Presentation on theme: "HBV/HIV co-infections: molecular and biological characterization of occult HBV strains or strains resistant to antiviral treatment ANRS N°12149 Selma de."— Presentation transcript:
1 HBV/HIV co-infections: molecular and biological characterization of occult HBV strains or strains resistant to antiviral treatmentANRS N°12149Selma de ANDRADE GOMESLab. de Virologia MolecularIOC-FIOCRUZAlan KAYCentre de Recherche en Cancérologie de Lyon (INSERM U1052)ANRS/Programa Nacional DST/AIDS Evaluation Meeting, Rio de Janeiro, 4-5 April 2011
2 History of the collaboration In the early 2000s, our laboratory in Lyon was actively working on the problem of occult Hepatitis B Virus (HBV) infections:Chemin, I., Zoulim, F., Merle, P., Arkhis, A., Chevallier, M., Kay, A., Cova, L., Chevallier, P., Mandrand, B., Trepo, C.,2001. High incidence of hepatitis B infections among chronic hepatitis cases of unknown aetiology. J Hepatol 34,Chemin, I., Jeantet, D., Kay, A., Trepo, C., Role of silent hepatitis B virus in chronic hepatitis B surface antigen(-)liver disease. Antiviral Res 52, Jeantet, D., Chemin, I., Mandrand, B., Zoulim, F., Trepo, C., Kay, A., Characterization of two hepatitis B viruspopulations isolated from a hepatitis B surface antigen-negative patient. Hepatology 35,In Rio de Janeiro at the same time, Selma GOMES was also working on occult HBV infections, and more specifically in HIV co-infected patients: Gomes, S.A., Yoshida, C.F., Niel, C., Detection of hepatitis B virus DNA in hepatitis B surface antigen-negativeserum by polymerase chain reaction: evaluation of different primer pairs and conditions. Acta Virol 40, Santos, E.A., Yoshida, C.F., Rolla, V.C., Mendes, J.M., Vieira, I.F., Arabe, J., Gomes, S.A., Frequent occulthepatitis B virus infection in patients infected with human immunodeficiency virus type 1. Eur J Clin MicrobiolInfect Dis 22, 92-98 Santos, E.A., Sucupira, M.V., Arabe, J., Gomes, S.A., Hepatitis B virus variants in an HIV-HBV co-infectedpatient at different periods of antiretroviral treatment with and without lamivudine. BMC Infect Dis 4, 29Selma contacted our laboratory, and I visited Selma’s lab in 2005We decided to apply for an INSERM/FIOCRUZ exchange agreement
3 HBV infection 350 million chronic HBV patients > 10 times the number of HIV patients 8%: High2-7%: IntermediateBrazil – Intermediate to high< 2%: Low
4 Geographic distribution of HBV genotypes Pujol and Devesa (2005) J Clin Gastroenterol. 39:611-8.
5 What is occult HBV infection? HBV DNAAcute infection: rapid loss (<6 months) of surface antigen (HBsAg) and HBV DNAChronic infection: persistence (>6 months) of HBsAg, persistence of HBV DNAOccult HBV infection: detectable HBV DNA, HBsAg undetectable DNA levels usually very low (<1000 copies/ml) the virus can be transmitted retains the oncogenic potential of chronic HBV infections
6 Occult HBV infection in the literature over yearsMoreThan 240Number of publications14012010080604020Chemin & Trepo J Clin Virol 2005, AdaptedRaimondo et al, J Hepatol 2008
8 In Vitro Complementation and Ligation Rolling circle amplification (RCA)A new tool for amplifying full-length HBV genomes by completion and ligation of HBV RC-DNA to cccDNAT4 DNA polymerase+T4 DNA ligaseHybridizationminus-strand primersDR1DR1DR2DR2HBV RC-DNAIn Vitro Complementation and LigationHBV ccc-DNAElongationminus-strand DNAStrandDisplacementHybridizationplus-strand primersEnd product –High molecular weightdouble-stranded DNAd.
9 Illustration of RCA Agarose gel of RCA products M H2OCopies / reactionAgarose gel of RCA productsSouthern blot of gel – HBV has beensuccesfully amplifiedUnit-length HBV genomes can beexcised from RCA products – canbe dirctly cloned and/or sequencedSensitivity can be increased by doingA genomic PCR on RCA products10 kb3 kbHBV genome (3.2 kb)3 kbHBV genome (3.2 kb)
10 1) Reactivation of an occult HBV infection in a HIV+ patient Applications1) Reactivation of an occult HBV infection in a HIV+ patientPatient HIV+, anti-HBc+, anti-HBs+ - profile resolved acute HBV infection?Reactivated occult HBV infection after glucocorticoid treatmentAmplified, cloned and sequenced full-length HBV genomes after RCAGenomes are of subgenotype A2 (European genotype A)No drug resistance mutations despite poor compliance of patientGenomes viable – replication competent, secrete viral particlesWhat causes immune-escape from anti-HBs?● all genomes contain 2 significant mutations in the S gene● K122R – very rare in genotype A● D144E – potential immune-escape mutant
11 Seroconversion to a-HBe What can the patient’s serum recognize?Genetically engineered genomes so that they express viral particles ofwild-type HBsAg sequence, with K122R, D144E or bothTransfected into cells, labeled with 35S-Met/Cys, immuno-precipitatedsecreted viral particlesK122/D144K122R/D144K122/D144EK122R/D144EC- C+ PC- C+ PC- C+ PC- C+ PC- = Normal human serumC+ = Serum from a vaccinated personP = Patient’s serumHBsAgAcuteInfection< 6 MoEmergence ofK122R.Low-levelHBV replicationHIV+Wild-typeVirusK122/D144Occult> 12 YrsGlucocorticoidtreatment6 Mo MoStimulation ofHBV replication.D144EReactivation2 MoK122R/D144EAntiviral treatmentSeroconversion to a-HBeHBV-
12 2) Mutations associated with HBeAg-negativity in subgenotype F2 Applications2) Mutations associated with HBeAg-negativity in subgenotype F2prevalent in BrazilHBeAg-negative mutants – important global health problemStop codon position 28 of precore region - not possible for genotype A or F2Anna KRAMVIS – subgenotype A1 – mutations in core promoter/precore region176217641809181218141858186218961901AGGCACATGTGTGGATGPrecoreHBcAgTATTCTACore promoterGenotype ASubgenotype F2HBV RC-DNADR1DR219011762Spe IRCASpe I1762Full-length HBV genome
13 Amplification and sequencing of genotype F genomes H20RCA cut Spe IHBV full-legth genomeRCA then genomicPCR using primersat Spe I siteHBV full-legth genome#1 GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGGCATGGAC#2 GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGGCATGGAC#3 GGGKGAGGAGASTAGGTTAATGGTTTATGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGACATGGAC#4 GGGKGAGGAGASTAGGTTAATGGTTTATGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGACATGGACF2 GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGGCATGGAC176217641809181218141858186218961901PrecoreHBcAgSubtype F2 genomes do not show the mutations found with subtype A1Some isolates (#3 & 4) have A1762T but not G1764AIsolates #3 & 4 have other mutations before and after 1762/1764Also have mutated codon 29 (GGC GAC, Glycine Tyrosine)More work needed (sequence complete genomes, study HBeAg expression)
14 HBV-HIV co-infectionHIV interferes with the natural history of HBV infection by enhancing HBV replication, leading to more severe liver disease, decreasing hepatitis B ‘e’ antigen seroconversion and increasing HBV DNA levelsDue to the shared modes of transmission of HBV and HIV, the prevalence of HBV infected patients is higher than in non-HIV infected individualsOccult HBV infection = Detection of HBV DNA in absence of HBsAgPossible explanations: Low rate of HBV replication, mutations that inhibit HBsAg expression or change HBsAg antigenicity, thus preventing detection by commercial assays.Among HIV-infected cohorts, occult HBV infection prevalence is widely divergent, ranging from 0% to 89%.Nós nos interessamos na co-infecção HBV HIV pois..slideJeantet et al., 2002
15 Demographical and serological data of HIV positive/ HBsAg negative patients characteristicsYearsn = 91n = 33Year2006n=432009n=144SexMaleFemale80 (88%)11(12%)21 (64%)12(36%)35 (81%)8 (19%)59 (41%)85 (59%)Age (years)< 3030-3940-49>492 (3%)21 (29.5%)32 (45%)16 (22.5%)3 (10%)16 (51.5%)8 (25.5%)4 (13%)0 (0%)7 (16.5%)19 (45.5%)16 (38%)24 (19%)42 (33.3%)40 (31.7%)20 (16%)Antiretroviral treatmentYesNo26 (31%)58(69%)26 (78%)7 (22%)39 (90%)4 (10%)105 (82%)23 (18%)Lamivudine33 (36%)58 (64%)23 (70%)10(30%)37 (95%)2 (5%)103 (80%)25 (20%)TDF10 (23%040 (30%)HBV markersAnti-HBc total87 (97%)26 (81%)39 (91%)23 (16%)Anti-HBc only34/89 (38%)8/30 (26%)11/42 (26%)4/144 (3%)Anti-HBs/Anti-HBc53 (59.5)18 (60%)28 (66.5%)19 (13.5%)Anti-HBs only2/89 (2%)1/32 (3%)3/42 (7%)37/144 (28%)No HBV serological marker3/32 (3%)84/144 (58%)HBV DNA Pre S or S region05 (5.5%)06 (18%)06 (14%)05 (3.3%)Estudamos coortes hbv/hiv ao longo do tempo (slide)HBsAg >10% ( ), 2%
16 Serological and molecular features of HBV isolates from cases of occult infection PatientSexYearAntiretroviral treatmentLAM/TVFAnti-HBs/Anti-HBcHBV loads(copies/mL)GenotypesSSTOP CODONSOther mutationsGM1998YN/NPos/Pos104A/A1S36-P36F2006Y/N106S216Y100CP1999NS187P51Neg/PosNoY100C,*E164D219672009NDQ101H F134LH1997P65A/A223818Neg/Neg2003102DT118V, A128VES136Y* LAM triple mutationHindIIIIsolados de HBV caracterizados de casos de infecção oculta: stop codon outras, algumas expressasEcoRVHindIIIEcoRVTransfection of eukariotic cells (CHO or HUH7)pre-S2SHindIIIEcoRVpcDNA3SBIOELISAHBsAg Colour (Biokit)
17 Expression of HBsAg containing Y100C variant frequently detected in occult HBV infection
18 Conclusions/Perspectives Ongoing studies at IPEC-Fiocruz should confirm or not the recent decrease of HBV prevalence in the HIV infected Brazilian population, observed by us.Drug resistance: In agreement with dr. Couto-Fernandez (member of National Network for HIV Genotyping Lab. AIDS, IOC) we had designed a doctoral thesis to study the lamivudine and tenofovir resistance mutations of both HIV and HBV (populations and subpopulations by pyrosequencing) in cases of co-infectin. Unfortunately, the collaboration did not happen. We are inviting co-infected patients from IPEC to initiate this study.RegiãPol HIV
19 Conclusions/Perspectives Studies of occult HBV infection in the sera and PBMC of HIV co-infected patients will also be carried out in FranceFuture studies are needed to monitor transmission of HBV isolates from cases of occult infections/drug resistanceThe most appropriate therapy for a particular genotype or a mutation type may be identified by in vitro phenotyping Test: Transfection of complete genomes into human cells in the presence of drugsThe study of mutations affecting HBeAg expression in Brazilian-specific genotypes/subtypes will also be continued
20 Valorization of the project (1) PublicationsMello, Francisco C. A. ; Martel, Nora ; Gomes, Selma A. ; Araujo, Natalia M. .Expression of Hepatitis B Virus Surface Antigen Containing Y100C Variant Frequently Detected in Occult HBV Infection. Hepatitis Research and Treatment, v. 2011, p. 1-4,Araujo, N. M. ; Branco-Vieira M ; Silva ACM ; Pilotto JH ; Grinsztejn B ; Trepo C; Gomes, SA . Occult hepatitis B virus infection in HIV-infected patients:Evaluation of biochemical, virological and molecular parameters.. Hepatology Research, v. 38, p , 2008Araujo NM, Waizbort R, Kay A. Hepatitis B Virus infection from an evolutionary point of view: how viral, host, and environmental factors shape genotypes and subgenotypes. Infectious, Genetics and Evolution, (under revision)2 other papers in preparation1 Brazilian patent deposited1 thesis in co-tutelle Instituo Oswaldo Cruz/Université Claude Bernard Lyon 1
21 PhD student, ANRS doctoral bursary Valorization of the project (2)A mini-symposium organized by Selma GOMES:“International Conferences and Debates on Relevant Aspects of Viral Hepatitis”IOC-FIOCRUZ, Rio de Janeiro, Brazil, February 26ty 2010Participants:Selma GOMES, IOC-FIOCRUZ, Brazil: IntroductionAnna KRAMVIS, Witwatersrand University, Johannesburg, South Africa:Phylogeny of Hepatitis B VirusStephen Locarnini, Victoria Infectious Diseases Reference Laboratory,Melbourne, Australia: Drug resistance of Hepatitis B VirusChristoph COMBET, IBCP, Lyon, France: Hepatitis C virus genomic variabilityAlan Kay, INSERM U871, Lyon, France:Cell-culture models for HBV infectivityulture models for HBV infectivityExchangesRio de Janeiro LyonLyon Rio de JaneiroNataliaARAUJOCarolineSOARESNora MARTELPhD student, ANRS doctoral bursaryJoint thesis IOC/UCBL1
22 Research team/ Acknowledgment Brazilian teamResearcherDr. Natalia M. AraujoPHD studentsKelly C. PereiraFrench teamResearchers/cliniciansDr Isabelle CheminPr. Christian TrepoDr. Laurent CotteDr. Sophie Allain (LimogesPHD studentNora MartelCollaborators (cohort of patients)IPEC/Fiocruz (Dr.Beatriz Grinsztejn, Dr.Juçara ArabeOther Financial SupportBrazil: CNPq/ France/Brazil: INSERM/FIOCRUZ
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