Antibodies Against Hepatitis G Virus and Their Use for the Diagnostic Detection of HGV and as a Therapeutic Agent

The present invention concerns antibodies against hepatitis G viruses and fragments thereof. The invention in addition concerns a conjugate which contains this antibody or an antibody fragment coupled to a biological molecule. Finally the present invention concerns the use of the antibody for the diagnostic detection of hepatitis G viruses and cell cultures.

In addition to hepatitis A virus (HAV) and hepatitis B virus (HBV) which have been known for a long time, further hepatitis-associated viruses have been characterized recently which belong to various virus families. They cause a number of quite different diseases some of which are very serious so that a differential diagnosis which is as early and unequivocal as possible is highly desirable. The hepatitis viruses are usually named by allocating a consecutive letter of the alphabet. Alternatively new hepatitis-associated viruses can also be named by exclusion of the known viruses. Thus hepatitis C virus (HCV) is also referred to as nonA/nonB hepatitis virus (Choo et al., Science 244 (1989), 359-362). The present invention concerns a virus which cannot be allocated to any of the virus families represented by HAV, HBV, HDV and HEV. The available information about this new virus suggests that it belongs, like HCV, to the family of flaviviridae [Chambers et al., Annu. Rev. Microbiol. 44 (1990), 649-688]. However, these data also clearly indicate that it differs significantly from HCV and thus belongs to its own virus group. Therefore this new virus is referred to as hepatitis G virus (HGV) (Linnen et al., Science 271 (1996), 505-508).

A hepatitis-associated virus is described in WO94/18217 which cannot be assigned to any of the groups HAV, HBV, HCV, HDV and HEV. However, the nucleotide sequence of this virus does not have any similarity to the sequence described in the present invention.

The nucleic acid and amino acid sequence of HGV is described in WO95/21922. The recombinant expression of HGV polypeptides in E. coli is disclosed in the examples 13, 19 and 20. Explicit reference is made to the sequences disclosed in this application.

The nucleic acid and amino acid sequence of HGV is also described in WO95/32291. The recombinant expression of HGV in E. coli, in insect cells and in vaccinia is disclosed in example 16. Explicit reference is made to the sequences disclosed in this application.

Viral infections are usually detected by the presence of antigens and/or antibodies against these antigens in body fluids such as blood serum. Antibodies are required to detect the antigens in an immunoassay which specifically detect the viral antigens. In order to carry out such an immunological test it is also necessary to provide suitable antibodies in an adequate amount.

New processes for the production of HGV antigens and their use for the diagnostic detection of HGV are known from the German Patent Application 196 13 406.4.

This state of the art provides new processes for the expression of HGV antigens. Suitable HGV antigens in this sense are polypeptides from the HGV genome which have at least one antigenic and/or immunogenic determinant. The DNA sequence regions of the HGV total genome are preferred which code for the putative envelope proteins E1 and E2. These envelope proteins are composed of an amino-terminal main section which is located on the outside of a functional virus particle and plays a decisive role when it docks with the host organism and a short carboxy-terminal hydrophobic section which is anchored in the membrane.

The above-mentioned patent application also discloses a recombinant cell which presents HGV antigens on its surface in a membrane-bound form, in particular the antigens E1 or/and E2 or immunologically relevant partial sequences thereof.

This cell can be used as a diagnostic reagent for the detection of HGV e.g. by FACS analysis or by ELISA. For this the reaction of a cell, which presents a HGV antigen on its surface, with a sample liquid e.g. human serum is determined. If a reaction occurs it can be assumed that anti-HGV antibodies are present in the tested sample.

However, there are still no antibodies against the HGV epitopes.

Classically antibodies are obtained by immunizing mice or other animals with antigens. Monoclonal antibodies, which have the advantage of unlimited production and exactly defined specificity, can then be selected from the at first polyclonal pool by hybridoma technology (Kohler and Milstein, Nature 256 (1975), 495). For this myeloma cells are fused with spleen cells of the immunized animal.

It was therefore the object of the present invention to produce a--preferably monoclonal--antibody against a HGV surface antigen in particular against the envelope protein E2 of HGV. A further object of the invention was to use this antibody as a diagnostic reagent for the detection of HGV.

The invention provides monoclonal and polyclonal antibodies against HGV surface antigens.

In addition a monoclonal or polyclonal antibody against the HGV E2 surface antigen is provided.

Suitable immunogens for obtaining the antibodies are polypeptides and in particular surface antigens from the HGV genome (WO95/21922 and WO95/32291) or partial peptide sequences thereof which have at least one antigenic or/and immunogenic determinant. The putative envelope protein E2 or an antigenic or/and immunogenic partial peptide section thereof is particularly preferred and especially a partial peptide section from the amino-terminal region. The putative envelope protein E1 or/and an antigenic or immunogenic partial peptide section thereof is also suitable as an immunogen which in particular is derived from the amino-terminal region. Immunization methods using viral envelope proteins are known cf. HBsAg (Michel et al., Bio/Technology 3 (1985), 561-566) and E2/NS1 of HCV (Lesniewski et al., J. Med. Virol. 45 (1995), 415-422).

However, the antibodies according to the invention are particularly preferably produced by a multistep immunization process which includes a DNA immunization (Davies et a., Ann., N.Y. Acad. Sci. 772 (1995), 21-29). In this manner it is possible to present to the animal's immune system the surface antigens in a form that is as authentic as possible i.e. in a form that is preferably correctly post-translationally processed and optionally glycosylated and exported from the cell. In the first step of the immunization process according to the invention the DNA sequence coding for the immunization antigen is cloned into a eukaryotic expression vector and this construct is injected directly into a suitable tissue of the experimental animal (e.g. mouse, rat, rabbit etc.) such as the skeletal muscle. The DNA sequence coding for the antigen is located on the expression vector under the control of a promoter which is known to be active in the tissue of the appropriate experimental animal that is used in each case.

It is particularly preferable to clone a DNA sequence coding for the envelope protein E2 or a section thereof in an expression vector such as pcDNA3 in the correct reading frame next to DNA sequences coding for an amino terminal signal peptide and optionally for a marker epitope e.g. the so-called FLAG epitope and to express it together with the two elements as a fusion protein. The signal sequence can for example be the erythropoietin signal sequence (Jacobs et al., Nature 313 (1985), 806-810). The FLAG epitope is an octapeptide (Hopp et al., Bio/Technology 6 (1988), 1204-1210) against which a monoclonal antibody is commercially available which can be used to identify and optionally to purify the desired expression product.

When the antigen is expressed in the tissue of-the experimental animal the protein biosynthesis takes place on the ribosomes in the cytosol. When the HGV antigens are expressed in operative linkage with amino-terminal signal sequences e.g. 20 to 30 amino acid long hydrophobic sequences which are recognized during protein biosynthesis in the cytosol by so-called signal recognition particles, the ribosomes are directed to the endoplasmic reticulum (ER). Here the polypeptide chains are channelled through the ER membrane until they are arrested in the membrane by stop transfer sequences. The proteins are optionally glycosylated in the lumen of the ER and subsequently further modified in the Golgi apparatus. Finally they are sorted for export in the direction of the plasma membrane. In this manner the antigen is presented to the immune system in as authentic form as possible and can lead to the formation of high quality anti-HGV antibodies.

The immunization process according to the invention preferably also includes a booster immunization in which eukaryotic cells which express the corresponding HGV surface antigen on their membranes are injected into the experimental animal. Reference is made to DE 196 13 406.6 with regard to the production of such cells.

Polyclonal antibody compositions or monoclonal antibodies against HGV surface antigens can then be isolated from the immunized experimental animal. In order to produce a polyclonal antibody composition that is specific for HGV the serum is preferably purified e.g. an affinity chromatography is carried out over a column coated with the appropriate antigen e.g. the E2 antigen.

In order to obtain monoclonal antibodies use is made of the hybridoma technology of Kohler and Milstein or subsequent developments thereof. Suitable antibody-producing hybridoma cells can for example be produced by fusing spleen cells from the immunized animals with myeloma cells according to Galfre and Milstein, Meth. Enzymol. 73 (1981), 3-46. The primary cultures of the fusion cells are then tested for the synthesis of specific antibodies. Specific primary cultures can be cloned in microtitre plates by means of fluorescence activated cell sorting (FACS).

Hence the invention concerns a polyclonal or monoclonal antibody against a hepatitis G virus surface antigen which can be produced by a process which comprises the following steps:

Immunization of experimental animals with an expression vector comprising: a promoter, a eukaryotic signal sequence, a DNA sequence coding for the hepatitis G virus surface antigen and optionally a marker sequence,

optionally a booster immunization of the experimental animals with cells which express the HGV surface antigen on their membranes and

obtaining polyclonal or monoclonal antibodies that are specific for the hepatitis G virus surface antigen from the immunized experimental animal.

The antibody against the HGV E2 surface antigen which is provided is preferably directed against a polypeptide which is coded by

(a) the nucleotide sequence between position 127 and 1290 shown in SEQ ID NO.1,

(b) a sequence corresponding to the sequence from (a) within the scope of the degeneracy of the genetic code or/and

(c) a nucleotide sequence hybridizing with the sequences from (a) or/and (b) under stringent conditions.

A further characteristic of the antibody against the HGV E2 surface antigen is that it is directed against a polypeptide which comprises

(a) the amino acid sequence between position 39 and 426 shown in SEQ ID NO.2 or

(b) an amino acid sequence which is at least 80% homologous with the sequence from (a).

In addition hybridoma cell lines are provided which produce monoclonal anti-E2 antibodies according to the invention. These hybridoma cell lines were deposited according to the rules of the Budapest Contract at the "Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)", Mascheroder Weg 1b, D-38124 Braunschweig. The depository data are as follows:

The invention also concerns a monoclonal antibody obtainable from the cell lines stated above as well as antibodies with an equivalent binding specificity which preferably recognize the same epitope as the deposited antibodies.

In addition the invention concerns any fragment of one of the antibodies described above.

Yet a further subject matter of the present invention is a conjugate which contains one of the antibodies described above or a corresponding antibody fragment which is coupled to a biological molecule.

In a preferred embodiment the biological molecule in the conjugate comprises a marker group. All known marker groups come into consideration as a marker group which can be detected in a test system i.e. directly or indirectly detectable marker groups. A directly detectable marker group is understood as a group which generates a directly detectable signal e.g. a radioactive group, an enzyme group or a luminescent group. Enzyme groups and luminescent groups are particularly preferred and in particular electrochemiluminescent groups. On the other hand the marker group can also be an indirectly detectable group e.g. a biotin or hapten group which can be detected by reaction with a suitable binding partner (streptavidin, avidin or anti-hapten antibody) which in turn carries a signal-generating group. Hapten or biotin groups can also be used as solid phase binding groups to immobilize the antibodies on a solid phase. The marker and solid phase binding group can be coupled to the antibody in a known manner.

A further subject matter of the invention concerns the use of the antibodies as diagnostic reagents for the detection of HGV e.g. by FACS analysis or by ELISA. For this the reaction of the antibodies with a sample liquid, e.g. human serum, is determined. If a reaction occurs it can be assumed that HGV antigens are present in the tested sample.

For use in a diagnostic test the antibodies are preferably provided with at least one marker group or solid phase binding group as described above.

The detection of HGV is carried out in particular by the immunological determination of HGV antigens in a sample liquid wherein the sample liquid is incubated with at least one antibody according to the invention and the binding is detected. This immunological method of determination can be carried out according to any known test format e.g. in a homogeneous immunoassay with a single reaction phase or in a heterologous immunoassay with more than one reaction phase. A heterogeneous test format is preferably used in which the presence of the antigen is detected in the presence of a solid phase.

One embodiment of this test format is the so-called bridge test (see example 5). In this embodiment the sample liquid is incubated with at least two antibodies A1 and A2 according to the invention in which A1 is bound to a solid phase or is present in a form capable of binding to a solid phase (so-called capture antibody) and A2 carries a marker group (so-called detection antibody). The antigen in the sample liquid is detected by determining the label in the solid phase or/and in the liquid phase, preferably in the solid phase, via an immobilized, i.e. a solid phase-bound immune complex. The test procedure preferably comprises mixing of the sample liquid with a labelled A2 as well as with A1 bound to or capable of binding to a solid phase in order to obtain a labelled immobilized complex of labelled antibody, antigen and solid phased antibody.

A further area of application for the antibodies according to the invention is a therapeutic application. For this the antibodies according to the invention are preferably produced in a purified form and then formulated as injectable liquids which can be solutions or suspensions. Further components are for example water, salt solutions, glucose or glycerol. The antibodies can also be enclosed in liposomes. The antibodies are usually administered parenterally by injection preferably subcutaneously or intramuscularly.

The present invention is described in more detail by the following examples, sequence protocols and figures, wherein:

SEQ ID NO. 1: shows a DNA nucleotide sequence which codes for HGV-E2 plus an amino terminal fusion part

SEQ ID NO. 2: shows an amino acid sequence of HGV-E2 plus an amino terminal fusion part

FIG. 1: staining of CHO cells that express FLAG-E2 with anti-FLAG-M1 and anti-E2 MABs 3, 5, 6, 11, 13, 17, 19, 30

FIG. 2: staking of CHO cells (filled in area) and CHO cells expressing FLAG-E2 (open area) with the anti-E2 MABs 3, 5, 6, 11, 13, 17, 19, 30.

Standard methods were used to manipulate the DNA such as those described by Sambrook et al. (1989) in Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York and Ausubel et al., (1989) in Current Protocols in Molecular Biology, John Wiley & Sons, New York.

The manipulation of the DNA was carried out in the E. coli K12 strain DH5.alpha..

A derivative of the vector pcDNA3 with a CMV promoter and BGH poly-adenylation signal (Invitrogen BV, NV Leek, Netherlands) was used as the expression vector for the DNA immunization. For this the vector pcDNA3 was modified by substituting the neomycin resistance gene by a dehydrofolate reductase (DHFR) gene. This was carried out by restriction cleavage of pcNDA3 with AvrII/Bst1107I, isolation of the ca. 4 kBp long vector fragment and insertion of a ca. 700 Bp long AvrII/-Bst1107I DHFR fragment [Setzer et al. (1982) J. Biol. Chem. 257, 5143-5147; Crouse et al. (1982) J. Biol. Chem. 257, 7887-7897].

The DNA sequence coding for the envelope protein E2 was cloned into the resulting vector pcDNA3-DHFR in operative linkage to a signal peptide sequence as well as to a DNA sequence coding for a so-called FLAG epitope. The erythropoietin signal sequence [Jacobs et al. (1985) Nature 313, 806-810] was used as the signal sequence as a 93 Bp long EcoRI/EheI fragment (position 1 to 93 of SEQ ID NO.1). The FLAG epitope is a short octapeptide [Hopp et al. (1988) Bio/Technology 6, 1204-1210] against which a monoclonal antibody anti-Flag-M1 (Kodak Eastman) is available which can be used to purify and identify the desired expression product. Two oligonucleotides were hybridized to each other to prepare the DNA sequence coding for the FLAG epitope and used as a linker after treatment by kinase (corresponding to position 94-126 of SEQ ID No. 1).

In order to produce the E2 expression vector, the plasmid pcDNA3-DHFR was digested with EcoRI and NotI and the ca. 5.9 kBp long vector fragment was isolated and ligated in a 3-way ligation with the EcoRI/EheI signal sequence fragment and the FLAG linker.

The HGV cDNA that was used was derived from Genelabs Technologies Inc., Redwood City, Calif., USA (WO 95/32291). The almost complete HGV genome was present as a ca. 9.3 kBp long XbaI/EcoRI fragment which had been cloned into the corresponding restriction sites of the vector pGEM-3Z (Promega Corp., Madison, Wis., USA). The DNA sequences coding for E2 (position 127-1290 of SEQ ID NO.1) were amplified by means of PCR using suitable oligonucleotides and cloned as NotI/XbaI fragments in frame next to the DNA sequences coding for the signal peptide and FLAG epitope.

2.1. DNA Immunization

Female BALB/c mice were used for the immunization. In order to increase the efficiency of DNA uptake which should be about ten-fold higher in regenerating muscle than in untreated muscle, the animals were treated with the snake venom Latoxan (Rosans, France) before the first immunization. For this the mouse was injected in each case with 80 .mu.l of a 10 .mu.M solution of the venom in both tibial muscles. Five days later the immunization was started, each mouse was injected five times (week 0, 5, 10, 11, 12) with plasmid DNA. In this process 50 .mu.g DNA was injected into each tibial muscle i.e. 100 .mu.g per animal and immunization. The plasmid DNA was purified over a Qiagen column according to the manufacturer's instructions.

The animals were boostered before removing the spleen in week 20. For this purpose 10.sup.7 CHO cells which expressed HGV-E2 on their membranes were injected intravenously into each animal.

2.2 Fusion and Cloning

Spleen cells of the immunized mice were fused with myeloma cells following the procedure of Galfre and Milstein (1981) Meth. Enzymol. 73, 3-46. In this process ca. 10.sup.8 spleen cells of the immunized mouse were mixed with 2.times.10.sup.7 myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged. The cells were then washed once in RPMI 1640 medium w/o FCS and again centrifuged at 400 g. The supernatant was discarded, the cell sediment was gently loosened by tapping, 1 ml PEG (molecular weight 4000, Life Technologies, Cat. No. 14030035) was added to this within one minute and mixed with the cells by gently swirling in a 37.degree. C. warm water bath. Subsequently 5 ml RPMI 1640 medium w/o FCS was added dropwise within 5 min and mixed in a 37.degree. C. warm water bath by continuous swirling. After the addition of 25 ml RPMI 1640 medium w/o FCS the cells were centrifuged for 10 min at 400 g. The cell pellet was taken up in RPMI 1640 medium, 10% FCS and inoculated into hypoxanthine azaserine selection medium (100 mmol/l hypoxanthine, 1 .mu.g/ml azaserine in RPMI 1640, 10% FCS). Interleukin 6 (Boehringer Mannheim, Cat. No. 1444 581) was added to the medium as a growth factor. After ca. 10 days the primary cultures were tested for the synthesis of E2-specific antibodies (see example 3). E2-specific primary cultures were cloned in microtitre plates by means of fluorescence activated cell sorting (FACS). For this purpose interleukin 6 was added to the medium as a growth additive. The purification of the antibodies from the ascites fluid of mice and the derivatization with biotin or digoxigenin was carried out according to standard methods in protein chemistry.

In order to determine the specificity of the antibodies in the culture supernatant of the hybridoma cells the reactivity with CHO cells which expressed on their membranes either the FLAG-E2 fusion protein or the human urokinase receptor, which is also provided with an NH.sub.2 -terminal FLAG sequence, was detenined in two parallel ELISA experiments. For this the cells were inoculated at a density of ca. 4.times.10.sup.4 per well of a microtitre plate on the day before the experiment. On the next day unspecific binding sites were firstly blocked by a two hour incubation with 200 .mu.l RPMI 1640 medium, 10% FCS, 1% BycoC per well. Subsequently 100 .mu.l of the cell culture supernatants was pipetted into each well and incubated for 60 min at room temperature. After washing three times with Dulbecco's PBS, 0.02% Tween 20, anti-mouse IgG-peroxidase Fab fragment (Boehringer Mannheim Cat. No. 1500 686) was added to the cells in a volume of 100 .mu.l and a concentration of 50 mU/ml. After 60 min incubation at room temperature and washing three times with Dulbecco's PBS, 0.02% Tween 20, ABTS.RTM. was used as a substrate and the colour change was measured in an ELISA reader at 405/490 nm after 30-60 min.

A total of 8 hybridoma clones were identified (anti-HGV-E2 clones 3, 5, 6, 11, 13, 17, 19, 30) whose monoclonal antibodies specifically recognize CHO cells expressing EGV-B2 (MABs 3, 5, 6, 11, 13, 17, 19, 30).

In this method CHO cells which express the FLAG E2 fusion protein on their membranes were stained successively with one of the E2-specific MABs and the anti-FLAG-M1. Since both epitopes are located on one molecule this experiment gives information about possible epitope overlaps and thus about the influence of the FLAG epitope on the binding of E2-specific antibodies.

The cells were detached from the culture vessel with 0.02% EDTA in PBS and washed in PBS. 2.times.10.sup.5 cells were resuspended in each case in 100 .mu.l Dulbeccol's PBS, 0.2% bovine serum albumin, 0.02% NaN.sub.3 and incubated for 15 min on ice with the MABs 3, 5, 6, 11, 13, 17, 19 or 30 (2 .mu.g/ml each case), washed twice with the same buffer and incubated for a further 15 min with anti-mouse Ig-fluorescein (Fab').sub.2 fragment (Boehringer Mannheim, Cat. No. 1295 750). After washing twice, the cells were incubated for 15 min on ice with mouse IgG (Sigma) at a concentration of 10 .mu.g/ml to block the free anti-mouse IgG binding sites. Subsequently biotinylated anti-FLAG-M1 was added to the mixture at a concentration of 0.35 .mu.g/ml. After incubating for 15 min. on ice it was washed twice and the second MAB labelled fluorescently this time with streptavidin-R-phycoerythrin (Boehringer Mannheim, Cat. No. 1428 560) was added for 15 min on ice. After washing twice, the cells were analysed in a flow cytometer (FIG. 1). The double staining resulted in each case in a straight line with a different gradient. A gradient of 45.degree. which is the case for the MABs 5, 17 and 30 is typical for the recognition of two independent epitopes. A gradient that differs from 45.degree. such as in the case of the MABs 3, 11, 13 and 19 could indicate a mutual steric hindrance of the binding.

In order to exclude an unspecific binding of the anti-E2-MABs to CHO cells, additional CHO cells were also stained as a negative reference. In this case the staining with anti-FLAG-M1 was omitted (FIG. 2). In this case only MAB 30 exhibited a slight unspecific reaction with CHO cells whereas no background staining was detectable for the other seven MABS.

The test was carried out as a bridge test using isolated HGV-E2 as the antigen to characterize the E2-specific MABs. The antigen was bound via a biotinylated E2-specific capture antibody which in turn was coupled to a streptavidin-coated ELISA plate.

a) Preparation of the ELISA Plates:

Streptavidin-coated ELISA microtitre plates (Microcode, streptavidin MTP F8) were incubated for 60-120 min with 50 .mu.l biotinylated MAB (2 .mu.g/ml in Dulbecco's PBS, 0.2% bovine serum albumin) and subsequently washed three times with 0.9% NaCl, 0.05% Tween 20.

b) Cell Lysis:

The cells expressing HGV-E2 were detached from the culture vessel with 0.02% EDTA in PBS and washed in PBS. Subsequently the cells were lysed in PBS, 0.5% Nonidet P 40, protease mix (Boehringer Mannheim, Cat. No. 1206 893). For this 10.sup.7 cells/ml solubilization solution were incubated in each case for two hours on ice. Undissolved material was separated by centrifugation.

c) Coupling of the Antigens and Assay:

The ELISA plates prepared according to a) were incubated for 60 min at room temperature with the cell lysate (diluted in PBS, 0.1% Nonidet P40). After washing three times with PBS, 0.1% Nonidet P40 the second MAB which was labelled this time with digoxigenin was added and incubated for 90 min at room temperature. After washing several times with PBS, 0.1% Nonidet P40 it was subsequently incubated for one hour with anti-digoxigenin IgG-POD. After washing intensively with PBS, 0.1% Nonidet P40 ABTS.RTM. was added as a substrate and the colour change was measured in an ELISA reader at 405/490 nm after 30-60 min.

The MABs 3, 5, 11, 13, 17, 19, 30 were used in all possible combinations as biotinylated capture antibodies and used as digoxigenin-labelled detection antibodies. Since in the ELISA format described above all reaction steps proceed sequentially, a low dissociation constant is essential for a good capture antibody. This prerequisite is fulfilled by the MABs 5, 11, 17 and 30. In contrast the MABs 3, 13 and 19 can only be used as detection antibodies. The simultaneous use of a MAB as a capture and detection antibody was not possible in this test which indicates a monovalent conformation of the antigen.

The test also enables a possible epitope overlap of the individual MABs to be examined. Apparently the MABs 5 and 17 recognize the same epitope since this combination did not lead to a signal in the bridge test. In contrast the MABs 5 and 17 did not compete with the MABs 11 or 30. This shows that at least three different epitopes on HGV-E2 are recognized by these MABs.