FI107804B - Hepatitis C virus asialoglycoproteins which are used in immunodeterminations - Google Patents

Description

107804

This invention relates to the general fields of expression and virology of recombinant proteins. The present invention relates to general domains of expression and virology of recombinant proteins. More particularly, the invention relates to glycoproteins which are useful for diagnosing hepatitis C virus (HCV) infection.

Non-A, non-B hepatitis (NANBH) is a transmissible disease (or family of diseases) believed to be viral and distinguishable from other viral forms of liver disease, such as those caused by hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV), or Epstein-Barr virus (EBV). Epidemiological evidence suggests that there may be three types of NANBH: an aqueous infectious type; blood or needle type; as well as a single occurrence purchased by the community. The number of causative agents 15 is unknown. However, a new viral species, hepatitis C virus (HCV), has recently been identified as the primary (if not the only) cause of the blood-derived NANBH (BB-NANBH). See. for example, PCT WO89 / 046699. Hepatitis C appears to be the major form of blood transfusion-related hepatitis in many countries or regions, including the US, Europe, and Japan. There is also evidence that HCV is involved in the development of hepatocellular carcinoma.

• · • ·· * * 20 • ·

Need for sensitive, specific methods for HCV carriers and HCV-contaminated blood or • · · *. ,,; is important for the screening and identification of blood products. Post - transfusion hepatitis:. (Post transfusion hepatitis ΡΊΉ) occurs in approximately 10% of patients receiving transfusion and * · ·. ·: ·. HC V accounts for up to 90% of these cases. The major problem with this disease is often the progression to chronic liver injury (25-55%).

• ·

Patient care, including preventing the transmission of HCV through blood and blood products or through close personal contact, requires reliable diagnostic and predictive tools for detection of nucleic acids, antigens, and antibodies associated with HCV.

h ··· 30 ψ ···· ....: HCV is present in the blood of infected individuals at very low levels with respect to other infectious viruses, which makes detection of the virus very difficult. The low viral load of 2 107804 is probably the main reason why the NANB hepatitis agent went undetected for so long. Although it has now been cloned, HCV remains difficult to cultivate and grow. Thus, there is a strong need for a combination tool for the production of diagnostic HCV proteins.

5

In addition, there is a great need for HCV vaccine. However, culturing HCV in cell lines is extremely difficult. Thus, it has not been possible to produce attenuated virus strains by transfection into tissue / cell culture.

io It has been observed that two HCV proteins, E1 and E2, appear to be membrane-associated asialoglycoproteins expressed in recombinant systems. This is astonishing, since glycoproteins do not usually remain in the mannose-terminated form to be absorbed, but are further modified by other carbohydrates: the mannose-terminated form is typically only a transition form. In the case of E1 and E2 (as expressed in our glacial systems), 15 asialoglycoproteins appear to be the final form. E1 (coat protein 1) is a glycoprotein with a molecular weight of about 35 kD read from the predicted E1 region of the HCV genome. E2 (coat protein 2) is a glycoprotein with a molecular weight of about 72 kD read from the predicted NS1 region of the HCV genome (non-structural protein 1) based on the flavivirus model of HCV. Because viral glycoproteins are often highly immunogenic, E1 and E2 # '.. | 20 are major candidates for use in immunoassays and • ·: * * *; therapeutic / prophylactic vaccines.

The invention that E1 and E2 are not sialylated is significant. The specific form of the protein often dictates which cells act as suitable hosts for recombinant expression. Prokaryotes such as Ecoli do not glycosylate proteins and are generally not suitable for the production of glycoproteins for use as antigens, since glycosylation is often important for the complete antigenicity, solubility and stability of the protein. Lower eukaryotes such as yeast and fungi glycosylate proteins, but are generally unable to add sialic acid residues. · · Carbohydrate complexes. Thus, yeast-derived proteins may be antigenically different from their natural (non-combination) counterparts. Expression in mammalian cells is considered to be the best for use, in which the antigenicity of the product is important because glycosylation of the recombinant protein *: * ·: should closely resemble that of wild-type viral proteins.

3, 107804

There is no evidence that HCV can enter host cells during infection, either through asialoglycoproteins found in hepatocytes or through mannose receptors and macrophages (especially Kupffer cells) found in liver endothelial cells. Surprisingly, it has been found that the natural sample of E1 and E2 does not contain terminal sialic acid residues but is merely crude glycosylated. The small fraction also contains terminal N-acetylglucosamine. Accordingly, it is an object of the present invention to provide HCV coat glycoproteins which lack all or substantially all of the terminal sialic acid residues.

already Asialo-E1 or -E2 can be produced under conditions that prevent the addition of terminal sialic acid, e.g., by expression in yeast or by expression in mammalian cells using antibiotics to facilitate secretion or release.

E1 or E2 can be purified by affinity for lectins that bind terminal mannose residues or terminal N-acetylglucosamine residues.

An immunogenic composition comprising a recombinant azialoglycoprotein selected from the group consisting of HCV E1 and E2 may be prepared in association with a pharmaceutically acceptable carrier. It may possibly contain an immunological booster • · ·. * ·· 20 if desired.

An immunoassay reagent may also be prepared comprising a * * recombinant asialoglycoprotein selected from the group consisting of HCV E1 and E2, in combination with a suitable carrier. . The second immunoassay reagent comprises a recombinant azialoglycoprotein selected from the group consisting of E1 and HCV. E2 in combination with a suitable detectable stamp.

HCV can be grown in cell culture comprising (a) reserving the cell, which expresses a receptor selected from the group consisting of mannose receptor and # *. * asialoglycoprotein receptor; (b) infecting the cell with HCV; and (c) culturing the infected cell. Preferably, the cell expresses a recombinant receptor.

4 107804 A. Definitions

The term "asialoglycoprotein" refers to a glycosylated protein that is substantially free of sialic acid moieties. Asialoglycoproteins may be prepared by recombinant methods or by purification from cell culture or from natural sources. Today, the preferred asialoglycoproteins are derived from HCV, preferably glycoproteins Eija and E2, most preferably recombinant E1 and -E2 (rE1 and rE2). A protein is substantially free of sialic acid according to this definition if the amount of sialic acid residues does not substantially interfere with the already binding of the glycoprotein to mannose-binding proteins such as GNA. This degree of sialylation is generally obtained when less than about 40% of the total N-linked carbohydrate is sialic acid, preferably less than about 30%, more preferably less than about 20%, more preferably less than about 10%, more preferably less than about 5%, and most preferably less than about 2%.

15

The term "E1" as used herein refers to a protein or polypeptide expressed within the first 400 amino acids of the HCV polyprotein, sometimes referred to as an E or S protein. In its natural form, it is a 35 kD glycoprotein which is found strongly associated with the membrane. In most natural HCV strains, the E1 protein is encoded. * ·. j 20 in the viral polyprotein, which follows protein C (core protein). The E1 protein extends • · from amino acid 192 to about aa383 at full-length polyprotein. The term "El"

• M

: * .. as used herein also includes analogs and truncated mutants which are immunologically cross-reactive with natural E1.

• · • · • · ♦ ♦ ♦ · :; ; The term "E2" as used herein refers to a protein or polypeptide expressed within the first 900 amino acids of the HCV polyprotein, sometimes referred to as the NS1 protein. In its natural form, it is a 72 kD glycoprotein that is found strongly attached to the membrane • · ·· /. In most natural HCV strains, the E2 protein follows the E1 protein. The E2- * ·: .i i protein extends from approximately aa384 to about aa820. The term "E2" as used herein also includes analogues and truncated mutants that are immunologically cross-reactive with natural E2 *: * ·.

• · 5 107804

The term "aggregate" as used herein refers to a complex of E1 and / or E2 containing more than one E1 or E2 monomer. E1: E1 dimers, E2: E2 dimers and E1: E2 heterodimers are all "aggregates" by this definition. The compositions may also contain larger aggregates and may have molecular weights greater than 800 kD.

5

The term "particle" as used herein refers to an E1, E2 or E1 / E2 aggregate visible to an electron microscope and having a size of at least 20 nm. Preferred particles are those having a roughly spherical appearance of approximately 40 nm in diameter as seen by electron microscope.

10

The term "purified" as used in proteins herein refers to a composition wherein the desired protein comprises at least 35% of the total protein component of the composition. The desired protein comprises at least 40%, preferably at least about 50%, more preferably at least about 60%, still more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95%. % of the total protein component. The composition may contain other components such as carbohydrates, salts, lipids, solvents and the like without affecting the definition of percent purity as used herein. "Isolated" HCV asialoglycoprotein means an HCV asialoglycoprotein composition which is at least 35% pure.

"Mannose Binding Protein" as used herein refers to lectin or other protein which specifically binds to proteins with mannose-terminated glycosylation (e.g.

♦ asialoglycoproteins), for example mannose-binding lectins, antibodies specific for mannose-terminated glycosylation, mannose receptor protein (R.A.B.

• · ** '* 25 Ezekowitz et al. J Exp Med (1990) 176: 1785-94). asialoglycoprotein receptor proteins (H.Kurata et al., J Biol Chem (1990) 265: 11295-98). serum mannose binding protein (I. Schuffenecker et al. Cvtoeenet Cell Genet (1991) 56: 99-102; K. Sastry et al. J Immunol '* (1991) 147: 692-97). serum asialoglycoprotein binding protein and the like.

* *

Mannose binding lectins include, for example, GNA, concavalin A (ConA) and other lectins having similar binding properties.

The term "GNA" lectin "refers to commercially available lectin Galanthus nivalus, which binds to mannose-terminated glycoproteins.

107804 The "compound" glycoprotein, as used herein, is a glycoprotein expressed from a recombinant polynucleotide, wherein the structural gene encoding the glycoprotein is expressed by control sequences that are not naturally occurring adjacent to the structural gene or where the structural gene has been modified. For example, a vector may be constructed where the E1 structural gene is inserted to direct a functional fragment of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. Today, the preferred promoter for use in yeast is the hybrid ADH2 / GAP promoter, which is described in U.S. Patent 4,880,734, which employs a fragment of the GAPDH promoter along with alcohol dehydrogenase-2, already from the upstream side with an activating. Variations in the structural gene may include replacement of different codons with degenerate codons (e.g., use of host-preferred codons, elimination or generation of restriction enzyme cleavage sites, control of hairpin structure formation, etc.) and codons encoding different amino acids (preferably no more than 5%, the amino acid sequence should be altered) with a limited number of substitution, insertion or deletion, and the like. Similarly, a "recombinant" receptor refers to a receptor protein expressed from a recombinant polynucleotide, wherein the structural gene encoding the receptor is expressed under the control of control sequences that are not naturally occurring adjacent to the structural gene or where the structural gene has been modified.

The term "isolated polypeptide" refers to a polypeptide that is substantially free of other HCV viral components, in particular polynucleotides. A polypeptide composition is " substantially free " from the other components if the polypeptide weight of the composition is at least 70% by weight; 1 ·. of polypeptides and other components, more preferably at least about 80%, more preferably about 90% and most preferably about 95% or more. For example, a composition containing 100 pg / ml E1 and only 3 Mg / ml other HCV components (e.g.

] / DNA, lipids, etc.) is substantially free of "other HCV viral components" and is thus · · *; 1 composition of an isolated polypeptide as defined herein.

The term "secretory leader" refers to a polypeptide which, when encoded at the N-terminus of a protein, causes secretion of the protein following reading into the culture medium of the host cell.

*: 1 1 · The Executive Director is usually derived from the host cell used. For example, suitable secretagogues 7,107,804 for use in yeast include the α-factor leader of Saccharomvces cerevisiae (see U.S. Patent 4,870,008).

The term "lower eukaryote" refers to host cells such as yeast, fungi and the like.

Lower eukaryotes are usually (but not necessarily) unicellular. Preferably, lower eukaryotes are yeasts, especially Saccharomves. Schizosaccharomyces. Kluveromvces. Pichia. Hansenula and similar species. Saccharomvces cerevisiae. S. carlsbereensis iaK.lactis are the most commonly used yeast hosts and are nice fungal hosts.

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The term "upper eukaryote" refers to host cells derived from higher animals such as mammals, reptiles, insects and the like. The presently preferred higher eukaryotic host cells are derived from guinea pig (e.g., CHO), monkey (e.g., COS cells), human and insect (e.g., Spodoptera frugiperda), host cells can be placed in suspension or flask cultures, tissue cultures, like that.

The term "calcium modulator" refers to a compound that is capable of sequencing or binding calcium ions within the cell membrane network or affecting the calcium ion concentration within the cell membrane network by affecting calcium regulatory proteins (e.g.

• · * · * ί 20 calcium channel proteins, calcium pumps, etc.). Suitable calcium modulators include ♦ ** • · * ··· *, for example, tigigargin, EGTA (ethylene glycol bis [B-aminoethyl ether] -N, N, N ', N'- • ·' ', tetraacetic acid). Today, the preferred modulator is tapigargin (see e.g.

* O.Thastrup et al., Proc Nat Acad Sci USA (9999-87: 2466-701).

The term "immunogenic" refers to the ability of a substance to elicit a fluid and / or cellular immune response, either alone or in association with a carrier, in the presence or absence of a enhancer. "Neutralization" • ·. · · ·. refers to an immune response that closes the infectious agent's ability to infect either partially or • · ·. ·. wholly. A "vaccine" is an immunogenic composition capable of eliciting protection against HCV, either partial or complete, and useful for the treatment of an individual.

• ·

The term "biological fluid" refers to a fluid obtained from an organism, such as serum, plasma, saliva, gastric secretions, mucus, and the like. Generally, biological fluids are screened for the presence of HCV-8 107804 particles. Some biological fluids are used as sources of other products, such as clotting factors (e.g. Factor VIII: C), serum albumin, growth hormone and the like. In such a case, it is important that the biological fluid used as the source is free from viral contamination such as HCV.

B. General Method The E1 region of the HCV genome is described as "E" in EP 388,232, while E2 is described as "NS 1". The E1 region comprises approximately amino acids 192-383 in the full-length 10 viral polyprotein. The E2 region comprises approximately amino acids 384-820. Complete sets of prototypes of these proteins (strain HCV-1) are available in the art (see EP 388,232), as are general methods for protein cloning and expression.

Both E1 and E2 can be expressed from a polynucleotide encoding the first 850-900 amino acids of the HCV polyprotein: post-reading treatment in most eukaryotic host cells cleaves the original polyprotein into C, E1 and E2. The 5 'end of the coding region can be truncated to reduce the amount of C protein produced.

Expression of asialoglycoproteins can be achieved by a variety of methods.

For example, expression can be achieved in lower eukaryotes (such as yeast) which • · •. * · · 20 do not normally add sialic acid residues to glycosylated proteins.

• · · * ... · In yeast expression systems, it is now preferred to use a special leader, such as the cerevisiaern α factor leader, so that the protein is expressed in the culture medium following ^] * reading. It is now also preferred to use glycosylation-deficient mutants, such as pmr 1, since these mutants only give kernel glycosylation and often secrete higher levels of heterologous proteins (HKRudolph et al., Cell (1989) 58). Alternatively, other yeast species, such as Pichia oatoris, expressing • · <# glycoproteins containing 8-9 mannose residues in a pattern believed to “resemble the yding glycosylation pattern observed in mammals and S. cerevisiae.

Alternatively, expression in mammalian cells can be sequenced and terminal * * glycosylation (addition of sialic acid) can be terminated. The composite structures preferably contain a secretory signal to ensure that the protein is targeted toward the cell membrane network. Transfer to Golgi itself seems to be closed by E1 and E2: high level expression of E1 or E2 in mammalian normal cells seems to restrain the secretion of all cellular proteins in the cell membrane network or cis -Golg. In addition, a glycosylation deficient mutant may be used. See. for example, P. Stanley, Ann Rev Genet (1984) J8: 525-52. In the case of glycosylation or transfer, the mutant expresses E1 or E2 with sialylation, the terminal sialic acid residues can be removed by treatment with neuraminidase.

The yield should be further increased using a calcium modulator to induce the protein to be released from the cell membrane network. Suitable modulators include tapigargin, EGTA, and A23817 (see, e.g., O.Thastmp et al., Proc Nat Acad Sci USA (1990) 87: 2466-1070). For example, a large amount of E1 or E2 can be expressed in intracellular mammalian cells (e.g., CHO, COS, HeLa cells and the like) by transfection with recombinant vaccinia virus vector. After allowing time for protein expression and accumulation in the cell membrane network, a calcium modulator is introduced into the cells at a sufficiently high concentration to cause the cell membrane network contents to be released.

The protein is then recovered from the culture medium, which is exchanged for the next cycle.

In addition, it may be advantageous to express the truncated form of the coat protein. Both E1 and E2 appear to have a very hydrophobic region which apparently anchors the protein inside the cell membrane network and prevents efficient release. Thus, it may be desirable to remove parts. \ · 20 sequences found in one or more of E1 regions aa170-190, aa260-290 or • · · ***: aa330-380 (numbering is from the beginning of the polyprotein) and E2 in aa660-830 (see Figs. e.g.

Figure 20-1 of EP 388,232). It is likely that at least one of these hydrophobic regions will form a transmembrane region, which is not necessary for the antigenicity of the protein, and ·· • * · · which can thus be removed without adverse effect. The best area to remove can be determined by performing a small number of removal tests that are within the skill of the art. Removal of the hydrophobic 3 'end of E2 results in the secretion of the resulting E2 * * moiety when the secreted protein is sialylated.

W, W · · ·,,,,,,:::: Can be used to obtain expression from different vectors. Lower eukaryotes, such as yeast, are typically transformed with plasmids using the calcium phosphate precipitation method: or transfected with a recombinant virus. The vectors may replicate independently within the host cell or may integrate into the host cell genome. Higher eukaryotes can be transformed with plasmids, but are typically infected with 10,107,804 recombinant viruses, such as recombinant vaccinia virus. Cow pox is considered to be particularly preferred because infection with cow poison stops the expression of host cell proteins. Today's preferred host cells include HeLa and plasma cell tumor cell lines. In this glacial system, this means that E1 and E2 5 accumulate as the major glycosylated species in the host cell membrane network. Because rE1 and rE2 will be predominantly glycoproteins that are mannose terminated, they can be readily purified from cells using lectins such as Galanthus nivalus agglutinin (GNA), which binds terminal mannose residues.

Proteins naturally expressed as mannose-terminated glycoproteins are relatively rare in mammalian physiology. In most cases, the lactating glycoprotein is mannose-terminated only as a short-term intermediate on the pathway of glycosylation. The fact that HCV coat proteins expressed in combination contain mannose-terminated glycosylation or (to a lesser extent) N-acetylglucosamine-15 means that HCV proteins and whole virions can be separated and partially purified from endogenous proteins using lectin mannose or N-acetylglucosamine. The recombinant proteins appear authentic and are believed to be substantially identical to the coat proteins found in the mature, free virion, or form of the cell-associated coat protein. Thus, lectins such as GNA for mannose-terminated proteins and WGA · · · · · · * (wheat germ-agglutinin) and its equivalent for N-acetylglucosamine-terminated proteins can be used.

• ·

Lectins attached to a solid phase (e.g., lectin-Sepharose®-column) can be used to separate E1 and E2 from cell culture fluids and other fluids, e.g., for purification during antigen production. or for immunoassay.

• ♦ · ·· ♦ 25 • · · * · #

Alternatively, a suitable lectin may be taken to isolate E1, E2, or HCV virions from fluid or tissue samples from subjects suspected of having HCV infection. Because mannose heads- ♦ ·. * * *. Secondary glycoproteins are relatively rare, such a procedure would work in a sample. to purify the proteins present, substantially reducing the background. Following lectin binding · · · 30, HCV protein can be detected using anti-HCV antibodies. Alternatively, if whole virions are present, HCV nucleic acids may be detected using PCR. techniques or other nucleic acid amplification techniques that target the conserved regions of the HCV • genome (e.g., the 5 'non-coding region). This method allows the isolation and characterization of different HCV-11 107804 strains without regard to antigenic anomaly or variation, e.g., in cases where the new strain is not immunologically cross-reactive with the strain used for antibody production. There are many other ways to benefit from the unique identification of mannose-terminated glycoproteins by specific lectins.

For example, samples suspected of containing HCV virions or proteins with biotin- or avidin-labeled lectins may be incubated and the protein-lectin complex precipitated using avidin or biotin. Lectin affinity for HCV proteins can also be used to target compounds to virions for therapeutic use, for example by conjugating the antiviral compound to GNA. Alternatively, suitable lectins may already be used to remove mannose-terminated glycoproteins from serum or plasma fractions, thereby reducing or eliminating the risk of HCV contamination.

It is now preferred to isolate E1 and / or E2 asialoglycoproteins from crude cell lysates by incubating with an immobilized mannose binding protein, particularly isectectin such as ConA or GNA. For example, cells are lysed by mechanical disruption in a hypotonic buffer, followed by spinning to obtain a postnuclear lysate and further centrifuged to obtain a crude microsomal membrane fraction. The crude membrane fraction is then dissolved in a buffer containing a surfactant such as Triton X-100, NP40 or the like. This surfactant change is further clarified from 20 insoluble particulate matter by centrifugation and the resulting clarified lysate is · · ·, 1 · · incubated on a chromatographic column comprising immobilized mannose-binding • · · protein: preferably fixed to the GNA, such as agarose or • ·: · · Sepharose ® for a period of time sufficient for binding, typically * 1 16-20 hours. The suspension is then applied to the column until E1 / E2 begins to appear in the eluate, then incubated in the column for a period of time sufficient for binding, typically 12-24 hours. The bound material is then washed with additional buffer containing a surfactant (e.g. Triton X-100, NP40 or the like) and eluted with mannose to provide purified asialoglycoprotein. When eluting, it is best to elute only so long as the protein begins to appear in the eluate, at which point elution is stopped and the column • · • · ♦; :: 30 Allow to equilibrate for 2-3 hours before proceeding with protein elution. This is believed to * ··· 1 provide sufficient time for the expected slow elimination rate of large protein aggregates.

In the case where E1 and E2 are expressed in one native form (i.e., without truncation of the membrane binding region of the membrane), a substantial portion of the asialoglycoproteins are present as E1: E2-12107804 aggregates. When examined by electron microscopy, a significant proportion of these aggregates appear as coarse spherical particles of about 40 nm in diameter, which is the expected size of the intact virus. These particles appear to be self-assembling sub-virus particles. These aggregates are expected to exhibit a quaternary structure, very similar to that of authentic HCV virion particles, and are thus expected to serve as highly immunogenic vaccines.

The E1 / E2 complexes can be further purified by gel chromatography in a basic medium, for example Fractogel-DEAE or DEAE-Sepharose®. Using Fractogel-DEAE-10 gel chromatography, E1 / E2 complexes can be obtained with a purity of approximately 60-80%. E1 can be further purified by treatment with lysine protease because E1 has 0-1 Lys residues. Treatment of the complex with lysine protease destroys E2 and allows easy separation of E1.

The tissue specificity of HCV, together with the observation that HCV coat glycoproteins are mannose terminated, suggests that the virus use a mannose receptor or an asialoglycoprotein receptor (ASGR) to enter host cells. Mannose receptors are found in macrophages and hepatic capillary cells, while ASGRs are found in basic hepatocytes. Thus, it should be possible to cultivate HCV using host cells that express one or both of these receptors. Can be used with either primary • · *; Cell cultures that naturally express the receptor under conditions where the receptor. "* * Is maintained or another cell line such as HeLa, CHO, COS and the like can be transfected with a vector · · · · · which gives expression of the receptor. The cloning of the mannose receptor and its transfection and expression in fibroblasts has been demonstrated by M.E.Taylor et al., J Biol Chem. # 25 (1990) 265: 12156-62. The cloning and sequencing of ASGR was described by K. Drickamer et al., J. Biol Chem (1984) 259: 770-78 and M. Spiess et al., Proc Nat Acad Sci USA (1985) 82: 6465- ... .: 69; transfection and expression of functional ASGR in HTC rat rats was described by M.

. * ··. McPhaul and P. Berg, Proc Nat Acad Sci USA (1986) 83: 8863-67 and M. McPhaul and P. Berg, · · ·. '. <Mol Cell Biol (1987) 7: 1841 -47. Thus, it is possible to transfect one or both of the receptors to appropriate cell lines and use the resulting cells as host cells to grow HCV in culture. HCV export to such cultures should be administered as a result. development of attenuated strains that are useful as live vaccines.

• ·

Either primary cell cultures which naturally express the receptor can be used using conditions that maintain the receptor or other cell lines such as HeLa, CHO, Cos and the like can be transfected with a vector to give expression of the receptor. The cloning of the mannose receptor and its transfection and expression in fibroblasts has been demonstrated by Taylor et al., While transfection and expression of functional ASGR in rat HTC cells was described by McPhaul et al., Supra. It is now preferred to use an immortalized cell line transfected with one or both recombinant vectors.

Immunogenic compositions may be prepared by methods known in the art. These compositions comprise an immunogenic amount of a polypeptide, e.g., Eritus, E2 or E1 / E2 particle compositions, usually in association with a pharmaceutically acceptable carrier, preferably further comprising an excipient. If a "cocktail" is desired, a combination of HCV poly peptides, such as E1 plus E2 antigens, may be mixed together for enhanced potency. Virus-like particles of E1 / E2 aggregates are expected to provide a particularly useful vaccine antigen. Immunogenic compositions may be administered to animals to induce antibody production, either to provide a source of antibodies or to induce protective immunity in the animal.

Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies, which is detrimental to the individual receiving the composition. Suitable carriers • · *. Are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, · · *; * * amino acid copolymers and inactive viral particles. Such carriers are well known to those of ordinary skill in the art t '*.

• · «· · • · · • ♦

Preferred excipients that enhance the effectiveness of the composition but are not ____. limited thereto: aluminum hydroxide (alum), N-acetylmuramyl-L-threonyl-D-isoglucan (thr-MDP), as disclosed in U.S. Patent 4,606,918, N-acetylnormuramyl-L-. . alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L- · · · · · · · · · · 1 alanine-2- (1′-2′-dipalmitoyl-sn-glycero-3 -hydroxyphosphoryloxy) ethylamine (MTP-PE) and * · · • · * * * RJOBI containing three components extracted from bacteria, monophospho [foryl lipid A, trehalose dicholate and cell wall framework (MPL + TDM + CWS) in a squalene / Tween® 80 emulsion. Additionally, additives such as 14,107,804 may be used

Stimuloid (Cambridge Bioscience, Worchester, MA). In addition, Complete Freund's Adjuvant and Incomplete Freund's Adjuvant (IFA) may be used for non-human use and for research purposes.

Immunogenic compositions typically include pharmaceutically acceptable vehicles such as water, saline, glycerol, ethanol, etc. In addition, such vehicles may include adjuvants such as wetting or emulsifying agents, pH buffering agents, and the like.

Immunogenic compositions are typically prepared for injection either as liquid solutions or suspensions; solid forms suitable for reconstitution or suspension in liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or encapsulated in liposomes for enhanced excipient action.

The immunogenic compositions used as vaccines comprise an immunologically effective amount of the HCV polypeptide and also any of the above components as required. "Immunologically effective amount" means that administering that amount to an individual, either as a single dose or as part of a Saija, is effective for treatment as defined above. This amount will vary depending upon the health and physical condition of the individual being treated, the taxonomic group of the individuals being treated (e.g., non-human primates, primates, etc.). *: the capacity of the immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the medical opinion of the attending physician, the infectious strain of HCV, and other relevant factors. It is expected that the amount will be relatively large, which can be determined by routine experimentation.

• · • · · 25 • · · · · · ·

Self-assembling E1 / E2 aggregates may also serve as vaccine carriers to express. heterologous (non-HCV) hapten in the same way as hepatitis B surface antigens (see EP-A-174,444). In this use, the E1 / E2 aggregates give an immunogenic • · ·. *. a carrier capable of stimulating an immune response to hapten or antigens conjugated to an aggregate. The antigen may be conjugated either by conventional chemical methods, or may be cloned into a gene encoding E1 and / or E2 at a position corresponding to the hydrophilic region of the protein.

• * 15 107804

Immunogenic compositions are conventionally administered parenterally, typically by injection, for example subcutaneously or intramuscularly. Other compositions suitable for other modes of administration include oral compositions and suppositories. Dosage treatment can be as per single dose plan or multiple dose plan 5. The vaccine may be administered in conjunction with other immunological regulatory agents.

C. Examples

The examples below are provided as additional guidance to one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

Example 1 (Cloning and Expression) is (A) Vectors were constructed from plasmids containing the HCV genome 5 portion as described in EP-318,216 and EP-388,232. The cassette HCV (S / B) contains a StuI-BglII DNA fragment encoding the 5 'end of the polyprotein from Meti to Leu from nucleotide -63 to Meti. This includes the core protein (C), the E1 protein (sometimes referred to as S), the E2 protein (also referred to as NS1r), and the 5 portion of the NS2a region. When construct 20 is expressed, the individual C, E1 and E2 proteins are produced by proteolytic treatment.

• · · · · · · · · · · ·

The cassette HCV (A / B) contains the ApaLI-Bg11 II DNA fragment which encodes the 5 'end of the polyprotein from Meti to Leu from nucleotide -6 to Meti. This includes the core protein '(C), the E1 protein (sometimes referred to as S), the E2 protein (also referred to as NS1) and the 5-region of the NS2a region. Upon expression of the structure, the individual C, E1 and E2 proteins are produced by proteolytic treatment.

• ·. · ... Cassette C-E1 (S / B) (StuI-BamHI portion) contains a 5 'end from Meti to Ile34.4 (BamHI site in the · gene). Expression of this cassette results in the expression of C and some truncated El • t · 1 (EΓ). The truncated portion at the 3 'end is a hydrophobic region which is believed to act as a - · · • · translocation signal.

• · «• ·

The cassette NS 1 (B / B) (BamHI-BglII portion) contains a small 3 'portion of E1 (from the Woods), all of E2, and a portion of NS2a (up to Leugoo). In this structure, the E1 fragment acts as a translocation signal.

16 107804 5 TPA-NS1 cassette uses the human tissue plasminogen activator (tPA) leader as a translocation signal instead of the 3 'part of E1. The cassette contains the truncated form of E2 from Gly 406 to Glu 4 'where the hydrophobic 3' end is removed.

Each cassette was inserted into vector pGEM3Z (Promega) with and without the 5 'non-coding sequence of the synthetic β-globin for copy and reading using T7 and rabbit reticulocyte expression in vitro. Recombinant vaccinia virus vectors (rVV) were prepared by inserting the cassettes into plasmid pSC11 (obtained from Dr. B. Moss, NIH), followed by fusion with vaccinia virus as described by Charkrabarty et al., Mol Cell Biol (1985) 5: 403-09.

is (B) An alternative expression vector was constructed by inserting HCV (A / B) between the StuI and SpeI sites of pSC59 (obtained from Dr. B. Moss, NIH), followed by fusion with vaccinia virus as described by Charkrabarty et al. Mol Cell Biol (1985) 5: 403-09.

(C) HeLa S3 cells were harvested by centrifugation for 7 minutes at 2000 rpm at room temperature in sterile 500 ml spinner flasks (JA-10 rotor). The pellets were suspended in a final concentration of 2 x 10 7 cells / ml in additional medium (Joklik modified MEM Spinner- *. * · · Medium + 5% horse serum and gentamicin) ("Mixing medium"). An ultrasound-crude batch of · · w / SC59-HCV virus was added at a multiple infection rate of 8 pfu / cell and the mixture was mixed at 37 ° C for 30 minutes. The infected cells were then transferred to a spinning flask containing 8 L of mixing medium and incubated for 3 days at 37 ° C.

• · • · · * ... 25 «· ·« · ·

The cultured cells were then harvested by centrifugation and the pellets resuspended in buffer (10 mM

# # φ 4. Tris-HCl, pH 9.0.152 mL). The cells were then homogenized using a 40 ml Dounce homogenizer. · · ·. (50 strokes) and the cores were pelleted by centrifugation (5 minutes, 1600 ipm, 4 ° C, JA-20 rotor).

· «·. *. The core pellets were resuspended in Tris buffer (24 mL), re-homogenized and? The pellet was again pelleted, transferring all the solutions into storage.

The stored ly was aliquoted into 10 ml aliquots and ultrasonicated for 3 x 30 minutes with a medium cup horn sonicator. The sonicated lysate (15 mL) was plated on a 17 17 107804 mL sucrose mattress (36%) in SW28 spin tubes and spun at 13,500 rpm for 80 minutes at 4 ° C to pellet the virus. The viral pellet was resuspended in 1 ml Tris buffer (1 nM Tris-HCl, pH 9.0) and frozen at -80 ° C.

Example 2 (Comparison of In vitro Irvivo Products) (A) E1 and E2 were expressed both in vitro and in vivo and 35S-Met was labeled using the vectors described in Example 1 above. BSC-40 and HeLa cells were infected with rW vectors in.

10 for in vivo expression. Both the culture medium and cell lysates were assayed for recombinant proteins. The products were immunoprecipitated using human HCV immune serum, while jn_in vitro proteins were analyzed directly. The resulting proteins were analyzed by SDS-PAGE.

The reticulocyte expression system (pGEM3Z with HCV (S / B) or HCV (A / B)) produced 15 C, E1 and E2 proteins with molecular weights of approximately 18 kD, 35 kD and 72 kD, respectively. Lysates from BSC-40 and HeLa cells were transfected with rW containing HCV (S / B), HCV (A / B), or C-E1 (S / B) showed the same proteins. Since the reticulocyte system does not provide efficient golgi treatment and therefore does not give sialic acid, the fact that both in vitro and in vivo products gave identical motions suggests that the proteins are not sialylated in vivo. Only the rVV vector containing TPA-NS1 resulted in · · extracellular E2 secretion with altered motility consistent with? · · · Sialylation.

(B) HCV (S / B) was expressed in vitro and incubated with a panel of biotinylated lectins: GNA, SNA, PNA, WGA and ConA. Following incubation, complexes of avidin · · ·, ·: · were collected. 25 acrylic beads, washed, eluted with Laemmli sample buffer and analyzed by SDS-PAGE.

• t ·

The results showed that E1 and E2 bound to GNA and ConA, indicating the presence of mannose. GNA binds to terminal mannose groups, whereas Con A binds v · “*; to any α-linked mannose. The lack of binding to SNA, PNA, and · * ·: WGA means that none of these proteins contained sialic acid, galactose-N- ··· · ·. 30 acetylgalactosamine or N-acetylglucosamine.

··· *. (C) Radiolabeled E1 and E2 were produced in BSC-40 cells by infection with rVV, resulting in? contained HCV (S / B) (w / SC11-HCV) and by immunoprecipitation of human HCV + immune seed- ♦ ·. whereby. Half of the immunoprecipitated material was treated overnight with neuraminidase to remove sialic acid 18 107804. Following treatment, treated and untreated proteins were analyzed by SDS-PAGE. No significant difference in motility was observed, indicating the absence of sialylation in vivo.

(D) Radiolabeled E1 and E2 were produced in BSC-40 cells by infection with rW containing HCV (A / B) (w / SC59-HCV) and either immunoprecipitated with human HCV * immune serum or was precipitated using biotinylated GNA-lectin coupled to acrylic spheres using w / SC11 free from HCV sequences as a control. The precipitates were analyzed by SDS-PAGE. The data show that E1 and E2 were the major species of mannose-terminated proteins in w / SC59-HCV-infected cells. GNA was as effective as human antiserum already in precipitating E1 and E2 from cell culture medium. A 25 kD component was observed but appeared to be specific for vaccinia infected cells.

Example 3 (Purification using lectin) 15 (A) HeLa S3 cells were infected with purified high titer w / SC59-HCV virus at 5 pfu / cell infection and mixed at 37 ° C for 30 minutes. The infected cells were then transferred to a mixing vessel containing 81 mixing media and incubated for 3 days at 37 ° C. Cells were harvested again by centrifugation and resuspended in hypotonic buffer 20 (20 mM HEPES, 10 mM NaCl, 1 mM MgCl 2, 120 mL) in an ice bath. Cells were homogenized • · 4 ': then in a Dounce homogenizer (50 strokes) and the nuclei pelleted by centrifugation (5 minutes, 1600 rpm, 4 ° C, JA-20 rotor). The pellets were pooled, resuspended in 48 ml of hypotonic buffer, resuspended, centrifuged, recombined and frozen at -80 ° C.

* ·· · ♦ · 25 • ♦ · • · ·

The frozen solutions were then thawed and the microsomal membrane fraction of the postnuclear lysate .... was isolated by centrifugation for 20 minutes in a JA-20 rotor at 13,500 rpm at 4 ° C. The solution was removed · ***; by suction.

· «· • · ♦ · · *« ·. · · ·. The pellets were taken up in 96 ml of surfactant buffer (20 mM Tris-HCl, 100 mM NaCl, 1-4 mM EDTA, 1 mM DDT, 0.5% Triton X-100, pH 7.5) and homogenized ( 50 strokes). Product • ·. clarified by centrifugation for 20 minutes at 13,500 rpm, 4 ° C, and the solutions were collected.

• ♦ 19 107804 GNA agarose column (1 cm x 3 cm, 3 mg GNA / ml spheres, 6 ml sphere volume, Vector Labs, Burlingame, CA) was pre-equilibrated with surfactant buffer. A sample of the solution was placed on the column by circulating at a flow rate of 1 ml / min for 16-20 hours at 4 ° C. The column was then washed with surfactant buffer.

5

Purified E1 / E2 proteins were eluted with α-D-mannose (0.9 M in surfactant buffer) at a flow rate of 0.5 ml / min. Elution was stopped when El / E2 appeared in the eluent and the column was allowed to equilibrate again for 2-3 hours. Fractions were analyzed by Westem blotting and silver staining. The peak fractions were pooled and UV irradiated to inactivate any remaining vaccinia virus already present.

(B) GNA agarose purified E1 and E2 asialoglycoproteins were sedimented through 20-60% glycerol gradients. Gradients were fractionated and proteins analyzed by SDS-PAGE and Westem blotting. Spots were probed with GNA to identify E1 and E2. The results indicate the presence of the E1: E2 heterodimer, which sedimentes at the expected rate (i.e.

js 110 kD protein position properties). Higher aggregates of HCV coat proteins are also evident. E2: E2 homodimers were also evident. E2 appeared to be over-represented in larger species with respect to E1, although distinct E1: E2 species were also shown. Larger aggregates sedimented significantly faster than the thyroglobulin marker.

(C) GNA agarose purified E1 and E2 were sedimented through 20-60% glycerol gradients containing 1 mM EDTA. Fractions were analyzed by SDS-PAGE for β-mercaptoethanol. *: (βΜΕ) with and without. Little or no difference was observed in the apparent ··· • · ... * abundance of E1 and E2 in the presence or absence of βΜΕ: η, indicating no disulfide bonds between the heterodimers.

\ (D) E1 / E2 complexes (approximately 40% pure) were analyzed in a Coulter DM-4 type • ,,,, 25 micron particle analyzer. Material was observed in the range of 20-60 nm.

(E) E1 / E2 complexes (approximately 40% pure) were analyzed by electron microscopy using negative staining with phosphotungstic acid. The electron micrograph revealed the presence of particles of · ·, · · · with a spherical appearance and a diameter of about 40 nm. The E1 / E2 complexes were incubated with human HCV + immune serum, then analyzed by · · · • · <1 electron microscopy for negative staining. Antibody complexes were detected which * · · · • · *! * contained large aggregates and smaller particles.

• · • «20 107804

Example 4 (Chromatographic purification) (A) GNA-lectin purified material (0.5-0.8 mL) prepared as described in Example 3 was diluted 10x with buffer A (20 mM Tris-HCl buffer, pH 8.0.1 mM EDTA). ) and applied to a 1.8 x 1.5 cm Fractogel EMD DEAE-650 column (EM Separations, Gibbstown, New Jersey, Cat. No. 16883) equilibrated with buffer A. The protein fraction containing E1 / E2 was eluted with the same buffer 0, At 2 ml / min and 1 ml fractions were collected. Fractions containing E1 and E2 (as determined by SDS-PAGE) were pooled and stored at -80 ° C.

(B) The purified material in Part (A) has 60-80% purity as assessed by SDS-PAGE. The identification of putative E1 and E2 bands was confirmed by N-terminal sequence analysis after using the transfer technique. For this purpose, Fractogel-DEAE-purified E1 / E2 was reduced by addition of Laemmli buffer, pH 6.8,0.06 M Tris-HCl, 2.3% is SDS, 10% glycerol, 0.72 M β-mercaptoethanol ) and boiled for 3 minutes. The sample was then loaded onto a 10% polyacylamide gel. After SDS-PAGE, the protein was transferred to a 0.2 µm polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratorie, Richmond, CA). The corresponding putative E1 and E2 protein bands were then cut from the spot and subjected to N-terminal amino acid analysis, although no particular care was taken to prevent amino-terminal blocking during material preparation. The first 15 cycles revealed, '. j that the E1 sample had the sequence Tyr-Gln-Val-Arg-X-Ser-Thr-Gly-X-Tyr-His-Val-X-Asn-Asp, while E2 had the sequence Thr-His- Val-Thr-Gly-XX-Ala-Gly-His-X-Val-X-Gly-Phe. This • · ·: '· .. amino acid sequence information is consistent with what was expected from the corresponding DNA ·: **: sequences.

25 · · ·, · * E1 / E2 product purified by Fractogel-DEAE chromatography above is believed to be aggregated, as evidenced by the fact that a large amount of E1 and E2 is eluted at the same time by * * gel permeation chromatography. Sil TSK-4000 SW column in the empty volume range. This indicates that, under natural conditions, a significant amount of the E1 / E2 complex has a molecular weight of at least 800 · 30 kD. El / E2 material having a molecular weight of about 650 O kD was also observed.

• · • m

Claims (3)

107804 1. Stympat asialoglycoprotein som används i immunobestämningar, transducer därav, att det har uttryckts frän HCVs E-omräde och omfattar en deletion sequences, 20 som forekommer et omräd, som tecker aminosyra 338-380 ,: numreringen startar frän böijan av HCV polyproteiner. •••••••••••••••••••••••••••• A truncated asialoglycoprotein for use in immunoassays, characterized in that it is expressed in the E1 region of HCV and comprises a deletion of a portion of the sequence which is present in the 5 region covering amino acids 338-380 of the E1 region at the start of the HCV polyprotein. A truncated asialoglycoprotein for use in immunoassays, characterized in that it is expressed from the E2 region of HCV and comprises a deletion of a portion of the sequence that appears in the region covering amino acids 660-830 of the E2 region, beginning with the numbering of HCV polyproteins. Composition for use in immunoassays, characterized in that it comprises an asialoglycoprotein of the hepatitis C virus according to claim 1 or 2. 15 2. Stympat asialoglycoprotein som används i immunobestämningar, kännetecknat därav, • att det har uttryckts frän HCVs E2-omen and deletion i del del se sequen, • 1 · s # 25 som forekommer ei omräy, som Täcker amy. i E2 omrädet när •; 1 · 1; numreringen startar frän böq an av HCV-polyproteiner. *: · 1: 3. Kompositionen som används i immunobestämningar, kännetecknad därav, att den 'omfattar et hepatit-C virus asialoglycoprotein enligt patentkrav 1 eller 2. • · • · · · · · · • ·