- Hepatitis B - treatment and resistance Prof. Dr. A. Erhardt Dr. - PowerPoint PPT Presentation

- Hepatitis B - treatment and resistance Prof. Dr. A. Erhardt Dr. J. Verheyen B. Beggel

Case report • 38 year old male, first presentation in 7/2004 • Intermittently elevated transaminases in 2003 • Blood transfusion in 1993 • Crohn`s disease since 1988 (ileocaecal resection, operations for fistula) • Latent hypothyroidism • Osteoporosis

Case report Laboratory results: AST 38 U/l ALT 186 U/l AFP 9.2 µg/l Serology: HBsAg +, anti-HBc +, anti-HBc IgM +, HBeAg + Anti-HBs -, anti-HBe- HBV DNA: >17 Mio IU /ml

Would you require any additional information? • Histology • Noninvasive fibrosis assessment • HBV genotype • HDV antibodies • HCV antibodies • HIV antibodies • Il28 status

How would you treat this patient? • Peg-Interferon alfa • Lamivudine • Telbivudine • Adefovir • Entecavir • Tenofovir • No treatment

HBV treatment Present options and results

HBV Hepadnaviridae behüllt, circular DNA-Genom (3,2kb) Reverse transcriptase (RNA intermediate) 8 Genotypen Glebe et al. 2007 Prävalenz: HBV s-Antigen Träger: WHO: 300-420 Mio (5-7%) D: 500.000 (0.5%)

HBV Verlauf Klinik: Akute Hepatitis (85%) Chronische Hepatitis (15%) (Liver cirrhosis, Hepatocellular carcinoma)

Reveal: High HBV viral load is associated with increased incidence of cirrhosis All participants (n=3,582) Cumulative incidence liver cirrhosis Baseline HBV DNA Level .4 ≥10 6 ≥10 4 –<10 5 10 3 –<10 4 .3 300–10 3 <300 .2 .1 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Year of follow-up Iloeje UH, et al. Gastroenterology 2006; 130:678–686

REVEAL: High HBV viral load is associated with increased incidence of HCC HBeAg negative, normal ALT, no liver cirrhosis at entry (n=2,925) Baseline HBV DNA Level .14 Cumulative incidence of HCC ≥10 6 .12 ≥10 4 –<10 5 10 3 –<10 4 .1 300–10 3 .08 <300 .06 .04 .02 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Year of follow-up Chen CJ, et al. JAMA 2006; 295:65–73

Therapieindikation HBV-DNA: positiv: + deutliche Leberfibrose/zirrhose od. Risikofaktoren für HCC >2x10 3 IU/ml + Leberenzyme: ALT > 2-fach od. Histo: >A1/F1 Kontrolle der HBV-DNA alle 3/6 Monate Therapierichtlinien der Hepatitis-B: Z Gastroenterol 2007; 45:525

HBV replication cycle and drug targets 3TC, ADF, TDF, ETV LdT Gerlich and Kann in Topley & Wilson

Response in HBeAg-pos. hepatitis Date after 48 weeks of treatment Undetectable Normal HBe- HBV-DNA ALT Serokonversion 77% 74% 69% 68% 67% 66% 60% 48% 39% 39% 30% 23% 25% 22% 21% 21% 21% 12 EASL Consensus, J Hepatol 2009

Response in HBeAg-neg. hepatitis Data after 48 weeks of treatment Undetectable HBV-DNA Normal ALT 91 % 90 % 88 % 78 % 77 % 74 % 74 % 72 % 72 % 63 % 51 % 38 % Daten nach einem Jahr Therapiedauer. Keine Head-to-Head Studien! EASL Consensus, J Hepatol 2009

HBV genotypes

HBV genotypes - heterogeneity of HBV Mutation rate 1.4-5.0 x 10 -5 per Nucleotid /year - missing proofreading of the RNA-dependent DNA polymerase - selection pressure - high replication rate Schaefer et al, J Viral Hep 2006 Andernach et al, Rev Med Virol 2009

HBV genotypes: molecular differences Genotype Lenght [ Bp] Molecular differences in the ORF A 3 2 2 1 I ns AS1 5 3 and AS1 5 4 in HBc B 3215 C 3215 D 3 1 8 2 Del AS1 -1 1 in preS1 E 3212 Del AS11 in preS1 F 3215 G 3248 Ins of 12 AA in HBc; Del AA11 in PreS1 H 3215 I? 3215 Schaefer et al, J Viral Hep 2006 Huy et al , J Virol 2008

Heterogenous geografical distribution of HBV genotypes A 2 D D B A 2 A 2 D 1 D B D D A> C> B> D D C D 1 D D B 2 D G F 1 E C 1 E D H C B 3 F 2 F 1 A 1 F 2 C 4 A 1 F 3/ 4 Erhardt; In: Hepatitis B, 2006

IFN response und HBV genotype HBeAg-positive patients Janssen et al . Lancet 2005 Ansprechen (%) Erhardt et al . Gut 2005 60 Lau et al . NEJM 2005 Wai et al . Hepatology 2002 50 Kao et al. J Hepatol 2000 40 30 20 10 0 A B C D HBV-Genotyp

HBV genotype and treatment Metaanalysis NA IFN Wiegand et al , Antivir Ther 2008

HBV genotypes E-H – response to IFN n= 23/ 49 treated with IFN-alfa Erhardt et al, JMV 2009

Dual Infection, Coinfection and Superinfection • Infection with at least two different strains Dual • Natural progression of the disease? Infection • Response to therapy? • Infection with two different strains • Either simultaneously or Coinfection • before the immune response Superinfection • Infection with a second strain after the immune (Reinfection) response has been established

Problem Statement: Subtyping with Ambiguities Surface Gene NT Position 322 364 365 370 376 377 390 Patient Y K W W Y R R Sequence Genotype A T G A A T A A Concensus Genotype D C T T T C G G Consensus • Ambiguities are explained by dual infection of genotype A and D • 9 genotypes and > 8% ambiguous positions ask for bioinformatic evaluation • Subtyping references ([Tedder et al. 2006] and [Tatusova et al. 2004]) do not explicitly cover dual infections

Model Evaluation - Synthetic Data 100% 90% 80% 70% 60% Prediction 50% 40% Dual Infection with 30% correct genotypes 20% Single Infection with 10% correct genotypes 0% Single Infection Dual Infection Dual Infection with identical with different genotypes genotypes Syntetic sequence - SHB SHB

Model Evaluation – Cologne HBV Cohort • The Cologne HBV cohort contains 242 sequences • 10 (4.1%) dual infections were identified • 7 of these are with genotype G • We validated three of these dual infections with clonal experiments • Dual infections with the predicted genotypes could be confirmed

IL28B - Polymorphisms

IL28B (rs12979860 ) and response Dongliang et al, Nature 2009

IL28B and ethnical distribution Dongliang et al, Nature 2009

IL28B in hepatitis B Martin et al, J Inf Dis 2010

HLA Polymorphisms

HLA DPB1* 301 – risk for persistence n=786 HBV; n=2201 controls; GWAS; rs9277535 Kamatani et al, Nature Gen 2009

IFNAR/ IL10R polymorphisms: virus clearance 318 microsatellites in 88 siblings and 61 Gambian families Frodsham et al, PNAS 2006

How was the patient treated? • Lamivudine 100 mg since 12/2004 • Reevaluation in 7/2005 • HBV DNA >17.8 IU/ml • HBeAg + • ALT 199 U/ml

How would you treat this patient? • Peg-Interferon alfa • (Lamivudine) • Telbivudine • Adefovir • Entecavir • Tenofovir • No treatment

How was the treatment continued? • Adefovir 10 mg from 7/2005 to 9/2008 • Reassessment in 7/2006: HBV DNA 1581 IU/ml; ALT 49 U/ml; HBeAg +; M204M/V; L180M; L173L • Reassessment in 5/2008: HBV DNA 5 IU/ml; HBeAg+; ALT 27 U/ml

HBV Resistence

HBV resistence Pol/RT RNaseH Terminal protein Spacer 692 349 845 a.a. 1 183 (rt 344) (rt1) GVGLSPFLLA YMDD I(G) II(F) A B C D E V173L LAM/ FTC L180M M204I/V A181V/T ADV A181V/T N236T I233V ? I169T V173L M250V ETV* T184G S202G/I LdT M204I A194T ? TDF *L180M + M204I/V als Baseline Allen et al. Hepatology 1998; Gish et al. J Hepatol 2005; Qi et al. J Hepatol 2004; Tenney et al. AAC 2004; Lai et al. Gastroenterology 2005; Sheldon et al. Antivir Ther 2005; Delaney et al. AAC 2006 ; Schildgen et al NEJM 2006 ; Curtis et al JID 2007

geno2pheno[ hbv] geno2pheno[hbv] • web-service for the analysis of HBV sequences • subtype • drug resistance: 3TC, ADF, LdT, ETV, TDF • escape mutations • rules based analysis • requires part of the RT domain (RT 80 - 252)

geno2pheno[ hbv] How to find the tool: http://www.genafor.org

geno2pheno[ hbv] Layout similar to geno2pheno[resistance] and geno2pheno[integrase] identifier upload files paste sequence align and predict

geno2pheno[ hbv] Reference Sequences and Mutation Lists • Currently we only support genotyping for genotypes A-H • We use one consensus sequence for each genotype • Shared with HIV-Grade (list of mutations are comparable) • Mutations are extracted w.r.t. the respective genotype • Therefore, genotype specific polymorphisms are filtered in the default output • Option “use genotype D consensus sequence” • Mutations are extracted w.r.t. genotype D

geno2pheno[ hbv] alignment sequence information drug resistance

geno2pheno[ hbv] – sequence information (sub)genotype completeness and similarity mutations escape mutations

geno2pheno[ hbv] – drug resistance drugs rated mutations evaluation

HBV rules (Jens Verheyen, Martin Däumer, Martin Obermeier, 07/ 2009) Drug Rule Definition Prediction 194T possible resistant TDF 173L resistant 3TC 180C or 180M resistant 3TC 204I or 204V or 204S resistant 3TC 181T resistant 3TC 80V or 80I possible resistant 3TC 181T or 181V resistant ADF 233V resistant ADF 236T resistant ADF (169T or 184A or 184G or 184I or 184S or 202G or 202I or resistant ETV 250V) and (180C or 180M) and (204I or 204V) 169T or 184A or 184G or 184I or 184S possible resistant ETV 202G or 202I possible resistant ETV 250V possible resistant ETV 204I resistant LdT 80I or 80V possible resistant LdT