1

HBV Genotype Panel

Michael ChudySection of Molecular Virolgy

Division of VirologyE-Mail: chumi@pei.de

SoGAT XXI MeetingBrussels, 28 - 29 May 2009

2

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

3

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 in the current WHO International Standard preparations

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

Expert Committee on Biological Standardization

4

International reference panels for HBV genotypes

HBV genotype panel (NAT tests)

HBV genotype panel (HBsAg tests)

5

HBV genotype panels

Efforts to collect plasma units worldwide- HBV DNA/HBsAg high titre plasma samples with sufficient volume

Characterization of 215 potential candidate materials- quant HBV DNA, quant HBsAg- Sequencing of entire S ORF,- genotyping, escape mutations, HBsAg subtyping

HBV genotypes A – G (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): 16 panel members 12 samples are member in both panels

Project in close cooperation with Prof Gerlich (Institute of Virology, University Giessen)

6

HBV genotype panel (NAT tests)Final characterization of the panel members

SampleNo

OriginHBsAg

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

7

HBV genotype panel (NAT tests)Final characterization of the panel members

SampleNo

OriginHBsAg

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 F2 or F3 1,10E+07 32,2 pos neg pos neg

15 Germany adw2 G G 1,40E+07 0,9 pos neg neg neg

*Concentration based on quantification by four different assays

8

HBV genotype panel (NAT tests)Dilution of panel members, filling and lyophilisation

Dilution matrix for panel members: Negative plasma pool- Testing of 117 negative pre-screened plasma units

• HBV serology: anti-HBs; anti-HBc• HBV/HCVHIV NAT: cobas Taqscreen MPX Test; Procleix Ultrio

Assay- Pooling of negative plasma units

If possible, dilution to a HBV DNA concentration of about106 IU/ml (12 / 15 samples)

Dilution to a volume of 1.2 litre per sample 15 x 2,000 vials Filling volume 0.5 ml per sample Filling and lyo by a certified Swiss company

9

HBV genotype panel (NAT tests)Characterization of the final product

Pre-study (PEI): Control of HBV DNA concentration before and after lyo- no loss of HBV DNA and HBsAg concentration

Stability testing programme Residual moisture content: 0.82% (SD ± 0.03%)

(method acc. EP) Collaborative study

10

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 18 labs 19 participants (incl. PEI)

- 5 NCLs: CBER, ISS, NIBSC, NIID Japan, PEI,- 6 special labs (special diagnostic expertise in viral hepatology): Argentina,

Brazil, Germany, South Africa, Spain, USA- 8 kit manufacturers: Germany, Korea, Sweden, Switzerland, Taiwan, USA (3)

HBV NAT: quant 15 labs (17 tests), qual 3 labs HBV DNA sequencing and genotyping: 1 lab Statistical analysis Draft report for circulating Report to ECBS in 2009

14 (16), 2

11

HBV genotype panel (NAT tests) – collaborative studyPreliminary results

2nd WHO IS 97/750

1,E+05

1,E+06

1,E+07

Lab - Quant HBV NAT

HB

V D

NA

(IU

/mL

)

Arithmetic mean values from 3 independent runs (replicate testing)

Mean 6.01 ± 0.17 log10 IU/ml

12

HBV genotype panel (NAT tests) – collaborative studyPreliminary results

Study Sample 14/F

1,E+03

1,E+04

1,E+05

1,E+06

Lab - Quant HBV NAT

HB

V D

NA

(IU

/mL

)

Arithmetic mean values from 3 independent runs (replicate testing)

13

Blast HBV genotyping tool – sequence comparison

WHO IS (GenBank AJ012207) vs. reference sequences

pre-S2 S C

PX

pre-S1

P

14

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 over sucrose cushion (decrease of infectivity about 99%)

Determination of HBsAg content in the supernatant by qCLIA (IU/ml)PEI Residual HBV-DNA by quant NATs 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

()

15

International reference panels for HBV genotypesConclusion

Two panels (NAT tests and HBsAg tests) Well characterized panels

- serological and molecular characterization, protein characterization- NAT: 16 different assays (13 quant; 3 qual),

overall good correlation in detecting most of the genotypes,

no unitage of panel members- HBsAg: results from pilot studies provide hints for different detection

efficiency Intended use

- Assay validation- Assay comparison- BV testing (NCLs, manufacturers)

Establishment by ECBS- NAT test panel probably 2009- HBsAg panel 2010

Global availability

16

Acknowledgements

- Prof Gerlich, Institute of Virology, University Giessen- Prof Yoshizawa & Prof Tanaka, Hiroshima University, Japan

- Biotest AG, Dreieich - Federal Blood Center, Moscow, Russia- Fundação Pró-Sangue Homocentro de São Paulo, Brazil- German Red Cross Frankfurt/Main- Iranian Blood Transfusion Organization, Tehran, Iran- South African National Blood Service

- WHO Collaborative Study Group

- Dr Ana Padilla, WHO, HSS / EMP / QSM, Geneva- NIBSC

- People from PEI- Section of Molecular Virology and IVD-Section- Administrative staff

17

Thank you for your attention!