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The Hepatitis B Virus Life Circle:

Achievements and Challenges


Stephan Urban, Prof. Dr. rer. nat.
UniversityHospitalHeidelberg,DepartmentofInfectiousDiseases,Molecular
Virology,Heidelberg,Germany
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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)
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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
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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
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Thereplication cycle of HBV;crucial steps for interference
Urbanetal.,J.Hepatology,2010
NTCP/SLC10A1*
*Yanetal,eLife,November2012
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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
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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
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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
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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
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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
?
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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 !
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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
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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
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How is reimport of nucleocapsids regulated inHBVinfected cells?
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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
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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.
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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.
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FinemappingofthesequencerequirementsforHBVinfectioninhibition:
DefinitionofMyrcludexBasaleadsubstanceforclinicaldevelopment
Schulzeetal.,J.Virol,2010
Myrcludex B
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transplanted cells
TransplantationofuPA/RAG2micewithprimaryhumanhepatocytes(PHH)
humanhepatocytes(HH)
immunedeficientuPAmice
transplantation
AtransplantedmousemodeltostudyinvivoinfectionofHBV
cimeric liver
mouse cells
NatBiotechnol.,26:335341(2008)
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MyrcludexBblocksHBVinfectionaftersubcutaneousadministrationinPHH
transplanteduPAmice
Petersenetal.,NatBiotechnol.,26:335341(2008)
Why is s.c.administration of HBVpreS/248
myr
soefficient?
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MyrcludexBaccumulatesintheliverofmiceafteri.v.injection
siteof
injection
Liver
Stearoyl-GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGy-I
125
conserved domain
Schieck etal.,Hepatology 2013)
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WT
G12E
Stearoyl-GQNLSTSNPLGFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGy-I
125
Stearoyl-GQNLSTSNPLEFFPDHQLDPAFRANTANPDWDFNPNKDTWPDANKVGy-I
125
WT
G12E
Asingleaminoacidexchangeinthehighlyconservedreceptorbindingsite
abolishespeptidehepatotropismandliveraccumulation
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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
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*Yanetal,eLife,November2012
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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
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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)
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Positronemissiontomography(PET)ofanintravenouslyinjectedHBV
unrelated
68
Galabeledpeptideinarat
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Positronemmissiontomographie(PET)ofintravenouslyinjected
68
Galabeledlipopeptideintoarat
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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
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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
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