RU2175657C2 - Hepatitis c virus (hcv) asialoglycoprotein, immunogenic composition, method of induction of immune response, method of immune assay - Google Patents

Abstract

FIELD: medicine, virology. SUBSTANCE: invention relates to methods of diagnosis, prophylaxis and treatment of patients with hepatitis C. Two envepole proteins of hepatitis C (E1 and E2) are expressed without sialylation reaction. Recombinant expression of these proteins in lower eucaryotic organisms or in mammalian cells where therminal glycosylation is blocked results to producing recombinant proteins exhibiting large analogy with natural hepatitis C virus glycoproteins. If their are isolated by method based on their affinity to lectin GNA then proteins E1 and E2 are assembled to virus-like particles. Cells carrying mannose or asialoglycoprotein receptors can be used for infection hepatitis virus C and to use for virus culturing. EFFECT: improved method of induction and immune analysis. 6 cl, 4 ex

Description

 This invention relates to the general fields of recombinant protein expression and virology. More specifically, the invention relates to glycoproteins suitable for the diagnosis, treatment and prevention of hepatitis C virus infection and to methods for producing such glycoproteins.

Prototypes of the invention
Hepatitis NIA-NIV (GNANV) is a vector-borne disease (or group of diseases), apparently of a viral etiology, different from other forms of liver damage associated with the virus, such as those caused by hepatitis A virus (HAV), hepatitis B virus (HBV) , hepatitis delta virus (IOP), cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Epidemiological data suggest that there can be three types of STDW: the epidemic type, which is transmitted through water, the type that is transmitted through blood or injections, and the type that sporadically appears in the population. The number of pathogens causing it is unknown. However, new types of viruses have recently been isolated, hepatitis C virus (HCV) as the main (if not the only) cause of blood-transmitted GNANV (BB-GNANV). See, for example, PCT WO 89/046699. Hepatitis C is the main form of transfusion-related hepatitis in many countries and regions, including the United States, Europe, and Japan. There is also evidence of HCV involvement in the development of hepatocellular carcinoma. Therefore, there is a need for an effective way to prevent and treat HCV infection.

 The need for sensitive specific techniques for screening and identifying carriers of HCV and infected HCV blood and blood products is significant. Post-transfusion hepatitis (PTT) occurs in approximately 10% of patients who have received a blood transfusion, and HCV causes up to 90% of these cases. The main problem of this disease is that it often causes chronic progressive liver damage (25-55%).

 Treatment of a patient, as well as preventing transmission of HCV with blood and blood products or in close contact with staff, requires available diagnostic and prognostic tools to detect nucleic acids, antigens, and antibodies associated with HCV. In addition, there is also a need for effective vaccines and immunotherapeutic drugs to prevent and / or treat this disease.

 HCV appears in the blood of infected individuals in very small amounts compared to other infectious viruses, which makes the virus very difficult to detect. A low concentration of the virus is probably the main reason that the causative agent of GNIV has remained undetected for so long. Even though it was currently cloned, HCV is still difficult to cultivate and propagate. Accordingly, recombinant methods for obtaining diagnostic (therapeutic) prophylactic HCV proteins are very necessary.

 In addition, a HCV vaccine is needed. However, growing HCV in cell culture is very difficult. Therefore, it was not possible to derive attenuated virus strains using serial passages on tissue / cell cultures.

SUMMARY OF THE INVENTION
When expressed in recombinant systems, it was found that two HCV proteins, E1 and E2, appear as membrane-linked asialoglycoproteins. This was unexpected, because glycoproteins usually do not remain in mammals in the form with mannose at the end, but undergo further modifications with other carbohydrates: the form ending in mannose, in typical cases, is only transitional. In E1 and E2 (how they were expressed in our systems), this asialoglycoprotein seemed to be the final form. E1 (envelope protein 1) is a glycoprotein with a molecular weight of about 35 kD, which is translated from the predicted E1 region of the HCV genome. E2 (envelope protein 2) is a glycoprotein with a molecular weight of about 72 kD, which translates with the predicted NSI region (non-structural protein 1) of the HCV genome based on the flavovirus model of HCV. Since viral glycoproteins are often highly immunogenic, E1 and E2 are the first candidates for use in immunoassays and therapeutic / prophylactic vaccines.

 The discovery that E1 and E2 are not sialylated is significant. The particular form of the protein often suggests which cells may serve as a suitable host for recombinant expression. Prokaryotes, such as E. coli, do not glycosylate proteins and, in general, are not suitable for the production of glycoproteins used as antigens, because glycosylation is often important for complete antigenicity, solubility and stability of the protein. Lower eukaryotes, such as yeast and fungi, glycosylate proteins, but are generally not able to add terminal sialic acid residues to carbohydrate complexes.

 Therefore, proteins obtained in yeast can be antigenically different from their natural (non-recombinant) analogues.

 Expression in mammalian cells is preferred in cases where the antigenicity of the product is important, since glycosylation of the recombinant protein should be very similar to that of proteins of natural viruses.

 New data indicate that, upon infection, the HCV virus can enter the host cells either through the asialoglycoprotein receptor found on hepatocytes or through the mannose receptor found on liver endothelial cells and macrophages (especially Kupffer cells). It was unexpectedly found that the mass of natural E1 and E2 does not contain terminal sialic acid residues, but only a glycosylated core. Small fractions additionally contain terminal N-acetylglucosamine. Accordingly, an object of the present invention is to provide HCV envelope glycoproteins lacking all or almost all of the terminal sialic acid residues.

 Another aspect of the invention is a method for producing asialo-E1 or E2 under conditions of suppressing the attachment of terminal sialic acid, for example, by expression in yeast or expression in mammalian cells, using antibiotics to facilitate secretion or excretion.

 Another aspect of the invention is a method for purifying E1 and E2 by affinity for lectins that bind terminal mannose residues or terminal N-acetylglucosamine residues.

 Another aspect of the invention is an immunogenic composition comprising a recombinant asialoglycoprotein selected from the group consisting of HCV E1 and E2 in combination with a pharmaceutically acceptable carrier. Optionally, an immunological adjuvant may be included.

 Another aspect of the invention is an immunoassay reagent comprising a recombinant asialoglycoprotein selected from the group consisting of HCV E1 and E2 in combination with a suitable carrier. Another immunoassay reagent of this invention consists of a recombinant asialoglycoprotein selected from the group consisting of HCV E1 and E2, in combination with a suitable detectable label.

 Another aspect of the invention relates to higher order dimers and aggregates E1 and / or E2. One type of invention is the E2 complex. Another variation of the invention is the heterodimer E1: E2.

 Another aspect of the invention is a HCV vaccine composition consisting of E1: E2 aggregates and a pharmaceutically acceptable carrier.

 Another aspect of the invention is a method for purifying E1: E2 complexes.

 Another aspect of the invention is a method for propagating HCV in a cell culture, comprising: (a) providing a cell expressing a receptor selected from the group consisting of mannose receptor and asialoglycoprotein receptor; (b) HCV cell infection; (c) growing an infected cell. Preferably, the cell expresses a recombinant receptor.

Methods of carrying out the invention
A. Definitions
The term “asialoglycoprotein” refers to a glycosylated protein that is substantially free of sialic acid residues. Asialoglycoproteins can be obtained recombinantly, either by purification from cell culture, or from natural sources.

 Asialoglycoproteins derived from HCV are currently preferred, preferably glycoproteins E1 and E2, most preferably recombinant E1 and E2 (pE1 and pE2). A protein "substantially free" of sialic acid falls within this definition if the amount of sialic acid residues does not significantly affect glycoprotein binding to mannose binding proteins such as GNA. This degree of sialylation is generally achieved if sialic acid is less than 40% of the total amount of N-linked carbohydrates, more preferably less than 30%, more preferably less than 20%, more preferably less than 10, more preferably less than 5% and most preferably less 2%

 The term “E1” is used herein to refer to a protein or polypeptide expressed within the first 400 amino acids of the HCV polyprotein, a site sometimes referred to as protein E or S. In its natural form, it is a 35 kD glycoprotein strictly bound to the membrane. In most natural HCV strains, protein E1 is encoded in the viral polyprotein after protein C (core).

 The E1 protein is located between approximately 192 and 383 amino acids of a full-sized polyprotein. The term "E1" also includes analogs and truncated mutants that give cross-immunological reactions with natural E1.

 The term “E2” is used herein to refer to a protein or polypeptide expressed within the first 900 amino acids of a HCV polyprotein, a site sometimes referred to as an NSI protein. In its natural form, it is a 72 kD glycoprotein strictly bound to the membrane. In most natural strains of HCV, protein E2 follows protein E1.

 Protein E2 is located between approximately 384 and 820 amino acids. The term "E2" also includes analogs and truncated mutants that give cross-immunological reactions with natural E2.

 The term “aggregate” is used herein to mean a complex E1 and / or E2 containing more than one monomer E1 or E2. In the framework of this definition, the E1: E1 and E2: E2 dimers and the E1: E2 heterodimers are “aggregates”. The compositions of the invention may also include larger aggregates with a molecular weight exceeding 800 kD.

 The term "particle" is used here to mean an aggregate E1, E2 or E1 / E2, visible by electron microscopy and having a size of at least 20 nm. Preferred particles are those that are approximately spherical in shape and about 40 nm in diameter by electron microscopy.

 The term “purified” is used herein to refer to the proteins in the composition, wherein the target protein is at least 35% of the total protein component of the composition. Preferably, the target protein is at least 40%, more preferably at least 50%, more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90% and most preferably at least 95% of the total protein component. The composition may contain other compounds, such as carbohydrates, salts, lipids, solvents, etc., without violating the determination of the percentage of purification used here. An “isolated” HCV asialoglycoprotein means a composition of the HCV asialoglycoprotein purified by at least 35%.

 "Mannose binding protein" here means a lectin or other protein specifically binding to proteins with glycosylation ending in mannose (e.g. asialoglycoproteins), for example, mannose binding lectins, antibodies specific for glycosylation ending in mannose, mannose receptor protein ( RAB Ezekowitz et al., J. Exp. Med. (1990) 176-1785-94), asialoglycoprotein receptor proteins (H. Kurata et al., J. Biol Chem. (1990) 265: 11295-98), whey protein binding mannose (1. Schuffenecker et al., Cytoqenet. Coll Genet. (1991) 56: 99-102; K. Sastry et al., J. Immunol. (1991) 147: 692-97 ), whey protein binding asialoglycoprotein, etc. Mannose binding lectins include, for example, GNA, concanavalin A (ConA), and other lectins with similar binding properties.

 The term "GNA lectin" refers to agglutinin of the plant Galanthus nivalus, a commercial lectin that binds glycoproteins ending in mannose.

 A "recombinant" glycoprotein is here referred to as a glycoprotein expressed from a recombinant polynucleotide in which a structural gene encoding a glycoprotein is expressed under the control of a regulatory sequence not naturally attached to the structural gene, or in which the structural gene is modified.

 For example, you can create a vector in which the structural gene E1 is placed under the control of a functional fragment of the promoter of glyceraldehyde-3-phosphate dehydrogenase yeast (GAPDH). Currently, the preferred promoter for use in yeast is the ADH2 / GAF hybrid promoter described in US Pat. No. 4,880.73, which uses a fragment of the HAFDH promoter in combination with the preceding activation sequence derived from alcohol dehydrogenase 2. Modification of the structural gene may include substitution of various codons by degenerate codons (e.g., use codons preferred by the host, eliminate or create restriction sites, control hairpin formation, etc. .), And substitution, insertion or deletion of a limited number of codons encoding different amino acids (preferably no more than 10%, more preferably less than 5% of the size of the natural amino acid sequence subject to change), etc. In the same way, a “recombinant” receptor is a receptor protein expressed from a recombinant polynucleotide in which a structural gene encoding the receptor is expressed under the control of regulatory sequences not naturally attached to the structural gene, or in which the structural gene is modified.

 The term "isolated polypeptide" refers to a polypeptide substantially free of other components of the HCV virus, in particular polynucleotides. A polypeptide composition is "substantially free" of other components if the weight of the polypeptide in the composition is at least 70% by weight of the polypeptide and the other component connected to it, more preferably at least 80%, even more preferably about 90%, and most preferably 95% or more.

 For example, a composition containing 100 μg / ml E1 and only 3 μg / ml of other HCV components (e.g. DNA, lipids, etc.) is substantially free of "other HCV components" and thus is an isolated polypeptide composition within of this definition.

 The term "secretion leader" refers to a polypeptide that, when encoded on the amino terminus of a protein, causes, after translation, secretion of the protein into the culture medium of the host cells. The secretion leader is typically obtained from the host cell used.

 For example, secretion leaders suitable for use in yeast include the alpha factor leader Saccharomyces cerevisial (see US Pat. No. 4,870.008).

 The term “lower eukaryotes” refers to host cells such as yeast, fungi, etc. Lower eukaryotes are typically (not necessarily) unicellular. Preferred eukaryotes are yeast, especially species related to Saccharomyces, Schizosaccharomyces, Kluveromyces, Pichia, Hansenuba and the like. Saccharomyces cerevisiae, S. carisbergensis, and K. lactis are the most commonly used yeast hosts and common fungal hosts.

 The term "higher eukaryote" refers to host cells obtained from higher animals, such as mammals, reptiles, insects, and the like.

 Higher eukaryotic host cells obtained from the Chinese hamster (e.g., Chinese hamster ovary CHO cells), monkeys (e.g. COS cells), humans and insects (e.g. spodoptera frugiperda) are currently preferred. Host cells can be presented as a suspension or culture in a mattress, tissue cultures, organ cultures, and the like.

 The term “calcium modulator” refers to a compound capable of sequestering or binding calcium ions in the endoplasmic reticulum or altering the concentration of calcium ions in the EPR by acting on proteins that regulate calcium metabolism (for example, calcium tubule proteins, calcium pumps, etc.). Suitable calcium modulators include, for example, thapsigargin, EGTA (ethylene glycol bis / beta-aminoethyl ether / - N, N, N ', N' - tetraacetic acid). Currently, the preferred modulator is thapsigargin (see, for example, O. Thastrup et al., Proc. Nat. Acad. Sci. USA (1990) 87: 2466-70).

 The term “immunogenic” refers to the ability of a substance to elicit a humoral and / or cellular immune response, either alone or in a state associated with a carrier, with or without an adjuvant. "Neutralization" refers to an immune response that blocks infectivity, either partially or completely, of an infectious agent. A "vaccine" is an immunogenic composition capable of providing partial or complete protection against HCV, useful for treating an individual.

 The term "biological fluid" refers to a fluid obtained from the body, such as serum, plasma, saliva, gastric juice, mucus, and the like. In general, body fluids are screened for the presence of HCV particles. Some body fluids are used as a source of other products, such as coagulation factors (for example, factor VIII: C), serum albumin, growth hormone, etc. In such cases, it is important that the source of body fluid is free from contamination with a virus, such as HCV.

B. General method
The E1 region of the HCV genome is described in EP 388.232 as region "E", while E2 is described as "NS1". Region E1 includes approximately amino acids 192-383 of a full-sized viral polyprotein. Region E2 includes approximately amino acids 384-820. Complete prototype sequences of these proteins (HCV-1 strain) are available to the person skilled in the art (see EP 388.232), as they are common 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-translational processing in most eukaryotic host cells cleaves the original polyprotein into C, E1 and H2. The 5 ′ end of the coding region can be truncated in order to reduce the amount of protein C produced.

 The expression of asialoglycoproteins can be obtained in many ways. For example, expression can be obtained in lower eukaryotes (such as yeast), which normally do not add sialic acid residues to glycosylated proteins. In yeast expression systems, it is currently preferred to use a secretion leader, such as S.cerevisial alpha factor leader, so that the protein is expressed after translation into the culture medium. It is also currently preferred to use glycosylation-deficient mutants such as pmr1, as these mutants only provide core glycosylation and often secrete heterologous proteins with high efficiency (N.K. Pudolph et al., Cell (1989) 58: 133-45 ) Alternatively, other types of yeast, such as Pichia pastaris, which express glycoproteins containing 8-9 mannose residues in a form resembling what is believed to be the type of core glycosylation observed in mammals in S. cerevisia, can be used.

 Alternatively, expression can be induced in mammalian cells and block terminal glycosylation (sialic acid addition). Recombinant constructs will preferably include a secretion signal to ensure that the protein is directed into the endoplasmic reticulum. Transport to the Golgi apparatus seems to be blocked by E1 and E2 themselves: the high level of E1 or E2 expression in mammalian cells seems to stop the secretion of all cell proteins in the endoplasmic reticulum or in the Golgi apparatus. You can additionally use a mutant with a glycosylation defect. See, for example, P. Stanlcy, Ann. Rev. Genet. (1984) 18: 525-52. In the case of glycosylation or transport, the mutant expresses E1 or E2 with sialylation, then the terminal sialic acid residue can be removed by treatment with neuraminidase.

 In the future, yeast should be stimulated with a calcium modulator in order to obtain protein secretion into the endoplasmic reticulum. Suitable modulators include thapsigargin and A23817 (see, for example, O.Thastup et al., Proc. Nat. Accd. Sci. USA (1990) 87: 2466-70). For example, a large amount of E1 or E2 can be expressed intracellularly in mammalian cells (e.g., CHO, COS, HeLa cells, etc.) by transfection with a recombinant vaccinia virus vector. After waiting until the protein is expressed and accumulates in the endoplasmic reticulum, the cells are treated with a calcium modulator in a concentration high enough to cause the release of ER contents. Then the protein is removed from the culture medium, which is replaced for the next cycle.

 Additionally, it may be advantageous to express a truncated form of the membrane protein. Both E1 and E2 appear to have a highly hydrophobic domain, which apparently retains the protein within the endoplasmic reticulum and inhibits efficient excretion. Therefore, you can optionally remove parts of the sequence found in one or more regions of amino acids 170-190, 260-290 or 330-380 in El (numbering from the beginning of the polyprotein) and 660-830 in E2 (see, for example, Figure 20-1 in EP 388.232). It appears that at least one of these hydrophobic domains forms a transmembrane region that is not essential for the antigenicity of the protein and therefore can be removed without damage. Which region is best to be removed can be determined by a few removal experiments carried out in the usual way. Removal of the hydrophobic 3'-end at E2 results in secretion of a portion of the expressed E2 with sialylation of the secreted protein.

 To obtain expression, you can use any of a number of vectors. Lower eukaryotes, such as yeast, are usually transformed with plasmids by precipitation with calcium phosphate or transfected with a recombinant virus. Vectors can independently propagate in the host cell or can integrate into the genome of the host cell. Higher eukaryotes can be transformed with plasmids, but they are usually infected with a recombinant virus, such as a recombinant vaccinia virus. The vaccinia virus is particularly preferred since infection with the vaccinia virus stops the expression of the host cell proteins. Preferred host cells currently include HeLa cell lines and plasmacytomas. In the presented system, this means that E1 and E2 accumulate as the main glycosylated species in the host ER.

Since pE1 and pE2 will be the predominant mannose-terminated glycoproteins, they can be easily isolated from cells using lectins such as agglutinin Galanthus nivalus (GNA), which binds terminal residues of mannose
Proteins that are naturally expressed as mannose-terminated glycoproteins are relatively rare in mammalian physiology. In most cases, the mammalian glycoprotein has a mannose termination only in a transient intermediate form, which is formed during glycosylation. The fact that recombinantly expressed HCV envelope proteins contain a mannose terminating glycosyl residue or (to a lesser extent) N-acetylglucosamine means that the HCV proteins and whole virons can be separated and partially purified from endogenous proteins using lectins specific for terminal mannose or N-acetylglucosamine. Recombinant proteins appear authentic and are considered to be essentially identical to envelope proteins found in the mature free virion or form of the protein bound to the cell wall. Therefore, lectins such as GNA for mannose-terminated proteins and WCA (wheat seed agglutinin) and their equivalents for N-acetylglucosamine-terminated proteins can be used. Solid phase fixed lectins (e.g., a lectin-sepharose column) can be used to separate E1 and E2 from the supernatant of the cell culture or from other fluids, for example, for purification during preparation of vaccine antigens or for use in immunoassays.

 Alternatively, a suitable lectin can be provided for the isolation of E1, E2 or HCV virions from the fluid or tissues of individuals with suspected HCV infection. Since glycoproteins with a mannose ending are relatively rare, this procedure should serve to purify the proteins present in the sample, significantly reducing the background intensity. After lectin binding, the HCV protein can be detected using anti-HCV antibodies.

 If whole virons are present, it is possible to alternatively detect HCV nucleic acids using polymerase chain reaction (PCR) techniques or other nucleic acid accumulation methods aimed at preserving regions of the HCV genome (e.g., non-coding region 5 ′). This method allows you to isolate and characterize various strains of HCV, regardless of deviations or antigenicity options, for example, in cases where the new strain does not cross-react with the strain used to produce the antibodies. There are many other ways to take advantage of the unique recognition of mannose-terminated glycoproteins using single lectins.

 For example, you can incubate samples presumably containing HCV virons or proteins with biotin- or avidin-labeled lectins, and precipitate the protein-lectin complex with avidin or biotin. You can also use the affinity of lectin for HCV proteins to target compounds to virons for therapeutic purposes, for example, by conjugating an antiviral compound with GNA. Alternatively, suitable lectins can be used to remove mannose-terminated glycoproteins from serum or plasma, thereby reducing or eliminating the risk of HCV contamination.

 It is currently preferred to isolate the E1 and / or E2 asialoglycoproteins from crude cell lysates by incubation with an immobilized mannose binding protein, especially with lectin such as ConA or GNA. Cells are lysed, for example, by mechanical disruption in a hypotonic buffer, then the lysate is centrifuged to prepare a post-nuclear lysate, and centrifuged again to obtain a crude fraction of microsomal membranes. The crude membrane fraction is sequentially solubilized in a buffer containing a detergent, such as X-100, Np40, or the like. This extract with a detergent is subsequently purified from insoluble particles by centrifugation, and the resulting clarified lysate is incubated in a chromatographic column containing an immobilized mannose binding protein, preferably GNA, fixed on a solid support such as agarose or sepharose for a sufficient time for binding, usually for 16-20 hours. Then the suspension is passed through the column until E1 / E2 begins to appear in the eluent, then they are incubated in the column for a time sufficient for binding, usually about 12-24 hours. The bound material is then washed with additional detergent buffer (for example, X-100, NP40, or the like), and eluted with mannose in order to obtain purified asialoglycoprotein.

 When eluting, it is preferable to elute only until a protein begins to appear in the eluate, at which point the elution stops and the column is equilibrated for 2–3 hours before proceeding with the elution of the protein. It is believed that this gives enough time for the remaining large protein aggregates to react. In cases where E1 and E2 are expressed together in their natural form (i.e., without processing the binding domain to the membrane), a substantial part of asialoglycoproteins appears as E1: E2 aggregates. When examined under an electron microscope, a significant part of these aggregates looks like particles of almost spherical shape with a diameter of about 40 nm, which corresponds to the estimated size of the intact virus. These particles appear to be self-organizing viral subunits.

 It is expected that these aggregates should form a quaternary structure very similar to the structure of authentic HCV virion particles, and therefore they are expected to serve as a highly immunogenic vaccine.

 The E1 / E2 complexes can be further purified by gel chromatography on a basic medium, for example, fractogel-DEAE or DEAE-Sepharose. Using fractogel-DEAE gel chromatography, E1 / E2 complexes can be obtained, purified to approximately 60-80%. E1 can be further purified by treatment with a lysine protease, because E1 has a 0-1 Lys residue. Processing the complex with a lysine protease destroys E2 and makes it easy to separate E1.

 The tissue specificity of the HCV, together with the observation that the HCV envelope glycoproteins have mannose endings, suggests that the virus uses the mannose receptor or asialoglycoprotein receptor (ACHR) in order to enter the host cell. Mannose receptors are found on macrophages and liver sinusoid cells, while ASHR is found on parenchymal hepatocytes. Therefore, it should be possible to grow HCV in host cells expressing one or both of these receptors. You can either use primary cultures of cells that naturally express this receptor under conditions that preserve the receptor, or you can enter into another cell line, such as HeLa, CHO, COS, etc., a vector that promotes the expression of this receptor.

 Cloning of the mannose receptor and its transfection and expression in fibroblasts have been demonstrated by M.E. Maylor et al. J. Biol. Chem (1990) 265: 12156-62.

 ASHR cloning and sequencing are 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 ASHR in NTS homologous typing rat cells is described by M. McPhaul and P. Bagg, Pros. Nat Acad. Sci. USA (1986) 83: 8863-67 and M. McPhaul and P. Berg, Mc1. Cell Biol. (1987) 7: 1841-47.

 Thus, it is possible to transfect one or both receptors into suitable cell lines and use the resulting cells as a host for propagating HCV in culture. Serial HCV passages in such a culture should result in attenuated strains suitable for use as a live vaccine. You can either use primary cell cultures that naturally express this receptor, creating the conditions under which the receptor is maintained, or you can transfect another cell line, such as HeLa, CHO, COS, etc., with a vector that allows expression of the receptor.

 Cloning of the mannose receptor and its transfection and expression in fibroblasts have been shown by Taylor et al. (see above), while transfection and expression of functional ASHR in rat homozygous typing cells is described by McPhaul et al. (see above).

 Currently, it is preferable to use the immortal line of cells transfected with one or both of the recombinant receptors.

 Immunogenic compositions can be prepared by known methods. The present compositions include an immunogenic amount of a polypeptide, for example a composition of particles of E1, E2 or E1 / E2, usually combined with a pharmaceutically acceptable carrier, preferably further comprising an adjuvant.

 If a “cocktail”, a combination of HCV polypeptides, is desired, for example, you can mix E1 and E2 antigens together to increase efficiency. Aggregates of virus-like particles of E1 / E2 are expected to present a particularly useful vaccine antigen. Immunogenic compositions can be administered to animals to cause the formation of antibodies, or to obtain a source of antibodies, or to create protective immunity in the animal.

 Pharmaceutically acceptable carriers include any carrier that does not itself produce antibodies that are harmful to the individual receiving the composition. Suitable carriers are typically large, slowly degradable macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers and inactive virus particles. Such carriers are well known in the art.

 Among the preferred adjuvants that enhance the effectiveness of the composition include, but are not limited to: aluminum hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr - MDP), found in US patent N 4.606.918, N-acetyl-norumramyl-L-alanyl-D-isoglutamine (hor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanyl-2- (1'-2'-dipalmitoyl-Sn-glycero- 3-hydroxyphosphoryloxy) ethylamine (MTP-PE) and R1B1, which contains three components extracted from bacteria, monophosphoryl lipid A, trialose dimicolate and cell wall skeleton (MPL + TDM + CWS) in a 2% squalene / twin emulsion 80.

 Additionally, adjuvants such as a stimulon (Cambridge Bioscience, Morcester, MA) can be used. Further, in preparations not intended for humans, and for research purposes, you can use Freund's complete adjuvant (PAF) and Freund's incomplete adjuvant (NAF).

 Immunogenic compositions will usually contain pharmaceutically acceptable excipients, such as water, saline, glycerin, ethanol, etc. Additionally, excipients, such as wetting or emulsifying agents, pH buffering agents and the like, may be included among such excipients.

 Typical immunogenic compositions are prepared in an injectable form, either as a liquid solution or as an emulsion, and dry forms suitable for dissolution in a liquid vehicle can also be prepared to form a solution or suspension before injection. Drugs can also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

 Immunogenic compositions used as vaccines include an immunologically effective amount of the HCV polypeptide, as well as other aforementioned components as needed.

 “Immunologically effective amount” means that administering such an amount to an individual, either once or as one of several subsequent doses, is effective for treatment as defined above. This amount varies, depending on the health and physical condition of the individual to whom it is administered, the taxonomic group of individuals exposed (e.g., primates, primates, etc.), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, and the composition of the vaccine , assessing the medical situation by the attending physician, the HCV strain that caused the infection, and other relevant factors.

 It is expected that this amount will fluctuate over a fairly wide range, which can be determined by routine testing.

 These self-assembled E1 / E2 aggregates can also serve as vaccine carriers for heterologous (non-HCV) haptens in the same way as hepatitis B surface antigen (see European Patent Application 174.444). In this application, E1 / E2 aggregates represent an immunogenic carrier capable of stimulating an immune response to haptens or antigens conjugated to the aggregate.

 The antigen can be conjugated either by conventional chemical methods, or it can be cloned into a gene encoding E1 and / or E2, with a localization corresponding to the hydrophilic region of the protein.

 Immunogenic compositions are customarily administered parenterally, usually by subcutaneous or intramuscular injection. Additional prescriptions suitable for other modes of administration include those for oral administration and suppositories. The therapeutic dosage can be administered once or in several doses according to the scheme. The vaccine can be administered in conjunction with other immunoregulatory agents.

B. Examples
The following examples are provided as further guidance to a practitioner and should not be construed as limiting the invention.

EXAMPLE 1 (Cloning and expression)
(A).

Vectors were constructed from plasmids containing the 5'-part of the HCV genome, as described in EP 318.216 and EP 388.232. The HCV cassette (S / B) contains a StuI-Bgt II DNA fragment encoding the 5'-end of the polyprotein from Met ₁ to Lei ₉₀₆ , starting with nucleotide-63, referring to Met ₁ . These include core protein (C), protein E1 (also sometimes referred to as S), protein E2 (also referred to as NSI), and the 5 ′ portion of the NS2a region. After expression of this construct, it is separated by proteolysis into individual proteins C, E1 and E2.

The HCV cassette (A / B) contains an ApaLI-BgIII DNA fragment encoding the 5'-end of the polyprotein from Met ₁ to Lei ₉₀₆ , starting from nucleotide-6, referring to Met ₁ . These include core protein (C), protein E1 (also sometimes referred to as S), protein E2 (also referred to as NSI), and the 5 ′ portion of the NS2a region.

 After expression of this construct, the individual proteins C, E1, and E2 are obtained by proteolytic treatment.

Cassette C-E1 (S / B) (part StuI - BamHI) contains the 5'-end from Met ₁ to Ile ₃₄₀ (BamHI site in the gene). Expression of this cassette leads to expression of C and, to some extent, truncated E1 (E1 '). The part cut from the 3'-end is a hydrophobic region, which is considered to serve as a broadcast signal.

The NSI (I / O) cassette (part BamHI - BgIII contains a small part 3 'from E1 (from Met ₃₆₄ ), all E2 and part HC2a (to Lei ₉₀₆ ). In this constructor, fragment E1 serves as a translocation signal.

The TPA-NSI cassette uses the human tissue plasminogen activator leader (tPA) as a translocation signal instead of the 3 ′ portion of E1. This cartridge contains a truncated form of E2 from Gly ₄₀₆ to Glu ₆₆₁ , in which the hydrophobic end 3 'is lost.

 Each cassette is inserted into the pGEM3Z vector (Promega) with or without a synthetic beta-globin non-coding sequence for in vitro transcription and translation using expression of T7 and rabbit reticulocyte.

 Recombinant vaccinia virus (rVV) vectors were prepared by inserting cassettes into plasmid pSC11 (obtained from Dr. B. Moss, NIH), followed by recombination with vaccinia virus, as described by Charkrabarty et al., Mol. Cell Biol. (1985) 5: 3403-09.

 (IN).

 An alternative expression vector is obtained by embedding HCV (A / B) in pS59 (obtained from Dr. B. Moss NIH) between the StaI and SpeI sites, followed by recombination with vaccinia virus, as described by Charkrabarty et al., Mol. Cell Biol. (1985) 5: 3403-09.

 (FROM).

 HeLa cells were harvested by centrifugation for 7 minutes at 2000 rpm at room temperature in 500 ml sterile centrifuge bottles (JA-10 rotor).

Precipitation was resuspended in additional culture medium (Joklik - modified MEM Spenner medium + 5% horse serum and gentamicin) ("centrifuge medium") to a final concentration of 2 × 10 ⁷ cells / ml. Ultrasound-destroyed untreated mass of vv / SC59-HCV virus was added to a plurality of infections in 8 plaque forming units / cell and the mixture was stirred for 30 minutes at 37 ^° C. Then, the infected cells were transferred to a centrifuge vial containing 8 L of centrifuge medium and incubated for 3 days at 37 ^o C.

Then, the cultured cells were collected by centrifugation and the pellets resuspended in buffer (10 mM Tris-HCL, pH 9.0, 152 ml). Then the cells were homogenized with a 40 ml Dounce homogenizer (50 strokes) and the nuclei were precipitated by centrifugation (5 minutes, 1600 rpm, 4 ^° C, JA-20 rotor). Nuclear pellets were resuspended in Tris buffer (24 ml), rehomogenized, and pelleted again, collecting all supernatants.

The collected lysate was divided into 10 ml portions and destroyed by ultrasound for 3 x 30 minutes in a sonicator in the form of a wine horn at medium power. The treated lysate (15 ml) was layered on 17 ml sucrose pads (36%) in SW 28 centrifuge tubes and centrifuged for 80 minutes at 13,500 rpm at 4 ^° C in order to precipitate the virus. The virus pellet was resuspended in 1 ml Tris-buffer (1 mm Tris-HCL, pH 9.0) and frozen at -80 ^o C.

EXAMPLE 2 (Comparison of products in vitro and in vivo)
(A)
E1 and E2 both expressed in vitro and in vivo and ³⁵ S-Meth methyls used the vectors described above in Example 1. BSC-40 and HeLa cells were infected with rVV vectors for in vivo expression. Nutrient medium and cell lysates were tested for recombinant proteins. Human HCV products were immunoprecipitated with immune serum, while in vitro proteins were analyzed by direct method. The resulting proteins were analyzed by SDS-PAGE chromatography.

 The reticulocyte expression system (pGFM3Z with HCV (S / B) or HCV (A / B) produced proteins C, E1 and E2 with a molecular weight of approximately 18 kD, 35 kD and 72 kD, respectively. BSC-40 and HeLa cell lysates after transfection rVVs containing HCV (S / B), HCV (A / B), or C-E1 (S / B) produced the same proteins because the reticulocyte system does not provide efficient processing in the Golgi apparatus and therefore does not provide sialic acid, the products, both obtained in vitro and obtained in vivo, exhibit the same mobility, suggesting that prot Eins do not sialylate in vitro Only the rVV vector containing TPA-NSI induces some extracellular secretion of E2 with altered mobility corresponding to sialylation.

(IN)
HCV (S / B) was expressed in vitro and incubated with a set of biotinylated lectins: GNA, SNA, PNA, WGA and ConA.

 After incubation, the complexes were collected on avidin-acrylic beads, washed, eluted with sample Lamley buffer and analyzed by SDS-PAGE chromatography. The results showed that E1 and E2 are fixed on GNA and ConA, which indicates the presence of mannose. GNA binds to terminal mannose groups, while ConA binds to any alpha-linked mannose. Lack of binding to SNA, PNA and WGA indicates that none of the proteins contains sialic acid, galactose-N-acetylgalactosamine or N-acetylglucosamine.

(FROM)
Radioactive-labeled E1 and E2 were obtained in BSC-40 cells by infection with rVV containing HCV (S / B) (vv / SC11-HCV) and immunoprecipitation with human HCV ⁺ immune serum was performed. Half the precipitated material was treated with neuraminidase overnight to remove any sialic acid.

 After treatment, the processed and untreated proteins were analyzed by SDS-PAGE chromatography. There was no significant difference in mobility, indicating a lack of in vivo sialization.

(D)
Radioactive-labeled E1 and E2 were obtained in BSC-40 cells by infection with rVV containing HCV (A / B) (vv / S C 59-HCV), and either HCV ⁺ human serum was immunoprecipitated or precipitated with biotinylated GNA lectin bound to acrylic beads using vv / S 11 free of HCV sequences as a control. Precipitates were investigated by SDS-PAGE chromatography. The data showed that E1 and E2 were the main types of mannose-terminated proteins in vv / SC 59-HCV infected cells. GNA, as well as human antiserum, precipitated E1 and E2 from cell culture medium. A 25 kD component was observed, but it seemed specific to cells infected with the vaccinia virus.

EXAMPLE 3 (Purification using lectin)
(A)
A mass of purified high-titer vv / SC 59-HCV virus was inoculated into HeLa cells with a plurality of infections in 5 plaque forming units / cell and the mixture was stirred for 30 minutes at 37 ^° C. Then, the infected cells were transferred to a centrifuge bottle containing 8 L of centrifuge medium and incubated 3 days at 37 ^° C. The cells were again harvested by centrifugation and resuspended in hypotonic buffer (20 mM HEPES, 10 mM NaCl, 1 mM MgCl ₂ , 120 ml) on ice. Then the cells were homogenized with a Dounce homogenizer (50 strokes) and the nuclei were precipitated by centrifugation (5 minutes, 1600 rpm, 4 ^° C, JA-20 rotor). Precipitation was collected, resuspended in 48 ml of hypotonic buffer, re-homogenized, centrifuged, collected again and frozen at -80 ^o C.

Then, the frozen supernatants were thawed and the fraction of microsomal membranes from the post-nuclear lysate was isolated by centrifugation for 20 minutes in a JA-20 rotor at 13,500 rpm at 4 ^° C. The supernatant was removed by suction.

The pellets were shaken in 96 ml of detergent buffer (20 mM Tris-HCI, 100 mM NaCl, 1 mM EDTA, 1 mM DDT, 0.5% Triton-X-100, pH 7.5) and homogenized (50 strokes). The product was clarified by centrifugation for 20 minutes at 13,500 rpm 4 ^° C. and supernatants were collected.

A column with GNA agarose (1 cm x 3 cm, 3 mg GNA / ml beads, 6 ml sorbent volume, Vector Lals, Burlingame, CA) was pre-equilibrated with detergent buffer. A sample of the supernatant was introduced into the column with recirculation at a flow rate of 1 ml / min for 16-20 hours at 4 ^o C. Then the column was washed with detergent buffer.

 The purified E1 / E2 proteins were eluted with alpha-D-mannoside (0.9 M in detergent buffer) at a flow rate of 0.5 ml / min. Elution was stopped when E1 / E2 appeared in the eluent and the column was allowed to equilibrate again for 2–3 hours. Fractions were analyzed by Western blotting and silver staining. Peak fractions were collected together and irradiated with UV to inactivate any residues of the vaccinia virus.

(IN)
Asialoglycoproteins E1 and E2, purified by GNA agarose, were sedimented in 20-60% glycerol. Gradients were separated and proteins analyzed by SDS-PAGE and Western blotting. Spots probed GNA to identify E1 and E2.

 The results indicate the presence of an E1: E2 heterodimer precipitating at the expected rate (i.e., a position characteristic of a 110 kD protein). Larger aggregates of HCV envelope proteins also appear. E2: E2 homodimers also appeared. It seemed that E2 prevailed over E1 in larger varieties, although individual varieties of E1: E2 were also detected.

 Larger aggregates precipitated much faster than the thyroglobulin marker.

(FROM)
E1 and E2 purified by GNA agarose were precipitated in a gradient of 20-60% glycerol with 1 mM EDTA. Fractions were analyzed by SDS-PAGE with and without beta-mercaptoethanol (ME). The amount of E1 and E2 was almost independent of the presence of ME, which indicated the absence of disulfide bonds between heterodimers.

(D)
E1 / E2 complexes (approximately 40% purity) were analyzed on a Coulter DM-4 submicron particle analyzer. Material was detected in the range of 20-60 nm.

(E)
The E1 / E2 complexes (approximately 40% purified) were examined under an electron microscope with negative contrast with tungsten phosphoric acid.

 Electronic micrograms revealed the presence of spherical particles with a diameter of about 40 nm. The E1 / E2 complexes were incubated with HCV + human immune serum, and then examined under EM with negative contrast. Antibody complexes were observed containing large aggregates and smaller particles.

EXAMPLE 4 (Chromatographic purification)
(A)
The lectin-purified GNA material prepared as described in Example 3 (0.5-0.8 ml) was diluted 10 times with buffer A (20 mM Tris-Cl buffer, pH 8.0; 1 mM EDTA) and applied to the column Fractogel EMD DEAE-650 1.8 x 1.5 cm (EM Separations, Gibbstown, New Jersey, catalog N 16883), balanced in buffer A.

The protein fraction containing E1 / E2 was eluted with the same buffer at a rate of 0.2 ml / min and 1 ml of fractions was collected. Fractions containing E1 and E2 (determined by SDS-PAGE) were combined and stored at -80 ^o C.

(IN)
The material purified by the above method (A) according to the results of SDS-PAGE, was purified by 60-80%. The identification of the bands of putative E1 and E2 was confirmed by analysis of the N-terminal sequence after the transfer procedure. For this, the E1 / E2 material purified by Fractogel-DEAE was reduced by the addition of Lammley buffer (pH 6.8, 0.06 M Tris-Cl, 2.3% SDS, 10% glycerol, 0.72 M beta-mercaptoethanol) and boiled 3 minutes. Then the sample was loaded into a 10% polyacrylamide gel.

 After chromatography, SDS-PAGE protein was transferred onto a 0.2 μm polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Richmond, CA). The strips corresponding to the putative proteins E1 and E2 were cut from the spot and subjected to N-terminal amino acid analysis, although no special measures were taken to prepare the material to prevent amino terminal blocking. The first 15 cycles showed that sample E1 has the sequence: Tir-Gln-Val-Arg-X-Ser-Tre-Gli-X-Tir-Gis-Val-X-Asn-Asp, whereas the sequence E2 was: Tre-Gis -Val-Tre-Gli-X-X-Ala-Gli-Gis-X-Val-X-Gli-Fen. These amino acid sequence determination data are consistent with what was expected from the corresponding DNA sequences.

 Product E1 / E2, purified as described above by Fractogel-DEDE chromatography, is considered aggregated on the basis that a large amount of E1 and E2 are co-eluted into the free volume region of the Bio-Sil TSK-4000 SW gel filtration column. This indicates that, in vivo, a significant amount of the E1 / E2 complex has a molecular weight of at least 800 kD. The presence of E1 / E2 material with a molecular weight of about 650 kD was also observed.

Claims (6)

 1. Hepatitis C virus (HCV) asialoglycoprotein selected from the group consisting of a truncated glycoprotein expressed from the HCV El region, which contains a deletion in the portion of the sequence found in the amino acid region 330-380 covering the El region numbered from the beginning of the HCV polyprotein and truncated glycoprotein expressed from the HCV E2 region containing a deletion in part of the sequence found in the region covering amino acids 660-830 of the E2 region numbered from the beginning of the HCV polyprotein.  2. The asialoglycoprotein according to claim 1, characterized in that said truncated glycoprotein is expressed from the E1 region of HCV and contains a deletion in part of the sequence found in the region covering amino acids 330-380 of the E1 region numbered from the beginning of the HCV polyprotein.  3. The asialoglycoprotein according to claim 1, characterized in that said truncated glycoprotein is expressed from the E2 region of HCV and contains a deletion in part of the sequence found in the region covering amino acids 660-830 of the E2 region numbered from the beginning of the HCV polyprotein.  4. An immunogenic composition for diagnosing, treating, or preventing hepatitis C virus infection, comprising hepatitis C virus asialoglycoprotein (HCV) according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.  5. A method of inducing an immune response with an antigen in an animal, comprising the steps of: obtaining a composition comprising an effective amount of an immunogenic substance in a pharmaceutically acceptable carrier; and administering said composition to said animal, wherein the immunogenic substance comprises HCV asialoglycoprotein according to any one of claims 1 to 3.  6. An immunoassay method for detecting antibodies to HCV in a biological sample, including obtaining a biological sample, obtaining a polypeptide reagent, reacting the biological sample with a polypeptide reagent under conditions providing for the formation of antigen-antibody complexes and determining the formation of an antigen-antibody complex containing said polypeptide reagent, characterized in that the composition according to claim 4 is used as the polypeptide reagent.