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Significance of the proposed WHO HBV genotype panel for quality control of HBsAg screening and monitoring

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Title: Significance of the proposed WHO HBV genotype panel for quality control of HBsAg screening and monitoring


1
Significance of the proposed WHO HBV genotype
panel for quality control of HBsAg screening and
monitoring
SoGAT XXI, Brussels May 29, 2009
Calibration of International HBsAg Units in
Nanogram HBs Protein Comparison with HBV DNA
levels
Wolfram H. Gerlich, Ulrike C. Wend, Christian G.
Schüttler Institute for Medical Virology (IMV),
Justus Liebig University Giessen,
Germany National Consulting laboratory for
Hepatitis B und D Micha Nübling, Michael
Chudy Paul Ehrlich Institute Langen, Germany
2
Why do we need quantitative determination of
HBsAg?
  • Quality control of HBsAg screening
  • Evaluation of testkits
  • Control of analytical sensitivity
  • Accurately adjusted working controls
  • Follow-up of HBV infection
  • Marker for activity of HBV gene expression
  • Surrogate of HBV cccDNA in liver
  • Prognosis of acute HBV infection
  • Monitoring of HBV therapy
  • Establishment of reference sera

3
Quantitative determination of HBsAg
  • There are three different units of HBsAg
  • WHO arbitrary International Units (IU)
  • Paul Ehrlich Institute Units (PEI-U)
  • equal to one nanogram (ng) HBsAg protein
  • Various nanogram units (ng)
  • Comparison of the 3 units showed
  • Incompatibility between PEI and most ng units
  • Large divergence between ng units in different
    reference preparations

4
The primary HBsAg Standards of the Paul Ehrlich
Institute (PEI)
  • Positive plasma units collected in the early
    1970ies
  • were screened for high HBsAg titer
  • and subtyped for HBsAg subtypes ad and ay
  • Subtypes were pooled separately and recalcified
  • HBs antigen (QIE or IEP) and protein (OD280)
  • was quantified as described by Gerlich
    Thomssen 1975
  • Adjusted to 50.000 ng HBs ad or ay protein / mL
  • Distributed to aliquotes and stored at -80 C
  • HBsAg with reactivity of
  • one nanogram HBs protein in the primary PEI
    standards
  • is one PEI unit of HBsAg

Bonin, Gerlich, Thomssen, 1975
5
Quantitative immune electrophoresis (QIE) of
HBs-antigen Laurell electrophoresis
  • Slides covered with antibody-containing agar
  • Anti-HBs/a antiserum raised with subsequent
    injections of subtypes ad and ay
  • Wells are filled with HBsAg pos. serum
  • HBsAg migrates to anode and diffuses laterally
  • At equivalence of antigen and antibody
    precipitates lines are formed
  • Length of figures increases with antigen

30
5
1
10
20
HBsAg µg/ml genotype D Internal reference serum
Gerlich and Thomssen, Devel Biol Standard
1975 Gerlich et al., 2005 Hepatitis B Virus
Protocols Book
6
Quantitative assay of HBs protein via optical
density (OD 280) of purified HBsAg in isopycnic
CsCl gradient ultracentrifugation
OD 280
7
Constant ratio of HBs protein (E280) to HBs
antigen amount (QIE units)
8
1mg / ml HBsAg protein has an OD 280 of 4.29
0.21
OD 280 and 320 of three pure HBsAg solutions and
µg protein in volume Vh used for acid hydrolysis
and quantitative amino acid analysis determinatio
n of specific lightscatter-corrected OD 280 of
10 mg/mL
9
The primary HBsAg Standards of the Paul Ehrlich
Institute (PEI) and the 1st WHO standard
  • 1 PEI unit of HBsAg
  • 1 ng HBs protein in the primary PEI standards
  • WHO disliked this material
  • The material is highly infectious
  • WHO generated an own HBsAg standard plasma
  • Low viremia, anti-HBe positive
  • Pasteurised to reduce infectivity further
  • HBsAg subtype ad
  • Arbitrary International Units (IU)
  • 1 IU corresponded to 0.55 PEI-U

10
The WHO 2nd International HBsAg standard
  • Starting material The Dutch Hepatitis B Vaccine
    lot 30
  • Purified from highly viremic HBeAg-positive
    plasma
  • PEG precipitation, ultracentrifugation steps
  • infectivity inactivated by heating to 102 oC for
    90 sec
  • Contained in the 80ies 86,000 PEI units/mL
    (Ausria)
  • Provided by Nico Lelie (formerly at CLB)
  • Characterisation studies done in Giessen
  • Assay of HBsAg activity in primary PEI units
    using QIE
  • Protein composition using PAGE and silverstain
  • Western blot for preS and S proteins
  • Size exclusion chromatography
  • Electron microscopy
  • Density gradient centrifugation

11
Reactivity of HBsAg in the VQC preparation
Anti-a antiserum raised with subsequent
injections of genotypes A, D, and C
Stock for WHO standard
IMV internal reference plasma
D E
1
Undil. 12 13 15 HBsAg
genotype A2 heated, semipurified, Ca 60,000 PEI
units/ mL
5
10
20
30µg
HBsAg µg/ml genotype D native plasma 1 µg is 1000
PEI units
12
Silver-stained SDS gel of VQC and purified
reference HBsAg
Paul Ehrlich units per lane
WHO HBsAg 60 120 240 480
Reference HBsAg 40 80 160 320
LHBs
MHBs
SHBs
SHBs
13
Size chromatography of native HBsAg containing
serum Column Superose 6 (Vt 117mL), TN-Buffer,
fraction size 1.7 mL OD from ELISA with C20/02
(red) and MA18/07 (green), 1100 diluted HBsAg
precipation figures in Laurell electrophoresis
are shown in the insert
Kav
Vo
23 nm-particles
14
Size exclusion chromatography of WHO HBsAg
preparation 1.0 mL, Column Superose 6 (Vt117
mL), TN-Buffer, fraction size 2 mL OD from ELISA
with C20/02 (red, 110 diluted) and MA18/07
(green) Precipitation figures from Laurell
electrophoresis are shown in the insert
Vo
Kav
23 nm-particles
aggregates
15
Broader density distribution of HBsAg in WHO
reference sample (left) than in a purified
native HBsAg reference sample Top QIE of the
density fractions. Center silver stained
SDS/PAGE of the density fraction. Bottom Sucrose
density ( w/w) of the fractions and kilo PEI
unit (KU) of HBsAg
41 31 - 45
37 35-38
WHO
native HBsAg
16
Summary on the source material for the 2nd WHO
HBsAg standard
  • Surprisingly stable
  • Contains ca 60,000 PEI units/mL by QIE
  • Previous estimate 86.000 by Ausria
  • Altered biochemical properties
  • Small HBs protein, P24 and GP27
  • Diffuse bands in PAGE
  • Most preS domains are removed
  • Heterogeneous particles
  • 23 nm diameter, single particles and aggregates
  • Floating density at 31 45 sucrose
  • HBsAg subtype adw2, HBV genotype A2
  • Central European variant
  • Inspite of differences to native HBsAg particles
  • suitable for common immune assays without preS
    antibodies
  • Successfully used in WHO trial with various
    immune assays

17
HBV genotypes A H of humans and of animals
18
Chimp HBV
19
Gorilla HBV
20
Orang Utan HBV
21
Gibbon HBV Old world
22
Woolly monkey New World Own species
23
Arctic squirrel New world Own species
24
Woodchuck New world Own species
25
Phylogenetic distance of human, primate and
rodent HBV
Humans and apes woolly monkey rodents
genotypes species genus
Genetic Distance 0.05
From Schaefer Gerlich, Textbook of Hepatology
2007, 825
26
Worldwide distribution of HBV Genosubtypes
, C, F
D
A2
C/D
From Schaefer Gerlich, Textbook of Hepatology
2007, 825
27
Estimated prevalences of HBV genotypes
Genotypes prevalence Number (millions) in
all 5 350 World
A2 A1, A3-6 0.5 ? 3 50? N. Europe, USA S.E. Africa
B/C 8 240 East Asia
D 2 40 Mediterranean Asia
E 8 20 West Africa
F/H Low 2 ? America
used for HBsAg vaccines and reference samples
28
Why we need a HBV genotype and variant panel
  • HBV genotypes react differently
  • WHO provides only heated HBsAg for genotype A2
  • Worldwide are genotypes A1, B, C and D
    predominant
  • Genotype F was not detected for years with some
    tests
  • Proposal for a genotype panel
  • for virion-bound HBV DNA in plasma
  • and native HBsAg in plasma
  • Accepted by the WHO group on blood safety
  • (Dr. Ana Padilla)
  • Realised by
  • Paul Ehrlich Institute together with
  • Drs. M. Chudy and M. Nübling
  • Institute of Medical Virology (IMV) Giessen





29
HBV geno(sub)type / HBsAg subtype reference panel
of WHO
  • 16 HBsAg highly positive samples from various
    parts of the world
  • 12 samples provided by M. Chudy and M. Nübling
    (PEI)
  • 4 from our collection.
  • Geno(sub)type determined by sequencing
  • of the entire S open reading frame
  • including the S, preS1, and preS2 domains
  • and phylogenetic analysis.
  • Nomenclature A1 H according to Norder et al.
    (2004)
  • HBsAg subtype alleles derived from the S gene
    sequence
  • Norder et al., 2004
  • d K122, or y R122 w K160, or r R160.
  • Determinant w is further divided into w1 to w4.
  • w1 P127, R122, F134, or A159 w2 P127, K122
  • w3 T127 w4 L or I127

30
HBV DNA geno(sub)type / HBsAg subtype reference
panel of the WHO/PEI
  • HBs protein concentration was determined after
    purification (modified after Gerlich and Thomssen
    1975).
  • Ultraviolet photometry yielding microgram HBsAg
    protein (µg).
  • The lipid and glycan content of HBsAg was not
    included.

31
HBV DNA geno(sub)type / HBsAg subtype reference
panel of the WHO/PEI
  • HBs antigen was determined
  • in PEI units (PU) by inhouse QIE
  • one PU should correspond to 1 ng HBs protein
  • HBs antigen was also determined
  • in International Units (IU)
  • by quantitative immune assay
  • Architect from Abbott
  • Calibrated with the 1st WHO standard

32
1st purification step of HBsAg from the IMV
internal reference plasma with 100 kPU by
sedimentation into a sucrose gradient
kIU/ml
OD 280
kPU/ml
33
2nd purification step of HBsAg from human plasma
by cesium chloride density gradient
ultracentrifugation and quantitation of HBsAg via
OD 280
HBsAg
OD 280
34
Silver stained PAGE of density gradient fractions
LHBs MHBs SHBs
35
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36
Assay of HBs antigen and protein in IMV internal
reference plasma
  • Immune assays
  • QIE 100 kPU/ml HBsAg
  • Architect 125.2 8.0 kIU/ml
  • Quantitative purification
  • Expected 100 µg/ml HBs protein
  • Measured 95.6 100.5 84.3 µg/ml
  • mean value 93.5 6.8 µg

37
1st purification of HBsAg from human plasma by
sucrose gradient ultracentrifugation
N4542 Genotype A1, South Africa
38
2nd purification step of HBsAg from human plasma
by cesium chloride density gradient
ultracentrifugation and quantitation of HBsAg via
OD 280
39
Silver stained PAGE of density gradient fractions
40
No quantitation of HBs protein for genotype F3,
25 kPU/ml, OD280 too low
41
Composition, origin and HBs protein contents of
the 16 WHO panel members
Internal reference plasma
HBs protein amount too low
HBs protein amount too low
HBs protein amount too low
42
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43
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44
H
G
45
Correlation between HBsAg units
H
G
46
Ratios of PEI-U or IU HBsAg to µg HBs protein in
the WHO reference plasmas
47
Conversion factors for nanogram HBsAg protein to
Paul Ehrlich Institute QIE-Units (PU)
  • One ng corresponded to
  • 0.62 PU for genosubtype B2, subtype adw2 to
  • 1.33 PU for genosubtype B1, subtype adw2
  • Mean value
  • 0.90 0.20 PU/ng
  • determined by QIE with anti-a serum generated
    with subsequent injection of genotype A2, D, and
    C2
  • no outliers

48
Conversion factors for nanogram HBsAg protein to
HBsAg International Units (Architect)
  • One ng was between
  • 0.84 IU for genotype C2, subtype adr to
  • 3.17 IU for genotype H, subtype adw2
  • Mean value
  • 1.35 0.45 IU/ng or
  • 1.08 0.13 IU/ng
  • if 4 outliers (B1, D3, F3, H) were excluded.

49
Ratio IU (Architect) to PEI-U (QIE)
  • Previous estimate 1.82 IU/PU
  • Or 1 IU equals 0.55 PEI-U
  • This study 1.73 0.98 IU/PU
  • All values included
  • This study 1.39 0.27 IU/PU
  • When 2 outliers (G and H) were excluded

50
Correlation between HBsAg units and viremia
H
30 µg/ml
G
51
Ratio between number of HBV genomes to pg HBs
protein
52
Ratio of HBV particles to subviral HBsAg particles
  • 1 pg HBsAg 37 18 HBV particles
  • Valid for high viremic plasmas with
  • gt108 ge/ml HBV DNA and 30µg/ml HBsAg
  • 1 pg HBsAg in pure virus ca. 105 particles
  • 240 subunits with 25 kD in one virus particle
  • 6 MD 10-17 g 10-5 pg
  • Ratio HBsAg in virus to HBsAg in subviral
    particles
  • ca. 1 2700 1300

53
Evaluation of Enzygnost HBsAg versions 5 and 6
with the proposed WHO HBsAg Panel
  • The specimens for the future WHO panel were
    prediluted to an estimated HBsAg concentration of
    50 PU/ml
  • then diluted further in 9 half log steps down to
    131,600, i. e., to 1.58 mPU/ml.
  • These nine dilutions were analysed with 3 lots of
    Enzygnost HBsAg 6.0
  • and one lot of Enzygnost HBsAg 5.0.

Siemens Health Care Products, formerly Dade
Behring, Marburg, Germany
54
Detection limit of Enzygnost v6
v6
v5
cutoff
Genotype A2, Germany
V6 7.2 pg/ml
half log dilution steps
55
Detection limit of Enzygnost v6
cutoff
V6 9.2 pg/ml
Genotype A1, South Africa
half log dilution steps
56
Detection limit of Enzygnost v6
cutoff
Genotype B2, Japan
V6 12.7 pg/ml
half log dilution steps
57
Detection limits of Enzygnost HBsAg version 6
with the future WHO panel
  • Picogram HBsAg protein
  • 7.2 pg/ml for gt A2 to
  • 12.7 pg/ml for gt B2
  • mean value 9.5 1.6 pg/ml.
  • Milli Paul Ehrlich Units (in house QIE, Giessen)
  • 5.8 for gt A1 to
  • 14.6 mPU/ml for gt D1
  • mean value 8.9 2.2 mPU/ml.
  • Milli International Units (WHO, Architect)
  • 8.3 for gt A2 to
  • 18.7 mIU/ml for gt F3
  • mean value 11.6 3.0 mIU/ml.

58
Conclusions
  • HBsAg protein amount of all genotypes correlates
    quite well with old PEI-units (QIE)
  • 1ng 0.90 0.20 PU (range 0.62 1.33), r20.84
  • More divergence with IU (Architect)
  • 1ng 1.35 0.45 IU (range 0.84 3.17), r20.65
  • 1ng 1.08 0.13 IU (range 0.84 1.18)
  • Without four outliers (B1, D3, G, H)
  • Most consistent data on detection limits with ng
    units
  • 9.5 1.6 pg/ml versus 11.6 3.0 mIU/ml for
    Enzygnost 6.0
  • Good correlation between HBs antigenemia and
    viremia
  • in high-viremic carriers (lt108 /ml HBV and gt 30
    µg/ml HBsAg)
  • 1pg 37 18 HBV particles (range 13 62)
  • Ratio virus to subviral particles ca. 1 2700
    1300

59
Proposal for the future
  • Remove majority of infectious virus from subviral
    HBsAg by ultracentrifugation
  • Dilute the supernatant of the 16 stock plasmas in
    negative plasma to 30 ng HBsAg protein/ml
  • Perform an international trial
  • If successful
  • Define 1ng HBsAg of these samples as new
    IU of the corresponding genosubtype
  • 1ng of HBV A2 is close (1.16) to 1 current IU

60
Many thanks to
Wulf Willems
Christian Schüttler
Ulrike Wend
Supported by DFG Collaborative Research Centre
SFB 535 Project A2 and WHO/PEI
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