Descriptive Epidemiology (1)

Hpatite BFabien ZoulimDpartement dhpatologie & INSERM U1052, Lyon

EPIDEMIOLOGIE DE LHPATITE B

Ott et al, Vaccine 2012EPIDEMIOLOGIE DE L'INFECTION A VHBHpatites aigues VHA : 40%VHB : 30%VHC : 20%incidence : 300 000 infections VHB / an30 000 nouveaux porteurs chroniques / an3 000 dcs / anAUX USAMODES DE TRANSMISSION DU VIRUS DE L'HPATITE B EN EUROPE

sexuelle34%htro23%homo11%drogue IV26%inconnue31%hmodialyss8%transfusions2%personnels de sant 2%contact avecporteur du VHB4%AsieTransmission verticaleDclaration obligatoire de lhpatite B en France : rsultats des 12 premiers mois de notificationDenise Antona, E Delarocque-Astagneau, D Lvy-Bruhldpartement des maladies infectieuses

Results158 acute hepatitis casesHospital doctor in 64% cases Sex ratio M/F : 2,95 (118/40)Median age: 37 yrs for males, 36yrs for females Jaundice : 69%Hospitalisation : 46%Fulminant hepatitis : 3 (2 death)

Risk exposure within 6 months preceding the acute case Source : obligatory declaration 2003-04

Source: obligatory declaration march 03- february 2004 N=145

Sexual 5940,6%No factor4329,6%IVDU 9 6,2%>1 factor3826,3%Invasive treatment 1510,3% Tatoo, piercing 5 3,4%Familial 14 9,7%Perinatal 2 1,4%Live in instiution 11 7,6%Travel in endemic 21 14,5% areas91/145 patients (63 %) had a vaccine indication (2 vaccinated 3 doses) Sentinel networks 91-96 N=195 sexual 35% IVDU 19%percutaneous 15%No factor 35%

Surveillance pidmiologique de linfection HBV14 446 adultes testsPrvalence de lAgHBs 0,65% (280 000 porteurs chroniques du VHB)Homme 1,1% versus 0,2% femmeNaissance en zone dendmie 4% versus 0,5%Prcarit, sjour en institution, homosexualit, usage de droguesMeffre et al, J. Med Virol 2004Hpatites virales B: pidmiologie- Vaccin mais 250 millions de porteurs chroniques dans le monde 280 000 porteurs chroniques en France (INVS) 45% ignorent leur statut 1 300 dcs par an en France 60 000 avec hpatite chronique active Environ 15 000 patients traitsVIROLOGIE FAMILLE: Hepadnaviridae, seul reprsentant humain

VIRUS RESISTANT :- 7 jours dans lenvironnement- pendant 5 mn 100C, 10 h 60C- la conglation.

LE VIRUS DE LHEPATITE B

Ssmall surface proteinMmiddle surface proteinLlarge surface proteincorecapsid proteinHBeAgsecreted e antigenpolpolymeraseHBxX protein (non-secreted)vvvspherefilamentDane particleHBeAgHBsAg

The HBV genomeTiollais, Nature 1985dterminant avaccin/IgHBsGne pol antivirauxMt pre-coreRponse anti-HBe?Mt du coreRponse CTL8 gnotypesA to H

The viral replication cycleZoulim & Locarnini, Gastroenterology 200915Model for sodium-dependent taurocholic cotransporting polypeptide (NTCP) binding to preS1 Seeger C , and Mason W S Gut 2013 in press; Yan H, et al. eLife 2012;1:e00049; Hu NJ, et al. Nature 2011;478:40811.

Sodium-dependent transporter for taurocholic acid Expressed at the basolateral membrane of hepatocytes Mediates the transport of conjugated bile acids 349 amino acid-long glycosylated transmembrane protein. Expression controlled by hepatocyte-specific transcription factors, including HNF3 and C/EBP

Model for sodium-dependent taurocholic cotransporting polypeptide (NTCP) binding to preS1. (A) Model for the topology of NTCP in the cytoplasmic membrane. The model was derived from the crystal structure of the bacterial homologue to SCL10A2 (apical sodium-dependent bile acid transporter), the intestinal bile acid receptor.22 The position of the first amino acid, K157, of the peptide conferring species specificity (see text) is indicated in red. (B) The figure shows an alignment of the amino acid sequences of the two external loop regions shown in (A) between human and mouse NTCP. The sequence of peptide 157165 is highlighted.Model for sodium-dependent taurocholic cotransporting polypeptide (NTCP) binding to preS1Seeger C , and Mason W S Gut 2013 in press; Yan H, et al. eLife 2012;1:e00049; Hu NJ, et al. Nature 2011;478:40811.

Model for sodium-dependent taurocholic cotransporting polypeptide (NTCP) binding to preS1. (A) Model for the topology of NTCP in the cytoplasmic membrane. The model was derived from the crystal structure of the bacterial homologue to SCL10A2 (apical sodium-dependent bile acid transporter), the intestinal bile acid receptor.22 The position of the first amino acid, K157, of the peptide conferring species specificity (see text) is indicated in red. (B) The figure shows an alignment of the amino acid sequences of the two external loop regions shown in (A) between human and mouse NTCP. The sequence of peptide 157165 is highlighted.

Transgenic miceHumanized miceHumanChimpanzeeGibbonbaboonsTupaaWoolley monkeyGround squirrelAmerican woodchuckPekin DuckGrey HeronSummers PNAS 1978, Mason J Virol 1981, Chisari Science 1985, Petersen PNAS 1998, Lanford PNAS 1998The animal models of HBV infection

Cell culture models for HBV infectionZeisel M Zoulim F, Gut 2015

Animal models for HBV infectionZeisel M Zoulim F, Gut 2015 Infection VHB et risque de CHCEtude de Beasley Taiwanrisque relatif = 100 chez les porteurs de l'AgHBsEtude de Tsukumarisque cumumatif de CHC 3 ans12,5% chez 240 patients avec cirrhose3,8% chez 677 patients avec hpatite chroniquerisque x 7 si AgHBs +risque X 4 si anti-HCV +Facteurs associs : alcool, tabac, aflatoxineDiminution incidence avec la vaccination de masse (Chen, NEJM 1995)CARCINOME HEPATOCELLULAIRE ET VIRUS DE L'HEPATITE B Co-incidence de rpartition gographique VHB / CHCPorteurs AgHBs : RR x 100 pour le CHCCHC dans les modles animaux de l'hpatite B :marmottecureuilPrsence d'ADN viral intgr dans les tumeurs

HBV replication and its role in HCC developmentWands, NEJM 2004Role du VHB dans loncognse hpatiqueVHBINFECTION CHRONIQUECARCINOGENESCO-FACTEURSREACTION INFLAMMATOIRE CHRONIQUEREGENERATION HEPATIQUEMUTAGENESE INSERTIONNELETRANSACTIVATION DE GENES CELLULAIRESINTERACTIONS PROTEIQUESINACTIVATION DE GENES SUPPRESSEURS DE TUMEURCHCPHYSIOPATHOLOGIE / IMMUNOPATHOLOGIEHBV and the immune responses

Current model of HBV pathogenesis

HPATOCYTE INFECTVHBCTLFasperforineHPATOCYTENON INFECT

IMMUNOPATHOGNIE DES HPATITES B CHRONIQUESAgHBc/eHLAIcytokinesRPONSE IMMUNITAIRECYTOKINESANTIVIRAUXANTICORPS NEUTRALISANTSIMMUNOPATHOLOGY OF HBV INFECTIONImmune toleranceClearance phaseChronic hepatitisSeroconversionRemission CD8+HBVCD8+HBVCD8+HBVImmunopathology Fulminant hepatitisCD8+HBVHpatocyte infectHBVHpatocyte non infect

Phase de tolrance immunitaireMarqueursAgHBe +HBV DNA +++ALAT = NFoie = NHBc/e AgHpatocyte infectHBVCD8FasperforineHpatocyte non infect

Phase de clairance immune(hpatite chronique)MarqueursAgHBe+HBV DNA > 2000 IU/mLALAT +++Foie: Hpatite chroniqueHBc/e AgHLAIcytokinesHpatocyte infectHBs AgHpatocyte non infect

MarqueursAgHBe-anti-HBe +HBV DNA < 2000 IU/mLALAT = NFoie = rmissionPhase de rmissionportage inactif de lAgHBs RactivationVirus sauvage ou mt pre-coreOncognseCD8CD4Hpatocytes infectsHpatocytes non infects

MarqueursHBsAg -anti-HBc +Anti-HBs +/-PCR srum (-) / foie (+)Clairance de lAgHBsMutants dchappementInfections occultesOncognseCD8CD4 BcccDNA (copies/cell)Total HBV DNA (copies/cell)cccDNA levels in the different phases of chronic HBV infectionHBeAg+ patients had significantly higher cccDNA (90-fold) and total HBV DNA (147- fold) levels compared to HBeAg- patients. (p lsions hpatocytaires : HCAsroconversion anti-HBe spontane (portage inactif) : 5-10% /an> diminution significative rplication virale> amlioration signes histologiquesvirus mut pr-C (-)slection au moment de la sroconversion anti-HBedpend du gnotype viralimmunopathologie ?svrit de l'hpatopathie : controverseassociation au CHC

ALTHBsAgHBeAgHBV DNANormal Months After ExposureALT and HBV DNA IU/L or million copies/mlLaboratory Diagnosis of Chronic Hepatitis B associated with wild type virus infectionSeeger, Zoulim, Mason, Fields Virology 2007ALT``HBsAgHBeAgHBV DNANormal Months After ExposureALT and HBV DNA IU/L and million copies/mlAnti-HBeLaboratory Diagnosis of Transition of Chronic Hepatitis B to The inactive Carrier State010020030040050060070080001234561224364860728092104Seeger, Zoulim, Mason, Fields Virology 2007

ALTHBsAgHBV DNANormal ALT levelsMonths ALT and HBV DNAIU/L and million copies/mlAnti-HBeHBeAgLaboratory Diagnosis of HBeAg negative Chronic Hepatitis BSeeger, Zoulim, Mason, Fields Virology 20070,0010,010,11101001000ALATADN-VHBAgHBe +anti-HBe +UI/mlpg/mlAgHBsTolrance hp chronique p. inactif mt pr-core VHB occultehybridationPCR9 log8 log7 log6 log5 log4 log3 log2 log1 logDynamic ranges of quantificationof HBV DNA assaysAmplicor HBV Monitor v2.0 (Roche)HBV Hybrid-Capture II (Digene)Ultra-sensitive HBVHybrid-Capture IIVersant HBV DNA3.0 (bDNA, Siemens)Cobas Taqman HBV(Roche)Abbot Real-time HBV(Abbott)Versant HBV DNA 1.0(kPCR, Siemens)**in development10102103104105106107108109RealArt HBV LC PCR(Artus Biotech)47JMFormes cliniquesMANIFESTATIONS EXTRAHEPATIQUES DU VHBPANComplexes immuns circulants HBs/anti-HBsDpots artres moyens et petit calibreTraitement : plasmaphreses, corticoides, antiviraux (vidarabine / IFN / famciclovir / lamivudine)GlomrulonphritesCryoglobulinmiesGuillain-BarrMyocarditeTRANSMISSION VERTICALE DU VHBmre AgHBe +transmission : 90%mre anti-HBe +transmission : 10-20%VHB mut pr-C (-) : hpatites fulminanteschronicit chez lenfant : 90%PRESENTATION CLINIQUEINFECTION PERI-NATALEALT normales ou subnormalesADN-VHB > 1000 pg/mlhistologie : lsions minimesINFECTION POST-NATALEALT levesADN-VHB < 1000 pg/mlhistologie : hpatite modre svreCARCINOME HEPATOCELLULAIRE : 30 ANSPathophysiologic Cascade of Chronic HBV InfectionHBV Replication(Measured by Serum HBV DNA)Liver InflammationWorsening Histology Necroinflammation Fibrosis CirrhosisDisease Progression Liver Failure Liver Cancer Transplant Death0Adapted from: Lavanchy D. Journal of Viral Hepatitis, 2004, 11, 97107. Chen JC, et al. JAMA. 2006;295:65-73. Iloeje U. H, et al. Gastroenterology. 2006;130:678-86.ALT ElevationThis describes the pathophysiologic cascade of the chronic HBV infection.

The early phase of CHB is characterized by the presence of hepatitis B e antigen (HBeAg) and high serum levels of HBV DNA (referred to as HBeAg-positive CHB). Following infection, the immune system attempts to clear the HBV by destroying infected hepatocytes.

This leads to increasing circulatory blood levels of alanine aminotransferase (ALT). However, the majority of patients will clear HBeAg (and produce anti-HBe antibodies) and achieve a state of nonreplicative infection, characterized by low or undetectable serum levels of HBV DNA and normal ALT levels.

High HBV DNA and ALT levels may persist in some anti-HBe-positive patients (referred to as HBeAg-negative CHB) because of the presence of an HBV variant that is unable to produce HBeAg (HBeAg-negative variant, also called HBV precore stop codon mutant).

Charge virale et incidence de la cirrhoseR.E.V.E.A.L. HBV StudyAnne de suiviIncidence cumulative de cirrhose.2.10123456789101112130.4.3P 104 - 106106This compares the cumulative hepatocellular carcinoma incidence at the end of the 13th year of follow-up derived from the stepwise analyses of different baseline viral levels. Approximately 15% of all participants with serum HBV DNA levels of 1 million copies/mL or greater at study entry developed hepatocellular carcinoma by the 13th year of follow-up compared with 1.3% of participants with undetectable levels of HBV DNA.The biological gradient of cumulative hepatocellular carcinoma incidence by serum HBV DNA level remained prominent in all stepwise analyses. Among the 2925 participants seronegative for HBeAg with a normal ALT level and no liver cirrhosis, the cumulative hepatocellular carcinoma incidence was 13.5% for HBV DNA levels of 1 million copies/mL or greater and 0.7% for those with undetectable levels of HBV DNA. Serum hepatitis B virus (HBV) DNA level is a marker of viral replication and efficacy of antiviral treatment in individuals with chronic hepatitis B.

The objective of the REVEAL study was to evaluate the relationship between serum HBV DNA level and risk of hepatocellular carcinoma.This was a prospective cohort study of 3653 participants (aged 30-65 years), who were seropositive for the hepatitis B surface antigen and seronegative for antibodies against the hepatitis C virus, recruited to a community based cancer screening program in Taiwan between 1991 and 1992.The main outcome measure was the incidence of hepatocellular carcinoma during follow-up examination and by data linkage with the national cancer registry and the death certification systems.

164 incident cases of hepatocellular carcinoma and 346 deaths during a mean follow-up of 11.4 years and 41 779 person-years of follow-up were reported. The incidence of hepatocellular carcinoma increased with serum HBV DNA level at study entry in a dose-response relationship ranging from 108 per 100 000 person-years for an HBV DNA level of less than 300 copies/mL to 1152 per 100 000 person-years for an HBV DNA level of 1 million copies/mL or greater. The corresponding cumulative incidence rates of hepatocellular carcinoma were 1.3% and 14.9%, respectively. The biological gradient of hepatocellular carcinoma by serum HBV DNA levels remained significant (P.001) after adjustment for sex, age, cigarette smoking, alcohol consumption, serostatus for the hepatitis B e antigen (HBeAg), serum alanine aminotransferase level, and liver cirrhosis at study entry. The dose-response relationship was most prominent for participants who were seronegative for HBeAg with normal serum alanine aminotransferase levels and no liver cirrhosis at study entry. Participants with persistent elevation of serum HBV DNA level during follow-up had the highest hepatocellular carcinoma risk.

Elevated serum HBV DNA level (10 000 copies/mL) is a strong risk predictor of hepatocellular carcinoma independent of HBeAg, serum alanine aminotransferase level, and liver cirrhosis.

High Baseline Serum HBV DNA Levels are Associated with Increased Risk of HCC Mortality in HBsAg-Positive Patients

HBV DNA NegativeHBV DNA Low< 105 copies/mL RR = 1.7 (0.5-5.7)HBV DNA High 105 copies/mL RR = 11.2 (3.6-35.0)p < 0.001 across viral categorieshttp://www.fccc.edu/docs/sci_report/Evans.pdf#search=%22haimen. Accessed 1/23/07.Chen G, et al. J Hepatology 2005; 42 (suppl 2):477A.Chen G, et al. Hepatology 2005; 40 (suppl 1):594A.This describes the relationship between high baseline serum HBV DNA levels with increased risk of HCC mortality.

In a prospective cohort study with 11 years of follow-up, Evans and colleagues assessed the relationship between past HBV viral load and mortality.

They measured HBV viral load by real-time PCR on stored samples from cohort entry (19921993) in 2763 hepatitis B surface antigen (HBsAg)-positive adults from a prospective cohort in Haimen City, China. Follow-up was completed through 2003, with information on deaths occurring during this interval abstracted from death certificates. Major endpoints were death from HCC or chronic liver disease (CLD). There were 447 deaths in total.

Viral load was divided into three categories: undetected ( AgHBe +ractivation virus mut pr-C (-)Corticothrapie, biothrapie, chimiothrapiesurinfection delta / VHC

COOH13714910799NH2 S - SS - SS - SS- SS-S138139147Tiollais P. et al., Nature 1985. Torresi J., J. Clin Virol 2002; Dryden KA. et al., Mol Cell 2006a determinantHBs Aga determinant induces the synthesis of anti-HBs neutralizing antibodiessG145R sP120T sD144H/A/E

PreS1PreS2SPolPr-CCBrin(+) 2,4kbBrin(-) 3,2kbXTATAAU5-likeDR1DR2Enh1Enh2GRE0/3221SHBs (S)MHBs (preS2+S)

LHBs (preS2+preS2+S)

76Variants de l'Ag HBschappement la rponse humorale anti-HBsnaturellevaccination (transmission mre-enfant)immunoprophylaxie (transplantation hpatique)infection active malgr Ac anti-HBssrologie AgHBs faussement ngative Risques : transmission virale + infections occultesVARIANTS DE L'AgHBsMutations ponctuelles dans le dterminant a de l'AgHBs (124-147)aa 145 : Gly -> Arg aa 126 : Ile -> Ser / Thr -> Asntransmission mre-enfant malgr la serovaccination (3%)infection du greffon hpatique malgr Immunoglobulines anti-HBshpatites chroniques avec anti-HBc et anti-HBs +Presence of HBV DNA in the liver ( serum) of individuals testing HBsAg negative by currently available assaysOccult HBV Infection (OBI)

Raimondo et al, J Hepatol 2008How to Detect Occult HBV InfectionCurrently there is no standardized diagnostic assay for occult HBV infection

Reported Prevalence of Occult HBV Infection in HIV Positive PatientsStudy

Country

N ofpatients

Occult HBVN (%)Methods

Hofer, 1998

Switzerland

57

51 (89%)

nested PCR(serial evaluation)Torres-Baranda, 2006

Mexico

35 7 (20%)nested PCRFilippini, 2006

Italy

86

17 (20%)

single step PCR

Mphahlele, 2006

South Africa

14031 (22.%)nested PCRPogany, 2005

Netherlands

93

4 (4%)

single step PCRNeau, 2005

France

160

1 (0.6%)

Santos, 2003

Brazil

101

16 (16%)

single step PCR

Wagner, 2004France 30 11 (37%)nested PCRGoncales, 2003Brazil159 8 (5%)nested PCRNunez, 2002

Spain

85

0

Cobas Amplicor HBV Monitor (Roche)Piroth, 2000

France 37

13 (35%) single step PCR Raffa, 2007

Italy

nested PCR (liver)Cobas Amplicor HBV Monitor (Roche) 101 42 (41%)Raimondo et al, J Hepaol 2007, modifiedOBICause(s) for the failure of HBsAg detectionSuppression ofHBV replication and gene expressionInfection byS gene Variantsfalse OBIOccult HBV infectionHBV cccDNAIntegrated HBV DNAHBV mutantsEpigenetic controlHBV replicationImmune surveillanceViral co-infectionsOBI Seropositive SeronegativeHBsAg lost during CHHBsAg lost after AHProgressive antibody disappearence Primary occult Schematic representation of HBV serum marker profile in OBI and false OBI false OBIS gene escape mutantsHBV DNA levels comparable to overt infectionHBV DNA levels < 200 UI/ml84

High prevalenceROLEin HCCDiagnosticTools ?Worsen HCVinfection ?Co-infections ?Therapy?To beimprovedSpecific treatments ?Not fully understood ?Occult HBV infections: unresolved issuesOrganization chart boxes will automatically fill with secondary color but you may want to change the box outlines and connectors to stand out on the page: Double click on the organization chart to enter Microsoft Organization Chart. Select Edit/ Select/ All in order to highlight all boxes.Select Boxes/ Border Color/ White.Select File/ Close and Return to Your Presentation Name.You will be prompted to update your presentation if you have not already done so.

AntivirauxPersistance viraleResistance aux antivirauxMonitoring des traitementsHBeAg(+)HBeAg(-) / anti-HBe(+)ALATHBV DNA Minimal CHModerate to severe CHModerate to severe CHRemissionCirrhosisImmunotolerantphaseImmuno-activephaseInactive phaseLow replication Reactivation phaseCirrhosis109-1012 IU/mL>2000- total intrahepatic HBV DNA > cccDNA Changes in HBsAg levels correlated with cccDNA changes-> 14 years of therapy to clear completely viral cccDNAWerle et al, Gastroenterology 2004

0.8 log10 (84%) decline in cccDNA, not paralleled by a similar decline in the number of HBcAg+ cellsSuggests cccDNA depleted primarily by non-cytopathic mechanisms or that cell turn-over occurred but was associated with infection of new cells during therapy Immunohistochemical Staining of Patient Biopsies at Baseline and After 48 Weeks ADV TherapyBaselineWeek 48

Maynard et al, J Hepatol 2005Persistence of cccDNA after HBs seroconversionClearance of viral infection versus selection of escape mutantsThe most important factors to consider: The rate of immune killing of infected hepatocytesThe rate of replication and spread of mutant virus in the chronically infected liver (I.e. fitness of the virus: the rate of spread to uninfected hepatocytes)Small changes in these factors may have profound effect on whether treatment response is durable or subject to rapid rebound (Litwin et al J Clin Virol 2005)These factors may be subject to therapeutic interventionKinetics of spread and emergence of drug resistant virus during antiviral therapyZhou T, et al. Antimicrobial Agents and Chemotherapy 1999; 43: 1947-1954.antiviralwtniFree liver spaceMutant fitnessIIIIIIIVINHIBITION OF WILD TYPE VIRUS REPLICATIONSDELAYED EMERGENCE OF DRUG RESISTANT VIRUSni = non-infectedwt = wild typemt = mutant type mtKinetics of HBV drug resistance emergenceSi Ahmed et al. Hepatology. 2000; Yuen et al Hepatology 2001; Locarnini et al Antiviral Therapy 2004; Villet et al Gastroenterology 2006 J Hepatol 2007 & 2008; Pallier et al J Virol 2007; Yim et al Hepatology 2006. Treatment beginsDrug-resistant variant

Drug-susceptible virusNaturallyoccurring viral variants Time HBV replicationPrimary resistance mutationsSecondary resistance mutations/ compensatory resistance mutationsFZ

cccDNA in the liver:Is propagated during the normal replication cycle of HBVCan serve as a template for the production of new virus

Archiving of viral variantsViral quasispeciescccDNA variantsLiverMajority populationMinority variants Resistant variants Blood circulation Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008KEY TAKEAWAYArchived covalently closed circular DNA (cccDNA) plays an important role in viral persistence.

This slide provides an overview of the archiving process. When HBV infects a hepatocyte in the liver, viral replication in the hepatocyte results in the formation of cccDNA within the cell. This cccDNA can serve as a template for the production of new virus, but can also remain within the hepatocyte, where it is said to be archived. Archived cccDNA plays an important role in viral persistence and in the reactivation of viral replication after the cessation of antiviral therapy.

cccDNA in the liver:Is propagated during the normal replication cycle of HBVCan serve as a template for the production of new virus

It is believed that viral variants with antiviral resistance may be archived in this wayArchiving of viral variantsViral quasispeciescccDNA variantsBlood circulation LiverMajority populationMinority variants Resistant variants Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008KEY TAKEAWAYViral variants with antiviral drug resistance may be archived in the form of cccDNA.

By drawing comparisons with animal models of HBV infection, it is believed that drug resistance may be archived in the form of cccDNA. In this slide, a viral variant with drug resistance (represented by the red circle) is formed via a point mutation during the replication of a sensitive variant (represented by the light blue circle). As described on the previous slide, during the normal course of replication cccDNA from this resistant variant may become archived in hepatocytes. Once archived in this way, drug resistance may persist for long periods in the absence of drug.Archived cccDNA molecules in hepatocytes act mainly as a reservoir for future viral replication and are therefore not inhibited by nucleos(t)ide analogues, which inhibit replication when they are incorporated into nucleic acid molecules during replication.

cccDNA in the liver:Is propagated during the normal replication cycle of HBVCan serve as a template for the production of new virus

It is believed that viral variants with antiviral resistance may be archived in this wayArchiving of viral variantsViral quasispeciescccDNA variantsLiverMajority populationMinority variants Resistant variants Blood circulation Zhou et al, AAC 1999; Zoulim F. Antivir Res. 2004. Zoulim F & Perillo R. J Hepatol. 2008Since cccDNA acts as a reservoir for future viral replication, the archiving of resistant variants may lead to the persistence and expansion of this population as demonstrated on this slide where the number of resistant variants (red circles) has increased to become the majority population. Definition of fitnessA parameter that quantifies the adaptation of an organism or a virus to a given environment

For a virus, ability to produce infectious progeny relative to a reference viral clone, in a defined environmentEsteban Domingo, In Fields Virology 2007Cross-resistance data for the main mutants and the commercially available drugsZoulim & Locarnini Gastroenterology 2009; Liver Int 2013PathwayAmino Acid Substitutions in the rt DomainLMVLdTETVADVTFVWild-typeSSSSSL-Nucleoside (LMV/LdT)M204I/VRRISSAcyclic phosphonate (ADV)N236TSSSRIShared (LMV, LdT, ADV)A181T/VRRSRIDouble (ADV, TFV)A181T/V + N236TRRSRRD-Cyclopentane (ETV)L180M+M204V/I I169 T184 S202 M250RRRSSMulti-Drug ResistanceA181T+N236T+ M250VRRRRR119119Phenotyping of HBV clinical isolates1. Durantel D, et al., Hepatology, 2004;40:855-64. 2. Yang H, et al., Antiv Ther, 2005;10:625-33. Lab Strain

Clone AClone AClone CClone DClone ESouthern blotanalysisPatient serumPCR cloningWhole genomeHBV clonesTransfectionHepG2Huh7

IC50 reference strainIC50 mutantFold resistance = Wild-type virusIncreasing antiviral concentrationCell culture platePatients virusSS -RC -

lamivudineadefovir

ADVrtN236T +/or rtA181V Wild-type virus ADV-resistant virus LAM-resistant virusLAMrtM204V/I rtL180M ETV-resistant virusrtT184 or rtS202 or rtM250ETVrtM204V/I rtL180M+/-TDFTDF: what can we expect?rtM204V/I +/- rtL180MLAMthen ETV rtT184 or rtS202 or rtM250LAM + TDF what do we see?Maximising the barrier to resistance?Multiple drug resistant mutants with complex pattern of mutations+ one mutation+ one mutationDrug ADrug BRisk of selection of MDR mutants by sequential therapy drugs sharing cross-resistance characteristics incomplete viral suppression liver transplantationThe problem of sequential therapy with nucleoside analoguesZoulim F, et al. J Hepatol. 2008;48:S2-19. Yim et al, Hepatology 2006; Villet et al Gastroenterology 2006 & 2009127103104105106107108109020406080100120Treatment (months)HBV DNA (copies/ml)entecavirIFNadefovirlamivudineGenotype HlamivudineDrugs sharing cross-resistance characteristics:Switching strategy emergence of MDR mutantL180M+S202G+M204VL180M+M204VVillet et al, J Hepatol 2007

Warner et al Hepatology 2009Kamili et al Hepatology 2009Villet et al Gastroenterology 2009Impact on virus infectivity and fitnessImpact on virion release (intracellular retention) and virologic monitoring of breakthroughImpact on vaccine prophylaxis efficacyVirologic Consequences of Persistent Viremia Infection of new hepatocytes slower kinetics of clearance infected cells and cccDNA

Increases the risk of occurrence and subsequent selection of HBV mutations responsible for drug resistance

On-treatment prediction of HBV drug resistanceLe Guerhier et al Antimicrob Agents Chemoter 2000;44:111-122; Delmas et al Antimicrob Agents Chemother 2002; 46:425-433; Kock et al Hepatology2003; 38:1410-1418; Richman Hepatology 2000;32:866-867Patients heavily exposed to NUCs with low barrier to resistance Risk of MDR selectionRisk of multidrug resistance by sequential accumulation of resistance mutations

Risk of partial response, even with the newest NUCs -> long-term impact ??Multiple drug resistant mutants with complex pattern of mutations+ one mutation+ one mutationDrug ADrug BRisk of selection of MDR mutants by sequential therapy drugs sharing cross-resistance characteristics incomplete viral suppression liver transplantationThe problem of sequential therapy with nucleoside analoguesZoulim F, et al. J Hepatol. 2008;48:S2-19. Yim et al, Hepatology 2006; Villet et al Gastroenterology 2006 & 2009133

Liu et al, Antivir Ther. 2010;15(8):1185-90.Sequential therapy with NUCs and the risk of MDRAccumulation of multiple mutations on the same viral genome

Complete change of the viral quasi-speciesA single a.a. substitution at position rt181 may be responsible for multidrug resistanceVillet S, et al. J Hepatol. 2008;48:747-55.wtA181VA181TA181V + N236TA181T + N236TN236TN236T + N238TM204VM204IL80VL80V + M204ILVDLVD+TDFLVD+ADV+TDFPatient #1(67 months)Patient #7(30 months)Patient #2(23 months)Patient #3(37 months)Patient #10(7 months)Patient #5(44 months)Patient #4(31 months)Patient #6(36 months)Patient #9(19 months)Patient #8(47 months)LVD+ADVADV135Impact of rtA181 and rtN236 mutations on antiviral drug efficacy and cross-resistance

Villet et al, J Hepatol 2008136

Viral loadBL viral load = 8.75logTreatment: TDFAdherence : 95.2%Patient 1051 data:LLODEvolution of viral genome during Tenofovir therapy in patients who previously failed ADVImpact of persisting low viremia levels on treatment outcome ?Impact of persisting resistant mutants ?Lavocat et al, AASLD 2010 & Ms submitted Virologic response to TDF according to ADV resistance mutations at baseline The Australian Experience

Patterson S J et al. Gut 2011;60:247-254Individual viral load profiles and proportion of patients achieving 10E9 copies/mL - Devrions nous raliser une biopsie lorsque la charge virale diminue sans lvation des ALAT ? Et penser un traitement antiviral ? Zoulim & Mason, W. S. Gut 2012Effective T-cells control virusExhausted T-cells lose control of virusCD8 T cellsInfected hepatocytesInfected hepatocytesINF-gTNF-aIL-2GranzymePerforinSpecific immunomodulation of existing T-cells e.g. PD-1 blockade1,2Patients who have resolved HBVPatients with chronic HBVRestoration of defective T-cell immune control1. Fisicaro P, et al. Gastroenterology 2010;138:68293. 2. Fisicaro P, et al. Gastroenterology 2012;143:157685Figure adapted from Nebbia G, et al. Q J Med 2012;105:10913 and Freeman G, et.al. J Exp Med 2006;203(10):22237. 148HBsAg clearance

Werle-Lapostolle B et al., Gastroenterology 2004;126: 1750-58.

Infected hepatocytesInfected liverCD8NKT CD4B cccDNAAntiviralsClearance of HBsAg?Blood circulationviral loadPerspectives / Prevention of drug resistanceFirst line therapyUse of antivirals with high antiviral potency and high barrier to resistanceCombination therapy with complementary drugs to increase the barrier to resistanceSecond line treatmentAdd-on strategies with complementary drugs preferred to sequential monotherapiesEarly treatment adaptation to prevent accumulation of mutationsChoice always based on cross-resistance data

Prevention of resistanceImpact of first line therapyChoose an antiviral drug withA potent antiviral activityA high barrier to resistance15163LVDADVLdTETVTDF0102030405060708023Proportion of patients (%)4655718001118295250.20.51.201234512345121234512300Option to add emtricitabine at week 72**Patients confirmed to be viraemic at Week 72 or beyond could add emtricitabine to TDF at the discretion of the investigator. Clinical data on the safety and efficacy of emtricitabine and TDF in CHB are pending

Rates of resistance with lamivudine (LVD), adefovir (ADV), telbivudine (LdT), entecavir (ETV) and tenofovir (TDF) among NA-nave patients40High barrier to resistance

50Gish, Jia, Locarnini, Zoulim, Lancet Infect Dis 2012152Zoulim & Locarnini, Gastroenterology 2009; EASL CPG J Hepatol 2009 & 2012Mangement of antiviral drug resistanceImpact of second line therapyEarly treatment adaptation to prevent accumulation of mutationsChoice always based on cross-resistance dataAdd-on strategy versus switch ?Good results with TDF switchSome cases of suboptimal responsesCombination to increase the barrier to resistance

153Cross-resistance data for the main mutants and the commercially available drugsZoulim & Locarnini Gastroenterology 2009; Liver Int 2013PathwayAmino Acid Substitutions in the rt DomainLMVLdTETVADVTFVWild-typeSSSSSL-Nucleoside (LMV/LdT)M204I/VRRISSAcyclic phosphonate (ADV)N236TSSSRIShared (LMV, LdT, ADV)A181T/VRRSRIDouble (ADV, TFV)A181T/V + N236TRRSRRD-Cyclopentane (ETV)L180M+M204V/I I169 T184 S202 M250RRRSSMulti-Drug ResistanceA181T+N236T+ M250VRRRRR154154ImmortalizationTransformationAvailabilityVariabilityRate of infectionDMSO for infectioncccDNA levels *HBV propagationInnate immunityMaintenance

Primary human hepatocytes--++++20-100 %1.8-2 %1-2 copies per nuclei-+++2-3 weeks

Differentiated HepaRG cell line+-+++++5-20 %1.8-2 %0.2-0.5 copies per nuclei-+++> 6 months

HepG2/Huh7cell lines++++++0 %0 %----

NTCP-HepG2cell line++++++50-100 %2.5-3.5%1-5 copies per nuclei--10 days

chimpanzeesmacaquesTupaa belangeriHuHep miceHis-HuHep miceAd-HBV or AAV-HBV mice

HBV entry + ? + + + -

HBV production + + + + + +

cccDNA establishment + ? + + + -

Chronic HBV infection - ? + + ? +

HCC development - ? ? ? ? ?

Adaptive immune responses + ? ? - + +

HBV tolerance - ? ? - + +

Antiviral drug testing + ? + + + +

Therapeutic vaccine development + ? ? - + +