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

Thimme, JVI (2003), 77(1), p.68-76

HBV-infected chimpanzees

CD8 CELL CD8 CELL CYTOPATHIC CLEARANCE BY LYSIS OF INFECTED CELLS CLEARANCE BY CYTOKINES IFN-gamma/TNF-alpha NON-CYTOPATHIC

Step 1 Step 2

1) Transgenetic mouse expressing HBV proteins (E. Chisari, L.Giudotti, review 2001 2) Chimpanzee L. Giudotti et al. Science (1999) 284

Control of virus infection by CTL1,2

Design A.Bertoletti

% LYSIS 10 20 30 40 50 ACUTE HEPATITIS CHRONIC HEPATITIS

• S. I.

In acute HBV infection, peripheral blood CD4 In acute HBV infection, peripheral blood CD4-

• and CD8

and CD8-

• mediated

mediated responses to HBV antigens are easily detectable in vitro and are responses to HBV antigens are easily detectable in vitro and are much much stronger than in stronger than in viremic viremic patients with chronic hepatitis B patients with chronic hepatitis B

20 40 60 80 PATIENTS

CD4 CD8

HBV core HBV core18-27

Accepted concepts

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

Resolved Woodchucks # 4862 # 4870 # 4864 # 4863 # 4872 # 4875 # 4874 # 4873 # 4876 # 4881 # 4877 WHV DNA [ge/ml] Stimulation Index WHcAg C97-110

6 4 2 6 4 2 6 4 2 1.0E+11 1.0E+10 1.0E+09 1.0E+08 1.0E+07 1.0E+11 1.0E+10 1.0E+09 1.0E+08 1.0E+07 1.0E+11 1.0E+10 1.0E+09 1.0E+08 1.0E+07

• nic Carrier Woodchucks

# 4878 # 4880 # 4871 # 4879

1.0E+11 1.0E+10 1.0E+09 1.0E+08 1.0E+07 6 4 2

Time [Weeks]

4 8 14 18 22 26 4 8 14 18 22 26 4 8 14 18 22 26 4 8 14 18 22 26

Acute PBMC Responses to WHcAg in Neonatal cWHV8P1 Infection

Summary

1.Control of HBV is achived by specific immune responses

• n 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 replication partial no partial partial Elimination of WHV no no no no

core particles (WHV) (1) WHsAg (2,4) WHsAg and Nucleosidanalogs (3) 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 replication partial no partial partial Elimination of WHV no no no no

core particles (WHV) (1) WHsAg (2,4) WHsAg and Nucleosidanalogs (3) 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

• f WHV carrier woodchucks

Vaccine Animals WHV Persistent clearance viremia WHV core 6 1 5 HBV core 4 4 WHsAg 7 7 No vaccine 301 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 replication partial no partial partial Elimination of WHV no no yes no

core particles (WHV) (1) WHsAg (2,4) WHsAg and Nucleosidanalogs (3) 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

1 2 3 4

anti WHs OD490

383

0,2 0,4 0,6 0,8 1 1,2

anti WH preS OD 490

• 20

20 40 60 80 100 120

day

1 2 3 4

anti WHs OD490

386

0,2 0,4 0,6 0,8 1

anti WHpreS OD 490

• 20

20 40 60 80 100 120

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

• f

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

• Antiviral treatment with

nucleoside analoga

• CTL inducers like DNA

vaccines

• Add proteins to enhance

specific antibody responses

Boni C. et al., JCI, 1998; Hepatology, 2001

Lamivudine enhances T-cell Proliferation and function

Immune Modulation in Chronic WHV Infection

Inhibition of replication partial no partial partial Elimination of WHV no no no no

core particles (WHV) (1) WHsAg (2,4) WHsAg and Nucleosidanalogs (3) 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

Vaccine/Treatment .

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

• 3 groups of chronic WHV-infected woodchucks
• IC: WHsAg (80 μg 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

Group 1 (n=2) Group 2 (n=4) Group 3 (n=4) Lamivudin 15mg/d + + + Immunization none IC plus DNA DNA vaccines

Immunizations

• f chronic carrier

woodchucks with IC/DNA induced anti-WHs antibody

Immunizations at week 8, 12, and 17 Anti-WHs antibodies were detected in 3 of 4 woodchucks after Immunizaitons

1 2

anti-WHs OD490

10 20 30 40

IC/DNA lamivudine

Week

Immune complex/DNA

17497 17494 17493 17492

1 2

• 2

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

week Anti-WHs(OD 490)

200 400 600 800 1000

WHsAg(ug/ml)

1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07 1,00E+08 1,00E+09

• 2

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

PCR

Transient decrease of titers of WHsAg and viral DNA in paralell with increase of anti-WHs titer in WH17493 I I I I I I

I=Immunization IC/DNA

lamivudine

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
• nly 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.

Acknowledgements

Institut für Virologie, Uni-Klinikum Essen

• Claudia Budde
• Melanie Fiedler
• Mengji Lu
• Yinping Lu
• Shihe Guan
• Yang Xu
• Thekla Kemper
• Barbara Bleekmann
• Gero Hilken (Animal

Center)

• Uta Dahmen (Surgery)
• Jörg Schlaak (Gastro)

Department of Molecular Virologie, Shanghai medical School, Fudan University, Shanghai, China

• Yumei Wen

Institut für Virologie, Uni Giessen, Germany

• Wolfram Gerlich

Department of Clinical Immunology, Tongji Medical College, Wuhan, China

• Dongliang Yang

Immunization Protocol of Chronic Carrier Woodchucks

day Vaccination 1 14 28 66 WHcAg C F A IFA I F A I F A n=6 50 µg 50 µg 50 µg 50 µg HBcAg C F A IFA I F A I F A n=4 50 µg 50 µg 50 µg 50 µg 1 20 41 62 102 WHsAg C F A IFA I F A I F A IFA n=7 100 µg 100 µg 100 µg 100 µg 100 µg

Ro/96/77

Experimental design

• Immunization protocol

– 20 μg purified WHsAg were mixed with adjuvants (0.5 ml)

• 10 woodchucks: AL and MPL
• 10 woodchucks: o/w, MPL, and QS21

– Six s.c. immunizations were carried out in time intervals of 3 weeks

• Monitoring of

– Antibodies to WHsAg and WHV PreS by ELISA – Viral load by spot-blot hybridization

Mengji Lu 2003

Immunizations of chronic carrier woodchucks induced antibodies to WHsAg/WHPreS

0,2 0,4 0,6 0,8 1

anti WH preS OD 490

• 28-14 0

14 28 42 56 70 84 98

day

B

1 2 3 4

anti WHsAg OD 490

• 28 -14 0

14 28 42 56 70 84 98

day

A

cut off 0.85 cut off 0.25

Mengji Lu 2003

Immunizations at Day 0,21,42,63,83 →Increasing reactivity to WHsAg (A) and PreS-GST fusion protein (B)