- 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 Prävalenz: HBV s-Antigen Träger: WHO: 300-420 Mio (5-7%) D: 500.000 (0.5%)

Glebe et al. 2007

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

HBV Verlauf

Reveal: High HBV viral load is associated with increased incidence of cirrhosis

Iloeje UH, et al. Gastroenterology 2006; 130:678–686

All participants (n=3,582) .4 .3 .2 .1 1 2 3 4 5 6 7 8 9 10 11 12 13 Cumulative incidence liver cirrhosis Year of follow-up Baseline HBV DNA Level ≥106 ≥104–<105 103–<104 300–103 <300

Chen CJ, et al. JAMA 2006; 295:65–73

HBeAg negative, normal ALT, no liver cirrhosis at entry (n=2,925) .14 .12 .1 .08 .06 .04 .02 1 2 3 4 5 6 7 8 9 10 11 12 13 Cumulative incidence of HCC Year of follow-up Baseline HBV DNA Level

REVEAL: High HBV viral load is associated with increased incidence of HCC

≥106 ≥104–<105 103–<104 300–103 <300

Therapieindikation

HBV-DNA: positiv: + deutliche Leberfibrose/zirrhose

• d.

Risikofaktoren für HCC >2x103 IU/ml + Leberenzyme: ALT > 2-fach

• d.

Histo: >A1/F1 Kontrolle der HBV-DNA alle 3/6 Monate

Therapierichtlinien der Hepatitis-B: Z Gastroenterol 2007; 45:525

3TC, ADF, TDF, ETV LdT

Gerlich and Kann in Topley & Wilson

HBV replication cycle and drug targets

Response in HBeAg-pos. hepatitis

Date after 48 weeks of treatment

EASL Consensus, J Hepatol 2009

Undetectable HBV-DNA Normal ALT

25% 39% 21% 67% 60% 74% 39% 66% 48% 68% 77% 69%

HBe- Serokonversion

30% 22% 12 21% 23% 21%

Response in HBeAg-neg. hepatitis

Data after 48 weeks of treatment

EASL Consensus, J Hepatol 2009

Undetectable HBV-DNA Normal ALT

63 % 72 % 51 % 90 % 88 % 91 % 38 % 74 % 72 % 78 % 74 % 77 % Daten nach einem Jahr Therapiedauer. Keine Head-to-Head Studien!

HBV genotypes

Schaefer et al, J Viral Hep 2006 Andernach et al, Rev Med Virol 2009

Mutation rate 1.4-5.0 x 10-5 per Nucleotid /year

• missing proofreading
• f the RNA-dependent

DNA polymerase

• selection pressure
• high replication rate

HBV genotypes - heterogeneity of HBV

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

• f HBV genotypes

Erhardt; In: Hepatitis B, 2006

B G F1 H F1 F2 F2 F3/ 4 A2 A2 A2 D D D D1 D D B C B2 C1 D D D1 C B3 C4 A1 A1 D E E D D A> C> B> D

IFN response und HBV genotype

HBeAg-positive patients

40 20

A

60

HBV-Genotyp

50 30 10

B C D Ansprechen (%) Janssen et al. Lancet 2005 Erhardt et al. Gut 2005 Lau et al. NEJM 2005 Wai et al. Hepatology 2002 Kao et al. J Hepatol 2000

HBV genotype and treatment

Metaanalysis

IFN NA

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

Dual Infection

• Infection with at least two different strains
• Natural progression of the disease?
• Response to therapy?

Coinfection

• Infection with two different strains
• Either simultaneously or
• before the immune response
• Infection with a second strain after the immune

response has been established Superinfection (Reinfection)

Problem Statement: Subtyping with Ambiguities

Surface Gene

NT Position 322 364 365 370 376 377 390

Patient Sequence Y K W W Y R R

Genotype A Concensus T G A A T A A Genotype D Consensus C T T T C G G

• 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

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Single Infection Dual Infection with identical genotypes Dual Infection with different genotypes SHB Dual Infection with correct genotypes Single Infection with correct genotypes

Syntetic sequence - SHB Prediction

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

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

RNaseH 845 a.a. Terminal protein Spacer

Pol/RT

A B C E D 1 183 349 692 YMDD

V173L L180M M204I/V A181V/T

GVGLSPFLLA I(G) II(F) (rt1) (rt 344)

LAM/ FTC ETV*

I169T V173L M250V

T184G S202G/I ADV A181V/T N236T I233V ? LdT M204I TDF A194T ?

*L180M + M204I/V als Baseline

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]

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] sequence information drug resistance alignment

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

geno2pheno[ hbv] – drug resistance drugs rated mutations evaluation

Drug Rule Definition Prediction

TDF 194T possible resistant 3TC 173L resistant 3TC 180C or 180M resistant 3TC 204I or 204V or 204S resistant 3TC 181T resistant 3TC 80V or 80I possible resistant ADF 181T or 181V resistant ADF 233V resistant ADF 236T resistant ETV (169T or 184A or 184G or 184I or 184S or 202G or 202I or 250V) and (180C or 180M) and (204I or 204V) resistant ETV 169T or 184A or 184G or 184I or 184S possible resistant ETV 202G or 202I possible resistant ETV 250V possible resistant LdT 204I resistant LdT 80I or 80V possible resistant

HBV rules (Jens Verheyen, Martin Däumer, Martin Obermeier, 07/ 2009)

geno2pheno[ hbv] – CSV output

Output options

geno2pheno[ hbv] – PDF output sequence information drug resistance place your own comments For mailing and archiving

geno2pheno[ hbv] – CSV output For further analysis

geno2pheno[ hbv] – CSV output

Group Colum n Nam e Description Subtype similarity_rt NT similarity to consensus reference sequence on RT domain genotype_rt Inferred genotype based on similarity to consensus reference on RT domain similarity_sp NT similarity to consensus reference sequence on SHB genotype_sp Inferred genotype based on similarity to consensus reference on SHB subgenotype_simi larity_sp Average NT similarity to subgenotype reference set (Norder et al. 2004) subgenotype_sp Inferred subgenotype based on average NT similarity to subgenotype reference set (Norder et

• al. 2004)

SHB Sequence start_pos_sp AA start position w.r.t. SHB of the input sequence end_pos_sp AA end position w.r.t. SHB of the input sequence mutations_sp List of SHB Mutations w.r.t. reference aa_insertions_sp List of AA insertions within SHB insertions_sp List of NT insertions within SHB

geno2pheno[ hbv] – CSV output

Group Colum n Nam e Description RT Sequence start_pos_rt AA start position w.r.t. RT of the input sequence end_pos_rt AA end position w.r.t. RT of the input sequence mutations_rt List of RT Mutations w.r.t. reference aa_insertions_rt List of AA insertions within RT insertions_rt List of NT insertions within RT Escape escape_sp List of rated escape Mutations w.r.t. SHB Drug Resistance Lamivudine_class Resistance Prediction for Lamivudin Lamivudine_muts Scored Mutations for Lamivudin Adefovir_class Resistance Prediction for Adefovir Adefovir_muts Scored Mutations for Adefovir Entecavir_class Resistance Prediction for Entecavir Entecavir_muts Scored Mutations for Entecavir Tenofovir_class Resistance Prediction for Tenofovir Tenofovir_muts Scored Mutations for Tenofovir Telbivudine_class Resistance Prediction for Telbivudin Telbivudine_muts Scored Mutations for Telbivudin

10 20 30 40 50 60 70 80 90 L A M L A M / P E G e

• L

A M / P E G e + A D V F T C L D T T D F E T V E T V / L A M

• R

L A M A D V / L A M T D F / L A M

1. 5 19 69 90 67 38 50 49 70 4 11 18 28 22 5 19 11 1.2 5 1 9 17 3.

Hadziyannis et al, NEJM 2005; Lai et al, Clin Inf Dis 2003; Qi et al, J Hepatol 2004; Hadziyannis et al, AASLD 2005; Colonno et al, AASLD 2006; Benhamou et al, Hepatology 1999; Tenney et al, EASL 2009;Manns et al EASL 2009; Snow-Lampart et al, AASLD 2008

Resistence developement with NUCs

HBV

HBV/HIV % Resistenz

Resistence: Pyrosequencing – tip of the iceberg

n= 17; naive patients

Margeridon-Thermet et al, JID 2009 additional 4.6 mutations/sequence detectable

HBV genotypische Resistenztestung

HBV Resistenzentwicklung nach x Jahren der Therapie

100 80 60 40 20 1. 2. 3. 4. 5. 1. 2. 3. 4. 5. 1. 2. 3. 4. 5. 1. 2.

80% 30% 1,2% 8% 20% 50% 20%

Lamivudin Adefovir Entecavir Telbivudin Resistenzentwicklung bei vorbestehender Lamivudinresistenz

JAHRE:

L80IV + M204I L180M + M204V genotype A genotype D 9,1% 68,2% 28,1% 45,3%

Svicher V et al. J Hepatology (2009)

HBV Resistenz und HBV Genotyp

M204I L180M + M204V Lamivudin Telbivudine Entecavir Adefovir R R R R I S S I

geno2pheno[ hbv] –genotype C (NCBI database)

13H 27A 35RN 40AP 55DKRQ 80I 80V 82M 85A 106C 118AN 123H 161Y 164FMP 173L 180K 180M 184L 191AI 198S 200V 223A 229V

Bastian Beggel, EHDRW 2010, Sorrento, Italien

positively correlated with M204I positively correlated with M204V

Bedeutung der Resistenzentwicklung

X X

• 1. Resistenzmutationen
• 2. Viruslast
• 3. Leberwerte
• 4. Krankheits-progression

How was the patient treated?

• Telbivudine 600 mg from 9/2008 to 2/2010
• Reassessment in 8/2008:

HBV DNA 15 IU/ml; HBeAg +; ALT mormal

• Reassessment in 1/2009:

HBV DNA 587 IU/ml; HBeAg +; ALT normal

• Reassessment in 9/2009:

HBV DNA neg; HBeAg -; anti-HBe -; ALT normal

• Reassessment in 1/2010:

HBV DNA 864; HBeAg -; anti-HBe-; ALT 46 U/ml L180IM; M204I; A181ACGS; N235NT; V173L; L180 IM

How was the treatment continued?

• Tenofovir 245 mg from 2/2010 still ongoing
• Reassessment in 5/2010:

HBV DNA neg; HBeAg -; anti- BeAg +; Alt normal

• Reassessment in 2/2011:

HBV DNA neg IU/ml; HBeAg -; anti-HBeAg +; ALT normal; HBsAg pos.

How long would you continue treatment?

• Stop now?
• Continue for 6 months?
• Continue for 12 months?
• Continue until HBsAg seroconversion?
• Continous therapy?

Tenofovir and HBs seroconversion

HBs-Seroconversion: Tenofovir

N=253; HBeAg+; quant. HBsAg unsing Abott Architect

Heathcote et al, EASL 2009 #909 Heathcote et al, Gastroenterology 2011

HBs-Seroconversion: Tenofovir

N=253; HBeAg+; characteristics

Heathcote et al, EASL 2009 #909 Heathcote et al, Gastroenterology 2011

NUCs and treatment response

NUCs: durability of response

AASLD Practice guidelines 2009

10.8% 8,0-16,8% 14,7% 17.6%

* *

* Consolidation treatment

NUCs: durability of HBeAg seroconversion

N=132; Seroconversion in 46 pts (35%) after a mean of 26 months treatment (51% Lam; 25% ADV; 17% ETV; 7% other)

Reijnders et al, Gastroenterology 2010

Nine patients that discontinued treatment:

HBsAg/HBeAg for monitoring therapy

Quantitative HBsAg and response

n=221;Peg-IFN±Lam; response: HBeAg loss and HBV DNA< 100000/cop/ml

Sonnevold et al, Hepatology 2010

HBsAg and response prediction

N=48; PEG-IFN2a 48 Wo; Response: HBV DNA <70 IU/ml Wo 72

Moucari et al, Hepatology 2009

HBsAg-Level at week 24

PEG-IFN: HBsAg and response

n=399; PEG-IFN±Lam; HBeAg+; quant. HBsAg with Abbott Architect

Lau et al, EASL 2009 #917

HBsAg-Level at week 12 HBsAg-Level at week 24 HBe-Serokonversion after 6 months

HBsAg and response prediction Overview

Liaw et al, Hepatology 2011

HBeAg and response prediction

Fried et al, Hepatology 2009

HBV DNA at week 24 HBeAg at week 24

Tenofovir: HBsAg and Response

N= 280; HBeAg+ ; factors associated with response

Heathcote et al, EASL 2009 #909

HBV Genom

Überlappende Leseraster der Polymerase (RT) und des s-Antigens (HBsAg)

HBV: Polymerase and s-Antigen

Polymerase Mutationen s-Antigen Mutationen

HBV drug resistance and immuno escape

Torresi et al.; Virology 2002 (A)

HBV drug resistance and immuno escape

Amini-Bavil-Olyaee et al.; J Virol 2009

rtW153Q/sG145R rtT128N/sP120T

Torresi et al.; Virololgy 2002 (B)

HBsAg mutants associated with escape

envelope

• 163

Y Q G M L P L P T C T T G S T G T P K T S I V T A C P Q P C G N M S F C C P P G T C K D C N T T F K Y P S I W L I C S W A A D L

98-

S

G145R

105L 109R/V/H/I *110M/R/H/I/V *114R 117T 118K/R/S/P 119R 120A/E/LP/Q/S/T/R 121S/Y 123A/N 124R/Y *126N/S/V/I/T *127R/S/T 128V 129H/K/P/R 130D/R *131D/I/S 132A/P 133I/L/T/V *134H/N/R/S/V 137R/S/W/Y 139S 140S 141E/I/R 142L/R/S *143L/P 144A/E/G 145A/K/L/R 146D/S 147S/T * Positions with genotypic specific polymorphisms

Schaefer et al., 2004

Increasing frequencies of HBsAg mutants after failure of antiviral treatment

Lacombe K et al., abstract 638, CROI 2010, San Francisco

308 HIV-HBV Co-infected patients, prospective study 3 years Antiviral Treatment: Lamivudine, Tenofovir

Emergence of HBsAg mutants due to:

• 1. HBV vaccination failure

rare, frequencies are declining

• 2. Immunglobulin treatment of chronic HBV

infection after liver transplantation frequent, low number of patients

• 3. Reactivation of HBV

more and more patients are at risk of HBV reactivation

• 4. HBV treatment

worldwide rising incidence of HBV resistant isolates

Polymerase Mutationen s-Antigen Mutationen

HBV: Polymerase and s-Antigen

polymerase mutations s-antigen mutations Stop-Codon

X X X X

HBV: Polymerase and s-Antigen

Gerlich and Kann in Topley & Wilson

HBV Replikation

Summary

• Choice of medication - has become easier:
• Pegylated interferon-alfa: susceptible HBV genotypes
• NUCS: - First choice: Tenofivir, Entecavir
• Alternatives: Telbivudine, (Lamivudine)
• Out: Adefovir
• „Resistence“ determination - will become easier:
• resistance mutations rarer with more potent drugs
• IL28B - polymorphisms? IL10R, HLA DPB….???
• Treatment monitoring – will become more complex
• HBV DNA, HBsAg?, HBeAg?

Ghany and Dool, Hepatology 2009