EP1144646A2 - Hepatitis b mutiertes oberflächeantigen und nachweis - Google Patents

Hepatitis b mutiertes oberflächeantigen und nachweis

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Publication number
EP1144646A2
EP1144646A2 EP00901186A EP00901186A EP1144646A2 EP 1144646 A2 EP1144646 A2 EP 1144646A2 EP 00901186 A EP00901186 A EP 00901186A EP 00901186 A EP00901186 A EP 00901186A EP 1144646 A2 EP1144646 A2 EP 1144646A2
Authority
EP
European Patent Office
Prior art keywords
neg
hepatitis
hbsag
antigen
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00901186A
Other languages
English (en)
French (fr)
Inventor
Frederick William Carman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Glasgow
Original Assignee
University of Glasgow
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Glasgow filed Critical University of Glasgow
Publication of EP1144646A2 publication Critical patent/EP1144646A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • 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.
  • HBsAg surface antigen
  • 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
  • MHR major hydrophillic region
  • 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).
  • 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 ⁇ 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.
  • HBV-specific vaccines led to complete down regulation of mRNA synthesis from the transgene by a post-transcriptional mechanism 9 .
  • 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)
  • 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.
  • 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 invention also relates to an immunoassay device wherein an antigen or antibody according to the present invention is immobilised on a solid substrate.
  • 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.
  • 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 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.
  • 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.
  • HBsAg negative but anti-HBs and anti-HBc positive 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
  • wild type sequences GlyY and GlyD themselves differ in amino acids at least at positions 122 and 160.
  • mutations are indicated as, for example, N3S; which indicates that normal amino acid N at position 3 is replaced by S in the mutant.
  • Hepatitis B serological markers HBsAg (Auszyme; monoclonal based), anti-HBs, HBeAg, anti-HBe, anti-HCV (EIA II), a ⁇ ti-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).
  • 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 ⁇ l of serum using the QIAamp Blood Kit (QIAGEN, Crawley, UK) according to the manufacturer's instructions.
  • QIAamp Blood Kit QIAGEN, Crawley, UK
  • 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 ⁇ l solution containing 1U Taq polymerase
  • 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 DH5 ⁇ .
  • 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, l ⁇ g plasmid was diluted in 50 ⁇ l Optimem 1 reduced serum medium (Life Technologies. Paisley, UK) and. in a separate vial. 7 ⁇ l of liposomes was added to 50 ⁇ l of Optimem 1.
  • the two solutions were mixed and allowed to stand for 15 min at room temperature, then further diluted with 500 ⁇ l 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 500 ⁇ l of COS7 medium added (Dulbeccos MEM Medium with 5% foetal bovine serum, 0.02 IU/ml Penicillin, 0.02 ⁇ l/ml Streptomycin and 4 ⁇ M 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.
  • COS7 medium Dulbeccos MEM Medium with 5% foetal bovine serum, 0.02 IU/ml Penicillin, 0.02 ⁇ l/ml Streptomycin and 4 ⁇ M L-Glutamine. All Life Technologies, Paisley, UK.
  • 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.
  • 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.
  • 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.
  • PBS phosphate-buffered saline
  • pAb goat anti-HBsAg polyclonal antibody
  • FITC anti-goat fluorescein isothiocyanate
  • IgG immunoglobulin
  • 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
  • the variants, origin and clinical background of each sample is detailed in Table 1.
  • 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.
  • 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].
  • 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
  • Figure 3 is the BMT nucleic acid sequence.
  • 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.
  • 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.
  • mAb for detection
  • assays 5 and 6 picked up 10.
  • the two assays which employed mouse mAb for both capture and detection 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.
  • 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).
  • sample BMT according to the invention 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.
  • 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.
  • 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
  • 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
  • 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.
  • COS7 cells 1.34 ⁇ 0.06 (8) 1.06-1.54

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
EP00901186A 1999-01-18 2000-01-18 Hepatitis b mutiertes oberflächeantigen und nachweis Withdrawn EP1144646A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9901046 1999-01-18
GBGB9901046.4A GB9901046D0 (en) 1999-01-18 1999-01-18 Hepatitis b testing
PCT/GB2000/000107 WO2000042194A2 (en) 1999-01-18 2000-01-18 Mutant hepatitis b surface antigen and testing

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Publication Number Publication Date
EP1144646A2 true EP1144646A2 (de) 2001-10-17

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EP (1) EP1144646A2 (de)
AU (1) AU2115000A (de)
GB (1) GB9901046D0 (de)
WO (1) WO2000042194A2 (de)

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Publication number Priority date Publication date Assignee Title
EP1696040A1 (de) * 2005-02-28 2006-08-30 Eurofins Viralliance, Inc. Verfahren zur Bestimmung des Genotypes und des Phenotypes von Hepatitis B Viren, die Resistenz gegen antivirale Moleküle zeigen

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JPS61158798A (ja) * 1984-12-28 1986-07-18 Japan Found Cancer Res Ｂ型肝炎ウイルス表面抗原の製造法

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See references of WO0042194A3 *

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AU2115000A (en) 2000-08-01
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GB9901046D0 (en) 1999-03-10

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