WO2000042194A2

WO2000042194A2 - Mutant hepatitis b surface antigen and testing

Info

Publication number: WO2000042194A2
Application number: PCT/GB2000/000107
Authority: WO
Inventors: Frederick William Carman
Original assignee: University Court Of The University Of Glasgow
Priority date: 1999-01-18
Filing date: 2000-01-18
Publication date: 2000-07-20
Also published as: AU2115000A; GB9901046D0; EP1144646A2; WO2000042194A3

Abstract

A mutant hepatitis B surface antigen useful in a screening assay or as a vaccine has amino acid changes at positions 3, 30, 53, 98, 101 and 210 relative to the wild type virus. Antibodies thereto are reactive with a wide variety of hepatitis B variants.

Description

HEPATITIS B TESTING

The present invention relates to a mutant hepatitis B surface antigen (HBsAg), and to monoclonal and polyclonal antibodies raised against the antigen, which exhibit an ability to detect a wide variety of hepatitis B virus (HBV) strains and antigenic variations. As such, it is particularly useful as the basis of a screening assay. The antigen may also be useful as a therapeutic (or prophylactic) vaccine.

The primary marker of current hepatitis B infection is the surface antigen (HBsAg), however HBsAg negativity does not exclude HBV infection. HBsAg antigenic variants can be responsible for such diagnostic failures.

Antigenic variation of HBsAg is clinically significant and has been discussed in several reviews (1,2). HBsAg variants can be divided into 2 aetiological classes. The first class occurs naturally and includes subtype variation and amino acid (aa) changes which may be poorly detected in diagnostic assays. Class 2 variants are selected by medically induced immune pressure, for example after vaccination and/or treatment with hepatitis B immunoglobulin (HBIG) (3,4). Class 2 antigenic variation tends to occur within the proposed neutralising epitopes but both classes show variation in the clusters of epitopes which constitute the external domain, referred to as the major hydrophillic region (MHR) of HBsAg

(1).

Much remains to be learnt about the frequency and clinical relevance of HBsAg variation, though it is clearly important to the development of commercial HBsAg assays. Sero-epidemiological surveillance will add to our understanding of the prevalance and geographical distribution of HBsAg (5). Though a range of commercial assays for HBsAg are widely available, not all are equally sensitive. It is thus imperative that the antibodies used for antigen capture in diagnostic assays are tested against a wide variety of variants in order to maximise their sensitivity and specificity. Some hepatitis B core antibody (anti-HBc ) positive. HBsAg negative sera are associated with variants of HBsAg that are not detectable by some commercial assays (10.1 1), but most appear to be due to the levels of HBsAg being below the level of detection. PCR is useful in differentiating between these two situations ( 16). One case in particular forms the basis for this patent application. She was HBsAg negative, but had antibodies to HBsAg (anti-HBs) and anti-HBc positive; usually this combination indicates past infection.

The invention also concerns therapy for HBV infection. Interferon α (IFN), the only drug licensed in the UK to treat HBV. is effective in only a third of those treated. The fundamental problem with chronic HBV is an ineffective HBV-specific immune response. There are a number of approaches which indicate that enhancement of specific immunity could be beneficial. For example, in transgenic mice, HBV-specific vaccines led to complete down regulation of mRNA synthesis from the transgene by a post-transcriptional mechanism⁹.

An object of the invention is to mitigate these problems.

The present invention provides a mutant hepatitis B surface antigen (HBsAg), which comprises wild type HBsAg having the following amino acid changes : Position Amino Acid (Wild Type)

3 S (N)

30 R (Q)

53 L (S)

98 V (L)

101 R (Q)

210 T (S) or the combination of any four or more of said changes. Generally speaking any amino acid change at any one or more of the six positions leading to substantially the same biological activity is included in the invention.

The mutant antigen having the four previously unknown amino acid changes at positions 30, 98. 101 and 210 is particularly preferred. Mutations at positions 3 and 53 have been noted previously in other strains. Of 1 13 hepatitis B surface antigen strains available from Norder (reference 18) and from Genbank (sequence database). 21 have S (serine) at amino acid position 3. the remainder have N (asparagine). At amino acid position 53, L (leucine) is present in 5 of the 1 13 strains only. N3S and S53L in combination are present in only 2 out of 1 13 strains.

The antigen may also have other sequence changes relative to the wild type at other positions provided that the antigenicity of the antigen of the present invention is substantially unaffected.

Conventional single amino acid codes are used herein.

The invention is based on the surprising finding that the mutant antigen is detected in all commercial immunoassays (employing immobilised antibodies from a variety of sources) tested, with a reactivity which was generally as good as and in some cases better than either of the hepatitis B wild types tested. The antigen bound to all monoclonal antibodies tested.

The mutant antigen therefore has utility in the production of monoclonal or polyclonal antibodies (possibly in higher titre than hitherto) having the ability to detect a wide variety of hepatitis B variants, and thus form the basis of a more complete type-independent screening assay. The invention extends to such monoclonal or polyclonal antibodies.

The production of antibodies is routine and can be carried out using techniques known in the art and referenced herein.

The invention also relates to an immunoassay device wherein an antigen or antibody according to the present invention is immobilised on a solid substrate. A variety of assay formats are known in the art. including solid substrates in the form of glass or plastic surfaces (such as microtitre plates), polymer beads or sticks. Alternatively, in more recent assay formats the antigen or antibody is first present in a liquid phase, where an initial reaction step occurs.

The antibodies may also carry a detectable label, such as a radiolabel, fluorescence label, enzyme label or other label known in the art. The labelled antibodies may be used to detect or visualise antigen from a test sample which has been captured by immobilised antibody in an immunoassay device.

The invention also extends to polynucleotide coding for the mutant antigen and which may be used to clone the antigen in a suitable expression system.

The invention also relates to the use of the antigen as a therapeutic or prophylactic vaccine. Post-infection therapeutic vaccination is believed to be effective in stimulating the immune system (particularly T-cells) and thereby clearing virus from infected patients. As a prophylactic vaccine, the present antigen would stimulate a wide range of antibodies, which would bind to and so protect against a variety of variant antigens. The antigen of the present invention may be formulated as a vaccine according to known techniques, including for example adjuvants such as aluminium hydroxide etc. The range of immune responses generated is beneficial.

Embodiments of the present invention will now be described by way of example only.

Detailed Description of Preferred Embodiments

13 different HBsAg variants were cloned into a mammalian expression system and the expressed protein tested using 7 commercially available diagnostic assays. Five samples displayed similar reactivity to the positive control sample (containing standard HBV surface (S) gene sequence) in most of the assays but six samples, containing various mutations across the entire major hydrophilic region, showed reduced reactivity. Two samples were not detected by any of the assays. Thus not all assays are equally able to detect HBsAg variants, implying that in order to show an acceptable level of sensitivity, the antibody repertoire of the

currently available assays should be extended. One particular example, which forms the basis for this invention, came from a female bone marrow transplant (BMT) donor, who was HBsAg negative but anti-HBs and anti-HBc positive. She was found to have four unique amino acid substitutions at positions 30, 98, 101 and 210 of the S gene. However, surprisingly, in vitro expressed HBsAg from the donor was detected by commercial kits at a similar or higher level than a standard sequence. Binding to monoclonal anti-HBs antibodies

was very strong, often better than a standard sequence, implying that an antiserum produced from this variant may detect all HBsAg variants. This was not the case for many of the other 13 variants, which bound at a low level or not at all and certainly no other variant bound to all monoclonal antibodies.

It is to be noted that the wild type sequences GlyY and GlyD themselves differ in amino acids at least at positions 122 and 160. In the Tables mutations are indicated as, for example, N3S; which indicates that normal amino acid N at position 3 is replaced by S in the mutant.

A) METHODS AND MATERIALS

1) Hepatitis B testing of the serum sample

Hepatitis B serological markers: HBsAg (Auszyme; monoclonal based), anti-HBs, HBeAg, anti-HBe, anti-HCV (EIA II), aπti-HDV and anti-HIV were all tested using commercially available enzyme immunoassays (Abbott Laboratories, North Chicago, IL. HBsAg was also tested by a polyclonal anti-HBs based assay (Murex, Dartford, UK).

2) Experimental approaches

(i) Summary: Cell culture supernatants. containing expressed protein from 13 diagnostically relevant HBsAg variants, were tested in 7 commercially available diagnostic assays. The reactivity of the variants was compared with the National Institute for Biological Standards and Control standard serum (0.5 IU/ml) and with an expressed standard HBV sequence. The supernatants were also tested in ELISA using a range of monoclonal antibodies against HBsAg (monoclonal-anti-HBs).

(ii) Derivation of variant sequences

Serum is rarely available in volumes sufficient for test against a multitude of capture antibodies, therefore we cloned variant HBsAg and obtained a significant quantity of protein using a mammalian expression system.

HBV DNA was extracted from 50 μl of serum using the QIAamp Blood Kit (QIAGEN, Crawley, UK) according to the manufacturer's instructions. A hot-start, nested polymerase chain reaction (PCR) was performed to amplify the surface (S) gene. Five microliters of extracted DNA was amplified in a 50 μl solution containing 1U Taq polymerase

TM

(Life Technologies, Paisley, UK), 1.4 μM TaqStart antibody (Clontech Laboratories Inc., Palo Alto, CA). 0.25 mM dNTPs (Pharmacia, St. Albans. UK), 2.5 mM MgCl2, 5 μl of 10X

PCR buffer, and 25 pM of primers SI [5'-CCTGCTGGTGGCTCCAGTTC-3'j and S2Na [5'- CCACAATTCKTTGACATA CTTTCCA-3'. where K= G or T] for 5 cycles of 95°C for 1 min. 55 C for lmin. and 72°C for 90 sec followed by 35 cycles with the denaturation temperature reduced to 90 C. One microliter of first round PCR product was re-amplified as

above with the nested primers S6C [5'- GCACACGGAATTCCGAGGACTGGGGACCCTG -3'] and S7 [5'- GACACCAAGCTTGGTTAGGGTTTAAATGTATACC -3'] for 5 cycles of 95°C for 1 min, 55°C for 75 sec. and 72°C for 90 sec followed by 25 cycles, with the denaturation temperature reduced to 90 C. DNA fragments of expected size were extracted from 1% agarose gel using Geneclean II kit (Bio 101, La Jolla, CA).

(Hi) Cloning and surface xzene sequencing

The S6C and S7D primers used in the nested PCR incorporate restriction sites for EcoRl and Hindlll respectively. The purified PCR product was ligated into the mammalian expression vector pRK5 using these restriction sites and transformed into the E.coli strain DH5χ. Plasmid DNA was purified using a Qiagen plasmid midi-kit (QIAGEN, Crawley, UK) and fluorescence-based sequencing of the whole surface gene was carried out using the ABI PRISM Ready Reaction dRhodamine Terminator Cycle Sequencing Kit (Perkin Elmer, Cheshire, UK) according to the manufacturer's instructions. The primers used for sequencing were S6C and S7D with the internal primers S3 [S'-AATGGCACTAGTAAACTGAGCC-S'J, S4 [5^'-GTATGTTGCCCGTTTGTCCTC-3'j and S8 [5'-AGAAGATGAGGCATAGCAGC- 3^*]. Sequence analysis was performed with the GCG programme (Wisconsin sequence analysis package, version 9.1. Genetics Computer Group, Madison, Wise.) (iv) Expression of BsAg

The plasmid. with its entire HBV surface gene insert, was transfected into subconfluent monolayers of COS7 cells on 16mm coverslips using either cationic liposomes, made from dioleoyl L- -phosphatidyl ethanolamine and dimethyldioctadecyl ammonium bromide (Sigma-Aldrich. Dorset. UK) (6,7). or the standard calcium phosphate method. Briefly, lμg plasmid was diluted in 50μl Optimem 1 reduced serum medium (Life Technologies. Paisley, UK) and. in a separate vial. 7μl of liposomes was added to 50μl of Optimem 1. The two solutions were mixed and allowed to stand for 15 min at room temperature, then further diluted with 500μl of Optimem 1 and added to pre-washed COS7 cells. The cells were incubated with the transfection mixture for 5h at 37°C in 5% CO2 and then 500μl of COS7 medium added (Dulbeccos MEM Medium with 5% foetal bovine serum, 0.02 IU/ml Penicillin, 0.02 μl/ml Streptomycin and 4μM L-Glutamine. All Life Technologies, Paisley, UK). Cells were incubated for 16h at 37°C in 5% CO2 when the transfection mixture was removed and 2ml of fresh COS7 medium added. Plasmid pRK5 containing standard HBV DNA S gene sequence (both adw and ayw subtypes) was used as a control for transfection and antigenic analysis. After 2 days, the culture medium was harvested and coverslips collected for immunofluorescence.

(v) Antigenic Analysis of Expressed HBsAg

(i) Commercial Diagnostic assay reactivity

Aliquots of culture supernatant were tested according to manufacturer's instructions in various commercially available diagnostic assays, which employ a combination of monoclonal and polyclonal antibodies for either capture or detection (Table 2). The signal to cut-off ratio was obtained for each sample in each assay and was divided by that of the standard serum, the working standard (0.5 IU/ml, National Institute for Biological Standards and Control, UK), to give an index of reactivity (Table 2). The reactivities of the variant sequences were also compared to that of the expressed standard HBV sequence taken as 100% activity (Table 3). The assays were repeated to assess intra-test variation of reactivities. To control for expression efficiency between experiments, supernatants from multiple transfections were tested using IMX HBsAg (V2) (Table 4).

The assays employed were - [1] bioELISA HBsAg colour. BIOKIT, Longfield, Kent, UK; [2JAUSRIA 11-125, Abbott Laboratories Ltd., Maidenhead. UK; [3] VIDAS HBsAg, bioMerieux SA. Marcy Etoile, France; [4]Enzymun-Test HBsAg ES300, Boehringer Mannheim GmbH. Mannheim. Germany; [5] IMX HBsAg (V2), Abbott Laboratories Ltd., Maidenhead, UK; [6]Murex HBsAg GE14, Murex Biotechnology Ltd., Dartford, UK and [7] Enzygnost HBsAg Monoclonal II, Behring Diagnostics GmbH, Marburg, Germany.

(ii) Immunofluorescence

COS7 cells on coverslips were methanol fixed, washed with phosphate-buffered saline (PBS) and incubated with goat anti-HBsAg polyclonal antibody (pAb) (Dako, High Wycombe, UK) for 45 min at room temperature. After washing, the cells were incubated with the secondary antibody, anti-goat fluorescein isothiocyanate (FITC) labelled immunoglobulin (IgG), for 30 min at room temperature (Sigma- Aldrich Company, Dorset, UK). Cells were examined under a Nikon Microphot-SA microscope.

(VI) Antibody Production

Monoclonal and polyclonal antibodies may be produced and labelled using conventional techniques as described, for example, in: 1. "Antibodies - a laboratory manual". Ed Harlow and David Lane, 1988

2. "Methods in immunology: a laboratory text for instruction and research". 2^nd edition (1970) by Dan Campbell, 3^rd edition by JS Garvey, NE Cramer and DH Sussdorf.

3. "Monoclonal antibodies - the second generation". Edited by H Zola. BIOS Scientific Publications Ltd., 1995.

4. "Monoclonal antibodies: principle and practice". James W. Goding (2^nd edition)

B) RESULTS

1) Sequencing

The variants, origin and clinical background of each sample is detailed in Table 1. In particular, the donor sequence that forms the basis of this application had 6 amino acid substitutions as compared with the sequence of an adr prototype [20]: N3S, Q30R, S53L, L98V, Q101R, and S210T. Among these amino acid substitutions, Q30R, L98V, Q101R and S210T present in the donor were not seen in the complete amino acid sequence of HBsAg from 88 HBV strains [18].

Amino acid and nucleotide sequences are given in the attached Figures, wherein: Figure 1 is the BMT amino acid sequence according to the present invention; Figure 2 compares the BMT sequence to the wild type (adr subtype) sequence; and Figure 3 is the BMT nucleic acid sequence.

2) Diagnostic assay reactivity

The diagnostic reactivity of the expressed HBsAg (Table 2) can be divided into three groups; those which displayed high reactivity in most assays, those with intermediate reactivity and those with poor reactivity. The first group includes samples GlyD and Gly Y (positive controls containing expressed HBsAg of standard sequence), 91-4696, BMT, AP3.1,

SA6 and SA7 which displayed mutations in HBs regions 1, 3 and 4 of the MHR (1,2). Samples SA6 and SA7 displayed high reactivity in 5 assays and reduced reactivity in 2 assays.

Six samples. Argl45. M5. 1056Sp, BA2.4, BA 3.4 and SA4 in the intermediate group were reactive in several assays but at a reduced level. Some of these samples displayed low level reactivity in certain assays : Argl45 was low in assays 2. 3 and 6 and negative in assay 7; M5 was negative in assays 3, 4, 6 and 7; 1056Sp. and BA2.4 were negative in assays 4 and 7; BA3.4 was negative in assays 3 and 5. This intermediate group contained mutations scattered across the entire MHR.

Two samples ,T5N and BA 3.2, containing mutations in HBs regions 2 and 4 could not be detected in any of the assays used .

When the results from the individual assays are compared, those which detected most samples (1 1 out of 13) employed polyclonal antibodies (pAb) for both capture and detection (assays 1 and 2) while two assays which had goat pAb for capture and mouse monoclonal

antibodies (mAb) for detection (assays 5 and 6) picked up 10. The two assays which employed mouse mAb for both capture and detection (assays 3 and 4) detected 9 and 8 samples respectively and the assay which detected the least number of expressed HBsAg samples (7 out of 13) was assay 7 which used sheep pAb for capture and mouse mAb for detection.

To control for inter-experiment expression efficiency supernatants from multiple transfections were tested using the Abbott IMX HBsAg assay (Table 4). 3) BMT results (invention)

As can be seen from tables 2 and 3 sample BMT according to the present invention displayed similar reactivity to the standard HBV sequence (Gly D) in 4 assays (assays 1,3,6 and 7). higher reactivity in 2 (assays 4 and 5) and slightly (but not significantly) reduced reactivity in one (assay 2).

4) Immunofluorescence

The transfection efficiency was similar for all clones, as a similar number of cells were positive by pAb immunofluorescence. Samples BA3.2 and T5N had a reduced number of cells fluorescing per field at xlO magnification.

5) Expression and antigenici y

The number of positive cells seen by immunfluorescence gives a rough guide to expression. Other work we have performed (not presented) using HBsAg cloned with an epitope tag to measure levels of HBsAg after expression revealed relatively equal levels between the variants described here and standard sequences.

6) Monoclonal antibody binding results

The reactivity of sample BMT according to the invention with each of the m-anti-HBs was greater than that of the standard HBV sequence (GlyD). This was not seen in any of the other variants tested (Table 5). Further it reacted with all m-anti-HBs.

C) DISCUSSION

The results shown in Table 3 (sample F) show that the BMT variant according to the present invention (having the sequence mutations set out in Table 5) shows a unique universal I

reactivity to all commercial assays tested, generally at a level at least as good as the standard

wild type virus. Antibodies derived therefrom are expected to show analogous universal reactivity. The amino acid substitutions at positions 30. 98, 101 and 210 are unique and not seen in sequences from 88 HBV published strains [18].

The variants seen in the BMT donor HBsAg sequence also corresponded to amino acids pN359H. pI437L. pS452C. pH470Y , pA474T, pE564D. and pS569A of the HBV polymerase gene. The changes at amino acids 437, 452 and 564 were not seen in the consensus sequence of the polymerase gene previously described [23] and are not those normally associated with resistance to famciclovir or lamivudine [24]. It is possible that they affect the replication competence of the virus.

References

I . Wallace LA, Carman WF. Surface gene variation of HBV : scientific and medical relevance. Viral Hepatitis Reviews. 1997, 3 (1 ); 5-16.

2. Carman WF. The clinical significance of surface antigen variants of hepatitis B virus. J Vir Hep. 1997. 4 (suppl 1 ); 1 1 -20.

3. Carman WF, Zanetti AR. Karayiannis P et al. Vaccine-induced escape mutant of hepatitis B virus. Lancet. 1990, 336; 325-9.

4. Hawkins A, Gilson R, Gilbert N et al. Hepatitis B virus surface mutations associated with infection after liver transplantation. J Hepatol, 1996, 24; 8-14.

5. Carman WF, Van Deursen FJ. Mimms LT et al. The prevalance of surface antigen variants of hepatitis B virus in Papua New Guinea, South Africa and Sardinia. Hepatology, 1999, 26(6); 1658-66.

6. Rose JK, Bounocore L, Whitt MA. A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques, 1991, 10 (4); 520-5.

7. Feigner PL, Gadek TR, Holm M et al. Lipofection : a highly efficient, lipid-mediated DNA transfection procedure. Proc Nat Acad Sci. 1987, 84 (21); 7413-17.

8. Carman WF, Korula J, Wallace, L MacPhee R, Mimms L, Decker R. Fulminant reactivation of hepatitis B due to envelope protein mutant that escaped detection by monoclonal HBsAg ELISA. Lancet, 1995, 345; 1406-1407.

10. Hino K, Newell A, Mphaleie J et al. Second episodes of hepatitis B: reinfection or reactivation? submitted.

I I. Ireland J, Hino K, Lau GKK et al. Failed adoptive immunity transfer: reactivation or reinfection? J Viral Hep, 1999, 6. 12. Mangold CMT. Unckell F, Werr M . Streeck RE. Secretion and antigenicity of hepatitis B virus small envelope proteins lacking cysteines in the major antigenic region. Virology, 1995,

21 1 : 535-543.

13. Mangold CMT, Streeck RE. Mutational analysis of the cysteine residues in the hepatitis B virus small envelope protein. J Virol. 1993. 67: 4588-4597.

15. Wallace LA. Echevarria JE. Echevarria JM. Carman WF. Molecular characterisation of envelope antigenic variants of hepatitis B virus from Spain. J Inf Dis, 1994, 170; 1300-1303.

16. van Deursen FJ. Hino K. Wyatt D et al. Use of PCR in resolving diagnostic difficulties potentially caused by genetic variation of hepatitis B virus. J Clin Path, 1998, 51 (2); 149-153.

17. Carman WF, Owsianka A. Wallace LA, Dow LA, Mutimer DJ. Antigenic characterisation of pre- and post-liver transplant hepatitis B surface antigen sequences from patients treated with hyperimmuneglobulin. J Hepatol (submitted)

18. Norder H, Hammas B, Lee S-D et al. Genetic relatedness of hepatitis B viral strains of diverse geographical origin and natural variations in the primary structure of the surface gene. J Gen Virol, 1993. 74: 1341-1348.

20. Ono Y, Onda H. Sasada R et al. The complete nucleotide sequence of the cloned hepatitis B virus DNA: subtype adr and adw. Nucleic Acids Research, 1983; 1 1; 1747-1757.

21. Hoofnagle JH. Seeff LB, Bales ZB et al. Type B hepatitis after transfusion with blood containing antibody to hepatitis B core antigen. N Engl J Med, 1978, 298:1379-1383.

22. Wachs ME, Amend WJ, Ascher et al. The risk of transmission of hepatitis B from HBsAg (-). HBcAb (+), HBIgM (-) donors. Transplantation, 1995, 59; 230-234.

23. Poch O, Sauvaget I, Delarue M, Tordo N. Identification of four conserved motifs among the RNA-dependant polymerase encoding elements. Eur Mol Biol Organisat J, 1989, 8; 3867- 3874. 24. Zoulim F, TrepoC. Drug therapy for chronic hepatitis B virus; antiviral efficacy of hepatitis B virus polymerase mutations on the outcome of therapy. J Hepatol, 1998, 29; 151- 168.

References List 2

1. Perillo, R.P. 1993. Interfer n in the management of chronic hepatitis B. Digestive Diseases and Science. 38: 4. 577-593.

2. Hoofnagle, J.H., A.M.D. DiBiscegiie. 1997. The treatment of chronic viral hepatitis. New England Journal of Medicine 336: 347-356.

3. Dienstag, J.L.. R.P. Perrillo, E.R. Schiff, M. Bartholomew, C. Vicary, M. Rubin.

1995. A preliminary trial of lamivudine for chronic hepatitis B infection. New England Journal of Medicine. 333: No.25, 1657-1661.

4. Lai, C.L.. R.N. Chien, N.W.Y Leung, T.T. Chang, R. Guan, D.I. Tai, K.Y. Ng, P.C. Wu, J.C. Dent, J. Barber, S.L. Stephenson, D.F. Gray. 1998. A one-year trial of lamivudine for chronic hepatitis B. New England Journal of Medicine.; 339: No.2, 61-68.

5. Niesters, H.G.M., P. Honkoop, E.B. Haagsma, R.A. deMan, S.W. Schalm. 1998. Identification of more than one mutation in the HBV virus polymerase gene arising during lamivudine treatment. Journal of Infectious Diseases; 111: 1382-1385.

6. Ilan, Y.. A. Nagler, R. Adler. R. TurKaspa, S. Slavin, D. Shouval. 1993. Ablation of persistent hepatitis B by bone marrow transplantation from a hepatitis B-immune donor. Gastroenterology; 104: 1818-1821.

7. Lau, G.K.K., A.S.F. Lok, R.H.S. Liang, C.L. Lai, E.K.W. Chiu, Y.L. Lau and S.K. Lam. 1997. Clearance of hepatitis B surface antigen after bone marrow transplantation: role of adoptive immunity transfer. Hepatology; 25: 1497-1501.

8. Wen, Y.M., X.H. Wu, D-C. Hu, Q-P. Zhang, S-Q. Guo. 1995. Hepatitis B vaccine and anti-HBs complex as approach for vaccine therapy. Lancet; Vol 345, 1575-1576.

9. Mancini, M., M. Hadchouel, H.L. Davis, R.G. Whalen, P. Tiollais, M.L. Michel.

1996. DNA mediated immunisation in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state. PNAS; 93: 12496-12501.

10. Guidotti, L.G., K. Ando, MN. Hobbs, T. Ishikawa, L. Runkel, R.D. Schreiber, FN. Chisari. 1994. Cytotoxic T lymphocytes inhibit hepatitis B virus gene expression by a noncytolytic mechanism in transgenic mice. Proceedings of the National Academy of Sciences USA; 91 : 3764-3768.

1 1. Dienstag. J.L.. C.E. Stevens, A. K. Bhan, W. Szmuness. 1982. Hepatitis B vaccine administered to chronic carriers of hepatitis B surface antigen. Annals of Internal Medicine; 96: 575-579.

12. Pol. S.. I. Couillin. M.L. Michel. F. Driss, B. Nalpas. F. Carnot, P. Berthelot. C. Brechot. 1998. Immunotherapy of chronic hepatitis B by anti-HBV vaccine. Acta Gastroenterol Belg; 61 : 228-233.

13. Zuckerman. J.N., C. Sabin. F.M. Craig, A. Williams, A.J. Zuckerman. 1997. Immune response to a new hepatitis B vaccine in healthcare workers who had not responded to standard vaccine: randomised double blind dose-response study. British Medical Journal; 314:32 '-333.

B. l056Sp Unable to subtype PI20S/ri25M/PI27T/SI43L 1,3.4 Spain (15) Subtype study. IVDU *

C. M5 Adw YI0ϋS/III8S/ π3I/ri34N/PI42SAπ 1.3.4 Arabia (16) Discrepant serology. Renal transplan

43L/GI45K patient.

D. I5N Λ>w I22NI I23/CM45R 2.4 Indonesian (8) Discrepant serology, vaccinee

K 91-46% Λd SM3I/1I43S 1.4 South Africa ( 10) Diagnostic failure r. BMI Λtlr L98V/Q10IR 1 Japan (11) Diagnostic failure, bone marrow don

G. BΛ32 Λ\U II23N/CI24R 2 Pakistan (17) Liver transplant -HBIG treated f

II BΛ24 Λ>w PI20T 1 Pakistan (17) Liver transplant -HBIG treated

1 BΛ34 Λyw 1I23N 2 Pakistan (17) Liver transplant -HBIG treated

I

CD J ΛP3.1 Λdw DI44Λ 4 UK(I7) Liver transplant -HBIG treated SΛ4 Λdw MI 3T 3 South Africa Acute hepatitis B

L. SΛ6 Λdw QI29R/G130N 3 South Africa Chronic Liver Disease

M SΛ7 Λyw MI33I 3 South Africa Chronic Liver Disease

* intravenous drug user t hepatitis B immunegiobulin

o o

- Immunofluo o

Gly D* 3 50 33 40 13 22 18 78 7 01 8 70 4 15 50-60

A Λrgl45 3 40 15 60 0 40 low 8 42 4 83 0 27 low 0 38 Ncg 50-60

B l (⁾56Sp 3 31 I I 88 1 36 0 52 Neg 1 16 6 17 0 35 Ncg 40-50

C M5 1 57 3 34 0 024 Neg 0 71 Neg 1 40 0 23 Ncg 0 30 Neg 40-50

1) I N 0 35 Ncg 0 16 Ncg 0 026 Neg 0 48 Neg 0 19 Neg 0 17 Ncg 0 27 Neg 5- 10

1 91-4696 3 66 29 88 12 97 14 50 5 76 8 35 5 I I 40-50

1 BM I 3 64 22 40 12 94 32 97 I I 52 8 51 5 01 40-50

G BΛ3 2 0 42 Neg 0 17 Neg 0 022 Ncg 037 Neg 0 196 Ntg 0 18 Neg 029 Neg 20-30

11 BΛ2 4 3 24 7 07 0 69 low 037 Neg 0 76 low 6 66 0 33 Neg 40-50

1 σs 1 BΛ3 4 2 20 4 08 0 029 Neg 5 67 0 27 Neg 2 20 2 47 40-50

J ΛP3 1 3 31 17 10 1 1 60 15 46 5 18 6 93 6 71 40-50

K SΛ4 1 89 2 91 3 46 6 06 4 58 2 31 1 38 40-50

L SΛ6 2 70 1 43 8 21 9 52 5 02 4 45 3 77 20-30

M SΛ7 2 79 5 20 9 59 4 27 7 59 8 04 5 59 40-50

PRK5 (plasmid) t 0 52 Ncg 0 1 1 Neg 0 024 Neg 0 32 Neg 0 196 Neg 0 16 Neg 027 Neg Neg

C 0S7 cells t ϋ 39 Ncg 0 16 Neg 0 063 Neg 046 Neg 0 20 Ncg 0 18 Neg 0 28 Neg Neg

W orkingStandard % 1 0 1 0 1 0 1 0 1 0 1 0 1 0 Not tested

Monitor Sample | 068 0 31 0 30 0 73 Neg 0 40 0 39 0 53 Not tested

C ut -oil 0 53 0 23 0 14 0 95 0 28 024 046

Capture Antibodv Guinea pig pAb Guinea pig pΛb Mouse mAb Mouse mAb Goat pAb Goat pΛb Sheep pΛb Goat pAb

Detection Antibody Goat pΛb Human pΛb Mouse mAb Mouse mAb Mouse mAb Mouse mAb Mouse mAb Rabbit pAb

Ncg - negative * positix c control t

control φ S/co set at 2 0/ modified from manufacturers instructions

% I lit working standard (0 5 IU/ml) and Ihe monitor sample (0 125 IU/ml) are solutions prepared by the National Institute of Biological Standards

(I ondon UK) and distributed nationalK to diagnostic laboratories as external controls

I- Tablc 3. Percentage reactivity of HBsAg variants in 7 assays compared to the positive control (100%). o O o

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Sample BΪoELΪSA AUSRΪA^~ϊi -Ϊ25~ VIDAS HBsAg Enzymun - IMX HBsAg HBsAg GEI4 Enzygnost HBsAg - H υ HBsAg colour Test HBsAg (V2) Monoclonal II φ

0. ES300 Gly Y* 100 100 100 100 100 100 100

Gly D* 100 100 100 100 100 100 too

A Λrgl45 97.1 46.7 3.0 44.8 68.9 3.2 (9.2 Neg) B l056Sp. 92.2 54.2 10.6 (5.8 Neg) 24.3 69.9 (7.4 Neg) C M5 44.9 10.0 (0.18 Neg) (3.8 Neg) 20.0 (2.6 Neg) (7.2 Neg) D 15N (9.7 Neg) (0.73 Neg) (0.20 Neg) (5.3 Neg) (4.0 Neg) (2.0 Neg) (5.7 Neg) E 91-4696 104.6 89.5 96.1 77.2 82.2 96.3 123.1

F BMT 104.0 67.1 97.9 175.6 164.3 97.8 120.7

G BΛ3.2 (1 1.7 Neg) (0.77 Neg) (0.17 Neg) (4.1 Neg) (4.1 Neg) (2.0 Neg) (6.13 Neg) II BΛ2.4 90.3 32.2 5.35 (4.1 Neg) 15.9 75.4 (6.98 Neg) 1 BΛ3.4 61.3 18.6 (0.23 Neg) 62.9 (5.7 Neg) 24.9 52.2

1 J A 3.1 94.6 51.2 87.7 82.3 73.9 79.7 161.7 σ K SΛ4 54.0 8.71 26.2 32.3 65.3 26.6 33.3

1 L SA6 77.1 4.28 62.1 50.7 71.6 51.1 90.8

M SA7 77.7 23.7 74.4 47.3 158.8 91.0 118.2

PRK5 (plasmid) t ( 14.5 Neg) (0.51 Neg) (0.19 Neg) (3.6 Neg) (4.1 Neg) (1 8 Neg) (5.8 Neg) COS7 cells t ( 10.9 Neg) (0.73 Neg) (0.49 Neg) (5.1 Neg) (4.2 Neg) (2-0 Neg) (6.0 Neg) WorkingStandard t 27.9 4.6 7.8 1 1.1 21.0 1 1.5 21.1

Monitor Sam iple t 18.9 1.4 2.3 (8.1 Neg) 8.4 4.5 11.2 Cut -off 14.8 1.0 I. I 10.5 5.9 2.8 9.8

Capture A .ntibody Guinea pig pAb Guinea pig pΛb Mouse mAb Mouse mAb Goat pAb Goat pAb Sheep pAb Detection An tibody Goat pAb Human pAb Mouse mAb Mouse mAb Mouse mAb Mouse mAb Mouse mAb

Neg - negative, * positive control, t negative control σs ψ S/co set at 2.0/modified from manufacturers instructions

© The working standard (0.5 IU/ml) and the monitor sample (0.125 IU/ml) are solutions prepared by the National o Institute of Biological Standards (London, UK) and distributed nationally to diagnostic laboratories as external controls.

Table 4 - Expressed surface antigen results of repeated transfection experiments using the IMX HBsAg assay

Sample Mean IMX HBsAg (sin)* ± s.et (N) J Range of reactivity

1. GlyD 46.4 ± 3.64(9) 29.10-67.93

2. GlyY 41.5 ± 4.09(7) 27.31-59.01

3. Argl45 34.28 ± 5.67 (4) 23.36-55.70

4. 1056Sp. 13.14 ± 1.98(3) 8.41-20.61

5. M5 11.68 ±2.99 (4) 4.76-27.41

6. T5N 1.12 ±0.06 (6) 0.90-1.44

7. 91-4696 34.65 ± 2.89 (4) 20.29-46.10

8. BMT 90.10 ±6.85 (6) 51.09-120.88

9. BA3.2 1.15 ±0.14 (2) 0.98-1.42

10. BA2.4 5.23 ± 0.67 (3) 4.58-7.63 11.BA3.4 1.36 ±0.30 (2) 1.06-1.95 12.AP3.1 30.46 ± 7.32 (3) 15.81-37.98

13. SA4 36.61 ± 3.41 (2) 33.20-40.02

14. SA6 35.92 ±3.89 (3) 28.96-42.40

15. SA7 55.99 ±0.91 (2) 55.08-56.90

16. pRK5 1.27±0.10(8) 1.16-1.47

17. COS7 cells 1.34 ±0.06 (8) 1.06-1.54

* signal /noise t standard error number of transfection experiments r- o

Gly Y Standard HBV sequence (ay subtype) '/. 14.5 18.5 17.5 14.2 16.7 13.3

PRK5 Plasmid (negative) control undiluted 0.6 0.4 0.4 0.3 0.4 0.6

1056 Sp PI20S/TI25M/l^,l27T/SI43I.(ay\v) 'Λ 1.2 2.6 2.1 0.9 0.7 4.0

Λrg 1.2 GI45R(adw) 'Λ 1.5 10.0 9.0 5.6 1.0 0.5

M5 Y100S/TII8V/KI33l/FI34N/PI42S/T143L/ϋl45K.(adw) '/» 0.3 2.0 2.3 1.2 0.3 0.4

T5N l22NTI23(insertion)/GI45R (ayw) 'Λ 0.3 0.4 0.4 0.3 0.4 0.4

BA 2.4 PI20T(ayw) '/. 2.6 2.6 2.1 1.3 0.7 4.0

ΛP3.I DI44Λ(adw) ^■/. 4.5 7.7 6.5 6.5 6.3 0.7

BA 3.2 TI23N/C124R(ayu) '/. 0.3 0.4 0.4 0.3 0.4 0.3

I f

( BA3.4 TI23N(ayw) '/. 0.6 3.7 3.0 2.8 3.1 0.6 I

SΛ6 QI29R/G130N/Λ166V(adw) '/. 4.6 6.3 5.6 5.0 7.6 3.9

SΛ7 Ml 331 3.7 4.8 5.1 2.7 5.2 4.2

91-4696 Sll3TΛII43S(adw) '/. 11.0 6.8 6.3 6.4 6.2 18.9

BMT (invention) N S/Q30R/S53L/L98 V/Q 101 R/S21 OT (adr) V. 19.2 21.6 22.9 17.9 21.4 34.9

Jl CI2IR(adr) undiluted 6.3 6.7 7.2 4.7 2.5 27.5

J2 FI34L(adr) '/, 2.9 2.2 2.0 2.1 2.8 4.2

Detection with biotinylated polyclonal antibody. PAb - Result expressed as signal/2CO. Values >1 = positive

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Claims

1. A mutant hepatitis B surface antigen (HBsAg), which comprises wild type HBsAg having the following amino acid changes :

Position Amino Acid (Wild Type)

3 S (N)

30 R (Q)

53 L (S)

98 V (L)

101 R (Q)

210 T (S) or the combination of any four or more of said changes.

2. An antigen according to claim 1 having five or six of said amino acid changes.

3. An antigen according to claim 1 having all six of said amino acid changes.

4. An antigen according to claim 1 having the combination of said four amino acid changes at positions 30, 98,101 and 210.

5. An antigen having the amino acid sequence of Figure 1 (sequence ID 1).

6. A monoclonal antibody against the antigen of any of claims 1 to 5.

7. A polyclonal antibody against the antigen of any of claims 1 to 5.

8. An antibody according to claims 6 or 7 which carries a detectable label.

9. An assay device for screening for the presence of hepatitis B infection, which comprises an antibody according to any of claims 1 to 6 attached to a solid substrate, or present in a liquid phase.

10. A polynucleotide coding for the antigen of any of claims 1 to 5.

1 1. A polynucleotide having the sequence of Figure 3 (sequence ID 2).

12. A vaccine for prophylactic or therapeutic treatment of hepatitis B infection which comprises the antigen of any of claims 1 to 5, or the polynucleotide of claim 10 or 11