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HBV/HIV co-infections: molecular and biological characterization of occult HBV strains or strains resistant to antiviral treatment ANRS N° 12149. Selma de ANDRADE GOMES Lab. de Virologia Molecular IOC-FIOCRUZ. Alan KAY Centre de Recherche en Cancérologie de Lyon (INSERM U1052).
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HBV/HIV co-infections: molecular and biological characterization of occult HBV strains or strains resistant to antiviral treatment ANRS N°12149 Selma de ANDRADE GOMES Lab. de Virologia Molecular IOC-FIOCRUZ Alan KAY Centre de Recherche en Cancérologie de Lyon (INSERM U1052) ANRS/Programa Nacional DST/AIDS Evaluation Meeting, Rio de Janeiro, 4-5 April 2011
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, 447-454. • Chemin, I., Jeantet, D., Kay, A., Trepo, C., 2001. Role of silent hepatitis B virus in chronic hepatitis B surface antigen(-) liver disease. Antiviral Res 52, 117-123 Jeantet, D., Chemin, I., Mandrand, B., Zoulim, F., Trepo, C., Kay, A., 2002. Characterization of two hepatitis B virus populations isolated from a hepatitis B surface antigen-negative patient. Hepatology 35, 1215-1224 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., 1996. Detection of hepatitis B virus DNA in hepatitis B surface antigen-negative serum by polymerase chain reaction: evaluation of different primer pairs and conditions. Acta Virol 40, 133-138 Santos, E.A., Yoshida, C.F., Rolla, V.C., Mendes, J.M., Vieira, I.F., Arabe, J., Gomes, S.A., 2003. Frequent occult hepatitis B virus infection in patients infected with human immunodeficiency virus type 1. Eur J Clin Microbiol Infect Dis 22, 92-98 Santos, E.A., Sucupira, M.V., Arabe, J., Gomes, S.A., 2004. Hepatitis B virus variants in an HIV-HBV co-infected patient at different periods of antiretroviral treatment with and without lamivudine. BMC Infect Dis 4, 29 Selma contacted our laboratory, and I visited Selma’s lab in 2005 We decided to apply for an INSERM/FIOCRUZ exchange agreement
8%: High 2-7%:Intermediate < 2%: Low HBV infection • 350 million chronic HBV patients • > 10 times the number of HIV patients Brazil –Intermediateto high
A D A2 D C B D A A2 D F/H A D E D A F/H A D F A D A1 F2 D A1 D C B Geographic distribution of HBV genotypes Pujol and Devesa (2005) J Clin Gastroenterol. 39:611-8.
HBV DNA What is occult HBV infection? Acute infection: rapid loss (<6 months) of surface antigen (HBsAg) and HBV DNA Chronic infection: persistence (>6 months) of HBsAg, persistence of HBV DNA Occult 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
2005-07 Occult HBV infection in the literature over years More Than 240 Number of publications 140 120 100 80 60 40 20 0 1980-89 1990-99 2000-04 2008-10 Chemin & Trepo J Clin Virol 2005, Adapted Raimondo et al, J Hepatol 2008
Occult HBV N° (%) Study Country N° patients Methods “nested” PCR (serial evaluation) 51 (89%) Hofer, 1998 57 Switzerland 7 (20%) 35 Torres-Baranda, 2006 Mexico “nested” PCR 17 (20%) Filippini, 2006 Italy single step PCR 86 South Africa “nested” PCR Mphahlele, 2006 31 (22.%) 140 93 Netherlands Pogany, 2005 4 (4%) single step PCR Cobas Amplicor HBV Monitor (Roche) Neau, 2005 France 1 (0.6%) 160 single step PCR 101 Brazil 16 (16%) Santos, 2003 11 (37%) 30 “nested” PCR France Wagner, 2004 8 (5%) “nested” PCR 159 Goncales, 2003 Brazil Cobas Amplicor HBV Monitor (Roche) 85 0 Nunez, 2002 Spain 37 Piroth, 2000 13 (35%) single step PCR France Italy 42 (41%) “nested” PCR (liver) 101 Raffa, 2007 Occult HBV and HIV co-infection Raimondo et al, J Hepaol 2007, modified
Rolling circle amplification (RCA) A new tool for amplifying full-length HBV genomes by completion and ligation of HBV RC-DNA to cccDNA T4 DNA polymerase + T4 DNA ligase Hybridization minus-strand primers DR1 DR1 DR2 DR2 In Vitro Complementation and Ligation HBV ccc-DNA HBV RC-DNA Elongation minus-strand DNA Strand Displacement Hybridization plus-strand primers End product – High molecular weight double-stranded DNA d.
HBV genome (3.2 kb) HBV genome (3.2 kb) 3 kb 3 kb Illustration of RCA Copies / reaction M H2O 103 102 10 1 0 Agarose gel of RCA products Southern blot of gel – HBV has been succesfully amplified Unit-length HBV genomes can be excised from RCA products – can be dirctly cloned and/or sequenced Sensitivity can be increased by doing A genomic PCR on RCA products 10 kb
Applications 1) Reactivation of an occult HBV infection in a HIV+ patient • Patient HIV+, anti-HBc+, anti-HBs+ - profile resolved acute HBV infection? • Reactivated occult HBV infection after glucocorticoid treatment • Amplified, cloned and sequenced full-length HBV genomes after RCA • Genomes are of subgenotype A2 (European genotype A) • No drug resistance mutations despite poor compliance of patient • Genomes viable – replication competent, secrete viral particles • What 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
C- C+ P C- C+ P C- C+ P C- C+ P Acute Infection < 6 Mo Occult Infection > 12 Yrs Glucocorticoid treatment Reactivation 2 Mo Antiviral treatment Seroconversion to a-HBe 6 Mo 1 Mo HBV- HIV+ Wild-type Virus K122/D144 HIV+ Emergence of K122R. Low-level HBV replication HIV+ Stimulation of HBV replication. Emergence of D144E HIV+ K122R/D144E HIV+ What can the patient’s serum recognize? • Genetically engineered genomes so that they express viral particles of wild-type HBsAg sequence, with K122R, D144E or both • Transfected into cells, labeled with 35S-Met/Cys, immuno-precipitated secreted viral particles K122/D144 K122R/D144 K122/D144E K122R/D144E C- = Normal human serum C+ = Serum from a vaccinated person P = Patient’s serum HBsAg
Spe I 1762 1762 DR1 RCA Spe I 1901 DR2 Full-length HBV genome 1762 HBV RC-DNA Applications 2) Mutations associated with HBeAg-negativity in subgenotype F2 prevalent in Brazil • HBeAg-negative mutants – important global health problem • Stop codon position 28 of precore region - not possible for genotype A or F2 • Anna KRAMVIS – subgenotype A1 – mutations in core promoter/precore region 1762 1764 1809 1812 1814 1858 1862 1896 1901 A G GCAC ATG T G TGG ATG Precore HBcAg T A T T C T A Core promoter Genotype A Subgenotype F2
1762 1764 1809 1812 1814 1858 1862 1896 1901 #1 GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGGCATGGAC #2 GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGGCATGGAC #3 GGGKGAGGAGASTAGGTTAATGGTTTATGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGACATGGAC #4 GGGKGAGGAGASTAGGTTAATGGTTTATGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGACATGGAC F2 GGGGGAGGAGATTAGGTTAAAGGTCTTTGTATT//CCAGCACCATGCAA//CCCACTGTT//CTTTGGGGCATGGAC Precore HBcAg • Subtype F2 genomes do not show the mutations found with subtype A1 • Some isolates (#3 & 4) have A1762T but not G1764A • Isolates #3 & 4 have other mutations before and after 1762/1764 • Also have mutated codon 29 (GGC GAC, Glycine Tyrosine) • More work needed (sequence complete genomes, study HBeAg expression) Amplification and sequencing of genotype F genomes H20 RCA cut Spe I HBV full-legth genome RCA then genomic PCR using primers at Spe I site HBV full-legth genome
HIV 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 levels • Due to the shared modes of transmission of HBV and HIV, the prevalence of HBV infected patients is higher than in non-HIV infected individuals • Occult HBV infection = Detection of HBV DNA in absence of HBsAg • Possible 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%. HBV-HIV co-infection Jeantet et al., 2002
Demographical and serological data of HIV positive/ HBsAg negative patients HBsAg >10% (1998-2003), 2% 2009-2010
pre-S2 S EcoRV EcoRV Serological and molecular features of HBV isolates from cases of occult infection * LAM triple mutation HindIII HindIII EcoRV Transfection of eukariotic cells (CHO or HUH7) HindIII pcDNA3 S BIOELISA HBsAg Colour (Biokit)
Expression of HBsAg containing Y100C variant frequently detected in occult HBV infection
Regiã Conclusions/Perspectives Pol HIV OngoingstudiesatIPEC-Fiocruzshouldconfirmornottherecentdecreaseof HBV prevalence in the HIV infectedBrazilianpopulation, observedby us. Drugresistance: 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.
Conclusions/Perspectives Studies of occult HBV infection in the sera and PBMC of HIV co-infected patients will also be carried out in France Future studies are needed to monitor transmission of HBV isolates from cases of occult infections/drug resistance The 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 drugs The study of mutations affecting HBeAg expression in Brazilian-specific genotypes/subtypes will also be continued
Valorization of the project (1) Publications Mello, 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. 1194-1203, 2008 Araujo 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, 2011. (under revision) 2 other papers in preparation 1 Brazilian patent deposited 1 thesis in co-tutelle Instituo Oswaldo Cruz/Université Claude Bernard Lyon 1
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 2010 Participants: Selma GOMES, IOC-FIOCRUZ, Brazil: Introduction Anna KRAMVIS, Witwatersrand University, Johannesburg, South Africa: Phylogeny of Hepatitis B Virus Stephen Locarnini, Victoria Infectious Diseases Reference Laboratory, Melbourne, Australia: Drug resistance of Hepatitis B Virus Christoph COMBET, IBCP, Lyon, France: Hepatitis C virus genomic variability Alan Kay, INSERM U871, Lyon, France:Cell-culture models for HBV infectivityulture models for HBV infectivity Exchanges Rio de Janeiro Lyon Lyon Rio de Janeiro Caroline SOARES Natalia ARAUJO Nora MARTEL PhD student, ANRS doctoral bursary Joint thesis IOC/UCBL1
French team Researchers/clinicians Dr Isabelle Chemin Pr. Christian Trepo Dr. Laurent Cotte Dr. Sophie Allain (Limoges PHD student Nora Martel Research team/ Acknowledgment • Collaborators (cohort of patients) • IPEC/Fiocruz (Dr.BeatrizGrinsztejn, Dr.JuçaraArabe • Other Financial Support • Brazil: CNPq/ France/Brazil: INSERM/FIOCRUZ Brazilian team • Researcher • Dr. Natalia M. Araujo • PHD students • Kelly C. Pereira