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400million are chronicallyinfectedwith HBV (170million with HCV) 1 million die (HBV-relatedliver cirrhosis, hepatocellular carcinoma) currently there are no curative treatment options for chronic HBV infections.
400million are chronicallyinfectedwith HBV (170million with HCV) 1 million die (HBV-relatedliver cirrhosis, hepatocellular carcinoma) currently there are no curative treatment options for chronic HBV infections.
400million are chronicallyinfectedwith HBV (170million with HCV) 1 million die (HBV-relatedliver cirrhosis, hepatocellular carcinoma) currently there are no curative treatment options for chronic HBV infections.
Stephan Urban, Prof. Dr. rer. nat. UniversityHospitalHeidelberg,DepartmentofInfectiousDiseases,Molecular Virology,Heidelberg,Germany M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g The global burden of Hepatitis B Virus (HBV) infection World wide ~2 billion people carryserological markers (HBsAg, anti-HBc) relatedto HBV infection. ~400million are chronicallyinfectedwith HBV (~170million with HCV). ~1 million die (HBV-relatedliver cirrhosis, hepatocellular carcinoma (HCC)). currentlythere are no curative treatment options for chronic HBV infections; vaccinationfor prevention Highly endemic regions : South East Asia, China, Central Africa. Risk of infection >60% vertical transmission to children Areas with medium endemicity: South- Eastern Europe, Middle East, Northern Africa, J apan, Mesoamerica, Russia. Risk of infection 20-60% Low prevalence areas: Northern America, West Europe, Australia, Parts of South America. Risk of infection <20% (horizontal transmission, sexual contacts) M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g HBVformsdifferenttypesofinfectiousandnoninfectiousparticles 10 4 10 10 /ml infectious virions of 4247 nm diameter with nucleocapsid (1) Long and short nucleocapsidfree filaments; 2225 nm in diameter (2) spherical, nucleocapsidfree particles with 2225 nm diameter (3); SVPs are synthesized in huge excess over virions HBsAg = SVPs (mostly spherical particles) HepatitisBvirions andsubviral particles Seitz et al., EMBO J., 2007 M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g Hepatitis B Virus Genome organization and particle morphology HBV encodes sevenproteins in a partiallydouble strandedhighlyoverlappinggenome (zip-genome) efficacyof transcriptiondepends on liver-specific transcriptionfactors (contributes to liver specificity) HBx, a regulatoryviral proteincontrols transcriptionof viral mRNAs fromcccDNA M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g Thereplication cycle of HBV;crucial steps for interference Urbanetal.,J.Hepatology,2010 NTCP/SLC10A1* *Yanetal,eLife,November2012 M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g cccDNA is synthesized de novo from incoming virus.(this pathway is not blocked by RT- inhibitors) .or after reimport of newly formed mature (!) rcDNA-containing nucleocapsids ( amplification ) cccDNA formation depends on the activity of cellular DNA-repair enzymes (most are unknown) There are no drugs that target and destroy cccDNA directly (innovative approach; site specific nucleases) cccDNA stability is assumed to be stable in resting (!) hepatocytes the ultimate goal of therapy is cccDNA eliminiation and HBsAg seroconversion. Formationof cccDNA inthe establishment of HBVinfection M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g Approvedtherapieswithnucleosideanaloguesareefficientinthesuppression ofviremia butgenerallynoncurative Why? Nucleosideanaloges:(Lamivudine,Adefovir,Entecavir,Telbivudine,Tenofovir) fastandefficientsuppressionofviraltitersintheserum,preventingdiseaseprogressionbut: onlyslowreductionofHBsAg titersduringtherapy developmentofresistantmutantswithsomedrugs(e.g.Lamivudine,Adefovir,Entecavir) naiive hepatocytes establish cccDNA inthe presence of NUCs undetectable serum levels dont necessarily mean complete virus suppression Morethan 5x10e6receptor molecules/hepatocyte (highvirus binding capacity of the liver) Novel intrahepatic infections of hepatocytes might occur even under strongvirus suppression Adefovir/ADV/Hepsera (Gilead,approved2002) AMPAnalog Entecavir/ETV/Baraclude (BMS,approved2006) GuanosinAnalog (HBVpolselective) Lamivudine/LMV/3TC/Zeffix (GSK,approved1998) CytidinAnalog Telbivudine/LThymidin/Sebivo (Novartis,approved2007) ThymidinAnalog Tenofovir/TDF/Viread (Gilead,approved2002/2008) AdenosinAnalog M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g cccDNA clearance in the course of a natural infection cccDNA isregularlyclearedfollowingacuteinfectionofimmuncompetentindividuals Incontrasttoretroviralinfectionswheregenomeintegrationismandatoryforvirusreplicationand inheritedtodaughtercellstheepisomal cccDNA canbecomeeliminatedfromhepatocytes 2-5 % 95-98 % 0,1 % 65-80 % asymptomatic 25-33 % progressive liver disease M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g Strong, polyclonal and multispecific CD4+ and CD8+ T-cell response How can cccDNA be cleared in the course of a natural infection? The current view: (1) Killing (apoptosis) of infected hepatocytes Division of infected cells, propagation and dilution of cccDNA Division of healty cells adopted from Anna S.F. Lok, EASL Monothematic conference 2005 M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g 2. Non-cytolytic mechanism by curation of cells cccDNA elimination without cell death through e.g. direct action of cytokines (curing of cells) TNF-, IFN- adopted from Anna S.F. Lok, EASL Monothematic conference 2005 ? M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g cccDNA depletion under NT-therapy depends on the number of cccDNA molecules/cell.. If this holds true the half-life time of infected hepatocytes. the rate of de-novo infection of naive or cleared hepatocytes under therapy and would probably take decades Hypothesis based on the assumption that cccDNA survives cell division ! M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g day 1 post plating 0 7 14 21 28 35 day 5 post plating plating DMSO addition (2% Endk.) day 9 post differentiation Infection with HBV 40 Det. of viral markers cultivation differentiation Infection HepaRG cells to study the authentic cccDNA-based HBV replication cycle in vitro Gripon, Rumin, Urban et al., PNAS, 99, 15655-15660 (2002) completely differentiated HBV infected M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g 1well infected HepaRG 2wells Split 1:2 d5p.i. 4wells P0 P1 P2 Split 1:2 d8p.i. P1atd8 P2atd13 P0atd8 AntiCore Rapid loss of intracellular HbcAg-expression following cell splitting existing cccDNA gets lost during cell division (no immune system) Ineffective replenisment of cccDNA by nucleocapsids reimport from the cytosol M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g How is reimport of nucleocapsids regulated inHBVinfected cells? M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g HBVinfected hepatocytes support superinfection and replication of HDV(notsurprising). HBVinfected hepatocytes support HBVentry butprevent nucelocapsid import and denovo cccDNA formation (unexpected). Thepresence of the Lproteinis sufficient to compromise infection. Hypothesis:reimport of nucleocapsids cannot occur inthe presence of Lproteinpresent!Replenishment of cccDNA is inefficient Therapeutical implications:Entryinhibition and the induction of hepatocyte proliferation might be akey for curative therapies. Conclusions and clinical consequences M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g A determinant in the preS1 region required for HBV entry Le Seyec et al. Infection process of the hepatitis B virus depends on the presence of a defined sequence in the pre-S1 domain. J Virol. (1999) 73(3):2052-7. Gripon et al. Myristoylation of the hepatitis B virus large surface protein is essential for viral infectivity. Virology (1995) 213(2):292-9. M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g Incubation o/n at 37C Collection of supernatant of days 8-12p.i. Measurement of secreted HBsAg/HBeAg etc. Infection of HepaRG cells or PHH Gripon et al., PNAS, 99 (24) 2002 Urban et al., J . Virol, 79 (3), 2005 Glebe et al., Gastroenterology, 129, 2005 Engelke et al., Hepatology, 43, 2006 Schulze et al., Hepatology, 46, 2007 AsyntheticpeptidederivedfromthelargeenvelopeproteinofHBVblocks HBVinfectioninHepaRGcellculture. M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g FinemappingofthesequencerequirementsforHBVinfectioninhibition: DefinitionofMyrcludexBasaleadsubstanceforclinicaldevelopment Schulzeetal.,J.Virol,2010 Myrcludex B M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g transplanted cells TransplantationofuPA/RAG2micewithprimaryhumanhepatocytes(PHH) humanhepatocytes(HH) immunedeficientuPAmice transplantation AtransplantedmousemodeltostudyinvivoinfectionofHBV cimeric liver mouse cells NatBiotechnol.,26:335341(2008) M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g MyrcludexBblocksHBVinfectionaftersubcutaneousadministrationinPHH transplanteduPAmice Petersenetal.,NatBiotechnol.,26:335341(2008) Why is s.c.administration of HBVpreS/248 myr soefficient? M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g MyrcludexBaccumulatesintheliverofmiceafteri.v.injection siteof injection Liver Stearoyl-GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGy-I 125 conserved domain Schieck etal.,Hepatology 2013) M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g WT G12E Stearoyl-GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGy-I 125 Stearoyl-GQNLSTSNPLEFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGy-I 125 WT G12E Asingleaminoacidexchangeinthehighlyconservedreceptorbindingsite abolishespeptidehepatotropismandliveraccumulation M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g Pharmakokinetics:Targeting to the liver within minutes Longhalflife timeat the hepatocyte Inhibitoryactivity:IC 50 90pM GMPMyrcludexB LeadSubstance:NterminallyderivedlipopeptideoftheHBVLprotein HepatitisBVirion StateofMyrcludex Bclinicaldevelopment GMPproductionof100gMyrcludex Baccomplished(stabilitystudiessuccessful) Longtermtoxicitystudiessuccessfullycompletedwithoutdrugrelatedsideeffects SingledoseandPKstudiesin3chimpanzeescompleted(livertargetingconfirmed) ApprovalbytheBfArM forPhase1aclinicaltrialinhealthyvolunteers(May2011) Successfulcompletionofthesingledoseescalatingphase1study(24individuals)inFebruary2012 Startof amultipledoses.c.PhaseIb study and aphase IIa efficacy study inNovember2012. Searchfor apharmaceutical company for further product development M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g *Yanetal,eLife,November2012 M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g ControlofcccDNA mightbeobtainedbyasustainedcombinationof inducedcellproliferationandentryinhibition Myrcludex BafirstinclassentryinhibitorofHBV/HDVcurrentlyinphaseIIa trialwillhelptoclinicallyprovethatconcept Novel NTCPbased cell culture systems willaccelarate future drug development including those that directly target cccDNA Summary,conclusionandoutlook HBVcccDNA isnotefficientlypropagatedtoprogenycellsafter inductionofhepatocytedivision. Futuretherapeutic approaches should aim at eliminating cccDNA,the key regulator of HBVreplication NTCPaddressing HBVpreSpeptidesare useful vehicles for liver specific drug targeting cccDNA clearence is possible and can be achieved by several means M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g PossibleapplicationsforHBVpreSlipopetidemediateddrugdelivery imagingofliverdiseases targetedHBVtherapies(e.g.Entecavir,Adefovir...) targetedHCVtherapies(e.g.Proteaseinhibitors,polymeraseinhibitors..) targetedtherapiesforhepatocellularcarcinoma(HCC)(e.g.apoptosis induction,inhibitionofangiogenesis,kinaseinhibitors Sorafenib) targetedMalariatherapies(e.g.primaquine) deliveryofpeptidesforhepatocytespecificantigenpresentation deliveryofsiRNAsbyhepatotropicliposomesornanoparticles (e.g.metabolicdiseases,infections...) targetedInterferontherapiestoavoidsideeffects(chronicHepatitisB,C) M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g Positronemissiontomography(PET)ofanintravenouslyinjectedHBV unrelated 68 Galabeledpeptideinarat M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g Positronemmissiontomographie(PET)ofintravenouslyinjected 68 Galabeledlipopeptideintoarat M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g People who did the work: Stefanie Held Matthias Engelke Stefan Seitz Kerry Mills Berit Lange Andreas Schulze Caroline Ghler Yi Ni Anja Meier Martina Spille Christa Kuhn Stefan Mehrle Jessica Sonnabend Walter Mier Alexa Schiek Isabell Janza Thomas Mller M o l e c u l a r
V i r o l o g y ,
H e i d e l b e r g Thankyouforyourattentionandthanksto: PhilippeGripon,INSERMU522,Rennes AlexanderAlexandrov,previously Vision7GmbH,now Myr GmbH RobertLanford,Texas,USA HeinerWedemeyer,MHHannover UlrikeEngel,ChristianAckermann,Nikkon ImagingCenter,HD ThomasWeiss,UniversityClinics Regensburg KarinLeotta,DKFZ DFG,EU,WHO,LandesstiftungBadenWrttemberg KompetenznetzHepatitis BMBF InnovativeTherapieverfahren DZIF(deutschesZentrumfrInfektionsforschung) RalfBartenschlager HeinzSchaller M o l e c u l a r