Escape mutations in occult and reactivated hepatitis B virus - PowerPoint PPT Presentation

AREVIR-GenaFor Meeting 10 April 2008 Stiftung caesar, Bonn Escape mutations in occult and reactivated hepatitis B virus infection Wolfram H. Gerlich Institute for Medical Virology German Consulting Laboratory for Hepatitis B

Definition of occult HBV infection: serum HBsAg non-reactive in spite of proven (or suspected) HBV infection often, but not always positive for HBV DNA gold standard: liver tissue

Entry of hepatitis B virus, start of replication slow replication Rapid immune response HBs antigenaemia: inapparent too low or transient infection duration too short immunity

Detection of transient occult HBV infection by single sample NAT • Blood donor positive in Ultrio NAT – 90 000 ge/mL HBV DNA – HBsAg negative: S/CO 0.6 in Prism – All HBV antibodies negative – ALT normal • 32 days later – ALT elevated – Anti-HBc and anti-HBs positive – HBV DNA positive, HBsAg negative • 57 days later – ALT normal, HBV DNA negative, anti-HBc/s positive Communicated by P. Torres, Centro de Transfusion, Madrid and P.N. Lelie, Chiron

HBV from transient occult infection • 90 000 ge/mL viral load, but HBsAg undetectable – On average: HBsAg detectable if >2000 ge/mL • Virus without HBsAg? No – Normal density before and after detergent treatment – Immune precipitated by anti-preS1, but not by anti-HBc • Mutated HBsAg? No – No mutations in the HBsAg loop • Low excess of subviral HBsAg particles ? Possibly – Frameshift in the S ORF in one of 3 clones • Rapid immune response ? Probably – Precore stop mutant: HBeAg negative – Quasispecies – Mutations in preS, in S/rt and in X – e. g. rt119, 149, 153, 213, 237, 257, 267

Entry of hepatitis virus, start of replication slow replication strong replication Delayed immune immune defence response HBsAg positive inapparent acute hepatitis transient infection immunity recovery

Acute resolving hepatitis B disease Anti-HBc HBV DNA Anti-HBs PCR HBsAg Recovery Occult months

Transmission of HBV by blood from donors with completely occult early phase infection Three independent cases from Germany • Multiple donors recognised by seroconversion to HBsAg positive: look back • Previous donations had been missed by minipool testing for HBV DNA • Retesting of stored single samples with realtime PCR, detection limit < 12.5 IU/mL negative • Recipients of red cell concentrates: infected, HBsAg +, HBV DNA +, anti-HBc -, same sequence, wildtype virus

Acute resolving hepatitis B disease Anti-HBc HBV DNA Anti-HBs PCR HBsAg Occult ? Recovery months

Acute resolving hepatitis B disease Anti-HBc HBV DNA Anti-HBs PCR HBsAg Occult, low level persistence Recovery months

Types of occult HBV infection: HBsAg non-reactive, but infected • Transient inapparent infection – Seemingly occult during normal acute infection • Early window period • Late acute window period • Low level persistence in occult carriers after – Acute „resolving“ hepatitis B – Inapparent infection

Course of hepatitis B virus infection weak replication strong replication immune defence Delayed, vigorous immune response inapparent acute fulminant infection immunity recovery Occult intrahepatic persistence Infectious blood and liver donations

Acute resolving hepatitis B disease Anti-HBc HBV DNA Anti-HBs PCR Escape mutants HBsAg Occult, low level persistance months

HBV escape mutants • Anti-HBs blocks int er cellular spread of HBV – can not stop int ra cellular pre-existing virus • HBV replication is inaccurate • Mutated HBsAg escapes recognition • Mutated HBV can spread within liver

Model of the HBsAg loop (aa 98 – 161): typical escape mutation after vaccination of HBV infected newborns a -determinant G145R mutation Carman et al., Lancet 1992;336:325

HBV transmissions by occult infected blood donors Summary of d ata from Germany and Denmark 2004 - 2005 • 5 donors – HBV DNA 9 – 240 ge/mL, no anti-HBs – In four donors several mutations of the HBs loop – Only one donor had wildtype HBsAg • 55 recipients, 68% anti-HBc pos. – 10 possible transmissions, asymptomatic – 22 probable transmissions, asymptomatic – 3 fatal hepatitis B cases (donors I – III) • cofactors: immunosuppression, sepsis – no normal acute hepatitis B after transfusion W. Gerlich et al. J Med Virol 2007; 79:S32-S36

Mutations in occult infected blood donor Fatal transmission I, 240 ge/mL, 14 clones sequenced a-determinant C121-C148 L L E G145K T L

Mutations in anti-HBc+/s- infectious blood donor Fatal transmission II, ca. 50 ge/mL a-determinant C121-C148 HBsAg subtype change I N F

No mutations in anti-HBc+/s- infectious blood donor Fatal transmission III, ca. 10 ge/mL a-determinant C121-C148

Occult infected blood donor IV • Ca 2000 IU/mL (10 000 ge/mL) HBV DNA – HBsAg in Abbott‘s Prism negative, <0.1 IU/ mL – Anti-HBc weakly positive, S/CO 0.4 – IgM anti-HBc negative • Anti-HBs negative, but • HBsAg loop mutated, quasispecies – Direct sequencing and 10 cloned sequences: – 5 of 6 mutations not detected by direct sequencing Communicated by M. Schmidt Red Cross BDS, Frankfurt

Model of the HBsAg loop IV Mutations in anti-HBc+/s-, HBV DNA pos. blood donor IV a-determinant C121-C148 2000 IU/mL 10 clones sequenced A I S I heterogeneous H N

Anti-HBc only : donor related look back • Anti-HBc screening in Germany since 2006 • One long-time blood donor has anti-HBc, no anti-HBs • HBV DNA in 5 of 5 consecutive donations pos.: 9 to 46 ge/mL • 5/13 red cell recipients anti-HBc positive • 11/11 recipients of fresh frozen plasma anti-HBc positive – probably all plasma donations infectious, – but no hepatitis • numerous mutations in the HBsAg loop • dynamic quasispecies A. Lattermann, Lahn-Dill-Klinik Wetzlar, S. Wienzek, et al., Institute of Clinical Immunology and Transfusion Medicine, Giessen, Germany

Mutations in anti-HBs negative, infectious blood donor V Model of the HBsAg loop IV 1st sample, 23 ge/mL, 10 clones sequenced F K N S Q H V homogeneous I heterogeneous

Mutations in anti-HBs negative, infectious blood donor V Model of the HBsAg loop IV 2nd sample, 9 weeks later, 9 ge/mL, 7 clones F K N S Q H V I transient S

Mutations in anti-HBs negative, infectious blood donor V Model of the HBsAg loop IV 3rd sample, 29 weeks later, 34 ge/mL, 12 clones F K N S R Q H I

Mutations in anti-HBs negative, infectious blood donor V Model of the HBsAg loop IV 4th sample, 38 weeks later, 46 ge/mL, 10 clones F K N S Q H P G V I R,stop

Pseudo-occult HBV infection in blood donor VI • Negative in Ortho‘s HBsAg screening assay • Strongly positive in Abbott‘s HBsAg assay • 24 IU/L anti-HBs and anti-HBc-IgG • 200 genomes /mL HBV DNA • Direct sequencing: escape mutations – 1st sample: L109V; M133T; D144G • Look back – 3 recipients not infected, 1 anti-HBc/s positive Communicated by Drs. Ullum and Dickmeiss, Copenhagen, DK

HBV escape mutations in blood donor VI • Negative in Ortho‘s HBsAg screening assay • Strongly positive in Abbott‘s HBsAg assay • 24 IU/L anti-HBs and anti-HBc-IgG • 200 genomes /mL HBV DNA • Direct sequencing: escape mutations – 1st : L109V…………………………………………………M133T D144G – 2nd : L109V P120L C121Y P127T G130D M133T • Look back – 3 recipients not infected, 1 anti-HBc/s positive Communicated by Drs. Ullum and Dickmeiss, Copenhagen, DK

Escape mutations in HBsAg and anti-HBs pos. blood donor VI Model of the HBsAg loop IV 2nd sample, 240 ge/mL, 19 clones sequenced T D stop A P127T G R C122Y P120L V, Q T heterogeneous Insertion T

Sequence of HBs loop Donor VI, Aa-sequence of S-loop immuno- selection wt M133T 19 mutant clones 13 different sequences

immuno- carrier, tolerant HBV Sequence of HBs loop Aa-sequence of S-loop

Elimination of the start codon (M)for the small HBs protein Donor VI, immune selection 9x M1R 3x M1L 1x M1G 1x M1K 3x M wt

preS1 preS2 SHBs Attachment Envelopment TM 1 Internal loop TM 2 HBsAg loop Variable topology a- determinant 300 200 400 0 100 Donor V 1 300 200 400 0 100 2 300 200 400 3 0 100 300 200 400 0 100 4 300 200 400 0 100 5 300 200 0 400 100 Donor I 200 300 400 0 100 Donor IV 300 200 400 0 100 Donor VI 200 300 400 0 100 reactivated homogeneous heterogeneous mutation 300

Variability of S ORF and major neutralising epitopes in 4 blood donors with (pseudo-) occult HBV infection Variation % a Type of HBV infection Ge/mL Ne/Nt a S ORF HBs loop b preS1 HBs/eAg+, control 10 9 25/12400 0.2% 0.2% 0.3% Anti-HBc+/s- 2000 63/4000 1,6% 4.4% 0.6% Anti-HBc+/s- 240 287/7600 3.6% 8.7% 2.0% Anti-HBc+/s- 1 23 164/4000 4.1% 10.3% 1.0% Donor C, 2 9 94/2800 3.4% 12.9% 2.2% Donor C, 3 29 168/4000 3.5% 10.5% 1.9% Donor C, 4 38 176/4000 4.4% 14.4% 2.9% HBsAg- c /+ Anti-HBc+/s+ 200 270/14400 3.8% 5.9% 2.5% a) number of exchanged aa/number of sequenced aa positions in 7 to 36 clones from amplified S ORF b)Aminoacid 99-170 of SHBs c) negative in screening test