NAs Induced Immune Response and Antiviral Therapy in Chronic Hepatitis B Gui-Qiang Wang Department of Infectious Diseases Center for Liver Diseases Peking University First Hospital

Chronic hepatitis B is hard to treat liver HBV immunity Other organs Control of infection Persistance of Infection Virus Host immunity Impaired immunity DCs, CTL, cytokines PD-1 in active T cells regulatory T lymphocytes cccDNA High viral load Mutation Replication out of liver Host immunity Viral replication

CD8 and CD4 response: Acute hepatitis B vs. Chronic hepatitis B 50 40 30 20 10 60 45 15 Specific lysis % In acute hepatitis, CD4 response targeting HBV core protein antigen are very vigorous. This can result in an clearance and possibly cure of the patient with acute disease. In chronic disease, you see CD4 responses were blunted. What about CD8 response ? Again in acute hepatitis, we are looking at targets, HBV POL, X, CORE and envelop proteins. Again may allow and dose allow clearance or cure of the disease. Chronic disease， that effects were blunted or non-existent. again, leading to viral persistence long term. Ferrari C et al. J Immunol. 1990; Penna A et al. J Exp. Med. 1991; Bertoletti A et al. Proc. Natl.Acad. Sci. USA,1991; Rehermann B et al. J Exp. Med. 1995; Jung C et al. Virology 1999;Maini et al. J Exp. Med. 2000

Goals of CHB therapy: Sustained immune control HBsAg clearance Reduction of HCC and cirrhosis, and improve survival rate Sustained immune control After the end of therapy HBeAg (+) patients: sustained HBeAg seroconversion HBeAg (-) patients: permanent HBV suppression lower quantitation of HBsAg Antiviral therapy Permanent suppression of HBV DNA replication ALT normalization Perrillo et al. Hepatology 2006; 17. EASL guidelines 2009; van Zonneveld et al. Hepatology 2004; 19. Marcellin et al. APASL 2010

Treatment Options Treatment combinations Immuno-modulators Antivirals Aiming for sustained remission IFNα Peg IFN Nx cytokines Vaccine therapy Antivirals Maintained remission Lamivudine Adefovir Entecavir Telbivudine Tenofovir CD8+ HBV Treatment combinations

Immune response during NAs treatment

Lamivudine Boni C, et al. J Hepatol, 2003, 39: 595-605.

CTL responses to individual HBV peptides containing the HLA-A2 binding motif . Each bar represents the percentage of significant cytotoxic responses to individual peptides (specific lysis . 15%) among the total number of assays performed at different time points before (left panel) and during the first 5 months (right panel) of treatment in the whole group of HBeAg1 patients with chronic hepatitis B. Each patient was tested with each individual peptide 4 or 5 times before therapy and 7 or 8 times during lamivudine treatment. Boni C, et al. HEPATOLOGY 2001;33:963-971

Number of IFN-γ+ lymphocytes/100,000 T cells Lamivudine induced the restoration of anti-viral T cell responses is transient  P<0.002 Treatment initiation 40 30 20 10 Number of IFN-γ+ lymphocytes/100,000 T cells Treatment termination -24-0 2-12 16-24 28-36 40-52 52-56 weeks Boni C, et al. J Hepatol, 2003, 39: 595-605.

HBV specific CTL response for Sustained HBeAg seroconversion after LAM treatment 􀁕􀁉􀁆􀀁 􀁇􀁓􀁆􀁒􀁖􀁆􀁏􀁄􀁊􀁆􀁔􀀁 􀁐􀁇􀀁 􀁄􀁊􀁓􀁄􀁖􀁍􀁂􀁕􀁊􀁏􀁈􀀁 􀀵􀁄􀀁 􀀒􀀙􀀎􀀓􀀘􀀁 􀁔􀁑􀁆􀁄􀁊􀁇􀁊􀁄􀀁 􀀤􀀥􀀙􀀌􀀁 􀀵􀀁 􀁄􀁆􀁍􀁍􀁔􀀁 􀁃􀁆􀁕􀁘􀁆􀁆􀁏􀀁 􀁔􀁖􀁔􀁕􀁂􀁊􀁏􀁆􀁅􀀁 􀁈􀁓􀁐􀁖􀁑􀀁 􀁂􀁏􀁅􀀁 􀁓􀁆􀁍􀁂􀁑􀁔􀁆􀁅􀀁 􀁈􀁓􀁐􀁖􀁑􀀏􀀁 􀀉􀀢􀀊􀀁 􀀷􀁊􀁔􀁖􀁂􀁍􀁊􀁛􀁂􀁕􀁊􀁐􀁏􀀁 􀁐􀁇􀀁 􀁄􀁊􀁓􀁄􀁖􀁍􀁂􀁕􀁊􀁏􀁈􀀁 􀀵􀁄􀀁 􀀒􀀙􀀎􀀓􀀘􀀁 􀁔􀁑􀁆􀁄􀁊􀁇􀁊􀁄􀀁 􀀤􀀥􀀙􀀌􀀁 􀀵􀀁 􀁄􀁆􀁍􀁍􀁔􀀁 􀁇􀁓􀁐􀁎􀀁 􀁓􀁆􀁑􀁓􀁆􀁔􀁆􀁏􀁕􀁂􀁕􀁊􀁗􀁆􀀁 􀁄􀁂􀁔􀁆􀀁 􀁂􀁎􀁐􀁏􀁈􀀁 􀁔􀁖􀁔􀁕􀁂􀁊􀁏􀁆􀁅􀀁 􀁈􀁓􀁐􀁖􀁑􀀁 􀁂􀁏􀁅􀀁 􀁓􀁆􀁍􀁂􀁑􀁔􀁆􀁅􀀁 􀁈􀁓􀁐􀁖􀁑􀀏􀀁 􀀢􀁔􀀁 􀁂􀀁 􀁏􀁆􀁈􀁂􀁕􀁊􀁗􀁆􀀁 􀁄􀁐􀁏􀁕􀁓􀁐􀁍􀀍􀀁 􀀱􀀣􀀮􀀤􀀁 􀁇􀁓􀁐􀁎􀀁 􀀩􀀭􀀢􀀁 􀀢􀀓􀀁 􀁏􀁆􀁈􀁂􀁕􀁊􀁗􀁆􀀁 􀁑􀁂􀁕􀁊􀁆􀁏􀁕􀁔􀀁 􀁘􀁆􀁓􀁆􀀁 􀁔􀁕􀁂􀁊􀁏􀁆􀁅􀀁 􀁘􀁊􀁕􀁉􀀁 􀀵􀁄􀀁 􀀒􀀙􀀎􀀓􀀘􀀁 􀁂􀁏􀁅􀀁 􀁂􀁏􀁕􀁊􀀎􀀤􀀥􀀙􀀁 􀁎􀀢􀁃􀀏􀀁 􀀯􀁖􀁎􀁃􀁆􀁓􀀁 􀁊􀁏􀀁 􀁕􀁉􀁆􀀁 􀁖􀁑􀁑􀁆􀁓􀀁 􀁓􀁊􀁈􀁉􀁕􀀁 􀁒􀁖􀁂􀁅􀁓􀁂􀁏􀁕􀀁 􀁊􀁏􀁅􀁊􀁄􀁂􀁕􀁆􀁔􀀁 􀁕􀁉􀁆􀀁 􀁑􀁆􀁓􀁄􀁆􀁏􀁕􀁂􀁈􀁆􀀁 􀁐􀁇􀀁 􀀵􀁄􀀁 􀀒􀀙􀀎􀀓􀀘􀀁 􀁔􀁑􀁆􀁄􀁊􀁇􀁊􀁄􀀁 􀀤􀀥􀀙􀀌􀀁 􀀵􀀁 􀁄􀁆􀁍􀁍􀁔􀀁 􀁘􀁊􀁕􀁉􀁊􀁏􀀁 􀁕􀁉􀁆􀀁 􀁕􀁐􀁕􀁂􀁍􀀁 􀀤􀀥􀀙􀀌􀀁 􀀵􀀁 􀁄􀁆􀁍􀁍􀀁 􀁑􀁐􀁑􀁖􀁍􀁂􀁕􀁊􀁐􀁏􀀏􀀁 􀀉􀀣􀀊􀀁 􀀧􀁓􀁆􀁒􀁖􀁆􀁏􀁄􀁊􀁆􀁔􀀁 􀁐􀁇􀀁 􀁄􀁊􀁓􀁄􀁖􀁍􀁂􀁕􀁊􀁏􀁈􀀁 􀀵􀁄􀀁 􀀒􀀙􀀎􀀓􀀘􀀁 􀁔􀁑􀁆􀁄􀁊􀁇􀁊􀁄􀀁 􀀤􀀥􀀙􀀌􀀁 􀁄􀁆􀁍􀁍􀁔􀀁 􀁃􀁆􀁕􀁘􀁆􀁆􀁏􀀁 􀁔􀁖􀁔􀁕􀁂􀁊􀁏􀁆􀁅􀀁 􀁈􀁓􀁐􀁖􀁑􀀁 􀁂􀁏􀁅􀀁 􀁓􀁆􀁍􀁂􀁑􀁔􀁆􀁅􀀁 􀁈􀁓􀁐􀁖􀁑􀀏􀀁 Lee CK, et al. Korea J Hepatol 2005; 11:34-42

Entecavir Zhang J, et al. PLoS ONE 2010 Nov 30; 5(11): e13869.

Treg/TH17 ratios and HBV DNA levels Frequency of Treg cells Entecavir yields transient decrease of Treg cells and ratios of Treg cells to Th17 cells Treg/TH17 ratios and HBV DNA levels 0 1 3 6 9 12 months 0.0 0.6 1.2 1.8 2.4 3.0 r=1.00 8 6 4 2 0 HBV DNA, log10 IU/mL Treg/Th17 ratios * *§ Frequency of Treg cells months 0 1 3 6 9 12 P=0.002 P=0.042 P=0.016 2 4 6 8 10 12 %FoxP3+ CD4+ T cells During treatment of entecavir in HBeAg-positive patients, Treg cells and Treg/Th17 ratios decreased and bottomed out at 3 months and then increased, exhibiting a reverse ‘‘V’’-type change. These transient changes of Treg cells and Treg/Th 17 ratios correlate with suppression of HBV DNA. Zhang J, et al. PLoS ONE 2010 Nov 30; 5(11): e13869.

Adefovir Dipivoxil .Jeroen N. et,al. Virology 361 (2007) 141–148

Adefovir induces transient decrease in expression of Treg cells in chronic hepatitis B patients After 6 months of treatment,the proportion of Treg increased somewhat, but remained lower than at baseline. These results concurred with the CD4, CD25, CD45RO and CTLA-4 Treg staining for all four patients The percentage of Treg defined as the percentage of CD4+CD25+FoxP3+ cells divided by the percentage of CD4+ cells (line and right y-axis) or the percentage of CD4+ CD25+CD45RO+CTLA-4+ cells divided by the percentage of CD4+ cells (bars and left y-axis) Jeroen N. et,al. Virology 361 (2007) 141–148

Comparison of CD4 T-cell reactivity to HBcAg in patients receiving ADV or the placebo ADV N=13 PLB N=6 there was a significant increase in the frequencies of IFN--producing CD4 T cells at TW16 compared to baseline values for ADV-treated patients (P 0.03). , at TW40 and TW48,patients receiving ADV had significantly higher frequencies of IFN--producing CD4 T cells than did patients receiving placebos (P 0.03). The bars represent means SEMs. Cooksley H, et al. Antimicrobial agents & chemotherapy, 2008, 312–320

HBcAg-specific T-cell proliferation according to virological response Group1: HBeAg seroconversion Group2: non-responder Group3: placebo . Patients in group 1 had a higher number of positive tests results for T-cell proliferation to HBcAg than did patients in group 3 (P 0.03) Cooksley H, et al. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 2008, 312–320

Telbivudine preserves Th1 cytokine production and down regulates PD-L1 in a mouse model of viral hepatitis

Telbivudine enhances the production of TNF-α in MHV-3-infected macrophages compared to lamivudine Vitro study 350 * * 300 * 250 200 TNF-α (pg/ml) 150 Vitro study: Peritoneal macrophages were harvested from BALB/cJ mice in vitro, and macrophages were divided into three groups: macrophages without stimulating factors, lipopolysaccharide (LPS)-stimulated macrophages and mouse hepatitis virus strain-3(MHV3)-stimulated macrophages. Changes of cytokine levels in MHV-3-stimulated macrophages without adding drugs or with telbivudine or with lamivudine were also investigated. 100 50 10 50 100 5 25 50 Lamivudine (ug/ml) Telbivudine (ug/ml) Q.Ning et al. J Viral Hepat., 2010, 17 (Suppl. 1), 24–33.

Effects of antiviral therapy with telbivudine on peripheral iNKT cells in HBeAg-positive chronic hepatitis B patients Shi TD, et al. Clin Exp Med. 2012 Jun;12(2):105-13.

Telbivudine continuously increased frequencies of IFN-γ iNKT cells in chronic hepatitis B patients CHB cells 0.3 0.2 0.1 0.0 0.4 0.3 0.2 0.1 0.0 P<0.05 IFN+iNKT cells frequency (%) iNKT cells frequency (%) 右侧的图纵坐标改为，细胞内合成IFN-γ的iNKT细胞 Baseline 12 weeks 24 weeks 52 weeks Healthy controls Baseline 12 weeks 24 weeks 52 weeks *P<0.05 (vs. healthy controls) P<0.05 (vs. baseline) This study included 29 HBeAg-positive and HBsAg-positive CHB patients. Telbivudine was orally administered at a dose of 600 mg per day, and heparinized venous blood was taken at four study visits: baseline and treatment weeks 12, 24 and 52 to detect the frequencies, function and PD-1 expression of peripheral iNKT cells. Shi TD, et al. Clin Exp Med. 2012 Jun;12(2):105-13.

Continually decreased PD-1 expression of iNKT cells in CHB patients was found during telbivudine treatment 50 40 30 20 10 * P<0.05(vs. healthy controls) P<0.05(vs. baseline) PD-1+ iNK cells (%) Healthy controls Baseline 12 weeks 24 weeks 52 weeks CHB patients This study included 29 HBeAg-positive and HBsAg-positive CHB patients. Telbivudine was orally administered at a dose of 600 mg per day, and heparinized venous blood was taken at four study visits: baseline and treatment weeks 12, 24 and 52 to detect the frequencies, function and PD-1 expression of peripheral iNKT cells. .Shi TD, et al. Clin Exp Med. 2012 Jun;12(2):105-13.

The frequencies of PD-1+ CD4+ and CD8+ T cells during treatment with LDT 29 HBeAg positive CHB treated with LDT for 52 weeks, 19 patients achieved virological response with HBV DNA less than 60IU/ml. Dynamic changes of PD-1 on total CD4 T (A) and CD8 T (B) cells. The frequencies of PD-1+ CD4+ and CD8+ T cells decreased more quickly in responders during the second 12 weeks’ treatment with LDT Unpublished data

Conclusion CHB is characterized by an impaired immune response to HBV. The host immune response is crucial to control HBV infection, Neither IFN nor NAs will not work without host immune response The immune modulatory effects of telbivudine have been broadly investigated because its higher HBeAg seroconversion rates, and shown promising data We need further study on immunological profiles with NAs based treatment to elucidate the whole story

Thank you! Gui-Qiang Wang wanggq@hotmail.com