Hepatitis B Virus RNA in serum and liver tissue ......HBsAg levels in serum. The results also...

HepatitisBVirusRNAinserumandliver

tissue–quantificationusingdigitalPCR

KasthuriPrakash

DepartmentofInfectiousDiseases,InstituteofBiomedicine

SahlgrenskaAcademyatUniversityofGothenburg

Gothenburg,Sweden

Gothenburg2020

Coverillustration:GenesisbyKasthuriPrakash

HepatitisBvirusRNAinserumandlivertissue–quantificationusingdigitalPCR©[email protected](PRINT)ISBN978-91-7833-725-5(PDF)PrintedinGothenburg,Sweden2019PrintedbyBrandFactory

TomydearestNanapaandChemmi

HepatitisBVirusRNAinserumandlivertissue–

quantificationusingdigitalPCR

KasthuriPrakash

DepartmentofInfectiousDiseases,InstituteofBiomedicine

SahlgrenskaAcademyatUniversityofGothenburg

Gothenburg,Sweden

ABSTRACT

Hepatitis B virus (HBV) infection is a global health issue that is responsible for

approximately900,000deathseachyear,byinducinglivercirrhosisandhepatocellular

carcinoma(HCC).AfewmarkersareusedtoclassifyHBVinfectionandmonitortreatment

efficacy,includingHBVDNA,surfaceantigen(HBsAg)andeantigen(HBeAg)inserumas

well asHBVDNA andRNA in liver tissue. The recent discovery of the receptorNTCP

facilitatesinvitrostudiesofHBV.

Theaimsofthisthesiswere(I)tocharacterizeanewmarkerofHBVinfection,HBVRNA

in serum (II) to investigate in vitro the neutralizing effect of HBV encoded subviral

(HBsAg) particles (III) to develop and apply a newmethod to discriminate viral and

integratedDNAinlivertissue(IV)toanalyzefocaldifferenceswithintheliverofHBVand

hepatitisDvirus(HDV)and(V) toexploreHBVRNAprofile in liverbiopsiesbydigital

PCR.

HighlevelsofserumHBVRNAwasfoundinthemajorityof95patientsamplesutilizedin

thisstudy.ThisRNAwasoffullgenomelength,appearedinfractionationtogetherwith

HBV DNA. Sequencing data supported that HBV RNA in serum represents virus-like

particleswithfailingreversetranscriptionofthepregenomicRNA(pgRNA).

Theroleofsubviralparticles(SVP)duringHBVinfectionwasexploredinHepG2-NTCP

cell line. The results support that SVP functions as a decoy to neutralize antibodies

synthesizedbythehost.

A novel droplet digital PCR (ddPCR) methodwas developed and applied on 70 liver

biopsies to quantify circular and linearHBVDNA, in order to estimate the amount of

integratedHBVDNAinthehumangenome.Acomplimentarystudyonthesamematerial

was performed to obtain an RNA profile using ddPCR to amplify six target regions.

Together, these results indicate that integrated DNA represents the majority of

intrahepaticHBVDNAinlatestagesofinfectionandisresponsibleformaintaininghigh

HBsAglevelsinserum.TheresultsalsosuggestthatreducedtranscriptionofpgRNAvia

anovelmechanismmaycontributetolowHBVreplicationinHBeAg-negativephase.

ddPCRanalysisofarangeofHBVmarkerswasusedtostudyfocaldifferencesininfection

in 15-30 pieces of liver explant tissue from six patients with HBV or HDV induced

cirrhosis.Largedifferencesinfocalitywasobservedespeciallyinpatientswithlowdegree

ofviralreplicationorwithHDVcoinfectionandtheresultsalsosupportexpressionofS

RNAfromintegratedHBVDNA.HDVinfectionwaslessfocalwithpresenceofhighHDV

RNAlevelsintheabsenceofHBV.

Insummary,thisthesiscompilationcontributestobetterunderstandingofHBVserum

andtissuemarkersandtheirrelationshiptoreplicationandintegration.

Keywords:hepatitisBvirus,NTCP,HBVDNA,HBVRNA,subviralparticles,integrations,

dropletdigitalPCR

ISBN:978-91-7833-724-8(PRINT)

ISBN:978-91-7833-725-5(PDF)

SAMMANFATTNINGPÅSVENSKA

InfektionmedhepatitB virus (HBV) är ett globalt hälsoproblemsom står för ungefär

900000dödsfallårligengenomattorsakaskrumpleverochlevercancer.Debiomarkörer

som idaganvänds förattklassificeraHBV-infektionenochövervakabehandlingseffekt

inkluderarHBV-DNA,ytantigenetHBsAg,e-antigenetHBeAgiserumsamtHBV-DNAoch

HBV-RNAilevervävnad.NyligenupptäcktesHBV-receptorn,NTCP,vilketunderlättarin

vitro-studieravHBV.

Syftetmeddennaavhandlingvaratt:(I)karakteriseraennybiomarkörförHBV-infektion

(HBV-RNA i serum); (II) undersöka den neutraliserande effekten av HBV-kodade

subviralapartiklar(HBsAg)invitro;(III)utvecklaochappliceraennymetodförattskilja

på viralt och integrerat HBV-DNA i levervävnad; (IV) analysera fokala skillnader i

förekomst av HBV och hepatit D-virus (HDV) i levervävnad; (V) utforska HBV-RNA-

profilerileverbiopsiermeddigitalPCR.

I enmajoritet av serumprover från95 patienter uppmätteshöga nivåer avHBV-RNA.

Detta RNA motsvarade hela HBV-genomets längd och förekom vid fraktionering i

partiklarmedsammadensitetsomdemedHBV-DNA.SekvenseringindikeradeattHBV-

RNA i serummotsvararviruslikapartiklardäromvänd transkriptionavpregenomiskt

RNA(pgRNA)ejfungerat.

FunktionenavHBsAg-bärande subviralapartiklar (SVP) iHBV-infektionundersöktes i

cellinjen HepG2-NTCP. Resultaten stöder hypotesen att HBsAg/SVP minskar den

virusneutraliseradeeffektenavantikropparriktademotvirusetsytprotein.

En ny metod, baserad på droplet digital PCR (ddPCR), utvecklades och användes för

analysav70leverbiopsier.MetodenkvantifieradecirkulärtochlinjärtHBV-DNAföratt

kunna uppskatta mängden integrerat HBV-DNA i det humana genomet. En

kompletterande studie på sammamaterial utfördes för att ta fram virus-RNA-profiler

genom att med ddPCR amplifiera sex olika målregioner. Sammantaget pekar dessa

resultatpåattintegreratHBV-DNAutgördenstörstaandelenavintrahepatisktHBV-DNA

isenarestadieravinfektionen,ochattdettaintegreradeDNAuttryckssåatthöganivåer

avHBsAgiserumbibehålls.ResultatentyderocksåpåattnedregleringavpgRNAviaen

nymekanismskullekunnabidratilllägrereplikationavHBViHBeAg-negativapatienter.

ddPCR-analysav fleraolikaHBV-marköreranvändes förattstudera fokalaskillnader i

HBV-infektioneni15-30bitaravleverexplantatfrånsexpatientermedHBV-ellerHDV-

orsakadskrumplever.Storaskillnader i fokalitetobserveradessärskilt ipatientermed

låggradig virusreplikation och resultaten stöder hypotesen att S-RNA uttrycks från

integrerat HBV-DNA. HDV-infektion (hos två av patienerna) var jämntare utspridd i

levervävnadenmedhöganivåeravHDV-RNAoberoendeavHBV.

Sammanfattningsvis bidrar denna avhandling till bättre förståelse av serum- och

levermarkörervidHBV-infektionochderaskopplingtillreplikationochintegration.

LISTOFPAPERS

The thesis is based on the following studies, referred to in the text by their roman

numerals.

I Kasthuri Prakash, Gustaf E. Rydell, Simon B. Larsson, Maria Andersson, Gunnar

Norkrans,HeléneNorder,MagnusLindh.HighserumlevelsofpregenomicRNAreflect

frequentlyfailingreversetranscriptioninhepatitisBvirusparticles.VirologyJournal

2018;15:86.

II GustafE.Rydell,KasthuriPrakash,HeléneNorder,MagnusLindh.HepatitisBsurface

antigenonsubviralparticlesreducestheneutralizingeffectofanti-HBsantibodieson

hepatitisBviralparticlesinvitro.Virology2017;509:67-70.

III Gustaf E. Rydell, Simon B. Larsson, Kasthuri Prakash, Maria Andersson, Heléne

Norder,KristofferHellstrand,GunnarNorkrans,MagnusLindh.Abundanceofnon-

circularHBVDNAsuggestsintegrationstobethepredominantformofviralDNAin

chronichepatitisB.Manuscript.

IV Kasthuri Prakash, Simon B. Larsson, Catarina Skoglund, Gustaf E. Rydell, Johan

Ringlander, Maria Andersson, Maria Castedal, Heléne Norder, Magnus Lindh.

IntrahepaticfocalityofhepatitisBinfectionanalysedbyquantificationofviralRNAin

multipletissuepiecesfromexplantedlivers.Manuscript.

V Kasthuri Prakash, Simon B. Larsson, Gustaf E. Rydell, Maria Andersson, Johan

Ringlander,GunnarNorkrans,HeléneNorder,MagnusLindh.AnalysisofHBVRNAin

liverbiopsiesbydigitalPCRrevealsdifferencesintranscriptlevels,lengthandorigin

betweeneantigenpositiveandnegativeindividuals.Manuscript.

ABBREVIATIONS

AuAg AustralianAntigen

HBV HepatitisBvirus

HCC Hepatocellularcarcinoma

NA Nucleoside/nucleotideanalogues

rcDNA RelaxedcircularDNA

HBcAg HepatitisBcoreantigen

HBsAg HepatitisBsurfaceantigen

HBeAg HepatitisB“e”antigen

VP ViralParticles

SVP Subviralparticles

NTCP Sodium-taurocholatecotransportingpolypeptide

cccDNA CovalentlyclosedcircularDNA

PreCRNA/PC PrecoreRNA/precore

PgRNA PregenomicRNA

dslDNA DoublestrandedlinearDNA

ssDNA SinglestrandedDNA

ALT Alanineaminotransferase

ORF Openreadingframe

BCP Basalcorepromoter

RT Reversetranscriptase/reversetranscription

HDV Hepatitisdeltavirus

PCR Polymerasechainreaction

qPCR QuantitativePCR

RT-PCR ReversetranscriptasePCR

ddPCR DropletdigitalPCR

cDNA ComplimentaryDNA

TableofContents

1 INTRODUCTIONTOHEPATITISBVIRUS....................................................................................................1

MOLECULARSTRUCTURE..............................................................................................................................................2 VIRALENTRYANDREPLICATION..................................................................................................................................4 SEROLOGICALANDINTRAHEPATICMARKERS..............................................................................................................7 NATURALHISTORYOFINFECTION................................................................................................................................9 INTEGRATIONS............................................................................................................................................................11 GENOMICVARIATIONS................................................................................................................................................12 TREATMENT................................................................................................................................................................15 CELLCULTURE&ANIMALMODELS...........................................................................................................................16

2 INTRODUCTIONTOHEPATITISDELTAVIRUS.......................................................................................18

HDVGENOMEANDREPLICATION.............................................................................................................................18 HBVSUPPRESSIONBYHDV......................................................................................................................................20 TREATMENT................................................................................................................................................................20

3 AIMS...................................................................................................................................................................21

4 MATERIALSANDMETHODS........................................................................................................................22

MATERIALS.................................................................................................................................................................22 METHODS....................................................................................................................................................................23

5 RESULTSANDDISCUSSION..........................................................................................................................28

PAPERI.......................................................................................................................................................................28 PAPERII......................................................................................................................................................................32 PAPERIII....................................................................................................................................................................34 PAPERIV.....................................................................................................................................................................38 PAPERV......................................................................................................................................................................46

6 CONCLUSIONS..................................................................................................................................................52

7 ACKNOWLEDGEMENTS.................................................................................................................................54

8 REFERENCES....................................................................................................................................................57

1

1 INTRODUCTIONTOHEPATITISBVIRUS

In1967,DrBlumbergandhisassociatediscoveredanewproteininserumofpatientsthat

underwentbloodtransfusions,specificallyinAustralianaboriginals.Thisproteinnamed

“Australian Antigen” (AuAg) became the first of many discoveries confirming the

presence of viral hepatitis [1]. In 1970, Dr Davis S Dane described 42nm virus-like

particlesinpatientswithAuAgandnamedthem“DaneParticles”[2]whichisnowknown

asHepatitisBVirus(HBV).

Theviral infection causedbyHBVstill remainsaglobalpublichealth issue in-spiteof

decadesofresearchandavailabilityofapreventivevaccinesince1982.HBVbelongsto

thefamilyofhepatotropicDNAvirusesthatcancausebothacute(infectionclearedwithin

6monthsofexposure)andchronic(infectionthatpersists>6months) infectionof the

liver.TheWorldHealthOrganizationasof2017estimatesthat2billionindividualsareor

have been infected with HBV of which 257 million have a chronic infection [3].

Approximately900,000deathseachyeariscausedbyHBVrelatedcomplicationssuchas

livercirrhosisandhepatocellularcarcinoma(HCC)[4].

HBVcanbe transmittedpercutaneouslyvia sharingofbloodproducts, throughsexual

transmissionorbymeansofvertical transmission, i.e., toanewborn froman infected

mother.AccordingtotheWorldHealthOrganization’s(WHO)statistics,about80-90%of

infantsand30-40%ofchildrenundertheageof6whoareinfectedwithHBVwilldevelop

a chronic disease, but when acquired as adults 95% will clear the infection. The

pathogenesisofcomplicationsinvolvesimmunemediatedkillingofhepatocytescausing

liverinjurywithsubsequentregenerationbyclonalexpansionofhepatocyteseventually

leading to scarification of the liver (fibrosis) and later on cirrhosis and HCC. Other

contributingfactorsforpathogenesisareintegrationofviralDNAintothehostgenome

and oncogenic effects of viral protein [5]. Patients showing signs of progressive liver

damagearegiven long-termtreatmentwithnucleoside/nucleotideanalogues(NA) in

ordertopreventthesecomplications,butwithitarisesthepossibilityforresistantstrains

toemergeviamutation[6-8].

2

DuringthecourseofthisPhD,variousaspectsofHBVinfectionwasinvestigatedinorder

to answer some pressing questions. Interesting associations were explored, and

intriguinghypothesesarepresentedasacompilationwiththehopethatthesestudieswill

aid better understanding of disease progression and assist the clinical community in

developmentofnewtreatmentsforpatientswithHBV.

Molecularstructure

HBVisclassifiedasoneofthesmallestknownDNAvirusesmeasuringapproximately42

nmindiameterbelongingtothefamilyofHepadnaviridaeviruses.Ithasa3.2kbsized

relaxedcircular(rcDNA)genomethatispartiallydoublestranded(ds)withacomplete

minus (-)strandandan incompleteplus (+) strand that is covalentlyboundtoaviral

polymeraseatits5’end.Thisgenomeisenclosedwithinanicosahedralnucleocapsidcore

protein (core antigen,HBcAg) that is surrounded by viral surface protein (hepatitisB

surfaceantigen,HBsAg).

Figure1:HBVparticlecontainingthepartiallydoublestrandedgenomewithcomplete–strandandanincomplete+strandcovalentlyboundtotheviralpolymerase

HBVgenomehasfouroverlappingreadingframes(ORF)thatcodesforviralproteins(i)

precore/coregenefornucleocapsidcoreandsecretory“e”protein(HBeAg)(ii)PreS/S

geneforsmall(SHBsAg),middle(MHBsAg)andlarge(LHBsAg)envelopeproteins(iii)

XgeneforregulatoryXproteinand(iv)polymerasegeneforviralpolymerase.

3

Figure2:HBVgenomethatispartiallydoublestranded.Representedhereare4overlappingreading

framesresponsiblefortranslationofviralproteins.

AlongwithHBVDNAcontainingviralparticles(VP),anexcessproductionof22nmsized

rodandcircularemptysubviralparticles(SVP)comprisedofonlyHBsAgcanbedetected

inthepatient’sserum.

Figure 3: Sphere and rod forms of HBV subviral particles with large, middle and small surface

proteinsthatlackaviralgenome.

4

Viralentryandreplication

Although presence of hepadnaviruses in extrahepatic tissues such as lymph nodes,

kidneys, skin and colon have been detected, the only target for these viruses are

hepatocytes [9] because of the presence of cell specific receptors expressed by the

hepatocytes.

The first step in viral entry involves interaction of the pre S1 domain of viral surface

antigen with hepatocyte specific heparin sulphate proteoglycan [10] followed by

attachment to the sodium taurocholate co-transporting polypeptide (NTCP) receptor

[11].Theuptakeofthevirusintothehepatocyteishypothesizedtotakeplaceviaclathrin

or caveolin-1mediated endocytosis throughwhich the encapsidated viral genome is

transportedintothehostcell[12,13].Themicrotubulesinthecytoplasmthentransport

thenucleocapsidcontainingtheviralgenomeandthepolymeraseintothecell'snucleus

where the capsid disassociates and the rcDNA is released [14]. A sequence of highly

precise steps to convert rcDNA into thehighly stablemini chromosomecccDNA takes

place.Theincomplete+strandiselongatedtothe5’endofthe–strand.Afterthisstep,

the polymerase is removed followed by the eliminationof eight terminally redundant

nucleotidesfromthe–strand.Atthisjuncturethe5’andthe3’endsofthe+andthe–

strandcanbeligatedandsupercoiledtoformcccDNA[15].

Ithasbeenestimatedthataround1-50copiesofthecccDNAremainwithineachinfected

hepatocyte as mini chromosomes that serves as a transcriptional template for viral

replication.TranscriptionofcccDNAresultsintheformationoffourmRNAsofsizes3.5kb,

2.4kb,2.1kband0.7kbthataretranslatedintosevenviralproteins(Figure4).The3.5kb

genomic mRNA includes precore (preC) mRNA that is translated into secretory “e

antigen”HBeAgproteinandpregenomicRNA(pgRNA)that is translated intocoreand

polymerase proteins needed for viral replication. They are larger that genome length

because of the presence of terminal redundancies on both 3’ and 5’ ends. The other

transcripts termed subgenomic RNAs are represented by PreS1 mRNA (2.4 kb) that

translatesintoLHBsAg,thePreS2/SmRNA(2.1kb)forMandSHBsAg,andXmRNA(0.7

kb)forregulatoryHBxprotein.

5

Figure 4 : Genomic placements of different ORFs and genomewith polyadenylation at 3’ and 5’

regionsarebothrepresentedinalinearform.AllRNAstranscriptsofdifferentlengthwitha5’cap

anda3’polyadenylation(An)isrepresented.

The pgRNA and precore RNA encompasses two highly conserved sequence regions

termedasepsilon(e)thatformsasecondarystemloopstructureatthe5’and3’regions.

The 5’ e structure directs encapsidation of RNA along with viral polymerase that

undergoesreversetranscriptiontoformfunctionallyinfectiousDNAcontainingvirions

[16-18].However,onlypgRNAisencapsidatedtoforminfectiousDNA[19]whileprecore

RNAiscleavedintheendoplasmicreticulumandissecretedasHBeAg.Figure5describes

highly precise steps required to convert pgRNA into infectious viral genome. These

replication steps generate encapsidated relaxed circular HBV DNA (rcDNA), that can

either(i)acquireHBsAgandbesecretedasviralparticlesor(ii)berecycledwithinthe

hepatocytetoincreaseormaintaincccDNApool[20].

Alongwith themature virion, secretionof empty nucleocapsids, RNA containing viral

particles and to some extent double stranded linear forms (dslDNA) has also been

observed[21,22].Duringplusstrandsynthesis,primertranslocationfailuremaycause

viral genome to remain linear instead of circularizing to form rcDNA. This double

6

strandedlinear(dslDNA)canrecirculateintothehepatocytenucleusandbeintegrated

intothehostchromosome[23].

Figure5:SequenceofstepsfollowingpgRNAencapsidationisrepresented.(A)showslinearpgRNA

within capsid. (B)Bindingof polymerase to thee loopat the5’ end. (C) Synthesis of short oligo

followedbytranslocationofthepolymeraseandtheoligotothedirectrepeat1(DR1)regionwhere

minusstrandsynthesisisinitiated.(D)Minusstrandsynthesisisaccompaniedbydegradationofthe

pgRNAtemplatewhereonlyDR1sequenceandtheshortoligofromthe5’endremains.Thisactsas

precursorfortheplusstrandsynthesisandafteratemplateswitch(representedinE)plusstrand

synthesisbeginsandsubsequentlycircularized(representedinF).

7

Figure6:HBVreplicationcycle

Serological and intrahepatic markers

Clinical staging of HBV infection is primarily done using quantifiable serological and

intrahepaticmarkers.HBViswhatisknownasastealthvirus,thatdependingontheage

atwhichinfectionisacquired,canreplicatefordecadesbeforeimmuneflareisinitiated.

ReplicationofHBVstarts immediatelypost infectionandmatureviralDNAalongwith

HBsAgbecomesdetectableintheblood.PresenceofHBsAgforlongerthan6monthsis

definedclinicallyas“chronicinfection”anditsclearanceisusuallyaccompaniedbythe

appearance of anti-HBs antibodies. Therefore, anti-HBs is an accepted marker to

determine end-point for treatment [24].HBeAg secreted into the blood from infected

hepatocytesisamarkertodetermineviralreplication[25,26].HighHBVDNAlevelsmay

howeverbepresentalsoinpatientsseronegativeforHBeAg,becausemutationsespecially

intheprecoreregion,mayemergeandprecludesynthesisofHBeAg,particularlyinHBV

genotypesB,DorE[27-29].PresenceofHBVRNAinserumfirstreportedin2001has

beensuggestedasadiagnosticmarkersincethen,inparticularformonitoringtheeffect

8

oncccDNAduringantiviraltreatment[30-33].DuringtreatmentofHBVinfectionusing

NA,HBVDNAisreducedtolevelsbelowthequantificationlimit,afterwhichtheeffectof

treatment cannot be assessed. Quantification of HBsAg in serum was proposed to

represent cccDNA,but itwas later found that thiswas found tonotbe true, since the

declineofHBsAglevelswasminimal.SinceHBVRNAisnotdirectlyreducedbydrugsthat

inhibitreversetranscription,itshouldbeusefulasserummarkerrepresentingcccDNA

levelsintheliver[34].

Intrahepatic cccDNA a highly stable mini-chromosome is the template needed to

synthesizeHBVRNAtranscriptsnecessaryforvirusproduction.Reductionofviralload

by two log10 units during HBeAg negative stage is caused by the loss of intrahepatic

cccDNA copies [35, 36]. Induckmodel, it hasbeen found that the number of cccDNA

moleculesmay vary over time,withmeannumber of cccDNAper infected hepatocyte

rangingbetween3and9[36,37].Becauseofitsroleinmaintainingchronicinfectionand

abilitytoavoiddruginducedclearance[38,39],quantifyingcccDNAandunderstanding

its relationship to other viral markers are important. Many approaches have been

designedtoquantifycccDNAintheliver[36,40,41]butbecauseofitspresenceinlow

concentrations, conventional approaches such as southern blot are not adequately

sensitive[42].AchallengefortheseassaysistobeabletodifferentiatebetweencccDNA

fromrcDNA,thereforeapolymerasechainbasedassaywasdesignedtotargetthegapin

plus strand region that would be present only in cccDNA [36, 43]. In addition to

intrahepaticmolecularmarkers suchas cccDNAandHBVDNA immunohistochemistry

canbeperformedtovisualizeHBsAg,HBcAg,HBxAgandpre-Speptidesonlivertissue.

Althoughliverbiopsiesprovidevaluabledata,thisinvasivesamplecollectionmethodhas

to a large extent been replaced by non-invasive techniques such as elastography

assessmentofliverfibrosis,atrendthatwillhamperfutureresearch.Adrawbacktousing

biopsy tissues is the riskof samplingerror,where thematerial analyzed isnota true

representationofthediseaseorinfectionstate.

9

Natural history of infection

DiversenomenclatureshavebeendefinedtodescribetheclinicalphasesofHBVinfection.

Discussed here are phases according to European Association for the Study of Liver

(EASL)guidelinesonHBVinfectionmanagement(i)HBeAgpositivechronicHBVinfection

(ii)HBeAgpositive chronichepatitisB (iii)HBeAgnegative chronicHBVinfection (iv)

HBeAgnegativechronichepatitisB(v)HBsAgnegativephase[24].

1.4.1 HBeAg positive chronic HBV infection / Immune tolerance

HBVinfectionthatisacquiredduringearlychildhood(<2years),usuallythroughvertical

transmission at birth or later horizontal transmission, usually inducesweak immune

responses for several decades. During this phase of HBV infection (often termed the

immunetolerancephase),HBVinfectsalmostall livercellsandreplicatesactivelywith

typically > 107 IU/mL HBV DNA detected in patient serum. The individual remains

asymptomatic at this stage and shows normal to mild elevation of alanine

aminotransferase(ALT)levelswithnormalliverhistologyanddetectableHBeAgdetected

in serum. So far, treatment interventions during immune tolerant phase has not been

recommended[44-48].

1.4.2 HBeAg positive chronic hepatitis B

Typicallyseentolastfromthe3rdto4thdecadepostinfection.Thisphaseischaracterized

byactiveimmunemediatedclearanceofHBVinfection,duringwhich,infectedlivercells

areeradicated,causingelevationinALTlevels.Thedurationandlevelofcellulardamage

totheliverdeterminesifthepatientgetsHBVmediatedfibrosisorcirrhosis.Eventually

most patients seroconvert to anti-HBewith suppressionofHBVDNA.However, some

patients fail to control viral HBV replication thereby progressing to HBeAg negative

hepatitis.

1.4.3 HBeAg negative CHB / inactive carrier

Thedetectionofanti-HBealongwithundetectableorsignificantreductionofviralDNA

andsubstantialreductionornormalizationofALTlevelsinserumofCHBpatientindicates

10

clinicalremission.HBVDNAlevelsvarybetween<2000–20,000IU/mLatthispointand

insomepatientsthiscanleadtoHBsAgseroconversion[49].

1.4.4 HBeAg negative chronic hepatitis

Progressionfrombeinganinactivecarriertoanindividualwithchronicinfectionishighly

variableand isassociatedwith factors likeageatwhich infectionwasacquired,ageof

HBeAgseroconversion,durationofinfectionandgenotype.IncreaseinserumHBVDNA

andALTlevelshavebeenobservedinlongtermfollowupstudiesintheabsenceofHBeAg

[49-53].This ismainlyduetothepresenceofmutations inprecoreregionof theviral

genome that survive as an escape mutant and causes relapse of chronic infection

(discussedindetailunderthetopicgenomicvariations)[54,55].

1.4.5 HBsAg negative phase

DuringthisphasepatientsundergolossofHBsAgwithorwithoutdevelopmentofanti-

HBsalongwithanti-HBcantibodiesandshownormalALTlevelsalongwithundetectable

HBVDNAandpresenceofcccDNAinlivertissue.Insomecasesdevelopmentof“Occult

HBV”takesplace,wherepatientremainsnegative forHBsAg inthepresenceoflowor

undetectablecirculatingviral load.LossHBsAgthatoccurseitherspontaneouslyorby

treatmentinterferencealthoughconsideredas“functionalcure”doesnoteliminatethe

probability of HCC. Development of cirrhosis before HBsAg seroclearance has been

associatedwithHCCincidencetherebyincreasingtheneedforsurveillanceafterreaching

treatmentendpoint[56-58].

11

Figure 7 : History of HBV infection as defined by EASL guidelines. There is no clear distinction

betweenthesephasesandonlyaroughestimateisdefinedhere.

Integrations

SimilaritiesbetweenHBVandretroviruseshavebeendiscussedincludingthepotential

forsuccessfullyintegrateintothehostchromosomes[59].Butunlikeretrovirusesthat

need integration forreplication,HBVintegrationsaredefinedasreplicativedeadends

12

withthepotentialtoproduceviralproteinsevenintheabsenceofactivereplication[60,

61].Primertranslocationfailureduringplusstrandsynthesisresultsintheformationof

double stranded liner DNA (dslDNA)which separates the precore/core gene from its

promoter. Therefore, this form can neither synthesize pregenomic RNA nor generate

capsids.SincedslDNAistheprecursorforintegration[62],onlySgenethatremainsintact

issuspectedtobeactiveandbecapableofproducingHBsAgevenintheabsenceofHBV

replication as indicated by low HBV DNA. This phenomenon was first observed in

hepatocellularcarcinomacell linePLC/PRF/5[63]andhasbeendescribed inarecent

studyconductedonchimpanzees[61].Integrationshavebeenobservedatearlystagesof

infectionandhasbeenassociatedwithincidenceofHCCandaffectsurvivalofinfected

individuals[64].Doublestrandedbreaksinhostchromosomeshavebeenestablishedas

thepreferentialsiteforintegrations[65],wherethehost’scellularmechanismsintegrates

viral into chromosomal DNA damage response (DDR). It has been accepted that DDR

pathways lead to HBV integrations, however the exact methodology by which this is

achievedisdebated.Somestudiessuggestnon-homologousendjoining(NHEJ)[65]for

doublestrandedbreakrepairareresponsibleforviralintegrationswhereasothersargue

thatitoccursviamicrohomology-mediatedendjoining(MMEJ)method[66].

Genomicvariations

1.6.1 Genotypes and Sub-genotypes

The polymerase present in mature HBV lacks proof reading ability causing the virus

withinthecapsidtocontaingenomicsequenceswithvariationsandhasresultedinan

array of viral strains. When the genomic sequences have a variation >8% they are

classified as genotypes andwith variationwithin a genotype between 4-8% they are

defined as sub-genotypes. Ten different genotypes and 30 sub-genotypes have been

identified so far with a distinct geographical distribution pattern. Variations in these

genotypesalsoextendtotheirclinicalandvirologicalfeatures.Largelydiscussedarethe

observationswithregardstodiseaseprogression[67],treatmentresponse,development

of cirrhosis and capability to advance toward HCC [68, 69]. Two studies comparing

genotypesBandCshowedthatthelatterhasthepotentialinassociationwithHBeAgto

cause severe liver disease [27, 70]. Results from a Taiwanese study observed higher

13

frequenciesofcirrhosisandHCCingenotypeCwhencomparedtogenotypeD[69].The

samestudyalsodiscussedlargerincidentsofHCCinnon-cirrhoticpatientscomparedto

genotypeB.InaEuropeanstudycomparinggenotypeAandD,theformerwasseento

causemorechronicinfections[71]andshowedlargerincidentsofremissionpostHBeAg

seroconversionthanthelatter[72].FrequenciesofHBVrelateddeathsarereportedtobe

higherinpatientswithgenotypeFwhencomparedtogenotypeA[72].

Thesestudies furtherpressonthe importanceofunderstandingdifferencescausedby

genotypesonviralload,clinicalmanifestationsandresponsetotreatment;supportbetter

comprehensionandaidinadvancementofclinicalpractices.

Figure8:GeographicaldistributionofHBVgenotypes

Inadditiontothelackofpolymerase’sproof-readingability,geneticvariabilitybecauseof

mutations are a product of immune selective pressure and drug resistance. A study

conductedonwoodchucksin1989approximated1erroroccurringevery107bases[73,

74].Selectionpressurefromimmuneresponsesthattargetwildtypevirusesmayleadto

emergenceofgenomeswithmutations.Mutationshavebeenobservedinall4ORFswith

14

theircontributiontoclinicaloutcomesandattributiontodiseaseprogressionextensively

researched[75].

1.6.2 S ORF mutation

Ahighlyconservedaminoacid(aa)regiontermedas“a”determinantinHBsAgisselected

as a target for vaccine against HBV aswell as for clinical diagnosis of disease status.

Mutationsinthisregionwasfirstdocumentedinapatientthathadverticallytransmitted

HBVevenaftervaccinationwithbothHBVDNAandanti-HBspresenceinserum[76].Lack

ofepitoperecognitioncanthusleadtovaccinefailure[76]andunreliableresultsfrom

diagnosticassays[77].

1.6.3 Precore / core ORF mutation

The precore and core regions are responsible for the production of twoproteins, the

structuralcoreandthesecretory‘e’protein.HBeAgisnotrequiredforviralproduction

buthasbeensuspectedtoserveasadecoytocurbimmuneresponseagainstcore[78],

butimmunetoleranceiseventuallylostandanti-HBeisproducedalongwithreduction

and inmany patients, clearance of HBV DNA. Failure to clear HBV DNA is seenwith

emergence of several mutations in the basal core promoter (BCP) and precore (PC)

regions suggesting selection due to immune pressure [79]. The most common PC

mutationoccursatposition1896whereguanine(G)isreplacedbyadenine(A)(G1896A)

which induces a stop codon in HBeAg sequence and abrogates HBeAg synthesis [80].

Another commonmutation is seenat1899whereguanine (G) is replaced by adenine

G1899AandthismutationwasfoundbothbyitselfandalongwithA1896Tmutation[80].

Mutations in the BCP region, 1762 (A1762T) and 1764 (G1764A) have also been

associatedwithcontributingtothereductioninHBeAgexpressionandiscorrelatedwith

higherHCCincidents[81].LowHBVDNAquantificationsareobservedinpatientswithPC

mutations alongwith lower ALT levels that suggest lesser liver damage compared to

infectionwithwildtypevirus.HoweverBCPmutationssuchasthyminereplacementwith

cytosine (C) at 1753 (T1753C) and also at C1766T reduce HBe secretion via

transcriptional mechanisms and have shown increase in HBV DNA and ALT levels

thereforesuggestinghigherprevalenceofprogressiontowardscirrhosis[82,83].

15

1.6.4 X ORF mutation

The X gene is only found in mammalian hepadnavirus and has important functions

includingabilitytoestablishaninfection[84],promoteviralreplication[85],andsupport

developmentofHCC[86].ThemutationsintheXORFhavebeencorrelatedtoenhanced

progressiontowardslivercirrhosis(LC)andHCC[83,87-89].

1.6.5 Polymerase ORF mutation

Polymerase ORF encodes for the polymerase protein that is encapsidated along with

pgRNAandisresponsibleformanystepsthatarenecessaryforsuccessfulcompletionof

HBVreplicationcycle[90-92].Amutation intheRTregionof thepolymeraseORFcan

dictate and alter replication events. Investigations regarding naturally occurring

mutationshavebeenundertakeninthepastfewyearsbecauseitcouldgiveinformation

about possible drug resistance [93, 94]. This may alter a clinician’s decision about

treatmentofapatientwiththesemutations.Associationsbetweendifferentmutationsin

theRT region and viral load,degree of liver disease have been published [94-96]. RT

inhibitors are commonly used for the treatment of HBV but can induce a selection

pressure that leads to proliferation of drug-resistant HBV strains. One of the most

commonmutations induced by the antiviral drug Lamivudine is seen in the tyrosine-

methionine-aspartate(YMDD)motifofHBVpolymerasearoundnucleotide552[97].This

mutation however is associated with low serum titers of the virus and shows low

probabilitytocauseadverseliverdisease[8].

Treatment

TwoclassesofdrugshavebeenapprovedforthetreatmentofCHB,immunomodulating

agents and nucleoside / nucleotide analogues in order to reduce viral load during

infection to achieve seroconversion of HBeAg and within short duration tominimize

immunemediatedliverdamageandpreventionofadverseeventssuchasLCandHCC.

The drugs used for management of HBV that exists now only manage to keep viral

replicationunder control, and does not eliminate the highly stablemini chromosome,

cccDNA[38,98-100].

16

1.7.1 Immunomodulators

Interferonsarenaturallyproducedproteinsthataresynthesizedbycellsinresponseto

viral infections to induce immune response in retaliation. Immunomodulators suchas

interferonalpha(IFN-a)andPegylatedinterferon(PEG-IFN)areusedinCHBtreatment

becauseofitspotentialtobindtocellularreceptorsandactivateproteinsynthesisneeded

for cellular defense against invading virus [101]. These drugs cannot however be

prescribedtopatientswhoareaffectedbydecompensationoftheliversuchascirrhosis

[102].

1.7.2 Nucleoside / Nucleotide analogues

Drugs under these classifications were initially developed for treatment against

herpesvirus and human immunodeficiency virus (HIV) because of their potential to

impairpolymerasefunctionandcanbeadaptedtoHBVtreatment.Therearecurrentlysix

drugs that are approved for the treatment of CHB, (i) lamivudine (ii) entecavir (iii)

adefovir(iv)tenofovir(v)tenofoviralafenamide(vi)telbivudine.Thesemedicationsare

takenorallyandaresafetousehowever,sincetheyonlyblockRT,longtermtreatmentis

required.Theformationofdruginducedresistantmutationswasamainproblemearlier,

sincelamivudineresistancedevelopedin15-32%ofpatientswithinoneyearoftreatment

[8].Thecurrentlyuseddrugstenofovirandentecavirhowever,havehighbarriersagainst

developmentofresistancemutations[103-105].

Cell culture & animal models

Cellcultureandanimalmodelsfacilitatebetterunderstandingofadiseaseandaidswith

treatmentdevelopment.DuetospecifichostrequirementsforHBV,therearelimitations

onanimalmodelsthatcanbeusedforresearch[106].Someanimalssuchaschimpanzees,

tupaias,ducksandhumanchimericmicehavebeenusedtostudyHBV[107].Ofthese,the

immune responseof chimpanzees are the closest tohumans and itwas precisely this

reasonthatledtotheuseofthismodeltotestefficacyofpreventiveHBVvaccine[108].

CellculturesystemssuchashepatomacelllinesHepG2andHuh7havebeenavailablefor

alongtimenowandhavesupportedresearchregardingtranscriptionandsynthesisof

17

new virion particles [109-111]. Even though they are hepatoma cell lines and are

availableinplenty,theydonotsupportinfectionduetothelackofthereceptorNTCP.

HepRG cells can support infection, however they require complicated differentiation

stepsinafashionsimilartotheinducedhepatocytes(iHep)[112].Comparingthesetwo

celllines,HepRGhaslowerefficiencyofinfectionthaniHepandcansupporttheentirelife

cycleofthevirus.Primaryhumanhepatocytes(PHH)isanothersystemthatisusedto

study HBV infection and although this is the closest condition possible tomimicking

naturalstateofinfection,itislessefficient,moreexpensiveandishugelydependenton

donoravailability[113].NTCPwasrecentlydiscoveredasthecellularreceptorforHBV/

HDVviralentry[11,114].ThereceptorwasthenexpressedintheHepG2hepatomacell

linetomakeitsusceptibletoHBV/HDVinfection.Thisdiscoveryopenedupnewavenues

toexploredifferentfacetsofinfectionandreplication.Althoughthesecellscanbeinfected

there are certain limitations that needs to be addressed. This system requires large

quantitiesofinputanddoesnotpromotelongterminfection;sincethisiscancerouscell

line, it lacksmany of the pathways needed to study host-virus interactions; and post

infectiononlyasmallamountofcccDNAisexpressed;althoughthecellsystemcanbe

infected,itneedsinadditionpolyethyleneglycol(PEG)toenhanceinfectionbypromoting

glycosaminoglycanbindinganddimethylsulphoxide(DMSO)toaidinfection.

18

2 INTRODUCTIONTOHEPATITISDELTAVIRUS

In1977anewantigenassociatedwithHBVwasdiscoveredinpatientswhowereHBsAg

positive [115]. It was later in 1980 using chimpanzees as a model system that delta

antigen was determined to be a defective infectious agent that interferes with HBV

replication[116].Sincethenmanystudieshaveshownhepatitisdeltavirus(HDV)tobea

satellitevirustoHBVthatrequiressurfaceantigenproductionfromHBVtoenvelopeits

genometogainaccessintohepatocytesforsuccessfulinfection[116].

HDVisthesmallestknownRNAvirusthatinfectsmammals[117]andistheonlymember

of the deltavirus genus. Transmission of HDV happens in the same routes as HBV,

percutaneouslyviasharingofbloodproductsorthroughsexualtransmissionorinrare

cases via vertical transmission. A study in 2003 estimated that 5% of the world’s

populationwithHBValsohasHDV[118],butaccordingtoWHOmanycountriesdonot

test for HDV co-infection in HBV positive patients and in countries such asMongolia

where60%ofthepopulationtestedpositiveforHBVmayalsobeinfectedwithHDV[3].

HDVmaybeacquiredasasimultaneousinfectionwithHBVcausingmildtosevereacute

hepatitis,wheredevelopmentintochronicHDVinfectionisrareoccurringinlessthat5%

of the cases [119]. If an individual with chronic HBV is superinfected with HDV,

developmentofchronicHDVinfectionisalmostinevitable,leadingtoLCmoreoftenand

fasterthaninapatientwithHBVmonoinfection[116,120].

HDV genome and replication

HDV is a small single stranded RNA virus with a circular genome containing 1679

nucleotides.With74%intramolecularbasepairing[121], thegenomeformsarodlike

structurethatcanbefoundalongwithdeltaantigen(small,S-dAgandlarge,L-dAg).

Forviralreplication,HDVengagesinamechanismtermedas“thedoublerolling-circle

amplification”whereitusesthehost’scellularpolymerasetosynthesizeacomplimentary

RNAstrandcalledtheantigenomicdRNAandanmRNAwith5’capandapolyadenylated

19

3’endthatcodesforthedeltaantigenprotein[122].Withtheproductionofantigenome,

viralreplicationcontinuesandthevirusissecretedwithanenvelopecontainingHBsAg.

Figure 9: (A) Single stranded HDV with large and small forms of d-antigen. (B) HDV genome

surroundedbyS,MandLsurfaceproteinsfromHBV.

Figure10:HDVreplicationcycleaidedbyHBsAgsynthesisbyHBV.Highlightedinpinkisthe“Rolling

circlemechanism”ofHDVreplication.

20

TwovariantsofdeltaantigenaresynthesizedfromthemRNAandtheyhavetwodistinct

functions. The S-dAg is responsible for viral replication where-as the L-dAg inhibits

replicationandpromotesvirionassembly.Therefore, thebalance in theproductionof

theseproteinsisveryimportanttoestablishasuccessfulinfection.

HBV suppression by HDV

IthasbeenrecordedincellcultureandanimalmodelsystemsthatthepresenceofHDV

reduces the expression ofHBV [123]. Themechanism throughwhich this is achieved

remainsunknown,butincellculturemodelsystemstheL-dAgandS-dAghaveshownthe

abilitytoactivateMxAgenetoincreaseIFN-aresponsetowardsHBVandactivatecellular

immuneproteinsynthesistocontrolinfection[124-126].

Treatment

SinceHBsAg isnecessary for successfulHDV infectionandsubsequent replication, the

currentpreventivevaccineforHBVthattargetsandneutralizesthesurfaceantigenworks

veryeffectivelyagainstHDV[127].Butonceinfectionisactivedthetreatmentoptionsare

quitebleak.SinceHDVonlyrequiressurfaceantigenfromHBV,blockingHBVreplication

isnoteffective[128],andtheonlytreatmentforHDVcurrentlyavailableisINF-a[129].

TreatmentwithINF-ahasshownsignificantimprovementsinhistologicalresponseand

lossofHDVRNAandHBsAginsomepatients[130],butlongtermstudieshavealsoshown

relapseinmostpatientswithfailuretoclearHDV[131].Afewnewtreatmentoptionsfor

chronicHDVinfectionsareunderway,includingMyrcludexBthatblocksHBV/HDVviral

entryintohepatocytes[132],butin-vivoithasbeenshownthatHDVcanpropagatevia

celldivisioneveninthepresenceofthisdrug[133].

21

3 AIMS

Theoverallaimofthisprojectwastoexploreandunderstanddifferentfacetsofchronic

hepatitis B virus infection by means of studying patient serum and liver tissue from

biopsiesandexplantedliver.Specificaimswereasfollows:

PaperI

ToquantifyandcharacterizeHBVRNA in serum in termsof sequence length,particle

association,concentrationandcorrelationswithHBVRNAinlivertissueandHBVDNAin

serum

PaperII

Toinvestigateinvitrotheinfluenceofsubviralparticleoninfectionandexploreitseffects

onanti-HBs.

PaperIII

TodevelopanewmethodtoanalyzeHBVDNAinlivertissueinordertodiscriminateviral

andintegratedDNA.

PaperIV

Toanalyzeintrahepaticfocaldifferencesinthepatientsundergoingtransplantationdue

toHBVorHBV/HDVrelatedliverdisease.

PaperIV

ToexploreHBVRNAprofilesinliverbiopsiesofpatientsrepresentingdifferentstagesof

chronicinfectionbyusingdropletdigitalPCRmethod.

22

4 MATERIALSANDMETHODS

Materials

PaperI:95patientserumfromapreviousstudythatincluded160sampleswasstored

andavailable for furtheranalysis [134].ThesesamplesrepresentbothHBeAgpositive

andnegativegroupsbelongingtogenotypesA,B,CandD.Oneanonymouspatientsample

withHBVDNAconcentrationof108IU/mLthatalsotestedpositiveforHBeAgandHBsAg

wasincludedinthisstudy.

PaperII:TwoanonymousserumsamplesonewithpositiveHBeAgandHBsAgwithHBV

DNAconcentrationwith108IU/mLandtheothernegativeforHBsAgandanti-HBcfrom

avaccinated individualwereselected.Boththesesampleswerealsonegative foranti-

HCV,anti-HIV,anti-HAVIgMandpositiveforanti-HAVIgG.

Paper III : 76 stored liver biopsies out of 160 thatwere included in a previous cross

sectionalstudywereavailableforfurtheranalysis[134]ofwhich70werepositivewith

digitalPCRmethod.ThesampleschosenrepresentedgenotypesA,B,CandD,withliver

damage ranging frommild to severe, either with or without HBeAg. They were also

negative for HIV, hepatitis C or D and were untreated for HBV when biopsy was

performed.

PaperIV:Thisstudyincludedtissuematerialobtainedfrom6patientsundergoingliver

transplantbecauseofHBVrelatedchronicliverdiseasewhereallpatientspresentedliver

cirrhosis. Two patients had HCC; one patient with cirrhosis developed acute liver

decompensationbroughtonbyacute-on-chronichepatitisandpresentedwithveryhigh

serumHBVDNA;twopatientswerealsoco-infectedwithHDV.Sampleswerecollected

duringsurgeryandsubsequentlystoredat-80°Cuntilitwaspreparedforanalysis.

PaperV:76ofthe77liverbiopsiesthatwereusedinpaperIIIwerealsousedforanalysis

inthiswork.

23

Figure11:Schematicrepresentationofpatientsamplesusedindifferentpaperscompiledinthis

thesis.

Methods

4.2.1 Nucleic acid extraction from serum & tissues

Total nucleic acid extractionwas carriedouton both tissue and serumsamples in an

automatedMagNAPureLCsystem(RocheAppliedScience)accordingtomanufacturer’s

protocol. For serum extractionMagNA Pure LC total nucleic acid isolation kit (Roche

AppliedScience)wasusedandfor tissuematerialMagNAPureLCDNAisolationkit II

(Roche)wasused.

4.2.2 DNase and RNAse treatment

A portion of the total nucleic acid extracted was treated with TURBO DNA-free kit

(ThermoFisherScientific)inarigoroustwo-stepprotocolaccordingtomanufacturer’s

instructiontoremovecontaminatingDNA,forRNAanalysis.

24

ASerumsamplewas treatedwithRNAse (ThermoFisherScientific)prior toandpost

MagNAPureLC(RocheAppliedScience)extractiontoquantifyfreeHBVRNAcontentin

serum.

4.2.3 Polymerase chain reaction

MolecularbiologytechniqueshavecomealongwaysinceestablishingtheWatson-Crick

modelin1953.Thepolymerasechainreaction(PCR)methodwasinventedin1983and

allowsamplificationofspecificDNAsegmentofinterest.Itwasbasedontheidentification

ofthespeciesThermusaquaticusin1969thatrequiresatemperatureof70–79°Cforits

growth[135],andtheisolationofitspolymerasetermedasthe“TaqPolymerase”in1976

[136],anenzymethatcouldwithstandhightemperatures.Thisenzymeisusedtocopya

specificDNA sequence in a PCR reaction that is supplementedwith (i) a target to be

amplified(ii)oligonucleotidesequencescalledprimerthatarespecifictothetarget(iii)

nucleotidesneededforcreatingnewtargetsequences(iv)Taqpolymerasetoplaceeach

nucleotideintherightorderforsequenceextension.

4.2.3.1 Reverse Transcriptase PCR

TheonlydifferencebetweenastandardPCRandareversetranscriptasePCR(RT-PCR)is

the template used for amplification. The former uses double stranded DNA (dsDNA)

whereasthelatterusesRNAasstartingmaterial.DuringanRT-PCR,anenzymetermed

asreversetranscriptaseisusedtocreateacomplimentaryDNA(cDNA)strandtotheRNA.

ThisenzymealsocontainstheRNAseHactivitywheretheRNAtemplateisdegradedafter

copying.Now,usingthenewcDNAastemplateasecondstrandissynthesizedtohavea

completedsDNA.Amplificationof thisnewlysynthesizedDNAiscarriedoutusingthe

principlesofstandardPCR.

4.2.3.2 Quantitative PCR and quantitative RT PCR

Aquantitative PCR (qPCR) is amethod that iswidely used in clinical diagnostics and

research alike. There are typically twomethods available to quantify the sequence of

interest,(i)dye-basedmethodand(ii)probe-basedmethod.Inthefirstmethod,agreen

fluorescent dye that intercalates with all double stranded products is used and the

increaseinfluorescenceismeasuredateachcycle[137].Thetechniquethatisextensively

used inthisstudy isprobebasedPCRmethodthat justlikethedye-basedmethodcan

25

measurein“realtime”theamplificationoftargetsequence.Thisproceduremakesuseof

astrandspecificprobethatistaggedwithafluorophoreononeendandaquencheron

theother.Whenkeptincloseproximity,thequencherquenchesthatfluorescenceemitted

bythefluorophore.Thepolymeraseinadditiontoaddingspecificnucleotidesforstrand

extensionhasa second responsibility to removeanydouble strandedsequences in its

pathand replace itwithavailablenucleotidesthis is termedas “exonucleaseactivity”.

Therefore, when it encounters the probe, it cleaves the probe thereby separating the

fluorophorefromthequencherandthefluorescencenowemittedcanbecapturedand

measured.Anadvantagetousingthistechniqueistheabilitytomeasuremultipletargets

inthesamereactionwhichwaspursuedonpaperIVandVusingthedigitalPCRmethod.

ForanRNA target, reverse transcriptase (RT)enzymewas included to create the first

cDNAstrandandthestandardPCRprotocolwasfollowed.

4.2.3.3 Digital PCR

ThetermDigitalPCR(ddPCR)wascoinedin1999whentworesearchersBertVogelstein

andKennethKinzlerstudyingraremutationsincolorectalcancerdevelopedaPCRsystem

wheremultiplereactionswereusedtostudyasingletargetfromthesamepatientsample

[138]. The disadvantage with a qPCR system is that it requires a standard curve to

determine the initial quantity that was present in the reaction which means small

variationsinthesamplesgounnoticed.Toavoidthis,VogelsteinandKinzlerhybridized

twofluorescentprobestotheamplifiedproductonethatbindstothespecifictargetand

thesecondthatbindstoallsequencesasacontrolandmeasuredthe fluorescence.By

doing this they obtained an absolute quantification of the mutation. This came with

certaindisadvantagesliketimerequiredfortheanalysisandtheneedforlargequantity

of starting material. The simplified commercialized procedure now enables the

separationofeachreactioninto20,000nanodropletsusingwater-emulsiontechnology.

FollowingastandardPCR,targetsamplifiedisassessedbyanautomatedsystemwhere

the fluorescence ismeasuredwithineachdroplet.UsingPoisson statisticsanabsolute

quantificationofthetargetcanbeobtained.

4.2.4 Nycodenz fractionation

FractionationofserumsampleinpaperItoshowthepresenceofviralRNAwithincapsids

andinpaperIItoseparateviralandsubviralparticleswascarriedoutusingNycodenz–

26

asubstanceusedcommonlyforisolationofmanycelltypes.Thissubstanceiswidelyused

becauseofitsnon-cytotoxicnature,abilitytobeautoclavedwithoutcompromiseandnon-

interferencewithdownstreamanalysis [139-141].Thismethodwasutilized inpaper I

and II where different products synthesized by HBV infection present in serum was

separated in different Nycodenz fractions depending on their density. The separated

fractions were then subjected to analysis such as TaqMan PCR, RT PCR and in-vitro

infectionofHepG2NTCPcells.

4.2.5 Sanger sequencing

ExtractedHBVRNAsequencefrompatientserumwasinthepresenceofRTenzymeand

areverseprimerthattargetsthepoly-Atailatthe3’end,usedtocreateacomplimentary

cDNAstrand.ThecDNAwasthensubjectedtotwoPCRsthattargetthe5’and3’regions

ofthecDNAandtheampliconsobtainedweresequencedusingSangermethodtoidentify

mutations that may contribute to the presence of RNA in serum where reverse

transcriptionhasnotoccurred.

4.2.6 In-vitro infection

Invitrocellculturetechniquesarebeingdevelopedrigorouslytoefficientlyreplicatean

infectionoutsidethehostinordertoimproveunderstandingofthecausativeagentand

developappropriatetreatment.HepG2NTCPcelllinewasrecentlyestablishedafterthe

discoveryofthereceptorNTCPneededforHBVtogainentryintothecells[114].HepG2

cellsstablytransfectedwithHBVspecificreceptorNTCPwasmaintainedat37°Cunder

5% CO2 in Dulbecco’s modified Eagles medium (DMEM) containing high glucose

(Invitrogen).InaccordancewiththeprotocoldefinedbyNietal.,in2014,themediaused

for culture maintenance and subsequent infection was substituted with 10% FBS,

100U/mL penicillin, 0.1 mg/mL streptomycin and 2mM L-glutamine [11]. Using a

hemocytometercellswerecountedandseededtogiveapproximately1.25*105cellsper

well.24hourslaterwhencellsreachedoptimumconfluency,HBVserumsamplewithor

withoutpriorincubationwithanti-HBsantibodieswasaddedtothecellsalongwith2.5%

dimethylsulfoxide(DMSO)(Sigma)and4%polyethyleneglycol800(PEG800)(Sigma)

andincubatedtostartinfectionexperiment.Thecellswerethenwashedthreetimeswith

DMEMafter24hoursandreplacedwithfreshculturemedia(CM).Everyseconddaythe

27

CMfromeachwellwascollectedandstoredfordownstreamanalysisandreplacedwith

freshCMspikedwith2.5%DMSOuntiltheendoftheexperiment.

4.2.7 Architect Assay

ThistechniquewasusedtoquantifyHBeAgsecretedbyinfectedHepG2NTCPcells.Inthis

two-stepimmunoassay,thesampleisfirstfloodedwithanti-HBecoatedmicroparticles

followedby introductiontoacridiniumlabeledconjugatedanti-HBe.Awashingstep is

includedafterbothstagestoremoveanyunboundproducts.Thechemiluminescencethat

isproducedbythebound,labelledtargetiscapturedandcomparedtosignalcutoffratio

measuredduringcalibrationoftheinstrumenttoobtainaquantificationofHBeAginCM

frominfectedcells.

28

5 RESULTSANDDISCUSSION

Paper I

NAsusedforCHBtreatment,blockpolymeraseactivitythatleadstorapidreductionof

HBVDNA,aserologicalmarkerquantifiedtomonitordiseaseprogression[24]aswellas

a predictor for liver cirrhosis andHCCdevelopment [142].HBVRNAhowever, is not

affectedbythetreatmentandthereforecanbeusedasamarkeroftheinfectioninthe

liver[31-33]inadditiontoserumHBsAg[143-145].

Inorder to characterizeHBVRNAdetected in serum,density-based fractionationwas

performedusingsixconcentrationsofNycodenzgradients(8,16,25,33,42,and50wt%)

followedbyquantificationusingrealtimePCR.HBVRNAandDNAparticlesweredetected

inthesamefractionssuggestingsimilardensities(figure12A).Anearlierstudyusing

sucrosegradienttofractionateHBVDNAandRNApostentecavirtreatmenthasshown

similar results [146]. Since mature HBV virions are present within nucleocapsids,

treatmentwithdetergentTween-80disrupted these capsids causing thepeak fraction

containingHBVDNA tomove toward higherNycodenz concentration (figure 12B). A

similarpatterninRNAsuggestedpresenceofRNAwithinenvelopedcapsids(figure12

B).

Figure12:(A)NycodenzgradientcentrifugationofserumsampleshowingseparationofHBVDNA

andRNA(B)NycodenzfractionationofHBVpositiveserumafterTween-80treatment.

HBVDNAandRNAwerequantifiedinserumcollectfrom95patientschronicallyinfected

withHBV.ThelevelsofHBVRNApresentweresimilartoHBVDNAinbothHBeAgpositive

andnegativepatientgroups(figure13A).

29

Figure13:(A)correlationbetweenHBVDNAandRNApresentinserum(B)GenotypeDshowing

significantlyhigherratiobetweenHBVDNAandRNAincomparisonwithnon-genotypeDsamples

(p=0.0001).

Sequencingwasusedtocharacterizethe3’partofHBVRNAfromserum.FirstcDNAfrom

wassynthesizedbyusingaprimerwithtagsequencetargetingthepolyadenylated3’end.

Thiswasfollowedupwithaforwardprimeratnt1550andareverseprimertargetingtag

sequence inserted during cDNA synthesis. Agarose gel electrophoresis of the PCR

products(figure14A) indicatedthatonlyasmallproportionofRNAwasprematurely

polyadenylated. In order to assess if the HBV RNA was of full length, real time PCR

targetingcoreat5’regionandXat3’regionwasperformedonthecDNAgeneratedbythe

poly-Treverseprimer(figure14B).Theresultsshowingsimilarquantitiessuggestthat

theHBVRNApresentinserumwasoffull(genome)length.

FromtheseresultsweconcludedthatHBVRNAinserumwaspgRNAinsidecapsidsthat

probablywere enveloped, likely because the reverse transcription step had failed. To

study if this might be due to mutations in the e sequence, Sanger sequencing was

performedon3samplesaftersynthesizingcDNAusingareverseprimertargetingthe3’

polyadenylationregion.Acorrectesequenceisnecessaryforencapsidation[17,147]and

toinitiatereversetranscription[18].Nomutationsthatwouldinfluencethefunctionof

the e sequencewas detected. Yet, sequence variationmight be of importance for the

failureofreversetranscription,becausetheratiobetweenHBVDNAandHBVRNAwas

higheringenotypeDincomparisontogenotypeA,BorC(figure13B).

30

Figure14:(A)AgarosegelelectrophoresisshowingPCRproductsobtainedfromserumsample(B)

Real time PCR targeting 5’ and 3’ genomic sequences (core and X) after cDNA targeting

polyadenylatedtailat3’region.

Figure15:LowcorrelationbetweenpgRNAinlivertoHBVRNApresentinserumforHBeAgpositive

andnegativepatients.

ThelevelsofHBVRNAinlivertissueandserumcorrelatedsignificantly,althoughlessin

theHBeAg-negativegroup(Figure15).Comparingresultsinfigure13Atodatainfigure

15,showsthatthecorrelationbetweenlevelsofHBVDNAandHBVRNAinserumwas

strongerthanbetweenpgRNAinliverandHBVRNAinserum.Thisshowsthatsecretion

of virus-like particles with HBV RNA is linked to secretion of mature viral particles

(containingHBVDNA),ratherthanrepresentingintrahepaticRNAlevels.

31

In conclusion, the results presented in this article represent high concentrations of

encapsidated, full genomic length RNA in serum and encourages further research to

explainfailureofreversetranscription.

32

Paper II

ExcessproductionofSVPduringHBVinfectionincomparisontogenomecontainingVP

hasbeenobservedpreviouslyattherateof2000SVPforeveryVP[141,148].BothVP

andSVPcontain theenvelopeproteinHBsAg,however the ratioofpreS1domainthat

recognizesthecellspecificreceptorNTCPishigherinVPincomparisontoSVP.Excess

SVPproductionhasbeenproposedtoactasadecoytoreduceeffectofanti-HBsantibody,

butnoexperimentalprooftosupportthishypothesisisavailable.

Inorder to study theeffectsof SVP,Nycodenz fractionationofpatient serumshowing

signsofhighviralreplicationwascarriedouttoseparateSVPfromVP, tobeusedfor

subsequentinfectiononHepG2-NTCPcells.Serumfromanindividualwithhighanti-HBs

levelsafterbeingvaccinatedforHBVwasalsousedinthisstudytoassesseffectsofSVP

onhost antibodies.HBeAgproducedandsecretedby infectedHepG2-NTCPcellswere

measuredtoquantifyinfectivity.

Anti-HBswasseriallydilutedandpreincubatedwith(i)unfractionatedserum(containing

bothVPandSVP),(ii)VPand(iii)mixtureofVP+SVPin1:1ratio.IncubationofVPwith

anti-HBsshowedamarkedreductionintheinfectionofHepG2-NTCPcells(lowerHBeAg

levelsforthestraightascomparedwiththedottedlinesinfigure16).Inthepresenceof

subviralparticles(VP+SVP), thisneutralizingeffectofanti-HBswasreduced, iehigher

concentration of anti-HBs was required to reduce infection. This effect was more

pronounced in unfractionated serum (which likely contained SVP in much higher

concentrationsthanVP(figure16AandB).

TostudyifSVPcanalsoblockVPentry,differentconcentrationsofSVPweremixedwith

fixedVPconcentrationsinthepresenceorabsenceofanti-HBsantibodies(table1Aand

B).SVPneutralizedactivityofanti-HBsby55%ataconcentrationof200IU/mL,whereas

SVP alone, in the absence of anti-HBs, showed minimum reduction in infectivity,

suggestingthatcompetitionbetweenSVPandVPforbindingandcellularentryisnotof

significant importance. This result is comparable to previously published datawhere

artificiallysynthesizedSVPdidnotblockVPentry[149],butafewstudiesconductedon

33

DuckHBVhaveexhibitedSVPspotentialtobothneutralizeanti-HBsandcompeteforviral

entry[150,151].

Figure16:Serialdilutionofanti-HBsconcentrationstartingfrom(A)10,000IU/Lor (B)30,000

IU/L. Straight and dotted lines represent presence or absence of anti-HBs antibodies. Infection

potentialwasquantifiedusingHBeAgsecretedbycells.

Table1:ExperimentaldesignandtabulatedresultshowinghighercapacityofSVPtoadsorbanti-

HBs(A)thanblockentryofVP(B).

In conclusion, observations from this study suggest that themain role of SVP during

infectionistoactasadecoyandneutralizeantibodiesproducedbythehost.

34

Paper III

Inthisarticleamethodwasfirstdevelopedtoquantifythecircularandlinearformsof

intrahepaticDNA fromCHBpatient biopsies. Nucleotide (nt) position 1830has been

recognizedasapreferentialsiteforHBVintegrationsandthereforeaPCRspanningthis

region would differentiate between integrated and non-integrated transcripts. A

discriminatingPCRassaywassetupusingtwosetsofprimers,onespanningnt1830(for

circularDNA)andanotherplacedintheXregionthatwouldamplifyallformsofHBVDNA

(circularandlinear).

Figure17 :DiscriminatingPCRusing forwardprimerat nt 1550 in combinationwitha reverse

primeratnt1627toamplifyallHBVDNAforms,andforwardprimeratnt1776incombinationwith

reverseprimeratnt1924toamplifyonlycircularformsofHBVDNA.

ThisdiscriminatingPCRwasappliedon70liverbiopsiesfrompatientsindifferentstages

ofchronicinfection.TheresultsrevealedthatlinearDNAconstitutedalargefractionof

thetotalintrahepaticHBVDNAinbothHBeAgpositive(54%)andnegativegroup(89%).

The variation between these two groups were statistically significant (P<0.0001)

indicatinghigherquantityoflinearDNAine-negativepatients(figure18).

Controlexperimentsonserumand liversamplesshowedthat linearHBVDNAin liver

tissue (but much less in serum) was sensitive to degradation by endonuclease

35

(benzonase), indicating that it was not encapsidated but rather represents HBV DNA

integratedinthechromosomalDNA.

Figure18:PercentageoflinearDNAinHBeAgpositiveandnegativeHBVpatients

Figure19:PercentageofintegratedHBVDNAoutoftotalintrahepaticDNA.

To calculate the fraction of integrated HBV DNA out of total intrahepatic DNA, two

assumptionweremade:(i)dslDNA(notintegrated)isapproximately20%ofrcDNAand

(ii)allcircularDNAisrcDNA(cccDNAismuchlowerthanrcDNA).Theseassumptionsare

supportedbypublisheddata, includingstudiesestimatingcccDNAtobe<10%of total

circularDNAinliver[36,39,142].ByusingtheformulaIntegratedDNA=totallinearDNA

–dslDNA(assumedtoconstitute20%oftotalrcDNA),integrationinHBeAgpositiveand

negativepatientswerecalculatedtoconstitute46%and87%oftotalintrahepaticlinear

36

DNA respectively (figure 19). Together, these experiments indicate presence of

integrationsinlargeproportionsoccurringveryearlyduringHBVinfection.

Previously published large scale study using inverse PCR technique has estimated

presenceof integrations inapproximatelyabout1%of total livercells[152].However

due to technical challenges, inverse PCR is said to detect only about 10% of total

integration events [153] which suggests approximately 10% of liver cells carry

integration.Thisislowerthanourestimates,butalmostatasimilarlevel.

Thehighdegreeof integration, inparticular inHBeAg-negativepatients,supports that

expressionofintegratedHBVDNAmightcontributetomaintaininghighlevelsofserum

HBsAginpatientswithlowrateofreplication.SuchhighHBsAglevelshavebeennoticed

in clinical diagnostics for a long time [60], and illustrates the striking difference in

immunemediatedreductionofHBVDNAandHBsAg.Inoursamplesthiswasobserved

asalarge(thousand-fold)differenceintheHBsAg/HBVDNAratioinHBeAg-positiveas

comparedwithHBeAg-negativepatients.AsshowninFigure20thisHBsAgtoHBVDNA

ratioinserumsignificantlycorrelatedwiththefractionoftotalHBVDNAinlivertissue

thatwaslinearHBVDNA,likelyintegratedinchromosomalDNA.Thisfindingsupports

that integrated DNA significantly contributes to HBsAg production in HBeAg-negative

patients.

Figure 20 : Significant correlation (p<0.0001) between ratio of HBsAg andHBVDNA in HBeAg

positiveandnegativepatientserumandproportionofintegrationindicativeoflinearDNAinbiopsy.

37

Inconclusion,thisstudydescribesanovelandreliabletechniquetoquantifyintegrations.

Inaddition,thisstudyalsocontributesexperimentalproofusingnaturalHBVinfectionin

patients,tosupportthehypothesisthatintegrationscontributetomaintenanceofHBsAg

evenduringlowreplicationrates.

38

Paper IV

Liverbiopsieswerewidelyusedinthepasttoestimateintrahepaticinjuryandanalyze

HBVmarkersbyimmunohistochemistrystainingormolecularmethods.Inthisstudy,15-

30livertissuefrom6patientsundergoingtransplantationforHBVrelatedliverdisease

wasanalyzed.TheaimofthestudywastoinvestigatetherelationbetweendifferentHBV

markers, in particular those representing integration in the host genome, that was

previously hypothesized to be variable by Lindh et al. [154]. This strategy was also

employedtoestimatetheriskofsamplingerror,thatis,theriskthatananalyzedpieceof

livertissueisnotrepresentativeoftheinfectionstatusinthewholeliver

Allpatientswereonantiviral therapyofshortorlongduration.Patients1-4hadHBV-

induced livercirrhosis,but the infectionactivitydifferedgreatly.Patients1and3had

recentlyexperiencedreactivationofhepatitiswithserumlevelsofHBVDNAat≈8log

IU/mL prior to initiation of entecavir or tenofovir treatment three weeks and four

months, respectively, before transplantation. Patient 2 had an infection thathad been

highlyactiveforalongtimeandhadrelativelyhighlevelsofHBVDNAinserumasaresult

ofantiviralresistance.Patient4hadlow-activeinfectionandhadbeenontenofovirfor>

twoyears.Patients5and6hadHBV/HDVco-infectionwithlowHBVDNAandrelatively

highHDV-RNAlevelsinserum.

As shown in Figure 21, the degree of infection focality differed markedly between

patients;betweenviruses;andbetweendifferentHBVviralmarkers.ForHBVinfection,

themostabundantmarkerwasSRNA,followedbycoreRNA,totalHBVDNAandcccDNA.

Patient 1 expressed the highest levels of all HBV markers, and had relatively small

variation in the distribution of infection (all markers detected in all pieces, with CV

rangingbetween4.4%and8.6%).Patients2and3(P3)hadlowerlevelsandhigherCV

values forbothHBVDNAandRNA,but thesemarkerswerestilldetected inallpieces.

Patient4(P4)(ontenofovir for>2yearsbeforetransplantation)had lowHBVDNAin

mostpiecesandmuchhigherCVvalues(Table2).

39

Figure21:Quantificationof(A)totalHBVRNAmeasuredbyStranscriptPCRand(B)coreRNA(C)DeltaRNAinpatient5(inblue)andpatient6(in

brown).

40

Patient1 Patient2 Patient3 Patient4Indicationfortransplantation Acuteliverfailure,

cirrhosisCirrhosis HCCandcirrhosis Cirrhosis

Ageattransplantation 43.6 50.5 54.8 34Geographicorigin EastAfrica Balkan MiddleEast EastAfricaAntiviraltreatment Entecavir, Lamivudine, Tenofovir Tenofovir

3weeks resistancea 4months >2yearsHBsAgserum(logIU/mL) 4.16 3.79 3.61 1.00HBVDNAserum(logIU/mL) 6.64b 5.55 2.09c NegLivertissuedata

Piecesanalyzed 20 30 25 20

Cellsperpiece,median(IQR) 2735 1962 2900 5940

HBVDNA,logcopies/1000cells Total(Starget) 3.69(3.07-4.24) 2.41(1.66-2.83) 1.32(0.59-1.72) 0.14(-0.32-1.72)Coefficientofvariation 8.6% 11% 22% 135%PiecesPCRpositive 20(100%) 30(100%) 25(100%) 16(80%)

cccDNA,logcopies/1000cells 3.27(2.78-3.66) 1.04(0.31-1.91) 0.84(-0.02-1.45) <-1

Coefficientofvariation 7.3% 33% 48%

PiecesPCRpositive 20(100%) 30(100%) 24(96%) 0

HBVRNA,logcopies/1000cells Starget 5.40(4.96-5.89) 4.24(2.75-4.83) 2.65(0.69-3.82) 0.15(<-1-2.54)Coefficientofvariation 4.4% 12% 30% 187%PiecesPCRpositive 20(100%) 30(100%) 25(100%) 12(60%)Coretarget 5.29(5.11-5.45) 2.78(2.55-2.97) 2.24(1.12-2.81) <-1Coefficientofvariation 4.7% 10% 40% 110%PiecesPCRpositive 20(100%) 30(100%) 25(100%) 9(45%)

aM204VmutationinthereversetranscriptaseregionofHBV. bTheHBVDNAlevelwas8.19logIU/mLwhenentecavirtreatmentwasinitiated3weeksbeforetransplantation.cTheHBVDNAlevelwas7.90logIU/mLwhentenofovirtreatmentwasinitiated4monthsbeforetransplantation.Medianand(range)values.CV,Coefficientofvariation=standarddeviation/mean.

Table2:CharacteristicsoffourlivertransplantedpatientswithHBVinfectionandresultsfromddPCRanalysesofliverexplanttissuepiec

41

Patient5 Patient6

Indicationfortransplantation Cirrhosis HCCandcirrhosis

Ageattransplantation 53.3 47.2

Sex Male Male

Geographicorigin MiddleEast MiddleEast

Antiviraltreatment Tenofovir2years Entecavir2months

HBsAgserum(logIU/mL) 3.27 2.86

HBVDNAserum(logIU/mL) Neg Neg

HDVRNAserum(logcopies/mL) 4.99 3.94

Livertissuedata

Piecesanalyzed 20 15

Cellsperpiece,median(IQR) 2130 4721

HBVDNA,logcopies/1000cells

Total(Starget) 1.03(0.43-2.29) <1(-0.08-1.87)

Coefficientofvariation 45% 86%

PiecesPCRpositive 19(95%) 7(47%)

cccDNA,logcopies/1000cells <-1 <-1

Coefficientofvariation

PiecesPCRpositive 0 0

HBVRNA,logcopies/1000cells

Starget 2.85(1.31-4.10) 1.91(1.12-3.74)



Coretarget 0.75(0.13-1.38) <-1



HDVRNA,logcopies/1000cells 6.70(5.26-7.28) 4.28(3.82-4.99)


PiecesPCRpositive 19(95%) 15(100%)Medianand(range)values.Coefficientofvariation,CV=standarddeviation/mean,calculatedforlevelsinsampleswithdetectedtarget.

Table3:CharacteristicsoftwolivertransplantedpatientswithHBV/HDVinfectionandandresults

fromddPCRanalysesofliverexplanttissuepieces

Patients5and6(P5andP6)hadco-infectionwithHDVlowHBVDNAandrelativelyhigh

HDVRNAinserum.Inlivertissue,cccDNAwasundetectedinallpieces,HBVDNAand

coreRNAwasloworundetected.Yet,SRNArangedfrommoderatetohighlevelsinall

pieces.HDVRNAwasdetectedathighlevelsinallpieces(figure21andtable3).

42

The findings in the HDV cases agrees relatively well with those in a previous study

comparingintrahepaticmarkersin21HBV/HDVco-infectedand22HBVmonoinfected

patients[155].Inthatstudy,livertissuefromHDV-infectedpatientshadlowerlevelsof

cccDNA, lower pgRNA/cccDNA and higher S RNA/cccDNA than patients with HBV

monoinfection.

TheresultsshowsignificantfocaldifferencesinthedistributionandactivityofbothHBV

and HDV and demonstrates the risk involvedwhen analyzing a single tissue piece to

quantitativelyrepresentintrahepaticlevelsespeciallyinpatientswithlowviralload.The

riskofobtainingfalsenegativeresultswasrelativelylowexceptforHBVmarkersintissue

withlowHBVload(suchasoftenseenwhenthereisHDVco-infection)

In the following figures,markersarepresentedpairwise inordertoshowcorrelations

andtodisplaydifferencesinmoredetailbyshowingvaluesforindividualpieces.Figure

22showsthatHBVcoreRNAandSRNAvaluescorrelatedstronglyinpiecesfromPatients

1 and 3. This probably reflects that in these patients, core and S RNA were mainly

transcribedfromcccDNA,andatsimilarratesindifferentpieces.Bycontrast,thelevels

didnotcorrelateinpiecesfromPatients2,4,5and6.InPatient2,coreandSRNAwere

bothpresentinrelativelyhighamounts,butwithoutcorrelation.InPatient4thelackof

correlationmighttosomeextentbeduetothepooreranalyticalprecisionforlowtarget

levels.InPatients5and6(withHDVco-infection)coreRNAwasundetectableormuch

lowerthanSRNA.

Figure23showsreflectionofcccDNAonHBVRNAlevels(in thethreepatientswhere

cccDNAwasdetected,P1-3).Overall,coreRNAcorrelatedstronglywithcccDNA,whereas

forSRNAacorrelationwasonlyobservedinP1andP3butnotinP2.

Infigure24thesamedataispresentedasRNAcopiespercccDNA.InPatient1whohada

highlyactiveinfectionthecoreandSRNAlevelspercccDNAweresimilarandatlevels

around100RNAcopiespercccDNAwithlittlevariationbetweenthepieces.InPatient2

thecoreRNAlevelswereslightly lowerwithrelatively littlevariation,whereasSRNA

levelsweremuch higher andwith large variation, from60 to8000 SRNA copies per

cccDNA.

43

Figure22.HBVcoreRNAandSRNAin15-30piecesoffromfourpatients(P1-P4)withHBV(A),and

twopatients(P5-P6)withHDV/HBVcoinfection(B).Thevaluesareinlog10copies/1000cells.

Figure23:Correlationsbetween(A)cccDNAandcoreRNAand(B)cccDNAandSRNAinpiecesof

explantedliverfromP1-3.Thevaluesareinlog10copies/1000cells.

SincecccDNAistheonlytemplateforcoreRNAwhereasSRNAcanbetranscribedfrom

cccDNA or from integrated sequences [63, 156], the results suggest that a large

proportionof theSRNA inPatient2 (andtosomeextent inPatient3)maybedue to

transcriptionfromintegratedHBVDNA.Thisobservationfitsbothwithourfindingsin

Paper 3 and with data from a recent publication on experimental HBV infection in

chimpanzees[61].

44

Figure24:LevelsrepresentedinP1-3ascopies/cccDNA.

In thetwopatientswithHDVco-infection, therewasnocorrelationobservedbetween

HDVRNAandHBVSRNA.PresenceofhighlevelsofHDVRNAwasobservedevenwhenS

RNAlevelswerelow,asshownin figure25A.Accordingly,asshownin figure25B,the

HDV RNA:S RNA ratio was highly different, spanning from 10:1 to >100,000:1. This

findingsuggeststhatproductionofHDVRNAisindependentofHBsAgandthatHDVRNA

might be produced also in cells that do not produceHBsAg. Thus, it seems as ifHDV

infectionmaybepresentinhepatocytesindifferentforms:(i)atrueco-infectiontogether

withreplicatingHBVwhichprovidesHBsAgfromcccDNA;(ii)incellswithoutreplicating

HBVbutwithintegratedHBVDNAthatprovidesHBsAgsothatHDVvirusparticlescan

beformedandsecreted;(iii)HDVRNAincellswithoutHBsAgproduction,replicatingand

translatedtodeltaantigenbutnotsecretedsinceHBsAgisnotprovided.Thismodelis

supportedbydatafrompreviouspublications.Inonestudy,HBsAgfromintegrationswas

showntosupportHDVreplicationinvitro[157],andarecentstudyreportedpersistence

of HDV during hepatocyte replication and its presence in daughter cells that did not

containHBV[133].

Patient 1 Patient 2 Patient 3

45

Figure25:(A)LackofcorrelationbetweenHDVRNAandHBVSRNAinP5andP6(B)Variedratio

betweenHDVRNAandHBVSRNAinP5andP6.

The model proposes that only a minority of cells capable of producing HBsAg (from

cccDNA or transcripts integration) are required for production of high quantities of

intrahepaticHDVRNA(asseenbythelackofcorrelationbetweenHBVSRNAandHDV

RNA).Thismayhaveclinicalimplications.ItwouldmeanthatproductionofHDVvirus

particlesmay occur in a small proportion of hepatocytes that produces HBsAg (from

cccDNAorintegrations),butthatHDVRNAfromtheseparticlesmightenteramuchlarger

number of hepatocytes and drastically increase the production of HDV antigens and

expressionoftheirepitopesonthecellsurfacetoprovokeharmfulimmuneresponses.

ThismodelwouldnotcontradicttheassociationbetweenHDVRNAinserumandliver

damage,butratherproposeamechanismbywhichthepathogeniceffectsofsecretedHDV

couldbeamplified.Iftrue,thismodelmayhelptounderstandmechanisminvolvedinthe

development of severe necroinflammation that is often seen during HDV infection.

Furtherresearchisrequiredtoclarifythesehypotheses.

Inconclusion,theanalysisofmultiplepiecesoflivertissueshowswiderangeoffocality

within the same infected liver especially in individuals with low viremia; shows

integrationstobeasourceofHBVSRNAespeciallyinHDVcoinfectedpatientswithlow

HBVviremia;andalsoproposesnovelmechanismstopavewayfor futureresearchto

understandaggravatedliverinjuryduringHBV/HDVco-infection.

46

Paper V In this article, biopsymaterial representing bothHBeAg positive andHBeAgnegative

infectionin76patientswereusedtodescribeRNAprofileofthedifferenttranscriptsseen

during HBV infection. Levels of the transcriptswere quantified to also distinguish its

source(cccDNAorintegratedHVDNA).

ThePCRtargetsareshowninfigure26.ThesePCRsinvolveamplificationofoverlapping

targets, anddropletdigitalPCR techniquegivesamethodological advantageover real

timePCRsinceabsolutequantificationcanbeobtainedwithoutamplificationefficiency

bias.

Figure26:PCRstrategytoquantifydifferentRNAspecies.

Figure27showslevelsofallRNAspeciesmeasuredinthe76biopsies,demonstratingthat

theRNAprofiledifferedsignificantlybetweenHBeAgpositiveandnegativepatients.In

particular,HBeAg-negativepatientshadlowerlevelsofpgRNA,coreRNAand3’precore

regions, a finding that agreeswellwith recent transcriptomedata from liver biopsies

takenfromexperimentallyinfectedchimpanzees[61].

47

Figure26:IntrahepaticHBVRNAprofilesinpatientspositiveornegativeforHBeAg.Theboxplots

showmedianlevelsandinterquartilerangesbydigitalPCRassaystargetingdifferentsegmentsof

thegenome.ThelevelsofHBsAgandHBVDNAinserumareshownforcomparison.Thewhiskers

show10thand90thpercentile.PC,precore;pg,pregenomic..***,p<0.0001;**,p<0.001.

Figure27showslevelsofcoreandSRNAfrom76patients.TheHBeAg-positivepatients

hadsimilarlevelsofcoreandSRNA.Bycontrast,HBeAgnegativepatientshad10-100

timeslowercorethanSRNA,illustratedinthefigurebythedistancefromthedottedline.

The difference could be due to specific transcriptional down- regulation of core RNA

synthesis.EvidenceofepigeneticregulationinHBVhasbeenpresented[158],butsofar

nodatasuggestsspecificcontrolofcoreRNA.

QuantificationofcoreRNAwasperformedusingtwoadditionalprimers,targetingits5’

part(“pgRNA”)oratargetregiondownstreamofthecoregene.Figure28Ashowslower

levelsofpgRNA(5’coreRNA)thancoreRNA,inparticularinHBeAgnegativepatients.

Figure 29 presents the levels from 28A as box plots, and with the core RNA values

obtained after subtraction of pgRNA (performed because the values by the core PCR

detectsalsothepgRNAmolecules).ThisplotclearlydemonstrateslowerpgRNAlevelsin

HBeAg-negativepatients.

48

Figure27:CorrelationbetweencoreandSRNAinHBeAgpositiveandnegativepatients.

Sincethetranscriptslackthe5’partcontainingtheeregionessentialforencapsidation

andinitiationofreversetranscription,thesecannotserveaspregenomicRNAandmay

contribute to explain lower level of HBV replication observed in HBeAg negative in

comparisonwithHBeAgpositivepatients[24].

Primers targeting the 3’ precore region (the 3’ redundancy) should amplifies the 3’

terminalpartofcoreRNA.Thefindingthatthelevelsby3’precorePCRwereloweras

comparedtocore(figure28B)suggestthatpartofthecoreRNAisshorterbecausean

upstreampolyadenylationsignalatnt1930wasused[159,160].Degradationofthe3´

partmightbeanalternativeexplanationtothisobservedreduction.

All RNA that are transcribed from cccDNA should contain the terminal 3’ redundancy

representingnt1830-1927.Therefore,ifcccDNAwasthesourceofSRNA,amplification

bytheSRNAand3’PCassaysshouldgivesimilarresults.AsshowninFigure30there

was a striking discrepancy,withmuch lower3’ RNA than and SRNA levels inHBeAg

negative than in HBeAg positive patients. The lower 3’ levels might be due to RNA

degradation,butthefindingthat3’RNAlevelswerealmostashighasSRNAinHBeAg-

positivepatientsargueagainst thispossibility.ThediscrepancybetweenSand3’RNA

wasgreatestinHBeAg-negativepatientswithlowerlevelsofreplication.Inthesecases,

49

3’RNAwas3log10unitslowerthanS,indicatingthat>99%oftheSRNAwasderivedfrom

integratedHBVDNA.

Figure28:RNAlevelsdetectedby(A)coreand5’core(pgRNA)assaysand(B)coreand3’PCRNA.

Figure29:Boxplotshowingthesamemarkersasinfigure27A,butwithcoreRNAvaluesobtained

aftersubtractionofpgRNAvalues.

pgRNA core RNA pgRNA core RNA0

2

4

6

8

Log

copi

es/1

000

cells

HBeAg+ HBeAg–

50

Figure30:Correlationbetween3’andSRNAtranscriptsinHBeAgpositiveandnegativepatients.

ToexploretheputativeimportanceofcccDNAandintegratedHBVDNAassourcesofHBV

DNAandHBsAg inbloodwecompared the ratiosof thesemarkers in liver tissueand

serum.Figure31AshowsratiobetweencccDNAandtotalintrahepaticDNAcorrelating

withratiobetweenpgRNA(thatcomesfromcccDNA)andXRNA(whichcancomefrom

bothcccDNAandintegratedDNA).Thesignificantcorrelationfitswiththeexplanationof

cccDNAbeingthesourceforpgRNAsynthesis.Figure30Bshowsthatinthenextstep,the

ratio between serumHBVDNA andHBsAg correlatewith intrahepatic proportions of

betweenpgRNAandXRNA.Theseresultsagreewellwiththeideathatalargeproportion

of HBsAg synthesized in HBeAg negative patients are a product of integrations, in

accordancewitharecentpublicationwheretranscriptomeanalysisinchimpanzeemodel

systemwasusedtostudyintegrations[61].

51

Figure 30. Correlations between (A) the core RNA/total HBV RNA ratio and the ratio between

cccDNAandtotalintrahepaticHBVDNA,and(B)thecoreRNA/totalHBVRNAratioandtheserum

HBVDNA/serumHBsAgratio.

AnalysesofmultiplebiopsiesandexplorationofRNAtranscriptratiobymeansofdigital

PCR has provided valuable support to the theory that most of the HBsAg in HBeAg

negative patients have integrated transcripts as their source. The results also show

presenceofintrahepaticHBVcoreRNAthatlacksthe5’orboth5’and3’end,eliminating

thepossibilitytofunctionaspgRNA.Thisishypothesizedtobecontributetothereduction

ofHBVreplicationinpatientswithHBeAgnegativeinfection.

A

B

y=0.75x+0.57R²=0.31p<0.0001

-4

-3

-2

-1

0

1

-6 -4 -2

pgRN

A/total(X)RNA

cccDNA/totalintrahepaGcHBVDNA

y=1.17x+3.86R²=0.57p<0.0001

-2

-1

0

1

2

3

4

5

6

-4 -3 -2 -1 0 1

SerumHBV

DNA/HB

sAgraGo

pgRNA/total(X)RNA

52

6 CONCLUSIONS

HepatitisBvirus(HBV)infectionhasthepotentialtocausesevereliverdamageincluding

cirrhosis and hepatocellular carcinoma (HCC) which is predicted using diagnostic

markerssuchasHBVDNA,HBsAgandHBeAginserumandHBVDNAandRNAinliver

tissue.Nucleoside/nucleotideanaloguetreatmentscurrentlyavailableeffectivelyblocks

viral replication but fails to clear reminiscent cccDNA in liver which can reactivate

replicationwhentreatmentisterminated.

Duetorapidreductioninviralreplication,HBVDNAinserumcannotbeusedtoevaluate

thelong-termeffectoftreatmentonintrahepaticcccDNA.HBVRNAinserumhasbeen

proposed as a potentialmarker for this purpose since it is not directly influenced by

treatment.Inpaper1,thismarkerwascharacterized.Analysisofserumfrom95patients

indicate that HBV RNA in serum is encapsidated and of full genomic length RNA and

possiblylessabundantingenotypeD.

Subviral particles (SVP) in the ratio of >10,000:1 to viral particles (VP) during active

replication has been proposed to act as a decoy to host antibodies (anti-HBs) against

HBsAg.Thefirstexperimentalsupportforthishypothesiswasdescribedinpaper2,in

resultsobtainedusingarecentlydevelopedcelllineHepG2-NTCP.ItwasshownthatSVP

significantly reduced theneutralizingeffectof anti-HBs in in vitro infection,buthad a

rathersmallcompetingimpactonbindingtotheviralspecificreceptor.

DuringHBVreplication,adoublestranded linear formof thegenome(dslDNA)rather

thanthe functionalcircular form, isproducedinminority,andmaybecomeintegrated

intothehostgenome.Anovelstrategytoestimate integrationbymeansofmeasuring

circularandlinearformsofHBVDNAinliverusingdigitalPCRwasdevelopedandapplied

on liver biopsies. A panel of digital PCR assays were also developed to obtain a

quantitativeprofileofHBVRNAinthesebiopsies.Theresultsfromtheseanalysessuggest

thatamajorityofHBVDNAfoundinliverduringlateinfectionstageisinintegratedforms

thatcontributestomaintaininghighlevelsofHBsAgintheserumevenduringlowHBV

replication. In addition, results suggesting a novelmechanism associating low pgRNA

transcriptiontoreducedreplicationduringHBeAgnegativestatewereobtained.

53

Inaseparatestudy,focaldifferencesinthedistributionofHBVinfectionintheliverwere

studiedusingliverexplantmaterialfrompatientsundergoingtransplantationduetoHBV

orHDVinducedliverdisease.Byanalyzingmultipletissuepiecesextensivedifferencesin

HBV viral load and transcription (a hundred-fold or more between some pieces),

especiallyinpatientswithlowviremiaorwithHDVcoinfectionwerefound.Highlevels

ofSRNAintheabsenceofcccDNAorcoreRNAsupporttheexpressionofHBsAgfrom

integratedHBVDNA.ThelevelsofHDVRNAweregenerallyhighwithlowerdegreeof

focaldifferences,andwithoutcorrelationtoSRNAlevels.WeproposethatpartialHDV

replicationandexpressionofdeltaantigensmayoccuralsoinhepatocytesthatarenot

co-infected by HBV or even express HBsAg from integrations, and that this might

contribute to the severe necroinflammation that is observed in many HDV infected

patients.

Thecompiledworkspresentedinthisthesisaddressimportantquestionsandproposes

novel mechanisms that hopefully will aid further research and contribute to the

understandingofinfectionscausedbyHBVandHDV.

54

7 ACKNOWLEDGEMENTS

Iwouldliketotakethisopportunitytothankeveryonewhohasbeenapartofmylifeand

madethisPhDpossible.

FirsttomysupervisorMagnusLindhforgivingmethiswonderfulopportunitytopursue

research,forallthevaluableguidanceandallthemidnightemailswithoutwhichthese

projectswouldhavebeenimpossible.Thankyoufortakingthetimetoteachandinspire

meinspiteofyourimpossiblybusyschedule!

Tomyco-supervisorHeléneNorder,thankyouforthesupport,wonderfulinsightsand

suggestionsonallmyprojects.YourpassionforresearchissomethingIadmire.Youare

awonderfulinspirationtoallresearchers.

Tomyco-supervisorSimonLarsson,fornotjusthelpingmewithmyprojectsbutalsofor

being therewhenever I needed help! Thank you for being awonderfulmentor and a

friend.

ToMariaAndersson,thankyouforintroducingmetowaysofVirologen,forteachingme

allthelabwork,foryourpatienceandhelpwithallmyprojects.Butmorethananything,

thankyouforbeingagoodfriend.

ToGustafRydell, forbeingagreatmentorand teachingme tobemeticulouswithmy

research.Thankyouforallthediscussions,foransweringmyquestionsnomatterhow

sillytheywereandforgreatcollaborationonallmyprojects.

ToEbbaSamuelsson,mypartnerincrimeatVirologenandtomyfellow“Indian”(well,

youaremoreIndianthanIcouldeverbe!)thankyouforeverything!Thememorieswe

sharedissomethingIwillcherishfortherestofmylife.Thankyouforallthechatsover

coffeeandforthe“Mondayicecream”tradition.These4yearsofmyPhDwouldnothave

beenpossibleifyouweren’tthereencouragingmeon.

ToGiorgiaOrtolani,itissaidthatgreatfriendsarehardtocomeby.Boy!AmIfortunate

tohavefoundyou!Thankyousomuchforbeingapartofmylife,forencouragingand

cheeringmeonalways!

55

ToSofiaBrunet,thankyouforbeingawonderfulfriend,forhearingoutallmyfrustrations

andnever-endingsupport.

ToRickardNordén,apersonwhoIhavegreatregardfor.Thankyouformakingtimeto

teachme,forallthegreatadvicesandforyourneverwaveringsmilesthatsays“you’re

gonnabefine!!!!”

ToJohanRinglander,thankyouforallthehelponmyprojectsandforlivelydiscussions.

Iwillcherishyourfriendshipforever!

ToMichaelKaan,thecoolestbossIhaveeverknown!Ithasbeenanabsolutepleasure

knowingyou.Theenthusiasmandvigoryouhaveforresearchisinfectious!Thankyou

fortheadvices,suggestionsandforallthefun!

ToThomasBergström,thankyouverymuchforallthesuggestionsandkindwords,for

encouragingbuddingresearcheslikemeandmotivatingustodobettereachday!

ToHaoWang,thepersonwhoIbuggedthemostthesefouryears!YoustartedyourPhD

onemonthbeforeIdid,whichmeansIwenttoyoutofindouteverythingonemonthin

advance!Forthoseofyouwhodon’tknow, that’sa lotttttttofquestionsheanswered!

ThankyouHao,forbeingsuchagreatfriendanddealingwithmycraziness!

Tomyco-authorsGunnerNorkrans,KristofferHellstrand,CatarinaSkoglundandallthe

staffattransplantationteam,althoughIhaven’tmetmostofyou,yourcontributionsto

thisprojectweretremendous!Thankyou!

My heartfelt thank you to Maria Johansson, Carolina Gustafsson, Anette Roth, Marie

Karlsson, Freddy Saguti, Johanna Said, Kristina Elfving, Edward Trybala, Theogene

Twagirumugabe,EricSeruyange,CharlsHannoun,PeterNorberg,NancyNenonen,Anna

Lundin, Charlotta Ericsson, Linn Persson, Kristina Nyström, Ka-Wei Tang, Hedvig

Engström,JacksonThomas,JoelGustafsson,SebastianMalmström,ShadiGeravandi,Priti

Gupta,GianlucaTripodi,YarongTian,MilaAdmekandsomanyothers!Thiswouldhave

beeninsurmountableifnotforthecontributionofeachoneofyou!Sayingthankyouonce

56

isnotenough.Youhaveallwelcomedmeintoyourlivesandmademefeelathomehere.

ForthatIwillbeeternallygrateful!

IwouldliketogivemyspecialthankstoeveryoneattheKvant,DetektionandHepatitlab

especially to Silviu Nitescu and Cvetla Spirovska taking the time to helpmewithmy

projectsandforMAKINGMESPEAKSWEDISH!TusenTackförallhjälpochuppmuntran!

TomydearestMaaruthyKumarYelleswarapuVenkataSathya(Yes,Ididwriteyourfull

name!),Ioweallmysuccesstoyoumybelovedfriend!Iwillforevercherishourgoodold

undergrad days! Phewwhat fun itwas! I don’t think I would have evermade it past

“ChemicalEngineering”ifnotforyou,I’dprobablystillbetrying.Thankyouforbeinga

partofmylifeandforeverythingelse!

ToKeerthana,Arjun,Sunith,NeethiandSiddharthyouareallmuchmorethanfriendsto

me,myfamilyfarawayfromhome.Forallthecrazydaysandwonderfulmemories,thank

youforbeingthereformethrougheverything!

Tomydearfamily,SushilaAmma,PrakashAppa,Gayu,sweetlittleYug,dearestAro,my

grandmotherSulo,mysecondparentsBalaAmmaandSureshAppaandtherestofmy

hugeeeeeefamilybackinIndia,thankyouisnotenoughtoexpressmygratitudetoyou.

Thestrengththatallofyougivemeisunmatchedandiswhatmadethispossible.

OneofmyfavoritequotesfromHarryPotterreads“Onewhowelovenevertrulyleaves

us”.IhavenowordstoexpresstheloveandadorationIhaveformydearestgrandparents

NanapaandChemmi.TheyarethetrueinspirationbehindeverythingIhaveachievedand

willachieveinthefuture.IfIgrowuptobehalfthepersonyouwere,Iwouldconsider

myselflucky!Thankyouforteachingmethevalueofeducation.

Lastbutnottheleast,mydearlovinghusbandAshwinwhohasbeenwaitingfouryears

to read this part! How can I ever begin to thank you for all that you have done?

Nevertheless,Iamgoingtotry.Thankyouforbeingwithmethroughallthegoodtimes

andthebad,foralltheencouragementandloveforgivingmeashouldertoleanon,for

sharingmyhappinessandsadnessalike!Thankyouforbeingyou.

57

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Hepatitis B Virus RNA in serum and liver tissue ......HBsAg levels in serum. The results also...

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