34pt Title

1
The Emergence of Hepatitis B Surface Antigen
Mutants and Their Impact on Diagnostic Detection.
Paul Coleman, Ph.D. Assoc. Research Fellow Life
Sciences Core RD Abbott Laboratories Abbott
Park, IL 60064
2
Webcast Objectives

• This seminar will explore the dynamics of HBV
  surface antigen mutants, what drives their
  occurrence, which marker profiles they may
  present, prevalence and their potential medical
  impact.
• - What causes HBsAg mutants to emerge?
• - Who is at risk for HBsAg mutant infection?
• - What type of screening can determine if an
  HBsAg mutant infection is present?

3
US Blood Bank Testing
Current Safety Estimates Risk of Infection
HBV 1 269,000 - 640,000 HCV 1
1,935,000 HIV 1 2,135,000 HTLV 1 514,000 -
2,993,000
Based on data from ARC repeat donations,
2000-2001, representing 4.2 million person-years
of observation
4
Hepatitis B Virus Major Global Public Health
Problem
More than two billion people have been infected
with HBV at some time in their lives 4 million
new acute clinical case annually. One million
carrier die each year from chronic active
hepatitis, cirrhosis or primary liver cancer. 350
400 million people remain chronic
carriers Incubation period range . . . . . . . .
. 45 180 days Acute case-fatality rate . . . .
. . . . . . 0.5 1.0 Chronic infection . . . .
. . . . . . . . . . . lt5 years, 30 90 gt5
years, 2 10 Premature mortality from chronic
liver disease . . . . . . . . . . . . 15 25
Immunization with HB vaccine is the most
effective means of preventing HBV infection and
its consequences Global HBV vaccination programs
started late 1980s
Sources WHO/CSR/LYO/2002Hepatitis B CDC
5
HBV Genome

• Small DNA virus (3.2Kb)
• 5 ORFs
• 3 promoters
• 2 enhancers
• At least 8 regulatory elements

6
HBsAg Gene Products
7
HBsAg a Determinant
Lys
Lys
Thr
Thr
Thr
Thr
Pro
Pro
Thr
Thr
Ala
Ala
160
Ala
Ala
Cys
Cys
Cys
Cys
Lys
Lys
Gln
Gln
Phe
Phe
124
Cys
Cys
Ala
Ala
Gly
Gly
121
Pro
Pro
Trp
Trp
Asn
Asn
Ser
Ser
Gly
Gly
Ser
Ser
Ser
Ser
Thr
Thr
Met
Met
Pro
Pro
Ser
Ser
Ile
Ile
Phe
Phe
Pro
Pro
Thr
Thr
Pro
Pro
Ile
Ile
Thr
Thr
Ser
Ser
149
Cys
Cys
Thr
Thr
Cys
Cys
Thr
Thr
138
Ser
Ser
Cys
Cys
Cys
147
Cys
Cys
Cys
107
Gly
Gly
CHO
Asn
Asn
Cys
Thr
Cys
Thr
Pro
Pro
Ile
Ile
Pro
Leu
Pro
Leu
Gly
Gly
Lys
Lys
Val
Val
3D epitopes stablized by conserved Cys bonding
Asp
Asp
Pro
Pro
Pro
Thr
Leu
Pro
Tyr
99
Thr
Gly
Leu
Gln
Tyr
Gly
Met
Gln
Met
Chen et al, PNAS (USA) 931997-2001 (1996)
8
(No Transcript)
9
HBsAg Structure
42 nm
104 excess of HBsAg vs. viron
CDC
10
HBV Life Cycle
Ganem, D., and Prince, A., NEJMed 350 1118-29
(2004)
HBV replication involves reverse transcriptase
activity. Poor transcription fidelity will
generate nucleotide mismatches in active
infection - leads to mutant emergence if a
selection pressure is applied. Inherent function
of replication - Allows virus to adapt to new
pressures in environment.
11
Viral Replication and Mutational Frequency

• High virion production 1012-13 virions per day
• High mutational rate lack of proofreading
  capacity of HBV polymerase reverse transcriptase
  activity 10-5 substitution/base/cycle
• 1010-11 point mutations produced per day
• All possible single base changes can be produced
  per day across the entire HBV genome
• Single double mutations pre-exist in patients
  prior to therapy

12
Darwinian Principles in Viral Evolution
An antiviral drug is a drug that selects for
resistance
Professor Douglas D. Richman. Hepatology.
200032866
13
Quasispecies
Fatal Error
Replication Defective
Adaptive Virus
Viral Diversity
14
HBV is divided into 8 genotypes based on
nucleotide divergence
Norder et. al. Intervirology 47289 (2004)
15
Mutants occur in each HBV gene.

• Polymerase gene Mutants
• Resistance to therapy
• Mainly Nucleoside Analogues (Lamivudine)

• Pre S and S gene Mutants
• Affects HBsAg immunogenecity
• Particularly a determinant
• Induced by vaccines/HBIg therapy

• X gene Mutants
• Involved in suppressionof HBV protein secretions

• Precore and Core Mutants
• Affects expression of HBe Ag
• HBeAg Neg. chronic HBV
• Low HBe, 2x replication ?

• Mutant Types
• Substitution
• Insertion
• Deletion
• Termination

16
Hepatitis B Genome
Viral Structure
HBsAg Polymerase overlap
3.2 Kb
Overlap of the polymerase gene and the surface
antigen gene in two different reading frames.
Selection of mutants in the polymerase gene can
cause a corresponding mutant selection in the
surface gene.
17
HBV Treatment Options
1. Vaccination (recombinant sHBsAg) 2.
Immunotherapy (HBIg) 3. Nucleoside analogs
Lamivudine Adefovir Entecavir Lobucavir Pencyclo
vir Famciclovir Tenofovir 4. Alpha interferon
18
HBsAg Mutants first reported case Italy.

• Initial description of a HBsAg mutant - Zannetti
  et al Hepatitis B variant in Europe. Lancet
  121132-3 (1988).
• Infant born to HBV positive mother. Vaccinated
  and anti-HBs response. Breakthrough infection
  had nt G587 to A mutation in the S gene which
  resulted in aa Glycine 145 to Arginine mutation.
• Stable infection has persisted for gt14 years.

19
Acute Hepatitis B Virus Infection with
RecoveryTypical Serological Course
Symptoms
HBeAg
anti-HBe
Window
Total anti-HBc
Titer
anti-HBs
IgM anti-HBc
HBsAg
0
4
8
12
16
24
28
32
52
100
20
36
CDC
Weeks after Exposure
20

• Do HBsAg mutants really impact health care?
• Prevalence
• Medical Impact

21
HBsAg Mutants in asymptomatic HBV Carriers
HBV carriers infected with HBsAg escape
mutants/variants have been found in
22
HBsAg Mutant Prevalence

• USA Nainan et al., J. Med. Virol. 68 p319
  (2002) HBsAg variants were detected in 51
  (47/93) of infants with chronic HBV infection.
  35 infants had Gly145Arg mutation. aa121-124
  mutations rare.
• Canada Osiowy, J. Clin. Micro. 40 p2566 (2002)
  The Gly145Arg mutation was observed in 18 of 28
  (64) HBV DNA positive samples. using Gap
  Ligase Chain Reaction.
• France Roque-Afonso et al., Path. Biol. 53
  p563 (2005) Studied 55 chronic hepatitis cases.
  Substitutions of the major absorbent area are
  found among 31 patients (56)... 6/31 patients
  had Gly145Arg mutation.
• Spain Avellon et al., J. Med. Virol. 78 p24
  (2006) Studied 272 chronic carriers. Analysis
  of amino-acid positions 112-157 revealed single
  or multiple substitutions in 39 of the carriers
  tested. low level of Gly145Arg mutation
• Korea Song et al., J. Med. Virol. 76 p194
  (2005) Studied 101 chronic HBV patients.
  Forty-seven MHR variants (46.5) of the 101
  patients were detected...

23
HBsAg Mutants - France
France has low prevalence for HBV infection. 55
highly selected samples out of a population of
chronically infected patients were sequenced with
the following results 56 (31/55) show mutants
in the major hydrophilic region of the
S-gene The most frequent substitutions involved
the following amino acids F/Y134H (7/31) M13
3I (6/31) D144A (6/31) G145R
(6/31) ...changes of the major hydrophilic
loop of HBs antigen are NOT rare and could have
important implications for transfusion safety and
diagnostic reliability.
Source Roque-Afonso, A.-M. et al., Pathologie
Biologie 53 563 (2005)
24
HBsAg Mutants - Spain
Spain has low prevalence for HBV infection. 272
unselected samples from chronic carriers of HBV
infection were sequenced. Single or multiple
substitutions were found in 39 of these
patients. The most frequently substitutions
involved the following amino acids M133T
2.2 Q129H, M133I, P/T134N 1.8 P/T134L,
G145A 1.5 P120T 1.1 (1)   ... that
a determinant HBV mutants seem to be extremely
common among chronic carriers from Spain, and
mutants with a potential impact on relevant
aspects of the HBV infection should be expected
in a significant proportion of carriers.
Avellon et al. Journal of Medical Virology,7824
(2006)
25
Prevalence of Naturally Occurring Surface
Antigen Variants of Hepatitis B Virus in Korean
Patients Infected Chronically. Byung-Cheol
Song, Sun-Hyun Kim, Hong Kim, Yuan-Hai Ying,
Hyun-Ju Kim, Yoon-Jun Kim, Jung-Hwan Yoon,
Hyo-Suk Lee, Chang-Yong Cha,Yoon-Hoh Kook, and
Bum-Joon Kim Journal of Medical Virology
76194202 (2005) In this study
(N101 chronic patients), an unexpectedly high
prevalence of naturally occurring MHR variants
was found in Korean chronic patients (n47)a
higher prevalence of MHR variants was associated
with HBeAg-negative serostatus and severe liver
disease, particularly HCC.
26
Prevalence of HBsAg mutants and impact of
hepatitis B infant immunisation in four Pacific
Island countries. Ashraf A. Basuni, Lesley
Butterworth , G. Cooksley , S. Locarnini , W.F.
Carman, Vaccine 22 (2004) 27912799 In this
study (N784 children), no a determinant
mutants were detected, indicating that
classical vaccine escape variants such as
sG145R and sP120T are not an important cause for
the failure to prevent HBV transmission in these
representative Pacific Island countries. Referrin
g to Oon Breakthrough infections in these
immunized infants were clearly due to other
factors. The most likely is the timing of the
three doses of hepatitis B vaccine Other
factors yet to be identified?
27
While the rate of HBV infection drops over time
in response to vaccination, the proportion of
HBsAg mutants increase in the breakthrough
infections.
28
HBsAg Mutant Prevalence (Singapore)
12/16 75 of mutants had Gly145 to Arg 12/345
3.5 prevalence in this population
Oon et al., Vaccine 13 699-702 (1995)
29
Detection of rHBsAg Singapore Mutants
Coleman et al, J. Med. Virol. 5919-24 (1999)
30
Assays Vary In HBsAg Mutant Detection
Assay C (S/CO)
Assay B (S/CO)
Assay A (S/CO)
AxSYM (S/CO)
MOERMAN, B, et al Clin Lab. 200450(3-4)159-62.
31
HBsAg Assay Format vs. Mutant Detection
Assay Format Mutant False Negative
(capture/detection) (relative
risk) Monoclonal/Monoclonal
High Monoclonal/Polyclonal Moderate Dual
Monoclonal/Polyclonal Low Polyclonal/Polyclonal
Very Low
32
Immunodetection of Gly145 to Arg mutant
33
Yeast expression and DNA immunization of
hepatitis B virus S gene with second-loop
deletion of a determinant region. Hui Hu,
Xiao-Mou Peng, Yang-Su Huang, Lin Gu, Qi-Feng
Xie, Zhi-Liang Gao World J Gastroenterol
200410(20)2989-2993 HBV S gene with deleted
second-loop, amino acids from 139 to 145, was
cloned and expressed. The deleted HBsAg could
not be detected by ELISA, ... but could be
detected by Abbott IMx HBsAg and immune dot
blotting... ELISA HBsAg kit was purchased from
Zhongshan Biological Engineering Company
(Guangdong, China).
34
Immunodetection of Artificial 139-145 Deletion
Construct
IMx
Lys
Lys
Thr
Thr
Thr
Thr
Pro
Pro
Thr
Thr
Ala
Ala
Ala
160
Ala
Cys
Cys
Cys
Cys
Lys
Lys
Gln
Gln
Phe
Phe
124
Cys
Cys
Ala
Ala
Gly
Gly
121
Pro
Pro
Trp
Trp
Asn
Asn
Ser
Ser
Gly
Gly
Ser
Ser
Ser
Ser
Thr
Thr
Met
Met
Pro
Pro
Ser
Ser
Ile
Ile
Phe
Phe
Pro
Pro
Thr
Thr
Pro
Pro
Ile
Ile
Thr
Thr
Ser
Ser
149
Cys
Cys
Thr
Thr
Cys
Cys
Thr
Thr
138
Ser
Ser
Cys
Cys
Cys
147
Cys
Cys
Cys
107
Gly
Gly
CHO
Asn
Asn
Cys
Thr
Cys
Thr
Pro
Pro
Ile
Pro
Ile
Pro
Leu
Leu
Gly
Gly
Lys
Lys
Val
Val
Asp
Asp
Pro
Pro
Leu
Pro
Tyr
Thr
Leu
Pro
Tyr
99
Thr
Gly
Gln
Gly
Met
Met
Gln
35
HBsAg Monoclonal Selection

• High Affinity
• Broad genotype recognition
• Good clinical specificity
• Mouse immune response to HBsAg
• Result Commercial monoclonal antibodies look
  very similar against 139-147 epitope.

36
Assays have different sensitivity endpoints
Information from assay package insert 2006
1 WHO IU 0.43 PEI units (current) or 1.93
French ng or 5.59 Abbott ng.
37
HBsAg assay formats
Information from assay package insert 2006 1
Moerman et.al Clinical Laboratory 3/4,2004 2
Flecheux et.al BioRad ISBT 2003 poster
1 WHO IU 0.43 PEI units (current) or 1.93
French ng or 5.59 Abbott ng.
38
HBsAg mutant medical impact MAUDE database
http//www.fda.gov/cdrh/maude.html

• MAUDE data represents reports of adverse events
  involving medical devices. The data consists of
  voluntary reports since June 1993, user facility
  reports since 1991, distributor reports since
  1993, and manufacturer reports since August 1996.
  MAUDE may not include reports made according to
  exemptions, variances, or alternative reporting
  requirements granted under 21 CFR 803.19.
• The on-line search allows you to search CDRH
  database information on medical devices which may
  have malfunctioned or caused a death or serious
  injury. MAUDE is scheduled to be updated
  quarterly and the search page reflects the date
  of the most recent update. FDA seeks to include
  all reports received prior to the update.
  However, the inclusion of some reports may be
  delayed by technical or clerical difficulties.

Manufacturers And Users Device Experience
FDA requires mutant disclosure in US assay
package insert
39
False Negative HBV Results in Blood Donors
Case 1 Blood donor in Slovenia. Tested positive
in PRISM HBsAg assay, tested negative in lower
volume assay (Ortho HBsAg 3.0). Subsequent
testing in other assays AxSYM HBsAg positive,
Ortho Vitros ECI negative. Markers HBsAg
confirmed positive, anti-HBc positive, anti-HBs
negative, PCR positive. DNA sequencing
Threonine 143 to Leucine substitution in a
determinant. We conclude that it is
absolutely necessary to use HBsAg assays with a
high sensitivity for mutant forms in blood donor
screening. S. Levicnik-Stezinar, Clin Lab.
5049-51 (2004)
Note Three independent occurrences of this same
mutant have recently been described. Ivanka
Mihaljevic in Zagreb, Croatia. T. Ly in Algeria.
T. Tallo in Estonia (J. Med. Virol. 74221-7
(2004)). Core RD detection in Macedonia 4/2006.
May be much more prevalent that originally
thought.
40
False Negative HBV Results in Blood Donors
Case 2 MAUDE 1319681-2004-00034 Blood donor in
Denmark his donation became part of a plasma
pool which tested HBsAg negative by a commercial
assay. During final processing, pool tested
positive in another lab by AxSYM HBsAg - result
disregarded as random error. Follow-up testing
by 3 blood banks using Prism or Architect systems
found positive HBsAg results. Four other blood
banks using Ortho HBsAg 3.0 microtiter, Ortho
Vitros ECi, or Bio Meriuex found the sample
negative. PEI and NTS both confirmed the sample
to be HBV positive. Markers HBsAg conf.
positive, anti-HBc positive, anti-HBs positive,
PCR pos. DNA sequencing Aspartic Acid 144 to
Glycine substitution in a determinant.
ongoing investigation Gerlich et al.
41
False Negative HBV Results in Diagnostic testing
Case 3 A kidney transplant recipient tested
negative for HBsAg prior to dialysis in October,
November, and December 2003 using Enzygnost Dade
Behring assay. The first sample was tested by
IMx HBsAg (V2) and showed a clear positive result
around 20 S/CO. Markers HBsAg AxSYM
confirmed positive, anti-HBc positive, anti-HBC
IgM negative, HBeAg positive, PCR positive (108
copies/ml). The sample contains 5 exchanges
compared to genotype A, subtype adw T115R, P120Q,
S154L, E164V, Q181R This dialysis patient was
an asymptomatic infectious chronic HBV carrier
who was misdiagnosed for almost two months due to
mutant HBsAg false negative results. Dr.
Sussitz, LKH Klagenfurt, Austria - Abstract at
5th Int. Symp. on Molecular Diagnostics in
Laboratory Medicine 2004
42
HBsAg mutant described with variable EIA detection
Carman et. al., Lancet 345 1406-7 (1995)
60 year old Indonesian lymphoma patient in USA
underwent chemotherapy for lymphoma. As
immunosuppresion therapy was withdrawn, ALT
levels increased. Presented 5 weeks later with
fulminate hepatitis, subsequently died from
hypotension. Markers Anti-HBc pos., HBeAg pos.,
HBV DNA pos., anti-HBs neg. Ausria pos.,
Auszyme neg.
wildtype aa sequence Cys121-Arg-Thr-Cys124
mutant aa sequence Cys121-Arg-Asn-Thr-Thr-Cys12
6
43
Figure 2. Amino acid positions of corresponding
Pol - HBsAg mutations.
Coleman et al. Poster 990 AASLD 11-05
44
6. Results
Graph 2. The epitope reactivity of the antiviral
resistance mutants is shown. Epitope reactivity
similar to the control is seen for all mutants
except for the triple HBsAg mutant F161L/
E164D/I195M which shows a 40 50 reduction in
reactivity for the H53 epitope (circled in red).
Coleman et al. Poster 990 AASLD 11-05
45
Conclusion The effect of multiple antiviral
mutations on HBsAg epitope presentation may
explain the emergence of HBsAg mutants in the
absence of immunoselective pressure.
Coleman et al. Poster 990 AASLD 11-05
46
Symposium on HIV Variants and Hepatitis B Surface
Antigen MutantsMay 22-24, 2005 Washington, D.C.
Detection of HBsAg mutant samples was
recommended for inclusion in the European Common
Technical Specification (CTS) required for CE
marking.
A study of HBV chronic carriers in Spain found
that an estimated 6.6 had vaccine-escape
mutants, and 9.2 had immunotherapy-resistant
mutants.
Surveillance studies to monitor the emergence
of HBsAg mutants are vital in the design of next
generation HBsAg assays to ensure detection of
all mutants.
Emerg. Infect. Dis. serial on the Internet.
2005 Oct. Available from http//www.cdc.gov/incido
d/EID/vol11no10/05-0856.htm
47
HBsAg Screening CE AssaysSeroconversion Score
(PEI 2005)
48
Acute Hepatitis B Infection
Transient surface antigenemia results in HBsAg
negative window periods (WP). HBV DNA may be
detectable by PCR during these WP.
Automated immunoassays for HBsAg detect samples
not detected with EIA technology
HBV DNA ()
HBsAg()
anti-HBc()
anti-HBs()
Late
Early
WP
WP
infection
49
HBsAg Mutant Surveillance

• What are possible screening markers for HBsAg
  mutant samples?
• HBsAg Immunoassay Discrepant Samples
• HBsAg , anti-HBs samples ? subset
• RD multi-epitope screening assay
• HBsAg -, anti-HBc , DNA
• Will this approach detect all HBsAg mutants?
• No, some mutants appear in the absence of
  obvious selective pressure. Must also consider
  affect of mutation on polymerase function.

50
HBsAg Mutants Summary

• HBsAg mutants result from the molecular mechanism
  of HBV reproduction and the application of a
  selective pressure - there is wide geographic
  prevalence of mutants.
• The most common HBsAg mutants (in 139-147
  eptiope) give false negative results in many
  commercial assays. These mutants are detected by
  assays using the capture epitope aa121-124.
• As mutants increase in prevalence so does the
  challenge to HBV diagnosis real risk of false
  negative results. Alternate false negative risk
  for false negative results due to assay
  sensitivity (window period).
• HBsAg testing algorithms should be designed to be
  capable of detecting mutants.

51
HBsAg Mutant Research Next Steps

• Quantitate mutant virus levels vs. wild-type
  virus (helper?) in escape mutants.
• How does patient anti-HBs response correlate with
  site of HBsAg mutation in escape mutants? Does
  type of vaccine influence escape mutant
  prevalence or mutation site?
• Is mutant distribution influenced by genotype or
  patient population?
• Are there mutants present/transmitted without
  obvious selection pressure (polymerase
  stabilized)?
• Can HBsAg epitope reactivity be changed w/o
  mutation?
• What is medical risk of 126, 133 vs. 145 mutants?