AREVIR-Meeting, 23th April 2009 Stiftung Caesar, Bonn Occult hepatitis B virus infections Dieter Glebe Institute for Medical Virology, Justus-Liebig Universität Gießen German Consulting Laboratory for Hepatitis B
Entry of hepatitis virus, start of replication slow replication immune defence inapparent transient infection immunity
Entry of hepatitis virus, start of replication slow replication strong replication immune defence Delayed, vigorous immune response inapparent acute fulminant transient infection immunity recovery
Entry of hepatitis B virus, start of replication slow replication strong replication immune defence Delayed, vigorous immunotolerance, immune response inapparent, chronic inapparent incomplete response acute fulminant transient efficient inefficient infection HBsAg chronic immunity recovery carrier hepatitis cirrhosis carcinoma
Entry of hepatitis B virus slow replication strong replication immune defence Delayed, vigorous immunotolerance, immune response inapparent, chronic inapparent incomplete response acute fulminant transient efficient inefficient infection HBsAg chronic immunity recovery carrier hepatitis cirrhosis carcinoma
1000 fold excess HBsAg HBcAg HBV DNA HBsAg
Acute resolving hepatitis B disease Anti-HBc HBV DNA Anti-HBs PCR HBsAg Recovery months
Definition of occult HBV infection (OBI): HBsAg non-reactive, but infected • Definition (true OBI): – Presence of HBV DNA in the liver, – With detectable or undetectable HBV DNA in serum – HBsAg tested negative by current assays – HBV DNA in serum very low (<200 IU/ml) • False OBI – Infection by HBV variants with SHBs mutations (escape mutants), not detectable by HBsAg tests Taormina expert meeting on occult hepatitis B virus infections. J.Hepatol 49 (2008) 652-657
Acute resolving hepatitis B disease Anti-HBc HBV DNA Anti-HBs PCR HBsAg Low level persistance Recovery months
Entry of hepatitis B virus, start of replication weak replication strong replication immune defence Delayed, vigorous immune response inapparent acute fulminant infection immunity recovery Continuous Occult intrahepatic infection selection of HBV mutants Infectious blood and organ donations
HBV transmissions by HBsAg negative, anti-HBc positive blood donors with occult HBV infection Summary of d ata from Germany and Denmark 2004 - 2005 • 5 donors – HBV DNA <10 – 240 ge/mL, no anti-HBs – In 4 donors several mutations of the HBsAg loop – 1 donor had wildtype HBsAg • 55 recipients, 68% anti-HBc pos. – 10 possible transmissions, asymptomatic – 22 probable transmissions, asymptomatic – 3 fatal hepatitis B cases • 2 with proven transmission • cofactors: sepsis, immunosuppression – no normal acute hepatitis B after transfusion W. Gerlich et al. J Med Virol 2007; 79:S32-S36
Definition of occult HBV infection (OBI): HBsAg non-reactive, but infected • Definition (true OBI): – Presence of HBV DNA in the liver, – With detectable or undetectable HBV DNA in serum – HBsAg tested negative by current assays – HBV DNA in serum very low (<200 IU/ml) • False OBI – Infection by HBV variants with SHBs mutations (escape mutants), not detectable by HBsAg tests Taormina expert meeting on occult hepatitis B virus infections. J.Hepatol 49 (2008) 652-657
Transient occult HBV infection with unusual high HBV DNA levels • Blood donor positive in Ultrio NAT – HBV DNA 90 000 ge/ml (15,500 IU/ml) – no anti-HBs, no anti-HBc – Normal ALT • 32 days later – HBV DNA + – Anti-HBc and anti-HBs positive – Elevated ALT • 57 days later – HBV DNA negative, – Anti-HBc and anti-HBs positive – ALT normal Bremer et al. Transfusion, in press
Transient occult HBV infection with unusual high HBV DNA levels • HBV DNA 90 000 ge/ml, but no HBsAg detectable – Escape mutations in HBsAg? – Altered epitopes for HBsAg ELISA? � Cloning and sequencing of the whole genome • No mutations in the HBsAg loop • No „diagnostic escape“, � true OBI • but: • Frameshift in the preS domain, � Possibly low secretion of subviral particles Bremer et al. Transfusion, in press
Transient occult HBV infection with unusual high HBV DNA levels • HBV DNA 90 000 ge/ml, but no HBsAg detectable – HBsAg and virions covered with anti-HBs? – Immune complexes? � Precipitation with Polyethyleneglycol (PEG) + addition of anti-HBs ? Bremer et al. Transfusion, in press
Transient occult HBV infection with unusual high HBV DNA levels • HBV DNA 90 000 ge/ml, but no HBsAg detectable – HBsAg and virions covered with anti-HBs? – Immune complexes? � Precipitation with Polyethyleneglycol (PEG) � Immune-precipitation with anti-human antibodies + addition of anti-HBs � No immune complexes present Bremer et al. Transfusion, in press
Transient occult HBV infection with unusual high HBV DNA levels • HBV DNA 90 000 ge/ml, but no HBsAg detectable – Altered topology of HBsAg on virions? – Virions without HBsAg? � Immune-precipitation with anti-preS1 and anti-SHBs antibodies � correct topology of LHBs and SHBs Bremer et al. Transfusion, in press
Transient occult HBV infection with unusual high HBV DNA levels CsCl gradient
Transient occult HBV infection with unusual high HBV DNA levels CsCl gradient
Transient occult HBV infection with unusual high HBV DNA levels CsCl gradient
Transient occult HBV infection with unusual high HBV DNA levels Unusual viral density profile � Association with plasma proteins possible CsCl gradient
Definition of occult HBV infection (OBI): HBsAg non-reactive, but infected • Definition (true OBI): – Presence of HBV DNA in the liver, – With detectable or undetectable HBV DNA in serum – HBsAg tested negative by current assays – HBV DNA in serum very low (<200 IU/ml) – Could be up to 15,500 IU/ml ! False OBI – Infection by HBV variants with SHBs mutations (escape mutants), not detectable by HBsAg tests Taormina expert meeting on occult hepatitis B virus infections. J.Hepatol 49 (2008) 652-657
Persistance of hepatitis B virus strong replication weak replication immune defence Delayed, vigorous immune response inapparent acute fulminant infection immunity recovery Immunosuppression Occult intrahepatic infection Reactivation
Persistance of hepatitis B virus strong replication weak replication immune defence Delayed, vigorous immune response inapparent acute fulminant infection immunity recovery Immunosuppression Occult intrahepatic infection Reactivation Continuous Growth of escape- selection of variants HBV mutants
HBV Reactivation • Induced by immunosuppression • Frequent in inactive HBsAg carriers • Possible in anti-HBc and/or anti-HBs positive persons – Rare in kidney transplantation – Frequent in bone marrow/stem cell transplantation and lymphoma therapy • Asymptomatic replication to high viremia – under immunosuppression • Hepatitis starts with immune reconstitution – May be fatal, but can be prevented • Pretesting for anti-HBc/s necessary • Monitoring with sensitive HBV DNA assays • Early or pre-emptive therapy necessary
Acknowledgements Institut for Medical Virology, Centro de Transfusión de Giessen, Germany la Comunidad de Madrid, Spain Corinna M. Bremer Pilar Torres Mona Saniewski Ulrike C. Wend Wolfram H. Gerlich Chiron, Novartis Vaccines Dieter Glebe and Diagnostics, Suresnes, France supported by DFG Nico Lelie Collaborative Research Center SFB 535
Transmission of HBV by anti-HBc only blood : donor related look back • Genotype A2 adw2, dynamic quasispecies – Many mutations in the RT-domain: – Conserved: V112I, N118H, R120S, L140I, Y151F – In donation 2: rtA181T � sW172stop – Occurs during during lamivudine, adefovir, telbivudine and clevudine therapy. – sW172stop mutation confers defect in HBsAg secretion and dominant negative effect on virion secretion (Warner et al., Hepatology 2008). – reduction of viral breakthrough during anti-viral therapy. � Genotypic profiling of HBV during reactivation A. Lattermann, Lahn-Dill-Klinik Wetzlar, S. Wienzek, et al., Institute of Clinical Immunology and Transfusion Medicine, Giessen, Germany
Model of the HBsAg loop : escape (?) mutations in an anti-HBs Model of the HBsAg loop IV negative blood donor IV F H Q V I
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