibody. In calves, colostrum from cows immunized with SA11

2/4/6/7VLP (P[2]G[3]) provided complete protection to calves against diarrhea when challenged with virulent IND BRV (P[5]G[6]) and partial protection against viral shedding (209). This cross protection of different G and P serotypes was the basis for monovalent G1 vaccine used for a human infants (594).

1.1.11.4 Individual RV proteins as potential vaccines

Individual RV proteins have been studied as candidate vaccines. The VP6, which is highly antigenic and the major component of the RV capsid is conserved among all group A RV was the first choice. Oral and IN administration of RV chimeric VP6- mannose binding protein (MBP) in combination with LT(R192G) adjuvants to mice induced between 95-99 % reduction in fecal viral shedding (130, 133-136). Other adjuvants were also tested with this chimeric protein for the induction of immune responses and protection in the mouse model (136). The VP6-MBP vaccines delivered with Adjumer, CpG-ODN and chimeric A1 subunit of CT induced a high titer of serum

IgG antibody when delivered IN, but not orally. Intranasal delivery with Adjumer increased Th2 responses, whereas CpG-ODN shifted the response more toward Th1. In contrast, adjuvants CTA1-DD, LT (R192G) and QS-21, did not alter the Th1/Th2 pattern.

All adjuvants, except QS-21, induced a high level of protection against virus shedding

(74-99%) when using the IN route. Intranasal delivery with QS-21 induced only 43% protection only, which was not significantly different from the administration of chimeric

59 VP6 alone. Oral immunization with QS-21 improved the protection rate (71%), whereas oral immunization with other adjuvants resulted in decreased protection rates. Thus in the mouse model, the IN route of immunization with the VP6 protein is more effective than the oral route.

Because mLT is not likely to be approved for human use via mucosal routes, skin immunization with this adjuvant was explored as an alternative to mucosal routes (137).

Needle-free transcutaneous (tci) delivery was achieved by gentle abrasion of the shaved skin of mice and the gauze pads containing the vaccine were applied to the shaved skin for 24h. Subcutaneous injection of the antigen was also tested using a biojet liquid jet injection apparatus. However, the tci delivery of VP6/mLT or VP6 alone did not induced significant protection against virus shedding whereas biojet injection induced low levels of protection in mice compared to IN administration, although VP6 specific serum IgG antibody was induced by using all methods. Delivery of the VP6 plasmid from the EDIM strain of RV using a Powerjet particle device induced serum IgG antibodies but failed to protect mice from homologous virus challenge (132). Similarly three doses of VP6 plasmid administered IM to Gn pigs did not induce protection against virus shedding and diarrhea (763). However, significant protection rates were induced when attenuated Wa

HRV was used as a priming dose followed by 2 IM booster doses of VP6 plasmid.

The use of other RV proteins has also been explored. Epidermal immunization with VP4 and VP7 genes alone did not induce protection in mice (131). The VP4 fused with maltose binding protein (MBP) expressed in the cytoplasm of

60 induced immune responses in mice, which suggested that the use of Shigella as a delivery

system can induce immune responses to both Shigella and RV (417). A single dose of

VP4 or VP7 DNA vaccines encapsulated in PLG microparticles delivered orally also

induced protection and intestinal and serum IgA antibody in mice when challenged with

the virulent virus 6 weeks later. The VP7 DNA vaccine was less effective than the VP4

vaccine (283). Using a tobacco mosaic virus (TMV) vector, the VP8 fragment of the

VP4 protein of BRV was expressed in plants. Immunization of dam with this VP8

fragment induced immune responses and provided protection to suckling pups against

challenge with BRV (529, 737). Maternal immunization of pregnant cows with the VP8

protein of BRV resulted in high VN antibody titers in colostrum and milk, which

neutralize in vitro BVR P5 serotype (B641) at significant level and P11 serotype (B223) moderately (760). Thus passive protection to calves against BRV by VP8* immune

colostrum and milk was suggested. Similarly the VP7 protein expressed in potato tubers

induced serum IgG and mucosal IgA antibodies specific for VP7 when fed to mice,

suggesting the possibility of employing the transgenic plant as an edible RV vaccine

(747). Yet in another study, particle bombardment by VP4 or VP7 did not

confer any protection, or induce serum and fecal IgA antibodies or neutralizing

antibodies in mice, indicating the importance of the immunization route in the induction

of protective responses against rotavirus in infants (131).

In summary, the expression of individual RV proteins as vaccines has been

studied extensively in the mouse model. In this model, the RV proteins VP4, VP6 or VP7

can be used as effective vaccines that induce protection when administered with

adjuvants or in expression vectors but not alone. There are few reports in other animal 61 models about the use of these individual proteins for RV vaccines. Current research results indicate the need for VP4 and/or VP7 for the induction of protection against RV challenge in animal models other than the mouse.

1.2 The mucosal immune system

1.2.1 General structure

The mucosal-associated lymphoid tissue (MALT) is the largest mammalian lymphoid organ system that provides protection of the respiratory, digestive and urogenital tracts, the eye conjunctiva, the inner ear and the exocrine glands (465). The highly compartmentalized MALT consists of defined lymphoid compartments such as the

PP, MLN, the appendix and follicles in the intestine and the tonsils and adenoids in the respiratory tract (369, 478). These compartments act as the site where immune responses are initiated. The MALT also contains the effector sites which are the diffuse accumulation of lymphoid cells in the parenchyma of the mucosal organs and in the glands (295).

The antigen is taken up by the absorptive epithelial cells and specialized epithelial cells, called M cells, that reside in the inductive sites and shuttle the antigen to the antigen presenting cells (APC) (295). The antigen can also be captured directly by the professional APC such as DCs, B cells and macrophages. The APCs present the antigen to the CD4 and CD8 α/β T cells in the inductive sites. Intraepithelial cells (IEL) can also present and process the antigen to T cells directly. Different types of responses can be followed: either Th1 or Th2 responses, or suppression responses leading to either oral tolerance or an inflammatory response which involves both humoral and cellular immune

62 responses. The sensitized B and T cells then leave the inductive sites, via the lymph, enter

the circulation and seed the mucosal effector sites where they differentiate into memory

or effector cells (581).

One of the properties of MALT is the ability of an immunocyte to be activated at

one site but travel to other remote mucosal tissues; this phenomenon is called the

”common mucosal immune system”. On the other hand, the MALT is also highly compartmentalized, i.e. it links specific inductive sites with specific effector sites, such as

the gut with the mammary gland, the nose with the respiratory and genital tracts. Thus consideration should be taken in regard of the route of mucosal immunization to induce

immune responses in distant sites. Oral immunization induced strong antibody responses

in the small intestine, colon, mammary and salivary glands, but induced only limited

responses in the genital tract, rectum or tonsils (194, 376, 588). Similarly, IN

immunization resulted in strong antibody responses in saliva, nasal secretions and the

genital tract but limited responses in the gut (334).

1.2.2 Innate immunity

Innate immune recognition is mediated by the molecules, pattern-recognition

receptors (PRR) which recognize pathogen associated molecular patterns (PAMPs)(12).

These PRR include LPS-binding protein, CD14, β2 integrins and TLRs.

Natural killer cells, phagocytic neutrophils and macrophages present in the

epithelium lining of mucosal tissues play important roles in the first line of defense

63 against pathogens. The NK cells are present in low numbers and carry CD16 and CD56

markers (humans, mice). These cells of the express TLRs that

mediate the recognition of pathogens in the mucosal system (12).

1.2.2.1 Mucosal DC.

The DCs are present in both the PP and LP, lining the villi, or in MLN and

peripheral lymphoid organs (479). Some DCs also enter the epithelium to sample antigen

in the lumen, similar to the M cell function (548). The prominent population of DCs in

PP express a high level of MHC class II, indicating their role as APC (491). There is also

a subset of DCs in PP with CD11c- phenotype associated with the induction of T cell

tolerance. Tolerance of T cells can be achieved by the production of IL-10 by DCs (318).

These special subsets of DC in the LP exhibit CD11clo and MHC class IIlo. In addition to

APC function, DCs also influence T cell homing. Furthermore mucosal DCs in MLN and

PP express the mucosal homing receptor α4β7 and CCR9, which guide the homing of the

effector/memory CD8 T cells to the intestine as mentioned previously.

1.2.2.2 Toll-like receptors

Members of the TLR family recognize PAMP leading to activation of NF-kB and

other signaling pathways, important for initiation of the immune response. The common structure of the TLR family includes an extracellular domain with leucine rich repeats, a cytoplamic domain called Toll-IL1 receptor homology domain with a signaling function

(439). In humans, ten members of the TLR family have been identified (7, 13, 559).

They are further classified into subfamilies TLR2 (consisting of TLRs 1, 2, 6 and 10),

TLR 3, TLR 4, TLR 5 and TLR 9 (consisting of TLRs 7, 8, and 9). The TLR 1, 2, 4, 5 64 and 6 are involved in the recognition of antigens of bacterial origin such as LPS,

peptidoglycan, lipoprotein and flagellin. The TLRs 7 and 8 recognize single-stranded

RNA and CpG whereas TLRs 3 and 9 recognize dsRNA. The TLR 9 also recognizes

CpG.

Previous studies of immunity to viral or bacterial infection have focused heavily

on the adaptive immune response, i.e. T and B cell responses. Only recently has

emphasis been put on the innate immune response which appears much sooner in the

course of an infection. The TLRs can be found on NK cells, monocytes/macrophages and

DCs. Binding of TLR4 to its ligand leads to the activation of DCs which in turn activate

B cells in the mouse mammary tumor virus (MMTV) infection (105). Direct binding of

MMTV to TLR4 induced maturation of bone marrow derived DCs (upregulated

expression of CD40 and CD80 markers) and induced them to secrete cytokines or

chemokines such as TNF-α, IL-6 and IL-12p40. These chemoattractants cause massive

recruitment of naïve B and T cells to PP on day 2 of infection (105) The activation of NK

cells via TLR3 in response to viral dsRNA or polyinosini-polycytidylic (poly-IC)

increases the cytotoxic effect of NK cells, independently of APC activation (602).

1.2.2.2.1 Roles of TLR in the recognition of microbial components

Different TLRs can bind to various ligands derived from microbial components or

can be activated by endogenous ligands causing autoimmune disease (552). The TLR4

responses to LPS via CD14 are expressed in monocytes/macrophages and neutrophils

which recognize LPS-LPS-binding protein complexes. The response of TLR4 to LPS can

be enhanced by a protein, MD-2, expressed in macrophages, DCs and B cells. The MD-2

65 acts as a to direct TLR4 from ER/cis Golgi to the cell surface (488). A protein

named RP105 on the B cell surface also associates with TLR4 to recognize LPS (504).

The TLR4 also recognizes various groups of ligands, e.g. HSP60 and 70, Taxol (354,

505, 689) and more importantly some viruses e.g. RSV, MMTV and MMLV (105, 281).

For RSV, the F glycoprotein of the virus induces TLR4 activation via the CD14

dependent pathway (389).

In the TLR2 family, TLR2 recognizes many types of microbial agents, e.g.

lipoprotein from Gram negative bacteria, mycoplasma and spirochetes, peptidoglycan

and lipoteichoic acid (LTA) from Gram positive bacteria (663). The TLR2 can also

associate with TLR1 and 6 for the recognition of distinct (666, 749).

The TLR5 which contains immunostimulatory activity at the highly conserved

amino and carboxyl termini responds to flagella of Gram negative bacteria(638).

The TLR3 recognizes dsRNA either as the viral genome, as intermediates during viral RNA synthesis or as byproduct of transcription of DNA virus or even as synthetic dsRNA e.g. poly IC (439). Stimulation by dsRNA leads to the production of type I IFN for antiviral and immunostimulatory activities. The dsRNA also promotes maturation of DCs. The TLR3 expresses several characteristics which are not found in

other TLRs, i.e. its preferential expression in mature DCs and its different genomic

organization and structure from other TLRs (439).

The TLR9 family consists of TLR7, 8 and 9. The TLR9 directly recognizes

unmethylated CpG DNA which is the motif found only in bacterial DNA and very

immunopotent (48). The CpG DNA activates DCs to produce IL-12 which favors Th1

66 like immune responses. The recognition of CpG DNA by TLR9 occurs in ,

whereas that of TLRs 1, 2 and 4 occurs at the cell surface (421). The presence of TLR9 in

the , as well as of that of TLR2 in the phagosomes suggests the possible use of

these receptors as vaccine adjuvants. The TLR7 identifies synthetic chemicals, nucleic

acid like structures, imidazoquinolines, R848 and others (340).

1.2.2.2.2 Distribution of TLRs in different tissues of humans and animals

The classification of DCs is rather complex; they can be classified according to

species, phenotype, tissue origin, function and location. In the previous lineage system,

DCs were classified into cells developed from myeloid restricted and plasmacytoid

restricted precursors. However, cells unable to form T cells still produce thymic

CD8+DCs, which were originally thought to be lymphoid derived (112). Rather each

lymphoid restricted or myeloid-restricted precursor can produce mature splenic and

thymic DC subtypes with some bias in the subset balance (618). Distinct DC subtypes

can be observed in mice differentiated by a number of markers. Currently, murine DCs

are classified into 5 subtypes based on the T cell markers CD4 and CD8, integrin CD11b

and interdigitating DC marker CD205. Human DCs are also classified into plasmacytoid

and myeloid lineages. In addition, another classification of human DCs is based on

pathways of development, since most of results came from studies of DC development in culture from immature DCs or mature DCs (618).

Human monocytes/macrophages express most TLRs except for TLR3 (485).

Human DCs express different TLRs depending on the DC subsets. Myeloid DCs express

TLRs 1, 2, 4 and 5 while plasmacytoid DCs express TLRs 7 and 9 (343). Expression of

67 TLR also depends on the maturation status of human DCs. Immature DCs express TLRs

1,2,4 and 5 whereas the expression of these TLRs decreases in the mature DCs; the

TLR3 is expressed only in mature DCs (485, 710). In mice, phagocytes express all

known TLRs, whereas B cells have several types of TLRs and mast cells express TLRs 2,

4, 6 and 8 (655).

The TLRs are expressed on a wide range of tissues. Each tissue expresses at least

one TLR in humans (773). However TLRs are also expressed by non-immune cells, e.g.

epithelial cells lining the respiratory and intestinal tracts at the basolateral surfaces,

possibly to provide the initial proinflammatory signals to attract professional immune

cells to the site of infection (481, 601). Thus TLRs can be expressed on either apical or

basolateral surfaces of the cells. Furthermore, TLR4 was localized in the Golgi apparatus

of the epithelial cells, which is also the final location of LPS for innate immune

recognition (299). The invasion of bacteria from apical to lateral sides of the intestine

elicits an inflammatory response. Human intestinal epithelial cells also express TLR4 in

the Golgi where LPS is delivered and induce an LPS response (113). Renal epithelial cells express TLRs 2 and 4 to prevent invasion of bacteria to the lumen of the renal tubules (744). Corneal epithelial cells express TLR4 leading to inflammatory responses to parasitic infections of the eyes (590).

The TLR 2, 4 and TLR9 homologous to those of humans have been found in pigs and guinea pigs (352, 617, 682). The TLR2 and TLR9 were preferentially expressed in

MLN and PP of adult swine, more than in the spleen. The TLR2 and TLR-9 expressing

68 cells were found both immune cells, such as T cells and B cells and in M cells in pigs.

The TLR2 was strongly expressed, not only in the cytoplasm, but also in the apical membrane of the pocket-like M cells. Thus the presences of TLR2 and TLR9 on the

MLN and PP enables the host defense to respond to a variety of bacterial cell wall components (682). In neonatal pigs, however, elevated expression of TLR9 was detected only in MLN (617).

1.2.2.2.3 Role of TLRs in the regulation of adaptive immunity

Because monocytes/DCs express at least one kind of TLR and these APCs participate in the signaling of adaptive immunity, TLRs no doubt play crucial roles in both innate and adaptive immunity. Immature DCs in the periphery are activated by microbial components to undergo maturation and increase the expression of TLRs, costimulatory molecules CD80/CD86 and inflammatory cytokines. The mature DCs migrate into the draining LN, and present the antigen to naïve T cells and thus initiate antigen specific responses. The type of T cell responses depends on the type of TLRs activated (540, 712). Activation of TLRs 4 and 9 in DCs induces IL-12 production and

Th1 type responses whereas TLR2 engagement leads to Th2 type responses. The activation of TLR7 induces the production of IL-12 in myeloid DCs and IFN-α in plasmacytoid DCs. Different lineages of DCs also determine the lineage of Th responses.

Myeloid DCs produce IL-12 in response to LPS whereas plasmacytoid DCs produce IFN-

α in response to viral antigen and CpG DNA (116, 621).

The activation of TLRs induces an antimicrobial activity via macrophages or by the production of antimicrobial peptides. The activation of TLR2 by Mycobacteria

69 tuberculosis (MTB) leads to nitric oxide (NO) dependent or independent killing by

macrophages (677). Intracellular infection of macrophages lead to , triggered

by LPS and lipoprotein-TLR2 interaction via FADD and caspase-8 pathways (15). In

addition, antimicrobial peptides can be produced by the epithelial surface of the small

intestine and in the lungs upon engagement of TLRs 4 and 2, respectively (61).

1.2.3 Adaptive immunity

1.2.3.1 CD4+ T cells

In the LP, CD4+ T cells represent 60-70% of the T lymphocytes, mainly

expressing αβ-TCR (84). Most of the CD4 T cells in the LP express mature or memory

phenotypes, CD45RO (humans) and the homing receptor α4β7 integrin (355). The LP

CD4 T cells have lower proliferative activity than the CD4 T cells in PBL, but are

capable of producing cytokines [IL-2, IL-4, IL-5 (Th2) and IFN-γ (Th1)], that provide

help to support B cell antibody production (322).

The CD4 T cells in PP contain different populations of T cells that play regulatory

roles in IgA antibody production, oral tolerance induction and inflammatory responses.

Both Th cell subsets regulate IgA production by the secretion of IL-6 and IL-10 (Th2)

and IFN-γ (Th1) (149, 275). Mucosal lymphoid tissues also contain an abundance of CD4

Treg cells which down regulate CD4 Th1 or Th2 responses. These regulatory cells express

CD25+, bind to CTLA-4 and produce regulatory cytokines such as IL-10 and TGF-β1.

These Treg cells produce TGF-β1, which is considered as a putative switch for T cells for

µ  α antibody isotype switching. The T cell clones from the murine intestine mixed

70 with noncommitted sIgM B cells induced isotype switching to B cells expressing surface

IgA (353). Evidence also indicates that CD3+, CD4+ CD8- T clones are responsible for

this switch of B cells as well as the terminal differentiation of sIgA+ B cells into IgA

producing plasma cells (51). The TGF-β1 induces Cα germ line transcripts and induces

mouse spleen B cells to switch to IgA upon LPS stimulation (149). However, TGF-β1

alone only induced 2-5% of B cells to switch to IgA antibody production (149, 400). The

TGF-β1 with IL-4 and IL-5 induced up to 15-20% of B cell switching to IgA antibody production (454).

+ The CD4 Treg but not CD8 cells play a role in tolerance induction through the

secretion of TGF-β1 to down regulate the host responses to low doses of oral antigen

(353). These suppressor T cells also carry the CD25+ marker and do not proliferate in

vitro (565). They downregulate the expression of the costimulatory signals CD80 and

CD86 on DCs, thus suppressing the APC activity of DCs (705). It appears that these cells

are derived from anergic T cells which have reduced IL-2R expression and the ability to

proliferate. Through production of IL-10, these cells develop into Treg cells that suppress

the immune responses of other cells (99). It is not known where these cells are located

but many cells with suppressor activity are found in the liver (723).

In pig LP, large proportions of cells are CD45+ of which 40% are T cells and 40%

are monocytes/granulocytes (700, 701). In 6-month-old pigs, there are 4-fold more T

cells in the villi than in the crypts, whereas B cell numbers are ten-fold more in the

crypts. In newborn pigs, in contrast, the T cells are more numerous in crypts than in the

villi, most of which are double negative (DN). During the first week of life, the number

71 of CD4 T cells increase rapidly whereas CD8 T cell numbers increase at much slower

rates. A significant increase in CD8 T cells occurs by 5-7 weeks of age. In germ free pigs

not exposed to any extraneous antigen, the differentiation of CD4 and CD8 subsets is not

clear even at 7 weeks of age (566). The CD25 (IL-2R-α) T cells (activated T cells) are

scattered throughout the LP, and the numbers are not influenced by age or environment.

The pig LP contains a significant population of activated cells (651).

1.2.3.2 CD8+ T cells

The CD8+ T cells account for 30-40% of LP T cells with CTL activity (501, 613).

Mucosal CTL are crucial for the clearance of enteric and respiratory viruses as well as

intracellular parasites. The CD8 T cells in the mouse PP are rare and are the source of

CTL in the LP. Studies showed that upon infection of mice with RV or of rats with

vaccinia virus, CD8-CTL were found in PP and MLN (414).

The IEL CD8 γ/δ T cells in the respiratory and intestinal mucosa play roles in innate immunity, IgA B cell differentiation and tolerance. In humans this γ/δ T cell subset

comprises 10-15% of IEL in the small intestine and 20-40% in the colon. The majority of

human CD3 IEL are CD45RO+, indicating that a high proportion of cells are memory or

recently activated cells (267).

In pigs, among IEL, 90% express the T cell marker CD2, 77% are expressed CD8

and only 5% express CD4 (39). The IEL T cells in pigs are recently activated cells,

expressing the CD25 marker (39).

72 1.2.3.3 B cells

The B cells in PP are organized into follicles and carry IgM+/IgD+ surface

markers surrounding germinal centers (GC) to form B cell centrocytes and centroblasts.

The GC B cells are associated with a network of follicular DC (FDC) and CD4 T cells.

Thus both direct B cell signaling and indirect B cell activation through T cell help are

present in the follicles for B cell isotype switching to IgA (642). In pig LP, there are only

a few IgM+ cells at birth and the number of these IgM+ cells exceeds IgA+ cell number

during the first 3 weeks of life. The IgA+ B cells become dominate afterwards (95). Pig

Ig delta genes have been identified and are transcriptionally active (776). The pig IgD H

chains are structurally similar to those of human IgD.

The B cells in the LP are highly differentiated into plasma cells, of which 80% are

IgA ASC (455). Mucosal B cells produce mainly sIgA, which is the major mediator of

humoral immune responses at the mucosal surface. The sIgA antibodies neutralize the

pathogens or act by molecular exclusion to prevent the pathogens from invading the

mucosal surface. The transcytosis of IgA antibody across the intestine removes RV and

prevents entry of the virus (572). The sIgA antibody is resistant to due to the

presence of the secretory component, making it most suitable for the effector functions in

the mucosal surface.

The presence of CD40L/CD40 interaction and TGF-β leads to the large

production of IgA antibodies (175). The T cell subset γ/δ IEL also has an impact on IgA

B cell differentiation (226). This subset has been shown to produce Th1 and Th2 cytokines as well as TGF-β for IgA class switching (660).

73 1.2.3.4 Biological roles of IgA antibody

The roles of sIgA antibody in the mucosal secretions have been demonstrated for

different pathogens.

1.2.3.4.1 Inhibition of adherence

From the structure of sIgA, it is postulated that sIgA antibodies surround the

microbes with a hydrophilic shell of the Fc portion –secretory components (Fc-SC) part

of the molecule, thus preventing the attachment of pathogens to the mucosal surface. The

sIgA can also inhibit colonization via agglutination as observed for H.infuenzae (350).

The IgA and sIgA antibodies can bind to bacteria and antigen via their carbohydrate

chains. The IgA2 can agglutinate E.coli through mannose rich chains (742).

Adhesion of S-fimbriated E.coli to human epithelial cells is inhibited by sialyloligosaccharides on s-IgA (605).

1.2.3.4.2 Neutralization of viruses, enzymes and toxins

The sIgA antibodies neutralize virus possibly by inhibiting viral binding to cellular receptors. High concentrations of sIgA to virus HA inhibit the cellular attachment (28). The polymeric IgA (pIgA), formed during the transepithelial transport of

IgA mediated by the polymeric immunoglobulin receptor (pIgR) can also provide virus neutralization activities. The pIgA antibodies to influenza HA inhibit the internalization

or intracellular replication of the virus (28). Transcytosis of IgA RV antibodies inhibits

RV replication in mice (572). Even IgA antibodies against the non-neutralizing

VP6 inhibits RNA transcription (208). Intraepithelial virus neutralization by IgA

74 antibodies was also observed for many other viruses e.g. HIV, measles, Sendai, etc (446,

741, 753). Enzymes such as neuraminidase glycosyltranserase of S.mutants, cholera toxin, heat labile enterotoxin or C.difficile toxin can be effectively neutralized by IgA

(335, 425, 637).

1.2.3.4.3 Inhibition of antigen presentation.

It was suggested that the pIgR mediates the transport of pIgA by enterocytes and helps to remove antigens if they form complexes with pIgA in the LP (527). In addition, sIgA inhibits the uptake of reovirus through M cells (629). The sIgA can interact synergically with other antimicrobial factors such as lactoferrin, causing antibody interference with channels of iron uptake, used by many mucosal pathogens (228). The sIgA can act in concert with lyzozymes and complement to lyse E.coli (8).

Other isotypes such as IgM and IgG antibodies, produced at the mucosal surface at lower levels, can contribute to the immune defense. The IgG antibodies can be transferred across the intestine via FcRn receptors, which will be discussed in detail later.

The numbers of IgG plasma cells in the LP increase during inflammation.

1.2.4 Leukocyte trafficking in the mucosal immune system

Both mucosal T and B cells, activated in PP, express α4β7 integrin and use this integrin for their entry into the mucosal tissues (20, 358). The counter receptor for this integrin is MadCAM-1, which is expressed on mucosal high endothelial venules (HEVs)

(52). In addition, L-selectin contribute to the mucosal homing process (81). Naïve cells in PP express CD45RA+ and IgG+ B cells express high levels of L-selectin but only

75 intermediate levels of α4β7. In contrast, mature T and B cells (CD45RO+ memory T cells

and sIgD-B cells) express both L-selectinhigh and α4β7 high (203). The expression of

adhesion molecules also depend on the route of immunization. Most circulating IgA and

IgG ASCs induced by oral or rectal route express α4β7, whereas the ASCs induced by IN route coexpress L-selectin and α4β7 (202, 541).

Trafficking patterns of IgG and IgA ASCs are different. The IgA B cells activated in the PP or MLN seed other mucosal sites including intestine, urogenital tract, mammary glands, salivary gland and respiratory tract, whereas cells activated in the lymphoid tissues of the upper aerodigetive tract e.g. oral cavity, oesophagus, bronchi and lungs migrate preferentially to the salivary gland and respiratory tract with less traffic to the intestine (385). The activated IgG B cells, on the contrary, preferentially migrate to non- mucosal lymphoid tissues regardless of whether they are originated from mucosal or peripheral lymph nodes

The CC chemokine receptors contribute to the trafficking of leukocytes in the mucosal system. The entry of B cells into PP involves contact with HEV via CXCR5,

CCR7 and CXCR4 (507). The CCR6 is expressed on most B cells, CD4 and CD8 T cells and CD11b+DCs (383, 410) and CCR6 is involved in the localization of DCs to PP (386).

On T cells, this receptor is mostly found on CD45RO+ memory cells (humans) or on

CD44hi and CD62Llo (mice), suggesting the role of this chemokine receptor in trafficking

of T memory cells. Mice deficient in CCR6 showed an abnormal expansion of LP T cells,

IEL T cells, smaller PP and defects in IgA antibody responses (739). The CCR9 is

present on T cells in IEL including the γ/δ subset and intestinal homing T cells and is

76 preferentially expressed on small intestinal T cells (387). The CCR9 T cells found in blood express the α4β7 integrins which are the mucosal homing receptors (772). Mice deficient in CCR9 showed an increase in γ/δ TCR T cells in peripheral LNs, but not in the IEL (748). The CCR10 is the receptor for chemokine CCL28, which is expressed by epithelial cells at various mucosal sites. The CCR10 directs IgA producing plasma cells into mucosal sites where the CCL28 expression takes place (385, 397).

1.3 Neonatal immune responses

1.3.1 Overview

The first challenge to neonatal vaccination is the immature immune system of the newborn. This immaturity is attributed to both the limited source of immune cells and the immature cell function. The newborn has a limited resource of immune cells. A germ- free animal has about 10% of ASC and very few T cells in the gut compared to a conventional animal. Because the gut immune system makes up 2/3 of total immune cells, this dictates the limited size of the neonatal immune system (270). The immaturity of immune cell function, exhibited by inadequate phagocyte and lymphocyte functions, leads to poor and delayed immune responses. There is insufficient help for B cell to produce antibody as well as for CTL and memory cells to develop.

The other challenge to neonatal vaccination is the Th2 bias in the immune response. It is postulated that the Th2 environment of the mother plays a role in this Th2 bias in infant immune response (270). This Th2 condition is produced to protect the from rejection by the mother’s immune system during . The activities of NK cells and macrophages which are enhanced by Th1 IFN- γ and CTL are down-regulated 77 to avoid destruction of a foreign body the fetus, by the mother (726). This down

regulation is achieved mostly via IL-10 which at the same time upregulates Th2

responses (121). The presence of FAS-ligand in the placenta induces CTL to undergo

apoptosis (309). The T cell suppressors in the placenta also inhibit the cytotoxic activity

of CTL (659). The human leukocyte antigen (HLA) expression in trophoblasts at the

placenta is inhibited. In addition, expression of HLA-G exclusively in placenta helps it to escape the attack by NK cells which is HLA-I and II independent (525). The function of

T regulatory cells in maternal tolerance to the fetus is also important. The T regulatory cells (CD4+CD25+), found in the human deciduas which are in direct contact with the

fetus, play an important role in the suppression of maternal immune responses against the

fetus (282). Aluvihare et al (17) showed that depletion of CD25 T cells in mice led to

gestation failure.

The following sections focus on the properties and functions of some immune

cells of the innate and adaptive immunity in neonates in comparison to those of adults in

humans and animals.

1.3.2 Innate immunity

1.3.2.1 Dendritic cells

Dendritic cells are central APC, essential to induction of non-specific or specific

immune responses. The decrease in antigen responses in neonates may reside in this APC

population and others (B cells and monocytes) (336). The function of neonatal DCs as

APC is reduced compared to adults and shows a bias against Th1 responses (396).

Human DCs from cord blood failed to produce IL-12 upon stimulation with 78 lipopolysaccharide (LPS). The kinetics of the upregulation of the DC surface markers

such as HLA-DR and CD86, CD25, CD83 are much reduced in cord DCs compared to

adult DCs. Cord blood DCs also fail to down-regulate the expression of CCR5 and

induce low levels of IFN- γ production. However cord blood DCs exhibit similar rates of

TNF-α and IL-10 production upon LPS stimulation compared to adult DCs. Thus human

neonatal DCs are programmed to favor the induction of Th2 immune responses (via

reduced IFN-γ and IL-12 production). This bias contributes to the overall Th2 bias of the

neonatal immune system in humans, which will be discussed in later sections.

A number of studies of mice have led to different conclusions from those of

humans about the characterization and function of neonatal DCs compared to adult DCs.

The murine neonatal DC subsets differ from adult DC subsets (653). In mice, neonatal

DCs exhibit higher a frequency of CD11clow marker compared to adult DCs which is

mainly CD11chigh (653). In regard to CD4 and CD8 markers, at birth, almost all DCs are

CD4-CD8- (DN) and remain so until the 2nd week of life. Neonatal DN DCs represent a transitional state, which first gives rise to CD8α+ and later generate CD4+ DCs. The CD4+

DC population continues to increase and becomes the major subset in the adult spleen.

Fifty per cent of adult murine DCs express CD4+, 25-30% express CD8α+ and the rest is

DN. The CD4+DC population in the adult spleen also express the myeloid markers e.g.

CD11b, F4/80 and 33D1, only low levels of CD1d and CD205, which are associated with

CD8α+CD4- DCs. In comparison, neonatal DCs, mainly DN, express CD205 and CD1d

of the CD8α+ but express poorly CD11b and 33D1. The CD205 marker which is rich in

carbohydrate recognition domains allows the binding and recognition of bacteria by DCs.

The CD1d marker facilitates the recognition of glycolipid antigens and represents the link

79 between DCs and NK T cells. However in spite of these subset differences, the murine

neonatal DCs are fully competent in their innate immune function. The neonatal DCs

produce IL-12 more efficiently than adult cells. The major producers of IL-12 are DN

DCs in neonatal mice compared to CD8α+ in adult mice. When stimulated through TLR9

and TLR3, neonatal DCs are more efficient in the production of IFN-α and IFN-γ for

antiviral activity and NO-mediated anti-bacterial activity. The murine neonatal DCs also

exhibit up-regulation of co-stimulatory signals including MHC class II, CD40, CD86 (a

costimulator for T lymphocyte activation, expressed in B cells, monocytes, DCs and some T cells) and CD25 (subunits of IL-2 receptor), which will be discussed subsequently (396).

1.3.2.2 Monocytes

Functional immaturity of neonatal immune responses could also reside in the reduced co-stimulation signals provided by monocytes to T cells during antigen uptake and presentation (336). These costimulation signals in monocytes are MHC class II molecules, CD40/CD40L, CD80/CD86 and CD28/CTLA-4. Fewer cord blood monocytes express MHC class II than adult cells. However, there is an increase in the percentage of

MHC class II monocytes during gestation, although still lower than the levels in adults.

The numbers of CD40+ and CD86+ monocytes in the fetus and neonate were comparable

to adults, yet the expression of CD80 is absent on fetal/neonatal cells (188, 189, 336).

Monocytes are also involved in the first line of defense against microorganisms.

Receptors on the monocyte surface involved in monocyte responses to microbial products

are CD11b, CD35 (type 1 complement receptor), CD14 and TLR4. The expression of

80 CD14 increases with gestational age: the CD11b and CD35 are also coexpressed with

CD14, although the intensity is not stable (336, 338). The TLR-4 expression in murine lung tissues also increases extensively after birth (271).

1.3.2.3 Natural killer (NK) cells

There are contradictory reports about the maturity of neonatal NK cells. In a study of cells from , the NK cytotoxicity in neonatal umbilical cord lymphocytes was found to be similar to that of maternal peripheral blood cells and correlated with a similar proportion of CD56 NK and CD8 CD56 LAK cells (185). In contrast, a study of preterm and term infants showed significant impairment of NK cytotoxicity activity compared to that of adult NK cells (232). It is not clear why there is such discrepancy in the neonatal NK activity between different studies.

1.3.3 Adaptive immunity

1.3.3.1 T effector and memory cell responses

There are significant differences between neonatal and adult T cell phenotypes, suggesting a reduction in the T cell responses of neonates (232). In humans and pigs, newborns exhibit a higher percentage of CD4 but a lower percentage of CD8 T cells resulting in higher CD4/CD8 ratios in blood. Neonates also exhibit lower numbers of

CD4 and CD8 IFN-γ producing cells. In human neonates, the CD4/CD8 ratio decreases with age, suggesting a gradual maturation of cytotoxic responses. In humans, all neonatal

T cells express high levels of CD38 (activation marker for B and T cells), similar to most thymocytes, indicating immature transition from thymocytes to mature adult T cells

81 (545). The CD45RA marker is expressed in more than 90% of neonatal T cells indicating

the unprimed state of the cells, whereas only 40-60% of adult T cells express this marker

(148). A higher percentage of CD4+/CD45RA+ (naïve T cell marker) but a lower

percentage of CD4+/CD45RO+ (memory T cell marker) were found in newborns than in

children and adult cells, indicating the naïve phenotype of T cells (232). However, almost

all newborn CD4 T cells express the dim isoforms of CD45 markers (~97%), whereas

28.4% and 53% of adult T cells express CD45RA+ bright and CD45RO+ bright markers,

respectively (276). Because cells with different CD45 isoforms reflect differences in T cell receptor signaling, these findings suggest differences between neonatal and adult T cell function (500). The CD40 ligand, whose expression is restricted to the activation status of CD4 T cells, is absent or reduced significantly on cord blood T cells compared

to adult T cells (182). This reduction correlates with the reduced antibody production in

newborns and infants.

Stimulation of human T cells with mitogens, alloantigens, mitogenic antibodies

and superantigens also demonstrated the differences between human adult and neonatal T

cell functions (545). The immune activities of neonatal umbilical cord are lower than of maternal peripheral blood cells with a lower degree of polyclonality and repertoire diversity, and lower T cell responses to phytohaemagglutinin (PHA) (185). Yet other studies showed that the response of T cells to PHA is similar between newborn and adult cells, whereas significantly lower responses to anti-CD3 or anti-CD2 antibodies occurs

(418, 762). The difference in the T cell stimulation by mitogens between the two populations, cord blood vs. newborn cells, was not clear, but could be due to the difference in proportion of T cells in each population. The IL-2 receptor expression, IL-2 82 synthesis and proliferation are also reduced in cord blood CD4 T cells (238). Neonatal T cells also respond strongly to IL-4 proliferation signals, whereas adult T cells remain unresponsive which is another example of Th2 bias in neonatal immune responses (183).

Thus many studies demonstrated a deficiency in neonatal T cell functions, whereas others documented that neonatal T cells, although naïve, can induce responses under appropriate stimuli. When stimulated with anti-CD3 antibody, neonatal T cells express similar levels of CD40 ligand as adult cells (545). Although the level of CD40 ligand expression depends on the concentration of the antigens, adult T cells required much lower phorbol myristate acetate (PMA) concentrations for CD40 expression compared to neonatal T cells. In response to TCR, neonatal cord blood T cells can be stimulated to produce cytokines of both Th1 and Th2 types (129). The neonatal T cell population, however, is composed of high numbers of self-reactive cells, which indicates the incomplete T cell tolerance process in neonates (3). For alloantigens presented on APCs, cord blood T cells appear unresponsive, even upon IL-2 stimulation, suggesting the anergy state of the cells and the activity of CD8+ suppressor cells (556).

In murine neonates, it has been shown that the Th1 function is impaired (9).

Many studies demonstrated the Th2 bias of neonatal immune responses, with different antigens, live or attenuated viruses and aluminum adjuvants, (75, 76, 375). The Th2 biased responses in neonates are not related to the antigen dose, the carrier and do not require the continual presence of the thymus or spleen to replenish the Th2 cell pool.

Splenectomized neonates showed no difference in Th2 bias memory responses in LN, which indicates that the spleen is not required for Th2 dominant memory responses in neonates, and thus these biased responses are more likely controlled by events in the 83 effector development (9). The major differences between neonates and adults reside in

IL-4 production and this difference persists until 5 weeks after immunization with keyhole limpet hemocyanin (KLH). At the same time, the level of INF-γ does not differ considerably between neonatal and adult LNs and spleens 1, 2 or 5 week after KLH immunization. This Th2 memory response induced by KLH immunization of neonates continues to develop when the same neonates were immunized with KLH as adults. Thus the increase in IL-4 production during primary immunization skews the development of memory effectors to a Th2 lineage and impaired Th1 function. It has been postulated that failure to develop Th1 memory effector function results in failure to develop Th1 effector cells, leading to the Th2 bias (9).

In young pigs, T cells exhibit reduced responses from birth up to 4 weeks, as indicated by low proliferative responses of blood mononuclear cells when exposed to a low antigen dose of a T-cell dependent antigen. The responses were not enhanced by the addition of IL-2 (606).The T lymphocytes from newborn pigs are immature in providing cognate help for B cells in antibody production. The IgG and IgM antibody production by the newborn B cells was enhanced when cocultured with adult T cells, but not with newborn T cells (652). The newborn porcine T cells also show suppressive activity during the suckling period, this activity; however, decreases with in adult pigs.

1.3.3.2 Neonatal B cell repertoire

The poor and restricted antibody responses of neonates can be partially explained by the immaturity of B cells, which possess restricted repertoires. The repertoire restriction was demonstrated in the biased expression of proximal V gene

84 families coding for the B cell receptor, the absence of N region diversity and the low rate

of mutations in V genes in humans and mice (539). The diversification of neonatal B cell

repertoires after immunization is mediated by activation of recombinase-activating genes

(RAG), which induce V(D)J rearrangement and light chain replacement (73). Upon

influenza infection, the human neonatal B cells expressed mainly one VH gene family in the first week of life, and diversification of the VH gene only started from the second

week with preferred usage of only a few VH genes (73).

However, a study by Weitkamp et al (728) suggested that adult B cells specific to

RV also exhibit bias in the use of VH gene segments, similar to cells from infants aged 2-

11 months. In particular, infant and adult RV specific B cells from blood showed frequent

usage of VH4 and VH1-46, a uniform bias toward the D3 and JH4 gene segments and

similar distributions of Vκ, Jκ and Jλ families. Thus the poor antibody responses to

viruses in infants can not be explained only by the residual fetal bias of the B cell

repertoires.

In addition, neonatal swine B cells are fully functional at birth. The repertoires of

oligoclonal IgA and IgM antibodies in a non-inductive site of the mucosal immune

system (parotid gland) become polyclonal in Gn pigs, suggesting that the expansion of

the fetal and neonatal B cells that undergo isotype switching is not driven by

environmental antigens (106). Neonatal isotype switch B cells can differentiate into ASC

in the presence of intestinal commensal bacteria in a T-cell dependent manner or via

direct stimulation by bacterial products in a T-cell independent manner (108). Upon

infection with PRRSV, Gn pigs showed dramatic increases in serum IgM, IgG and IgA

85 antibody responses which were not dependent on bacterial colonization (407). The serum

antibody levels of PRRSV infected Gn pigs were 42-94% of adult levels depending on

isotypes, similar to those of bacteria colonized pigs. The pigs have B cells that produce

antibodies even 2 days before birth (60). The generation of Ig-producing cells in PBL

from suckling pigs increased with age, and reached about half the adult mean numbers at six weeks of age (652).

1.3.3.2.1 Activation of neonatal B cells- in vitro system

The activation of B cells involves the interaction of many types of cells,

cytokines, growth factors, etc. Replication of this process in vitro allows assessment of

individual components in this interaction. The lack of adequate numbers of effector cells

and accessory cells and the antigen naivety of B cells make it difficult to activate

neonatal B cells. Stimulation of neonatal B cells with antigen is even more difficult due

to the low number of antigen specific B cells in the population (a few cells per thousand).

Fayette et al (206) successfully established an in vitro system for the differentiation of human naïve B cells into plasma cells. In this system, the role of DCs was strongly emphasized. The B cell primary responses take place in the T cell-rich area of the GC of

secondary lymphoid tissues in which DCs also reside. Upon activation, 12% of naïve B

cells differentiated into plasma cells and this proportion increased in the presence of IL-2

and IL-10 without CD40 signaling and DCs. Addition of DCs to this system enhanced the

plasma cell differentiation to 57% B cells, secreting IgM, and IgA and IgG antibodies

(206). It was shown that DCs act in synergy with IL-2 in the early stage of differentiation

which is CD40 dependent, whereas in the later stage, IL-2 and IL-10 act together to

86 promote plasma cell differentiation in a CD40 independent manner. Therefore a two-step culture was developed in which B cells were cultured with DCs, CD40-L transfected cells and IL-2 in the first step for 7 days. After that the B cells were harvested and seeded with

DCs in the presence of IL-2 and IL-10 for 4 days (206).

The IL-12 was also found to contribute to DC-induced differentiation of naïve B cells, demonstrated in in vitro systems with cells from different animal origins. The IL-

12, produced by CD40-activated DCs was essential for promoting human plasma cell differentiation and IgM antibody secretion (181). Similarly, B, T cells and DCs of porcine origin cocultured in the presence of IL-12 induced plasma cell differentiation, whereas the formation of memory B cells was IL-12 independent (163). In addition, interaction of T cells with the CD11c+CD4+CD3- DC subset was crucial in providing

signals for B cell differentiation into memory cells. The bovine B cells and DCs can be

cocultured in the presence of a CD40L transfected cell line to induce IgG1 antibody

responses (40)

Although antibody secretion can be measured in these systems, it is not clear in

any of these studies whether antigen-specific antibody production can readily be detected

due to the low percentage of the specific antigen responsive cells in the antibody

producing pool. Measurement of antigen-specific antibodies from naïve B cells was

accomplished by Colino et al. in which the mouse DCs pulsed with S.pneumonia antigen

in vitro were introduced into recipient mice to induce antigen specific IgM and IgG

antibodies 14 days after cell transfer (150).

The reason for the difficulty in stimulating naïve B cell differentiation into

antigen specific plasma cells was proposed by Tangye et al (667). In regard to naïve and

87 memory B cells, the human memory B cells entered the first cell division 20-30h earlier

than naïve cells in response to stimulation with CD40 ligand alone or with IL-10,

whereas subsequent cell division time was similar in both groups. In addition, increased

proliferation of memory B cells can be mediated by IL-2, whereas this cytokine did not

improve proliferation of naive cells. Naïve B cells remained undivided for 72h, and only

a small portion (up to 20%) entered cell division after this period.

1.3.3.3 Mucosal immune system in neonatal pigs

In pigs the mucosal immune system doesn’t develop before birth, whereas the systemic immunity has already developed. However, two days before birth, most organs in pigs contain some ASC, suggesting the immunocompetence of the pig immune system

(60). The T cells represent 70% of lymphocyte population in MLN and IEL of neonatal

pigs (733). The IEL in neonatal pigs do not proliferate well in response to Con A, yet

they exhibit a high proliferative response to exogenous IL-2, in relation to the change in

T cell phenotype from CD2- CD4- CD8- cells to CD2+ CD4- CD8- cells as the age of pigs

increases. In contrast to IEL T cells, T cells in MLN proliferate in response to Con A, but

become unresponsive to IL-2, indicating the different stages of activation and

responsiveness in neonatal pigs. Mucosal T cells in neonatal pigs are more susceptible to

apoptosis, which is one of the regulatory mechanism to eliminate the excess mucosal

effector cells produced by the active thymus in the young (38). However unlike adults,

the involvement of T suppressors as a regulatory mechanisms doesn’t exist in neonates

because by 5 weeks the T suppressive activity declines (652).

88 In conventionally raised neonatal pigs, PP follicles and T cell areas expand

rapidly within 12 days after birth to reflect the structures in the mature animal (38). The

T cells that enter the intestinal villi mainly express the CD2 marker but not CD4 and CD8

markers. Expression of MHC class II antigen in the intestinal villi increases with age. The

CD4 T cells enter the intestine by 2-4 weeks, at the same position as in the intestine of

adult pigs. The movement of CD8 T cells takes place following the CD4 movement to the

epithelium and LP of the intestinal villi. The normal distribution of T cells as in adult

animals is as follows: T cells are confined in the villi, not around the crypts, CD8 T cells reside immediately bellow the epithelium of the villi, whereas CD4 cells are located deep in the villi (38).

1.3.3.3.1 Gnotobiotic pig model for human enteric viruses and mucosal immunity

The Gn neonatal pig model represents a good model to study the mucosal immune responses to enteric viruses, in particular RV. The pig gastrointestinal physiology is comparable with that of humans (38). The Gn pigs are susceptible to infection and diseases induced by several HRV strains for up to 8 weeks of age (589). The histopathologic lesions in the small intestine of pigs after HRV infection are similar to those in humans (715). Because of the impervious nature of the sow placenta, the Gn pigs, derived by hysterectomy, are devoid of MatAb, but are immunocompetent, allowing the study of the true neonatal immune responses. In addition MatAb, cytokines and colonizing bacteria can be added to study the effects of these components on neonatal immune responses (32, 522, 589). Similarly, Gn pigs infected with PRRSV developed bronchial and submandibular lymph nodes that were 5-10 times larger than those in

89 E.coli colonized animals (407). Typical lesions, infiltration of inflammatory cells, deposition of antibody to the basement membrane and vascular of kidney were observed in Gn pigs similar to conventional pigs infected with PRRSV. Thus Gn pigs were demonstrated to serve as a useful model to study virus-induced immunopathology.

Despite these advantages, there are disadvantages in using the Gn pig model.

Under the Gn conditions, little development of the mucosal immune system is observed, which indicates a role for the normal intestinal flora or exposure to common enteric viruses (, RV, etc) to mature the immune system (516). In and newborn pigs as in human infants, limited Vh and Dh segments are used, leading to a limited B-cell repertoire (654). This limitation persists longer for the Gn pigs. In comparison, there is an increase in V segment usage resulting in increasing B cell repertoires in conventional pigs and in germfree pigs colonized with intestinal flora

(107). In the Gn pigs, the T cell population is also limited. The T cell population of 49- day old the Gn pigs is similar to that of 5-day old conventional pigs (566).

However, all the above mentioned studies of Gn pigs are limited to pigs that were not exposed to any extraneous antigen. When stimulated with RV antigen (live virus infection or vaccine regimens), the immunocompetent neonatal Gn pigs developed strong intestinal and systemic T and B cell effector and memory responses (589, 716, 770).

Similarly, Gn pigs infected with PRRSV showed elevated serum antibody responses, independent of bacterial colonization and hyperplastic lymph nodes of larger size than

90 those of bacterial colonized pigs (407). Thus viral antigen stimulation can also substituted

for the effects of microflora colonization in promoting maturation/development of the

mucosal immune system in neonatal Gn pigs.

1.4 Maternal interference with neonatal active immune responses: sources and

mechanisms of interference

1.4.1 Sources of maternal antibodies (MatAb)

Depending on the species, MatAb transferred to neonates can be derived from different sources, as summarized in Table 1.3 (259). In , MatAb are transferred across the placenta prenatally or supplied postnatally as colostrum and milk or by both routes. In particular, the hemendothelial and hemochorial structures of the placenta of humans, primates and rodents allow passive transfer of immunoglobulins mainly IgG across the placenta to the fetus (Table 1.3). In guinea pigs and rabbits, MatAb are transferred to fetuses via yolk sacs whereas postnatal transfer of MatAb is not available in these species. In and other animals such as pigs, horses and donkeys, on the contrary, no transfer of MatAb occurs across the placenta and therefore the newborns acquire passive immunity entirely from colostrum and milk. In , reptiles and birds,

MatAb are deposited in eggs to be transferred to the offspring via yolk sac.

The persistence of MatAb in neonates depends on the species; from 10 days in fish to 9 months in humans, as a function of body size and catabolic rates (Tables 1.3 and

1.4). In addition, the half-life (t1/2) of MatAb specific to a particular pathogen might vary

91 according to the pathogen, the specificity and the amount of antibodies. For some viruses,

specific antibodies from immunized cows transferred to neonatal calves have longer half-

lives than those from unimmunized animals (227).

1.4.1.1 Maternal antibodies from milk

In humans and most animals except cattle, IgA antibodies are the major

immunoglobulin in colostrum and milk of these species. However in pigs and horses, IgG

antibody is the major isotype in colostrum, but later changes to the predominance of IgA

antibodies in milk after the transition from colostrum to milk. In cattle, IgG1 antibodies

persist as the major isotype in both colostrum and milk and they are selectively

transported from serum into the colostrums and milk.

These antibodies play important roles in the early immune defense of neonates.

The secretory IgA and IgM antibodies present on the mucosal surface of the intestine

mask the surface receptors on the enterocytes and prevent adherence and entry of enteric

pathogens. These antibodies can also neutralize infectious virus and bacterial enterotoxins. They activate the complement pathway and exhibit bactericidal action. The

presence of sIgA antibodies was shown to prevent the entry of viruses such as RV to the

cytosol (445).

Antibodies can be either transferred from blood to mammary secretions or

synthesized by local ASC in the mammary glands. The IgG is transudated to the

mammary secretions via the Fcγ receptors on the surface of epithelial cells, similar to the 92 neonatal receptor (FcRn) for epithelial cell transport of IgG through the placenta. The

structure and the role of FcRn in the transfer of maternal IgG antibodies to neonates will

be discussed subsequently.

The sIgA antibodies secreted in milk play an important role in passive immunity

of infants. The IgA dimer-J chain-producing lymphocytes migrate to the mammary gland from the intestinal lymphoid tissues. Secretion of IgA antibodies into milk occurs via the pIgR. These sIgA antibodies are specific to many pathogens that the mothers have been exposed to in their lives, thus they provide protection to infants. Milk also has prolonged effects even after breast feeding is terminated, as confirmed by higher numbers of hypersensitivity reactions and respiratory infections in non breast-fed infants compared to breast-fed infants beyond the period of breast-feeding (103, 627). Antibodies, e.g. anti- idiotypic antibodies from milk can also enhance infant responses to vaccines; the detailed mechanisms will be discussed in a later section.

The following sections explain the mechanisms of transfer of antibodies (IgA) into mammary secretions.

1.4.1.2 Transfer of sIgA antibodies into mucosal secretion

1.4.1.2.1 Structure of the pIgR The polymeric Ig receptor (pIgR) is an important receptor for the transfer of IgA and IgM antibodies into mucosal secretions. The pIgR is a glycosylated membrane protein, a homolog of the Ig superfamily. The extracellular portion of the molecule consists of five homologous domains, resembling the V domain of the Ig superfamily

(542) (Figure 1.2). Interaction between the pIgR and IgM is high with Ka=6x10-8 to

93 2x10-9M, whereas the IgA binds to the pIgR via both covalent and noncovalent bonds

(253, 464). Interaction between this receptor and IgA occurs via the extracellular Ig domains 1 and 5 of the pIgR and Cα2 and Cα3 domains of both IgA molecules (234,

542).

1.4.1.2.2 Functions of pIgR

The pIgR performs several important functions including protection of IgA from proteolysis by adding the secretory components (SC) to mediate immune exclusion by sIgA at the mucosal surface. Secretory component can be released as the free form into the lumen and serves as a scavenger to prevent the interaction between the pathogen and intestinal epithelial cells. Yet some pathogens can utilize pIgR to invade the mucosal defense, such as pneumoniae (89). However the inefficiency of the apical to basolateral movement of pIgR and the cleavage of pIgR at the luminal surface prevent the recycling of this receptor, and thus reduces the chance for the pathogens to use it for internalization (533).

In mice, milk sIgA antibodies are produced locally in the mammary gland. During the initial stage of lactation, milk IgA antibody may originate from blood, although the derivation and contribution of milk IgA antibody derived from blood remains controversial (89). In sows, 70% of colostrum/milk IgG and >90% of IgM and IgA antibodies were synthesized in the mammary gland (78). In addition, IgA ASCs that arise in the intestinal lymphoid tissue after oral immunization with transmissible gastroenteritis virus (TGEV) were the first description of trafficking of IgA ASC to populate the mammary gland (70, 582, 695).

94 The levels of pIgR in the mammary gland vary according to the stages of pregnancy and parturition. In the mammary gland, the pIgR is expressed on the secretory epithelial cells surrounding the ducts and alveoli. In mice, the pIgR levels increase gradually during mid-pregnancy, peak at parturition then drop to minimal levels around post parturition day 7 (691). Then the pIgR level increases again and maintains a high level even at the end of lactation period, corresponding to the increased level of sIgA in milk.

Similarly the number of ASC in the mammary gland also differs according to the pregnancy and lactation stage (691). In mice, the IgA ASC number is low in the mammary gland during late pregnancy, increases around parturition and increases substantially during mid and late lactation. When labeling lymphocytes from the MLN and inguinal (ILN), it was observed that lymphocytes in the mammary gland were derived from both the MLN and ILN (593). In particular, T cells and large B cells

(plasma cells) migrated from the MLN whereas small and medium B cells (memory cells) migrated from the ILN. The ASC from the MLN also traveled to the mammary gland via interaction with MadCAM-1, which together with the pIgR was expressed in early-mid pregnancy. The levels of MadCAM-1 declined during lactation whereas the levels of pIgR and the IgA-ASC numbers continued to rise until the end of lactation

(691). However, MadCAM-1 was not considered as the rate limiting step for the B cell traffic from blood to the mammary gland since the rise in the MadCAM-1 level was out of phase with numbers of IgA ASC. In contrast, the rise of the pIgR level was sufficient to explain the increase sIgA levels in milk. Of interest, expression of the pIgR is regulated by lactogenic hormones and IFN-γ (555).

95 1.4.1.3 Transfer of IgG across the placenta and from milk to neonates

The only antibody which is able to across the placenta of mammals is IgG, which

mediated by the neonatal Fc receptor, called FcRn. The following section describes the

structure of this receptor and the mode of transfer of the IgG antibody across the placenta

using this receptor. This receptor is also postulated to play role in the transfer of milk IgG

antibodies to neonatal ruminants and in the maintenance of the passive IgG antibody

levels in the gut and in serum for passive protection. In ruminants, pigs and horses, this is

the only method of acquiring passive immunity in these animals due to the

impermeability of their placenta. Of note, transport of IgG differs from that of sIgA

antibodies as the FcRn is recycled whereas the pIgR is degraded after transcytosis.

1.4.1.3.1 Structure of the FcRn

The FcRn was first isolated from rats in 1989 (632) but its function in maintaining the homeostatic status of serum IgG antibody was postulated in 1970 by Brambell (83,

179). The FcRn is encoded for by a gene lying outside of the MHC region, splitted into two exons for the cytoplasmic and transmembrane domains, respectively (631). The

FcRn, an MHC class I homolog, is a heterodimer of β2 microglobulin and a larger subunit α (45-53 kDa) with 3 extracellular domains (631) (Figure 1.2). The β2m

subunit, similar to one found in MHC class I molecules, mediates contact with IgG. The

larger α subunit is glycosylated at 4 different sites and alteration of these glycosylation

sites lowers the binding of FcRn to IgG. The FcRn binds to IgG at a ratio of 2:1; that is

FcRn is required to form a dimer to bind to IgG with high affinity. The FcRn

96 extracellular and transmembrane domains share homology with MHC class I respective

regions, whereas the cytoplasmic domain differs, corresponding to differences between

functions of MHC class I and FcRn molecules.

Consistent with the conserved role of FcRn across different animal species, the amino acid residues in the binding region of FcRn with IgG antibodies are also highly conserved between different species. There is between 50-60% homology between humans and rats in the α and β chain domains of the FcRn (631). Thus most functions of

FcRn derived from studies in rodents are implicated for humans.

The FcRn interacts with IgG antibody via the Fc residues at the CH2-CH3 interface. This interaction is pH dependent: a pH of 6.0-6.5 is the maximum condition for binding. This is due to the presence of a single highly conserved histidine residue at

position 433 (or 435, depending on the studies). Other residues at the CH2-CH3 interface

are also involved in the interaction with the Fc region of IgG antibody, which explains

the variation in affinity of FcRn with different IgG isotypes.

1.4.1.3.2 Functions of FcRn

(i). The FcRn has been suggested to play a major role in transfer of IgG antibody to the

fetus via the placenta (633). The FcRn is also found on the epithelial layer of the placenta

in humans. The FcRn found in the syncytiotrophoblast cells tends to be expressed only 2-

3 weeks before parturition and drops considerably after birth (634). In humans, the

concentration of maternal IgG concentrations in fetal blood increase from early in the

second trimester and peak at term; most antibodies are transferred during the third

trimester. These findings coincided with the observation that pre-term infants have lower

97 concentrations of anti- IgG antibodies compared to those of full-term infants

(180). An in vitro placental model was used to analyze the materno-fetal transfer using recombinant IgG1 antibody with a mutation at His 435 site (H435A). The mutated form of antibody showed reduction in the amount transferred across the placenta compared to the wild-type antibody indicating that binding of an IgG molecule to FcRn is a prerequisite for transport across the placenta (214). However the mechanism of IgG transport across the endothelium of fetal blood vessels is not understood (630).

(ii).Role of FcRn in general homeostasis of serum levels of IgG in different species.

Brambell suggested the existence of a protection receptor for serum IgG (FcRp), which explained why serum IgG can survive longer than other isotypes in blood (82, 339).

Brambel proposed that serum IgG is bound to FcRp in pinocytic vacuoles for its protection from degradation and redirection of the antibody into the circulation. When the

FcRp is saturated, any excess IgG is directed to for degradation. Later FcRn was identified in the intestinal epithelium of neonate mice for transfer of IgG from ingested milk to serum, which was later also found in other species including humans, pigs, cows and sheep (242, 337). The proposed FcRp has a similar role and structure to

FcRn.

The roles of FcRn receptor in IgG homeostasis and transfer of passive immunity to neonates were demonstrated in various species, including humans, primates, rodents, marsupials, ruminants etc (560). The expression of FcRn can be temporal or life long, depending on the species. In rodents, the FcRn is expressed at high levels in intestinal epithelial cells during suckling to mediate the absorption of IgG antibodies from milk.

Expression decreases with time and is completely lost at weaning (56, 241). Thus in 98 mice, FcRn is not expressed throughout life long. In humans, on the contrary, the intestinal FcRn is expressed in both neonates and adults (612). In human fetal intestinal

cells (18-22 weeks of age), similar to the adult intestine, FcRn is found in the apical

region of both villous and crypt enterocytes and in stomach and colon. The expression of

FcRn was enhanced in the ileum of children and adolescents (612). In fetal tissues, it was also suggested, but not proven that enhanced expression of the FcRn occurred in the fetal

ileum with reduced expression in the fetal stomach. Using human malignant intestinal

epithelial cell lines, FcRn was shown to mediate IgG transport in both directions.

However, uni-directional transfer (from basolateral to apical surface) of IgG antibody,

which was demonstrated in neonatal rodents, was suggested to human FcRn. The receptor

can also be found on the bronchial epithelial cells of adult humans, non-human primates

and mice in which FcRn gives rise to a steady and dynamic distribution of IgG across the

respiratory epithelium (643). The FcRn has been also found in human skin, muscle and

liver (178).

The receptor was also found in the intestine of the brushtail possum (732), non-

human primates (342, 643) and cows (342). The FcRn transcripts were also found in

adult possums, suggesting a role in regulation of serum IgG in this species. In possums,

FcRn α and β2m expression is biphasic; high level occur shortly after the birth and after

110th days in the mother’s pouch, whereas low or undetectable levels were observed from

birth until the 110th day (732).

The FcRn also is expressed in the bovine intestine (342). Newborn lamb FcRn is

expressed by intestinal crypt cells mainly at the apical surface (444). However, FcRn was

not detectable in lamb duodenal enterocytes. Thus the initial uptake of IgG antibody from 99 colostrum in lamb during the first 18-24h is not receptor mediated and may be due to a non-specific mechanism(444). However in calves, the bovine FcRn was still hypothesized to play an important role in colostral IgG transport because different haplotypes of the bovine FcRn genes (FCGRT) are associated with differences in serum

IgG levels in newborn calves (392). The haplotype 3 of FCGRT in dams was associated with an increased likelihood of failure of passive transfer of antibody to calves; the haplotype 2 was less likely to have high level of passive transfer. The expression of FcRn has not been demonstrated in the porcine intestine.

(iii). The FcRn plays a role in transport of IgG antibody during colostrum formation

(443). In cattle and sheep, the FcRn expressed by epithelial cells of the mammary gland selectively binds and transports IgG1 into the mammary gland’s lumen, contributing to mucosal immunoprotection (341, 342, 444). Bovine FcRn expression was found in different tissues including the intestine and the mammary gland, suggesting its involvement in IgG transcytosis (342). In sheep, FcRn was localized in the epithelial cells of the acini and ducts in the mammary gland around parturition (443, 444). The presence of FcRn in the mammary gland was demonstrated in pigs (108, 603), mice (138), brushtail possums (6), lambs (443) and cows (342). Expression of the receptor in the sheep’s mammary gland is time dependent. Before parturition, the receptor is expressed homogeneously in the cytoplasm of epithelial cells. After lambing, more FcRn molecules move to the apical side of the cells (443) .

The regulation of FcRn expression by hormones has not been extensively studied.

Using the rat alveolar epithelial cell lines, Kim et al showed that dexamethasone caused a decreased mRNA level of rat lung FcRn α -subunit and the apical to basal (but not basal 100 to apical) flux of IgG antibody (362). A study of rat intestine showed that spermidine and cortisone decreased the neonatal Fc receptor expression in the intestine (111).

Dihydrotestosterone decreased FcγRIIB2, but not FcRn in patients with autoimmune

Grave’s disease (198).

(iv). Function of FcRn in neonatal immunity.

The binding of IgG to the FcRn receptor could have both positive and negative consequences (316). The receptor allows the passage of IgG antibody from colostrum/milk into the circulation of the neonate, providing systemic passive immunity to animals especially ruminants, horses and pigs. The reverse transfer of IgG from serum to the intestines of calves accounts for clearance of up to 70% of passively acquired antibodies (59). This transport is mediated by FcRn on crypt cells of the intestine. If the level of serum IgG antibody is sufficiently high, at least short term protection can be achieved at the mucosal surface (59, 492). Because the absorption of colostrum/milk in rodents can last up to 19 days, the FcRn is expected to a play role in absorption of milk to the neonatal circulation of mice (680). However the presence of passive antibodies in the intestine could interfere with vaccine responses; more detailed discussion about this topic is in the following section.

(v). Function of FcRn in active immunity.

A new function for FcRn has been proposed recently, that FcRn mediates transport of IgG antibody across the intestine in active immunity (562). In this model,

IgG antibody is endocytosed at the basolateral side of the cells, transported by FcRn to the apical surface and released into the intestinal lumen. In the presence of antigen in the

101 lumen, antigen-antibody complexes are formed. Via their interaction with FcRn or via

internalization by fluid-phase endocytosis, the complexes are transcytosed back to the

basolateral surface and delivered to the LP for immune activation or tolerance. The model

is supported by a study in which mouse Fc-eythropoetin chimeric molecules can be

shuttled by FcRn across the neonatal mouse intestinal epithelium or the adult mouse

respiratory lining and stimulate production of red blood cell progenitors (643).

1.4.1.3.3 Mechanisms of FcRn mediated IgG homeostasis

The endothelial cells, which line the blood vessels may be responsible for the

regulation and maintenance of a steady state of serum IgG in mice and humans (242). In

humans, expression of FcRn was found in endothelial cells of various tissues such as in

intestine (612), liver (673), kidney (280) and renal proximal epithelial cells(372). Thus

cells throughout the body are involved in IgG catabolism.

There are two proposed models for the mechanism of maintaining serum IgG

homeostasis but neither is fully proven (242). In both models, IgG antibodies are

internalized nonspecifically into endosomes. The low pH of the endosome (pH=6) allows the tight binding of IgG antibody and FcRn. Once the receptor is saturated, any excess unbound IgG antibodies would be directed to the . The release of IgG antibodies is mediated by the fusion of the endosome with the plasma membrane. The two models differ in the movement of the endosome inside the cell. In one model, the IgG antibodies in the endosome do not traverse the cell, instead they cycle back to the membrane from which they were pinocytosed (called the circular recycling model). In the second model

(trancellular recycling model), IgG molecules traverse the cells so that serum IgG

102 antibodies can be transported to the interstitial space and vice versa. The exact events that might occur are not yet known, but the second model is more likely to be the case as endothelial cells exhibit bi-directional transport of vesicles and endosomes can be found in these cells (242). These two models of IgG transversing the cells were derived from studies using a wild type human IgG and a mutant type (at one of the Histidine residues) that can’t bind to FcRn in the polarized cell lines (713).

These models also suggest a mechanism of regulation of IgG antibody levels. The

IgG antibody levels might depend considerably on FcRn receptor levels which could be up or down regulated by cytokines (suggested but not proven). It was also shown that phosphorylation of FcRn at the serine residue 313 regulates the transcytosis of the receptor across the rat inner medullary collecting duct cells. Mutation at this site reverse transcytosis from apical to basolateral surface of the cells (447). The stability of the cell and network also regulates the transcytosis of FcRn as drugs that destabilize the network result in blocking of FcRn trancytosis in one or both directions

(187, 650).

The mechanism of IgG transport across mucosal surfaces is unknown.

Transudation of IgG from the serum could be one possible mechanism which would result in the same proportion of IgG isotypes in the secretion and in the serum. As mentioned previously, FcRn is found in the apical region of both villous and crypt enterocytes and in stomach and colon in humans. The FcRn has not been identified in porcine intestine. However evidence has shown that IgG is locally produced in the intestine and transported to the lumen mediated by a receptor, rather than simply by transudation. It was shown that the pattern of IgG subclasses in serum and mucosal 103 secretions differed; the IgG1 is more prevalent in mucosal secretions than in serum (53).

In peridontitis individuals, the concentration of IgG4 antibodies in gingival crevicular fluid was higher than that in the serum (53).

1.4.2 Cells in passive immunity

1.4.2.1 Origin of plasma cells in the mammary gland

There are two major inductor/effector axes that are responsible for the presence of

antigen specific antibodies in breast milk. In the first axis (the gut mammary axis),

primed cells formed following antigenic stimulation in the gut can home to the mammary

gland (70, 582). These cells then mature into plasma cells in the mammary gland and

secrete dimeric IgA (567). The IgG-ASC, although present in small numbers in the

mammary gland, also may originate from the gut (451). The second inductor/effector

axis, the broncho -mammary axis, can also transfer the maternal encounter of mucosal

pathogens or maternal immunization into breast-milk antibodies (269). The gut-

mammary immunologic axis was first suggested by Bohl et al (70) and Saif et al (582) in

studies of lactogenic immunity to TGEV in swine. In these studies, antigenic stimulation

at one mucosal site (intestine) led to sIgA antibody responses at distant site(s) such as the

mammary gland. Maturation of these B cells into IgA ASC occurs at the mucosal effector

sites in response to antigens, T cells and cytokines (580). In pigs, the contribution of

immunocytes sensitized in the intestine to the IgA ASC pool in the mammary gland

appears to be more important than local production of IgA antibodies because IMm

injections of live attenuated TGEV failed to produce any appreciable level of TGEV IgA

antibodies in milk (70). These important findings, confirmed by subsequent studies in

104 mice (727), pigs (273) and humans (453) were an important tenet of the common

mucosal immune system. However, there is little evidence for the existence of a gut-

mammary gland immunologic axis in cattle and sheep. Antibody responses in milk were

not detected in cattle orally vaccinated with E.coli K99 (469). Harp et al (274) found that

most lymphocytes from the intestinal lymph nodes of cattle and sheep returned to the

intestine. At the same time, in another study by the same authors, trafficking of cells to

the mammary gland of sows was observed (273). It was suggested that the plasma cells in

the mammary gland of ruminants may be derived from cells stimulated in the draining

lymph nodes for the mammary gland and not derived from gut (109).

1.4.2.2 Transfer of functional immune cells in milk to neonates

Milk also contains many functional immune cells, billions of which are provided to neonates during the first few days. Sow milk contains 2x105 to 2x107 cells/ml (592).

The proportion of different cells are in sow milk as follows: epithelial cells: 31%,

neutrophils: 47%, lymphocytes: 12% and macrophages: 9% (604). The T cells account

for 70-90% of the sow colostral lymphocytes. The presence of epithelial cells, NK cells,

neutrophils and macrophages in milk are responsible for innate immune responses,

providing early protection to the neonate. Activities of sow NK cells have been shown to

provide resistance to TGEV when transferred to newborn pigs (117). The CD8 and CD4

T cells are both found in the mammary gland with CD8 being more dominant, however,

these cells exhibit properties of memory cells: weak reactivities to mitogens but strong

proliferative responses to enteric antigens.

105 The lymphocyte population in mammary secretions is dynamic during the

pregnancy and lactation period. In dairy cows, 80-90% of lymphocytes in the involuting gland are CD2+, and proportions of CD4 and CD8 are 55% and 40%, respectively.

Numbers of CD4+ decrease strongly at birth and always remain lower than 20%, whereas

the numbers of CD8 cells remain constant during lactation (755).

The absorption of colostral cells has been demonstrated in neonatal pigs (399,

685, 740). The neonatal pigs were estimated to ingest on average 500-700 million

colostral cells/days (398). Twenty-four hours post feeding, the maternal lymphocytes can

be found in liver, lung, spleen, lymph nodes (LN) and gastrointestinal tract of pigs (740).

These cells carrying immunological information such as antigen specificity or memory

from previous exposure of the mothers can be passed on to neonates.

1.4.3 Lactoferrin

Lactoferrin, the major protein in milk, about 1-4g/L, together with sIgA make up

30% of milk proteins (only 5% in cow’s milk). Lactoferrin possesses microbicidal,

immunostimulatory and anti-inflammatory properties. Human and bovine

inhibit IL-6 production of a monocytic cell line as early as 15-30 minutes after

stimulation with LPS (441). Lactoferrin exhibits antiviral activity against both DNA- and

RNA-viruses, including RV, respiratory syncytial virus, adenovirus, herpes viruses and

HIV(693). Lactoferrin from bovine milk was shown to prevent RV attachment to

intestinal cells by binding to viral particles and inhibiting a post adsorption step of the

virus (609). Lactoferrin interferes with an early infection step of and together

106 with Zn+2 ions inhibits viral replication after the viral adsorption phase. Lactoferrin also competes for common glycosaminoglycan receptors to prevent the attachment of adenovirus to cell membranes.

1.4.4 Cytokines

Human milk contains high levels of TGF-β, TNF-α, IL-1, IL-6, IL-8 and IFN-γ

(247). In human milk, the cells expressed transcripts for TGF-β (1 and 2) and IL-6 and production of these cytokines by mammary epithelial cells were detected (518, 646). The

TGF-β synergistically enhances the secretion of IL-6 by epithelial cells (452).

Interferon-γ was shown to be secreted by T cells from human milk in vitro (58). Table 1.5 summarizes the levels of different cytokines in human colostrum/milk.

Limited data are available on the levels of cytokines as well as the cytokine producing cells in porcine colostrum and milk (711). The level of the latent form of TGF-

β is high only in the sow colostrum period and the concentration of this form rapidly declines and is partially replaced by the free form.

These milk cytokines have been suggested to play roles in immunomodulation of neonatal immune development. The TGF-β, present at high concentrations in milk, helps to control inflammation in neonates due to its immuno-down regulatory function (526).

Uncontrolled inflammatory responses were shown to be fatal in TGF-β knock-out mice after weaning. When provided as supplement, TGF-β also helps to restore the level of pro

IL-18 in the intestine. Of note, IL-18 has been shown to play a role in homeostasis by influencing the development of Th1 or Th2 responses depending on the cytokine environment at the time of antigen priming (489, 751). In another study, the important

107 immunoregulatory role of TGF-β was demonstrated in a study where TGF-β1 supplied to the fetus by injection into the mother’s circulation during gestation or to the neonate via milk during suckling was shown to rescue TGF-β1-/- newborn pups from severe cardiac abnormalities (408). The presence of TGF-β1 in the fetus could play a role in limiting the development of fetal primary immune responses and favoring the neonatal Th2 responses, which explains the Th2 bias responses in human infants and murine neonates as mentioned in previous sections (420, 538). Milk cytokines such as TGF-β, IL-4 and

IL-10, which can also influence antigen priming (526). The immunomodulatory anti- inflammatory activity of IL-10 in milk has also been suggested (231). However the effects of these cytokines have not been clearly validated in human milk in vivo (690).

Certain cytokines such as IL-4, IL-5 and IL-13 were elevated in colostrum of allergic mothers compared to non-allergic mothers, which might contribute to the Th2 bias of neonatal immunity development in breast-fed children (49). However, it is also suggested that the presences of IFN-γ and IFN-γ producing cells also enhance Th1-IgG2 production in neonates (628). Thus milk cytokines and cytokine secreting cells can modulate both types of Th responses. The IL-18 was present in milk of women with complication in their pregnancy (661). The acute phase cytokines (TNF-α and IL-6) were minimal in human whey in one study using ELISA (646), but other studies (569,

570) reported much higher levels of TNF-α (620pg/ml) and IL-6 (151pg/ml) using radioactive .

Cytokines in milk also function in trafficking of immune cells. Higher levels of chemoattractant factors IL-8 and RANTES (regulated on activation, normal T cell expressed and secreted) were found in colostrum of allergic mothers which may enhance 108 the traffic to breast milk of specific cell populations involved in allergic reactions, e.g.

IgE secreting cells and their transfer to neonates (77). A high level of TGF-β in milk has

also been associated with high levels of serum IgA and allergen specific IgA ASC

responses in infants (344, 574). Other cytokines that are present in colostrum and milk

are IL-6, which is known to enhance IgA antibody production. Milk TNF-α enhances the

production of secretory component associated with sIgA (246).

1.4.4.1 Transfer of cytokines via the placenta

In humans, TGF-β1 and IL-10 can be detected in human umbilical cord fluid

(538). Elevation of TGF-β levels in maternal and fetal circulations from pregnancy to birth have been reported in humans (538). In humans, TGF-β1 was present at the maternal-fetal interface including the placenta. However there is also no correlation between maternal and fetal TGF-β1 levels in humans suggesting that either restriction of its passage across the placenta or its polarized secretion by the placenta at the fetal side

(538). It has also been shown that IL-4, but not IFN-γ and IL-12 are constitutively produced by cord blood mononuclear cells upon stimulation with PHA (94). In mice, there was no transfer of maternal interferon to the fetus even when the level of interferon injected into the mothers was high (199).

Transfer of cytokines via the sow placenta has not been reported. It is well established that the sow placenta which consists of 6 tissue layers doesn’t support transfer of immunoglobulins (382). Uteroferrin, the iron-containing glycoprotein composed of a single oligosaccharide chain has been shown to be transported across the pig placenta via a receptor (598). Monomers such as glucose and amino acids are actively transported to

109 the fetus as nutrients whereas the transfer of fatty acids is limited (678). In chapter 5, we

showed that cytokines such as IFN-γ, IL-4, IL-6 and IL-10 were not detectable in serum

of germ-free pigs or in pre-suckling pigs. However, IL-12 and TGF-β were present in

serum of these pigs. The presence of these cytokines in piglet serum can be constitutive

or via ingestion of placental fluids or possible transfer via the placenta or colostrum/milk.

1.4.5 CD14

CD14, the co-receptor for bacterial LPS on mature monocytes, macrophages and neutrophils, can be found in colostrum and milk in the soluble form. These CD14 molecules in milk were shown to stimulate B cell growth and differentiation in neonates

(212). The soluble form of CD14 (sCD14) in mammary secretions was enriched 100-

1000-fold compared to the serum levels, and this level persists up to 400 days post partum in humans (300 days in cows). The LPS-induced soluble CD14 signaling and B cell activation are mediated by TLR-4. Intramammary administration of soluble CD14 with E.coli was shown to induce an early increase of sCD14 levels in milk, resulting in rapid clearance of bacteria and reduction of the severity of E.coli infection in the mammary gland (404). It is postulated that the sCD14 might induce early recruitment of neutrophils to milk, which help with the clearance of bacteria. Similarly, the presence of the soluble form of TLR2 produced by blood monocytes in breast milk and plasma can modulate the activation of monocytes (401)

1.4.6 Levels of MatAb transferred and factors influencing the transfer

The level of MatAb transferred and its variation between individuals are partially hereditary. It was reported that the level of IgG antibody in the colostrum of female cattle 110 is constant overtime (167). Chicks born to lines selected for an antigen-specific antibody

response leads to similar consequences in the offspring as in the parent (68, 101). The

environment experienced by the mother also plays a role in the variation of antibody

transmission from mothers to offsprings. If the mother is exposed to a particular pathogen

and develops antibody responses to the pathogen with the specific antibody transmitted to

her offspring, these offspring are more likely to and more efficiently mount similar specific antibody responses (259).

The MatAb level is also dietary dependent. A study of rats showed that restriction of the dietary proteins resulted in 2-fold lower IgG antibody levels in colostrum (466).

Similarly, birds raised under restriction of vitamin E showed decreased antibody transmission to chicken eggs (319).

The age of the mother is also an important factor for MatAb level transference to neonates. Offspring from 3 year-old cows had significantly higher serum IgG levels than those from younger (2 year-old) or older (more than 3 year-old) cows. The numbers of

IgG ASC in the ovary of hens at 50 days of age or more than 450 days of age were significantly lower than those of mature but young hens (180 days).

The influence of MatAb on neonatal and infant immune responses is long-term, persisting even after being catabolized completely in the offspring. This influence is inducible early during the development of infants (259). The effects of MatAb on both the humoral and cellular arms of the immune system will be discussed in next section.

The MatAb influence is not only applied to that particular individual but also to the next

generation (423). In the first generation, there was no difference in antibody responses to

111 E.coli in normal pups vs. pups inoculated with mouse antibodies against E.coli antigen.

In the next generation, however, the responses to E.coli were significantly higher in pups

from the mouse dams with antibodies than those from dams which were not inoculated

with antibodies. This is partially due to the expansion of B and T cell repertoires, as was

similarly observed during vaccination of an individual with multiple antigen doses.

The MatAb also improves the intensity of the offspring immune responses (259).

Offspring from B cell deficient mice expressed 2- 3-fold lower pre-B and B cell numbers

in bone marrow and spleen (429). The frequency of MHC II expressing cells also

decreased in these mice (756). The antibody level after primary immunization of the F2 offspring of immunized mothers was equal to that obtained after secondary responses in

offspring from unimmunized mothers.

Infants may have different expression levels of the IgG receptor (FcRn) and the

receptor affinity to IgG, different metabolic rates of antibodies, as well as incubation/gestation periods (411, 480). Thus the level of MatAb transmitted is also a function of interaction between maternal genome (antibody transferred) and offspring genome (antibody absorbed). However the ability to absorb MatAb by the offspring tends to co-adapt with the level of MatAb transmitted (743).

1.4.7 Interference of neonatal responses by MatAb

1.4.7.1 Inhibition by Mat Ab

Inhibition of B cell responses by MatAb has been documented for many vaccines.

The humoral responses in infants were reduced or inhibited following vaccination with measles, , the combined diphtheria, pertusis and (DPT) and 112 Hemophilus influenza vaccines (63, 190, 213, 499, 595, 758). Again, the inhibitory effects of MatAb can be long lasting: after 3 doses of Hepatitis A vaccine in children before 6 months of age, only 24% of children born to Hepatitis A antibody positive mothers showed protective antibody levels at 6 years of age, compared to the 68% in children born to Hepatitis A antibody negative mothers. Feeding or IP injection of rats as neonates with monoclonal IgG2a or IgG1 antibodies led to suppression of humoral responses in these rats as adults; thus, the suppression by MatAb lasted for up to 5 months (528). This suppression was only observed when the rat received the before weaning or gut closure. Feeding at a subsequent time did not cause suppression. Thus the maternal IgG antibodies determine the immune repertoire of the offspring.

The T cells responses to vaccines appear to be more resistant to the suppression by MatAb. The T cell proliferation, IFN-γ and IL-12 production specific to measles vaccine were not affected by the presence of MatAb (230). In the murine model, the in vitro T cell responses to measles and tetanus vaccine in pups were unaffected by high titer MatAb (623). However, there are many other studies which have described a suppressive impact of MatAb on T cell responses. Both humoral and CTL responses were decreased in newborn rhesus macaques immunized with recombinant vaccinia viruses expressing measles virus hemagglutinin and fusion proteins in the presence of measles immunoglobulin (780). Similarly, pups from virus-immune dams showed a decrease in the specific B- and T-cell responses to immunization, measured by serum antibody level and cytokine release from in vitro lymphocyte cultures,

113 respectively, leading to the failure of the vaccine. The degree and duration of the vaccine

failures was inversely correlated with the amount of maternally transferred antibodies

(750).

1.4.7.2 Enhancement of the immune responses by MatAb

Maternal antibodies can also enhance vaccine responses. It has been demonstrated

that IgG antibodies in the immune complex form can stimulate the formation of GC,

activate somatic mutation of B cells and suppress differentiation of ASC simultaneously

(641). Antibody dependent immune enhancement has been reported previously for both

vaccines and infectious agents. Maternal tetanus IgG antibodies enhanced the T cell

responses by increasing IL-4, IL-5 and IL-13 responses to tetanus vaccine in infants

(568). Low or moderate levels of pneumococcal polysaccharide-specific MatAb not only

provided protection against pneumococcal infections but also enhanced the immune

responses elicited by pneumococcal vaccine in neonatal and infant mice (551).

The immune enhancement by passive antibodies is dose dependent. In vivo and in

vitro studies showed that mice receiving Japanese virus-immune mouse

serum at dilutions of 1:10 and 1:100 but not undiluted serum, had a significant reduction in average time to death when challenged with the related Murray Valley Encephalitis virus (90). Low titer MatAb present in conventional pigs was shown to enhance PRRSV virus infection, as indicated by an increase in viremia as well as the tissue distribution of virus likely mediated by an antibody dependent enhancement mechanism of PRRSV infection of macrophages (616).

114 The immune enhancement is not only applicable for antigen specific antibodies but also to natural antibodies. Chicken given pooled plasma from non-KLH immunized hens (containing natural antibodies), then immunized with KLH antigen showed increased IgM and IgY antibody titers to KLH compared to those that received phosphate buffered saline (PBS) (734).

1.4.7.3 Mechanisms of MatAb interference

1.4.7.3.1 Neutralization of live viral vaccines

It is hypothesized that MatAb neutralize and hence reduce the , preventing the induction of effective B and T cell responses. It has been demonstrated that MatAb reduced the replication of human RSV in the lower respiratory tract of mice

(164). Oral vaccination of young foxes with a modified live vaccine against rabies in the presence of MatAb did not protect the animals from infection with virulent virus (482).

The lack of protection against rabies in these animals was correlated with the failure of this vaccine to induce active antibody responses. Homologous but not heterologous

MatAb to Marek’s Disease virus delayed the development of viremia induced by the live vaccine virus and also inhibited immune responses to the vaccine in chicks (367). The level of inhibition of MatAb to infant vaccine responses is the function of MatAb levels at the time of vaccination and the antigen load, as proposed by Siegrist et al (622),

Figure1.3. Interference by MatAb is dose dependent; some studies suggest micrograms of

MatAb may lead to suppression whereas nanogram amounts lead to enhancement of an infant’s immune response (665, 738).

115 1.4.7.3.2 Interference of passive antibodies via the Fc receptor

At least 3 mechanisms have been proposed for IgG mediated suppression: (1) IgG masks antigenic epitopes and prevents B cells from recognizing and responding to the antigen; (2) the immune complexes are removed by phagocytoxic cells by FcγR before they can activate B cells and (3) IgG inhibits B cell activation by cross-linking the B cell receptors (BCR) and FcγRIIB (284). The FcγRIIB, expressed on the macrophages and B cells, acts as an inhibitory receptor that terminates the activation signals initiated by the

BCR (2). At the same time, enhancement by IgG antibody has also implied the involvement of complement and FcγR.

The first mechanism, epitope masking by IgG, helps to explain the inhibition of B cell responses, but not T cell responses in the majority of studies as mentioned in the previous sections. Because epitope masking is not expected to prevent the uptake of the

IgG/antigen complexes by the APC, the presentation of antigen to T cells would be unaffected which was demonstrated by Karlsson et al (348). By this mechanism, the induction of memory and secondary responses may not be effected by passive antibody

(284). The epitope masking but not the suppression via FcR is likely the mechanism of immune response suppression because non- antigen specific antibody also causes similar suppression (348). Thus antibody bound to one epitope on an antigen can sterically block the recognition of the neighboring epitopes (284).

There has been no significant support for the second mechanisms of suppression.

The role of FcγRII in feedback inhibition in the third mechanism is still controversial.

First, IgG mediated suppression is often observed for particulate antigens whereas

116 stimulation is observed for protein antigens (284). Secondly, co-ligation of the BCR and of FcγRIIB results in an inhibitory signal (534). The FcγRIIB has an immunoreceptor tyrosine-based inhibitory motif (ITIM), which is the predominant form in both cord blood and adult B cells (325). However this suppression was always observed in adult cells, but was not seen or was less pronounced in cord blood cells, suggesting a lower level of

CD32 (human FcγRII) on cord blood cells, making cord blood B cell less susceptible to feedback inhibition by antibody (325). In addition, studies showed that mice lacking

FcγRII still showed IgG mediated suppression and suppression was still observed with

Fab fragments. Furthermore, FcγRIIB deficient mice given the IgG1/antigen complexes

(anti-TNP/TNP-BSA) still produced BSA antibody (662). It is postulated that FcγRIIB only prevents the IgG/antigen complexes from reaching abnormal levels in the body.

Thus most data suggested that epitope masking is the most important mechanism of suppression of antibody responses by passive antibodies.

The IgG mediated enhancement of antibody responses involves complement or

FcγR or both. The role of complement was demonstrated in complement depleted mice in which the ability of IgG to prime DNP (2,4 Dinitrophenyl) specific B cells is abolished

(192). Monoclonal antibody to DNP complexes with DNP-KLH can be found in splenic follicles and the presence of these complexes in these sites correlated with enhancement of the memory cell responses (160, 735). However other studies suggest that IgG antibody can enhance antibody responses without involvement of the complement system. Mutant IgG, which fails to activate complement, still enhanced the efficiency of antibody responses to KLH (736).

117 On the contrary, the FcγR mediated antibody enhancement is considered to be the

major mechanism. Mice deficient in this receptor showed impaired responses to antigen/IgG complexes (729). In FcγR deficient mice, where FcγRIIB was normally

present, the responses to antigen/IgG complexes were impaired.

1.4.7.3.3 Interference via idiotypic interaction

Maternal antibodies result in an altered primary antigen-specific antibody

repertoire of the offsprings via altering the idiotypic composition of the offspring’s

immune responses (394). Somatic mutations that produce a pool of antibodies specific to

different antigens can be regarded as a step in the learning process of the mother.

Maternal immunological experience in turn educates the nascent immune system of the

newborn. The transfer of antibodies from mother or grandmother influences the

repertoires in the early primary response of the offspring.

When a panel of monoclonal antibodies (Mab) to hen egg-white lyzozyme (HEL)

was administered to the mother, these antibodies did not have equal effects on infant’s

immune responses (640). Only Mab that are idiotypically connected in the anti-HEL

Mab panel induced suppression of the offspring’s responses. The findings suggest that

idiotypic suppression occurs through the reaction of anti-idiotopes with idiotopes on the

surface of newly generated B cells. There are two types of suppression: either direct

blocking of B cell maturation which is short-lived, or induction of regulatory cells via the

idiotope-anti-idiotype complexes, which is long-lived (665).

118 Transfer of protective idiotopes from mother to the child could both prime the neonate for a booster effect of antigen and provide passive protection against infection

(19). Of note, antibodies against antigen are labeled Ab1; antibodies raised against Ab1 antibodies are called Ab2. The Ab2 that recognizes an idiotope outside the antigen binding site of Ab1 is called Ab2α. The Ab2 that recognizes the paratope inside the antigen binding site is called Ab2β and is an internal image antibody since it mimics the shape of the original antigen. Antibodies against Ab2 are called Ab3 and so on and so forth. Some of these Ab3 can cross react with the original antigen and can be labeled

Ab1. This chain of reaction is called a network response (19). This network has been found in young animals. Any given antibody species will bind to more than 20% of other antibodies of the same repertoire (291, 292). Immunization of mice with monoclonal

Ab1 to Schistosoma mansoni induces high levels of Ab2 and Ab3 whereas Ab2 can inhibit 33-100% of Ab1 binding (277).

This immune network can be formed when MatAb are transferred to neonates.

This network could be responsible for translating the maternal signal Ab1 into Ab2 and antibodies Ab3 that cross react with the original antigen or Ab1. Okamoto et al (508) measured both Ab1 and Ab2 in neonatal rat serum, indicating that neonates do mount antibody response against the incoming Ab1, but these levels decline shortly after birth.

By the fifth month, the level of Ab2 becomes immeasurable whereas the suppression still persists in rats (528). It is hypothesized that idiotypic specific T suppressor cells are responsible for the suppression (47, 664, 665).

This immune network is found largely in neonates, whereas the antibodies don’t bind to each other at a similar frequency in adult animals (293). It is possible that the

119 adult T cells undergo extensive selection to avoid the anti-self responses, thus there is no help available to develop this network of B cell responses in adults. It is also hypothesized that the expansion of non-self reactive clones will overtake other clones with age.

Anderson et al. built a model for maternal-neonatal idiotypic interaction, namely the molecular attention hypothesis (19). The neonatal immune network is said to amplify and translate the maternal signal Ab1 into a dynamic activity for all clonal species. There will be thousands of copies of id and anti-id. The id and anti-id complex can be taken up by APC and the critical region of Ab1 and Ab2β can be presented in association with

MHC II. The Ab1-Ab2β complex can also bind to Fcγ receptor on the surface of FDCs.

These FDCs can retain the complexes over long periods of time, then travel to spleen and

LN and deposit in GC. Thus these cells help to maintain the dynamic state of the anti-id

B cell clones.

Alternatively, Th cells for internal image antibodies can be formed. First, there is an overlap between B cell epitopes and T cell determinants. Fragments of Ab1 can be processed and associated with MHC molecules. If the fragments contain the VH-VL parts, they would trigger binding of T cells (115).

1.5 Vaccine strategies to overcome the immaturity of neonatal immunity and

interference by MatAb

The following sections focus on the types of vaccines, modes of vaccine delivery and adjuvants, which have demonstrated efficacy or have shown the potential for effective vaccination of neonates in the presence of MatAb

120 1.5.1 Type of vaccines

If neutralization by MatAb remains a limitation of live vaccines, the use of non-

replicating vaccines, such as virus-like particles and DNA plasmids may be

advantageous.

1.5.1.1 Virus-like particles (VLPs)

The VLPs have been produced for more than 30 different viruses (Table 1.6), including viruses with single capsid proteins ( and Retroviridae family), viruses with multiple capsid proteins (poliovirus, infectious bursa disease virus etc), enveloped viruses (Hepatitis , influenza virus etc) and non-enveloped viruses (RV, parvovirus etc) (497). The VLP possess the overall structure of the virus, but without the infectious genetic material. The use of non-replicating vaccines is much safer than live vaccines even attenuated vaccines because recombination, reversion to virulent strains or

re-assortant can not occur with the VLPs. The VLPs can be used alone without adjuvants

to induce both humoral and cellular responses in some cases. The VLP or

foreign epitopes expressed as chimeric proteins on hepatitis E VLP induces both systemic

and mucosal IgA antibody responses (495). Use of the VLP generated from lymphocytic

choriomeningitis virus (LCMV) with CpG in a prime/boost strategy demonstrated

effectiveness in induction of CTL responses (571, 608).

However, the suppressive effects of MatAb were also observed using non-

replicating vaccines including VLPs. In Chapters 3 and 4, the pre-challenge IgG ASC

numbers and the serum IgA and IgG antibody responses induced by 3 doses of RV 2/6

VLP with ISCOM were suppressed in the presence of high and low titer passively

121 transferred RV specific antibodies. Thus interference by MatAb is not restricted to neutralization of live viral vaccines. An inactivated pertussis toxin vaccine is a clear example in which infant immune responses can only be observed in infants without

MatAb (104). In addition, larger amounts or higher concentrations of the non-replicating vaccines will be required for induction of effective immune responses. Therefore an antigen delivery system or adjuvant is normally required to reduce the antigen dose, to prevent the degradation of the vaccine as well as to avoid tolerance when the vaccine is delivered via oral route. Thus the use of non-replicating vaccines alone might not be sufficient to overcome the interference by MatAb. A combination of different strategies will be necessary to improve vaccine efficacy in the presence of MatAb.

1.5.1.2 DNA vaccines

The DNA vaccines can be administered with adjuvants or supplied with cytokines that might deviate from the Th2 bias of the neonate. vaccines have been shown to be strong inducers of Th1 responses, which enhance Th1 CD4 T cells and induce CD8 CTL. The DNA vaccination might also reduce the number of injections required.

Substantial efforts have been made in developing an effective way to deliver

DNA vaccines. The DNA vaccines are normally administered via systemic or subcutaneous routes. Mucosal DNA vaccines are less common. Most such studies used mucosal DNA vaccines targeted to human immunodeficiency virus (HIV) infection; only a limited number of vaccines for other mucosal infections. In BALB/c mice, protective immune responses were obtained after oral immunization with one oral dose of RV VP6

122 DNA vaccines encapsulated in poly(lactide-coglycolide) (PLG) microparticles, indicated by RV-specific serum and intestinal IgA antibody responses and significantly reduced fecal RV antigen after challenge with homologous RV(124). Similarly, one dose of VP4

DNA and VP7 DNA vaccines, given to BALB/c mice by oral gavage induced RV serum antibodies and intestinal IgA antibodies 6 weeks after immunization and protection against viral shedding at 12 weeks post-immunization. Unencapsulated VP7 DNA vaccines delivered orally also induced protection against viral shedding in these mice but to a lesser degree (283). Priming by a mucosal route followed by boosting via a systemic route can induce both mucosal and systemic immunity which are important for vaccines against pathogens such as human virus (HSV) and RV (193, 763). Yuan et al (763) demonstrated protection against RV induced diarrhea (30%) and shedding

(70%) in Gn pigs using a prime/boost vaccine strategy consisting of an oral dose of

AttHRV and two intramuscular doses of VP6-DNA vaccine. High levels of IgA ASC responses in ileum and intestinal antibody responses were induced by this vaccine regimen.

The DNA vaccines overcame the Th2 bias of neonatal immune responses as mentioned previously. Intramuscular injection of murine VP6 DNA vaccines raised a

Th1-like antibody response, high titers of RV-specific serum IgG and serum but not fecal

IgA antibodies in BALB/c mice (754). In addition, both partial homologous and heterologous protection against murine RV challenge as measured by reduction of RV antigen shedding in feces were achieved by murine and bovine VP6 DNA vaccines, respectively. Immunization of VP6 DNA vaccines via the abdominal epidermis or anorectal epidermis using a gene-gun (PowderJet, Inc) induced protection against 123 challenge virus, yet stimulated Th-2 type responses even in adult BALB/c mice (123).

Thus the routes of DNA vaccination are important in the induction of the Th1/Th2 balance in neonatal immune responses.

Mucosal DNA vaccines have been delivered by other routes. The most interesting approach is via the oral route in utero to the fetus, which has been tried in lambs (237) against HSV and in baboons against (724). In these studies, 75-80% of the animals produced both systemic and mucosal immune responses. Strong memory responses were still detected 3 months after birth. The reason for the success of the in utero approach is that the fetal T cells might have a longer life span than the neonatal T cells. In addition, mucosal epithelial cells in the fetus might have much lower turnover rates, which allows sufficient time for transfection and after oral DNA immunization of the fetus (236). Thus the rate of survival of memory T cells can be improved in fetuses or for at least 3 months in this study. Several drawbacks of this method of DNA vaccine delivery were noted. The presence of high levels of cortisol in serum of the mammalian fetus might have significant effects on the peripheral T cell function, T cell trafficking or T cell commitment to Th1 or Th2 responses, as well as a reduction in T cell survival rates. Also because DNA vaccines often require long time to develop immune responses, in utero immunization should be performed weeks before the delivery date, for examples 4 weeks in the lamb study (237), which can pose a danger to the fetus.

Other modes of administration have been tried for mucosal DNA vaccines. The

DNA vaccines administered via eye-drops preferably induced IgA antibodies in tear and bile of young chickens (573). The DNA vaccines can also encode antigens together with 124 cytokines e.g. IL-12 and GM-CSF, which exert immunomodulating effects or enhance

antigen presentation, useful for the induction of immune responses in early life. The

DNA vaccines containing genes encoding both IL-12 and IL-18 have been tested to enhance the immune responses and protection in vaccines against HSV and Leshmania

(668, 779). A DNA vaccine codelivered with IL-2 or GM-CSF increased the antibody and lymphoproliferative responses to bovine viral diarrhea virus (498).

The common concern with DNA vaccines is that if the vaccine is expressed in a bacterial genome or plasmid, the presence of the CpG motif can have significant effects on both innate and adaptive immune responses. The CpG can promote the development of autoimmune disease, or cause strong local inflammatory reactions. Although the half- life of the plasmid is short (2-4h in amniotic fluid, degraded completely within 8h), there is still a brief time for bacterial DNA to interact with the innate immune system (237).

The DNA plasmid vaccines also pose a potential risk of integration into the chromosomal

DNA (375).

In spite of these concerns, DNA vaccines are the most promising vaccines for use in infants to overcome MatAb suppression. This type of vaccine has been tried in different animal models and against different pathogens. The DNA vaccines have been demonstrated to induce adult-like Th1 and CTL responses in newborn and young mice against measles, Sendai, influenza, LCMV, and rabies viruses (75, 278, 434,

596). Some formulations of DNA vaccines such as those used for HSV do not require adjuvants. Naked DNA encoding for a HSV protein could be administered intramuscularly and induced effective humoral and T cell responses in the presence of

MatAb (426). The DNA immunization targets APCs to increase their uptake of antigen. 125 However, there are contradictory studies concerning whether DNA vaccines can escape interference by MatAb. The gD glycoprotein gene of the pseudorabies virus

(PRV) inoculated in pigs born and suckled by immune mothers did not induce immune response or protection against PRV. On the contrary, Fischer et al. described a DNA vaccine that induces both humoral immune responses and protection against PRV in pigs suckled by PRV seropositive sows (215). The difference between these studies in the time of challenge of the pigs (16 weeks in the former vs. 20 weeks in the latter) and the duration between priming and boosting (6 weeks in the former vs. 11 weeks in the latter), which led to further declining MatAb titers in the pigs might explain the conflicting results. In turkeys, vaccination with DNA plasmids expressing the major outer membrane protein of avian Chlamydophila psittaci induced protective immunity against homologous challenge in the presence of MatAb. Although the antibody responses induced by the vaccine were affected, the cell-mediated-immunity was unchanged (694).

The effects of MatAb are not the same for different proteins of the same virus.

The MatAb inhibited the antibody responses induced by DNA vaccines encoding HA but did not have impact on the antibody responses to the nucleoprotein (NP) nor the generation of cellular immune responses to HA or NP (532). A DNA vaccine against

Plasmodium yoelii induced tolerance rather than immunity in neonatal suckling mice at

2-5 days of age (470).

Thus not all DNA vaccines are effective in preventing the disease in the presence of MatAb. Further studies are needed to analyze the mechanisms by which DNA vaccines and the various modes of delivery/adjuvant which can escape MatAb interference.

126 1.5.2 Antigen delivery system

1.5.2.1 Aluminum salts

Aluminum salts form antigen depots at the site of injection from which the

antigen is slowly released. This is an adjuvant that favors Th2 responses in mice and

humans; therefore it is only advantageous to any infection that needs strong antibody

responses e.g. tetanus, diphtheria toxoid or to viral agents such as poliovirus vaccine.

Therefore its role in neonatal vaccination to overcome MatAb effects is limited although

this adjuvant is the only one licensed for human use. However alum can be combined

with other adjuvants to enhance the immune responses. Immunization of newborn (1-7-

day-old) BALB/c mice against HBsAg with alum and CpG ODN induced HBsAg-

specific CTL responses in the presence of high levels of MatAb (anti-HBs) (725).

However, the B cell responses to HBsAg/alum/CpG ODN remained weak.

1.5.2.2 Liposomes, virosomes and Archaeosomes

Vaccines encapsulated within liposome microspheres have the tendency to move to the cytosol and be processed by the MHC class I pathway and presented to CD8 T cells. Virosomes are a special form of liposomes whereby the virus fusion protein is inserted into the liposome bilayer. These methods of antigen delivery enhance cell binding, endocytic uptake of the antigen and delivery into the cytosol. A liposome-based vaccine against Hepatitis A virus (Epaxal, Swiss Serum and Vaccine Institute), induced

100% seroconversion after 2 doses and being well tolerated by vaccinees, has been

licensed for human use (18). In addition, liposomes have been combined with cholera

toxin, lipid A or cytokines to enhance adjuvancity (272, 391, 549). 127 Another lipid based adjuvant recently being investigated is Archaeosomes.

Archaeosomes are a polar lipid structure or triphospholipid (TPLs) derived from

Achaebacteria which is able to form stable liposomal vesicles (645). Unlike the conventional liposomes which are made from ester phospholipids, often with unsaturated lipid chains of variable length, Archaesomes are composed of fully saturated, branched chains attached via ether bonds to a glycerol backbone. This unique ether structure may facilitate the formation of unique head group structures that interact with the specific receptor on APCs. Archaeosomes induced antibody responses, CD4+ and CD8+CTL responses, recruited and activated DCs and macrophages and attracted NK cells to tumor sites (379-381). Protection of mice against monocytogenes was induced after immunization with the protective antigen entrapped in Archaeosomes.

Several studies indicated that Archaeosome-based vaccines are safe (379, 380).

An Archaeosome species such as Methanobrevibacter smithii is a nonpathogenic inhabitant of the human colon, which suggests a high tolerance when used as vaccine

(467). However the safety of this adjuvant group is still in question as other

Archaeosomes of species such as Planococcus still evoked unnecessarily non-specific inflammatory cytokine secretion in the absence of antigen, whereas the liposomes of

H.volcanii species did not (644). In addition, it is unknown whether this lipid can enter the circulation of the host, which helps to accelerate immune responses. These adjuvant systems have not been tested in the presence of MatAb.

128 1.5.2.3 Microspheres

Several studies have documented the use of microspheres to deliver vaccines. In this system, antigens are delivered by microspheres of biodegradable polymers. The polymers can be manipulated to release antigens gradually or as pulses. Microspheres have been shown to retain antigens for up to 9 days. Presentation of antigen-loaded microspheres to macrophages can extend over 7 days whereas the presentation of soluble antigen has a half-life of 18.6h (31). Rotavirus VP6 encapsulated in alginate microspheres delivered orally to BALB/c mice induced high level of fecal IgA antibodies, similar to those induced by live virus (359). In contrast, RV VP6 in incomplete Freund’s adjuvant induced only IgG, but not IgA antibodies. Microspheres were observed in the follicles-associated-epithelium of PP (359, 360). Microspheres of more than 5um which are taken up and remain in the dome area of PP are likely to induce strong mucosal immune responses. Branched aliphatic oligoester microspheres with incorporated RV administered to mice IP or orally resulted in the production of systemic

IgG and IgA antibodies Repeated oral vaccination with an increased dose was required to obtain mucosal antibody responses (537). Neither the liposome (previous section) or microsphere adjuvants have been evaluated in the presence of MatAb.

1.5.2.4 Vectored vaccines

There are numbers of microorganisms including bacteria and viruses which can be used as vaccine vectors. The BCG strains can be used as the vector for vaccine delivery in the newborn due to its safety record. They have been shown in newborn mice to induce preferentially Th1 responses (384). They also induce mucosal responses in

129 macaques and guinea pigs (286, 351, 395, 463). The normal flora such as ,

Streptococcus and Staphylococcus can also be used as vaccine vectors (357, 405, 600).

Non-replicating live viral vectors such as genetically engineered poxvirus NY-

VAC or naturally attenuated vectors such as AL-VAC, TRO-VAC or vectors from avipoxviruses e.g. canarypox, which are unable of replicating in mammalian cells, have been under investigation as a possible safe vaccine vectors (519). The deletion of virulence and host range genes in the NYVAC vector prevents reversion to the virulent phenotype. Canarypox-carrying rabies vaccines induced CD4 and CTL responses and conferred protection against rabies in both young and adult dogs and in humans (222,

670). However it has been shown that this vector is not able to overcome the Th2 bias of neonatal immune responses (626). When used with the HA of measles virus in young mice, high levels of IL-5, but lower IFN-γ and poor CTL responses were obtained, which differed quantitively from the Th1/CTL-prone responses in adult (623). This vaccine strategy was also unable to induce measles specific responses in pups born to immune mothers (623). However, high titers of MatAb did not affect the induction of vaccine- specific Th1/Th2 responses, as assessed by proliferation and levels of IFN-γ and IL-5 productions nor the CTL responses in infant mice.

1.5.2.5 Monophosphoryl lipid A (MPL)

Monophosphoryl lipid A (MPL) is another promising adjuvant for use in infant vaccines. The MPL is the product derived from bacterial cell walls, a detoxified form of lipid A from the LPS of Salmonella Minnesota R595 (41). Its immunostimulating effects include activating APCs to produce IFN-γ, TNF-α, IL-1β (Th1 response) and inducing

130 IgG antibody production (an implication for long term immunity). A study of mice using an inactivated RV vaccine administered IM with MPL reported high levels of serum IgG and VN antibody titers, but low fecal IgG and IgA antibody titers even after the third dose (333). The MPL can also be a good inducer of mucosal immunity or of IgA antibody responses in particular. Intranasal immunization of mice with HIV-1 oligomeric gp160 (o-gp160), formulated with liposomes containing MPL induced strong antigen specific IgG and IgA responses in serum, vaginal, lung, and intestinal washes and in feces (698).

1.5.2.6 Saponins and derivatives

Saponins have been used in veterinary vaccines for a long time. Examples of vaccines using saponins are the vaccines against foot and mouth disease, Rhipicephalus sanguineus ticks in dogs, Fusobacterium necrophorum in steers, feline virus in cats and rabies vaccine in mice (290, 310, 428, 576, 658). The more frequently used saponin is the one derived from the bark of the Quillajia saponaria trees. The mode of action of saponins includes intercalating with cholesterol in the cell membranes, forming pores to allow the antigen transport across the membranes and facilitating antigen uptake and processing (636).

Several derivatives/purified forms of saponins have been tested for use in mucosal vaccines. The Quil A, the fragments from saponins with immunostimulating activity can either be mixed with cholesterol and phospholipids to create immunostimulating complexes (ISCOM), or further purified as QS-21 both of which have been tested as adjuvants for mucosal vaccines for humans. These forms of saponins have been known to

131 induce CTL, both Th1 and Th2 types of responses and increase the magnitude of

antibody responses. The ISCOM associated with RV 2/6-VLP (Figure 1.4) induced more

balanced Th1/Th2 responses in Gn pigs (489).This immunity enhancing ability is the

result of the ability of ISCOM to recruit accessory cells e.g. DCs, macrophages to the PP,

MLN, evident at 3.5 and 7h and peaked at 48h after feeding of the vaccine (229). The

ISCOM increased the uptake of protein significantly and induced an earlier peak of

antigen uptake into the circulation after feeding, compared to vaccination with the

antigen alone (30min vs. 60min) (229).

Especially, ISCOM have been suggested to overcome MatAb suppression (515).

Calves immunized with the bovine respiratory syncytial virus (BRSV) vaccine with

ISCOM, but not with the inactivated vaccine were protected against challenge with

virulent virus even in the presence of BRSV-specific MatAb (264). In addition,

significantly higher BRSV-specific nasal IgG, serum IgG1 and IgG2 antibody titers were

detected before and after challenge in animals immunized with ISCOM compared to

those immunized with inactivated vaccine.

The ISCOM has a higher potential for human use compared to QS-21 due to its

safety record. The ISCOM has been pronounced safe in field trials in several animal

models e.g. pigs, cattle, horses, macaques, etc (307, 314, 483) and in human adult

volunteers (554). The use of QS-21, on the other hand, has reported several side effects

such as moderate to severe pain in many of the volunteers with vasovagal episodes and

hypertension (200). A combination of QS-21 and other adjuvants such as MPL, CpG,

CRL-1005 (a non-ionic block copolymer), Titermax, Titermax plus CpG have been tried to improve the adjuvancity (363). The GPI-0100, a new semi-synthetic saponin adjuvant 132 containing the docecylamide derivative of the hydrolyzed saponins, was found to be more

potent than QS-21 for antibody responses, delayed-type hypersensitivity reactions and

IFN-γ production in the mouse model (363). Further testing to confirm a similar

adjuvancity of GPI-0100 as ISCOM in humans is needed.

1.5.2.7 Muramyl dipeptide derivatives (MDP)

In the search for a safe but effective adjuvant for clinical use, the N-

acetylmuramyl-L-alanyl-D-isoglutamine, the minimal active component of the Freund’s complete adjuvant (FCA) was found. The antigens with MDP in emulsion (Syntex

adjuvant formulation SAF) has shown efficacy against several viruses such as influenza

virus, , HSV and (16).

1.5.2.8 Hormones

It is possible to use hormones such as Vitamin D3 to enhance both mucosal and

systemic immune responses. Vitamin D3 has been observed to induce DC migration from the skin to PP (191). There is still controversy about the beneficiary effects of Vitamin

D3. Positive effects to the systemic and mucosal immune responses of Vitamin D3 have been reported in pigs and cattle but not in humans receiving influenza vaccines (378, 546,

692). In the study by Kriesel et al (378), an unusual mode of vaccination was employed.

Instead of mixing the vaccine with the adjuvant, Calcitriol was injected at 1cm from the site of influenza antigen injection. Administering antigens with Vitamin D3 on the skin can induce both systemic and mucosal immune responses due to migration of DCs from skin to PP (191).

133

1.5.2.9 Cytokines

Cytokines which have immunomodulating activities such as IL-12 or pro- inflammatory cytokines such as IL-1 and IL-6 have been tested as adjuvants.

Administration of Leshmania antigen with rIL-12 at 200ng/dose or more induced 2-fold or more increases in antigen specific IgG antibody titers, whereas no increase in antibody titer was observed in the absence of rIL-12 (356). Partial protection against the parasite was observed in the presence of either rIL-12 or alum as adjuvants, whereas complete protection was found when both alum and 2ug rIL-12 were used in combination.

Interleukin-1, IL-6 and IL-12 promote either Th2 or Th1 while IL-1 and IL-12, not IL-6 can be used as adjuvants to induce mucosal immune responses (80). Interleukin-1 promotes IgG1 and IgG2b, and little IgG2a, an indication of Th2-type response.

Interleukin-18, similar to IL-1β, which plays an important role in Th1 responses and often acts synergistically with IL-12, has also been demonstrated as a potential adjuvant

(80).

Cytokine adjuvants are especially effective when given IN with little or low side effects as compared with systemic injection (80). The nasal route also requires much less cytokine than the systemic route to achieve similar responses, yet the kinetics of these responses achieved via the IN route are also faster (431). Interleukin-12 is also able to induce IFN-γ, more efficiently by the IN than the parental route. Mice immunized IN with IL-1α or IL-1β produced specific IgG and sIgA antibody responses, similar to those induced by cholera toxin (647). A combination of different cytokines can also

134 enhance the vaccine responses. A DNA vaccine against HIV induced increased antibody responses and T cell proliferative responses when coinjected with IL-18 and INF-γ (361).

However, cytokine adjuvants have not yet been documented in vaccine strategies to overcome MatAb effects.

1.5.2.10 CpG Oligodeoxynucleotides

The unmethylated cytosine-guanine dinucleotides (CpG motifs) in the synthetic oligodeoxynucleotide (ODN) or in bacterial DNA possess immunostimulatory properties

(377). The TLR-9 in DCs and B cells recognize CpG motifs which trigger a series of signaling cascades that cause B cell proliferation and antibody production. The CpG also activates cytokine secretion by macrophages, monocytes and DCs, which in turn stimulate T cells to secrete other cytokines and NK cells to increase cytotoxic activity

(370, 450). More importantly, CpG induces predominantly Th1 responses (IFN-γ and IL-

12) and very little Th2 responses; thus, this adjuvant has the potential to overcome the

Th2 bias in neonatal immune responses.

The CpG was demonstrated to have adjuvant effects when delivered IN, orally or intrarectally with Hepatitis B surface antigen (HBsAg), tetanus toxoid and killed influenza virus (448, 449). The HBsAg-CpG vaccine induced antibodies with higher antigen-binding affinity but with minor side effects in humans (625). The HBsAg/CpG

ODN vaccine induced CTL responses in young mice, but weak B cell responses in the presence of high levels of MatAb against HBsAg (725). Thus more optimization is necessary to improve CpG for use in humans in the presence of MatAb.

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Sites of studies Age of Subject Serum IgA Serum IgG Virus Neutralizing Antibody Methods References antibody antibody titers (RV strain specificity) titers titers Bangladesh 0-4 months 200-600a ELISA (147) 4-7 months 50-110a 0-24 months 40- 81920 (64) Bangladesh 0-6 months >200 NA 8 Complement (575) fixing antibody Brazil - Belem 1-7 months ≤50 (anti-Wa) VNb test (27) China- x = 8 months NAb NA 67c VN test (777) Guangzhou 3426d Venezuela- 0-1 month 20 NA 90 VN, ELISA (530) 204 Caracas 2 months 20 70 3 months 25 50 4 months 30 30 5 months 40 30 6-11 months 80 100 Venezuela- Newborn 88 (anti-RRV), 49 (anti-P) (553) Caracas 4-10 months 30 (anti-RRV), 27 (anti-P)

1-6 months 13 (anti-RRV), 14 (anti-P)

Table 1.1: Level of circulating rotavirus specific antibody of infants in some developing countries

a titers range from children with no infection to infection with non-severe or severe diarrhea b abbreviation: NA; Not available, VN: virus neutralizing c antibody titer measured in children with rotavirus in stool d antibody titer measured in children without rotavirus in stool

204

Sites of studies Subject (age) Serum IgA Serum IgG Virus Neutralizing Methods References antibody antibody titers Antibody titers titers (RV strain specificity) Australia Mother 91 212 (serum) NAa ELISA (456) 313 (cord blood) Germany-Bochum Mother NA NA 490 (anti-Wa) VNa (97) 330 (anti-SA11) Germany-Bochum Infants NA NA 80-200 (anti-Wa) VN (97) (1-4 months) 40 (anti-SA11) Texas Infants 25-6400b 25-6400 NA ELISA (513) -United States (< 18 months) 205

United States Infants NA NA 5-600 VN, (719) (2-6 months) ELISA United States Infants NA NA 52-61 VN (547) (1-6.5 months)

United States Infants 100-1600 NA NA ELISA (118)

Table 1.2: Level of circulating rotavirus specific antibody of mothers and infants in some developed countries

a abbreviation: NA; Not available, VN; virus neutralizing b preexisting antibody titers range from children infected to children not infected with rotavirus during the following season

205 Species Type of placenta Route of transfer of Duration of immunoglobulin passive immunity prenatal route Postnatal route Fish NA Yolk sac Yolk sac ~10 days Reptile NA Yolk sac Yolk sac <1 year Chicken/duck NA Yolk sac Yolk sac 2 weeks (<5 days for chicken Ruminant Syndesmochorial None gut (24-36h) Pig Epitheliochorial None gut (24-36h) 3-4 weeks Horse Epitheliochorial None gut (24h) 32-39 days Dog, cat Endotheliochorial placenta (+) gut Rat/mouse Hemendothelial placenta (+++) gut (20 days) 4-10 weeks Primates Hemochorial placenta gut Human Hemochorial placenta (+++) gut 9 months

Table 1.3: Routes of transmission of MatAb across taxa

[modified from Grindstaff et al (259)]

Species IgM IgA IgG t (½)a 97%b t (½) 97% t (½) 97% loss loss loss Chicken ? ? 1.7 9 4 20 Bovine 4.8 24 2.8 14 20 100 Sheep 4.1 21 1.8 9 10 50 Pig 4.7 24 2.7 14 13 65 Horse 4 20 2 10 20 100 Dog 4 20 2 10 6 30

Table 1.4: Expected loss (days) of MatAb in serum of the newborn in different species a t(½): half-life (days) of MatAb in newborn’ serum b time for 97% MatAb loss=5x half-lives [modified from Hines, H.C. (285)]

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Colostrum/milk TGF-β1 TGF-β2 IL-10 IL-6 TNF-α IL-8 IL-12 IL-1β Mean ± Range Mean ± SD Mean ± SD Mean Range (mean) Mean±SD Mean±SD Mean±SD range SD (mean) Latent Free Latent Free Preterm Colostrum 240 a NDb 2300a ND 0-10 (8.5)a 0-24 (10)a mother (0-5days) Transitional milk 440a ND 3560a ND 0a (6.8)a (6-30days) Mature milk 520a ND 5400a ND 0-1.5(0.4)a 0-32 (14.3)a (>30days) At-term 0-6 (3.5)a mother 3280a 8-34 (21)a 620±183g 3684 ± Colostrum a d h i 1408 ± k 19200 130 ± 108 66 - 9301 ± 2910 j 1-400

2 99 10 (0-5days) c c d f 2256 0 1366 ± 243 728 ± 249 (3304) 151 ± 89 7

Transitional milk 435a 3560a 0a 0-14 (6.6)a (6-30days) 328a Mature milk 5310a 0-200 (13.5)a 0-25 (3.5)a (>30days) 953 ± 213c 179 ± 157 c

a (646) a (646) a (646) a (646) a (646) g (569) Ref. d (518) i (518) j (98) k (279) c (591) c (591) e (231) f (570) h (484)

Table 1.5: Concentrations of cytokines in human colostrum and milk (pg/ml)

b: ND: not detectable (646)

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Virus family Representative Genome Envelope Major Minor

virus proteins proteins

Caliciviridae Norwalk Ss RNA No 1 1-2

Picornaviridae Poliovirus No 4 0

Flaviviridae Yes 1 0

Retrovirus Human Yes 1 0

immunodeficiency

virus

Paramyxoviridae Newcastle disease Yes 4 0

Bunyaviridae Hantaan Yes 1 0

Orthomyxoviridae Influenza A Yes 2 3

Birnaviridae Infectious bursal No 3 0

disease

Reoviridae Rotavirus No 4 2-4

Parvoviridae Parvovirus No 1 2

Papillomarividae Papillomavirus Ds DNA No 1 1

Polyomaviridae SV40 No 1 2

Hepadnaviridae Hepatitis B Discontinuous Yes 1 0

Ds DNA

Table 1.6: Virus-like particles for various virus families

Modified from Noad and Roy (497).

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VP7 VP4 VP2 VP3 VP4

Figure 1.1: Rotavirus structures

Modified from Jayaram et.al., 2004 (324).

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pIgR FcRn

Figure 1.2: Structure of FcRn and pIgR

Modified from Simister and Ahouse, 1996 (631)

The pIgR is a glycosylated membrane protein, homolog of Ig superfamily. The extracellular portion of the molecule consists of five homologous domains, resembling to the V domain of Ig superfamily. The FcRn, a MHC class I homolog, is a heterodimer of

β2 microglobulin and a larger subunit α (45-53 kDa) with 3 extracellular domains.

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100 MatAb concentration

50

Antigen load Inhibition (%) Inhibition

0 Excess MatAb Insufficient MatAb Insufficient Ag Equivalent Excess Ag

Inhibition of Preservation of Preservation T and B cell responses T +/- B cell of T and B cell responses responses Enhancements?

Figure 1.3: Expected influence of maternal antibody (MatAb) on neonatal immune responses to vaccines

[Modified from Siegrist et al. 2003, (622) ]

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ISCOM

2/6-VLP

Figure 1.4: Association of RV 2/6VLP with immunostimulatory complexes (ISCOM)

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CHAPTER 2

PROTECTION AND ANTIBODY RESPONSES TO ORAL PRIMING BY ATTENUATED HUMAN ROTAVIRUS FOLLOWED BY ORAL BOOSTING WITH 2/6-ROTARUS-LIKE-PARTICLES WITH IMMUNOSTIMULATING COMPLEXES IN GNOTOBIOTIC PIGS

2.1. SUMMARY We evaluated antibody responses and protection induced by attenuated Wa human rotavirus (AttHRV) and VP2/6 rotavirus-like particles (VLP), 100 or 250µg/dose, with immunostimulating complexes (ISCOM) (VLP/ISCOM) each given orally, alone or sequentially to gnotobiotic pigs. The AttHRV-VLP250µg/ISCOM and 3-dose-AttHRV

(AttHRV3x) groups had significantly higher serum IgA, IgG and intestinal IgA antibody titers to HRV pre-challenge than the 3-dose-VLP100µg/ISCOM group (VLP/ISCOM3x) and controls (diluent/ISCOMmatrix). Protection rates against viral shedding and diarrhea were highest in the AttHRV-VLP250µg/ISCOM and AttHRV3x groups, lower in the AttHRV-VLP100µg/ISCOM group, with no protection in the VLP/ISCOM3x group and controls. Thus VLP/ISCOM boosted antibody titers and protection after priming with AttHRV.

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2.2. INTRODUCTION

Rotavirus is the major cause of dehydrating diarrhea in children worldwide, resulting in high morbidity and mortality rates in developed and developing countries (21). Rotaviruses consist of a core viral protein VP2 surrounding double stranded RNA, an inner capsid of VP6 and an outer capsid of VP7 with VP4 spikes (21). The major protein VP6 comprises 51% of the total viral protein and determines viral group and subgroup specificity, whereas the outer capsid proteins VP7 and VP4 contain neutralizing epitopes and determine G and P serotype specificity, respectively. In the baculovirus expression system, interaction of VP2 and VP6 leads to the spontaneous formation of double-layered virus-like-particles (VLP); addition of VP7 and VP4 leads to triple layered VLPs resembling the structure of native rotavirus (12). In addition to high yields of VLP from this system, the VLP, which are devoid of nucleic acid, are potentially safe, non- replicating vaccines compared to live vaccines.

The immunogenicity and protective efficacy of VLP have been evaluated in various animal models. In adult mice, 2/6/7-VLP, but not 2/6VLP of bovine RF strain, administered intranasally (IN) with or without cholera toxin (CT) adjuvant induced high virus neutralizing (VN) antibody titers in milk, which provided passive protection against viral shedding and diarrhea to rotavirus challenged nursing pups (10). Similarly, rotavirus seropositive cows vaccinated with simian SA11 2/4/6/7 VLP had significantly increased VN and IgG1 antibody titers in colostrum compared to controls and this colostrum completely protected calves against diarrhea after inoculation with virulent bovine rotavirus. In comparison, colostrum from SA11 2/6-VLP vaccinated cows provided only partial protection to calves against diarrhea and none against virus shedding (14). In

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studies of active immunity to VLP, oral administration of 2/6-VLP to adult mice at high doses (100µg) with CT resulted in a 50% reduction in virus shedding in half of the animals, whereas IN administration of 2/6-VLP or 2/6/7-VLP protected the mice completely from virus shedding upon challenge (28). Intramuscular administration of different VLP formulations from simian SA11 rotavirus strains to rabbits induced high serum IgG and VN antibodies (6). In gnotobiotic pigs, intranasal boosting with two doses of 2/6-VLP and mLT (mutant heat labile toxin from Escherichia coli) after oral priming with one dose of attenuated Wa human rotavirus (AttHRV) induced significantly higher mean numbers of intestinal IgA antibody secreting cells (ASC) compared to one dose of AttHRV, leading to partial protection against viral shedding (58%) and diarrhea (44%); the converse (2/6VLP/mLT IN2x followed by AttHRV orally induced only low protection rates (51). Moreover, in contrast to studies of adult mice and rabbits, IN administration of 2-3 doses of 2/6VLP/mLT to pigs failed to induce protection against viral shedding or diarrhea. These studies confirm the antigenicity of VLP and their vaccine potential, but illustrate the differences seen in their protective efficacy in adult versus neonatal animal models of infection and disease. Another factor for a safe and effective vaccine is the adjuvant. To date, the only adjuvants registered for humans use are aluminium hydroxide and calcium salts (13, 39). Adjuvants like CT, LT or mLT have been widely tested for many mucosal vaccines including rotavirus. However the toxic effects of CT and LT adjuvants have limited their use in humans and even for mLT, its potential safety for infants is unclear. On the other hand, the immunostimulating complexes (ISCOM), have been used for human clinical trials with no or low toxicity reported (34, 39). The ISCOM, consisting of cholesterol, phospholipid and Quillaja saponin, have been widely used with various vaccines, especially enveloped viruses such as influenza virus, measles virus, Japanese encephalitis

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virus, human immunodeficiency virus, respiratory syncytial virus (RSV), but also with rotavirus, malaria, etc. (17, 33, 38, 40). In these studies, ISCOM-associated antigens induced strong local and systemic immune responses, activated both Th1 and Th2 and cytotoxic T lymphocyte (CTL) responses and stimulated the production of all immunoglobulin classes. Therefore, further studies of ISCOM-based vaccines and optimization for use in humans are warranted. In the present study, we examined the antibody responses and protection against diarrhea and infection induced by 2/6VLP with ISCOM alone, or as a sequential regimen with AttHRV, administered orally to gnotobiotic pigs. We also studied the effect of the VLP dose (100µg vs. 250µg) in the AttHRV-VLP/ISCOM regimen on antibody responses and protection of pigs against challenge with virulent Wa HRV.

2.3. MATERIALS AND METHODS

2.3.1. Production and purification of 2/6-VLP: Double-layered 2/6 VLP composed of Wa strain HRV VP6 and bovine rotavirus RF strain VP2 were produced by co-expression of recombinant baculoviruses containing the WaVP6 and RFVP2 genes in Spodoptera frugiperda 9 (Sf9) insect cells according to procedures described previously (12, 51). The integrity of the particles was verified by immunoelectron microscopy (IEM) immediately before use (36, 51). The protein composition of the VLP was verified by polyacrylamide gel electrophoresis (PAGE) and Western blotting using hyperimmune porcine anti-rotavirus serum (51). Sterility and endotoxin tests (Associates of Cape Cod, Inc., Woods Hole, Mass.) were also performed for each lot of VLP. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad, Hercules, Calif.) using bovine serum albumin as the standards.

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2.3.2. Association of 2/6VLP with ISCOM matrix. The lyophilized ISCOM matrix consisted of cholesterol, phospholipid and saponin (semipurified Quillaja saponin) in the ratio of 1:1:3.5 (w/w), respectively. The VLP/ISCOM and ISCOM matrix used for the control group was prepared according to previously published procedures (19). The formation of 2/6VLP-ISCOM matrix and ISCOM-matrix was verified by EM (36).

2.3.3. Viruses and cells. The virulent Wa HRV strain was passaged in gnotobiotic pigs and the pooled intestinal contents of pigs from the 18th passage were used for challenge

6 6 at a dose of ~10 fluorescent forming units(FFU) (10 50% infectious dose, ID50) (37, 45, 53). The cell culture adapted Wa AttHRV used for vaccine was propagated in the MA104 monkey kidney cell line and virus titers were determined by a cell culture immunofluorescence (CCIF) assay in MA104 cells (2).

2.3.4. Preparation of inactivated double-layered Wa HRV. As a control for the double- layered 2/6 VLP, double-layered particles were prepared from Wa AttHRV. The infected MA104 cell lysates were centrifuged at 100,000 x g on 30% sucrose (w/v, in PBS, pH7.4) for 2hr. The virus pellet was suspended in PBS and then treated twice with 50mM EDTA at 37oC for 1h with occasional shaking to dissociate the triple-layered virus particles. The virus suspension was purified through CsCl gradients as described earlier for the VLP. The bands containing double-layered virus particles, verified by density measurements

and IEM, were pooled and inactivated with 0.01M binary ethylenimine (the activated product of 2-bromoethylamine hydrobromide, Aldrich Chemical Co., St. Louis, Mo.) as described previously (51). The loss of viral infectivity was confirmed by plaque and CCIF assays (2). Loss of VP7 was confirmed by PAGE and Western blotting with porcine anti- rotavirus hyperimmune serum and monoclonal antibody specific for rotavirus VP7 (Common 60) provided by Dr Greenberg, Stanford University, USA (Figure 2.1).

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2.3.5. Gnotobiotic pigs. The hysterectomy-derived near-term pigs were obtained and maintained as described previously under an approved animal use protocol (45, 53). Pigs were maintained in sterile isolation units and fed a commercial infant formula (Ross laboratories, Columbus, Ohio).

2.3.6. Experimental design. The pigs were assigned to one of 7 vaccine regimens consisting of 3 vaccine doses administered orally, 9-10 days apart (Table 2.1). At 3-5 days of age, pigs from each group received 5ml of 100 µM sodium bicarbonate to reduce

stomach acidity followed by the oral vaccines. Groups 1 (16 pigs) and 2 (8 pigs) received AttHRV (5x107 FFU/ml) for the first dose, followed by 2 doses of 2/6VLP-ISCOM (AttHRV-VLP/ISCOM): 100µg VLP + 500µg ISCOM for group 1 (AttHRV- VLP100/ISCOM); 250µg VLP + 1250µg ISCOM for group 2 (AttHRV- VLP250/ISCOM). Group 3 (8 pigs) received 3 doses of 100µg 2/6 VLP + 500ug ISCOM

(VLP/ISCOM3x). Group 4 (4 pigs) received 3 doses of 100µg inactivated double-layered Wa HRV with 500µg ISCOM matrix (dl-inact-HRV/ISCOM3x). Groups 5 (13 pigs) and

6 (20 pigs) were inoculated with 1 dose and 3 doses of AttHRV (AttHRV1x and AttHRV3x), respectively. Group 7 (21 pigs) received 3 doses of 500µg ISCOM matrix or diluent (ISCOM/diluent). At post-inoculation day (PID) 28, subsets of pigs

(approximately half) from each group were euthanised (post-challenge day, PCD 0) and

6 6 the others were challenged with 10 FFU (~10 ID50) of virulent Wa HRV and euthanised 7 days later (PCD7) (45, 53). Serum samples were collected from all pigs at each inoculation. The small (SIC) and large (LIC) intestinal contents were collected at euthanasia and diluted 1:2 in diluent containing 250µg/ml trypsin inhibitor (Sigma) and

50ug/ml leupeptin (Sigma) to inhibit proteolytic enzymes and stored at - 20oC until tested.

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2.3.7. Analysis of clinical signs. All pigs were monitored for clinical signs after challenge. Rectal swabs were collected at PCD 1 to PCD6. Fecal consistency was scored as follows: 0, normal; 1, pasty; 2, semi-liquid and 3, liquid. Pigs with fecal scores of ≥2 were considered to have diarrhea. Rotavirus shedding was detected in rectal swab fluids using antigen capture ELISA (37) and CCIF (2).

2.3.8. Plaque-reduction virus-neutralization assay. The VN antibody titers were determined by a plaque-reduction virus neutralization assay described previously (37). The VN titers were expressed as the reciprocal of the highest sample dilution in which the number of plaques was reduced by ≥80%.

2.3.9. Isotype-specific antibody ELISA. The Wa HRV specific isotype antibody (IgA, IgG and subclasses IgG1 and IgG2) titers in serum, LIC and SIC samples were determined by an indirect isotype-specific antibody ELISA as previously described (14, 31, 32, 41). IgG1 and IgG2 antibody responses to Wa HRV were analysed in serum at PID28 (at challenge) using secondary antibodies to swine IgG1 (diluted 1:1000) and IgG2 (diluted 1:500) produced in mice (Serotec, UK), followed by anti-mouse IgG (H+L) (KPL, Gaithersburg, Maryland, USA) conjugated to horseradish peroxidase (diluted

1:10,000). The absorbance at 414nm (A414) was corrected for background by subtraction of sample absorbances in the mock antigen coated from those in the virus-coated wells. The titer was expressed as the reciprocal of the highest dilution having a corrected

A414 greater than the cut-off value (mean corrected A414 of negative controls + 3 times of standard deviation (SD) or 0.05 whichever was higher). The initial serum or intestinal content dilution tested was 1:4 followed by serial 4-fold dilutions; samples negative at this dilution were assigned a titer of 2 to calculate the geometric mean antibody titers (GMT).

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2.3.10. Statistical analysis. Statistical analyses were performed on log10 transformed, isotype-specific ELISA antibody titers and VN antibody titers, with a p-value of ≤ 0.05 denoting statistical significance. One-way ANOVA (SAS Institute Inc., Cary, N.C) followed by Duncan's multiple-range test was used to compare significance between samples at each time point. A Spearman correlation test was used to correlate protection against virus shedding and diarrhea with VN, serum and intestinal IgA antibody responses of individual pigs and to correlate the IgA and IgG antibody titers in serum to that in the intestinal contents. Differences in proportions of pigs with diarrhea and virus shedding were determined by Fisher’s exact test.

2.4. RESULTS

2.4.1. The AttHRV-VLP250/ISCOM and AttHRV3x vaccines induced the highest protection against diarrhea and viral shedding among all vaccines

The protection rates against viral shedding and diarrhea in Table 2.1 were presented in part previously by Iosef et al.(19). Complete protection against virus shedding was obtained in 50% of pigs receiving AttHRV-VLP100/ISCOM, which was significantly higher than that obtained in the AttHRV1x group (14%). Increasing the VLP dose to 250µg conferred a higher protection rate (75%) against shedding, but this was not statistically different from the 100µg VLP group (50%) or compared to the AttHRV3x group (62%). However, significantly higher protection rates against diarrhea were observed in the AttHRV-VLP250/ISCOM and AttHRV3x groups compared to the 100µg

VLP group (50% vs. 14%, respectively). Neither the VLP/ISCOM3x nor dl-inact- HRV/ISCOM3x conferred any protection against virus shedding or diarrhea.

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2.4.2. Serum Wa HRV neutralizing antibody responses correlate with protection against diarrhea and viral shedding The VN GMTs in the serum of the inoculated gnotobiotic pigs are summarized in Figure 2.2. The VN GMTs were highest in the AttHRV3x group at all times tested before challenge with virulent Wa HRV (PID10 and PID20, data not shown) and were significantly higher at PID28 (Figure 2.2). There were no statistically significant differences between the pre-challenge VN antibody titers in the groups inoculated with AttHRV-VLP/ISCOM (100µg or 250µgVLP) and the AttHRV1x vaccine groups suggesting lack of boosting of VN antibodies by 2/6VLPs that contain no VN antigens (present only on VP4 and VP7). After challenge, pigs in all groups had 4-fold or greater increases in VN GMT. The two groups receiving AttHRV-VLP/ISCOM (100µg or 250µg VLP) had VN titers statistically similar to those of the AttHRV1x (group 5) and AttHRV3x (group 6). There was a positive correlation between the VN titers pre-challenge and protection rates

against viral shedding and diarrhea (rs=0.65, p<0.001 for both correlations, Spearman correlation test). Interestingly, the VLP/ISCOM3x and dl-inactHRV/ISCOM3x, (served as a comparative intact virus, double-layered control for the VLP/ISCOM vaccine) had statistically similar post-challenge VN titers to those of the control group

(ISCOM/diluent), suggesting that double-layered rotavirus or VLP failed to prime pigs for increased serum VN antibodies.

2.4.3. Serum rotavirus-specific isotype antibody responses and their correlation with protection against diarrhea and viral shedding The development of rotavirus specific IgA and IgG antibodies in serum during the course of immunization and after challenge are depicted in Figure 2.3 and Table 2.2.

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2.4.3.1. IgA antibody responses. The IgA antibodies were undetectable until PID28 in the VLP/ISCOM3x and dl-inact-HRV/ISCOM3x groups. The IgA antibodies appeared earlier (PID20) and in higher titer in all 4 groups receiving AttHRV as the priming vaccine. Among these groups, the two groups that also received VLP/ISCOM booster vaccine had significantly higher pre-challenge IgA antibody titers than the AttHRV1x group, but not significantly higher than in the AttHRV3x group. The correlation coefficients (r) between serum IgA antibody titers at challenge and protection rates against virus shedding and diarrhea were 0.62 and 0.49, respectively (p<0.001). After challenge, all groups except the ISCOM/diluent control had elevated IgA antibody titers. No significant difference in the post-challenge IgA titers was observed among vaccinated groups except between the AttHRV-VLP250/ISCOM and the VLP/ISCOM3x group (p<0.05).

2.4.3.2. IgG antibody responses. The IgG antibodies were detected by PID10 for group 2 (AttHRV-VLP250/ISCOM) and group 6 (AttHRV3x). The IgG antibody titers were highest in the two groups receiving AttHRV-VLP/ISCOM compared to groups receiving AttHRV3x or AttHRV1x at PID28. Slower kinetics of IgG antibody responses were observed in the VLP/ISCOM3x and dl-inactHRV/ISCOM3x groups (antibodies appeared

20 days after the first dose). The pre-challenge IgG GMTs of these 2 groups were the lowest among the vaccine groups. After challenge the IgG GMT increased in all vaccinated groups, and no statistical differences in antibody titers were evident. The correlation coefficients between pre-challenge serum IgG titers and protection against

shedding and diarrhea were low but significant (rs=0.44 and 0.33, respectively, p<0.001).

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2.4.3.3. IgG subclass responses (Figure 2.4). At PID28 the two groups with AttHRV- VLP/ISCOM had the highest IgG antibody titers for both subclasses compared to the other groups. Regarding the IgG subclass distribution, IgG1 antibodies were prevalent over IgG2 antibodies in the AttHRV1x and AttHRV3x groups (mean IgG1/IgG2 ratios of 5.3), whereas both IgG1 and IgG2 antibodies were induced to a similar extent in the VLP/ISCOM3x and dl-inact-HRV/ISCOM3x groups (mean IgG1/IgG2 ratios of 2 and 1, respectively). The IgG1/IgG2 ratio was intermediate in the AttHRV-VLP/ISCOM groups (ratios of 3.0 to 3.2).

2.4.4. Isotype antibody responses in intestinal contents and their correlation with protection against viral shedding and diarrhea. The intestinal antibody titers were quantitated in intestinal contents (LIC and SIC) of pigs before challenge (PID28) or after challenge (PID35/PCD7) and are shown in Figure 2.5.

2.4.4.1. The IgA antibody responses in SIC were highest in the AttHRV- VLP250/ISCOM and AttHRV3x groups and were highly correlated with protection against diarrhea and viral shedding The AttHRV-VLP250/ISCOM and AttHRV3x groups (groups 2 and 6, respectively) had higher IgA GMT in SIC and LIC compared to in the AttHRV1x (group 5), and the

difference was statistically significant between groups 2 and 5. The AttHRV- VLP100/ISCOM (group 1) had elevated SIC IgA antibody titers at PID28, but titers did not differ significantly from the other vaccine groups. After challenge the IgA antibody GMT in the intestinal contents increased 4-fold or more in all vaccine groups. The highest titers occurred in the groups receiving AttHRV as priming vaccine. In the VLP/ISCOM3x or dl-inactHRV/ISCOM3x, the IgA antibody titers also increased

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significantly post-challenge (8-14-fold in SIC and 5-16-fold in LIC). Moderate to high significant correlations were found between IgA titers in serum and intestinal contents

before (rs=0.64-0.74) and after challenge (rs =0.86-0.9) (Spearman’s correlation, p<0.001), in agreement with the trafficking of IgA ASC from intestinal tissues into the circulation and back to the effector sites in the intestine. The IgA GMT at PID 28 in the SIC and not in the LIC showed a strong significant correlation with protection rates

against viral shedding and diarrhea (for SIC, rs=0.9 and 0.85, respectively, p<0.05).

2.4.4.2. Intestinal IgG antibody responses were low in all vaccine groups except

AttHRV-VLP100/ISCOM and were not correlated with protection.

IgG antibody titers were generally low in all groups, except in SIC of pigs receiving AttHRV-VLP100/ISCOM. After challenge intestinal IgG titers generally did not increase and remained low except in intestinal contents (SIC and LIC) of the AttHRV- VLP250/ISCOM group. Interestingly, even though IgG antibody GMTs at PID28 in SIC of the AttHRV-VLP250/ISCOM group were lower compared to those of the AttHRV- VLP100/ISCOM group, the former group had the highest increase in IgG antibody titers after challenge, suggesting induction of stronger memory responses, whereas no increase in IgG antibody titers were observed in the latter. Moderate to low correlations were found between serum and intestinal content IgG antibody titers before and after challenge

(rs ranges between 0.4-0.56, p<0.001), suggesting some transudation of serum IgG into the intestinal lumen may occur, especially after gut damage from virus infection. No correlation was found between IgG from SIC or LIC and protection rates against rotavirus shedding and diarrhea.

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2.5. DISCUSSION

The disease burden caused by rotavirus in children has prompted a need for safe and effective vaccines. The VLP may potentially reduce the risk of intussusception related to live vaccines, and also are potent non-replicating immunogens. Strong boosting effects of

VLP/ISCOM or dl-inactHRV/ISCOM were manifested by the increase seen in serum and intestinal IgA and IgG antibody titers after challenge (similar to levels of other vaccine groups). The VLPs remained intact and were not incorporated into the cage-like structure of ISCOMmatrix as described for soluble membrane proteins of influenza virus, measles virus or RSV (18, 30, 33, 34, 38, 40). In this study, ISCOMmatrix was observed by IEM to aggregate to the VLPs (19), and yet serum IgA, IgG and intestinal IgA antibody responses were enhanced considerably compared to those of 2/6-VLP alone which did not induce significant serum and intestinal antibody responses (50). The use of ISCOMmatrix with antigen upregulates specific immune responses. Mixing of ISCOMmatrix with influenza antigen stimulated lymphocyte expansion and proliferation as well as increased antibody levels (49). The ISCOM may also alter the rate and efficiency of protein uptake by increasing intestinal leakiness to allow transport of larger moieties (15). The ISCOM may also alter the processing mechanism of peptide presented to T-helper cells (15).

Future studies of uptake of antigen by gut lymphoid tissues in the presence or absence of

ISCOMmatrix are needed to identify how the VLPs with or without ISCOMmatrix are taken up during transport via the gut epithelium or into Peyer’s patches.

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For the delivery of subunit antigens like VLP via mucosal routes, safe and

effective adjuvants are important. The ISCOMs have been used for human clinical trials

with low or no toxicity reported. Another advantage of ISCOM includes the escape from

suppressive effects of maternal antibodies as evident using an ISCOM-based measles

vaccine administered to human infants (30). Similarly, for rotavirus, because infections

occur in children as early as 3 months of age, especially in developing countries, the

vaccines need to be given early to induce immunity, and consequently will be affected by

the presence of maternal antibodies. Therefore ISCOM could be a potential adjuvant for

rotavirus vaccines to be used in human infants.

In this study we investigated protection against disease and infection induced by

different vaccine regimens consisting of AttHRV and 2/6VLP with ISCOM. A number

of ISCOM-based vaccines have been tested, e.g. influenza, measles and Japanese encephalitis and in a number of species including humans, but there are few reports for rotavirus and none for rotavirus VLP vaccines (19, 42, 43). Gnotobiotic lambs given one

dose of inactivated rhesus rotavirus (RRV)/ISCOMmatrix orally had low levels of

protection and no serum and intestinal IgA and IgG antibodies (42). Intramuscular

immunization of mice with one dose of inactivated EDIM murine rotavirus adjuvanted

with Quil A or QS21 (the saponin components of ISCOM) induced high serum IgG,

moderate serum IgA, but low fecal IgA and IgG antibody titers (26). Results of these

studies suggest that a multiple dose vaccine may be required to achieve better protection.

In our study, no protection was obtained in pigs given 3 doses of VLP/ISCOM, yet when

combined with one oral priming dose of with AttHRV, moderate levels of protection

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were achieved. Increasing the dose of VLP from 100µg to 250µg increased the protection rates (from 50% to 75% protection against shedding and from 14% to 50% protection against diarrhea), which were similar to those obtained with AttHRV3x (62% and 50% respectively). The observed lack of protection against shedding and diarrhea in the oral

VLP/ISCOM3x group agreed with the results of our previous studies using 2/6VLP with mLT in neonatal gnotobiotic pigs (51). Our results are also consistent with studies of mice in which no protection was provided to neonatal suckling mice by vaccinating the dams with 2/6VLP with CT adjuvant (9). In another study by O’Neal et al. of adult mice orally inoculated with 2/6VLP at a 100µg dose with CT, only a 50% reduction in virus shedding in 50% of the mice was observed (28). In our current study and our previous reports (19, 51) a priming dose of AttHRV was needed before boosting with 2/6VLP to achieve partial protection against virus shedding and diarrhea. With this strategy, we minimized the use of multiple doses of attenuated vaccines, and this approach may reduce the possibility of intussusceptions related to the second dose of live vaccine (27). In addition, the results of this study provide a basis for future studies to test if attenuated rotavirus vaccines may be replaced with non-replicating 2/4/6/7VLP for priming, which contain VP4 and VP7 antigens which induce neutralizing antibodies.

We also found that the combined oral regimen of AttHRV-VLP/ISCOM, especially with the 250µg dose of 2/6VLP induced the highest intestinal and systemic isotype antibody titers before challenge, comparable or higher than those induced by

AttHRV3x. The high intestinal IgA antibody titers found in the two AttHRV-

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VLP/ISCOM groups and the AttHRV3x group coincided with the high numbers of IgA

ASC detected in the intestinal lamina propria of these same groups (19). The IgA antibody titers in the intestinal contents were enhanced after challenge in all groups, but more so in the AttHRV-VLP/ISCOM groups, suggesting that these regimens induced more memory B cells producing IgA antibodies post-challenge. In addition serum dimeric

IgA might also be transported via the polymeric Ig receptor across intestinal epithelial cells, explaining the strong correlation between serum and intestinal IgA antibodies. Thus even though the 2/6VLP-ISCOM induced both intestinal and systemic antibody responses, a priming dose of AttHRV was necessary to stimulate and enhance the initial intestinal antibody responses and induce VN antibodies.

The combined oral vaccine regimen AttHRV-VLP/ISCOM induced the highest serum IgG1 and IgG2 antibody responses compared to other vaccine groups. The high

serum IgG1 antibody titers are consistent with the IgA serum and intestinal antibody titers

(Th2) found in these groups. The IgG1 and IgG2 responses were enhanced in serum of pigs in the VLP/ISCOM3x and dl-inactHRV/ISCOM3x groups, indicating a more mixed

Th2 and Th1 response induced by oral VLP/ISCOM, whereas the IgG1 antibody response was more dominant in the AttHRV1x and AttHRV3x groups, consistent with Th2 responses. This observation is consistent with previous findings about the immunomodulating ability of ISCOM (1, 18, 22). The ability of ISCOM to induce both

Th types of responses supercedes other adjuvants approved for human use like Al(OH)3 which induces mainly IgG1 and IgE antibodies (Th2). Studies of the cytokines secreted

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by the lymphocytes from mucosal or systemic tissues are underway in our laboratory to

further complement the IgG isotype findings and to understand the effect of ISCOM on T

cell responses.

A correlation between serum rotavirus neutralizing antibodies and protection was observed in this study and was also observed in a rotavirus vaccine study in infants (47) and a passive immunity study in neonatal mice (10). In the former study, induction of neutralizing antibodies specific to serotypes 1 and 3 were correlated with protection against rotavirus diarrhea. In the latter study, neutralizing epitopes of VP7 were needed to induce protection against diarrhea: pups born to mothers vaccinated with VLP2/6/7 but not 2/6VLP were protected against disease when challenged with rotavirus. However, in adult mice, active protection against virus shedding was independent of neutralizing antibody titers (25, 28, 48). Intranasal immunization of adult mice with 2/6VLP with CT induced complete protection in a similar manner to 2/6/7VLP with CT. These discrepancies may be explained by the use of different animal models: neonatal pigs and mice vs. adult mice (51, 52). The later model allows the assessment of reduction in viral shedding only, whereas the former models permit analysis of protection against diarrhea and infection. In addition, the virus dose required for infection and diarrhea induction in adult mice is 103 - 104 times higher than that in gnotobiotic pigs (3, 44). Thus the types and magnitude of immune responses including VN antibodies or serum/ intestinal IgA antibodies that are required to protect piglets against rotavirus infection and diarrhea may differ from those required to protect mice and rabbits against rotavirus infection (52).

We found higher correlations between serum and intestinal IgA antibody GMT with protection against viral shedding and diarrhea. These observations agree with reports by

229

Matson et al (24) comparing children not infected or asymptomatically infected or

symptomatically infected with rotavirus. Pre-exposure fecal IgA antibody titers were higher in subsequently uninfected children than those in subsequently infected ones.

Several VP7-specific IgA monoclonal antibodies assessed as backpack tumors also

protected newborn mice from diarrhea after challenge with RRV (35). Coulson et al (11)

showed that high titers of fecal IgA antibodies were maintained after rotavirus reinfection

and directly correlated with protection against rotavirus illness. Although most data

confirm the role of fecal IgA antibodies in protection, the contribution of serum

antibodies to protection remains controversial. Contradicting results about the correlation

were found in studies of naturally infected children and vaccine trials (4, 5, 7, 8, 16, 20,

23, 29, 46, 47) . Jiang et al (20) postulated that these discrepancies may result from

differences in sample size, vaccine type and dose, demographics and laboratory

diagnostics used. Nevertheless the majority of investigators have reported a positive

correlation between serum and intestinal IgA and protection.

In summary, we compared the antibody responses and protection induced by

multiple doses of oral VLP/ISCOM alone or combined with AttHRV, with multiple doses

of AttHRV. We found that the combined regimens of AttHRV-VLP/ISCOM (especially

the high dose 250µg of 2/6VLP) and AttHRV3x induced higher intestinal and systemic

IgA antibody responses than the other vaccines. To our knowledge, our strategy of using

oral rotavirus VLP vaccine associated with ISCOM matrix is unique and hasn’t been

tested previously in humans or animals except in our prior gnotobiotic pig study assessing

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the ASC responses (19). These results may be applied to rotavirus vaccines and other

ISCOM-related vaccine studies. Further studies are needed to test 2/4/6/7 VLP, which elicit VN antibodies using this model. Our findings once again support the potency of

2/6-VLP/ISCOM as an oral vaccine, but only when combined with oral AttHRV for priming, to induce moderately high rates of protection against rotavirus.

2.6. ACKNOWLEDGMENTS

We thank Dr. Juliette Hanson, Rich McCormick and Veronica Costantini for technical assistance. We also thank Bert Bishop for help in statistical analysis. This work was supported by grants from the National Institutes of Health (RO1AI33561 and RO1AI37111). Salaries and research support were provided by state and federal fund appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Marli Azevedo is a fellow of Conselho Nacional de Desenvolvimento Cientifico and Tecnologico (CNPq), Brazil.

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Protection rates (%)b Group Treatment a Priming dose a Boosting doses a n Virus Diarrhead sheddingc 1 AttHRV-VLP100/ISCOM AttHRV VLP/ISCOM (100ug VLP) 8 50Ae 14Be 2 AttHRV-VLP250/ISCOM AttHRV VLP/ISCOM (250ug VLP) 4 75A 50A 3 VLP/ISCOM3x VLP/ISCOM VLP/ISCOM 5 0C 0C 4 dl-inactHRV/ISCOM3x dl -inactHRV/ISCOM dl-inact HRV/ISCOM 2 0C 0C 5 AttHRV1x AttHRV Diluent 7 14B 0C 6 AttHRV3x AttHRV AttHRV 8 62A 50A 7 ISCOM/diluent ISCOMmatrix/ ISCOMmatrix/ 15 0C 0C diluent diluent

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Table 2.1: Summary of protection rates against virus shedding and diarrhea in gnotobiotic pigs receiving different vaccine treatments a Gn pigs were orally inoculated at 3-5 days of age with one of the different vaccine regimens in 3 doses, 9-10 days apart. A subset of pigs within each treatment group were challenged with virulent Wa HRV (106 FFU). These data have been presented in part by Iosef et al. (19). b Protection against diarrhea was calculated as (1-percentage of Wa HRV inoculated pigs in each group with diarrhea/ percentage of ISCOM/diluent control pigs with diarrhea). Protection against shedding was calculated similarly. The shedding and diarrhea rates of the ISCOM/diluent group were both 100%. c Determined by ELISA and cell culture immunofluorescence infectivity assay. d Pigs were positive for diarrhea if daily fecal consistency score was ≥2 at any day during 7-8 days post-challenge. Fecal consistency was scored daily as follows: 0, normal; 1, pasty; 2,semi-liquid; and 3,liquid. e Percentages in the same column, with different superscript letters differ significantly (Fisher’s exact test).

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GMT a b (PID28/PCD0) Correlation coefficient ( rs ) IgA - Virus IgA - IgG - Virus IgG - Group TREATMENT IgA IgG shedding Diarrhea shedding Diarrhea AttHRV- 1 VLP100/ISCOM 1956A 94848A 0.62 0.49 0.44 0.33 AttHRV- 2 VLP250/ISCOM 1248A 44102A 3 VLP100/ISCOM3x 23DC 2896D 4 dl-inact-HRV/ISCOM3x 64C 4096DC 5 AttHRV1x 285B 7767BC 6 AttHRV3x 724AB 16384B 7 ISCOM/diluent 2E 2E

Table 2.2. Isotype antibody responses to Wa HRV in serum of pigs at PID 28/PCD0 and the correlation between antibody titers and protection against virus shedding and diarrhea at this time point.

a The GMTs with different superscript letters differ significantly (one-way ANOVA,

followed by Duncan grouping on log10-transformed titers).

b Spearman’s correlation between serum antibody titer at PID28/PCD0 and protection

against viral shedding and diarrhea. (p<0.001)

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MW MW markers 1 2 3markers 1 2 3

124k

83k VP2

42k VP6

VP7 32k

Swine anti-WaHRV serum Mouse anti-VP7 monoclonal antibody (Common 60)

Figure 2.1: Immunoblot of protein components in 2/6VLP and double-layered inactivated HRV (dl-inact-HRV). Membrane was probed with porcine anti-Wa HRV hyperimmune serum (a) or with mouse anti-VP7 ascites Common 60 (b). Attenuated Wa

HRV (lane 1), 2/6-VLP (lane 2) and dl-inact-HRV (lane 3).

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T 100000

a a a 10000 a

A PID28/PCD0 1000 B B B PID35/PCD7 100 b b b

10

C C C rs shedding=0.65 eometric mean VN(GMeometric antibody titers

G 1 rs diarrhea =0.65 1234567 (p<0.001) Vaccine groups

Figure 2.2: Geometric mean virus neutralizing (VN) antibody titers in sera of pigs that received different vaccine regimens and their correlation with the protection rates against viral shedding and diarrhea. Pigs were inoculated with the vaccine at PID0, boosted twice, at 9-10 day interval and challenged at PID28. VN antibody titers were assayed in sera at PID28/PCD0 (open bars) and at PID35/PCD7 (solid bars). Error bars represent standard error of the means. Bars, with different upper-case letters A,B,C (PID28/PCD0) and lower-case letters a,b (PID35/PCD7), differ significantly (one-way ANOVA, followed by Duncan grouping on log10 transformed titers). Correlation between pre- challenge serum VN titers (PID28/PCD0) and protection rates against viral shedding and diarrhea is indicated (Spearman's correlation). Group1: AttHRV-VLP100/ISCOM, Group2: AttHRV-VLP250/ISCOM, Group3: VLP/ISCOM3x, Group4: dl-inact- HRV/ISCOM3x, Group5: AttHRV1x, Group6: AttHRV3x, Group7: ISCOM/diluent.

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Figure 2.3: GMT of isotype-specific antibody to Wa HRV in serum of gnotobiotic pigs at various time-points from each vaccine treatment group. Pigs were inoculated with the

vaccine at PID0, boosted twice at 9-10 day interval and challenged with virulent Wa

HRV at PID28. Pigs were euthanised at PID28/PCD0 and PID35/PCD7. Data represent

the GMT of each isotype-specific antibody (IgA, 2.3a; IgG, 2.3b) at each time point. The

GMTs at a particular time-point with different upper-case letters A, B, C (PID28/PCD0)

and lower-case letters a,b (PID35/PCD7), differ significantly (one-way ANOVA,

followed by Duncan grouping on log10-transformed titers). Pigs were assigned to different vaccine groups, AttHRV-VLP100/ISCOM (square), AttHRV-VLP250/ISCOM (filled- square), VLP/ISCOM3x (triangle), dl-inact-HRV/ISCOM (filled-triangle), AttHRV1x

(circle), AttHRV3x (filled-circle), ISCOM/diluent (asterisk).

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a. 1,000,000 IgA

100,000

a AttHRV-VLP100/ISCOM 10,000 ab AttHRV-VLP250/ISCOM VLP100/ISCOM3x A b 1,000 dl-inact-HRV/ISCOM3x AB B AttHRV1x 100 AttHRV3x C ISCOM/diluent DC 10

Geometric mean antibody titers (GMT) E c 1 PID0 PID20 PID35/PCD7

b. 1,000,000 IgG a b A bc 100,000 c B AttHRV-VLP100/ISCOM BC 10,000 DC AttHRV-VLP250/ISCOM

D VLP100/ISCOM3x 1,000 dl-inact-HRV/ISCOM3x AttHRV1x 100 AttHRV3x ISCOM/diluent 10 E d Geometric mean antibody(GMT) titers 1 PID0 PID20 PID35/PCD7

Figure 2.3

243

Ratio IgG1/IgG2 3.0 3.2 2.0 1.0 5.3 5.3

100000 A a AB D BC 10000 ab bc Ig G 1 CD D bc Ig G 2 bc c

1000

100 VLP100/ISCOM VLP250/ISCOM VLP/ISCOM3x HRV/ISCOM3x AttHRV1x AttHRV3x Geometric mean antibody titer (GMT) dl-inact- AttHRV- AttHRV-

Figure 2.4: The IgG subclass responses to Wa HRV in serum samples at PID28/PCD0 from different vaccine groups. Rotavirus-specific IgG subclass antibody IgG1 (opened bars) and IgG2 (solid bars) were assayed by ELISA. Data represent the GMT of each antibody isotype at PID28/PCD0. Error bar represents standard error of the means. For each IgG subclass, columns with the same letter do not differ significantly (upper-case letters for IgG1, lower-case letters for IgG2, one-way ANOVA of log10-transformed titers followed by Duncan's multiple-range test).

244

(a): Intestinal IgA antibody SIC LIC

10000 a ab ab a a ab a ab ab A A b PID28/PCD0 1000 ab b AB PID35/PCD7 B AB B B BC BC 10 0 BC C B For SIC- IgA at (GMT) c PID28/PCD0 10 c C D rS shedding =0.9

1 rs diarrhea = 0.85 Geometric mean antibody titer titer antibody mean Geometric 12345671234567 (p<0.05) Groups (b): Intestinal IgG antibody SIC LIC 10000

1000 A a a a PID28/PCD0 ab ab A B B ab PID35/PCD7 10 0 ab ab ab ab B AB (GMT) AB b AB B B AB 10

Bb B b B b Geometric meanGeometric antibody titer 1 12345671234567 Groups

Figure 2.5: GMT of isotype-specific antibodies to Wa HRV in small (SIC) and large (LIC) intestinal contents of gnotobiotic pigs euthanised at various time-points from each vaccine group (1-7). Pigs were given the vaccine at PID0 and boosted twice for 9-10 day- interval and challenged with virulent Wa HRV at PID28 as described in Materials and Methods section. Rotavirus-specific antibody isotypes (IgA, Figure 2.5a; IgG, Figure 2.5b) of intestinal contents collected at PID28/PCD0 (open bars) and at PID35/PCD7 (solid bars) were assayed by ELISA. Data represent the GMT of each antibody isotype at each time-point. Error bars represent standard error of the means. For each time point, columns with the same letter do not differ significantly (upper-case letters for PID28/PCD0, lower-case letters for PID35/PCD7, one-way ANOVA of log10-transformed titers followed by Duncan's multiple-range test). Spearman’s correlation between SIC IgA antibody titers at PID28/PCD0 and protection against viral shedding and diarrhea is indicated. Group1: AttHRV-VLP100/ISCOM, Group2: AttHRV-VLP250/ISCOM, Group3: VLP/ISCOM3x, Group4: dl-inact-HRV/ISCOM3x, Group5: AttHRV1x, Group6: AttHRV3x, Group7: ISCOM/diluent.

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CHAPTER 3

HIGH TITER SERUM MATERNAL ANTIBODIES IMPACT

PROTECTIVE IMMUNITY AND B CELL RESPONSES INDUCED BY

ATTENUATED ROTAVIRUS PRIMING AND A ROTAVIRUS-LIKE PARTICLE

–ISCOM BOOSTING VACCINE REGIMEN

3.1. SUMMARY

We investigated maternal antibody (MatAb) effects on protection and immune responses induced by attenuated (Att) human rotavirus (HRV) priming and immunostimulating complex (ISCOM)-rotavirus-like-particle (VLP) booster vaccine (AttHRV/VLP) or a

VLP-ISCOM prime/boost vaccine (VLP3x). Passive circulating MatAb (hyperimmune sow serum) injected into newborn gnotobiotic piglets contributed to partial protection against HRV challenge; however, MatAb interference led to no or low intestinal IgM,

IgA and IgG antibody titers and significantly reduced intestinal IgA and IgG effector and memory B cell responses in the AttHRV/VLP piglets pre- and post-challenge. The suppression was not alleviated and was actually enhanced after extending the

246 vaccination/challenge interval from 28 to 42 days. In piglets vaccinated with non-

replicating VLP-ISCOM alone, the MatAb suppressed intestinal IgG but not IgA and

IgM antibody secreting cells, serum and intestinal IgA antibodies and pre-challenge

memory B cell. Alternative strategies are needed to overcome MatAb suppression of

immune responses to vaccines in neonates.

3.2. INTRODUCTION

Vaccination of neonates faces many challenges due to the immaturity of the

neonatal immune system and interference by maternal antibodies (MatAb) present at

vaccination. Interference of vaccine-induced immune responses by MatAb has been

reported for live vaccines such as measles and Sabin oral poliomyelitis vaccine as well as

for non-replicating vaccines (i.e. inactivated, subunit or DNA vaccines) such as tetanus, diphtheria, hemophilus influenza, hepatitis A, classical swine fever and measles virus (5,

6, 10, 12, 16, 21). The degree of interference may depend on the ratio between the

amount of MatAb and the antigen present (28). Optimal amounts of MatAb can enhance

B cell responses by forming antigen-antibody complexes which induce complement

deposition, in turn engaging the B cell receptor and the complement receptor CD21,

thereby producing costimulation via CD19/CD81. On the other hand, neutralizing MatAb

neutralize live vaccines and reduce the antigen mass available for immune response

induction. Some studies suggest that micrograms of MatAb lead to suppression whereas

nanograms result in enhancement of infant’s immune responses (29, 36). Thus the

outcomes of MatAb effects, whether positive (enhancing) or negative (suppressing) are 247 dependent on the MatAb titers, the vaccine, and the dose and type of antigen (26). The

outcomes of MatAb interference can not be reliably predicted; experimental models and

field trials are required to study the MatAb effect to a vaccine.

Rotavirus infections occur as early as under 3-months of age in infants even when

the levels of MatAb in the circulation (acquired from placental transfer) and intestine

(acquired from breast milk) remain high. The variable efficacies and seroconversion rates

in many rotavirus vaccine trials in human infants were influenced by the effects of

MatAb on vaccine take and immunogenicity (22). Nonetheless few efforts have been

made to define the mechanism of interference of MatAb to rotavirus vaccines, which is

the focus of the present study. Various titers of rotavirus specific MatAb at vaccination have been shown to influence the outcome of active immunity. Hodgins et al, 1999 demonstrated that in gnotobiotic piglets, induction of antibody secreting cell (ASC) responses after virulent (Vir) Wa human rotavirus (HRV) primary infection and challenge was suppressed in the presence of circulating high titer MatAb and that milk antibody added to the suppression (9). In addition, the intestinal IgA antibody responses were also suppressed (18). At low titer MatAb, only IgG ASC, and not IgA ASC numbers in duodenum and mesenteric lymph nodes, were significantly reduced after challenge (9). Thus the effect of different levels of MatAb on different antibody isotypes varies. Similarly, calves fed with colostrum before inoculation with bovine rotavirus showed an inverse relationship between the IgG1 antibody titers in colostrum and the

ASC responses (17). These studies, demonstrating the effects of different titers of MatAb

248 (circulating or local) to natural infection by rotavirus, established the basis for our current

study to develop rotavirus vaccine regimens that can overcome the effect of MatAb.

Oral priming and intranasal boosting strategies used in rotavirus vaccine studies

of gnotobiotic piglets were highly effective in inducing antibody responses, presumably

due to exploiting multiple mucosal inductive sites. This strategy of avoiding mucosal sites where booster vaccines may be neutralized by local antibodies induced by the priming vaccine assures that booster vaccination will evoke effective antigenic stimulation. A vaccine regimen consisting of one oral priming dose of attenuated HRV

(AttHRV) and 2 intranasal booster doses of 2/6-virus-like-particles (2/6-VLP) associated

with immunostimulating complexes (ISCOM) induced high protection rates against viral shedding and diarrhea, and comparable or higher IgA antibody and ASC responses than 3 oral doses of AttHRV [Azevedo & Saif, unpublished, (8)]. However it is unknown whether this vaccine scheme (AttHRV/VLP) induces similar immune responses and protection in the presence of rotavirus specific MatAb. For other disease models such as measles and influenza vaccines in macaques, ISCOM were successfully used to overcome the inhibition of immune responses by MatAb (4, 31).

The goal of this study was to explore the effects of high titered passive circulating

MatAb on the immune responses induced by a sequential live HRV priming/non- replicating 2/6-VLP-ISCOM boosting vaccine regimen and on the non-replicating

2/6VLP-ISCOM vaccine alone. The antibody responses, ASC and memory B cells

induced by both types of vaccines were investigated to elucidate if MatAb interfere with

249 the development of B cell responses after rotavirus vaccination and with protection

against VirHRV challenge. We also evaluated the effect of declining MatAb by

comparing the B cell responses and protection in piglets given the AttHRV/VLP regimen and challenged with VirHRV at a longer interval PID42, versus in piglets vaccinated and challenged at PID28, the same time frame as in Gonzalez et al (8).

3.3. MATERIALS AND METHODS

3.3.1. Viruses

A tissue-culture adapted Wa strain AttHRV derived from the twenty-seventh

passage in African Green monkey kidney cells (Ma104) was used for vaccination, sow

immunization and ELISA and ELISPOT. The VirHRV derived from stools of an infected

infant was maintained by serial passage in gnotobiotic piglets (37, 41). Pooled intestinal

contents of the VirHRV infected gnotobiotic piglets were used for challenge at 106

median infectious doses (ID50). The ID50 of the VirHRV inoculum for Gn piglets was

previously determined to be at least 1 fluorescent-focus-forming units (FFU) and the Wa

AttHRV was previously determined to be 1.3x106 FFU (23, 33, 34). The titers of both

AttHRV and VirHRV were determined by cell culture immunofluorescence assay (CCIF)

(23, 24).

3.3.2. Preparation of maternal serum pools: Serum with high HRV antibody titer

(MatAb) was produced by intramuscularly immunizing rotavirus-seropositive sows (n=3)

with 5 doses of Wa AttHRV inactivated with 0.01M binary ethylenimine (Aldrich

Chemical Co., St. Louis, Mo.) and mixed with incomplete Freund’s adjuvant (IFA) (25).

250 The pre-inactivation titer of the virus was 1x108 FFU/dose. Serum was collected and

pooled after the last immunization, heat inactivated at 56oC for 30min and filtered

through Seitz micromedia filter pads, (Ertel/Alsop, Kingston, NY) followed by 0.22µm

membrane filters (Millipore, Bedford, Mass.). The IgG and virus-neutralizing (VN)

antibody titers to Wa HRV were measured by ELISA and a plaque-reduction assay,

respectively, as described (24).

3.3.3. Gnotobiotic piglets injected with maternal serum to mimic infants with passive

circulating MatAb

The hysterectomy-derived near-term piglets were obtained and maintained in

isolation units as described previously (15) under an approved animal use protocol.

Newborn unsuckled piglets are devoid of MatAb due to the impervious nature of the sow placenta to immunoglobulins (11). The MatAb administered via the intraperitoneal (IP) route is transferred to lymphatic vessels and enters the circulation (9), to mimic the effect of circulating passively-derived MatAb. Piglets were given 30ml of the MatAb, twice IP within the first 24h after birth as determined by previous studies (9, 18) and confirmed in this study to result in serum IgG concentrations in piglets (mean= 27mg/ml, data not shown) similar to those reported for naturally suckled piglets (32). The total serum IgG concentration in piglets was measured by porcine IgG kits (Bethyl Laboratories Inc.,

Montgomery, Tex.).

3.3.4. Experimental groups

3.3.4.1. Experiment (Exp) I. The vaccination schemes are summarized in Table 3.1.

Piglets in the groups designated as MatAb-AttHRV/VLP, MatAb-VLP, and MatAb-

251 ISCOM were given maternal serum; piglets in AttHRV/VLP, VLP, and ISCOM groups

did not receive maternal serum. At 3-5 days of age, piglets in the MatAb-AttHRV/VLP or

AttHRV/VLP groups were orally inoculated with AttHRV (5x107FFU/dose) followed by

2 intranasal doses of 2/6-VLP/ISCOM (250µg of 2/6-VLP associated with 1250µg of

ISCOM), 10 days apart at PID10 and 21. Piglets in MatAb-VLP or VLP groups were

inoculated intranasally with 3 doses of 2/6VLP/ISCOM, 10 days apart starting also from

3-5 days of age. Piglets in MatAb-ISCOM and ISCOM groups were inoculated with

diluent and ISCOM matrix (ISCOM) as controls within the same time frame as the

vaccinees. Subsets of piglets from each group were challenged with VirHRV at PID 28.

In a subsequent study (Exp.II), piglets were challenged at PID 42 as described below.

3.3.4.2. Experiment (Exp) II. To study the longer-term effect of the maternal serum

(LTMatAb), in a subsequent experiment, the same combined vaccine AttHRV/VLP and

control ISCOM were administered over a longer time frame and the piglets were

challenged with VirHRV at PID 42 (instead of PID 28) when the titer of MatAb had

declined further. This challenge time point falls within the 8-week interval during which

the gnotobiotic piglets are susceptible to infection and disease with HRV. Piglets in

LTMatAb-AttHRV/VLP and LT-AttHRV/VLP vaccine groups (the letters “LT”

preceding the group name indicate the longer term groups in Exp.II) were inoculated with

one oral dose of AttHRV at 3-5 days of age and boosted with 2/6VLP at PID 14 and 28,

instead of PID 10 and 21. Piglets in LTMatAb-ISCOM and LT-ISCOM groups were

inoculated with diluent and ISCOM matrix (ISCOM) as controls with the same time

frame as the vaccine groups.

252

3.3.5. Assessment of protection

Challenge and protection studies were done as described previously. At PID 28

(for Exp.I) and PID 42 (for Exp.II), subsets of piglets (5-7 piglets/group) were challenged

orally with ~106 ID50 of VirHRV. Rectal swabs were collected and diarrhea scores were

observed for 6 days after challenge for assessment of viral shedding and diarrhea (Fecal

scores of 0, normal; 1, pasty; 2 semi-liquid; and 3, liquid). Scores of greater or equal to 2 were considered as diarrhea. The same two researchers scored all the piglets throughout the study to minimize scoring variation introduced by subjectivity from different individuals. In addition, susceptible age-matched mock-vaccinated control piglets

(ISCOM group) were included in each experiment as reference to evaluate the challenge inoculum and the susceptibility of piglets to rotavirus induced diarrhea. Infectious virus and viral antigen were detected in the rectal swab fluids using CCIF and antigen-capture

ELISA, respectively, as previously described (3, 24). The piglets were considered as completely protected against shedding or diarrhea upon challenge with VirHRV only when they did not shed virus or have diarrhea respectively, during the entire observation period.

3.3.6. Plaque reduction assays for VN antibodies. This assay was performed on serum samples of piglets and sows, as described previously, and the VN antibody titers were expressed as the reciprocal of the serum dilution which reduced the plaque numbers by

>80% (24)

253 3.3.7. Isotype-specific antibody ELISA. The Wa HRV-specific IgM, IgA and IgG

antibody titers in sows’ sera and piglets’ sera and small intestinal contents were

determined by an indirect isotype-specific antibody ELISA as previously described (30).

3.3.8. ELISPOT assay for rotavirus-specific ASC

Subsets of piglets were euthanized at PID28/PCD0 (pre-challenge) and

PID35/PCD7 (post-challenge) in Exp.I and at PID42/PCD0 and PID49/PCD7 in Exp.II.

The small intestine (duodenum and ileum), spleen and peripheral blood lymphocytes

(PBL) were collected for the isolation of mononuclear cells (MNC) as previously

described (38, 40, 41). The ELISPOT assays to enumerate rotavirus-specific ASC of

different isotypes (IgM, IgA and IgG) were conducted based on previously published

methods (41). The assays were performed for the freshly isolated lymphocytes (effector

ELISPOT, to measure the effector B cell responses) and for the cells stimulated in culture

with AttHRV antigen (memory ELISPOT) to measure the short-term memory B cell

responses (39). Briefly, for the memory ELISPOT assay, 3.75 x106 MNC were incubated

for 5 days with attenuated Wa HRV, 24µg (for ileum) and 12µg (for spleen and blood),

and semi-purified by ultracentrifugation through 30% sucrose (100,000xg, 2h). On day

5, the cells were collected, washed to remove residual virus, counted and loaded onto the

96-well plates containing the rotavirus-infected MA104 cell monolayers fixed with 80%

acetone to detect memory B cells. Mock-stimulated cells were included in each test to

account for residual effector B cells. The numbers of ASC were expressed as per 5x105

MNC. The numbers of memory B cells were expressed as per 5x105 MNC (viable cells

254 after 5 days in vitro stimulation with the antigen). Only the memory B cell responses of

the MatAb-AttHRV/VLP and AttHRV/VLP, LT-MatAb-AttHRV/VLP and LT-

AttHRV/VLP vaccine groups (pre- and post-challenge) and the memory responses in

VLP and MatAb-VLP groups (pre-challenge) are presented.

3.3.9. Statistical analyses. For all the parameters, statistical comparisons were made (1) between piglet groups with and without MatAb for the same vaccine; (2) between

AttHRV/VLP and VLP vaccine groups with and without MatAb within the same experiment; (3) between Exp.I and II for the same vaccine and control groups pre- and post challenge; and (4) between pre- and post challenge for the same isotype ASC and memory B cell responses. For antibody titers (isotype-specific ELISA and VN antibodies), statistical analyses were performed on log10 transformed titers. One-way

ANOVA (SAS Institute Inc., Cary, N.C) followed by Duncan's multiple-range test was

used to reveal significant differences (marked by different capital letters: A, B, C) among

groups at each time point. The mean number of ASC was calculated for each treatment

group at PID28/PCD0 and PID35/PCD7 (Exp.I) and at PID42/PCD0 and PID49/PCD7

(Exp.II). Kruskal-Wallis rank sum (nonparametric) tests were used to first compare the

ASC and memory B cell numbers among groups at each time point. If significant

differences were detected among groups, then differences between particular group pairs

were further tested using Kruskal-Wallis rank sum tests. Differences in proportions of

piglets with diarrhea and virus shedding were determined by chi-square test; when

significant differences were present among groups, pair-wise comparisons were made by

Fisher’s exact test. Unless the p value is specified, a p value of ≤ 0.05 was used to denote

statistical significance. 255

3.4. RESULTS

3.4.1. Intraperitoneal injection of newborn piglets with maternal hyperimmune serum

to HRV mimics the passive circulating MatAb titers to HRV in infants in developing

countries

The titers of circulating Wa HRV specific VN and IgG antibodies in piglets at the

first vaccination [post inoculation day (PID) 0, 3-5 days after maternal serum injection]

were within the range of rotavirus antibody titers in human infants less than 4-6 months

of age, typically of those in developing countries. In particular, the rotavirus specific

antibody titers in infants/young children in developing countries are 50-3,000 (for rotavirus VN antibody titers) and 160-80,000 for rotavirus specific IgG antibody titers (1,

20, 42). Thus the VN and rotavirus-IgG antibody titers in the sera of MatAb piglets

(2,200 and 65,000-110,000, respectively) at PID0 represent the range of high titers of

rotavirus specific antibodies in children in the developing countries. A subsequent study

focuses on the effect of lower titer MatAbs which mimick the passive circulating MatAb

titers in infants from developed countries (Chapter 4). Although the piglets in the MatAb

groups did not receive the antibodies from their own mothers directly, the serum pool derived from the lactating sows (14 days post-farrowing) and injected into the piglets closely mimics the level of serum MatAb passively transferred from the mother to the fetus.

256 3.4.2. The moderate protection against viral shedding and diarrhea in the MatAb-

AttHRV/VLP group was contributed by both passive antibodies and active immune

responses

At PID28 (Exp.I) or PID42 (Exp.II), 5 to 7 piglets within each group were

challenged with VirHRV and protection against viral shedding and diarrhea were

assessed (Table 3.1). In Exp.I, protection rates were highest in the AttHRV/VLP group

(71% protection against viral shedding and diarrhea) without MatAb but decreased to

50% for both viral shedding and diarrhea with MatAb illustrating the trend for MatAb

interference with vaccine efficacy. Protection in the MatAb-AttHRV/VLP group were

likely partially due to the remaining level of MatAb at challenge and the active immunity

induced by vaccination because neither the MatAb-VLP vaccine nor the MatAb-ISCOM

control groups were protected against virus shedding. However there were low partial

protection rates against diarrhea in these latter two groups (29-33%, respectively). This

asymptomatic infection was probably due to the protective effect of the remaining MatAb

which remained high at challenge (VN titers of 1621 and 1015, respectively, Figure

3.1A).

In Exp.II, the highest level of protection was similarly observed in the LT-

AttHRV/VLP group (80% protection against both viral shedding and diarrhea) without

MatAb (Table 3.1). In comparison, protection against both viral shedding and diarrhea induced by the AttHRV/VLP vaccine was reduced (67% and 33%, respectively),

although not significantly, by the presence of MatAb in the LTMatAb-AttHRV/VLP group. The LTMatAb-ISCOM control group had a significantly lower protection rate

against diarrhea (20%) than the vaccinated groups consistent with the reduction in VN 257 and IgG antibodies in serum by 3.2 and 12-fold from PID0 to PID42/PCD0, respectively in this group (Figure 3.1B). In the LTMatAb-AttHRV/VLP group, the serum VN antibodies decreased 6-fold from PID0 to PID42 (compared to 2-fold reduction in the

MatAb-AttHRV/VLP, Exp.I). The decreased protection rate against diarrhea in the

LTMatAb-AttHRV/VLP group (compared to the MatAb-AttHRV/VLP in Exp.I) was likely due to this decrease in passive antibody level. The inclusion of the 2/6VLP group in Exp.I illustrated the effect of circulating MatAb on the immunogenicity of the ISCOM- based non-replicating intranasal vaccine. Because the 2/6-VLP vaccine did not induce protection in Exp.I, this vaccine was not included in Exp.II.

3.4.3. Active IgA antibody responses in serum to the AttHRV/VLP and the VLP vaccines before and after challenge in both Exp.I and II were inhibited by high titer

MatAb

In both experiments, high VN and IgG antibody titers and low IgA antibody titers were present in serum of all maternal serum-injected piglets (MatAb- and LTMatAb groups) and the respective titers did not differ among groups at PID0 before vaccination

(Figure 3.1). After vaccination at PID28/PCD0 and PID42/PCD0, in the absence of

MatAb, the piglets in the AttHRV/VLP and VLP vaccine groups developed significant

IgA and IgG antibody responses in serum compared to controls. In the presence of

MatAb, the serum antibody titers measured in the AttHRV/VLP and VLP vaccine groups were the combination of passive circulating MatAb and active antibodies induced by the vaccines. Serum IgA antibodies in these groups became undetectable at PCD0 (PID28 or

258 PID42), indicative of suppression of active IgA responses by MatAb compared to the significantly elevated titers in the non-MatAb vaccine groups. The serum VN and IgG antibody GMT in the MatAb-vaccine groups decreased slightly (2-4 fold) compared to

PID0 but remained high at PID28 and these antibody titers decreased further decreased at

PID42 (6 and 23-fold lower, respectively). However, the remaining titers of passive VN and IgG antibodies were still high, masking the detection of active serum VN and IgG antibody responses in these vaccine groups. The ISCOM control group with MatAb in

Exp.I and II showed a reduction in the VN, IgA and IgG antibody titers of similar magnitude to the vaccine groups.

After challenge (PID35/PCD7 or PID49/PCD7), the AttHRV/VLP and VLP groups without MatAb developed significantly increased serum VN, IgG and IgA antibody responses. (Figure 3.1). The ISCOM control groups without MatAb also developed low VN and IgG antibody responses. However, in Exp.I, antibody titers

PID35/PCD7 in the respective vaccine and control piglets with MatAb remained similar to or lower than the pre-challenge (PID28/PCD0) antibody levels at, indicating no discernable induction of active serum VN, IgA and IgG antibody responses in the vaccinated and control piglets in the presence of MatAb post-challenge.

In Exp. II, in the LTMatAbAttHRV/VLP group the serum VN antibody titers decreased further, whereas the serum IgG antibody GMT increased about 2-fold post- challenge; a probable result of the increasing active antibodies. In contrast, the serum

IgA antibody GMT in the LTMatAb-AttHRV/VLP group increased significantly (8-

259 folds), yet it was still significantly lower than the IgA titers in LT-AttHRV/VLP group without MatAb. Thus although maternal inhibition of serum IgA antibody responses remained in this vaccine group at PID49/PCD7; an anamnestic response indicative of IgA priming in the presence of MatAb was evident.

The IgM antibody titers in the serum of all the piglet groups with or without

MatAb in Exps. I and II were measured but are not presented. However they showed trends similar to those of IgA and IgG in both Exps. I and II with IgM antibody titers in the AttHRV/VLP and VLP groups without MatAb were significantly higher than the corresponding vaccine groups with MatAb both pre- and post-challenge, indicating suppression of IgM responses by MatAb.

3.4.4. Small intestinal antibody responses to the AttHRV/VLP and VLP vaccines were inhibited by circulating MatAb regardless of the time of challenge (Exp. I and II)

3.4.4.1. Experiment I

In the MatAb-AttHRV/VLP, MatAb-VLP and MatAb-ISCOM groups, the only antibody isotype observed in the intestinal contents (SIC) before challenge

(PID28/PCD0) was IgG, at low titer, likely due to transudation into the gut of the IP inoculated maternal serum (Figure 3.2A). In contrast, in the absence of MatAb, high intestinal IgM and IgA antibody titers were measured in the AttHRV/VLP group, whereas the IgG antibody titers in these groups were low or undetectable. This confirms that MatAb significantly suppressed the IgM and IgA antibody responses to both vaccines in the intestine. Lower IgA and significantly lower IgM antibody titers occurred in the intestine of the VLP group compared to the AttHRV/VLP group.

260 After challenge (PID35/PCD7), the intestinal IgM antibody titers increased

significantly (4- to 64-fold) in all MatAb- groups, but remained significantly lower than

the corresponding groups with no MatAb. In contrast IgA and IgG antibodies were absent

or of low titer in the MatAb groups and similar in magnitude to responses in the non-

vaccinated challenged controls with or without MatAb, consistent with only a primary

immune response to the VirHRV. Like IgM, the IgA antibody titers of the MatAb-

vaccine groups were significantly lower than the corresponding groups with no MatAb.

Increased (3-25 fold) intestinal IgA and IgG antibody responses occurred in the vaccine groups but not controls without MatAb (AttHRV/VLP and VLP) at PID35/PCD7

compared to PID28/PCD0, indicative of anamnestic responses to the VirHRV challenge.

3.4.4.2. Experiment II

Similar to Exp.I, there were no IgM and IgA antibody responses and low IgG

antibodies in the intestinal contents of piglets in the LTMatAb-AttHRV/VLP groups pre- challenge at PID42/PCD0 (Figure 3.2B), whereas the LTAttHRV/VLP group had significantly higher intestinal IgM, and IgA antibody titers compared to the LTMatAb-

AttHRV/VLP and the control LT-ISCOM groups.

After challenge, mainly intestinal IgM antibody titers were detected in both the

LTMatAb-AttHRV/VLP and the LTMatAb-ISCOM control groups (GMT= 81 and 91), again suggesting a primary immune response to VirHRV. The intestinal IgA but not IgG antibody responses increased slightly after challenge in the LTMatAb-AttHRV/VLP group, yet the titers of both antibodies remained significantly lower than the same vaccine group without MatAb, suggesting MatAb suppression of antibody class

261 switching. In the absence of MatAb, the LTAttHRV/VLP vaccine induced antibody titers

of similar magnitude for IgM, IgA and IgG post-challenge compared to the same vaccine

administered shorter term (28 days).

3.4.5. Isotype specific ASC responses in vaccinated piglets before and after challenge

3.4.5.1. IgM ASC responses induced by AttHRV/VLP vaccine post-challenge were

generally suppressed by MatAb in both short-term (Exp.I) and long-term (Exp.II)

vaccinated piglets

Higher pre-challenge IgM ASC responses in intestinal tissues (duodenum and ileum) than in systemic tissues (spleen and blood) occurred in all vaccine groups with or without MatAb. In Exp.I (Figure 3.3A), the pre-challenge (PID28/PCD0) IgM ASC responses in the AttHRV/VLP and MatAb-AttHRV/VLP groups were low (≤20 ASC per

5 x105MNC) for all tissues and did not differ significantly. The IgM ASC responses in

the VLP and MatAb-VLP groups were minimal (<3 ASC per 5 x105MNC) for all tissues

and as expected no ASC were detected in the MatAb-ISCOM and ISCOM controls. The

IgM ASC numbers in the intestine of the AttHRV/VLP group were higher (ileum) or

significantly higher (duodenum) than those induced by the VLP vaccine, indicating the

lower antigenicity of the 2/6-VLP vaccine.

After challenge, the IgM ASC numbers increased slightly (not significantly) in all

tissues in the AttHRV/VLP group without MatAb, but not in the MatAb-AttHRV/VLP

groups. Among all vaccinated groups, the IgM ASC responses after challenge were

higher in the intestine than in the systemic tissues. The mean numbers of intestinal IgM

ASC were 2- to 5-fold lower in the MatAb-AttHRV/VLP group compared to the

262 AttHRV/VLP group suggesting that the suppressive effects of MatAb remained at

PID35/PCD7. The mean numbers of IgM ASC among all the tissues between the

MatAb-VLP and VLP vaccine groups were not significantly different, indicating that the suppressive effect of MatAb on the VLP vaccine was less substantial (except for the duodenum). Like pre-challenge, the intestinal IgM ASC numbers in the AttHRV/VLP group were higher (ileum) or significantly higher (duodenum) than that induced by the

VLP vaccine, indicating the lower antigenicity of 2/6-VLP vaccine. Low, but significant,

IgM ASC responses were detected in all tissues of the ISCOM control groups with or without MatAb as a result of primary immune responses to VirHRV challenge.

Based on the observation that MatAb suppression of the ASC responses induced by the AttHRV/VLP vaccine remained at PID35/PCD7, Exp.II was designed to challenge the AttHRV/VLP vaccinated piglets at a later time when the MatAb titer had further declined. The VLP vaccine group was not included in this experiment because less

MatAb effect on this vaccine was found in Exp.I and the VLP vaccine did not induce protection in piglets.

In Exp.II, pre-challenge (PID42/PCD0), the LTMatAb-AttHRV/VLP group had even greater suppression of the IgM ASC responses as evident by significantly lower

IgM ASC responses in the duodenum and ileum compared to the LT-AttHRV/VLP group

(Figure 3.3B). The highest IgM ASC responses were found in the duodenum of the

AttHRV/VLP vaccine group. The pre-challenge IgM ASC responses in spleen and blood were low in the AttHRV/VLP vaccine and ISCOM control groups with or without

MatAb.

263 After challenge (PID49/PCD7), IgM ASC numbers in the LT-AttHRV/VLP group increased significantly in the duodenum and ileum and slightly in the spleen with the highest numbers in the duodenum. The IgM ASC numbers in the LTMatAb-

AttHRV/VLP increased only slightly in the duodenum, ileum and spleen, but significantly in the blood with the highest IgM ASC in the blood. The IgM ASC in the duodenum and ileum of the LTMatAb-AttHRV/VLP group were again significantly lower than that in the LT-AttHRV/VLP group. In the latter group, the mean numbers of

IgM ASC in the duodenum and ileum were significantly higher than the short-term

AttHRV/VLP group post-challenge. In contrast, the IgM ASC numbers in the LTMatAb-

AttHRV/VLP group post-challenge were similar (duodenum, spleen) or slightly lower

(ileum) than that of the short-term MatAb-AttHRV/VLP group. Thus the extended vaccination scheme increased the magnitude of IgM ASC responses without the presence of MatAb; however, the suppressive effect of MatAb was not alleviated by extending the vaccination/challenge period to PID42. Consequently, the differences in the intestinal

IgM ASC numbers between the groups with and without MatAb were increased in Exp.

II compared to Exp.I leading to the appearance of more severe suppression. This phenomenon was observed also for IgA and IgG ASC responses (see below). High numbers of IgM ASC were detected in all the tissues of the LT-ISCOM group, but were very low in the LTMatAb-ISCOM group. The IgM ASC numbers in the duodenum and ileum of the LTMatAb-ISCOM group were significantly lower compared to the LT-

ISCOM group, suggesting the neutralization of the challenge VirHRV and inhibition of primary immune responses by the remaining MatAb.

264 3.4.5.2. Intestinal IgA ASC responses were strongly suppressed in the AttHRV/VLP

vaccine group with MatAb pre- and post challenge (Exp. I and II), but not in the

VLP vaccine group

Prechallenge (PID28/PCD0), in Exp.I (Figure 3.4A), the IgA was the predominant

isotype of ASC detected in the intestinal lymphoid tissues of the AttHRV/VLP and VLP

vaccine groups both with and without MatAb. The IgA ASC numbers in the duodenum

and ileum of the MatAb-AttHRV/VLP group in were significantly lower compared to the

AttHRV/VLP groups, indicative of the suppressive effect of MatAb on intestinal IgA

ASC responses. On the other hand, higher but not significantly higher numbers of IgA

ASC in the duodenum (40 vs 16) and ileum (59 vs 35) occurred in the MatAb-VLP group

compared to the VLP group, suggesting that the antigenicity of the non-replicating

vaccine (administered IN) was less affected by high titers of MatAb than the combined

replicating (oral route) and non-replicating (IN route) vaccine. The intestinal IgA ASC numbers in the VLP group were lower (duodenum) or significantly lower (ileum) than

those in the AttHRV/VLP group, indicating that the antigenicity of the VLP vaccine was

significantly lower than the AttHRV/VLP vaccine in the absence of MatAb (Figure

3.4A). The IgA ASC responses in the systemic tissues were low and did not differ

between vaccine groups with or without MatAb. No IgA ASC responses were observed

in the MatAb-ISCOM and ISCOM control groups before challenge.

After challenge, intestinal IgA ASC responses increased slightly in the MatAb-

AttHRV/VLP group (Figure 3.4A), but remained significantly lower than those of the no

MatAb (AttHRV/VLP) group, indicating the suppressive effect of MatAb remained post-

challenge. Like pre-challenge, post-challenge intestinal IgA ASC responses did not differ 265 significantly between the MatAb-VLP and VLP groups. However there was no increase in the IgA responses in any tissues of the MatAb-VLP piglets after challenge with

VirHRV suggesting inhibition of memory B cell response. The IgA ASC responses in systemic tissues were significantly lower in the MatAb-AttHRV/VLP (spleen and PBL) and MatAb-VLP (spleen) vaccine groups compared to the respective vaccine groups without MatAb. The IgA ASC responses in the intestinal and systemic tissues of the

MatAb-ISCOM and ISCOM control groups were low and did not differ significantly.

In Exp.II (Figure 3.4B), pre-challenge (PID42/PCD0) the LTMatAb-

AttHRV/VLP group showed even more pronounced suppression by MatAb of the IgA

ASC responses in the intestinal lamina propria than that observed in Exp.I (Figure 3.4A).

There were significant decreases in the duodenum IgA ASC numbers: an 87-fold decrease between LTMatAb-AttHRV/VLP and LT-AttHRV/VLP ASC numbers (Figure

3.4B) versus a 5-fold decrease between MatAb-AttHRV/VLP and AttHRV/VLP ASC numbers (Figure 3.4A). The pre-challenge IgA ASC numbers in duodenum were higher

(p=0.086) in the LT-AttHRV/VLP (Exp.II) compared to the AttHRV/VLP groups

(Exp.I). Similar to Exp.I, only low IgA ASC numbers were detected in spleen and blood of the LT-AttHRV/VLP group at PID42/PCD0. No IgA ASC responses were observed in the ISCOM and MatAb-ISCOM control groups pre-challenge.

After challenge (PID49/PCD7), the intestinal IgA ASC numbers increased moderately (6 to 10-fold) in the LTMatAb-AttHRV/VLP group, indicative of active immune responses, yet they remained significantly lower than in the LT-AttHRV/VLP group without MatAb, suggesting that the suppressive effect of MatAb continued even when MatAb titers had declined at PID49/PCD7 (Figures.3.1B and 3.4B). In contrast the 266 post-challenge IgA ASC numbers in the ileum of the LT-AttHRV/VLP group increased

significantly compared to pre-challenge. The IgA ASC numbers in systemic tissues of the

AttHRV/VLP vaccine group and all the tissues of the ISCOM control group with or

without MatAb were low and did not differ between groups with or without MatAb.

3.4.5.3. Intestinal and spleen IgG ASC responses were suppressed in the

AttHRV/VLP (pre- and post-challenge) and VLP vaccine (post-challenge) groups in the presence of MatAb

In Exp.I (Figure 3.5A), pre-challenge (PID28/PCD0), IgG ASC responses were significantly higher in duodenum, ileum and spleen and higher in PBL of the

AttHRV/VLP group without MatAb compared to the MatAb-AttHRV/VLP groups, indicative of suppression of active IgG responses by MatAb in both the intestinal and systemic lymphoid populations (Figure 3.5A). Similarly to IgM, for the MatAb-VLP and

VLP groups, the IgG ASC responses in the intestinal and systemic lymphoid tissues were low pre-challenge, but significantly higher numbers of IgG ASC in spleen and slightly higher numbers in the ileum and PBL occurred in the VLP group compared to the

MatAb-VLP group. Similar to ASC responses of other isotypes, no IgG-ASC responses were detected in the ISCOM control groups with or without MatAb before challenge.

After challenge (PID35/PCD7), the numbers of intestinal and spleen IgG ASC increased significantly in the MatAb-AttHRV/VLP group compared to pre-challenge (7- to 30-fold) (Figure 3.5A). The substantial increase in splenic IgG ASC numbers coincided with the high numbers of splenic memory IgG ASC in this group (see the memory response section). Nevertheless, the numbers of IgG ASC in all tissues

267 remained significantly lower in the MatAb-AttHRV/VLP group than in the AttHRV/VLP vaccine group confirming MatAb suppression of the IgG ASC responses. In the latter group and the VLP vaccine group with and without MatAb, IgG ASC numbers were higher in the intestine compared to the spleen and PBL. In the MatAb-AttHRV/VLP vaccine group, the numbers of IgG ASC were similar among all the tissues. The post- challenge intestinal and systemic IgG ASC numbers in the MatAb-VLP group were also significantly lower than those in the VLP group indicative of MatAb suppression of IgG

ASC post-challenge to VLP vaccine. Low IgG ASC responses were detected in all the tissues in the ISCOM control group with and without MatAb. Interesting to note, significantly lower numbers of intestinal IgG ASC were detected in the MatAb-ISCOM group compared to the ISCOM group, suggesting that the MatAb significantly suppressed the intestinal IgGASC responses to the VirHRV at PID35/PCD7.

In Exp.II, pre-challenge (PID42/PCD0), in the absence of MatAb, the

AttHRV/VLP vaccine induced moderate or low intestinal and systemic IgG ASC responses, respectively (Figure 3.5B). In the presence of MatAb, the LTMatAb-

AttHRV/VLP group showed significantly lower (suppressed) IgG-ASC responses in intestinal tissues compared to the LT-AttHRV/VLP group, similarly to the suppression observed in Exp.I for this vaccine group. The IgG ASC numbers in the spleen and blood were lower in both AttHRV/VLP groups with and without MatAb and did not differ significantly. No IgG ASC numbers were detected in LT-ISCOM or LTMatAb-ISCOM groups in any tissues pre-challenge.

After challenge (PID49/PCD7), intestinal and spleen IgG ASC numbers were significantly increased (3-18-fold) in the LT-AttHRV/VLP group compared to pre- 268 challenge. For the LTMatAb-AttHRV/VLP group, the numbers of IgG ASC increased in all tissues (significantly in intestine), but they remained significantly lower than those in the LT-AttHRV/VLP group in the intestine and spleen. The increase in IgG ASC numbers in the LTMatAb-AttHRV/VLP group agreed with the 2-fold increase in serum

IgG antibody titers after challenge in this group (Figure 3.1B), suggestive of active immunity. There were no significant differences in the intestinal IgG ASC responses in the LTMatAb-AttHRV/VLP and the MatAb-AttHRV/VLP groups post-challenge, once again suggesting that the suppressive effect of MatAb was not alleviated by extending the vaccination/challenge scheme from PID28 to PID42, similar to the IgM and IgA ASC responses. No IgG ASC responses were observed in the MatAb-ISCOM group after challenge in any tissues and the numbers of IgG ASC in the intestine of the LT-ISCOM group were significantly higher than those of the MatAb-ISCOM, again suggesting the

MatAb suppression on IgG ASC responses at PID49/PCD7.

3.4.6. Memory B cell responses induced by the AttHRV/VLP and VLP vaccines were also inhibited by circulating MatAb

3.4.6.1. Exp.I: MatAb suppression of memory B cell responses was most pronounced for IgG memory B cells in spleen and blood both pre- and post-challenge

Interestingly, the IgA and IgG memory B cells in the MatAb-AttHRV/VLP and

AttHRV/VLP groups at PID28/PCD0 were more numerous in the spleen and blood than in the intestine (Table 3.2) and IgA memory B cell numbers were highest in the blood pre-challenge but in the ileum post-challenge of piglets with MatAb. Because there were

269 no significant IgA and IgG memory B cell responses in ISCOM and MatAb-ISCOM

control groups pre- and post-challenge, these responses were not included in Table 3.2.

Before challenge (PID28/PCD0), there were few IgA memory B cells in the ileum

of all vaccine groups with or without MatAb and the numbers did not differ between the

Mat-AttHRV/VLP and AttHRV/VLP groups did not differ significantly. The IgA memory B cell numbers were higher or significantly higher in the spleen and blood of the

AttHRV/VLP and VLP vaccine groups in the absence of MatAb compared to the MatAb-

AttHRV/VLP and MatAb/VLP groups, which agrees with the suppression of serum IgA

antibody responses induced by both vaccines in the presence of MatAb both pre- and post

challenge. Similarly, the pre-challenge IgG memory B cell responses in ileum were low

for all vaccine groups with or without MatAb. The numbers of IgG memory B cells in the

spleen and PBL were, however, significantly higher in the AttHRV/VLP and VLP groups

than the MatAb-AttHRV/VLP and MatAb-VLP groups. A striking difference regardless

of MatAb status was the significantly lower numbers of IgA and IgG memory B cells in

spleen and PBL of the VLP vaccine groups v.s. the AttHRV/VLP groups, which likely

had a major impact on the lack of protection seen in the VLP vaccine groups.

After challenge, in the AttHRV/VLP group without MatAb, the numbers of IgA

memory B cells increased in the ileum (significantly) and spleen (not significantly), but

decreased in the blood. The IgA memory B cell numbers in the MatAb-AttHRV/VLP

group also increased (not significantly) after challenge in all tissues, but the numbers in

the ileum and blood remained lower than those in the AttHRV/VLP group. The IgG

memory B cell numbers in the MatAb-AttHRV/VLP group increased after challenge in

the blood and were significantly increased in the ileum and spleen, yet these numbers 270 were still lower (ileum) or significantly lower (spleen and blood) than those in the

AttHRV/VLP group. Thus suppression by high titer MatAb existed not only for the

effector B cell responses, but also for induction of memory B cells, and especially pertaining to IgG memory B cells.

3.4.6.2. Exp.II: Both intestinal and systemic IgA and IgG memory B cell responses

were suppressed by MatAb

The numbers of IgA and IgG memory B cells in the LTMatAb-AttHRV/VLP

group were lower or significantly lower than that in the LT-AttHRV/VLP group before

challenge in all tissues (Table 3.2). Even without MatAb, the numbers of IgA and IgG

memory B cell in the LT-AttHRV/VLP group were significantly higher in ileum but

significantly lower in blood compared to the short term AttHRV/VLP group, suggesting a

different distribution/dynamics of memory B cells in the long term versus short term

experiments. One possible scenario is that a major portion of the IgG and the majority of

the IgA memory B cells were still in the circulation at PID28; whereas by PID42, these memory B cells have reached their resident sites (ileum and spleen). The magnitude of memory B cell responses in spleen induced by AttHRV/VLP vaccine did not differ between the two experiments.

After challenge, the mean numbers of IgA memory B cells in the ileum and spleen did not increase in the LT-AttHRV/VLP group. However the IgA memory B cell numbers increased in the ileum and blood of the LTMAb-AttHRV/VLP group, yet the numbers of IgA memory B cells in all the tissues remained significantly lower compared to the no MatAb group. There were significant increases in the numbers of IgG memory

271 B cells in the spleen and blood of the LTMatAb-AttHRV/VLP group post-challenge;

however these numbers remained significantly lower than those in the LT-AttHRV/VLP

group. Thus, both IgA and IgG memory B cell responses induced by the AttHRV/VLP

vaccine were affected by high titers of MatAb and the suppressive effects for the ileum

and spleen, which are the resident sites for memory B cells, were more pronounced at

PID42/PCD0 than at PID28/PCD0. The memory B cell responses in the VLP and MatAb-

VLP groups after challenge in Exp. II were not done.

3.5. DISCUSSION

We studied the effects of high titer MatAb on the protective efficacy and B cell

responses induced by two candidate rotavirus vaccines that were previously evaluated in

colostrum-deprived seronegative gnotobiotic piglets (8). Although protection rates

induced by the AttHRV/VLP vaccine in the presence of high titer MatAb were reduced, they did not differ statistically from those induced by this vaccine without MatAb.

However, the mechanism of protection differed based on the magnitude of the immune responses we observed. The higher protection rates observed in the MatAb-AttHRV/VLP group were likely due to the combination of active and passive immunity, because the

MatAb-ISCOM and LTMatAb-ISCOM control groups were also partially protected (but with much lower protection rates) from diarrhea upon challenge due to the remaining

MatAb in the serum. The remaining MatAb however did not completely protect the

piglets from virus shedding which is consistent with previous findings by Hodgins et al

(9). In addition, intravenous infusion of rotavirus seronegative macaques with serum

272 from experimentally and naturally infected macaques with high rotavirus specific IgG

antibodies (titer 800-1,600) 18 hrs before challenge with 106FFU simian rotavirus strain

YK-1 led to reduction of duration and titer of virus shedding, but not to complete

protection against infection (no diarrhea occurs in this model) (35). In our study, the

serum IgG titers at the time of challenge in the MatAb-ISCOM and MatAb-VLP piglets

were greater than those in macaques (~16,000-32,000), resulting in partial protection against diarrhea but no protection against virus shedding. Thus mechanisms other than only the passive antibodies likely conferred the protection seen against rotavirus shedding in the MatAb-AttHRV/VLP group.

Because the B cell responses were suppressed (but not completely) in the MatAb-

AttHRV/VLP group as indicated by the lower or significantly lower antibody titers, ASC and memory B cell responses, the T cell responses might also contribute to the protection induced by the AttHRV/VLP vaccine in the presence of MatAb. It has been shown that in the presence of MatAb, B cell responses were depressed, but the T cell responses were not affected (2). In infants given 2 doses of measles vaccine, booster led to higher seroconversion rates and enhanced T cell responses even in the presence of

MatAb, suggesting that the infants would benefit from the additive effects mediated by both MatAb and active T cell responses (7).On the other hand, cellular immune responses induced by a DNA vaccine for HIV did not improve the protection conferred by circulating antibodies (13). Thus the role of T cell responses is still controversial as a determining factor for the success of a vaccine in the presence of MatAb.

273 In this study, the suppressive effects of MatAb on the numbers of ASC, antibody production and memory B cell responses were observed in both AttHRV/VLP and VLP vaccine regimens, but especially for the AttHRV/VLP vaccine regimen at both PID28 and PID42. Furthermore, the suppression was higher with the longer vaccination/challenge interval (42 days) as compared to the shorter interval (28 days).

This observation may be attributed to the effects of MatAb on the homing of memory B cells in addition to their suppressive effects on induction of effector and memory B cells.

It is likely that this vaccine regimen in the absence of MatAbs was able to induce effector responses more effectively at the 42-day interval than at the 28-day interval, as indicated by the higher duodenum (effector site) IgM, IgA and IgG ASC responses pre- and post- challenge.

In addition, we found that the interference of MatAb on the memory B cell responses, which has not been previously recognized, differed according to the time and the lymphoid tissues assessed. In Exp.I, the highest memory B cell responses without

MatAb were mostly IgG and found mostly in spleen and blood. In Exp.II, when the vaccine was administered in the absence of MatAb, the highest IgA and IgG memory B cell responses were found in ileum and spleen. Thus a major portion of the IgG and the majority of the IgA memory B cells remained in circulation (PBL) at PID28; whereas by

PID42, these memory B cells had reached their resident sites (ileum and spleen). In the presence of MatAb, the homing of the IgA and IgG memory cells to the resident sites in the long term piglets (Exp.II) were strongly inhibited. Thus the stronger inhibition of the memory B cell response in the LTMatAb-AttHRV/VLP group may be explained by the differences in distribution of memory B cells in the short-term (Exp.I) versus long-term 274 (Exp. II) experiments. The long-term suppressive effect of MatAb observed in this study

agrees with previous findings. Feeding or IP injection of rats as neonates with

monoclonal IgG2a or IgG1 antibodies led to suppression of humoral immune responses

in these rats as adults; thus the suppression by MatAb lasted for up to 5 months (19)

In addition, the presence of low levels of passive antibody in the intestine may

provide feedback inhibition to the secretion of antibody by plasma cells in the intestine,

which explains the failure to detect IgA antibodies in the intestinal contents of piglets in

the MatAb-AttHRV/VLP group, although low numbers of IgA-ASC were detected at the

same time point. The feed back inhibition of antibody secretion has been observed in

hybridomas with low antibody production which generally display high initial rates of monoclonal antibody production, however, after a few hours the monoclonal antibody secretion was reduced to low concentrations (14). Furthermore, the ELISPOT assay allows enumeration of activated B cells capable of antibody production, whereas the

ELISA assay measures both the amount of antibodies actively produced by these cells plus the remaining passive MatAb. Some discordance between the presence of low

numbers of IgA and IgG ASC in the intestinal lymphoid tissues and the low titers or

absence of these antibodies in the intestinal contents suggests a possible inhibition of the

level of antibody production by these plasma cells. Of interest, the sizes of the spots

generated by the ASC in the ELISPOT assay appeared smaller from piglets with MatAb

than those from piglets without MatAb. A similar observation was reported in a previous study using one dose of VirHRV in the presence of low titer MatAb (18). In a recent study of calves, the calves that received control colostrum (with low titers of antibodies to bovine rotavirus) exhibited lower IgA and IgG1 antibody titers in intestinal contents 275 compared to the colostrum-deprived calves after challenge with bovine rotavirus, yet the intestinal IgA ASC numbers did not differ between these groups (17). Thus in the presence of MatAb, although the induction of antibody containing plasma cells may not be affected, the generation of functional plasma cells or their levels of antibody secretion may be affected.

In this study, the maternal mainly IgG antibody clearly exerted a strong inhibition of IgG ASC responses both in intestinal and systemic tissues of the MatAb-AttHRV/VLP and LTMatAb-AttHRV/VLP groups. The MatAb in the circulation or transudated into the intestinal lumen may have produced negative feedback inhibition effects on lymphocyte homing to the intestinal lymphoid tissues. Similarly, the IgG ASC response was suppressed in intestinal tissues before and after challenge in piglets inoculated with 3 doses of VLP vaccine, whereas the intestinal IgM and IgA ASC responses were low but they appeared to be less affected by MatAb. This finding coincides with the observations by Hodgins et al (9), whereby post-challenge IgG ASC, but not IgA ASC numbers in piglets inoculated with VirHRV were affected by low titer MatAb. Taken together, the observations that MatAb suppression in the AttHRV/VLP and VLP3x groups appears to be more severe for IgG than for IgM or IgA antibody and ASC induction suggests that

MatAb suppression (including negative feedback) may be isotype specific, because IgG antibody titers were the highest antibody titers in the maternal serum.

The use of the non-replicating 2/6VLP vaccine associated with ISCOM may have partially overcome the intestinal suppression by MatAb, as evident by the comparable

IgM and IgA ASC responses in the MatAb-VLP and VLP groups. On the other hand, the significantly reduced IgG ASC responses and the reduced serum and intestinal IgA 276 antibody responses in the MatAb-VLP vaccine group suggest that: (i) the challenge virus

dose may have been partially neutralized by MatAb, resulting in lower booster responses

after challenge; (ii) lower numbers of pre-challenge virus-specific IgA and IgG memory

B cells were induced by the non-replicating VLP vaccine with the presence of MatAb

leading to a reduced magnitude of anamnestic responses post-challenge.

Alternatively, oral delivery and replication of the AttHRV may be differentially

affected by circulating MatAb compared to the IN delivery of the VLP/ISCOM. The

intranasal route of VLP delivery may lead to less impact by circulating MatAb on

intestinal IgA and IgM ASC but a greater impact on IgG ASC and serum IgA antibodies.

Thus the inhibition by high titer MatAb also extended to the non-replicating VLP

vaccine, which further supports the hypothesis that mechanisms other than neutralization

of the virus replication by MatAb are involved (27). Although the 2/6VLP vaccine alone

was not sufficiently immunogenic and did not induce any protection in piglets, perhaps

higher doses of VLPs with VP4 or/and VP7 associated with ISCOM or other effective

adjuvants may improve immunogenicity, overcome MatAb interference and induce

protective neutralizing antibody responses.

In summary, our study confirmed once again that the presence of high titer MatAb has substantial effects not only on effector, but also, although not previously recognized, on memory B cell responses to rotavirus vaccines in neonates. The B cell responses

induced by the sequential AttHRV priming and 2/6VLP boosting regimen were greatly suppressed by MatAb and were not alleviated but were actually enhanced by the extended vaccination/challenge interval tested. The moderate protection rates against viral shedding and diarrhea in the group receiving AttHRV/VLP vaccine in the presence 277 of MatAb may be attributed to a combination of immune factors: residual MatAb, a low active B cell response and a possible role for T cell responses. Low and significantly reduced intestinal IgA ASC responses were induced in the presence of MatAb and serum and intestinal IgA production was suppressed pre- and post-challenge, possibly due to feedback inhibition by MatAb in the AttHRV/VLP regimen. The suppressive effect on the B cell responses continued through PID 42 and although the MatAb level had declined at this time (Figure 3.1B), memory B cell and intestinal ASC responses were the most suppressed. The antibody responses induced by the non-replicating 2/6VLP vaccine were also inhibited by high titer MatAb, but in ways different from the MatAb inhibition of the AttHRV/VLP vaccine. In particular, the intestinal IgM and IgA ASC were low, but not affected, whereas the intestinal IgG ASC, the memory B cells and intestinal and serum IgA antibody responses were suppressed. Thus our study suggests that at least three mechanisms of inhibition by MatAb are involved, including neutralization of the virus, feedback inhibition function of antibody secretion by plasma cells and inhibition of the homing of memory B cells to effector sites.

To our knowledge, this is the first study to confirm the effect of MatAb on both the effector and memory aspects of B cell responses to rotavirus vaccines. The titers of

MatAb used in this study were within the range of rotavirus antibody titers in human infants less than 4-6 months of age mostly in developing countries and in some populations in developed countries; thus, our study mimics rotavirus immunity in infants as influenced by the universal presence of MatAb. Our study also demonstrated the effects of MatAb on replicating versus non-replicating vaccines to facilitate the design of vaccines to overcome MatAb suppression. Understanding the immune mechanisms for 278 protection conferred by AttHRV/VLP and other vaccine regimens will enable us to

develop alternative strategies to further improve the efficacy of rotavirus vaccines given

to infants in the presence of MatAb.

3.6. ACKNOWLEDGMENTS

We thank Dr. Juliette Hanson, Rich McCormick, Severin Pouly and Marcela

Azevedo for technical assistance. We thank Dr. Viviana Parreno (Instituto de Virologia,

CICU y A, INTA, Castelar, Bs.As, Argentina) for helpful comments. We also thank

Hong Liu for help in statistical analysis.

This work was supported by grants from the National Institutes of Health

(RO1AI33561 and RO1AI37111). Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development

Center, The Ohio State University.

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31. van Binnendijk, R. S., M. C. Poelen, G. van Amerongen, P. de Vries, and A. D. Osterhaus. 1997. Protective immunity in macaques vaccinated with live attenuated, recombinant, and subunit measles vaccines in the presence of passively acquired antibodies. J Infect Dis 175:524-32.

282 32. Ward, L. A., E. D. Rich, and T. E. Besser. 1996. Role of maternally derived circulating antibodies in protection of neonatal swine against porcine group A rotavirus. J Infect Dis 174:276-82.

33. Ward, L. A., B. I. Rosen, L. Yuan, and L. J. Saif. 1996. Pathogenesis of an attenuated and a virulent strain of group A human rotavirus in neonatal gnotobiotic pigs. J Gen Virol 77 ( Pt 7):1431-41.

34. Ward, L. A., L. Yuan, B. I. Rosen, T. L. To, and L. J. Saif. 1996. Development of mucosal and systemic lymphoproliferative responses and protective immunity to human group A rotaviruses in a gnotobiotic pig model. Clin Diagn Lab Immunol 3:342- 50.

35. Westerman, L. E., H. M. McClure, B. Jiang, J. W. Almond, and R. I. Glass. 2005. Serum IgG mediates mucosal immunity against rotavirus infection. Proc Natl Acad Sci U S A.

36. Wilke, J., G. Lehle, and E. Weiler. 1987. Isogeneic monoclonal antibodies against anti-alpha(1----3)dextran idiotypes. II. Neonatally induced idiotope-specific suppression: a comparative analysis. Eur J Immunol 17:261-7.

37. Wyatt, R. G., W. D. James, E. H. Bohl, K. W. Theil, L. J. Saif, A. R. Kalica, H. B. Greenberg, A. Z. Kapikian, and R. M. Chanock. 1980. Human rotavirus type 2: cultivation in vitro. Science 207:189-91.

38. Yuan, L., A. Geyer, D. C. Hodgins, Z. Fan, Y. Qian, K. O. Chang, S. E. Crawford, V. Parreno, L. A. Ward, M. K. Estes, M. E. Conner, and L. J. Saif. 2000. Intranasal administration of 2/6-rotavirus-like particles with mutant Escherichia coli heat- labile toxin (LT-R192G) induces antibody-secreting cell responses but not protective immunity in gnotobiotic pigs. J Virol 74:8843-53.

39. Yuan, L., A. Geyer, and L. J. Saif. 2001. Short-term immunoglobulin A B-cell memory resides in intestinal lymphoid tissues but not in bone marrow of gnotobiotic pigs inoculated with Wa human rotavirus. Immunology 103:188-98.

40. Yuan, L., S. Y. Kang, L. A. Ward, T. L. To, and L. J. Saif. 1998. Antibody- secreting cell responses and protective immunity assessed in gnotobiotic pigs inoculated orally or intramuscularly with inactivated human rotavirus. J Virol 72:330-8.

41. Yuan, L., L. A. Ward, B. I. Rosen, T. L. To, and L. J. Saif. 1996. Systematic and intestinal antibody-secreting cell responses and correlates of protective immunity to human rotavirus in a gnotobiotic pig model of disease. J Virol 70:3075-83.

42. Zheng, B. J., G. Z. Ma, J. S. Tam, S. K. Lo, M. H. Ng, B. C. Lam, C. Y. Yeung, and M. Lo. 1991. The effects of maternal antibodies on neonatal rotavirus infection. Pediatr Infect Dis J 10:865-8.

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Table 3.1: Rotavirus shedding and diarrhea in gnotobiotic pigs after challenge with Vir HRV

a MatAb: maternal antibody, Exp.I; LTMatAb, maternal antibody in Exp.II

b PID, post inoculation day

c Protection rate=[1-(percentage of vaccinated pigs in each group with diarrhea or shedding/percentage of ISCOM control pigs with

diarrhea or shedding)]x100

d Proportions in the same column with different superscript letters differ significantly (Fisher’s exact test) 284

Vaccine Protection rates (%)c Exp Vaccine regimena Day of Maternal Priming dose Boosting dose N Viral Diarrhea challenge serum shedding (PID)b I MatAb-AttHRV/VLP 28 Yes AttHRV VLP/ISCOM 6 50A d 50A d I AttHRV/VLP No AttHRV VLP/ISCOM 7 71A 71A B A I MatAb-VLP Yes VLP/ISCOM VLP/ISCOM 7 0 29 B B I VLP No VLP/ISCOM VLP/ISCOM 5 0 0 I MatAb-ISCOM Yes Diluent ISCOM matrix 6 0B 33A I ISCOM No Diluent ISCOM matrix 6 0B 0B

285 II LTMatAb-AttHRV/VLP 42 Yes AttHRV/VLP VLP/ISCOM 6 67A 33A

II LT-AttHRV/VLP No AttHRV/VLP VLP/ISCOM 5 80A 80A II LTMatAb-ISCOM Yes Diluent ISCOM matrix 5 0B 20A II LT-ISCOM No Diluent ISCOM matrix 7 0B 0B

Table 3.1

Table 3.2: Comparison of memory B cell responses in piglets given AttHRV/VLP vaccines in the presence or absence of MatAb

and between vaccine groups in Exp.I and II

a MatAb: maternal antibody, Exp.I; LTMatAb, long term maternal antibody in Exp.II

b Mean number of HRV-specific memory B cells (n=4-5) after in vitro stimulation with Wa HRV for 5 days and subtraction of

numbers of effector B cells stimulated similarly with mock . Statistical analyses were performed using Kruskal-Wallis rank sum

test (p<0.05). Single asterisks denote significant differences between MatAb and no MatAb respective vaccine groups in the

28 same experiment. The double asterisks denote significant differences between AttHRV/VLP and VLP vaccine groups with or 6

without MatAb in Exp.I pre-challenge.”#” denotes significant differences between pre- and post challenge of the same vaccine

group with or without MatAb. “♦” denotes significant differences between Exp.II vs Exp.I with the AttHRV/VLP vaccine group

in the presence or absence of MatAb.

c SEM: standard error of the means.

d ND; not done

Number of memory B cells (per 5x105 MNC)b PID28/PCD0 PID35/PCD7 Ileum Spleen PBL Ileum Spleen PBL

a Exp Vaccine regimen IgA IgG IgA IgG IgA IgG IgA IgG IgA IgG IgA IgG I MatAb- Mean b 3 1 5 65* 12* 96* 19 34# 13 235*# 14 116* AttHRV/VLP (SEM)c (2) (1) (2) (19) (4) (32) (8) (14) (5) (73) (8) (49) ♦ ♦ I AttHRV/VLP Mean 2 2 18 1924 81 585 64# 94# 24 2631 11 2786 (SEM) (1) (1) (9) (438) (39) (203) (27) (22) (3) (227) (2) (410) d I MatAb-VLP Mean 3 2* 0*** 1*** 1*** 0*** ND ND ND ND ND ND SEM (1) (1) (0) (0) (0) (0) ND ND ND ND ND ND

I VLP Mean 6 10 3** 9** 7** 11** ND ND ND ND ND ND

28 SEM (6) (3) (1) (5) (4) (6) ND ND ND ND ND ND 7 PID42/PCD0 PID49/PCD7

♦ ♦ ♦ ♦ II LTMatAb- Mean b 2* 8* 1* 2* 0* 5 10* 8* 1* 492*# 9* 580*# AttHRV/VLP (SEM) (1) (5) (1) (1) (0) (3) (4) (4) (1) (160) (5) (256) ♦ ♦ ♦ ♦ ♦ II LT-AttHRV/VLP Mean 129 2276 20 1793 3 28 42 633 15 2145 46 6547# (SEM) (58) (914) (7) (744) (2) (19) (12) (181) (6) (459) (15) (2008)

Table 3.2

Figure 3.1: Virus neutralizing (VN) and isotype specific geometric mean antibody titer

of piglets vaccinated and challenged in the presence or absence of MatAb. Maternal

serum was given to piglets in MatAb groups IP at birth. Piglets were vaccinated at PID 0,

10, 21 (Exp.I) and PID 0, 14, 28 (Exp.II) and challenged at PID28/PCD0 (Exp.I) and

PID42/PCD0 (Exp.II). Approximately half of the piglets were euthanized pre-challenge

at PID28 or PID42 and the rest of the group were euthanized post-challenge at PID

35/PCD7 or PID49/PCD7. Blood samples were collected at the first inoculation and at euthanasia. Different letters A, B and C indicate significant differences among vaccine and control groups with or without MatAb for the same time point in the same

experiment (one-way ANOVA, followed by Duncan grouping on log10 transformed

titers). The “#” denotes significant difference between antibody responses pre-challenge

(PID28 or PID42/PCD0) and post challenge (PID35 or PID49/PCD7) for the same

isotype in the same vaccine or control group with or without MatAb.

288 Serum VN Serum VN

10000 10000 A # A A A A A # 1000 1000 B A A

A 100 100 AB B # B # B 10 10 C B B B 1 1 0 28/0 35/7 0 42/0 49/7 PID/PCD PID/PCD

Serum IgG Serum IgG

1000000 1000000

A 100000 A 100000 A A# A A # A A 10000 AB A# 10000 B A # A 1000 1000

100 100 B B 10 10 B C C B 1 1 0 28/0 35/7 0 42/0 49/7 Geometric mean antibody titers PID/PCD PID/PCD

Serum IgA Serum IgA 100000 100000 A # A # 10000 A # 10000 A 1000 1000 A

100 100 A A B # 10 10 B B B B B B B 1 1 0 42/0 49/7 0 28/0 35/7 PID/PCD PID/PCD MatAb-AttHRV/VLP AttHRV-VLP LTMatAb-AttHRV/VLP LTAttHRV-VLP MatAb-VLP VLP LTMatAb-ISCOM LTISCOM MatAb-ISCOM ISCOM Figure 3.1A Figure 3.1B Figure 3.1 Experiment I Experiment II

289

Figure 3.2: Isotype specific geometric mean antibody titers in small intestinal contents of piglets following vaccination and

challenged in the presence or absence of MatAb. Maternal serum was given to piglets in MatAb groups IP at birth.

Approximately half of the piglets were euthanized pre-challenge at PID28/PCD0 (Exp.I) or and PID42/PCD0 (Exp.II) and the

rest of the group were euthanized post-challenge at PID35/PCD7 or PID49 /PCD7, respectively. The single asterisks denote

significant differences between groups with MatAb and no MatAb receiving the same vaccine at the same time point in each

experiment (one-way ANOVA, followed by Duncan grouping on log transformed titers, p<0.05). The double asterisks denote

290 10

significant differences between AttHRV/VLP and VLP vaccine groups at the same time point (p<0.05). The “#” denotes

significant difference between ASC responses pre-challenge (PID28 or PID42/PCD0) and post challenge (PID35 or

PID49/PCD7) for the same isotype in the same vaccine or control group with or without MatAb (p<0.05). Open bars, vaccine

without MatAb; solid bars, vaccine with MatAb.

290 NoAb LTNoAb SIC-IgM MatAb SIC-IgM LTMatAb

10000 * # 10000 # 1000 * * * 1000 * 100 ** 100 10 10 1 1 0.110 0.110 # 0.01 100 100 # 0.01 # # 0.0011000 0.0011000 # AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD 0 PID35/PCD7 PID42/PCD 0 PID49/PCD7

SIC-IgA SIC-IgA

10000 # 10000 * # # 1000 * 1000 * * * 2 100 100

9 * 1 10 10 1 1

0.110 0.110 10 0.01100 0.01100 0.0011000 0.0011000 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD 0 PID35/PCD7 PID42/PCD 0 PID49/PCD7 Geometric mean antibody titers antibody mean Geometric

SIC-IgG SIC-IgG 10000 10000 1000 1000 # 100 # 100 ** * 10 10 1 1 0.110 0.110 0.01100 * * * 0.01100 0.0011000 10000.001 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD 0 PID35/PCD7 PID42/PCD 0 PID49/PCD7

Figure 3.2 Figure 3.2A Figure 3.2B Experiment I Experiment II

Figure 3.3: IgM ASC responses to Wa HRV in piglets vaccinated in the presence or absence of MatAb. The MNC from duodenum, ileum, spleen and PBL of piglets were collected and assayed on PID28/PCD0 and PID35/PCD7 (Figure 3.3A) for Exp.I and

PID42/PCD0 and PID49/PCD7 (Figure 3.3B) for the longer term Exp.II piglets. The data represent the mean number of ASC per 5x105 MNC. The single asterisks denote significant differences between groups with MatAb and no MatAb receiving the same vaccine at the same time point in each experiment (Kruskal-Wallis rank sum test, p<0.05). The double asterisks denote significant differences between AttHRV/VLP and

VLP vaccine groups at the same time point (Kruskal-Wallis rank sum test, p<0.05). The

“#” denotes significant difference between ASC responses pre-challenge (PID28 or

PID42/PCD0) and post challenge (PID35 or PID49/PCD7) for the same isotype in the same vaccine or control group with or without MatAb. The “♦” denotes significant difference in the ASC responses between LT-AttHRV/VLP (Exp.II) and AttHRV/VLP

(Exp.I) of the same time point in the same tissue for the same antibody isotype. Open bars, vaccine without MatAb; solid bars, vaccine with MatAb.

292 NoAb LTNoAb Duodenum IgM Duodenum IgM MatAb LTMatAb #♦ 75 75 * # * 50 ** ** 50 25 25 # 0 0

-2525 # -2525

-5050 -5050 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7

Ileum IgM Ileum IgM

50 50 * #♦ * #

25 25 # * 0 0 MNC 5 # -2525 -2525

-5050 -5050 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7

Spleen IgM Spleen IgM

50 50 # 25 25 #

0 0 # -2525 -2525

-5050 -5050

Mean numbers of HRV-specific ASC/5x10 of HRV-specific numbers Mean AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7

PBL IgM PBL IgM 50 50 # 25 25 # 0 0 # -2525 # -2525

-5050 -5050 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7 Figure 3.3 Figure 3.3A Figure 3.3B Experiment I Experiment II

293

Figure 3.4: IgA ASC responses to Wa HRV in piglets vaccinated in the presence or absence of MatAb. The MNC from duodenum, ileum, spleen and PBL of piglets were collected and assayed on PID28/PCD0 and PID35/PCD7 (Figure 3.4A) for Exp.I or

PID42/PCD0 and PID49/PCD7 (Figure 3.4B) for the longer term Exp.II piglets. For an explanation of symbols, see the legend to Figure 3.3. Note the change of vertical scale from Figure 3.3

294 NoAb LTNoAb Duodenum IgA Duodenum IgA MatAb LTMatAb ♦ 400 * 400 *

300 300

200 200

100 * * 100

0 0 100 -100 -100100 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID 28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7

Ileum IgA Ileum IgA

400 MNC 400 5 300 300 * # ♦ 200 * 200 * * 100 100

** * * 0 0

-100100 -100100 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7

Spleen IgA Spleen IgA 50 50 *# 25 * 25 Mean numbers of HRV-specific ASC/5x10 0 0

-2525 -2525

-5050 -5050 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7

PBL-IgA PBL IgA 50 50

25 25 * # 0 0

-2525 -2525

-505050 -5050 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7 Figure 3.4A Figure 3.4 Figure 3.4B Experiment I Experiment II

295

Figure 3.5: IgG ASC responses to Wa HRV in piglets vaccinated in the presence or absence of MatAb. The MNC from duodenum, ileum, spleen and PBL of piglets were collected and assayed on PID28/PCD0 and PID35/PCD7 (Figure 3.5A) for Exp.I or

PID42/PCD0 and PID49/PCD7 (Figure 3.5B) for the longer term Exp.II piglets. For an explanation of symbols, see the legend to Figure 3.3. Note the change of vertical scale from Figure 3.3. No significant difference between AttHRV/VLP and VLP vaccine groups were noted.

296 NoAb LTNoAb Duodenum IgG Duodenum IgG MatAb LTMatAb 200 200 * # * # 100 # 100 * * * * 0 * 0 # #

100-100 -100100 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7

Ileum IgG Ileum IgG # 200 200 * * # # MNC 100 * 100 5 * * 0 * 0 * # #

-100100 -100100 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7

Spleen IgG Spleen IgG 200 200 * #

100 * # 100 * * * 0 0

Mean numbers of HRV-specific ASC/5x10 of HRV-specific numbers Mean # -100100 -100100 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7

PBL IgG PBL IgG 100 100

50 50 # * * 0 0

-5050 -5050 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM AttHR/VLP ISCOM AttHRV/VLP ISCOM PID28/PCD0 PID35/PCD7 PID42/PCD0 PID49/PCD7

Figure 3.5A Figure 3.5B Figure 3.5 Experiment I Experiment II

297

CHAPTER 4

LOW TITER MATERNAL ANTIBODIES CAN BOTH ENHANCE AND

SUPPRESS B CELL RESPONSES TO A COMBINED LIVE ATTENUATED

HUMAN ROTAVIRUS AND IMMUNOSTIMULATING COMPLEX-BASED

ROTAVIRUS-LIKE PARTICLE VACCINE

4.1. SUMMARY

High titer maternal antibodies suppress effector and memory B cell responses to rotavirus vaccines. We investigated the effect of low titer circulating maternal antibodies

(LoMatAb) on protection and immune responses induced by live attenuated (Att) human rotavirus (HRV) priming and immunostimulating complex (ISCOM)-2/6-virus-like particle (VLP) booster vaccines (AttHRV/VLP) or VLP vaccines alone. Protection rates in the AttHRV/VLP groups with and without LoMatAb were similar against viral shedding (67% and 71%, respectively) and diarrhea (50% and 71%, respectively) after challenge with virulent HRV. The LoMatAb had both enhancing and suppressive effects on B cell responses, depending on antibody isotype, tissue and vaccine. For the

298

AttHRV/VLP (replicating) vaccine, LoMatAb enhanced intestinal IgM antibody secreting cell (ASC) numbers pre-challenge and intestinal IgM and IgA ASC numbers post-challenge but it suppressed systemic (spleen and blood) IgA ASC numbers and intestinal IgA antibody responses pre- and post- challenge. For the VLP (non-replicating) vaccine, LoMatAb also enhanced intestinal and splenic IgM ASC numbers post- challenge, but it suppressed IgG ASC numbers pre-challenge in all tissues. Pre-challenge

IgA memory B cells in ileum and spleen were unaffected, but they were suppressed in blood of both vaccine groups. The differential effects of LoMatAb on the IgA responses suggests that LoMatAb did not suppress the induction of IgA ASC and memory B cells at the induction site (ileum) but it impaired the homing of activated B cells to secondary lymphoid or effector tissues, reducing IgA ASC and antibodies at these sites. Thus, even

LoMatAb exerts different effects on B cell responses to replicating versus non-replicating vaccines. Nevertheless the AttHRV/VLP vaccine partially overcame the inhibitory effect of LoMatAb and represents a new approach for rotavirus vaccines.

4.2. INTRODUCTION

Rotaviruses (RV) are the major cause of dehydrating gastroenteritis in children worldwide (8) and cause an estimated 600,000 deaths annually (6). Rotavirus vaccines have been in development for the past 2 decades with clinical trials conducted in human infants worldwide. In many cases, inconsistent results were seen for vaccine efficacy in different countries or regions. A tetravalent human-rhesus RV reassortant vaccine (RRV- TV) was effective in reducing RV-induced diarrhea in trials in the US and Finland but

299

was less effective in trials in some developing countries such as Peru and Brazil (2, 19, 23, 25, 33). The variable seroconversion rates and the low protection rates in some trials were attributed to the presence of maternal antibodies (MatAb) (11, 21).

The licensed RRV-TV live vaccine (Rotashield®) was associated with a higher risk of intussusception which led to its withdrawal from the market (29), although a follow-up study in the US showed a reduction in intussusception cases among RRV-TV vaccinated infants (39). A study of Mexican infants further reported a lack of association between natural RV infection and intussusception (42). The risk of intussusception following Rotashield is now estimated as 1 in 10,000 to 1 in 32,000 vaccinated children (3). The risk increases in children older than 3 months of age (80%) at the time of the first vaccination compared to that associated with infants less than 3 months of age (20%) (16). Thus, the recommendation was to give RV vaccines to infants less than 3 months of age at the first dose. However, administration to infants at this age poses the likelihood of a greater impact of MatAbs on the vaccine. Currently, live vaccines other than RRV-TV such as a bovine RV based pentavalent vaccine (RotaTeq, Merck Inc.) or the live attenuated (Att) monovalent human RV (HRV) vaccine (Rotarix, GlaxoSmithKline) are undergoing Phase III clinical trials (1, 14) with the latter vaccine now licensed in Mexico (7, 46). With the renewed interest in the use of live RV vaccines and the shift in their use to younger infants, it is important to understand the impact and mechanism of MatAb interference on RV vaccines, either replicating (Att vaccines) or non-replicating (i.e. virus-like particle [VLP] vaccines) or combined live virus and VLP vaccine regimens. Because VLP vaccines are non-replicating, but immunogenic, they may be safer with a potential to replace live RV vaccines, such as Rotarix and RotaTeq in the future.

In our previous study, when a vaccine containing the Wa strain AttHRV followed by 2 intranasal (IN) doses of 2/6VLP with immunostimulating complexes (ISCOM) was

300

administered to pigs in the presence of high titer MatAb, long-term suppressive effects on antibody secreting cells (ASC), antibody production and memory B cell responses were observed (Chapter 3). A non-replicating vaccine consisting of 3 doses of 2/6 VLP with ISCOM did not induce protection again viral shedding or diarrhea upon challenge, but it was less affected by high titer MatAb and it induced intestinal IgM and IgA, but not IgG ASC responses. The MatAb titers in the previous study were high, falling within the range of RV antibody titers found in developing countries where repeated exposures to RV occur (4, 32, 51). Because the effect of MatAb may be dose dependent (38), the purpose of this study was to investigate the effect of lower titer MatAb on B cell responses (effector and memory B cell numbers and serum and intestinal antibody responses) induced by the above two vaccines. Knowledge of the differential effects of different levels of MatAb on B cell responses induced by different vaccine regimens and the potential mechanisms involved will facilitate the development of appropriate vaccination strategies to overcome the negative influence of MatAb.

4.3. MATERIALS AND METHODS

4.3.1. Preparation of serum pools

The serum with low HRV antibody titers (LoMatAb) was produced as control serum for the high titer maternal serum in a previous study by intramuscularly inoculating RV-seropositive sows (n=2) with 5 doses of uninfected (mock) cell culture supernatants mixed with incomplete Freund’ adjuvant (Chapter 3). Serum was collected and pooled after the last immunization, heat inactivated at 56oC for 30min and filtered by

Seitz micromedia filter pads, (Ertel/Alsop, Kingston, NY) followed by 0.22µm

301

membrane filters (Millipore, Bedford, Mass.). The IgG and virus-neutralizing (VN)

antibody titers to Wa HRV were measured by ELISA and a plaque-reduction assay,

respectively, as described previously (37).

4.3.2. Virus

The tissue-culture adapted Wa strain of AttHRV derived from the twenty-seventh

passage in African Green monkey kidney cells (Ma104) was used for vaccination (5x107 fluorescent focus forming units, FFU), ELISA and ELISPOT. The virulent (Vir) HRV derived from stools of an infected infant was maintained by serial passage in gnotobiotic

(Gn) pigs (48, 50). Pooled intestinal contents of the VirHRV infected Gn pigs were used

6 for challenge at 10 median infectious doses (ID50). The ID50 of the VirHRV inoculum for

Gn pigs was previously determined to be at least 1 FFU and the Wa AttHRV was

previously determined to be 1.3x106 FFU (36, 44, 45). The titers of both AttHRV and

VirHRV were determined by a cell culture immunofluorescence assay (CCIF) (36, 37).

4.3.3. Experimental groups

Gnotobiotic pigs were injected with low titer sow serum (LoMatAb) to mimic infants

with low titer passive circulating MatAb. The hysterectomy-derived near-term pigs were

obtained and maintained in isolation units as described previously (28) under an approved

animal use protocol. Newborn unsuckled pigs are devoid of MatAb due to the impervious

nature of the sow placenta to immunoglobulins (22). The LoMatAb administered via the

intraperitoneal (IP) route is transferred to lymphatic vessels and enters the circulation

(18), to mimic the effect of circulating passively-derived LoMatAb. Pigs were given

30ml of the LoMatAb, twice IP within the first 24h after birth as determined by previous

302

studies (18, 31) and confirmed in this study to result in serum IgG concentrations in pigs

(mean= 27mg/ml, data not shown) similar to those reported for naturally suckled pigs

(43). The total serum IgG concentration in pigs was measured by porcine IgG ELISA kits

(Bethyl Laboratories Inc., Montgomery, Tex.).

Pigs given the low titer serum IP were randomly assigned into three groups, the

LoMatAb-AttHRV/VLP, LoMatAb-VLP and LoMatAb-ISCOM groups. Pigs in the

AttHRV/VLP, VLP and ISCOM groups did not receive the MatAb serum. The vaccine

groups are summarized in Table 4.1. Briefly, at 3-5 days of age, pigs in the LoMatAb-

AttHRV/VLP and AttHRV/VLP groups were inoculated using a vaccine regimen

consisting of an oral dose of Wa AttHRV (5x107FFU/dose) followed by 2 IN doses of

2/6VLP/ISCOM (250µg of VLP associated with 1250µg of ISCOM), 10 days apart. Pigs

in LoMatAb-VLP and VLP groups were inoculated IN with 3 doses of 2/6VLP/ISCOM.

Pigs in LoMatAb-ISCOM and ISCOM groups were inoculated with diluent and ISCOM

matrix (ISCOM) as controls within the same time frame as the vaccines.

4.3.4. Assessment of protection

Challenge and protection studies were done as described previously (50). At PID

28, subsets of pigs (5-7 pigs/group) were challenged orally with ~106 ID50 of VirHRV.

Rectal swabs were collected and diarrhea scores were observed for 6 days after challenge for assessment of viral shedding and diarrhea (Fecal scores of 0, normal; 1, pasty; 2 semi- liquid; and 3, liquid). Scores of greater or equal to 2 were considered as diarrhea.

Infectious virus and viral antigen were detected in the rectal swab fluids using the CCIF assay and antigen-capture ELISA, respectively, as previously described (5, 37). The pigs

303

were considered as completely protected against shedding or diarrhea upon challenge with VirHRV only when they did not shed virus or have diarrhea during the entire observation period.

4.3.5. Plaque reduction assays for VN antibodies. This assay was performed on serum samples of pigs and sows, as described previously (37). The VN antibody titers were expressed as the reciprocal of the serum dilution which reduced the plaque numbers by

>80%.

4.3.6. Isotype-specific antibody ELISA. The Wa HRV specific IgM, IgA and IgG antibody titers in the sera of sows and pigs and in pig intestinal contents were determined by an indirect isotype-specific antibody ELISA as previously described (41).

4.3.7. ELISPOT assay for RV-specific ASC

Subsets of pigs were euthanized at PID28/PCD0 (pre-challenge) and

PID35/PCD7 (post-challenge) and the small intestine (duodenum and ileum), spleen and peripheral blood lymphocytes were collected for the isolation of MNC as previously described (50) . The ELISPOT assays to enumerate HRV-specific ASC of different isotypes (IgM, IgA and IgG) were conducted using previously published methods (50).

The assay was performed on the freshly isolated lymphocytes (in vivo, to measure the effector B cell responses) and on the cells stimulated in culture with AttHRV (in vitro) for 4 days to measure the short-term memory B cell responses (49). The numbers of

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ASC were expressed per 5x105 MNC. The numbers of memory B cells were expressed per 5x105 MNC that remained viable after in vitro stimulation. Memory B cell responses at PID 28/PCD0 were presented in this study.

4.3.8. Statistical analyses. For all the parameters, statistical comparisons were made: (1) between pig groups with and without MatAb for the same vaccine at the same time point;

(2) between AttHRV/VLP and VLP vaccine groups with and without LoMatAb at the same time point and (3) between pre- and post-challenge for the same isotype ASC and antibody responses within the same vaccine group with or without LoMatAb. For antibody titers (isotype-specific ELISA and VN antibodies), statistical analyses were

performed on log10 transformed titers. One-way ANOVA (SAS Institute Inc., Cary, N.C)

followed by Duncan's multiple-range test was used to test for significant differences

(marked by different capital letters: A, B, C) among groups at each time point. The mean

number of ASC was calculated for each treatment group at PID28/PCD0 and

PID35/PCD7. Kruskal-Wallis rank sum (non-parametric) tests were used to compare the

ASC numbers between all group pairs and between the ASC responses pre- and post-

challenge within the same vaccine group with or without MatAb. Differences in

proportions of pigs with diarrhea and virus shedding were determined by chi-square test;

when significant differences were present among groups, pair-wise comparisons were

made by Fisher’s exact test. Differences in duration and time of onset of diarrhea and

virus shedding between groups were evaluated by survival analysis. Unless the p value is

specified, a p value of ≤ 0.05 was used to denote statistical significance.

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4.4. RESULTS

4.4.1. Intraperitoneal injection of newborn pigs with the low titer MatAb serum mimics

the passive circulating RV-specific low titer MatAb in infants in developed countries

The geometric mean titers (GMT) of circulating Wa HRV specific VN and IgG

antibodies in pigs at the first vaccination (post inoculation day [PID] 0, 3-5 days after

maternal serum injection) were 97 and 1942, respectively (Figure 4.1), which are within

the range of RV VN (5-600) and IgG (800-6400) antibody titers in human infants less

than 4-6 months of age, especially those in developed countries (10, 34). Although the

pigs in the LoMatAb groups did not receive the antibodies from their own mothers

directly, the serum pool derived from the lactating sows and injected into the Gn pigs

closely mimics the level of passive circulating MatAb transferred from the mother to the

fetus.

4.4.2. The AttHRV/VLP vaccine induced similar protection rates against viral shedding

and diarrhea in the presence and absence of LoMatAb

To assess the protection rates against viral shedding and diarrhea, approximately half of the pigs (5 to 7 pigs) within each group were challenged with VirHRV at PID

28/PCD 0 (Table 4.1). Protection rates against viral shedding and diarrhea after challenge were higher, but not significantly, in the AttHRV/VLP group (71% for each) than those in the LoMatAb-AttHRV/VLP group (67% and 50%, respectively). The protection rates in the latter group were likely due to active immunity as the LoMatAb-ISCOM and

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LoMatAb-VLP groups were not protected against shedding or diarrhea. In the LoMatAb-

AttHRV/VLP and AttHRV/VLP groups, the duration of virus shedding and diarrhea were shorter or significantly shorter (less than 1 day) than those of the LoMatAb-VLP, VLP,

LoMatAb-ISCOM and ISCOM groups (2-3 days). The LoMatAb-AttHRV/VLP and

AttHRV/VLP groups also showed significant delays in the onset of shedding (PCD 4 or

5) and diarrhea (PCD 3 or 4) compared to each of the other groups (PCD 1 or 2). Both the

LoMatAb-AttHRV/VLP and AttHRV/VLP groups showed lower virus shedding titers

(3.5-30 fold, respectively) post-challenge compared to the respective ISCOM control groups.

4.4.3. Serum and intestinal IgA antibody responses were inhibited in the AttHRV/VLP and VLP vaccine groups with LoMatAb both pre- and post-challenge

Moderate VN, IgM and IgG antibody titers and low IgA antibody titers were present in serum of all LoMatAb pigs and the respective titers did not differ among groups before vaccination at PID0 (Figure 4.1). After vaccination at PID28/PCD0, in the absence of LoMatAb, the pigs in the AttHRV/VLP and VLP vaccine groups developed significant IgM, IgA and IgG antibody responses in serum compared to controls. In the presence of LoMatAb, the serum VN antibody titers increased 11-fold in the LoMatAb-

AttHRV/VLP group pre-challenge (PID28/PCD0) compared to PID0, to the same level measured in the AttHRV/VLP group without MatAb suggesting induction of active immunity. This increase in the serum VN antibody responses coincided with a 6-fold increase in the IgM antibody responses in the LoMatAb-AttHRV/VLP group at

PID28/PCD0. The serum IgG antibody titers increased 2-fold in the LoMatAb-

AttHRV/VLP group whereas the serum IgG antibody responses did not change in the 307

LoMatAb-VLP group. The IgG antibody titers in both groups were significantly lower

than those in the respective AttHRV/VLP and VLP groups without MatAb, suggesting

partial suppression of active immune responses by LoMatAb. The VN and IgM antibody

titers declined in both LoMatAb-VLP and LoMatAb-ISCOM groups from PID0-PID28.

The serum IgA antibody responses in the LoMatAb-AttHRV/VLP and LoMatAb-VLP groups also declined to low or undetectable levels after vaccination (PID28/PCD0) indicative of suppression of active IgA antibody responses by LoMatAb compared to the significantly elevated titers in the AttHRV/VLP group without MatAb.

After challenge (PID35/PCD7), all groups had significant increases in serum IgM antibody responses (Figure 4.1). The AttHRV/VLP and the LoMatAb-AttHRV/VLP groups developed significant increases in serum VN, IgG and IgA antibody responses.

However, the IgG and IgA, but not IgM antibody titers in the LoMatAb-AttHRV/VLP group were lower (IgG) or significantly lower (IgA) than those of the AttHRV/VLP group without MatAb. The LoMatAb-VLP group exhibited no increase in serum VN antibody titers, whereas the post-challenge IgA and IgG antibody levels increased but not significantly, and were significantly lower than the levels in the VLP group. The ISCOM control groups without MatAb also developed low VN antibody responses after challenge. The LoMatAb-ISCOM group showed reduced IgG antibody and no increase in the VN and IgA antibody responses. Thus maternal inhibition of serum IgA antibody responses remained for both vaccine groups at PID35/PCD7. However active immunity as indicated by a significant increase in the VN, IgA and IgG antibody responses after challenge occurred in the LoMatAb-AttHRV/VLP group.

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The intestinal antibody responses were measured, and as in the previous study

(submitted for publication), the intestinal IgA antibody responses were suppressed pre and post- challenge in the LoMatAb-AttHRV/VLP and LoMatAb-VLP vaccine groups

(Figure 4.2). The IgG antibodies were present at low levels pre-challenge in the

LoMatAb-groups and they did not increase significantly post-challenge. Of the groups receiving LoMatAb, only IgM antibodies were detected in the LoMatAb-AttHRV/VLP group pre-challenge, whereas IgM antibody responses occurred in all LoMatAb groups after challenge, indicative of inhibition of intestinal IgA and IgG antibody production of the LoMatAb vaccine groups pre- and post-challenge.

4.4.4. Isotype specific ASC responses

Circulating LoMatAb enhanced the IgM ASC responses in the LoMatAb-

AttHRV/VLP and LoMatAb-VLP vaccine regimens pre- and post-challenge

Pre-challenge (PID28/PCD0), the IgM ASC responses in duodenum and spleen of the LoMatAb-AttHRV/VLP group did not differ significantly from those of the

AttHRV/VLP group without MatAb (Figure 4.3). The IgM ASC numbers in the ileum and blood of the LoMatAb-AttHRV/VLP group were 3 to 4-fold higher (but not significantly) than those in the AttHRV/VLP group, suggesting enhancement of IgM

ASC responses by LoMatAb. The ileal IgA ASC and the IgG-ASC responses in all tissues were similar between the AttHRV/VLP vaccine groups with or without LoMatAb

(see below), indicating that impaired isotype switching (µ to α/γ) was not the main reason for the enhanced numbers of IgM ASC in the LoMatAb group. These IgM ASC

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responses were minimal in the intestinal and systemic tissues of the VLP and LoMatAb-

VLP groups. However, the LoMatAb-VLP group showed significantly higher IgM ASC numbers in the spleen compared to those in the VLP group, indicative of enhancement of systemic IgM ASC responses in the VLP vaccine group by LoMatAb. These observations imply that circulating LoMatAb did not suppress the IgM-ASC responses induced by the

AttHRV/VLP and VLP regimens; on the contrary, LoMatAb enhanced the IgM-ASC responses induced by both vaccine regimens. No IgM ASC responses were observed in the ISCOM and LoMatAb-ISCOM control groups pre-challenge. The IgM ASC numbers in the intestine of the AttHRV/VLP group were higher (ileum) or significantly higher

(duodenum and spleen) than those induced by the VLP vaccine pre-challenge, indicating the lower antigenicity of the 2/6-VLP vaccine.

After challenge (PID35/PCD7), the IgM ASC responses did not increase in any tissue of the AttHRV/VLP vaccine group without MatAb (Figure 4.3). The IgM ASC responses however increased after challenge in the intestine of the LoMatAb-

AttHRV/VLP group, and become higher (ileum) or significantly higher (duodenum) compared to the AttHRV/VLP group (2-8-fold). Similarly, the post-challenge IgM ASC responses increased in intestine (significantly) and spleen of the LoMatAb-VLP group compared to those of the VLP group (3-8-fold higher). The IgM ASC responses in blood induced by both vaccines were low regardless of the presence or absence of MatAb. The

ISCOM group developed low but significant IgM ASC responses in the ileum, spleen and blood post-challenge; but the IgM ASC responses did not differ significantly between the

ISCOM and LoMatAb-ISCOM groups.

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Circulating LoMatAb enhanced the intestinal IgA ASC responses post-challenge but suppressed these responses in spleen of the LoMatAb-AttHRV/VLP pigs pre- and post-challenge

Pre-challenge (PID 28/PCD0), IgA was the main isotype of ASC in the intestine in the AttHRV/VLP vaccine group with or without LoMatAb (Figure 4.4). The IgA ASC numbers in the duodenum and spleen of the LoMatAb-AttHRV/VLP pigs were lower and significantly lower, respectively, compared to the AttHRV/VLP group, indicating the suppressive effects of LoMatAb. However, there was no difference in the ileal IgA ASC numbers between the two groups. The differential effects of LoMatAb on the IgA ASC responses induced by the AttHRV/VLP regimen between the ileum and duodenum or spleen suggests that LoMatAb did not suppress the induction of IgA ASC responses in the ileum (induction site), but only impaired the homing of the activated B cells from ileum to the secondary lymphoid tissues where the activated B cells develop into ASC

(duodenum or spleen).

The pre-challenge intestinal IgA ASC numbers between the LoMatAb-VLP and

VLP groups were low and did not differ significantly, although the mean numbers of ileal IgA ASC in the LoMatAb-VLP group were 3-fold higher than those of the VLP group. The IgA ASC responses in the blood were low and there was no difference between the LoMatAb and no MatAb groups regardless of the vaccine. No IgA ASC responses were observed in the ISCOM and LoMatAb-ISCOM groups pre-challenge. The

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intestinal IgA ASC numbers in the VLP group were lower (duodenum) or significantly lower (ileum) than those in the AttHRV/VLP group, indicating that the antigenicity of the

VLP vaccine was significantly lower than the AttHRV/VLP vaccine.

After challenge (PID35/PCD7), intestinal IgA ASC responses increased significantly in the duodenum (13-fold) and moderately in the ileum (2-fold) of the

LoMatAb-AttHRV/VLP group, indicating that active immune responses were primed pre-challenge by the AttHRV/VLP vaccine in the presence of LoMatAb (Figure 4.4). The

LoMatAb-AttHRV/VLP group had higher but not significantly higher intestinal IgA ASC responses compared to the AttHRV/VLP group. However, the IgA ASC numbers in the spleen and blood of the LoMatAb-AttHRV/VLP group did not increase after challenge and were significantly lower than those of the AttHRV/VLP group, coinciding with the significantly reduced post-challenge serum IgA antibody responses in the former compared to the latter group (Figure 4.1). These differential affects of circulating

LoMatAb on the ASC responses in different tissues may be due to different mechanisms of inhibition by maternal serum in the intestinal versus systemic tissues. Similarly to pre- challenge, the post-challenge intestinal and systemic IgA ASC responses in the VLP vaccine groups did not differ significantly between LoMatAb and no MatAb groups. The

LoMatAb-ISCOM group had IgA ASC responses after challenge in all tissues that were higher (blood) or significantly higher (duodenum and ileum) than the ISCOM control group without LoMatAb, suggesting enhancement by LoMatAb of the IgA ASC responses to VirHRV.

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Pre-challenge circulating LoMatAb suppressed the IgG ASC responses to the

LoMatAb-VLP vaccine, but not to the LoMatAb-AttHRV/VLP vaccine

Pre-challenge (PID28/PCD0), IgG ASC responses in intestinal and systemic

tissues were low in the no MatAb and LoMatAb groups receiving both AttHRV/VLP and

VLP vaccines (Figure 4.5). There were no significant differences in the IgG ASC

responses between the LoMatAb-AttHRV/VLP and the AttHRV/VLP groups in all

tissues. However, pre-challenge IgG ASC numbers in all the tissues were significantly

lower in the LoMatAb-VLP group compared to the VLP group without LoMatAb,

suggesting isotype specific inhibition by LoMatAb of IgG ASC responses induced by this

vaccine. No IgG ASC responses were observed in the ISCOM and LoMatAb-ISCOM

groups pre-challenge.

After challenge (PID35/PCD0), there were no significant differences in the IgG

ASC responses in the intestine and spleen between the LoMatAb-AttHRV/VLP and

AttHRV/VLP groups or between the LoMatAb-VLP and VLP groups (Figure 4.5). Both

vaccine groups in the presence or absence of LoMatAb showed more than 3-fold

increases in the IgG ASC numbers post-challenge in all tissues (except for the IgG ASC

responses in spleen of the VLP group without MatAb). In particular, the LoMatAb-

AttHRV/VLP group had increases or significant increases (3- to 12-fold) whereas the

AttHRV/VLP group had significant increases (4- to 6-fold) in intestinal and systemic IgG

ASC numbers. The increased IgG ASC numbers in the AttHRV/VLP groups with or without MatAb post-challenge coincided with the high numbers of memory IgG B cells in spleen of these groups at PID 28 (608 and 1900 per 5x105 MNC, respectively, Figure

4.6). The IgG ASC numbers after challenge increased in all tissues of the VLP and

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LoMatAb-VLP groups (7- to 53-fold) with significant increases in the post-challenge IgG

ASC numbers only in the intestine of the VLP group and in the spleen and blood of the

LoMatAb-VLP group. Isotype specific inhibition also occurred in the ISCOM control

group in the presence of LoMatAb as significantly lower IgG-ASC responses occurred in

the intestine and spleen of the LoMatAb-ISCOM group compared to the ISCOM control

group without LoMatAb.

4.4.5. The IgA memory B cell responses in blood of both vaccine groups were

significantly inhibited by circulating LoMatAb; the IgG memory B cell responses in

the systemic tissues of the AttHRV/VLP vaccine were also affected, but not

significantly

Figure 4.6 depicts the HRV-specific IgA and IgG memory B cell responses in

ileum, spleen and blood induced by AttHRV/VLP and VLP vaccines and the ISCOM

control with or without MatAb, pre-challenge at PID28/PCD0, which also corresponded

with the post-challenge IgA and IgG ASC responses.

The magnitude of the memory B cell responses in ileum in the vaccine groups

was similar for the IgA and IgG isotypes. In contrast, the IgA memory B cell responses in

spleen and blood were much lower than those of the IgG isotype (note the scale

difference in the y-axis, Figure 4.6). There were no differences in the IgA memory B cell

responses in ileum of the AttHRV/VLP and VLP vaccines with or without LoMatAb, corresponding to a lack of differences in intestinal post-challenge IgA ASC responses in these groups (Figure 4.4). There were also no differences in the spleen IgA memory B cell responses between the vaccine groups with or without LoMatAb. However, the

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numbers of IgA memory B cell numbers in the blood were significantly lower in the

LoMatAb-AttHRV/VLP and LoMatAb-VLP groups than those for the AttHRV/VLP and

VLP groups, corresponding to the significantly lower systemic IgA ASC (Figure 4.4) and

serum IgA antibody responses (Figure 4.1) post-challenge in the LoMatAb groups

compared to the no MatAb group, again indicative of LoMatAb suppression of these IgA responses in the blood. Thus, the circulating LoMatAb did not affect the IgA memory B cell responses in the ileum, but it significantly suppressed the IgA memory B cell responses in the blood induced by the AttHRV/VLP and VLP vaccines.

The IgG memory B cell responses in the ileum were higher, but not significantly

higher in the LoMatAb-AttHRV/VLP group than the AttHRV/VLP groups (Figure 4.6).

The IgG memory B cell responses were present at very high numbers in spleen and

blood of the AttHRV/VLP vaccine group (mean numbers of 585-1900/5x105MNC). In

contrast to the ileum, the IgG memory B cell responses in the systemic tissues were

higher (3-4 fold), but not significantly, in the AttHRV/VLP group compared to the

LoMatAb-AttHRV/VLP, corresponding to the higher (but not significantly higher)

systemic IgG antibody responses post-challenge (Figure 4.1). The magnitude of the IgG

memory B cell responses was low in the LoMatAb-VLP and VLP groups in all tissues.

Lower but not significantly lower numbers of IgG memory B cells were detected in the

ileum of the LoMatAb-VLP group. There were also no significant differences in the very

low IgG memory responses in spleen and blood between the LoMatAb-VLP and VLP

groups, corresponding to the lack of differences in the post-challenge IgG ASC

responses in spleen of these groups (Figure 4.5).

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4.5. DISCUSSION

We studied the effects of low titers of MatAb on the immunogenicity and protective efficacy induced by two candidate RV vaccines. The titer of MatAb in this study was 1.3-1.8 logs (20-60-fold) lower compared to the high titer MatAb used in our previous study (Chapter 3). The sequential vaccine regimen of oral AttHRV priming and

IN 2/6VLP boosting was able to partially overcome the suppressive effects of the low titer MatAb and this regimen induced partial protection against both RV associated diarrhea and infection, similar to the protection rates induced in the absence of MatAb.

Protection was associated with the induction of the HRV-specific intestinal IgM and IgA

ASC responses before challenge, with increased virus-specific IgA and IgG ASC numbers in intestinal lymphoid tissues after challenge. Furthermore, protection in the

LoMatAb-AttHRV/VLP group was also associated with substantial increases in serum

VN antibody titers pre-challenge and increases in serum VN, IgA and IgG antibodies post-challenge. However lower (VN and IgG) and significantly lower (IgA) serum antibody responses were observed in the LoMatAb-AttHRV/VLP group compared to the

AttHRV/VLP group pre-challenge, which corresponded with the lower, but not significantly lower, protection rates against shedding and diarrhea. In comparison, the low titer MatAb also exerted a neutralizing effect on the VirHRV challenge as evident in the controls by a reduction of serum (VN and IgG) antibody titers (2-3-fold) after challenge of the LoMatAb-ISCOM group compared to the ISCOM group. Thus, the

AttHRV/VLP vaccine partially overcame the neutralizing effect of low titer MatAb pre- challenge and effectively primed for enhanced responses post-challenge. Similar

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observations were reported in a study of mice immunized with live attenuated respiratory

syncytial virus (RSV) vaccines in the presence of circulating MatAb (12). Mice

vaccinated with either one of the two attenuated vaccines after IP injection with undiluted

or 1:10 diluted RSV immune serum (equivalent to our high and low titer MatAb sera),

were highly protected against challenge with wild-type RSV. In the mice injected with

the lower RSV antibody titer serum (1:10 dilution), although the primary systemic and

mucosal IgM, IgA and IgG antibody responses to the vaccines were suppressed at

challenge (PID 28), the B cells were primed for strong secondary antibody responses at

PID 56, i.e. 28 days after challenge with wild type RSV.

Differential effects of low titer MatAb on the AttHRV/VLP vaccine responses

were observed in different tissues (intestinal vs. systemic). In the intestine, where there

was a lower level of the transudated serum MatAb, the intestinal IgM and IgG ASC

responses induced by the AttHRV/VLP vaccine were not affected before or after

challenge, but a slight transient suppression of duodenal IgA ASC responses was

observed pre- but not post-challenge. This lack of inhibition of intestinal antibody

responses by low titer MatAb was further indicated by the similarity in the pre-challenge

IgA effector and memory B cell responses in ileum between the AttHRV/VLP groups

with and without LoMatAb. Similarly, there was no significant difference in intestinal

IgM, IgA and IgG ASC responses between the low RV specific MatAb and no MatAb

groups when Gn pigs were inoculated with VirHRV (18).

In addition, the low titer MatAb enhanced the duodenal IgA and IgM ASC

responses post-challenge in both the LoMatAb-AttHRV-VLP and the LoMatAb-VLP groups. Thus, the low titer MatAb enhanced various intestinal ASC responses induced by

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both vaccines. It is possible that transfer of protective idiotopes from the mother to the

child could prime the neonate for a booster effect to the antigen previously experienced by the mother (24). In addition, it is possible that the low titer of MatAb enhanced the

uptake, processing and presentation of the viral proteins when complexed with low

amounts of antibodies. This antibody dependent immune enhancement has been reported

previously for vaccine responses in different animal models. In Gn pigs, Hodgins et al

(18) found higher (but not significantly) IgA ASC responses to HRV in the intestine of pigs inoculated with VirHRV in the presence of passive low titer serum MatAb at PCD4, but not in systemic tissues (spleen and blood). The authors also observed higher or significantly higher total IgM secreting cell numbers pre- and post-challenge in all tissues of Gn pigs that received the HRV-specific serum MatAb than in pigs that did not received. We observed similarly enhanced virus-specific IgM ASC responses, in ileum pre-challenge and in both duodenum and ileum post-challenge in the LoMatAb-

AttHRV/VLP group. Richter et al (35) found that low or moderate levels of

pneumococcal polysaccharide-specific MatAb not only provided protection against

pneumococcal infections, but also enhanced the immune responses elicited by a

pneumococcal vaccine in neonatal and infant mice. The immune enhancement is not only

applicable for antigen specific antibody but also to natural antibody. Chicken given

pooled plasma from non-keyhole limpet hemocyanin (KLH) immunized hens (containing

natural antibody), then immunized with KLH antigen showed increases in IgM and IgY

antibody titers to KLH compared to those that received phosphate buffered saline.

Although enhancement and not significant suppression of B cell responses by the

LoMatAb was found in the intestine, some suppression was observed in systemic tissues

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(spleen and PBL) after both vaccine regimens. Suppression of spleen IgA ASC responses

in the LoMatAb-AttHRV/VLP group was observed pre-challenge. The suppression of

immune responses in systemic tissues was also evident by the lower or significantly

lower post-challenge IgA and IgG antibody responses in serum of the LoMatAb-

AttHRV/VLP and LoMatAb-VLP groups compared to the no MatAb groups. Thus, this

study and our previous study (Chapter 3) suggest that a broad suppression of humoral

immune responses was induced by a wide range (low and high) of MatAb. The difference

between the effects of LoMatAb on IgA ASC responses in intestinal and systemic tissues

may be because a T–cell independent pathway is involved in induction of intestinal IgA

antibody responses (27) whereas a T-cell dependent pathway may be involved in

development of the systemic IgA antibody responses (26, 27, 30). The significantly

lower systemic IgA ASC and antibody responses post-challenge are consistent with our previous observations that the post-challenge T cell responses in blood and spleen are lower but not significantly lower, in the LoMatAb-AttHRV/VLP group compared to

AttHRV/VLP group (Yuan and Saif, unpublished).

The low titer MatAb was shown to have little suppressive effect on the ileal IgA and IgG memory B cell responses induced by the AttHRV/VLP vaccine. Yet the IgA and

IgG memory responses in blood and the IgG memory responses in spleen were decreased or significantly decreased in the LoMatAb-AttHRV/VLP group. It appears that the effect

of LoMatAb may be restricted to the traffic of activated B cells in the circulation, and not

on the development of B memory cells locally (intestine and spleen). This effect of low

titer MatAb differs from that of high titer MatAb in our previous study (Chapter 3). High

titer MatAb also suppressed memory responses induced by the AttHRV/VLP vaccine in

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all tissues examined (ileum, spleen and blood). The induction of memory responses in the presence of low titer MatAb observed in this study has also been documented elsewhere.

Previous studies showed that T and B cell memory responses were generated in the presence of MatAb (9, 15, 20). Calves developed a memory antibody response to bovine viral diarrhea virus (BVDV) when vaccinated with BVDV modified live vaccine twice at

7 and 14 weeks after the high circulating BVDV antibody titers (via colostrum ingestion at birth) declined (15). Low titers of IgG antibodies in the maternal serum may actually enhance antigen presentation, and stimulate memory B cell differentiation in germinal centers in the presence of follicular dendritic cells (40).

In the presence of low titer MatAb, the antibody responses induced by the 2/6VLP vaccine were also partially affected, showing that low titer circulating MatAb may also exert significant effects on B cell responses induced by a non-replicating (VLP) vaccine.

Similar to the effects of high titer MatAb in our previous study (Chapter 3), the systemic and/or intestinal IgG ASC responses and the serum and intestinal IgA and IgG antibody responses induced by the 2/6-VLP vaccine were effected, whereas the intestinal IgM and

IgA ASC responses pre- and post-challenge were not. Moreover the IN route of the VLP vaccine delivery may lead to less impact by the circulating LoMatAb on intestinal IgA and IgM ASC, but a greater impact on systemic IgG ASC and serum IgG and IgA responses. In the NALT, IgA isotype switching, for differentiation and maturation of

IgA-ASC may occur before these cells migrate out of NALT, whereas IgG ASC responses may require transit of the B cells through the draining lymph nodes of the

NALT (47). The suppressive effects on IgG ASC responses induced by the VLP vaccine continued after challenge in the presence of high titer MatAb (Chapter 3), whereas these

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effects were not observed post-challenge in the intestine and spleen of the LoMatAb-VLP

group. In particular, the VLP vaccine increased the intestinal IgG ASC responses post-

challenge of similar magnitude to the responses in the VLP group without MatAb.

In summary, our study demonstrated that LoMatAb had both enhancing and

suppressive effects on the effector and memory B cell responses to RV vaccines in

neonates. The sequential AttHRV priming and 2/6-VLP boosting regime partially

overcame the suppressive effect of low titer MatAb and conferred moderate protection

rates against RV shedding and diarrhea upon challenge. High intestinal IgM and IgA

ASC responses were associated with protection in this vaccine group. The results of our

study suggest the possibility of using a high titer (5x107FFU) attenuated vaccine as an

oral priming dose with the 2/6-VLP boosters to overcome the suppressive effect of low

titer MatAb. Of note, the oral live attenuated rotavirus vaccine used in a vaccine trial in the US was lower containing either 105.2FFU or 106.4 FFU of RIX4414 strain human

rotavirus P[8]G1 (13) and the impact of MatAb on this two-dose oral vaccine remains

uncertain.

Enhancement of intestinal IgM and IgA ASC responses at various times (pre- and

post-challenge) by LoMatAb was observed for both the AttHRV/VLP and VLP vaccines.

However, there was evidence for suppression of systemic antibody responses by low titer

MatAb in both the AttHRV/VLP and VLP vaccine regimens, especially systemic IgA

ASC (for the AttHRV/VLP vaccine), IgA memory B cells in peripheral blood and serum

IgA antibody responses (for both vaccines). The low titer MatAb also caused transient

suppression of the pre-challenge IgG ASC responses in all tissues of the LoMatAb-VLP

group.

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Our current and previous studies have documented the differential effects of

MatAb at different titers on both effector and memory B cell responses to RV vaccines in different tissues. Our findings may at least partially explain the variable efficacies observed in vaccine trials between developing (high titer MatAb) and developed (low titer MatAb) countries. Our study contributes toward an understanding of the mechanism of interference by different levels of MatAb on different types of vaccines, both replicating and non-replicating, facilitating the design of more effective vaccines to

overcome the suppressive effects of MatAb in the different targeted populations.

4.6. ACKNOWLEDGMENTS

We thank Dr. Juliette Hanson, Rich McCormick, Marcela Azevedo and Severin

Pouly for technical assistance. We thank Dr. Viviana Parreno (Instituto de Virologia,

CICU y A, INTA, Castelar, Bs.As, Argentina) for helpful comments. We also thank

Hong Liu for help in statistical analysis.

This work was supported by grants from the National Institutes of Health

(RO1AI33561 and RO1AI37111). Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development

Center, The Ohio State University.

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327

Virus sheddingc Diarrhea Protection rates (%)h

Group a Mean Average peak Mean Vaccine regimen b % Mean days % with Mean days Viral n duration titer shed duration Diarrhea sheddingd to onset e diarrhead to onsete shedding dayse (FFU/ml)f dayseg 1 LoMatAb-AttHRV/VLP 6 50A 4.5A 0.5B 4.8x103 33A 0.8A 3.3AB 67 50 2 AttHRV/VLP 7 29A 5.1A 0.4B 6.25x102 29A 0.6AB 4.3A 71 71 B 3 LoMatAb-VLP 7 100 1B 3A 4.36x103 100B 1.6ABC 1.4B 0 0 B 4 VLP 5 100 1.6B 2A 8x104 100B 2.4BCD 1B 0 0 5 LoMatAb-ISCOM 6 100B 1B 3.2A 1.7x104 100B 3.6CD 0.6B 0 0 6 ISCOM 7 100B 1.2B 2.6A 2x104 100B 2.8D 1.5B 0 0

32 8

Table 4.1: Rotavirus shedding and diarrhea in gnotobiotic pigs after challenge with VirHRV

a - Abbreviation LoMatAb; low titer maternal antibody b - Number of pigs challenged with VirHRV per group. Part of the data in the AttHRV/VLP, VLP and ISCOM groups were from pigs done in study by Gonzalez et al 2004 (17) and Chapter 3. c - Determined by ELISA and cell culture immunofluorescence infectivity assay. d - Proportions in the same column, with different superscript letters differ significantly (Fisher's exact test); e - Number in the same column with different superscript letters differ significantly (survival analysis) f - FFU, fluorescent focus forming units. g - Duration of diarrhea determined by number of days with fecal scores greater or equal to 2: feces were scored as follows: 0=normal; 1=pasty; 2=semiliquid; 3=liquid h - Protection rate = [1-(percentage of vaccinated pigs in each group with diarrhea/percentage of control pigs with diarrhea)] x100 328 VN IgM A # 10000 100000 A # AB # ABC # 10000 BC # 1000 A A C # 1000 AB 100 A B B 100 B A 10 C 10 B C D 1 1 B 0 28/0 35/7 028/035/7 PID/PCD PID/PCD

IgA 100000 IgG 100000 A # A # A AB # 10000 10000 B B A BC 1000 A 1000 C C 100 B # 100

Geometric mean antibody titers A 10 10 B BC D B D 1 B C 1 0 28/0 35/7 0 28/0 35/7 PID/PCD PID/PCD LoMatAb-AttHRV/VLP AttHRV/VLP LoMatAb-VLP VLP LoMatAb-ISCOM ISCOM

Figure 4.1: Virus neutralizing (VN) and isotype specific geometric mean antibody titers in serum of pigs vaccinated and challenged in the presence (solid lines) or absence (dashed lines) of LoMatAb. Low titer maternal serum was given IP to pigs at birth in the LoMatAb groups IP. Pigs were vaccinated at PID 0, 10, 21 and challenged at PID28/PCD0. Approximately half of the pigs were euthanized pre-challenge at PID28 and the rest of each group were euthanized post-challenge at PID35/PCD7. Blood samples were collected at the first inoculation and at euthanasia. Different letters A- D indicate significant differences among vaccine and control groups with or without LoMatAb for the same time point (one-way ANOVA, followed by Duncan's multiple- range test on log10 transformed titers). The “#” denotes significant difference between antibody responses pre-challenge (PID28/PCD0) and post-challenge (PID35/PCD7) for the same isotype in the same vaccine or control group with or without LoMatAb (one- way ANOVA, followed by Duncan's multiple-range test on log10 transformed titers).

329 SIC-IgM NoMatAb 10000 10000 LoMatAb ## 1000 1000 * 100 100 10 ** 10 1 1 0.1 0.110 10 ** 0.01 0.01100 100 0.0011000 # 0.0011000 0.000110000 # 0.000110000 Att H R V/VL P VLP I SC O M AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7

10000 SIC-IgA 10000 # * # 1000 * 1000 * * 100 100

10 10 1 1

0.110 0.110

0.01100 0.01100 AttHRV/VLP VLP ISCOM Att H R V/VL P VL P IS C O M PID28/PCD0 PID35/PCD7 SIC-IgG 10000 10000

1000 1000 # 100 100 # * Geometric mean antibody titers 10 10

1 1

0.110 0.110

0.01100 0.01 100 At tH R V/VLP VLP IS C O M AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7

Figure 4.2: Isotype specific geometric mean antibody titers in small intestinal contents of pigs following vaccination and challenged in the presence (solid bars) or absence (open bars) of LoMatAb. Maternal serum was given to pigs in MatAb groups IP at birth. Approximately half of the pigs were euthanized pre-challenge at PID28/PCD0 and the rest of each group were euthanized post-challenge at PID35/PCD7. The single asterisks denote significant differences between groups with LoMatAb and No MatAb receiving the same vaccine at the same time point (one-way ANOVA, followed by Duncan's multiple-range test on log10 transformed titers, p<0.05). The double asterisks denote significant differences between AttHRV/VLP and VLP vaccine groups with or without LoMatAb at the same time point (p<0.05). The “#” denotes significant difference between antibody responses pre-challenge (PID28/PCD0) and post challenge (PID35/PCD7) for the same isotype in the same vaccine or control group with or without LoMatAb (p<0.05).

330 Figure 4.3: IgM ASC responses to Wa HRV in pigs vaccinated in the presence (solid bars) or absence (open bars) of LoMatAb. The MNC from duodenum, ileum, spleen and

PBL of pigs were collected and assayed on PID 28 (pre-challenge) and PID35 (post- challenge). The data represent the mean number of ASC per 5x105 MNC. The single asterisks denote significant differences between groups with LoMatAb and no MatAb receiving the same vaccine at the same time point (Kruskal-Wallis rank sum test, p<0.05).

The double asterisks denote significant differences between AttHRV/VLP and VLP vaccine groups with or without LoMatAb at the same time point (Kruskal-Wallis rank sum test, p<0.05). The “#” denotes significant difference between ASC responses pre-challenge

(PID28/PCD0) and post challenge (PID35/PCD7) for the same isotype in the same vaccine or control group with or without LoMatAb (Kruskal-Wallis rank sum test, p<0.05).

331 Duodenum-IgM NoMatAb LoMatAb 50 ** 50 0 0

-5050 -5050 -100 100 -100100 -150150 -150150 * # -200200 -200200 # AttHRV/VLP VLP ISCOM AttHRV/VLP* VLP ISCOM PID28/PCD0 PID35/PCD7 Ileum-IgM

50 50 # 0 0 MNC 5 -5050 -5050 # -100 -100100 100 150 -150150 ** -150 -200200 -200200 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM PID35/PCD7 PID28/PCD0 Spleen-IgM

50 50

25 25 # ** #

0 0

-25 * 25 -2525 * -5050 -5050 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7 Mean numbers HRV-specific Mean ASC/5x10 numbers Peripheral blood -IgM 50 50

25 25 # 0 0

-2525 -2525

-5050 -5050 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7

Figure 4.3

332 Duodenum-IgA 200 NoMatAb 200 100 LoMatAb 100 0 0 -100100 -100100 * -200 200 -200 200 -300300 # -300300 -400400 # -400400 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7 Ileum-IgA 200 200

100 ** 100

0 0 MNC

5 -100100 -100100

-200 200 -200 200 * -300300 -300300 -400 400 -400400 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7 Spleen-IgA

100 100 50 50 * # * 0 0

-5050 -5050

-100100 -100100 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7 Mean numbers HRV-specific Mean ASC/5x10 numbers Peripheral blood-IgA 100 100

50 50 * # 0 0

50 -50 -5050

100 -100 -100100 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7

Figure 4.4: IgA ASC responses to Wa HRV in pigs vaccinated in the presence or absence of LoMatAb. For an explanation of symbols, see the legend to Figure 4.2. Note the change of vertical scale from Figure 4.3. 333 Duodenum-IgG 200 200 NoMatAb LoMatAb # 100 100 # # * * 0 0

-100100 ** -100100

200 -200200 -200200 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7 Ileum-IgG 200 200 #

100 100 # # * 0 0 *

MNC -100100 5 -100100

-200200200 -200200200 # AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7 Spleen-IgG 100 100 #

50 * 50 # 0 0 *

-5050 ** -5050 # -100100 -100100 # Att HR V/VLP VLP IS CO M AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7 Peripheral blood-IgG 100

Mean numbers HRV-specific Mean ASC/5x10 numbers 100

50 50 # * # * 0 * 0 # -5050 -5050

-100100 -100100 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM PID28/PCD0 PID35/PCD7

Figure 4.5: IgG ASC responses to Wa HRV in pigs vaccinated in the presence or absence of LoMatAb. For an explanation of symbols, see the legend to Figure 4.2. Note the change of vertical scale from Figure 4.3.

334 Ileum IgA IgG NoMatAb 100 100 LoMatAb

50 50

0 0

-5050 50 AttHRV/VLP VLP ISCOM -50 AttHRV/VLP VLP ISCOM MNC 5 Spleen IgA IgG 100 2000

1500

50 1000

500 0 0

-500500 -5050 -1000 AttHRV/VLP VLP ISCOM 1000 AttHRV/VLP VLP ISCOM Peripheral blood IgA IgG 150 2000 * 1500 100 1000

50 500 * 0 0 -500500 Mean numbers HRV-specific Mean numbers memory B cells /5x10

-5050 -10001000 AttHRV/VLP VLP ISCOM AttHRV/VLP VLP ISCOM

Figure 4.6: Memory B cell responses in pigs vaccinated with AttHRV/VLP, VLP vaccine regimens or ISCOM control in the presence and absence of LoMatAb at PID28/PCD0. The MNC from ileum, spleen and PBL of pigs were collected and assayed on PID 28 (pre- challenge) and stimulated with semi-purified Wa HRV. The data represent the mean numbers memory B cells per 5x105 MNC. The single asterisks denote significant differences between groups with LoMatAb and No MatAb receiving the same vaccine (Kruskal-Wallis rank sum test, p<0.05).

335

CHAPTER 5

CYTOKINES TRANSFERRED FROM MOTHER TO NEONATES IN SWINE:

IMPLICATIONS FOR IMMUNOMODULATION OF NEONATAL IMMUNITY

BY MATERNAL CYTOKINES

5.1. SUMMARY

The effects of maternally-acquired cytokines on development of neonatal immunity are

undefined. Swine provide a unique model to study lactogenic cytokines because our data

indicate most maternal cytokines are not acquired transplacentally by piglets. We

investigated IL-6 and TNF-α (pro-inflammatory), IFN-γ and IL-12, (Th1), IL-10 and IL-

4 (Th2) and TGF-β1 (Th3) cytokine concentrations in sow serum and colostrum/milk and in serum and intestinal contents of their suckling piglets at 0-13 post-partum days (PPD)

or post-weaning. All cytokines were detected in sow colostrum/milk, and except for

possibly mammary-derived TNF-α and TGF-β1, their concentrations correlated with

those in sow serum. No IL-6, TNF-α, IFN-γ, IL-4 and IL-10 were detected in piglet sera at PPD0 documenting absence of transplacental transfer, whereas IL-12 and TGF-β1

present at birth, may be constitutively produced or maternally-derived. The peak mean cytokine concentrations in piglet sera were detected at PPD1-2 (IL-4>TGF-β1>IL-6>IL-

336 12>IFN-γ>IL-10) with high concentrations of IL-4 and TGF-β1 likely contributing to the neonatal Th2 bias. Increased concentrations of intestinal IL-6 and IL-12 at PPD5-13 and of TNF-α at PPD2-3 were potentially induced by intestinal colonization with commensal bacteria. In weaned piglets, serum IL-6 and IL-12 concentrations were increased compared to suckling piglets, also suggesting acclimation to the post-suckling microbial flora. Low concentrations of IFN-γ in suckling and weaned piglets may be due to its down-regulation by TGF-β. In summary, we comprehensively documented the transfer of maternal cytokines from colostrum/milk to neonates and have provided new evidence for their potential role in the Th2 bias of neonatal immune responses.

5.2. INTRODUCTION

Neonates depend on transfer of immune factors from their mothers via the

placenta and/or breast feeding to be protected from pathogens until the maturation of

their immunity. The maternal immune factors transferred to the neonates include

antibodies, growth factors, cytokines etc. as well as lymphoid and non-lymphoid cells. A

few studies, mainly of humans, of the transfer of cytokines via the placenta and the

components of colostrum/milk have suggested possible roles for maternal cytokines such

as TGF-β, IL-1, IL-6, TNF-α, etc in immunologic protection of neonates and in

modulating the neonatal immune system development during colonization by commensal

bacteria (5) but most did not follow up on the cytokine profiles over time in their infants.

In mammals, three isoforms of TGF-β (β1, β2 and β3) have been identified of which

TGF-β1 is the most abundant form in tissues whereas TGF-β2 is more abundant in body

337 fluids (21). However, the TGF-β1 is of particular interest as it has been reported to play

an immunoregulatory role during pregnancy and at birth in humans as well as a role in

the Th2 bias of neonatal immune responses (14, 24). Both IL-10 and TGF-β1 were detected in the maternal and fetal circulation in humans (24). The latter cytokine could favor Th2 memory responses by suppression of memory Th1 cells in the fetus predisposing neonates to the observed Th2 bias (17, 31). Few studies have investigated the role of maternal cytokines in the development of the immune system of neonates. The

TGF-β1 supplied to the fetus by injection into the mother’s circulation during gestation or to the neonate via milk during suckling was shown to rescue TGF-β1 -/- newborn mice from severe cardiac abnormalities (16). In suckling rats, feeding of formula lacking

TGF-β2 led to inflammatory responses to food antigens including accumulation of IL-18 and recruitment of high numbers of activated dendritic cells, eosinophils and mast cells to the intestine (23). These inflammatory responses in suckling rat pups could be alleviated by addition of TGF-β2 to the feeding. The IL-10 is mainly produced by both Th2 and T regulatory type 1 (Tr1) CD4+ T cells and plays an important role in anti-inflammation and antibody production (9). The possible roles of other cytokines in the development and function of the neonatal immune system were suggested, but not previously analyzed or documented.

There is little information concerning the level and function of cytokines transferred from milk to suckling pigs or their impact on neonatal immune development of swine (30). Porcine milk contains abundant TGF-β, which has been suggested to play a role in regulating the intestinal immune system in neonatal pigs (33). Other cytokines 338 have not been demonstrated in porcine colostrum/milk. There is also a lack of information for humans and animals about the persistence of these maternal cytokines in the neonate after transfer by suckling.

The goals of this study were to investigate the transfer efficiency of various cytokines from the colostrum and milk of the sow to the serum and intestines of neonatal piglets, to determine the persistence of the passive cytokines in the intestine and serum of neonatal piglets, and to provide a basis for comparison of milk cytokine components between pigs and humans to establish their universal roles in neonatal immune development. The analysis of the cytokine components of porcine mammary secretions and their transfer and persistence in suckling and weaned neonates has important implications for immunomodulation of the neonatal immune system.

5.3. MATERIALS AND METHODS

5.3.1. Experimental design and sample collection

Near-term sows (n=5) were closely monitored and allowed to farrow naturally.

The animal use protocols employed in this study were reviewed and approved by the

Agricultural Animal Care and Use Committee, The Ohio State University. Blood was collected from the sows at post partum days (PPD) 2,7,11 and at 3 days pre-partum

(PPD-3) on average. Colostrum/milk samples were collected at days 0, 1, 2, 3, 5, 7, 9, 11 and 13 after farrowing. In the suckling experiments, piglets (n=47) were allowed to suckle the sows for 14 days. In the weaning experiment, a group of piglets (n=16) derived from a sow suckled the sow for the first two days. At PPD3, a subset of piglets (n=10)

339 were removed from the sow and weaned onto infant formula (Similac, Abbott

laboratories, Columbus, Ohio) from PPD3 to 13 (post weaning day, PWD 0 to 10),

whereas the rest of the litter remained on the sow. Piglets (n=20) derived by

hysterectomy from 4 sows were maintained in gnotobiotic and colostrum-deprived

conditions for up to 33 days (2).

Blood from piglets was collected at farrowing, presuckling (PPD0) and at PPD 1,

2, 3, 5, 7, 9, 11 and 13. For PPD 1-3, blood was collected from different piglets in both

suckling and weaning experiments on alternative days. Blood was also collected from

hysterectomy-derived gnotobiotic piglets through 33 days post-derivation (2). Serum was

collected and stored at -20oC until tested. In the suckling experiment, three to four piglets

were euthanized at each time point and intestinal contents were collected. Piglets derived

by hysterectomy and maintained in gnotobiotic and colostrum-deprived conditions were

also euthanized for the collection of intestinal contents at various time points. Intestinal

contents were diluted 1:2 (v/v for the small intestinal contents, SIC, and w/v for the large

intestinal contents, LIC) in phosphate buffered saline (PBS 0.5mM, pH 7.2) with 1% bovine serum albumin fraction V (BSA), with 250µg/ml trypsin inhibitor and 50ug/ml leupeptin (Sigma, St. Louis, Missouri) to inhibit proteolytic enzymes. The intestinal contents were then clarified and supernatants were stored frozen at -20oC. After

collection, colostrum/milk samples were immediately centrifuged at 1000xg for 30min to

remove cells and debris. Colostrum/milk supernatants were collected and stored at -20oC.

340 5.3.2. Enzyme-linked immunosorbent assay (ELISA) for porcine cytokines.

Concentrations of IL-6 and TNF-α (pro-inflammatory), IFN-γ and IL-12 (Th1), IL-4 and

IL-10 (Th2) and TGF-β1 (Th3) were measured using capture sandwich ELISA following procedures developed in our laboratory (2). Briefly, 96-well microtitre plates (Nunc-

Maxisorp, Rochester, New York) were coated with purified goat (polyclonal) antibodies to porcine cytokines IL-4, IL-6 or IL-12 (R&D system, Minneapolis, Minnesota) or purified mouse (monoclonal) antibodies to porcine IL-10 or IFN-γ or to human TGF-β1

(Biosource, Camarillo, California) or human TNF-α (Endogen, Rockford, Illinois) at concentrations of 0.75-4.0µg/ml overnight at room temperature (RT). The purified antibodies to human TNF-α and TGF-β1 were indicated as cross-reactive with porcine

TNF-α and TGF-β1 respectively, by the manufacturers (Endogen and Biosource, respectively). The plates were then blocked for 2h at RT with 0.5% (w/v) BSA in PBS pH 7.2 (for IL-4, IL-6, IL-10, IL-12 and IFN-γ) or with PBS/0.5%BSA/1% sucrose for

TGF-β1 or with PBS/4%BSA/5% sucrose for TNF-α. Samples, negative control (buffer) and cytokine standards were added to the wells in duplicate in a volume of 50µl +50µl of

PBS/BSA1% (4% BSA for TNF-α) and incubated for 2h at RT. The cytokine standards were diluted 2-fold, from the initial concentration of 4000 pg/ml (for IL-4, IL-6, TNF-α and TGF-β1) and of 1000pg/ml (for IL-10, IL-12 and IFN-γ). The detection antibodies

(biotinylated goat antibodies against porcine IL-4, IL-6 or IL-12 or biotinylated mouse monoclonal antibodies against porcine IFN-γ, IL-10, human TNF-α or human TGF-β1) were then added at concentrations of 0.2-0.75ug/ml, followed by streptavidin-HRP conjugate (Biosource, 0.1µg/ml or Endogen 1:400 for TNF-α). Tetramethylbenzidine

341 (TMB) and H2O2 (KPL, Gaithersburg, Maryland, USA) were used as substrates.

Reactions were stopped by 1.2N H2SO4 within 5-30 min depending on the cytokines and

the absorbance at 450nm (A450) was read with an ELISA reader.

The A450 was corrected for background by subtraction of the absorbances of the

buffer wells from those of the sample wells. The detection concentration for the TNF-α and TGF-β1 assay was 15.6pg/ml. The detection concentrations for the other cytokines were 7.8pg/ml. Samples below these detection concentrations (7.8pg/ml or 15.6pg/ml) were assigned a concentration of 4 or 8 pg/ml, respectively for calculation of the mean and for statistical analysis.

The ELISA for total (latent and free) TGF-β1 was performed after acid treatment of the samples (29). Briefly, colostrum and milk samples were diluted 1:2 with PBS/ 1%(w/v) BSA, then treated with 10µl of 1.2N HCL/100µl milk supernatant for 15 min at RT, followed by neutralization with 25µl 0.5M HEPES (N-2-Hydroxyethylpiperazine- N´-2-Ethane Sulfonic Acid)/0.72N NaOH. Both untreated and acid treated colostrum/milk samples were tested on the same ELISA plates. The latent TGF-β1 concentration was calculated by subtracting the free form (untreated samples) from the total amount (treated samples), including adjustment of the reading from treated samples with a factor of 1.35x to account for the dilution by acid treatment.

Analysis of cytokine concentration data. Standard curves for each cytokine were generated on a 4-parameter plot for each assay, and the cytokine concentration in each serum sample was calculated from the corresponding curve fitting equation. Each sample was tested in duplicate, and the mean values were calculated and reported. The cytokine concentrations between different days in sows’ colostrum/milk samples and in piglets’ 342 sera and intestinal contents were compared by the Wilcoxon rank-sum test (SAS 9.1,

SAS institute, NC). The cytokine concentrations in serum and intestinal contents between

piglets derived by hysterectomy at PPD3-5 and by natural birth at PPD0 (presuckling)

were also compared (Wilcoxon rank-sum test). Significant differences were considered

as p<0.05 unless indicated. The cytokine concentrations between serum and

colostrum/milk of the same sow at PPD 2, 7 and 11 were compared using the binomial

proportion test (SAS 9.1, SAS Institute, NC). The mean cytokine concentrations in sera

and intestinal contents of piglets derived from the same sows and the cytokine concentrations in serum and colostrum/milk samples of the corresponding sows were evaluated for correlation using Spearman coefficient (r) with p values.

5.4. RESULTS

5.4.1. Various concentrations of pro-inflammatory (IL-6), Th1 (IFN-γ and IL-12), Th2

(IL-4 and IL-10) and Th3 (TGF-β1) cytokines were present in colostrum/milk and were

correlated with the concentrations in sow serum, except for much higher TGF-β1 in

colostrum/milk and a lack of TNF-α in serum

In general all cytokines exhibited a wide range of concentrations in colostrum

and/or milk among different sows, leading to an often wide standard error of the mean

(Figure 5.1). The peak concentrations of cytokines in colostrum/milk (IL-4>TGF-β1>

IL6=IFN-γ >IL-12>IL-10=TNF-α) were generally correlated with their respective

concentrations in serum except for the much higher TGF-β1 in colostrum/milk and the

absence of TNF-α in sow’s serum. The concentrations of all cytokines decreased at

343 different rates in the transition from colostrum to milk (PPD0-3 versus PPD3-13). The

IL-6, IL-12, IL-4 and TGF-β1 concentrations in colostrum at PPD0-2 were higher than

those in sow serum, suggesting local production of these cytokines in mammary glands or

selective transport of these cytokines from sow serum to colostrum.

In assessing pro-inflammatory cytokines, the mean IL-6 concentrations in colostrum/milk ranged from a high of 2.7 ng/ml at PPD0 to a low of 550 pg/ml at PPD7

(Figure 5.1A). At PPD7-13, the IL-6 concentrations were significantly lower than at

PPD0. The IL-6 concentrations in sow serum were generally lower than the concentrations in colostrum/early milk (PPD0 and 2) but higher than the concentrations in later milk (PPD7 and 11). The correlation between the serum and colostrum/milk IL-6 was moderate (r=0.68, p=0.008). Of interest, the concentration of IL-6 correlated with

the concentration of TGF-β1 in colostrum/milk (r=0.46, p=0.0043). The TNF-α was

only detectable in colostrum (PPD0-2), then was present in one sow at PPD3 and

decreased to undetectable from PPD5 (Figure 5.1B). The peak TNF-α concentration in

colostrum of different sows ranged from 24-500 pg/ml. No TNF-α was detected in sow

serum at any time point, suggesting the local production of this cytokine in mammary

gland. Thus a correlation could not be deduced between the concentration of TNF-α in

serum and colostrum/milk.

For the Th1 cytokines, the concentration of IFN-γ in colostrum (PPD0-1) was

higher, but not significantly higher than the concentration in later milk (PPD5-13) (Figure

5.1C). The IFN-γ concentration in sow serum was higher than the concentrations in

colostrum and milk throughout lactation (significantly higher at PPD 11). The

concentration of IL-12 in colostrum peaked at PPD0 and then decreased significantly 344 after PPD1 in the transition to milk (Figure 5.1D). Moderate correlations in the IFN-γ and

IL-12 concentrations were found between sows’ serum and colostrum/milk (r=0.63 and

0.69, respectively, p<0.05).

From the analysis of the Th2 cytokines, the mean concentrations of IL-10 in colostrum/milk were low, ranging from 157 pg/ml (PPD0) to 30 pg/ml (PPD13), and were 74- to 83-fold lower than the IL-4 concentrations (ranging from 11.5 ng/ml at PPD0 to 2.50 ng/ml at PPD13), (Figure 5.1E and 5.1F). The IL-4 had the highest peak mean concentrations among all the cytokines. The IL-4 concentrations in milk decreased significantly from PPD7-13 compared to PPD1. The IL-10 concentrations in colostrum/milk exhibited a moderate correlation with the serum concentrations (r=0.75, p=0.002), whereas the correlation between the serum and colostrum/milk IL-4 was lower

(r=0.52, p=0.056)

For the Th3 cytokine, TGF-β1, the mean concentrations in colostrum/milk ranged from 4.5 ng/ml at PPD0 to 2.3 ng/ml at PPD13 and they were significantly higher (3- to

8-fold) than those in sow serum (except for PPD11), suggesting local production of this cytokine in the mammary glands and secretion into colostrum/milk (Figure 5.1G). In further support of local production, there was no correlation between the TGF-β1 concentrations in colostrum/milk and in sow serum. The TGF-β1 concentration decreased significantly by PPD5 compared to PPD0.

5.4.2. The latent form of porcine TGF-β1 was gradually substituted by the free form in the transition of colostrum to milk

The dynamics of latent and free forms of TGF-β1 in colostrum/milk are depicted in Figure 5.2. The latent form was activated by acid treatment of the colostrum/milk 345 samples. The concentrations of both latent and free form were highest at PPD0 then decreased significantly from PPD5 through PPD11 and increased slightly at PPD13

(significantly increased for the free form). The concentration of latent form TGF-β1 was higher than that of the free form TGF-β1 during the colostrum period (PPD0-1). The concentration of latent form TGF-β1 then decreased significantly to lower concentrations than the free TGF-β1 in the transition of colostrum to milk.

5.4.3. The mean peak cytokine concentrations in piglet sera were detected at PPD1-2 and correlated with those in colostrum/milk (except TNF-α, TGF-β1 and IL-10)

All cytokines detected in the colostrum/milk were present in piglet sera except for

TNF-α (Figure 5.3). The mean peak cytokine concentrations (IL-4>TGF-β1≥IL6>IL-

12>IFN-γ>IL-10) in piglet sera were detected at PPD1-2, (time of gut closure) in low but

proportional concentrations to those in colostrum/milk (except for TGF-β1).

The pro-inflammatory cytokine IL-6 was undetectable at birth, peaked at PPD1-3,

and decreased significantly by PPD9 (Figure 5.3A). The estimated half life for IL-6 was

5-6 days. There was a moderate correlation between the IL-6 concentrations in piglet

serum and sow colostrum/milk (r=0.70; p<0.0001). In contrast, there was no detectable

TNF-α in piglet serum at all times, throughout the suckling period and presuckling

(Figure 5.3B), which coincide with the undetectable TNF-α concentration in piglet SIC in this period (Figure 5.5C).

Comparing Th1 cytokines, IFN-γ was undetectable at birth, peaked at PPD1-2, decreased from PPD3-9, and became significantly lower than the peak concentrations by

PPD5 and thereafter. The estimated half life of IFN-γ was about 2-4 days (Figure 5.3C).

346 Although serum IL-12 was present at birth, it increased significantly after suckling at

PPD1, then decreased from PPD1-5, and increased thereafter, indicating active

production (Figure 5.3D). Thus the half-life of IL-12 in piglets’ serum could not be

determined in this study. Moderate but significant correlations were found for IFN-γ and

IL-12 between piglets’ sera and colostrum/milk (r=0.53 and 0.54, respectively, p≤0.001).

For the Th2 cytokines, both IL-10 and IL-4 were undetectable at birth and

concentrations in piglets’ sera then peaked post-suckling at PPD1. The serum IL-10

concentration was low even at the PPD1 peak (mean of 56 pg/ml), and it quickly

decreased from PPD2 (Figure 5.3E). By PPD5, the concentration of IL-10 was

significantly lower than the concentration at PPD1. There was no correlation between the

IL-10 concentrations in the piglet’s sera and the sows’ colostrum/milk. In contrast, the

serum IL-4 concentration was high at PPD1 (mean of 4.5ng/ml), and decreased at a

slower rate than IL-10, indicative of a longer half life or active production (Figure 5.3F).

Only by PPD9 was the serum IL-4 concentration significantly lower than the peak concentration at PPD1. At PPD13, the IL-4 was still present at relatively high concentrations (mean=547pg/ml) in piglets’ sera. The estimated half life for IL-4 was 5-6 days. There was a strong correlation between the IL-4 concentrations in piglets’ sera and the sows’ colostrum/milk (r=0.87; p<0.0001).

For the Th3 cytokine, a substantial concentration of TGF-β1 was present (mean

=754 pg/ml) in sera of all piglets at birth (Figure 5.3G). Thus it was not possible to determine the half-life of TGF-β1 in piglets’ sera. The serum TGF-β1 concentration increased only ~2-fold from PPD1-13 compared to the concentration at birth, yet the 347 increase was significant at PPD5-13. There was no correlation between the TGF-β1 concentrations in piglets’ sera and the concentration of free form TGF-β1 in colostrum/milk.

5.4.4. The serum IL-12 present in presuckling piglets born by natural birth and in piglets derived by hysterectomy may be constitutively produced, whereas the significantly higher serum TGF-β1 in the former piglets may be both maternally- derived and constitutive.

Before suckling, IL-6, TNF-α, IFN-γ, IL-10 and IL-4 were not detectable in the sera of piglets born by natural birth, like the piglets derived by hysterectomy (2). The IL-

12 was present in the sera of piglets at low concentrations at birth after both methods of delivery, indicating that the IL-12 was actively produced by the piglets (Table 5.1). In addition, the serum IL-12 concentrations remained constant in hysterectomy-derived piglets maintained in the gnotobiotic condition throughout the 33 days tested (2), supporting the hypothesis that IL-12 is constitutively produced and may not be derived transplacentally. Similar concentrations of IL-12 were detected in sera of piglets derived by both methods (113 vs.84 pg/ml). Interestingly, however, at birth, an approximately 7- fold higher mean concentration of TGF-β1 was present in sera of piglets born naturally

(754 pg/ml) compared to piglets derived by hysterectomy (112pg/ml), suggesting that maternal TGF-β1 may be acquired by the piglet during the process of natural birth. Of interest, the serum TGF-β1 concentrations in hysterectomy-derived piglets maintained in the gnotobiotic condition remained constant throughout 33 days (2), suggesting that this cytokine is also constitutively produced. At PPD0, there were also low concentrations of 348 TGF-β1 in the SIC of piglets born naturally and by hysterectomy. They may be maternal

cytokines acquired by fetal piglets orally, possibly by ingestion of amniotic fluid in utero or they may be actively produced by piglets.

5.4.5. Increased serum IL-12 and IL-6 concentrations in weaned piglets at various times post-weaning (PWD2-10) compared to those in suckling piglets suggests acclimation to the altered commensal microflora post weaning

To study the persistence of passive cytokines in piglet serum, piglets from one sow were weaned at PPD3 and the serum cytokine concentrations at PPD5 (post weaning day, PWD2), PPD7-9 (PWD4-6) and PPD11-13 (PWD8-10) were measured (n=10)

(Figure 5.4). Suckling piglets from the same sow were included for comparison (n=6).

The serum cytokine concentrations in piglets weaned at PPD7 and 9 were combined and the concentrations in piglets weaned at PPD11 and 13 were combined because no significant differences were observed between the two time points.

From PPD5-13, concentrations of IL-10, IL-4 and TGF-β did not differ

significantly between suckling and weaned piglets. These results suggest that there was

no significant absorption of these cytokines from milk into piglet serum after gut closure

(PPD2). The TNF-α was not detectable in serum of weaned or suckling piglets at any

time (data not shown).

At PPD7-9 (PWD 4-6), the IL-12 concentration was significantly higher in the

weaned piglets compared to the suckling piglets. The IL-6 concentration was also higher,

but not significantly higher, in the weaned piglets at PPD5 (PWD2) and PPD11-13 (PWD

8-10), suggesting that weaning (acclimation to altered commensal microflora after loss of 349 sow’s milk diet) stimulates production of pro-Th1 (IL-12) and pro-inflammatory (IL-6)

cytokines. In contrast as the time post-weaning progressed, by PPD7-9 and PPD11-13

(PWD 4-6 and 8-10, respectively), the IFN-γ concentrations in the suckling piglets were

significantly higher than in the weaned piglets; the reason for this observation is unclear.

However the decreased IFN-γ concentrations in both suckling (Figure 5.3C) and weaned

(Figure 5.4C) piglets may be due to the high passively acquired or actively produced

TGF-β1 concentrations, respectively in these piglets, suggesting negative regulation by

TGF-β1 of IFN-γ, but not of IL-12 (see Discussion).

5.4.6. Although all cytokines exhibited low stability during transit in the intestinal

tract, active production of pro-inflammatory (IL-6 and TNF-α), Th1 (IL-12) and Th2

(IL-4) cytokines in the intestine were observed, likely stimulated by colonization of the intestine with commensal bacteria

Cytokine concentrations were measured in the small intestinal contents (SIC) of suckling piglets, which represented the initial site for maternal cytokine trancytoses from colostrum to serum prior to gut closure. The cytokine concentrations measured in the large intestinal contents (LIC) allowed assessment of the stability of the cytokines during transit in the intestinal tract.

Peak cytokine concentrations in SIC (IL-4>TGF-β1>IL-6>IL-12>IFN-γ>IL-10)

were detected at PPD1-2 (Figures 5.5-5.7) and generally coincided with their

corresponding peak concentrations in sow colostrum. The TNF-α was not detectable in

SIC of suckling piglets at any time. The positive correlations between the cytokines in

piglet SIC and sow colostrum/milk were significant, except for TNF-α, IFN-γ and IL-10. 350 For the pro-inflammatory cytokine IL-6, the concentrations were highest in both

LIC and SIC at PPD1-3, then decreased significantly to low concentrations from PPD 5-

11 in SIC and from PPD5-13 in LIC (Figure 5.5A and 5.5B). There was a significant

correlation between IL-6 concentrations in the SIC and in colostrum/milk (r=0.7, p=0.0003). In SIC, although IL-6 concentrations decreased significantly at PPD5, 9 and

11 compared to PPD1, there were gradual increases at PPD5-7 and 11-13, possibly related to bacterial colonization. No TNF-α was detectable in SIC of piglets in spite of the presence of TNF-α in sow colostrum, suggesting the instability of this cytokine in the piglet intestine (Figure 5.5C). However there was a transient increase in TNF-α concentrations in piglet LIC at PPD2 and 3, followed by a rapid decrease after PPD5

(Figure 5.5D), suggesting a rapid inflammatory response to colonization of the large intestine by the commensal flora.

The concentrations of the Th3 cytokine, TGF-β1, were highest in both SIC and

LIC at PPD2, with much higher concentrations in the SIC and they remained relatively high until PPD13 in the SIC; whereas TGF-β1 decreased significantly to low concentrations in LIC after PPD3 (Figure 5.5E and 5.5F). There was a significant correlation between TGF-β1 concentrations in the SIC and colostrum/milk (r=0.78, p<0.0001).

From analysis of the Th1 cytokines, the IFN-γ concentrations were higher in SIC than those in serum of piglets from PPD5-13 and remained relatively constant in SIC through PPD1-13, indicating the constant supply of maternal IFN-γ in piglets from the

suckling of colostrum/milk or active local production (Figure 5.6A). However, there was

351 no correlation between IFN-γ concentrations in piglet SIC with the concentrations in sow

colostrum/milk. The IFN-γ concentrations in LIC decreased by PPD7 from its peak titer at PPD3, indicating the instability of this cytokine in intestine (Figure 5.6B).

The concentrations of IL-12, another Th1 cytokine, in SIC and LIC were highest

at PPD1-2, corresponding to the highest IL-12 concentration in colostrum (Figure 5.6C

and 6D). Strong correlations (r=0.73-0.78; p≤0.0001) were found between the IL-12

concentrations in SIC and LIC with the IL-12 concentrations in the colostrum/milk. The

IL-12 concentrations in both SIC and LIC decreased by PPD5 compared to the peak

concentrations, also indicating low stability of IL-12 in the intestinal tract. The gradually

increasing concentrations of IL-12 in the serum and SIC during PPD5-13 suggest that the

IL-12 in piglet serum was actively produced by a systemic source and the later peak of

IL-12 in the SIC was actively produced in the gut, possibly stimulated by colonization of

the intestine with commensal bacteria.

For the Th2 cytokines, there were low and relatively constant concentrations of

IL-10 in SIC (Figure 5.7A) corresponding to the low and constant IL-10 concentrations in

sow colostrum/milk from PPD2-13. The IL-10 concentrations in the LIC were similar to

SIC at PPD1-3, but decreased significantly by PPD7 compared to the peak concentrations

(Figure 5.7B). The IL-4 concentrations in SIC and LIC were highest at PPD1-2 (Figure

5.7C and 5.7D). The IL-4 concentrations in SIC and LIC were moderately to highly

correlated with the concentrations in sow colostrum/milk; they were also highly

correlated with the IL-4 concentrations in the piglet serum (r=0.77 and 0.84 for SIC and

LIC, respectively, p<0.0001, data not shown). By PPD5, the IL-4 concentrations in both 352 SIC and LIC decreased significantly compared to the peak concentrations, and become

very low in the LIC. However, similar to IL-6, the IL-4 concentration in piglet SIC increased at PPD13, again suggestive of local active cytokine production.

5.5. DISCUSSION

In this study we determined the concentrations of various pro-inflammatory (IL-6

and TNF-α), Th1 (IFN-γ and IL-12), Th2 ( IL-4 and IL-10) and Th3 (TGF-β) cytokines

in sow serum and colostrum/milk We demonstrated the absorption of the cytokines from

colostrum/milk into the gut and serum of piglets, assessed the persistence (estimated half-

life) of most of the cytokines (IFN-γ, IL-10, IL-4 and IL-6) in the piglet serum and intestinal contents and demonstrated possible constitutive cytokine production (IL-12 and

TGF-β) in serum of presuckling piglets. Our study is unique because the limited number of studies of colostral cytokines in sows explored only growth factors including TGF-β

(12, 30, 33).

Various concentrations of pro-inflammatory, Th1, Th2 and Th3 cytokines were

present in colostrum/milk. The moderate correlations observed between sow blood and

colostrum/milk for all cytokines except TGF-β1 suggest that the cytokines are

transudated or transported from the circulation to the mammary secretions. The observed

higher TGF-β1 concentrations in colostrum/milk than in serum and a lack of correlation

between TGF-β1 concentrations in serum and colostrum/milk of sows suggests that local

production of TGF-β1 occurs in the mammary glands. The IL-6, IL-12 and IL-4 might

also be locally produced in the mammary gland. The TNF-α was only detectable in

colostrum and early milk but not in the sow serum, confirming the local production of

353 this cytokine (11, 25). The local production of these cytokines can be determined by analyzing the cytokine producing cells in colostrum and milk which is the subject of another ongoing study in our laboratory. Only limited information is available on the concentrations of cytokines and the cytokine producing cells in porcine colostrum and milk (30). In human breast milk, cells expressed transcripts for TGF-β (1 and 2) and IL-6 and production of these cytokines by mammary epithelial cells was detected (22, 26).

Interferon-γ is synthesized by T cells from human milk in vitro (3). Human milk contains high concentrations of TGF-β, TNF-α, IL-1, IL-6, IL-8 and IFN-γ (10). In sows, in addition to TGF-β1, IL-6 and IFN-γ, we also found a high concentration of IL-4, a moderate concentration of IL-12 and a low concentration of TNF-α and IL-10 in the colostrum and milk. Further studies are required to identify the cytokine production profiles of sow colostrum and milk cells and to determine the contribution of the cytokines in serum to those appearing in mammary secretions.

Our findings agree with a previous study of sows that the concentration of the latent form of TGF-β was high only in the colostrum and the concentration of this form of TGF-β declined rapidly and was partially substituted by the free form TGF-β (33). In addition, the moderate correlation between the concentrations of TGF-β1 and IL-6 in colostrum/milk (r=0.46) agrees with the concept that TGF-β synergistically enhances the secretion of IL-6 by epithelial cells (19). The TGF-β1 down-regulates immune activation of intestinal epithelial cells and lamina propria immune cells (20). Thus the high concentrations of TGF-β1 in the colostrum/milk may play an important homeostatic role in the intestinal mucosa of neonates.

354 To our knowledge, our study is the first to assess the transfer of maternal

cytokines transplacentally and via milk to neonatal piglets. The transfer of molecules across the placenta can occur via normal diffusion or can be receptor-mediated.

Monomers such as glucose and amino acids are actively transported to the fetus as nutrients whereas the transfer of fatty acids was limited (27). It is well established that the sow placenta which consists of six tissue layers does not allow the transfer of immunoglobulins to the fetus (13). There are more limited studies on the transfer of other macromolecules such as polysaccharides, proteins, lipids and nucleic acids across

the sow placenta. In mice (placenta has only two tissue layers), there was no transfer of

IFNs (type not specified) from the mother to the fetus even when the concentration of

interferon injected into the mother was high (8). Our study showed there was no

detectable transplacental transfer of IL-6, TNF-α, IFN-γ, IL-4 and IL-10 to the porcine

fetus. Thus the placenta, either hemochorial (humans, mice), syndesmochorial

(ruminants) or epitheliochoral (pigs) shows a high degree of selection in transferring

macromolecules to the fetus, especially immunoregulatory cytokines.

We compared cytokine transfer in piglets after natural birth (equivalent to

vaginally derived human infants) with those born by hysterectomy (equivalent to

cesarean-delivered human infants) (2) because differences in cytokine profiles were

observed in human cord blood between infants born by these two methods. The

concentrations of IFN-γ, TNF-α, IL-1β and IL-6 in human umbilical cord blood were

significantly higher in cases of vaginal delivery than in cases of cesarean section (18). In

addition, cord blood mononuclear cells derived from vaginally delivered infants

responded to mitogens and produced higher concentrations of IFN-γ and IL-12 than those 355 from cesarean-derived infants (6). Elevation of TGF-β concentrations in maternal and

fetal circulation from pregnancy to birth have been reported in humans (24). In our study

and the previous study by Azevedo and Saif (2), only TGF-β1 and IL-12 were present in the presuckling piglets born naturally and derived by hysterectomy. Furthermore, the IL-

12 concentrations in piglets born by both methods were similar whereas TGF-β1 was almost 7-fold higher in the piglets born naturally compared to piglets derived by hysterectomy. Yet the TGF-β1 concentration in serum of hysterectomy-derived piglets remained constant during the 33 days of study (2). Consequently, the TGF-β1 in the sera of these presuckling piglets is likely both maternally-derived and constitutively produced.

However, it is unclear how this elevation of TGF-β1 occurs in piglets during natural birth; it may be due to ingestion of the amniotic fluids, because low concentrations of

TGF-β1 were also detected in the SIC of those piglets. The IL-12 in the presuckling piglets’ sera was likely constitutively produced because the concentrations of this cytokine did not decrease in piglets’ sera even after weaning and were constant in serum of hysterectomy-derived piglets maintained for 33 days in gnotobiotic conditions (2).

Interleukin-10 was present at low concentrations in colostrum/milk and in the piglets’ serum; however it remained at a relatively constant concentrations in the piglets’ serum and SIC. The reported IL-10 concentrations in the colostrum and milk and serum of mothers and newborn infants were also low (4, 26), agreeing with the observations in piglets. However, the IL-10 concentrations reported in human milk in one study were much higher (~3300pg/ml, range 66-9301) (10). It is unknown whether in the pigs and in the studies of humans, the low IL-10 concentrations are sufficient to provide the 356 important immunoregulatory effects (anti-inflammation), or if it is possible that the

regulatory function of IL-10 was superseded by the high concentration of TGF-β, which did not decrease in the piglets’ serum through PPD13.

Regarding the profile of passive cytokines in the fetus, piglets exhibit some similarities and differences with humans. In humans, TGF-β1 and IL-10, but not IL-12 can be detected in human umbilical cord blood (24). Low concentrations of IL-6

(7.7±3.8 pg/ml), TNF-α (6.2 ± 2.3 pg/ml) and IFN-γ (0.2-10.46 pg/ml) were detected in human umbilical cord during vaginal delivery; these cytokine concentrations were at the detection limit or lower in our assays for hysterectomy-derived and naturally-derived piglets (18). It has also been shown that IL-4, but not IFN-γ and IL-12 are constitutively produced by mitogen stimulated cord blood mononuclear cells (6). Thus based on these studies, the known similarities between pigs and humans reside in the presence of TGF-

β1 and the low/undetectable concentrations of IL-6, TNF-α and IFN-γ in the fetal pigs and human cord blood. The source of TGF-β1 in the porcine fetus is unclear. In humans,

TGF-β1 is present at the maternal-fetal interface including the placenta. There was also no correlation between maternal and fetal TGF-β1 concentrations in humans suggesting either restriction of TGF-β1 passage across the placenta or polarized secretion by the placenta on the fetal side (24).

The presence of TGF-β1 in fetal pigs may play an important role in the development of the neonatal immune system before and after birth. The TGF-β1 in the circulation of the fetus and newborns could also explain the Th2 biased immune

357 responses in human neonates (17, 24). In addition, the presence of TGF-β1 inhibits inflammatory responses due to activated macrophages, thus playing an important immunoregulatory role against inflammation and allergies.

The highest absorption of cytokines from colostrum into the piglets’ circulation

(except for TNF-α) occurred at PPD1-2, coinciding with the gut closure period. The high cytokine concentrations in piglets’ sera can be attributed to the absorption of cytokines and possibly the colostral cells transferred to the piglet across the intestinal epithelium with the latter demonstrated in neonatal piglets (15, 28, 32). Twenty-four hours post colostrum feeding, maternal lymphocytes were found in liver, lung, spleen, lymph nodes and the gastrointestinal tract of piglets (32). The piglets were estimated to ingest an average of 500-700 million colostral cells/day (15). The sow colostral lymphocytes account for 10-25% of the total cells, 70-90% of which are T cells which could account for the presence of IFN-γ, IL-10, IL-4 and TGF-β1. The IL-4 and IL-6 showed the highest correlations between the cytokine concentrations in piglet serum with the concentrations in colostrum/milk (r=0.70-0.87; p<0.0001), indicating that the maternal cytokines are the main source of these cytokines in the neonatal suckling piglets. The epithelial cells which comprise 20% of the sow colostral cells, together with the lymphocytes and macrophages might explain the high TGF-β1 concentrations present in piglet sera which increased not only throughout the suckling period in suckling piglets but also in the weaning piglets (30). However, the piglet serum TGF-β1 did not correlate with the concentrations in colostrum/milk, suggesting that colostrum/milk was not the sole source of piglet TGF-β1.

358 The colostral cytokines that were absorbed and that persisted in the piglets’ serum

during suckling and weaning might have important implications for immunomodulation

of the neonatal immune system. Beginning from PPD3, the IFN-γ and IL-10 cytokine

concentrations in suckling piglets’ sera decreased gradually whereas the concentrations in

SIC remained relatively constant indicating that absorption of these two cytokines was

minimal at this stage. It is noteworthy that IL-4 remained elevated in piglets’ sera at high

concentrations through PPD13 (547 pg/ml). Interleukin 4 can inhibit B cell proliferation

and differentiation in piglets (M.Murtaugh, 2005, personal communication), which may contribute to the weaker B cell immune responses induced in neonates compared to adults. In addition, IL-4 is an important Th2 cytokine that antagonizes Th1 related IFN-γ production, and likely contributes to the Th2 biased immune responses in neonatal pigs, similar to those observed in human infants and neonatal mice (1, 7). The presence of high concentrations of TGF-β1 in piglets’ sera throughout the suckling and weaning periods may contribute to the reduced responsiveness of neonates. Concurrently, the maternal IFN-γ concentrations in piglets’ sera decreased significantly from PPD5, in both weaned and suckling piglets, which may further contribute to the Th2 bias in neonates.

The reason for the lower serum IFN-γ concentrations, but not the other Th1 cytokine, IL-

12, in weaned piglets compared to concentrations in suckling piglets is unclear.

However, it is possible that the increased serum TGF-β1 in both suckling and weaned piglets from PPD1-13 may contribute to the reduction in serum IFN-γ concentrations. It has been shown in mice that TGF-β1 negatively regulates Th1 differentiation through the regulation of IFN-γ production by natural killer (NK) cells (14). Blockade of TGF-β

359 signaling in NK cells caused the accumulation of NK cells secreting IFN-γ, responsible

for Th1 immune responses and protection from Leishmania infection. In contrast, blockade of TGF-β signaling did not affect the IL-12 production in the same study. Thus the TGF-β may negatively regulate the IFN-γ but not the IL-12 production.

Apart from being a Th1 cytokine, the IL-12 also functions in innate immunity and is produced during the early innate immune response. The observed increase of serum

IL-12 production stimulated by weaning (acclimation to altered microflora) suggests the development of innate immune responses in these neonatal weaned piglets. The IL-6 concentrations in the serum of weaned piglets also tended to be higher than in the suckling piglets, suggesting that weaning stimulated production of both pro-Th1 (IL-12) and pro-inflammatory (IL-6) cytokines likely in response to a newly acquired commensal microflora after loss of the milk diet. In suckling piglets, the serum IL-6 concentrations decreased 11-fold by PPD13 compared to peak concentrations. Because IL-6 functions in induction of pro-inflammatory responses, this decreased concentration of IL-6 may be important to avoid unnecessary hypersensitivity or allergic reactions in neonates.

All cytokines exhibited low stability in the LIC, including IL-6, IFN-γ, IL-12, IL-

4 and TGF-β. Although there was a constant supply of cytokines from milk and the concentrations of these cytokines in SIC were high and constant throughout the suckling period, high concentrations of these cytokines were present in the LIC only in the first 3 days of suckling and decreased significantly after PPD5. The TNF-α was unstable even in the SIC. Incubation of porcine recombinant cytokines (IFN-γ, IL-10, IL-4 and TNF-α) at 37oC with intestinal contents from gnotobiotic piglets led to loss of ≥90% of initial

cytokine concentrations (2). However in that study, IL-12 was the most stable cytokine 360 with a 90% recovery rate. In our study the gut of conventional piglets may contain

higher concentrations of proteolytic enzymes than the gut of the gnotobiotic piglets,

which would accelerate the degradation of all cytokines including IL-12 during passage

in the large intestine. Yet increased concentrations of IL-6 and IL-12 (from PID5-13) and

IL-4 (PID13), occurred in SIC probably due to colonization of the gut by commensal

bacteria. The time course for the increased concentrations of these 3 cytokines was later

than that of TNF-α in LIC (PPD2 and 3), consistent with the acute phase responses for

TNF-α.

In summary, this is the first study to comprehensively assess the various cytokines present in sow colostrum and milk and to document the transfer of maternal cytokines from colostrum/milk of mothers to neonates. Knowledge of the transfer of cytokines from

sow colostrum/milk to their suckling offspring and the persistence of cytokines in the

offspring provides a basis for future studies of the influence of maternally-derived

cytokines on development of the neonatal immune system. The findings also allow a

comparison of cytokine components in milk between pigs and humans to facilitate a

better understanding of species similarities and differences using pigs as models for

human diseases.

5.6. ACKNOWLEDGMENTS

We thank Dr J.Hanson, Greg Myers, Todd Roots, Richard McCommick, Peggy

Lewis, Terry Meek, Marcela Azevedo and Stacie Shafer for their technical assistance.

We also thank Hong Liu for help with statistical analysis. This work was supported by a

361 grant from the National Institutes of Health, NIAID (RO1AI37111-05). Salaries and

research support were provided by state and federal funds appropriated to the Ohio

Agricultural Research and Development Center, The Ohio State University.

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364

Delivery method Samples N IL-12 TGF-β1

Hysterectomya Serum 20 84c ±17 112c ±18

Natural birthb Serum 12 113±7 754±83*d

Hysterectomya SIC 4 4±0 15±4

Natural birthb SIC 3 7±3 50±21

Table 5.1: Concentrations (pg/ml) of IL-12 and TGF-β1 in serum and small intestinal contents (SIC) of neonatal piglets derived by hysterectomy or natural birth a Data summarized from Azevedo and Saif (2). Serum and SIC samples were collected from piglets from 4 different sows, at 3-5 days after piglet derivation by hysterectomy and piglets were maintained under gnotobiotic and colostrum-deprived conditions

b Serum and SIC samples were collected from piglets before suckling born naturally from 3 different sows

c Mean cytokine concentration ± standard error of the mean. The concentrations of IL-6, TNF-α, IFN-γ, IL-10 and IL-4 in serum and SIC of piglets derived by hysterectomy (at 3- 5 days after derivation) or by natural birth (before suckling) are not shown because they were below the detection concentrations (4-8pg/ml).

d Single asterisk denotes significant difference in the concentration of TGF-β1 between piglets derived by hysterectomy and piglets born naturally (Wilcoxin rank-sum test, p<0.0001).

365 Figure 5.1: Cytokine concentrations in colostrum/milk and the relationship with the cytokine concentrations in sows‘serum samples.

Spearman correlation coefficients were calculated with p values. Open bars represent the cytokine concentrations in colostrum/milk; triangles represent the cytokine concentrations in sows’ sera. The “#” indicates significant difference in cytokine concentrations in sows’ milk at different days compared to the colostrum concentration at

PPD1 (Wilcoxon rank-sum test, p<0.05). Single asterisks indicate significant difference in the cytokine concentrations between the sow serum and colostrum/milk at the same time point (binomial proportion test, p<0.05). Note the differences in vertical scales among the different cytokines.

366 α IL-6 B TNF- 4000 A 1000 Col/milk

Sow serum 800 3000

r=0.68 600 2000 (p=0.008) pg/ml pg/ml 400 # # 1000 # # 200

0 D 0 PPD -301235791113 PPD -301235791113

IL-12 IFN-γ 1000 6000 C # r=0.63 800 r=0.69 (p=0.007) (p=0.017) # 4000 600 # # # # # pg/ml

pg/ml 400 2000 * 200

0 0 PPD -301235791113 PPD -3 0 1 2 3 5 7 9 11 13

F 1000 E IL-10 12000 IL-4

10000 800 r=0.75 r=0.52 (p=0.002) 8000 (p=0.056) 600 6000 pg/ml

pg/ml # 400 ### 4000

200 2000

0 0 PPD -3 0 1 2 3 5 7 9 11 13 PPD -301235791113

6000 G TGF-β1 r=0.3 * (p=0.296) 4000 #* # pg/ml #

2000 #

0 PPD -301235791113

Figure 5.1

367 12000

10000

free 8000 latent

6000 pg/ml *

4000 * # * 2000 # * # #

0 PPD01235791113

Figure 5.2: Different forms of TGF-β1 in sows’colostrum/milk.

Solid bars represent the free TGF-β1, open bars represent the latent form. Asterisks indicate significant difference in free TGF-β1 concentrations in sows’ milk at different days compared to the colostrum concentration at PPD 1 (Wilcoxon rank-sum test, p<0.05). The “#” indicates significant difference in latent TGF-β1 concentrations in sows’ milk at different days compared to the colostrum concentration at PPD1

(Wilcoxon rank-sum test, p<0.05)

368 Figure 5.3: Cytokines in sera of suckling piglets and correlations with the concentrations in sows’ colostrum/milk.

Spearman correlation coefficients were calculated with p values. Open bars represent the cytokine concentrations piglets’ sera; lines represent the cytokine concentrations in sows’ colostrum/milk; closed triangles represent the cytokine concentrations in sows’ sera. The

“#” indicates significant difference in cytokine concentrations in piglets’ sera at different days compared to the concentration at PPD1 (IL-10) and PPD 1 and 2 for other cytokines

(Wilcoxon rank-sum test, p<0.05). Asterisks indicate significant difference in TGF-β and

IL-12 concentrations in piglets’ sera at different days compared to presuckling concentration (PPD0). Secondary axis was used to depict IFN-γ concentrations in sows’ serum and colostrum/milk samples. Note the difference in vertical scales between cytokines. Samples below detection concentrations of the ELISA were assigned a concentration of 4 pg/ml (IL-6, IFN-γ, IL-12, IL-4 and IL-10) or 8 pg/ml (TNF-α and TGF-

β).

369 TNF-α A B 4000 IL-6 200 pig serum Col/milk r=0.70 Sow serum 3000 (p<0.0001) 150

2000 100 pg/ml # pg/ml

1000 50 # # # 0 0 PPD -301235791113 PPD -301235791113

1000 C IFN-γ 5000 1000 IL-12 D

800 4000 800 r=0.53 r=0.54 (p=0.001) (p=0.0008) 600 3000 600 * pg/ml * * * 400 2000 400 * *

Pig IFN-g (pg/ml) IFN-g Pig * # Sow IFN-g (pg/ml) * 200 # # # 1000 200 # # # 0 0 0 PPD -3 0 1 2 3 5 7 9 11 13 PPD -301235791113

F 200 12000 IL-4 E IL-10 10000 r=0.87 150 (p<0.0001) r=0.1 8000 (p=0.58) 100 6000 pg/ml pg/ml 4000 # 50 2000 # # # # # # # # # 0 0 PPD -301235791113 PPD -301235791113

TGF-β1 G 5000 r=0.24 (p=0.15) 4000

3000

pg/ml # 2000 # # * * * * * 1000 #

0 PPD -301235791113 Figure 5.3 370 IL-6 B TGF-β 1000 A 1200

800 1000

800 600 600

pg/ml 400 pg/ml 400 200 200

0 0 PPD 357-911-13 PPD 3 5 7- 9 11- 13 PWD 0 2 4-6 8-10 PWD 0 2 4-6 8-10

10 0 0 C IFN-γ 1000 D IL-12 800 800

600 600 pg/ml 400 pg/ml 400 *

200 * * 200 0 0 PPD 357-911-13 PPD 3 5 7-9 11-13 PWD 0 2 4-6 8-10 PWD 0 2 4-6 8-10

10 0 E IL-10 1000 F IL-4

80 800

60 600

pg/ml 400 pg/ml 40

200 20

0 0 PPD 357-911-13 PPD 357-911-13 PWD 0 2 4-6 8-10 PWD 0 2 4-6 8-10

Figure 5.4: Comparison of cytokine concentrations in sera of weaned and suckling piglets.

The piglets were weaned at PPD3. Open bars represent mean serum cytokine concentrations in suckling piglets. Striped bars represent mean serum cytokine concentrations in weaned piglets. Asterisks represent the significant difference in cytokine concentrations of weaned piglets compared to the concentrations in suckling piglets of the same day (Wilcoxon rank-sum test, p<0.05). Note the difference in vertical scales between cytokines. The X-axis indicates the PPD and the respective PWD.

371 Figure 5.5: The concentrations of IL-6 and TNF-α (pro-inflammatory cytokines) and

TGF-β (Th3 cytokine) in the intestinal contents and serum of suckling piglets.

Correlations between cytokine concentrations in intestinal contents of piglets with the concentrations in colostrum/milk were calculated using Spearman correlation coefficients with p values (only significant correlations with p<0.05 were included). Striped bars represent the cytokine concentrations in piglets’ intestinal content (SIC or LIC), open bars represent the cytokine concentrations in piglets’ serum; lines represent the cytokine concentrations in sows’ colostrum/milk. The “#” indicates significant difference in cytokine concentrations in piglets’ intestinal contents at different days compared to the concentration at PPD1 and 2 (Wilcoxon rank-sum test, p<0.05). Note the difference in vertical scales between cytokines. Samples below detection concentrations of the ELISA were assigned a concentration of 4 pg/ml (IL-6) or 8 pg/ml (TNF-α and TGF-β).

372 IL-6

Pig LIC Pig SIC 4000 4000 Pig serum A Pig serum B Col/milk Col/milk

3000 3000 r=0.7 (p=0.0003)

2000 2000 pg/ml pg/ml

1000 1000 # # # # # # # # # 0 # 0 PPD01235791113 PPD0 1 2 3 5 7 9 11 13 TNF-α # 300 C Pig SIC 300 D Pig LIC Pig serum Pig serum Col/milk 250 Col/milk 250

200 200

150 150 pg/ml pg/ml

100 100

50 50

0 0 PPD01235791113 PPD01235791113

TGF-β1 Pig LIC 5000 E Pig SIC 5000 F Pig serum Pig serum Col/milk Col/milk 4000 4000 r=0.78 (p<0.0001) 3000 3000 pg/ml pg/ml 2000 2000

1000 1000 # # # # # # # 0 0 PPD01235791113 PPD0 1 2 3 5 7 9 11 13

Figure 5.5

373 IFN-γ IFN-γ A 1000 5000 1000 Pig SIC 5000 B Pig LIC Pig serum Pig serum 800 4000 800 Col/milk 4000 Col/milk

600 3000 600 3000

400 2000 400 2000 Pig IFN-g (pg/ml) IFN-g Pig Sow IFN-g (pg/ml) Sow IFN-g Pig IFN-gPig (pg/ml) 200 1000 (pg/ml) IFN-g Sow 200 1000

# 0 # 0 0 0 PPD01235791113 PPD01235791113

C IL-12 D IL-12

1000 1000 Pig SIC Pig LIC Pig serum Pig serum Col/milk 800 800 Col/milk r=0.73 r=0.78 600 (p=0.0001) 600 (p<0.0001) pg/ml 400 pg/ml 400 # # 200 200 # # ### # # # 0 0 PPD01235791113 PPD0 1 2 3 5 7 9 11 13

Figure 5.6: The concentrations of Th1 cytokines (IFN-γ and IL-12) in the intestinal contents and serum of suckling piglets.

See Figure 5.5 for explanations of symbols. Secondary axis was used to depict IFN-γ concentrations in sows’ colostrum/milk samples. Samples below detection concentrations of the ELISA were assigned a concentration of 4 pg/ml.

374 IL-10 A B IL-10 200 Pig SIC 200 Pig LIC Pig serum Pig serum Col/milk Col/milk 150 150

100 100 pg/ml pg/ml

50 50 ## # # # 0 0 # PPD01235791113 PPD01235791113

C IL-4 D IL-4 12000 12000 Pig SIC Pig LIC Pig serum Pig serum 10000 Col/milk 10000 Col/milk r=0.69 8000 8000 r=0.82 (p=0.0004) (p<0.0001) 6000 6000 pg/ml pg/ml 4000 4000 #

2000 2000 # # # # # # # # # 0 0 PPD01235791113 PPD01235791113

Figure 5.7: The concentrations of Th2 cytokines (IL-10 and IL-4) in the intestinal contents and serum of suckling piglets.

See Figure 5.5 for explanations of symbols. The “#” indicates significant difference in cytokine concentrations in piglets’ intestinal contents at different days compared to the concentration at PPD1 (IL-10) and PPD1 and 2 (IL-4) (Wilcoxon rank-sum test, p<0.05).

Samples below detection concentrations of the ELISA were assigned a concentration of 4 pg/ml.

375

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