HBV Genotype Panel AREVIR-GenaFor-Meeting Bonn, 23. 24. April 2009 - - PowerPoint PPT Presentation

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HBV Genotype Panel

Michael Chudy Section of Molecular Virolgy Division of Virology E-Mail: chumi@pei.de

AREVIR-GenaFor-Meeting Bonn, 23. – 24. April 2009

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Background

During the ‘WHO Consultation on Global Measurement Standards and their use in the in vitro Biological Diagnostic Field’ in June 2004 concern was raised that HBsAg and HBV NAT test kits might be less sensitive for some HBV genotypes other than A2, which is represented by the in the WHO reference preparation (2nd International Standard) During the ECBS meeting in October 2005 the PEI proposed a project to establish WHO International Reference Panels representing different HBV genotypes/HBsAg subtypes This project was assigned as high priority by ECBS

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HBV genotype reference panels

HBV genotype panel (NAT tests) HBV genotype panel (HBsAg tests)

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Genotype Subgenotype HBsAg subtype Frequency Main Geographical Distribution A A1 adw2, ayw1 high Africa A2 adw2 high Europe, North America, Australia A3 Cameroon, Democratic Republic of Congo, Gabon B B1 adw2 high Far East (Japan, China, Taiwan) B2 adw2/adw3, high/low Far East (China, Japan, Viet Nam/Thailand) B3 adw2 high Far East (Indonesia, Sumatra, Sulawesi) B4 ayw1 high Viet Nam C C1 adr/ayr/adw2 high/high/low Far East (Japan, China) C2 adr/ayr; ad high/high Thailand, China/Viet Nam C3 adrq-/adw2 high/low Pacific (New Zealand to Polynesia), Micronesia/Far East C4 ayw3 low Northeast Australia D D1 ayw2 high Mediterranean, Middle East, India D2 ayw3/ayw4 high/low worldwide/USA D3 ayw2/ayw3 high/high South Africa, Alaska/Europe, Costa Rica D4 ayw2, high Oceania, Somalia, not identified adw3 low Eastern Europe Spain E

• ayw4

high Africa F F1 adw4q-/ayw4 high/low Cental America, Argentina, Spain, Alaska/Nicaragua F2/F3/F4… adw4q-/ayw4 high/low South America, Polynesia, France/Venezuela G

• adw2

low USA, Mexico, Europe H

• adw4

low Central America (Nicaragua, Mexico), California Recombinant Strains A/D adw2 India C/D ayw2 Tibet C/? adw2 Viet Nam

HBV - genotypes/subtypes, frequency and geographical distribution

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HBV genotype reference panels

Efforts to collect plasma units worldwide

• HBV DNA/HBsAg high titre plasma samples with sufficient volume
• NAT testing, HBsAg-testing, and HBV genotyping of about 215

potential candidate members

HBV genotypes A – H

• One genotype H sample received recently
• This sample could not be considered for the NAT panel

HBV genotype panel (NAT tests): 15 panel members HBV genotype panel (HBsAg tests): 15 panel members 12 samples are member in both panels Project in close cooperation with Prof Gerlich (Institute of Virology, University Giessen)

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HBV genotype panel (NAT tests)

Final characterization of 15 panel members

• Sequencing of entire S ORF
• Genosubtype, HBsAg subtype, escape mutations

Characterization of negative plasma Dilution Filling and lyophilisation Collaborative study Report to ECBS in 2009

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HBV Genotype Panel (NAT Tests) Characterization of the panel members

Sample No Origin HBsAg Subtype HBV Genotype HBV Genotype HBV DNA HBsAg anti-HBc HBeAg anti-HBe HIV1/HCV RNA

INNO-LiPA Sequencing (IU/mL) ARCHITECT* ARCHITECT Elecsys ARCHITECT Procleix

1 South Africa adw2 A A1 6,08E+08 131,9 pos pos neg neg 2 Brazil adw2 A A1 6,53E+08 94,0 pos pos neg neg 3 Germany adw2 A A2 6,87E+08 74,3 pos pos neg neg 4 Japan adw2 B B1 1,48E+08 51,4 pos pos neg neg 5 Japan adw2 B B2 2,84E+08 95,3 pos pos neg neg 6 Viet Nam ayw1 B B4 6,29E+06 4,6 pos pos neg neg 7 Japan adr C C2_Ce 3,99E+08 70,2 pos pos neg neg 8 Japan adr C C2_Ce 1,25E+08 47,0 neg pos neg neg 9 Russia adr C C2_Ce 2,92E+08 54,4 neg pos neg neg 10 Germany ayw2 D D1 1,17E+09 130,4 pos pos neg neg 11 South Africa ayw2 D D3 1,04E+08 63,8 pos pos neg neg 12 Iran ayw2 D D1 1,00E+08 17,7 pos pos neg neg 13 West Africa ayw4 E E 9,45E+08 82,6 pos pos neg neg 14 Brazil adw4 F F3 1,10E+07 32,2 pos neg neg neg 15 Germany adw2 G G 1,40E+07 0,9 pos neg neg neg

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HBV Genotype Panel (NAT Tests) Characterization of the panel members

Sample No Origin HBsAg Subtype HBV Genotype HBV Genotype HBV DNA

(IU/mL)

HBsAg

(KIU/mL)

anti-HBc HBeAg anti-HBe HIV1/HCV RNA

INNO-LiPA Sequencing qNATs ARCHITECT ARCHITECT Elecsys ARCHITECT Procleix

1 South Africa adw2 A A1 6,08E+08 131,9 pos pos neg neg 2 Brazil adw2 A A1 6,53E+08 94,0 pos pos neg neg 3 Germany adw2 A A2 6,87E+08 74,3 pos pos neg neg 4 Japan adw2 B B1 1,48E+08 51,4 pos pos neg neg 5 Japan adw2 B B2 2,84E+08 95,3 pos pos neg neg 6 Viet Nam ayw1 B B4 6,29E+06 4,6 pos pos neg neg 7 Japan adr C C2_Ce 3,99E+08 70,2 pos pos neg neg 8 Japan adr C C2_Ce 1,25E+08 47,0 pos pos neg neg 9 Russia adr C C2_Ce 2,92E+08 54,4 pos pos neg neg 10 Germany ayw2 D D1 1,17E+09 130,4 pos pos neg neg 11 South Africa ayw2 D D3 1,04E+08 63,8 pos pos neg neg 12 Iran ayw2 D D1 1,00E+08 17,7 pos pos neg neg 13 West Africa ayw4 E E 9,45E+08 82,6 pos pos neg neg 14 Brazil adw4 F F3 1,10E+07 32,2 pos neg pos neg 15 Germany adw2 G G 1,40E+07 0,9 pos neg neg neg

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HBV Genotype Panel (NAT Tests) Characterization of the panel members - Genotyping INNO-LiPA HBV Genotyping Assay

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HBV Genotype Panel (NAT Tests) Characterization of the panel members – qNAT testing

1,000E+06 1,000E+07 1,000E+08 1,000E+09 1,000E+10 1/A1 2/A1 3/A2 4/B1 5/B2 6/B4 7/C2 8/C2 9/C2 10/D1 11/D3 12/D1 13/E 14/F3 15/G HBV Genotype Panel - Candidate Panel Members HBV DNA (IU/mL CAP/CTM Abbott RealTime artus HBV LC PCR Kit CA HBV Monitor Mean

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HBV genotype panel (NAT tests) – Dilution of panel members Dilution matrix for panel members: Negative plasma pool

• Testing of 117 negative pre-screened plasma units (CSL Behring)
• HBV serology: anti-HBs; anti-HBc
• HIV/HCV NAT: cobas Taqscreen MPX Test; Procleix Ultrio Assay
• Pooling of negative plasma units

If possible dilution to a HBV DNA concentration of about 106 IU/ml Dilution to a volume of 1.2 litre per sample

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HBV genotype panel (NAT tests) – Filling and lyophilisation

Certified company in Switzerland 4-ml screw-cap glass vials with rubber stopper Filling volume 0.5 ml per sample 15 x 2,000 vials Add 144 vials filled with 0.5 ml of negative plasma pool (for determination of residual moisture content) Instrument CHRIST Epsilon 2-25 D (LPC-16/NT process documentation) Five lyo batches, no mixing of genotypes per tray Characterization of the final product: Pre-study (PEI): Control of HBV DNA concentration before and after lyo; no significant loss of HBV DNA concentration Collaborative study Programme of stability testing Residual moisture content: 0.82%; SD ± 0.03% (method acc. EP)

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HBV genotype panel (NAT tests) – Collaborative study

Study purpose: evaluation of the HBV genotype panel using different NAT assays; parallel testing of the 2nd HBV DNA IS (97/750) Invited 23 labs Reply from 19 labs 20 participants (incl. PEI)

5 NCLs: NIBSC, CBER, ISS, PEI, NIID Japan 6 special labs (special diagnostic expertise in viral hepatology): Brazil, Argentina, South Africa, Spain, USA, Germany 9 kit manufacturers: Korea, Taiwan, USA (4), Sweden, Switzerland, Germany

HBV NAT quant: 15 labs (18 tests) HBV NAT qual: 5 labs HBV NAT sequencing whole genome (data to GenBank) and genotyping: 1 lab Report to ECBS in 2009

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HBV Mutation Assays

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HBV Mutation Assays - G1896A (Pre-C)

Detection with Real-Time PCR and Melting Curve Analysis (LightCycler)

Fluorescein

Anchor Probe

Red 640

Sensor Probe

Forward Primer Reverse Primer

5‘ 3‘

WILD TYPE

5‘ 3‘

Mutation in the sensor sequence (preferential on the 3’)

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HBV Pre-C 1896: Mixture of Wild-Type and Mutation Samples

1 3 4 NC 2 5

sample 1 Wild-Type (N2810) 4*103 copies/reaction 2 Mutation (N2875) 7*103 copies/reaction 3 Wild-Type/Mutation 75:25 4 Wild-Type/Mutation 50:50 5 Wild-Type/Mutation 25:75

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NC N2810 HBV plasmid N2811 N3595 N3761 N3596 N2875 N2876 Mutation Wild-Type

HBV Pre-C 1896: Analysis of Clinical HBV Samples

• N2876: mixture of mutation and wild-type
• N3761, N2811: show a hint for an additional mutation

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Acknowledgements

• Prof Wolfram Gerlich, Institute of Virology, University Giessen
• Dr Michael Schmidt, German Red Cross Frankfurt/Main
• Dr Christoph Jochum, Biotest, Dreieich
• Dr Eugene Zhiburt, Federal Blood Center, Moscow, Russia
• Prof Majid Cheraghali, Iranian Blood Transfusion Organization, Tehran,

Iran

• Prof Hiroshi Yoshizawa, Hiroshima University, Japan
• David J. Watts, South African National Blood Service
• Dr Márcia Otani, Fundação Pró-Sangue Homocentro de São Paulo, Brazil
• Dr Ana Padilla, WHO, Geneva
• People from PEI
• Section of Molecular Virology and IVD-Section
• Administrative staff

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HBV genotype panel (HBsAg tests)

• Prof. Gerlich, Univ. Giessen

Cloning and sequencing of entire S ORF

• Genosubtype, HBsAg subtype, escape mutations

Determination of HBs antigen concentration by

• Laurell immune electrophoresis (PEI-units)
• qCLIA (Architect, IU)

Determination of HBsAg protein by

• UV photometry after purification (ng)

Removal of virions from the HBsAg subviral particles by ultracentrifugation

• ver sucrose cushion (decrease of infectivity about 99%)

Determination of HBsAg content in the supernatant by qCLIA (IU/ml) PEI Residual HBV-DNA by qtNATs Dilution in negative plasma pool (HBsAg concentration about 30 IU/ml) Pilot study to investigate the effects of lyophilisation on the consistency of HBsAg detection Filling and lyophilisation Collaborative study Report for establishment to ECBS 2010 √ √ √ √ (√)