WO1997035204A1 - Procede de pronostic de l'evolution d'une hepatite b - Google Patents

Procede de pronostic de l'evolution d'une hepatite b Download PDF

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Publication number
WO1997035204A1
WO1997035204A1 PCT/EP1997/001351 EP9701351W WO9735204A1 WO 1997035204 A1 WO1997035204 A1 WO 1997035204A1 EP 9701351 W EP9701351 W EP 9701351W WO 9735204 A1 WO9735204 A1 WO 9735204A1
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hbeag
infection
antibodies
hbe
sample
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PCT/EP1997/001351
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English (en)
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Wilhelmina Petronella Paulij
Markus Hendrikus Van Roosmalen
Rudolf Arnold Heijtink
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Akzo Nobel N.V.
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Priority to KR1019980707348A priority Critical patent/KR20000064637A/ko
Priority to AU20282/97A priority patent/AU2028297A/en
Priority to JP9533145A priority patent/JP2000506735A/ja
Priority to EP97908256A priority patent/EP0888551A1/fr
Publication of WO1997035204A1 publication Critical patent/WO1997035204A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/082—Hepadnaviridae, e.g. hepatitis B virus

Definitions

  • the present invention is concerned with a method for predicting and monitoring the course of an infection with the Hepatitis B virus (HBV). Furthermore the invention is concerned with antibodies and amino acid sequences that can be used in such methods.
  • HBV Hepatitis B virus
  • Hepatitis B virus causes acute and chronic infection in man.
  • the infection can be subclinical, lead to minimal or severe acute hepatitis and can progress to chronic infection eventually leading to liver cirrhosis and primary liver carcinoma.
  • Hepatitis B virus is a small DNA virus which consists of a lipid envelope and an inner core containing the viral genome and a DNA polymerase.
  • the HBV core gene (nucleotides 1814-2449) is divided in a pre-core and a core region composed of 29 and 183 codons respectively.
  • HBeAg nucleocapsid particles
  • pre-core protein which is directed to the endoplasmic reticulum, subsequently N- and C- terminally processed and secreted as e-antigen (HBeAg).
  • HBeAg was firstly identified in 1972. It is present in a nonparticulate state in the serum of HBV infected patients. The presence of HBeAg is generally considered to be an indication of active viral replication in the infected host.
  • HBeAg may be a complex of antigens and up to three precipitin lines, designated e1 , e2 and e3, have been detected by agar gel diffusion in sera from individual patients. Both IgG-bound HBeAg having a molecular size of approximately 300 kDa and a smaller HBeAg component with a molecular weight of approximately 16 kDa have been detected in the sera of HBV-infected patients.
  • HBcAg is related to HBeAg on basis of the fact that HBcAg can be converted into HBeAg upon denaturation and/or limited proteolysis. Immunological distinction between HBcAg and HBeAg has become possible with the introduction of monoclonal antibodies specific for either antigen. HBcAg has been proposed to have one major discontinuous epitope but also the presence of linear HBcAg determinants and of an internal determinant have been suggested.
  • HBe/alpha, or HBe1 is a linear determinant and resides in the amino acid sequence (76) L-E-D-P-A-S-R-D-L-V-V-S-Y (89).
  • the HBe1 determinant overlaps with the conformational HBc determinant and appears to be also exposed in an HBe conformation on the core particle surface (Salfeld et al., J.Virol., 63, 798-808, 1989).
  • HBe/beta or HBe2 is a discontinuous determinant and requires for its correct conformation not only amino acid sequences ending around amino acid 138 but also the indirect or direct intramolecular participation of aminoterminal amino acid sequences.
  • the HBe2 determinant is either absent or inaccessible on the core particle surface (Salfeld et al., 1989).
  • Sallberg et al. murine monoclonal antibodies and human sera may recognize the HBe2 epitope as a linear determinant residing around aa 130.
  • Two anti-HBe2 monoclonal antibodies were found to recognize the sequence T-P-P-A-Y-R at residues 128-133.
  • the PP was shown to be essential for the recognition by both monoclonal antibodies.
  • Monoclonal antibodies that recognized a peptide containing the linear HBe2 epitope as a single peak were those with the most efficient inhibiting activities in the anti-HBe RIA.
  • patient sera reactivity to the HBe2 epitope containing peptide was mainly found in patient sera that gave strong inhibition in an anti- HBe RIA (95%).
  • HBeAg The function of HBeAg is not clear but it may protect the virus infected hepatocytes by blocking cytotoxic T cells during infection.
  • Milich et al. Proc.Natl.Acad.Sci., USA, 87, 6599-6603, 1990
  • HBeAg may induce immunologic tolerance in utero.
  • Expression of HBeAg may represent a viral strategy to guarantee persistence after perinatal infection. When patients come nearer to seroconversion to anti-HBe they are more likely to recover from the infection and to respond to antiviral therapy. Little is known about the mechanisms and factors which determine the course of infection. Therapy of chronic HBV infected patients usually involves treatment with interferon.
  • the present invention is concerned with prognosis of the development of hepatitis B infection.
  • the method according to the invention may also be used to monitor antiviral therapy with, for example, antiviral compounds like alpha- interferon.
  • HBV X antigen has been reported to be preferentially produced in chronic HBV infections (Moriarty et al., Science, 227, 429-433, 1985). It has also been described that higher levels of IgM anti-HBe are generally produced during the acute phase as compared with chronic infection and this quantitative difference has become the only serological means of differentiating an acute HBV infection of an acute elevation of a chronic infection (Gerlich et al., J.CIin. Microbiol. ,2 , 228-293, 1986).
  • Maruyama et al. J. Immunol. Meth., 155,65-75, 1992
  • This assay consists of a solid phase coated with antibodies raised to an HBeAg/ayw derived peptide comprising amino acids 73-87 (exclusive of the precore sequence) designated as HBe73-87:GVNLEDPASRDLWSC.
  • HBe73-87:GVNLEDPASRDLWSC an HBeAg/ayw derived peptide comprising amino acids 73-87 (exclusive of the precore sequence) designated as HBe73-87:GVNLEDPASRDLWSC.
  • the anti-peptide antibodies do not compete with antibodies raised against native HBeAg.
  • the present invention is based on the finding that the presence of specific biomolecules able to form complexes with HBeAg is a prognostic marker for the further development of an acute Hepatitis B infection. It has been found that based on the presence or absence of said specific biomolecules or complexes of said biomolecules and HBeAg during the acute phase of infection it can be predicted whether patients will recover and clear the virus after acute infection or will develop a chronic infection.
  • the present invention therefore involves the use of an assay for the detection of circulating complexes comprising specific biomolecules bound to HBeAg in a method for predicting the course of infection in patients with an acute HBV infection.
  • the biomolecules which are detected with the method according to the present invention are presumably antibodies of the IgG type.
  • Said biomolecules are capable of masking an epitope on the HBeAg that is located in the region comprising amino acids 85-109 of the complete HBeAg amino acid sequence (When the numbering started at the beginning of the HBeAg sequence). With masking is meant that, when the biomolecules have formed a complex with HBeAg, certain antibodies that normally recognize the epitope are prevented from binding to the HBeAg when the HBeAg is present as a complex with the biomolecules.
  • the method according to the invention can be performed in several ways. The presence or absence of the biomolecules can be determined in several ways.
  • An assay can be based on the capability of the biomolecules to mask an epitope on the HBeAg for binding of certain antibodies specific for said epitope. Said antibodies will only be capable of binding to the HBeAg in case the biomolecules are absent. In case biomolecules are bound to the HBeAg the antibodies will not be able to bind to their epitope. Thus, by detecting whether any HBeAg will bind to said antibodies, the presence or absence of the biomolecules can be determined.
  • the antibodies used are preferably monoclonal antibodies.
  • the antibodies used with the method of the invention are antibodies preferably having a low affinity towards the HBeAg present in the sample compared to the affinity of the biomolecules optionally complexed to the HBeAg. As a result of their lower affinity towards the HBeAg, the antibodies only bind to HBeAg in the absence of biomolecules capable of masking their epitope.
  • the antibodies can be used, for example, coated on a solid support. Immune complexes formed on the solid phase may, for example, be detected using another anti-HBe antibody recognizing HBeAg in all forms or anti-human antibodies. This second antibody might be conjugated to any label.
  • a negative reaction in such an assay will indicate that HBeAg is present as a complex in the sample and thus not capable of binding to the antibodies on the solid phase.
  • HBeAg is present as a complex in the sample and thus not capable of binding to the antibodies on the solid phase.
  • an assay detecting HBeAg positivity for example, the Abbott HBeAg EIA, Organon Teknika Hepanostika HBeAg/anti-HBe or the HBe MAB assay (see example 2) or using alternative techniques such as SDS-page or immunoblotting.
  • a positive reaction in the assay will indicate that the sample is HBeAg positive and that the HBeAg epitope can be approached by antibodies.
  • HBeAg is present in non-complexed form or as a complex in which the epitope recognized by the antibody is not masked.
  • Preferred antibodies used with the method according to the invention to detect the presence or absence of biomolecules bound to HBeAg are monoclonal antibodies having the same reactivity towards HBeAg as monoclonal antibodies produced by the cell line deposited at the ECACC under no. 95090611.
  • Monoclonal antibodies produced by the cell line deposited at the ECACC under no. 9509611 recognize an epitope on HBeAg located around the sequence RDLWNYVNTN. This epitope overlaps with the sequence defined as HBe1 by Salfeld et al. (J.Virol., 63(2), 798-808, 1989).
  • the sequence of the recombinant HBeAg to which HB.OT95A antibodies were raised has a substitution of an Asparagine (N) residue for the Serine (S) residue at position 87 in the HBe1 sequence as described by Salfeld et al. (The numbering adhered to by Salfeld et al.
  • HBeAg including a Serine (S) residue at position 97 will be referred to as "S-type" HBeAg and HBeAg including an Asparagine (N) residue at position 97 will be referred to as HBeAg of the "N-type".
  • step (c) determining from the ratio between signals obtained in step (a) and (b) whether the Hepatitis B e antigen present in the sample is of the N- or the S- type.
  • Serum samples containing HBeAg or the "S-type” will produce a lower signal in step (a) than serum samples containing HBeAg of the "N-type".
  • the ratio between signals obtained in step (a) and (b) will be higher in case of a sample containing "N-type" HBeAg.
  • the exact values of the ratio's characteristic for S- and N-type HBeAg will be dependent on the format of the assay systems used.
  • Biomolecules can be determined by contacting sera with a solid support coated with a peptide, a recombinant protein or any other biomaterial mimicking at least the minimal sequence to which the biomolecules bind of the epitope.
  • the biomolecules may be derived from dissociated HBeAg complexes, from anti-HBe positive sera or any other serum derived from HBV infected patients after clearance of the virus.
  • HBeAg positive sera in which no biomolecules are present or alternative forms of HBeAg such as recombinant HBeAg can be used as positive sample.
  • This sample can be preincubated with samples unknown of including epitope masking biomolecules. Inhibition of the signal in HBeAg assays based on antibodies like, for example, HB.OT95A indicate the appearance of masking biomolecules in the sample.
  • Biomolecules forming complexes with HBeAg which can be related to the clinical outcome of the HBV infection can be purified for example by affinity chromatography. Subsequently, these molecules can be used as reagent in HBeAg assays. The molecules will only react with non-complexed HBeAg and can thus also be used to detect the presence or absence of complexes in the sample of a patient with an acute HBV infection.
  • Solid supports which can be used with the method according to the invention are known in the art, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a membrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a dye sol, a metal sol or metal compound as sol particle, a carrier protein such as BSA or KLH.
  • Labeling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal compound as sol particle.
  • Monoclonal antibodies that can be used with the method according to the invention are also part of the present invention.
  • Monoclonal antibodies according to the invention are monoclonal antibodies having the same reactivity towards HBeAg as monoclonal antibodies derived from the cell line deposited at the ECACC under No. 95090611. Immortalized cell lines capable of excreting monoclonal antibodies according to the invention are also part of the present invention.
  • the preparation of cell lines producing monoclonal antibodies may occur by, for example, by the Kohler and Milstein technique (Kohler and Milstein devised the techniques that resulted in the formation of monoclonal antibody-producing hybridomas (G. Kohler and C. Milstein, 1975, Nature 256:495-497; 1976, Eur. J. Immunol.
  • transformation with Epstein-Barr Virus or a direct transformation technique of B-lymphocytes with oncogenic DNA, or a direct fusion of human B-lymphocytes with a fusion partner being either a human or a mouse-human hybrid myeloma cell line, or a direct fusion of an EBV-transformed B cell line with said myeloma cell lines.
  • a preferred cell line according to the invention is the cell line deposited at the European Collection of Animal Cell Cultures, Porton Down (UK) under deposit No. 95090611.
  • This hybridoma cell line was produced by the fusion of a mouse myeloma cells with a splenic lymphocyte derived from a mouse previously inoculated with recombinant HBeAg from E.coli.
  • HBe MAB assay Reactivity of sera mentioned in Figure 1 A. All samples were tested HBeAg positive. Sera obtained from patients who became chronically infected could not be discriminated from sera obtained from patients who recovered between 6 (recovery) or 9 (late recovery) months after infection.
  • PS Positive control
  • Serum 343b was obtained from a HBV infected donor four years after infection.
  • Serum 162a was obtained during the acute phase of infection from a donor who cleared the virus within 6 months.
  • Serum 162b is obtained from the same donor as serum 162a after viral clearance ten months after primary infection.
  • FIG. 3 Determination of HBe-S and HBe-N types by using the HBe MAB assay and HBe selection assay.
  • the total amount of arbitrary units (one arbitrary unit reciprocal value of dilution) associated with an absorbance of 1000 was determined for each serum (AU1000) in both assays.
  • AU 1000 determined in the HBe MAB assay is divided by AU 1000 resolved the HBe selection assay (y-ax). Ratios below 0.03 indicate the HBe-S type. Ratios above 0.13 indicate the HBe- N type. S and N types were checked by determination of the complete HBeAg DNA sequence.
  • This Example illustrates how antibodies according to the invention were produced and the corresponding epitope was mapped.
  • Murine monoclonal antibodies were produced by injecting Balb/c mice with E.coli derived recombinant HBeAg or HBcAg in Freund's complete adjuvans. After 2 months mice were boosted with the antigen mixed in Freund's incomplete adjuvans, which was repeated after two weeks. The best responding mouse received an intravenous dose of the recombinant antigen dissolved in PBS. Three days later the spleen was removed and splenic lymphocytes were fused with P3x63Ag8.6.5.3 mouse myeloma cells using polyethylene glycol 1000 according to standard methods. Hybridoma cells were examined for the production of HBeAg or HBcAg specific antibodies using human sera and/or recombinant antigens. Reactive clones were recloned to 100% clonality by the technique of single-cell cloning.
  • Peripheral blood was taken from a human chronic HBsAg carrier.
  • 10.9 ml dextran (MW 200.000) 0.37 ml NaCl solution (0.27 g.ml" 1 ) and 1 ml heparin solution (510 IU ml -1 ) were added.
  • the mixture was incubated for 1 h at room temperature to allow erythrocytes to settle.
  • the upper Iayer containing the leukocytes was isolated and spun down for 10 min at 2000 N kg -1 .
  • the cell pellet was resuspended in complete culture medium (Dulbecco's modified Eagles medium (DMEM) 10% foetal calf serum) supplemented with 10% dimethylsulphoxide (DMSO), frozen and stored in liquid nitrogen.
  • DMEM Dulbecco's modified Eagles medium
  • DMSO dimethylsulphoxide
  • EBV Epstein-Barr virus
  • 1x10 8 viable lymphocytes were isolated, diluted to 1x10 4 cells mM in culture medium and distributed over ten 96-well cluster dishes containing human fibroblasts as feeder cells. Cultures then were incubated for 4-6 weeks: medium was refreshed twice a week. From wells containing microscopically visible transformed lymphocytic clones, supernatant was harvested to be tested for the presence of specific antibodies to native HBeAg. Positive results were confirmed after 7 days. The cultures were continued and frozen in liquid nitrogen.
  • the solid-phase synthesis of the peptides was carried out in a semi-automated manner.
  • the Fmoc amino acid derivatives were obtained from Bachem (Bubendorf, Switzerland).
  • the peptides were synthesized on a TentaGel S RAM Fmoc resin (RAPP Polymere, Tubingen, Germany) via the Fmoc tBu chemistry.
  • the linker is of a Rink-amide type, which automatically yields a C-terminally amidated peptide.
  • the amino acid side- chains were protected with acid-labile protecting groups: the Epsilon- aminogroup of lysine with Boc, the delta-guanidino group of arginine with 2,2,5,7, 8-pentamethylchroman-6-sul ⁇ honyl (Pmc), the gamma-carboxyl group of glutamic acid and the ⁇ -carboxyl group of aspartic acid with OtBu, the gamma- amide group of glutamine and the ⁇ -amide group of asparagine with trityl (Trt), histidine and cysteine with Trt, the ⁇ -hydroxyl group of serine and threonine with tBu, and tyrosine with tBu.
  • acid-labile protecting groups the Epsilon- aminogroup of lysine with Boc, the delta-guanidino group of arginine with 2,2,5,7, 8-pentamethylchroman-6-sul ⁇ honyl (Pmc), the
  • the fully protected peptides were cleaved from the resin during a 2-hr reaction at room temperature under nitrogen with 5% thioanisole (vol/vol), 3% ethanedithiol (vol/vol), 2.5% water (vol/vol), and 2% anisole (vol/vol) in trifluoroacetic acid (87.5% vol/vol) followed by precipitation in diethylether.
  • the crude peptides were washed twice with diethylether, dried at the air, dissolved in water/acetonitrile (3:1 ) and lyophilized.
  • HPLC analysis and purification were carried out on a Beckman Gold HPLC system.
  • HPLC analyses were performed on a RP-C2/C18 column (Superpack prepS, 4x250 mm, Pharmacia) at a flow rate of 1 ml/min, using a 3 min isocratic elution with 0.1 % followed by a 30 min linear gradient from in water (100%) to 75 % 0.1 % trifluoroacetic acid in acetonitrile.
  • Peptides were detected by UV measurement at 206 nm.
  • OTP-145 H-NLEDPASRDLW N YVNTNMGLKIRG-NH2
  • OTP-146 H-NLEDPASRDLW S YVNTNMGLKIRG-NH2
  • the synthesis of the peptides was carried out on a Perkin Elmer/Applied Biosystems Inc. 433A peptide synthesizer, using standard FastMoc 0.25 mmol procedures with UV-monitoring and feedback option.
  • the Fmoc amino acid derivatives were obtained from Bachem (Bubendorf, Switzerland).
  • the peptides were synthesized on a TentaGel S RAM Fmoc resin (RAPP Polymere, Tubingen, Germany) via the Fmoc/tBu chemistry.
  • the linker is of a Rink-amide type, which automatically yields a C-terminally amidated peptide.
  • the amino acid side-chains were protected with acid labile protecting groups: the epsilon-aminogroup of lysine with Boc, the delta-guanidino group of arginine with 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc), the gamma- carboxyl group of glutamic acid and the ⁇ -carboxyl group of aspartic acid with OtBu, the gamma-amide group of glutamine and the ⁇ -amide group of asparagine with trityl (Trt), histidine and cysteine with Trt, the ⁇ -hydroxyl group of serine and theonine with tBu, and tyrosine with tBu.
  • acid labile protecting groups the epsilon-aminogroup of lysine with Boc, the delta-guanidino group of
  • Peptides were dissolved in DMSO (minimal concentration 0.5 mg/ml) and diluted in 0.05 M Na 2 CO3 (pH 9.6) to a final concentration of 5 ⁇ gr/ml. Diluted peptides were coated ovemight at room temperature in microtiterplates (96-wells). Each well was post-coated during 30 minutes using 0.05 M TRIS/HCI including 0.2% casein.
  • MAB HB.OT95A was diluted in 20% normal goat serum and 1% Triton in PBS pH 7.4. Dilution series between 1 :500 to 1:16.000 were tested by adding 100 ⁇ l of each diluted sample per well.
  • Example 2 This example illustrates how, based on antibodies produced as described in Example 1 an immunoassay was constructed which will be referred to as the " HBe selection assay". A second assay was constructed to ensure HBeAg positivity of a sample (referred to as HBe MAB assay). HBe selection assay.
  • the assay is based on two monoclonal antibodies.
  • MAB HBe.OTHu03-HRP horse-radish peroxidase
  • HRP is conjugated to IgG according to the SPDP conjugation procedure (Pharmacia) Briefly, HBe.OTHu03 was desalted using PD-10 chromatography (Pharmacia).
  • HRP-SH was prepared by adding HRP-PDP, 1 M HAC pH 4.3 and 1.6 M DTT in a ratio of 25:2.5: 1. After incubation for 15 minutes the mix was desalted as mentioned above.
  • IgG-PDP and HRP-SH were coupled by mixing both components in a ratio of 13.4:1 mol/mol and incubating during 30 minutes at AT followed by overnight incubation at 2-8°C. The conjugate was stored at -20°C until needed.
  • Murine MAB HB.OT95A is coated on the solid phase.
  • HB.OT95A is diluted to a final concentration of 40 ⁇ gr/ml in coatbuffer (0.05M NaHCO3 pH 9.6).
  • coatbuffer 0.05M NaHCO3 pH 9.6
  • the mix is incubated overnight with the solid phase (microtitre plates Greiner 96-wells) at AT (135 ⁇ l/well).
  • the MAB solution was replaced by the same volume buffer (0.2 M Tris/HCI, 0.2 M NaCl, 0.05% Tween 20 pH 7.4) and incubated for 10 minutes.
  • the buffer was then replaced subsequently by above mentioned buffer excluding Tween 20, a 0.2 M TRIS/HCI buffer including 0.2 M NaCl pH 7.4 and a 0.05 M TRIS/HCI buffer pH 7.4.
  • the plates are dried using nitrogen gas.
  • EIA was performed to standard procedures. Briefly, 100 ⁇ l sample is incubated for 1 hour at 37°C followed by four wash-steps using PBS-Tween pH 7.4. After washing 100 ⁇ l conjugate (diluent 20% goatserum and 1% Triton X100 in PBS pH 7.4) is added, incubated and removed as above mentioned. Hereafter 100 ⁇ l TMB ready-for-use substrate (Organon Teknika UniForm II substrate) is added and incubated during 30 minutes at room temperature. The reaction is terminated using 1 M sulfuric acid (H2SO4). Absorbance is measured at 450 nm.
  • This assay is based on two monoclonal antibodies.
  • MAB HBeOT.Hu03-HRP is used as conjugate in a 1 :8000 dilution.
  • Sorin MAB 364C8 is coated on the solid phase (5 ⁇ g/ml).
  • Monoclonal 364C8 was directly obtained from the Sorin Biomedica company (Italy) and was used for practical purposes.
  • the assay was merely constructed, since an assay was needed including the HBeOT.Hu03-HRP conjugate to ensure that specificity of the selection assay was due to the HBOT95A monoclonal antibodies and not due to the monoclonal used in the conjugate.
  • the choose of the Sorin monoclonal is not essential for the performance of the assay for this particular purpose.
  • This monoclonal can be replaced by any other anti-HBeAg monoclonal antibody recognizing HBeAg complexes. EIA is performed according to standard procedures as mentioned above.
  • Example 3 The example describes how using the HBe selection assay and the HBe MAB assay HBeAg positive samples were tested. From the results can be concluded that using the HBe selection assay the clinical outcome of the infection could be predicted in the acute phase of infection. HBeAg positive sera.
  • HBe heterogeneity was resolved by determination of the complete HBe sequence.
  • DNA was isolated according to the Boom protocol (Boom et al., J.CIin.Microbiol., 28, 495-503, 1990). Briefly, to nine volumes of lysis buffer one volume of serum was added (225 ⁇ l and 25 ⁇ l respectively). To this mixture 70 ⁇ l of silica suspension was added. The solution was incubated for 10 minutes at room temperature and regularly vortexed. After incubation the silica was spun down by centrifugation for at least 15 sec. at highest speed. The supernatant was removed. The pellet was dissolved in 1 ml of L2 wash buffer (0.05 M Tris/HCI pH 6.4, 5M GuSCH).
  • the silica was spun down again and the pellet washed once more with 1 ml of L2 wash buffer. After the removal of the supernatant the pellet was washed twice with 70% ethanol and one time with acetone (in the same way as the previous steps). The pellet was dried in a heating block at 56°C for 10 minutes. The nucleic acid was eluted by adding 100 ⁇ i water and incubated at 56°C for 10 minutes (vortex once after 5 minutes). After centrifugation for 2 minutes at 10.000 g +/- 80 ⁇ l nucleic acid solution was transferred into a new eppendorf tube. After another centrifugation step, in order to remove all the silica, +/- 70 ⁇ l nucleic acid solution was transferred to a new tube and stored at -20°C.
  • PCR was performed according to normal PCR protocol (total volume 50 ⁇ l). The input of the DNA was 2 ⁇ l of the isolated fraction. If no product after PCR was obtained another PCR was performed with a higher DNA input.
  • the primers used for the PCR were primers existing of specific HBe sequence elongated with M13 forward or M13 reverse sequence.
  • HBe M13 forward 5'CGA CGT TGT AAA ACG GCC AGT AGG AGG CTG TAG GCA TAA AT 3' HBe M13 reverse 5'CAG GAA ACA GCT ATG ACC TTC TGC GAC GCG GCG ATT GA 3'
  • sequence analysis was carried out according to the Sequitherm long read cycle sequencing kit (Epicentre Technologies, Madison USA). About 300 to 500 ng of purified PCR fragment was used as DNA input. The primers used for the sequence analysis where M13 forward and M13 reverse. The reactions where run on an A.L.F. DNA sequencer (Pharmacia Biotech) according to the standard protocol. Results
  • the discrimination is based on variation in immunoreactivity of HBeAg which is appearant in all sera according to reference assays.
  • the differences in clinical outcome can not be related to variation of the virus because it was shown that the HBV HBeAg had the same sequence in all cases.
  • This example shows how, by using an inhibition assay, it was confirmed that biomolecules able to mask the epitope of HB.OT95A are present in samples tested negative in the Hbe selection assay and positive in another HBeAg assay. Inhibition assay.
  • HBeAg immunoreactivity could therefore not have been caused by variation in HBeAg itself. It was suggested that one or more host derived factors present in the serum must play an important role. This was investigated by an inhibition experiment.
  • Serum from patient no. 343 was obtained during the acute phase of the infection (see Figure 2A). This sample was used as positive control (PS). First the PS was diluted 2/3 times using normal human serum. Subsequently, different sera, 343b, 162a/b and the positive control were diluted 1 :1 with NHS and measured in the l fc>
  • Example 5 This example shows that sera HBeAg positive in the Abbott HBeAg EIA but negative in the selection assay contain complexes of HBeAg and biomolecules and that these biomolecules presumably are anti-HBeAg antibodies of the IgG type.
  • IgG and IgM molecules from HBeAg positive sera were purified using protein A chromatography according to conventional procedures.
  • HBeAg in the elution fraction was determined by using the Organon Teknika Hepanostika HBeAg/anti- HBe assay.
  • IgG and IgM molecules bound to HBeAg were detected by using an adapted EIA assay.
  • the monoclonal 364C8 obtained from Sorin was coated on the solid phase.
  • HBeAg-lgG complexed HBeAg positive fraction was diluted 1/30 or 1/100 times (Dilution medium 20% normal goat serum in PBS (7.2) and 1 % Triton-X100) and 100 ⁇ l of the sample was contacted with the solid phase.
  • Mouse anti-Human IgG or IgM HRP conjugate was used as second antibody.
  • various undiluted sera from different HBV infected donors were tested individually in the EIA without foregoing purification steps.
  • HBeAg complexes can inhibit HBeAg reactivity in the selection assay.
  • HBeAg complexes were purified by affinity chromatography.
  • MAB Sorin 364C8 (5 mg/ml Sepharose) was coupled to CNBr-Sepharose 4B (Pharmacia) according the method described by Pharmacia (In: Affinity Chromatography Principles and Methods: Methods for coupling ligands to CNBr-activated Sepharose 4B).
  • HBeAg was purified by adding 20 ml HBeAg positive serum (assays Organon Teknika Hepanostika HBeAg/anti-HBe (positive) and selection assay (negative)) to 1 ml Sepharose including MAB Sorin 364C8. The mixture was incubated overnight at 4°C under rotation.
  • Fraction 3 was diluted (1 :10) in normal human serum or mixed directly 1 :1 with a human serum HBeAg positive in the HBe selection assay (for example Serum nr 343 see example 3) or normal human serum (positive control). The mixture was incubated ovemight at ambient temperature and tested in the selection assay as described before.
  • Results showed that signals of HBeAg positive sera declined in the selection assay after incubation with fraction 3. This in contrast to the positive control in which normal human serum was used. In total 15.3 % inhibition was obtained if fraction 3 was used without previous dilution steps. Higher inhibition can be expected if fraction 3 is concentrated before incubation with the HBeAg positive serum.
  • fraction 3 contained biomolecules which are able to reduce the HBeAg signal in the selection assay.
  • biomolecules are derived from HBeAg complexes which naturally appear in human sera HBeAg positive in various HBeAg assays but negative in the HBe selection assay. Apparently, the biomolecules remain competent of epitope masking after partial purification.
  • This example illustrates how HBeAg complexes can be dissociated using NaSCN.
  • HBeAg positive sera (HBe MAB assay) were obtained from HBV infected patients. The sera were diluted with 5M NaSCN or PBS pH 7.2. Normal human serum was used as negative control. Sera were mixed and incubated on a shaker for 1 h at ambient temperature or overnight at 4°C. After incubation the sera were diluted using normal human serum (1 :5, 1:10, 1 :20, 1 :30 and 1 :100).
  • HBeAg complexes were purified as described in example 5. Non-purified sera were used as positive control. After purification the elution fractions were diluted with NaSCN or PBS pH 7.2 (1 :1 ) and tested in the HBe selection assay (1 :50).
  • HBeAg complexes could be identified in the elution fraction using the HBe MAB assay. It was also found that these complexes remained negative in the HBe selection assay after dilution with PBS but became positive after treatment with NaSCN. In conclusion, dissociation of HBeAg complexes with NaSCN results in the recognition of HBeAg epitopes in the HBe selection assay.
  • This example illustrates a new method which can be used to distinguish HBe N- type from HBe S-type.
  • HBeAg sequence was determined as described in example 3. Sera were grouped as a HBe S-type or N-type on basis of aminoacid 97 (serine (S) or asparagine (N).

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Abstract

La présente invention concerne le pronostic du développement de l'hépatite B. Le procédé de l'invention convient également au suivi d'une thérapie antivirale à base par exemple de composés antiviraux tels que l'interféron α. L'invention repose sur la découverte que la présence de biomolécules aptes à former des complexes avec le HBeAg constitue un indicateur de pronostic du développement ultérieure de la phase aiguë de l'hépatite B. On a découvert la présence ou l'absence de ces biomolécules spécifiques ou de complexes de ces biomolécules et de HBeAg pendant la phase aiguë de la maladie, permet de prévoir si le patient guérira et se débarrassera du virus après la phase aiguë ou s'il développera une infection chronique. La présente invention fait donc intervenir un dosage permettant de détecter des complexes circulants comprenant les biomolécules spécifiques liées au HBeAg, et ce, en utilisant un procédé permettant de prévoir l'évolution de la maladie chez des patients atteints d'hépatite B en phase aiguë. L'invention concerne également un procédé de pronostic du développement de l'hépatite B qui convient également au suivi d'une thérapie à base d'agents antiviraux et qui permet d'indiquer l'effet produit par une telle thérapie.
PCT/EP1997/001351 1996-03-19 1997-03-17 Procede de pronostic de l'evolution d'une hepatite b WO1997035204A1 (fr)

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KR1019980707348A KR20000064637A (ko) 1996-03-19 1997-03-17 비(b)형 간염의 감염 결과를 예측하는 방법
AU20282/97A AU2028297A (en) 1996-03-19 1997-03-17 Method for predicting the outcome of hepatitis b infection
JP9533145A JP2000506735A (ja) 1996-03-19 1997-03-17 Ｂ型肝炎感染の転帰を予測するための方法
EP97908256A EP0888551A1 (fr) 1996-03-19 1997-03-17 Procede de pronostic de l'evolution d'une hepatite b

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EP96200758.9 1996-03-19

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002014871A1 (fr) * 2000-08-11 2002-02-21 Advanced Life Science Institute, Inc. Procede servant a detecter ou a determiner vbh
US20130084584A1 (en) * 2010-03-31 2013-04-04 Anna Blom Method to detect tissue degradation leading to inflammation
WO2019018629A1 (fr) * 2017-07-19 2019-01-24 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Anticorps et procédés de diagnostic et de traitement d'infection par le virus de l'hépatite b
WO2019213619A1 (fr) * 2018-05-04 2019-11-07 Abbott Laboratories Méthodes et produits de diagnostic, de pronostic et de thérapie du vhb
CN111879923A (zh) * 2020-08-06 2020-11-03 深圳科隆生物新材料有限公司 一种可消除hama效应的试剂盒

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090280474A1 (en) * 2008-05-08 2009-11-12 Abbott Laboratories Method for detecting a virus

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EP0075395A2 (fr) * 1981-09-02 1983-03-30 Biogen, Inc. Produits montrant l'antigénéité de l'antigène E du virus de l'hépatite B et méthode de production de ces antigènes
EP0080109A1 (fr) * 1981-11-19 1983-06-01 New York Blood Center, Inc. Immunoassays sensibles d'antigènes ou d'anticorps séquestrés dans des immunocomplexes
WO1994008597A1 (fr) * 1992-10-14 1994-04-28 The Scripps Research Institute Procede de detection de complexes immuns specifiques d'antigenes
JPH06321991A (ja) * 1993-05-14 1994-11-22 Mitsubishi Kasei Corp Ｂ型肝炎ウイルス由来のポリペプチドおよびそれをコードする遺伝子
WO1995027083A1 (fr) * 1994-03-31 1995-10-12 The Scripps Research Institute Procede de diagnostic de l'infection chronique par le virus de l'hepatite b
JPH0827185A (ja) * 1994-07-11 1996-01-30 Fujirebio Inc 新規ポリペプチド及びそれを用いた抗Ｂ型肝炎ウイルスｅ抗原抗体の免疫測定方法

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EP0075395A2 (fr) * 1981-09-02 1983-03-30 Biogen, Inc. Produits montrant l'antigénéité de l'antigène E du virus de l'hépatite B et méthode de production de ces antigènes
EP0080109A1 (fr) * 1981-11-19 1983-06-01 New York Blood Center, Inc. Immunoassays sensibles d'antigènes ou d'anticorps séquestrés dans des immunocomplexes
WO1994008597A1 (fr) * 1992-10-14 1994-04-28 The Scripps Research Institute Procede de detection de complexes immuns specifiques d'antigenes
JPH06321991A (ja) * 1993-05-14 1994-11-22 Mitsubishi Kasei Corp Ｂ型肝炎ウイルス由来のポリペプチドおよびそれをコードする遺伝子
WO1995027083A1 (fr) * 1994-03-31 1995-10-12 The Scripps Research Institute Procede de diagnostic de l'infection chronique par le virus de l'hepatite b
JPH0827185A (ja) * 1994-07-11 1996-01-30 Fujirebio Inc 新規ポリペプチド及びそれを用いた抗Ｂ型肝炎ウイルスｅ抗原抗体の免疫測定方法

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002014871A1 (fr) * 2000-08-11 2002-02-21 Advanced Life Science Institute, Inc. Procede servant a detecter ou a determiner vbh
US7323331B2 (en) 2000-08-11 2008-01-29 Advanced Life Science Institute, Inc. Method for detecting or assaying HBV
US20130084584A1 (en) * 2010-03-31 2013-04-04 Anna Blom Method to detect tissue degradation leading to inflammation
WO2019018629A1 (fr) * 2017-07-19 2019-01-24 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Anticorps et procédés de diagnostic et de traitement d'infection par le virus de l'hépatite b
US11827669B2 (en) 2017-07-19 2023-11-28 The Usa, As Represented By The Secretary, Dept. Of Health And Human Services Antibodies and methods for the diagnosis and treatment of hepatitis b virus infection
WO2019213619A1 (fr) * 2018-05-04 2019-11-07 Abbott Laboratories Méthodes et produits de diagnostic, de pronostic et de thérapie du vhb
CN111879923A (zh) * 2020-08-06 2020-11-03 深圳科隆生物新材料有限公司 一种可消除hama效应的试剂盒

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