IMMUNODOMINANCE AND IMMUNOPROTECTION OF ANTI-VIRAL SPECIFIC CD8+ T CELL RESPONSE DURING HBV INFECTION TAN ANTHONY TANOTO (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (YONG LOO LIN SCHOOL OF MEDICINE) DEPARTMENT OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2012 i ACKNOWLEDGEMENTS Firstly, I would like to thank Dr. Antonio Bertoletti, my supervisor, for his constant guidance and patience throughout the entire duration of the project. More importantly, his trust in my decisions has allowed me to mature in thought and as a scientist. For that I am extremely grateful. I would also like to thank Dr Adam Gehring for his input in the project and the initial laboratory training that he has given several years back, which forms the foundation for the skills I have learned through the years; Mr Ho Zi Zong and Miss Adeline Chia for their numerous assistance; and all the members (both past and present) of the laboratory who have contributed in one way or another. Special thanks also go out to Kelly for her support and understanding as I pursue my PhD degree. ii TABLE OF CONTENTS Content Acknowledgements . Page i Summary iii List of Tables vi List of Figures vii List of Abbreviations viii 1. Background . 1.1. Hepatitis B virus (HBV) . 1.2. HBV Genotypes 1.3. HBV-specific Immune Response 1.3.1. Latent Phase . 1.3.2. Viral Replication Phase 1.3.3. Adaptive Immunity Phase 1.4. Current Work 1.5. Tables and Figures 10 2. Chapter – Immunodominance of HBV-specific T cells 12 2.1. Introduction . 13 2.2. Materials and Methods 16 2.3. Results . 23 2.4. Discussion . 32 2.5. Tables and Figures 37 2.6. Supplementary Tables and Figures . 46 iii 3. Chapter – Immunoprotection of HBV-specific T cells during hepatic flares 48 3.1. Introduction . 49 3.2. Materials and Methods 51 3.3. Results . 56 3.4. Discussion . 64 3.5. Tables and Figures 70 3.6. Supplementary Tables and Figures . 80 4. Concluding Remarks . 83 References . 85 Appendix . 94 Relevant Publications  Tan, A. T., E. Loggi, C. Boni, A. Chia, A. J. Gehring, K. S. Sastry, V. Goh, P. Fisicaro, P. Andreone, C. Brander, S. G. Lim, C. Ferrari, F. Bihl, and A. Bertoletti. 2008. Host ethnicity and virus genotype shape the hepatitis B virus-specific T-cell repertoire. J Virol 82:10986-10997.  Tan, A. T., S. Koh, W. Goh, H. Y. Zhe, A. J. Gehring, S. G. Lim, and A. Bertoletti. 2010. A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B. J Hepatol 52:330-339.  Tan, A. T., S. Koh, V. Goh, and A. Bertoletti. 2008. Understanding the immunopathogenesis of chronic hepatitis B virus: an Asian prospective. J Gastroenterol Hepatol 23:833-843.  A. Bertoletti, Tan A. T. and A. J. Gehring. 2009. HBV‐specific adaptive immunity. Viruses 1:91‐103. iv  Watanabe, T., A. Bertoletti, and T. A. Tanoto. 2010. PD-1/PD-L1 pathway and T-cell exhaustion in chronic hepatitis virus infection. Journal of viral hepatitis 17:453-458. Other Publications  Gehring, A. J., Z. Z. Ho, A. T. Tan, M. O. Aung, K. H. Lee, K. C. Tan, S. G. Lim, and A. Bertoletti. 2009. Profile of tumor antigen-specific CD8 T cells in patients with hepatitis B virus-related hepatocellular carcinoma. Gastroenterology 137:682-690.  Sandalova, E., D. Laccabue, C. Boni, A. T. Tan, K. Fink, E. E. Ooi, R. Chua, B. Shafaeddin Schreve, C. Ferrari, and A. Bertoletti. 2010. Contribution of herpesvirus specific CD8 T cells to anti-viral T cell response in humans. PLoS Pathog 6(8): e1001051. v SUMMARY The Hepatitis B virus (HBV) is a non-cytopathic hepatotropic DNA virus, with a host range limited to humans and chimpanzees. Despite the availability of a prophylactic vaccine, approximately 350 million individuals are chronically infected worldwide and it is one of the leading causes of hepatocellular carcinoma (HCC). Depletion of T cells in experimentally infected chimpanzees and the longitudinal analysis of T cell responses in both acute and chronic patients have shown the crucial importance of T cells in the control and clearance of HBV infection. This thesis will focus on two aspects of T cell responses during HBV infection, namely: 1) the factors that influence the immunodominant hierarchy of HBV-specific T cells and 2) immunoprotection of HBV-specific T cells during hepatic flares (HF). Chapter – Immunodominance of HBV-specific T cells Repertoire composition, quantity and qualitative functional ability are the parameters that define virus specific T cell responses and are linked with their potential to control infection. By taking advantage of the segregation of different HBV genotypes in geographically and genetically distinct host populations, we were able to directly analyze the impact that host and virus variables exert on these virus-specific T cells parameters. T cell responses against the entire HBV proteome was analyzed in a total of 109 HBV infected Chinese or Caucasian subjects. We demonstrate that HBV-specific T cell quantity is determined by the virological and clinical profile of the patients, which outweighs any influence of race or viral diversity. In contrast, HBVspecific T cell repertoire is divergent in the two ethnic groups with T cell vi epitopes frequently found in Caucasian patients seldom detected in Chinese patients, demonstrating the ability of HLA micro-polymorphisms to diversify the T cell response. In conclusion, we provide a direct biological evaluation of the impact that host and virus variables exert on the immunodominance of virusspecific T cell response. Chapter – Immunoprotection of HBV-specific T cells during hepatic flares The pathogenesis of HF in patients chronically infected with HBV is controversial. Since HBV is not a directly cytopathic virus, an increase in virusspecific T cell response has been conventionally thought to occur during HF, even though experimental evidence to support such a scenario is scarce. Therefore, we studied the kinetics of innate and adaptive immune activation during HF in chronic hepatitis B to answer the following questions of immunoprotection: a) Is the HBV replication rebound that precedes HF associated with activation of innate or adaptive immunological events?; b) Are HF associated with the recovery of HBV-specific immunity? We analyzed longitudinally soluble (IFN-α, IL-1β, TNF-α, IL-6, IL-8, IL-10, CCL-2, CCL-3, CXCL-9, CXCL-10) and cellular (HBV-specific T, NK and T-regulatory cells) immunological parameters in patients (n=5) who developed HF after anti-viral therapy withdrawal, and cross-sectionally in chronic (n=29) and acute hepatitis B patients (n=5). A progressive increase of HBV replication precedes HF but occurs without detection of innate immune activation, with the exception of increased serum CXCL-8. Despite the absence of increased circulatory HBVspecific T or activated NK cells, HF were temporally associated with high serum levels of IFN-γ inducible chemokines CXCL-9 and CXCL-10 (but not CCL-2 or vii CCL-3). CXCL-9 and CXCL-10 also displayed different in vitro requirements for activation and are differentially produced in liver injury present in acute or chronic patients. In conclusion, we demonstrate that both HBV replication rebound and HF were not associated with a recovery of peripheral HBV-specific T cell immunity and confirm that CXCL-9 and CXCL10 are major mediators of liver inflammation. Their differential expression in acute versus chronic patients also suggests the presence of different mechanisms that govern liver injury during acute and chronic hepatitis B. viii LIST OF TABLES Tables Page Chapter Table 1. Tabulated summary of CD8+ T cell responses against known HLA-A2 restricted epitopes in HLA-A2+ Chinese and Caucasian patients 37 Chapter Table 1. Longitudinal clinical data of patients who withdrew from Remofovir treatment 70 ix LIST OF FIGURES Figures Page Background Figure 1. Geographical distribution of HBV genotypes. . 10 Figure 2. Immunological progression of HBV infection . 11 Chapter Figure 1. Ex vivo quantitative profile of HBV-specific T cells in Chinese and Caucasian HBV patients 38 Figure 2. Quantification of HBV-specific T cells after in vitro expansion . 39 Figure 3. Quantification of IFN-γ production in acute and chronic Chinese patients . 40 Figure 4. Induction of CD8+ T cell response against known A2restricted epitopes in HLA-A2+ Oriental and Caucasian patients . 41 Figure 5. Hierarchy of HBV-specific CD8+ T cell response in a HLAA0206 and a HLA-A0203 Chinese acute HBV patient . 42 Figure 6. Functional presentation of Core18-27 by HLA-subtypes common in the Chinese population 43 Figure 7. Time course analysis of Core18-27 epitope presentation by HLA-A2 subtypes 44 Figure 8. Influence of amino acid variations within different genotypes in T cell recognition . 45 Chapter – Immunoprotection of HBV-specific T cells (Supplementary Table 1) 80 SUPPLEMENTARY TABLES AND FIGURES Antiviral withdrawal n =5 Spontane ous n = 10 Me an ALT (IU/ml) 369 845.5 Me an C XC L-9 C once ntration (pg/ml) 2543 179.4 Me an C XC L-10 C once ntration (pg/ml) 2458 202.4 Supplementary Table 1. Comparison of serum ALT, CXCL-9 and CXCL-10 levels in patients with spontaneously occurring and antiviral withdrawal induced hepatic flares. The mean values of each parameter in the respective patient groups are shown. Chapter – Immunoprotection of HBV-specific T cells (Supplementary Figure 1) 81 Supplementary Figure 1. Slight elevation of serum CCL-2 during hepatic flares. Serum cytokine concentrations at the different time points were quantified by the cytometric bead array system and the results were grouped into the corresponding disease phases. Each dot represents a single time point of the longitudinal measurements from chronic patients at a total of 41 time points. The mean cytokine concentrations at each disease phase are indicated. Differences between groups were tested using non-parametric Mann-Whitney test and only P-values below 0.05 are shown. Chapter – Immunoprotection of HBV-specific T cells (Supplementary Figure 2) 82 Supplementary Figure 2. CXCL-9 and CXCL-10 profile of in vitro cytokine stimulated HepG2 cells. HepG2 cells were cultured in the presence of various combinations of IFN-γ, IFN-α and TNF-α. The concentration of CXCL-9 and CXCL-10 in the supernatants was quantified after 24 hours. Dark and light bars represent the mean concentration of CXCL-9 and CXCL-10 from experiments. Concluding Remarks 83 CONCLUDING REMARKS The immunology of HBV infection is a complex issue with many still unanswered aspects that require extensive investigation. This thesis fills up these gaps in the understanding of the virus-specific T cell responses and pathogenesis by providing; 1) the first comparative study between two HBV infected patient populations; and 2) clinical evidence of the pathogenetic mechanisms of HBV-associated liver injury. We showed that the dichotomy of HBV-specific T cell responses is a universal phenomenon observed in HBV patients infected with different HBV genotypes and of different genetic backgrounds. On the other hand, the repertoire of the T cell response appears to be altered by variations in both the viral proteome sequence and the HLA haplotypes, suggesting that the analysis of virus-specific T cell responses have to be tailored to the specific patient population. These findings clearly have important implications for the protective role of the immune response against HBV. However, though it is intuitive to consider the HBV immune response as a solely protective mechanism against the virus, the non-cytopathic nature of HBV and the occurrence of liver inflammation during the natural history of HBV infection automatically places the HBV immune response at the other end of the spectrum where it could also mediate hepatic damage. Delineating the mechanisms of liver damage during HBV infection then becomes a topic of interest. Using a cohort of patients where hepatic injury and viral replication were uniquely separated, we demonstrate that the HBV-specific T cell response Concluding Remarks 84 is an unlikely factor responsible for the direct damage of the liver, and that hepatic injury during HBV reactivation is likely to be mediated by the hepatic secretion of chemokines CXCL-9 and CXCL-10. Common to both studies, these analyses were primarily focused on the peripheral compartment. Even though such work can provide very important insights into the immunology of HBV, the hepatotropism of the virus could result in distinct immunological characteristics specific only to the liver and not in the periphery. Future studies would have to attempt to move the analysis into the physiologically relevant liver compartment, a task both daunting and exciting at the same time. References 85 REFERENCES 1. Ganem, D. & Prince, A.M. Hepatitis B virus infection--natural history and clinical consequences. N Engl J Med 350, 1118-1129 (2004). 2. Lok, A.S.F. & McMahon, B.J. Chronic hepatitis B. Hepatology 45, 507-539 (2007). 3. WHO World Health Organization Weekly Epidemiological Record. 79, 253-264 (2004). 4. Mast, E.E. et al. A comprehensive immunization strategy to eliminate transmission of hepatitis B virus infection in the United States: recommendations of the Advisory Committee on Immunization Practices (ACIP) part 1: immunization of infants, children, and adolescents. MMWR Recomm Rep 54, 1-31 (2005). 5. McQuillan, G.M. et al. Prevalence of hepatitis B virus infection in the United States: the National Health and Nutrition Examination Surveys, 1976 through 1994. Am J Public Health 89, 14-18 (1999). 6. Lok, A. Chronic hepatitis B. Hepatology 34, 1225-1241 (2001). 7. Bosch, F.X., Ribes, J., Cleries, R. & Diaz, M. Epidemiology of hepatocellular carcinoma. Clin Liver Dis 9, 191-211, v (2005). 8. Mohanty, S.R., Kupfer, S.S. & Khiani, V. Treatment of chronic hepatitis B. Nat Clin Pract Gastroenterol Hepatol 3, 446-458 (2006). 9. Beck, J. & Nassal, M. Hepatitis B virus replication. World J Gastroenterol 13, 4864 (2007). 10. Locarnini, S. Molecular virology of hepatitis B virus. Semin Liver Dis 24 Suppl 1, 3-10 (2004). 11. Pawlotsky, J.M. Virology of hepatitis B and C viruses and antiviral targets. J Hepatol 44, S10-13 (2006). 12. Orito, E. et al. Host-independent evolution and a genetic classification of the hepadnavirus family based on nucleotide sequences. Proc Natl Acad Sci U S A 86, 7059-7062 (1989). 13. Steinhauer, D.A. & Holland, J.J. Direct method for quantitation of extreme polymerase error frequencies at selected single base sites in viral RNA. J Virol 57, 219-228 (1986). 14. Kramvis, A., Kew, M. & Francois, G. Hepatitis B virus genotypes. Vaccine 23, 2409-2423 (2005). 15. Fung, S.K. & Lok, A.S.F. Hepatitis B virus genotypes: Do they play a role in the outcome of HBV infection? Hepatology 40, 790-792 (2004). 16. Kidd-Ljunggren, K., Miyakawa, Y. & Kidd, A.H. Genetic variability in hepatitis B viruses. J Gen Virol 83, 1267-1280 (2002). References 86 17. Orito, E. et al. A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV Genotype Research Group. Hepatology 33, 218-223 (2001). 18. Sumi, H. et al. Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease. Hepatology 37, 19-26 (2003). 19. Wai, C.T., Chu, C.J., Hussain, M. & Lok, A.S. HBV genotype B is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C. Hepatology 36, 1425-1430 (2002). 20. Bertoletti, A. & Gehring, A.J. The immune response during hepatitis B virus infection. J Gen Virol 87, 1439-1449 (2006). 21. Thimme, R. et al. CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol 77, 68-76 (2003). 22. Wieland, S., Thimme, R., Purcell, R.H. & Chisari, F.V. Genomic analysis of the host response to hepatitis B virus infection. Proc Natl Acad Sci U S A 101, 66696674 (2004). 23. Visvanathan, K. et al. Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology 45, 102-110 (2007). 24. Menne, S. et al. Deficiencies in the acute-phase cell-mediated immune response to viral antigens are associated with development of chronic woodchuck hepatitis virus infection following neonatal inoculation. J Virol 76, 1769-1780 (2002). 25. Nakamura, I. et al. Pathogenesis of experimental neonatal woodchuck hepatitis virus infection: chronicity as an outcome of infection is associated with a diminished acute hepatitis that is temporally deficient for the expression of interferon gamma and tumor necrosis factor-alpha messenger RNAs. Hepatology 33, 439-447 (2001). 26. Asabe, S. et al. The Size of the Viral Inoculum Contributes to the Outcome of Hepatitis B Virus Infection. J Virol 83, 9652-9662 (2009). 27. Ferrari, C. et al. Cellular immune response to hepatitis B virus-encoded antigens in acute and chronic hepatitis B virus infection. J Immunol 145, 3442-3449 (1990). 28. Maini, M.K. et al. Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T cells associated with the control of infection. Gastroenterology 117, 1386-1396 (1999). 29. Penna, A. et al. Predominant T-helper cytokine profile of hepatitis B virus nucleocapsid-specific T cells in acute self-limited hepatitis B. Hepatology 25, 1022-1027 (1997). 30. Tan, A.T. et al. Host ethnicity and virus genotype shape the hepatitis B virusspecific T-cell repertoire. J Virol 82, 10986-10997 (2008). References 87 31. Webster, G.J.M. et al. Longitudinal Analysis of CD8+ T Cells Specific for Structural and Nonstructural Hepatitis B Virus Proteins in Patients with Chronic Hepatitis B: Implications for Immunotherapy. J Virol 78, 5707-5719 (2004). 32. Webster, G.J. et al. Incubation phase of acute hepatitis B in man: dynamic of cellular immune mechanisms. Hepatology 32, 1117-1124 (2000). 33. Boni, C. et al. Characterization of Hepatitis B Virus (HBV)-Specific T-Cell Dysfunction in Chronic HBV Infection. J Virol 81, 4215-4225 (2007). 34. Fisicaro, P. et al. Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B. Gastroenterology 138, 682-693, 693 e681-684 (2010). 35. Chisari, F.V. & Ferrari, C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 13, 29-60 (1995). 36. Alberti, A., Diana, S., Sculard, G.H., Eddleston, A.L. & Williams, R. Detection of a new antibody system reacting with Dane particles in hepatitis B virus infection. Br Med J 2, 1056-1058 (1978). 37. Grady, G.F. et al. Hepatitis B immune globulin for accidental exposures among medical personnel: final report of a multicenter controlled trial. J Infect Dis 138, 625-638 (1978). 38. Urbani, S. et al. Acute phase HBV-specific T cell responses associated with HBV persistence after HBV/HCV coinfection. Hepatology 41, 826-831 (2005). 39. Kalams, S.A. & Walker, B.D. The critical need for CD4 help in maintaining effective cytotoxic T lymphocyte responses. J Exp Med 188, 2199-2204 (1998). 40. Ciurea, A., Hunziker, L., Klenerman, P., Hengartner, H. & Zinkernagel, R.M. Impairment of CD4(+) T cell responses during chronic virus infection prevents neutralizing antibody responses against virus escape mutants. J Exp Med 193, 297305 (2001). 41. Chen, M.T. et al. A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci U S A 101, 1491314918 (2004). 42. Wieland, S.F. & Chisari, F.V. Stealth and Cunning: Hepatitis B and Hepatitis C Viruses. J Virol 79, 9369-9380 (2005). 43. Reignat, S. et al. Escaping high viral load exhaustion: CD8 cells with altered tetramer binding in chronic hepatitis B virus infection. J Exp Med 195, 1089-1101 (2002). 44. Stoop, J.N. et al. Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infection. Hepatology 41, 771-778 (2005). References 88 45. Franzese, O. et al. Modulation of the CD8+-T-Cell Response by CD4+ CD25+ Regulatory T Cells in Patients with Hepatitis B Virus Infection. J Virol 79, 33223328 (2005). 46. Wang, F.S. et al. Dysfunction of peripheral blood dendritic cells from patients with chronic hepatitis B virus infection. World J Gastroenterol 7, 537-541 (2001). 47. Beckebaum, S. et al. Hepatitis B virus-induced defect of monocyte-derived dendritic cells leads to impaired T helper type response in vitro: mechanisms for viral immune escape. Immunology 109, 487-495 (2003). 48. Lohr, H.F. et al. Reduced virus specific T helper cell induction by autologous dendritic cells in patients with chronic hepatitis B - restoration by exogenous interleukin-12. Clin Exp Immunol 130, 107-114 (2002). 49. van der Molen, R.G. et al. Functional impairment of myeloid and plasmacytoid dendritic cells of patients with chronic hepatitis B. Hepatology 40, 738-746 (2004). 50. Gehring, A.J. et al. The Level of Viral Antigen Presented by Hepatocytes Influences CD8 T-Cell Function. J Virol 81, 2940-2949 (2007). 51. Merican, I. et al. Chronic hepatitis B virus infection in Asian countries. J Gastroenterol Hepatol 15, 1356-1361 (2000). 52. Tan, A.T. et al. A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B. J Hepatol 52, 330-339 (2010). 53. Yewdell, J.W. Confronting complexity: real-world immunodominance in antiviral CD8+ T cell responses. Immunity 25, 533-543 (2006). 54. Moore, C.B. et al. Evidence of HIV-1 adaptation to HLA-restricted immune responses at a population level. Science 296, 1439-1443 (2002). 55. de Campos-Lima, P.O. et al. HLA-A11 epitope loss isolates of Epstein-Barr virus from a highly A11+ population. Science 260, 98-100 (1993). 56. Gaudieri, S. et al. Evidence of Viral Adaptation to HLA Class I-Restricted Immune Pressure in Chronic Hepatitis C Virus Infection. J Virol 80, 11094-11104 (2006). 57. Frahm, N. et al. Consistent cytotoxic-T-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities. J Virol 78, 2187-2200 (2004). 58. Wentworth, P.A. et al. Identification of A2-restricted hepatitis C virus-specific cytotoxic T lymphocyte epitopes from conserved regions of the viral genome. Int Immunol 8, 651-659 (1996). 59. Bertoni, R. et al. Human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic T lymphocyte responses in patients with acute hepatitis. J Clin Invest 100, 503-513 (1997). 89 References 60. Thimme, R., Chang, K.M., Pemberton, J., Sette, A. & Chisari, F.V. Degenerate immunogenicity of an HLA-A2-restricted hepatitis B virus nucleocapsid cytotoxic T-lymphocyte epitope that is also presented by HLA-B51. J Virol 75, 3984-3987 (2001). 61. Sette, A. & Sidney, J. Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics 50, 201-212 (1999). 62. Frahm, N. et al. Extensive HLA class I allele promiscuity among viral CTL epitopes. European Journal of Immunology 37, 2419-2433 (2007). 63. Sidney, J., Peters, B., Frahm, N., Brander, C. & Sette, A. HLA class I supertypes: a revised and updated classification. BMC Immunology 9, (2008). 64. Bertoletti, A. et al. Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope. J Exp Med 180, 933-943 (1994). 65. Seifert, U. et al. Hepatitis C virus mutation affects proteasomal epitope processing. J Clin Invest 114, 250-259 (2004). 66. Le Gall, S., Stamegna, P. & Walker, B.D. Portable flanking sequences modulate CTL epitope processing. Journal of Clinical Investigation 117, 3563-3575 (2007). 67. Middleton, D., Menchaca, L., Rood, H. & Komerofsky, R. New allele frequency database: http://www.allelefrequencies.net. Tissue Antigens 61, 403-407 (2003). 68. Barouch, D. et al. HLA-A2 subtypes are functionally distinct in peptide binding and presentation. J Exp Med 182, 1847-1856 (1995). 69. Sudo, T. et al. Differences in MHC class I self peptide repertoires among HLA-A2 subtypes. J Immunol 155, 4749-4756 (1995). 70. Tynan, F.E. et al. The immunogenicity of a viral cytotoxic T cell epitope is controlled by its MHC-bound conformation. J Exp Med 202, 1249-1260 (2005). 71. Chang, J.J. et al. Reduced Hepatitis B Virus (HBV)-Specific CD4+ T-Cell Responses in Human Immunodeficiency Virus Type 1-HBV-Coinfected Individuals Receiving HBV-Active Antiretroviral Therapy. J Virol 79, 3038-3051 (2005). 72. Tsai, S.L. et al. Purification and characterization of a naturally processed hepatitis B virus peptide recognized by CD8+ cytotoxic T lymphocytes. J Clin Invest 97, 577-584 (1996). 73. Penna, A. et al. Cytotoxic T lymphocytes recognize an HLA-A2-restricted epitope within the hepatitis B virus nucleocapsid antigen. J Exp Med 174, 1565-1570 (1991). References 90 74. Rehermann, B. et al. The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis. J Exp Med 181, 1047-1058 (1995). 75. Jung, M.C. et al. Virus-specific lymphokine production differs quantitatively but not qualitatively in acute and chronic hepatitis B infection. Virology 261, 165-172 (1999). 76. Sobao, Y. et al. Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*2402, the most common HLA class I allele in East Asia. J Hepatol 34, 922-929 (2001). 77. Hwang, Y.K. et al. HLA-A2 restricted peptides from the HBx antigen induce specific CTL responses in vitro and in vivo. Vaccine 20, 3770-3777 (2002). 78. Krausa, P. & Browning, M.J. HLA-A2 polymorphism and immune functions. Eur J Immunogenet 23, 261-274 (1996). 79. Lindh, M., Gonzalez, J.E., Norkrans, G. & Horal, P. Genotyping of hepatitis B virus by restriction pattern analysis of a pre-S amplicon. J Virol Methods 72, 163174 (1998). 80. Addo, M.M. et al. Comprehensive epitope analysis of human immunodeficiency virus type (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load. J Virol 77, 2081-2092 (2003). 81. Chen, L. et al. B7-H1 up-regulation on myeloid dendritic cells significantly suppresses T cell immune function in patients with chronic hepatitis B. J Immunol 178, 6634-6641 (2007). 82. Hesse, M.D., Karulin, A.Y., Boehm, B.O., Lehmann, P.V. & Tary-Lehmann, M. A T cell clone's avidity is a function of its activation state. J Immunol 167, 1353-1361 (2001). 83. Bertoletti, A. et al. HLA class I-restricted human cytotoxic T cells recognize endogenously synthesized hepatitis B virus nucleocapsid antigen. Proc Natl Acad Sci U S A 88, 10445-10449 (1991). 84. Nayersina, R. et al. HLA A2 restricted cytotoxic T lymphocyte responses to multiple hepatitis B surface antigen epitopes during hepatitis B virus infection. J Immunol 150, 4659-4671 (1993). 85. Sette, A. et al. The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes. J Immunol 153, 5586-5592 (1994). 86. Stevens, C.E., Beasley, R.P., Tsui, J. & Lee, W.C. Vertical transmission of hepatitis B antigen in Taiwan. N Engl J Med 292, 771-774 (1975). References 91 87. Sugimoto, K. et al. Influence of ethnicity in the outcome of hepatitis C virus infection and cellular immune response. Hepatology 37, 590-599 (2003). 88. Riedl, P. et al. Distinct, cross-reactive epitope specificities of CD8 T cell responses are induced by natural hepatitis B surface antigen variants of different hepatitis B virus genotypes. J Immunol 176, 4003-4011 (2006). 89. Depla, E. et al. Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections. J Virol 82, 435-450 (2008). 90. Leslie, A. et al. Differential selection pressure exerted on HIV by CTL targeting identical epitopes but restricted by distinct HLA alleles from the same HLA supertype. J Immunol 177, 4699-4708 (2006). 91. Brander, C., Frahm, N. & Walker, B.D. The challenges of host and viral diversity in HIV vaccine design. Curr Opin Immunol 18, 430-437 (2006). 92. Dienstag, J.L. Hepatitis B virus infection. N Engl J Med 359, 1486-1500 (2008). 93. Perrillo, R.P. Acute flares in chronic hepatitis B: the natural and unnatural history of an immunologically mediated liver disease. Gastroenterology 120, 1009-1022 (2001). 94. Maruyama, T., Iino, S., Koike, K., Yasuda, K. & Milich, D.R. Serology of acute exacerbation in chronic hepatitis B virus infection. Gastroenterology 105, 11411151 (1993). 95. Tsai, S.L. et al. Acute exacerbations of chronic type B hepatitis are accompanied by increased T cell responses to hepatitis B core and e antigens. Implications for hepatitis B e antigen seroconversion. J Clin Invest 89, 87-96 (1992). 96. Maini, M.K. et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med 191, 1269-1280 (2000). 97. Kakimi, K. et al. Blocking chemokine responsive to gamma-2/interferon (IFN)gamma inducible protein and monokine induced by IFN-gamma activity in vivo reduces the pathogenetic but not the antiviral potential of hepatitis B virus-specific cytotoxic T lymphocytes. J Exp Med 194, 1755-1766 (2001). 98. Sitia, G. et al. MMPs are required for recruitment of antigen-nonspecific mononuclear cells into the liver by CTLs. J Clin Invest 113, 1158-1167 (2004). 99. Iannacone, M., Sitia, G., Ruggeri, Z.M. & Guidotti, L.G. HBV pathogenesis in animal models: recent advances on the role of platelets. J Hepatol 46, 719-726 (2007). 100. Dienstag, J.L. New antiviral agents for viral hepatitis: our cup runneth over but is only half full. Gastroenterology 117, 525 (1999). References 92 101. Khabar, K.S. et al. The alpha chemokine, interleukin 8, inhibits the antiviral action of interferon alpha. J Exp Med 186, 1077-1085 (1997). 102. Kakimi, K., Guidotti, L.G., Koezuka, Y. & Chisari, F.V. Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med 192, 921-930 (2000). 103. Dunn, C. et al. Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell-mediated liver damage. J Exp Med 204, 667-680 (2007). 104. Xu, D. et al. Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B. J Immunol 177, 739-747 (2006). 105. Borsellino, G. et al. Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression. Blood 110, 1225-1232 (2007). 106. Lang, K.S. et al. Immunoprivileged status of the liver is controlled by Toll-like receptor signaling. J Clin Invest 116, 2456-2463 (2006). 107. Sitia, G. et al. Depletion of neutrophils blocks the recruitment of antigennonspecific cells into the liver without affecting the antiviral activity of hepatitis B virus-specific cytotoxic T lymphocytes. Proc Natl Acad Sci U S A 99, 13717-13722 (2002). 108. Yang, P.L., Althage, A., Chung, J. & Chisari, F.V. Hydrodynamic injection of viral DNA: a mouse model of acute hepatitis B virus infection. Proc Natl Acad Sci U S A 99, 13825-13830 (2002). 109. Kakimi, K., Lane, T.E., Chisari, F.V. & Guidotti, L.G. Cutting edge: Inhibition of hepatitis B virus replication by activated NK T cells does not require inflammatory cell recruitment to the liver. J Immunol 167, 6701-6705 (2001). 110. Biron, C.A. Initial and innate responses to viral infections--pattern setting in immunity or disease. Curr Opin Microbiol 2, 374-381 (1999). 111. Fong, T.L. et al. High levels of viral replication during acute hepatitis B infection predict progression to chronicity. J Med Virol 43, 155-158 (1994). 112. Salazar-Mather, T.P., Hamilton, T.A. & Biron, C.A. A chemokine-to-cytokine-tochemokine cascade critical in antiviral defense. J Clin Invest 105, 985-993 (2000). 113. McCaffrey, A.P. et al. The host response to adenovirus, helper-dependent adenovirus, and adeno-associated virus in mouse liver. Mol Ther 16, 931-941 (2008). 114. Dunn, C. et al. Temporal Analysis of Early Immune Responses in Patients With Acute Hepatitis B Virus Infection. Gastroenterology 137, 1289-1300 (2009). References 93 115. Taub, D.D., Anver, M., Oppenheim, J.J., Longo, D.L. & Murphy, W.J. T lymphocyte recruitment by interleukin-8 (IL-8). IL-8-induced degranulation of neutrophils releases potent chemoattractants for human T lymphocytes both in vitro and in vivo. J Clin Invest 97, 1931-1941 (1996). 116. Taub, D.D., Longo, D.L. & Murphy, W.J. Human interferon-inducible protein-10 induces mononuclear cell infiltration in mice and promotes the migration of human T lymphocytes into the peripheral tissues and human peripheral blood lymphocytes-SCID mice. Blood 87, 1423-1431 (1996). 117. Shields, P.L. et al. Chemokine and chemokine receptor interactions provide a mechanism for selective T cell recruitment to specific liver compartments within hepatitis C-infected liver. J Immunol 163, 6236-6243 (1999). 118. Larrubia, J.R. et al. The role of CCR5/CXCR3 expressing CD8+ cells in liver damage and viral control during persistent hepatitis C virus infection. J Hepatol 47, 632-641 (2007). 119. Crane, M. et al. Immunopathogenesis of hepatic flare in HIV/hepatitis B virus (HBV)-coinfected individuals after the initiation of HBV-active antiretroviral therapy. J Infect Dis 199, 974-981 (2009). 120. Bone-Larson, C.L., Hogaboam, C.M., Evanhoff, H., Strieter, R.M. & Kunkel, S.L. IFN-gamma-inducible protein-10 (CXCL10) is hepatoprotective during acute liver injury through the induction of CXCR2 on hepatocytes. J Immunol 167, 7077-7083 (2001). 121. Hokeness, K.L. et al. CXCR3-dependent recruitment of antigen-specific T lymphocytes to the liver during murine cytomegalovirus infection. J Virol 81, 1241-1250 (2007). APPENDIX Appendix 95 APPENDIX Relevant Publications  Tan, A. T., E. Loggi, C. Boni, A. Chia, A. J. Gehring, K. S. Sastry, V. Goh, P. Fisicaro, P. Andreone, C. Brander, S. G. Lim, C. Ferrari, F. Bihl, and A. Bertoletti. 2008. Host ethnicity and virus genotype shape the hepatitis B virus-specific T-cell repertoire. J Virol 82:10986-10997.  Tan, A. T., S. Koh, W. Goh, H. Y. Zhe, A. J. Gehring, S. G. Lim, and A. Bertoletti. 2010. A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B. J Hepatol 52:330-339.  Tan, A. T., S. Koh, V. Goh, and A. Bertoletti. 2008. Understanding the immunopathogenesis of chronic hepatitis B virus: an Asian prospective. J Gastroenterol Hepatol 23:833-843.  A. Bertoletti, Tan A. T. and A. J. Gehring. 2009. HBV‐specific adaptive immunity. Viruses 1:91‐103.  Watanabe, T., A. Bertoletti, and T. A. Tanoto. 2010. PD-1/PD-L1 pathway and T-cell exhaustion in chronic hepatitis virus infection. Journal of viral hepatitis 17:453-458. Other Publications  Gehring, A. J., Z. Z. Ho, A. T. Tan, M. O. Aung, K. H. Lee, K. C. Tan, S. G. Lim, and A. Bertoletti. 2009. Profile of tumor antigen-specific CD8 T cells in patients with hepatitis B virus-related hepatocellular carcinoma. Gastroenterology 137:682-690.  Sandalova, E., D. Laccabue, C. Boni, A. T. Tan, K. Fink, E. E. Ooi, R. Chua, B. Shafaeddin Schreve, C. Ferrari, and A. Bertoletti. 2010. Contribution of herpesvirus specific CD8 T cells to anti-viral T cell response in humans. PLoS Pathog 6(8): e1001051. [...]... the surface of the infected cells Virally infected cells can produce thousands of potentially immunogenic peptides, but CD8+ T cells are usually directed against only a few peptides and CD8+ T cells specific for different viral determinants can possess different anti- viral activity 53 The information regarding virus -specific T cell repertoire and the potential antiviral efficacy of CD8+ T cells with... chronic HBV infection shows the presence of multi -specific CD8 T- cells with the absence of CD4 T- cells 38 This suggests that the absence of CD4 cytokine help prevented the proper maturation and subsequent functioning of the CD8 T- cells Clearly, these 7 Background studies support the necessity of a functional and coordinated CD8 and CD4 T cell response for HBV control and clearance In addition to their... discrete antiviral role, the humoral and cellular components of the adaptive immune system are also interconnected in a way that the failure of one of them clearly affects the expansion and protective efficacy of the other A lack of CD4 T cell help can impair CD8 T cell activity and antibody production 39 , while the inability to mount a virus -specific CD8 T cell response results in a level of circulating... effect on the eventual development of the adaptive immune response and the outcome of the infection 6 Background Adaptive Immunity Phase The important role of the adaptive immunity in determining the outcome of a HBV infection has been demonstrated through various studies, both in experimentally infected chimpanzees and in patients 21, 26-34 This antiviral ability requires both the humoral and cellular... differing antigen specificity is essential to understand viral pathogenesis and develop vaccines Such information is limited in the great majority of viral and bacterial infections due to cumbersome methods that are required for the detection and characterization of new MHCclass I restricted epitopes 53 In addition, the identification of the T cell repertoire against viruses infecting different ethnic... immediately after the rapid expansion of HBV is an important link between the innate immunity and the activation of adaptive immunity against HBV The same study by Wieland et al (2004) showed that chimpanzees with a typical acute HBV infection profile had a robust induction of T helper type 1 (Th1) genes including IFN-γ, tumour necrosis factor α (TNF-α) and RANTES immediately after the latent phase However,... lesser than 0.05 were considered statistically significant Chapter 1 – Immunodominance of HBV -specific T cells (Results) 23 RESULTS Comprehensive analysis of HBV -specific T cell responses in genotype B (HBVgenB) and genotype D (HBVgenD) HBV infected patients To identify similarities/differences in the breadth and magnitude of HBVspecific T cell responses between Caucasian and Chinese patients, HBV -specific. .. these two aspects Chapter 1 will discuss how host and virus variables influence the immunodominant hierarchy of the T cell response 30 More specifically, it describes the quantitative, functional and repertoire profile of HBV -specific T cells in a wide population of HBV infected Chinese and Caucasian patients This comparison demonstrates for the first time the similarities and differences in the HBV -specific. .. experimentally infected woodchucks that develop chronicity seem to lack the initial large production of Th1 cytokines 24, 25 In line with observations in the woodchuck model of HBV infection, the development of chronicity is clinically associated with the absence or mild symptoms of hepatitis, while patients who resolve the infection often experience acute hepatitis 20 This led to the suggestion that the... protein (containing the core antigen, HBcAg) and the three variants of the surface envelope proteins (containing the surface antigen, HBsAg) with the preS2 section, with both pre-S1 and pre-S2 sections or without both sections, the non-structural viral polymerase, the secretory form of the pre-core protein (containing the antigen, HBeAg) and the X protein 9, 10 The core and envelope proteins have a structural . qualitative functional ability are the parameters that define virus specific T cell responses and are linked with their potential to control infection. By taking advantage of the segregation of. direct biological evaluation of the impact that host and virus variables exert on the immunodominance of virus- specific T cell response. Chapter 2 – Immunoprotection of HBV -specific T cells during. control and clearance of HBV infection. This thesis will focus on two aspects of T cell responses during HBV infection, namely: 1) the factors that influence the immunodominant hierarchy of HBV- specific