Therapeutic Immunization in Chronic Hepatitis B: Preclinical - PowerPoint PPT Presentation

Therapeutic Immunization in Chronic Hepatitis B: Preclinical Studies in the woodchock Michael Roggendorf Institut für Virologie Universitätsklinkum Essen Bonn, 19.04.2007

Effect of CD8+ T cells on viral clearance HBV-infected chimpanzees Thimme, JVI (2003), 77(1), p.68-76

Control of virus infection by CTL 1,2 Step 1 Step 2 NON-CYTOPATHIC CYTOPATHIC CD8 CELL CD8 CELL CLEARANCE BY CYTOKINES CLEARANCE BY LYSIS IFN-gamma/TNF-alpha OF INFECTED CELLS 1) Transgenetic mouse expressing HBV proteins (E. Chisari, L.Giudotti, review 2001 2) Chimpanzee L. Giudotti et al. Science (1999) 284 Design A.Bertoletti

Accepted concepts In acute HBV infection, peripheral blood CD4- - and CD8 and CD8- -mediated mediated In acute HBV infection, peripheral blood CD4 responses to HBV antigens are easily detectable in vitro and are much responses to HBV antigens are easily detectable in vitro and are much stronger than in viremic viremic patients with chronic hepatitis B patients with chronic hepatitis B stronger than in ACUTE HEPATITIS CHRONIC HEPATITIS 50 S. I. HBV core 40 30 CD4 20 10 0 HBV core18-27 80 % LYSIS 60 CD8 40 20 0 PATIENTS Penna et al. (1996) J. clin Invest 98, 1168

Woodchuck (Marmota monax) a mammalian animal model for Immunological approaches to treat hepatitis B virus infection:

WHV/Woodchuck (Marmota monax) • WHV and HBV have high similarities in morphology, genome structure, replication cycle, etc. • WHV and HBV infections resemble in • natural history of infection: chronicity, HCC • host immune respones at B- and T-cell levels • Woodchuck has been used to study: • Pathogenesis of hepadnavirus infection • New vaccines and therapeutic vaccination • Antiviral drugs • T cell response to WHV infection

Acute PBMC Responses to WHcAg in Neonatal cWHV8P1 Infection # 4862 # 4863 # 4864 # 4870 1.0E+11 Resolved Woodchucks 6 1.0E+10 4 1.0E+09 2 1.0E+08 1.0E+07 0 # 4872 # 4873 # 4874 # 4875 1.0E+11 6 1.0E+10 4 1.0E+09 2 1.0E+08 1.0E+07 0 Stimulation Index WHV DNA [ge/ml] # 4876 # 4877 # 4881 WHcAg C97-110 1.0E+11 6 1.0E+10 4 1.0E+09 2 1.0E+08 1.0E+07 0 # 4878 # 4879 # 4871 # 4880 1.0E+11 onic Carrier Woodchucks 6 1.0E+10 4 1.0E+09 2 1.0E+08 1.0E+07 0 4 8 14 18 22 26 4 8 14 18 22 26 4 8 14 18 22 26 4 8 14 18 22 26 Time [Weeks]

Summary 1.Control of HBV is achived by specific immune responses on B-and T-Cell level 2.Absence of T-cell response in the early phase of infection results in viral persistence (Menne et al. J.Virol 2003,Webster et al. Hepatology 2000) 3.Antibodies (anti-HBs)prevent reinfection of hepatocytes

The aim of therapeutic immunization: to achieve long term control of HBV by stimulating specific immune responses on B-and T-Cell level in chronic HBV carriers

Clinical experiences: published clinical trials – Conventional HBsAg vaccines • Pol S. et al., Acta Gastroenterol. Belg., 1998, J. Hepatol., 2001 • Couline et al., J. Infect. Dis., 2001 • Jung C. et al., Vaccine, 2002 • Ren F., et al., J. Med. Virol., 2003 • Yalcin et al., J. Clin. Gastroenterol., 2003 • Safadi R. et al., American J. Gastroenterol., 2003 • Dikici et al., J Gastroenterol. Hepatol., 2003 – HBsAg vaccine+lamivudine • Horiike N., J Clin. Virol., 2005 – CTL-peptide • Heathcote et al., Hepatology, 1999 – DNA vaccine • Mancini-Bourgine et al., Hepatology, 2004 – HBsAg-anti-HBs immune complexes • Wen Y. et al., Lancet, 1995, • Xu D. Z. et al., Vaccine, 2005

Immune Modulation in Chronic WHV Infection Immunization with proteins, DNA vaccine to induce a specific T cell (CD4, CD8) or B-cell response. Inhibition of Elimination of replication WHV core particles (WHV) (1) partial no no WHsAg (2,4) no partial no WHsAg and Nucleosidanalogs (3) partial no WHV immune complexes and nucleosidanalogs(5) 1) Roggendorf et al. 1995 Intervirology 2) Lu et al. J Hepatol,2003 3) Menne et al. J. Virol (2002) 76, 5305 4) Hervas-Stubbs et al., J Hepatol., 1997; 2001 5) Lu et all unpubl. data

Immune Modulation in Chronic WHV Infection Immunization with proteins, DNA vaccine to induce a specific T cell (CD4, CD8) or B-cell response. Inhibition of Elimination of replication WHV core particles (WHV) (1) partial no no WHsAg (2,4) no partial no WHsAg and Nucleosidanalogs (3) partial no WHV immune complexes and nucleosidanalogs(5) 1) Roggendorf et al. 1995 Intervirology 2) Lu et al. J Hepatol,2003 3) Menne et al. J. Virol (2002) 76, 5305 4) Hervas-Stubbs et al., J Hepatol., 1997; 2001 5) Lu et all unpubl. data

Summary of therapeutic vaccination of WHV carrier woodchucks Vaccine Animals WHV Persistent clearance viremia WHV core 6 1 5 HBV core 4 0 4 WHsAg 7 0 7 30 1 No vaccine 0 30 1 25 historical controls (observation period 3 years) Ro/96/76

Immune Modulation in Chronic WHV Infection Immunization with proteins, DNA vaccine to induce a specific T cell (CD4, CD8) or B-cell response. Inhibition of Elimination of replication WHV core particles (WHV) (1) partial no no WHsAg ( 2,4) no partial yes WHsAg and Nucleosidanalogs (3) partial no WHV immune complexes and nucleosidanalogs(5) 1) Roggendorf et al. 1995 Intervirology 2) Lu et al. J Hepatol,2003 3) Menne et al. J. Virol (2002) 76, 5305 4) Hervas-Stubbs et al., J Hepatol., 1997; 2001 5) Lu et all unpubl. data

Kinetic of antibody induction by WHsAg immunization in two representative animals 383 386 anti WHs OD490 anti WHs OD490 4 4 3 3 2 2 1 1 0 0 anti WHpreS OD 490 anti WH preS OD 490 1 1,2 0,8 1 0,8 0,6 0,6 0,4 0,4 0,2 0,2 0 0 -20 0 20 40 60 80 100 120 -20 0 20 40 60 80 100 120 day day Mengji Lu 2002

Why are the recent therapeutic approaches not successful? The vaccines used in these studies did not effectively induce immune responses that are required for control of WHV replication. The high WHV replication may prevent the induction and maintainance of effective immune responses. The WHV proteins may interfere with the induction of immune responses.

New therapeutic appraoches Boni C. et al., JCI, 1998; Hepatology, 2001 • CTL inducers like DNA vaccines • Add proteins to enhance specific antibody responses • Antiviral treatment with nucleoside analoga Lamivudine enhances T-cell Proliferation and function

Immune Modulation in Chronic WHV Infection Inhibition of Elimination of Vaccine/Treatment . replication WHV core particles (WHV) (1) partial no no WHsAg (2,4) no partial no WHsAg and Nucleosidanalogs (3) partial no WHV immune complexes and nucleosidanalogs(5) 1) Roggendorf et al. 1995 Intervirology 2) Lu et al. J Hepatol,2003 3) Menne et al. J. Virol (2002) 76, 5305 4) Hervas-Stubbs et al., J Hepatol., 1997; 2001 5) Lu et al. unpubl. data

Combination Therapy • Lamivudine reducing viral replication/stimulating T-cells • DNA-based Vaccines inducing CTL responses • Antigen-antibody Complexes Immune complexes (IC) of anti-HBs and HBsAg were tested in a pilot trial (Wen et al., Lancet, 1995) and in transgenic mice (Zheng et al., Vaccine, 2001). IC may be efficiently taken up by antigen- presenting cells. Mengji Lu 2005

Experimental design Group 1 (n=2) Group 2 (n=4) Group 3 (n=4) Lamivudin + + + 15mg/d Immunization none IC plus DNA vaccines DNA • 3 groups of chronic WHV-infected woodchucks IC: WHsAg (80 μ g of per ml) in complexes with woodcuck anti- • WHs, add 1 mg of plasmids (pWHsIM) per ml • DNA vaccine: Combination of three plasmids expressing WHsAg, WHcAg, and woodchuck IFN-gamma (as adjuvant Mengji Lu 2005

Immunizations Immune complex/DNA of chronic carrier lamivudine woodchucks 2 IC/DNA with IC/DNA anti-WHs OD490 induced anti-WHs antibody 1 Immunizations at week 8, 12, and 17 Anti-WHs antibodies 0 were detected in 3 of 4 0 10 20 30 40 woodchucks after Week Immunizaitons 17492 17494 17497 17493

Transient decrease of titers of WHsAg and viral DNA in paralell with increase of anti-WHs titer in WH17493 I 2 1000 Anti-WHs(OD 490) 800 WHsAg(ug/ml) I I 600 1 400 200 0 0 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 I week 1,00E+09 1,00E+08 I 1,00E+07 PCR I 1,00E+06 I=Immunization IC/DNA 1,00E+05 lamivudine 1,00E+04 1,00E+03 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

What we learnt from this experiment? Good news: • Therapeutic immunizations are able to – induce anti-WHs antibody responses in chronic carriers – Induce specific lyphoproliferative responses (for DNA vac) – achieve a transient reduction of WHV DNA titer and WHsAg concentration • Liver samples of woodchucks in this study showed only mild hepatitis. Bad news: • The induced responses and therapeutic effects were not sustained. • DNA vaccination alone was not effective in terms of induction of anti-WHs antibodies.