Asialoglycoprotein of hepatitis c virus (НСv), immunogenic composition, the method induction of the immune response, the method immunoassay


(57) Abstract:

The invention relates to diagnosis, treatment and prevention of hepatitis C. Two envelope protein of hepatitis C (E1 and E2) are expressed without sililirovanie. Recombinant expression of these proteins in lower eukaryotes or in mammalian cells blocked at the terminal glycosylation, gives recombinant proteins that are more similar to natural glycoproteins of hepatitis C virus When they are isolated by means of affinity to the lectin GNA, proteins E1 and E2 are connected in virus-like particles. Cells bearing the mannose receptor or receptor asialoglycoprotein, HCV can infect and be used for the cultivation of the virus. 4 S. and 2 C.p. f-crystals.

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

Prototypes of the invention

Hepatitis NIA-NIV (GNNV) is a vector-borne disease (or group of diseases), apparently, viral etiology, as distinct from other forms of paragem hepatitis Delta (IOP), cytomegalovirus (CMV) or Epstein-Barr (EBV). Epidemiological data suggest that GNAW can be of three types: epidemic-type, water-borne type, blood-borne, or by injection, and type, sporadically appearing in the population. The number of pathogens causing it is unknown. However, recently, have been highlighted new types of viruses, the hepatitis C virus (HCV) as a major (if not the only) cause blood-borne GNAW (HV GNNV). See, for example, PCT WO 89/046699. Hepatitis C is the main form of hepatitis associated with blood transfusion in many countries and regions, including the United States, Europe and Japan. There is also information about the involvement of HCV in causing hepatic cell carcinoma. There is therefore a need for an effective method of prevention and treatment of HCV infection.

The need for sensitive, specific methods for screening and identifying carriers of HCV and HCV infected blood and blood products is significant. Post-transfusion hepatitis (ITL) occurs in approximately 10% of patients receiving a blood transfusion and HCV causes up to 90% of these cases. The main problem of this disease is that it often causes peredachi HCV from blood and blood products or in close contact with the staff, requires available diagnostic and prognostic tools for the detection of nucleic acids, antigens and antibodies associated with HCV. In addition, there is also the need for effective vaccines immunotherapeutics therapeutic drugs for the prevention and/or treatment of this disease.

HCV appears in the blood of infected individuals in very small quantities compared to other infectious viruses, because of what this virus is very difficult to detect. Low concentration of the virus is probably the main reason that the causative agent of GNAW so long remained undetected. Even though at present it has cloned, HCV is still difficult to cultivate and multiply. Accordingly, a very desired recombinant methods of obtaining diagnostic (therapeutic) prophylactic HCV proteins.

In addition, you really need a HCV vaccine. However, to grow HCV in cell culture very difficult. Therefore, it was not possible to deduce weakened strains of the virus through serial passages in tissue culture/cell.

The invention

When ekspressirovannoj in recombinant systems, it was found that two proteins HCV E1 and E2 appear as linked is melting in the form of mannose on the end, and undergo further modifications other carbohydrates: form ending in mannose, in typical cases, is only a transition. The E1 and E2 (as they expressibility in our systems) this asialoglycoprotein, it seemed, was the ultimate form. E1 (envelope protein 1) is a glycoprotein with a molecular weight of about 35 KD, which is broadcast with the predicted region E1 of the HCV genome. E2 (envelope protein 2) is a glycoprotein with a molecular weight of about 72 KD, which is broadcast with the predicted region NSI (non-structural protein 1) of the HCV genome, based on flaviviruses model of HCV. Since the 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 do not sililirovany, is significant. A special form of the protein is often dictates which cells can serve as a suitable host for recombinant expression. Prokaryotes, such as E. coli, not glycosilated proteins and, in General, are not suitable for receiving glycoproteins used as antigens, due to the fact that glycosylation is often important to fully antigenicity, solubility and stabilnosti complexes of terminal sialic acid residues.

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

Expression in mammalian cells is preferred in cases where important antigenicity of the product, since glycosylation of the recombinant protein should really like it the natural proteins of viruses.

New data indicate that during infection with the HCV virus can penetrate into the host cells or through asialoglycoprotein receptor found on hepatocytes or via the mannose receptor, found on endothelial cells of the liver and macrophages (especially on kupperbusch cells). It has been unexpectedly discovered that the mass of natural E1 and E2 does not contain terminal sialic acid residues, but only glycosylated core. A small fraction optionally contain terminal N-acetylglucosamine. Accordingly, an object of the present invention to provide a membrane glycoproteins HCV deprived of all or almost all of the terminal sialic acid residues.

Another aspect of the invention is a method for asialo-E1 or E2 in terms of suppression of attaching a terminal sialic acid, for example, SIP or selection.

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

Another aspect of the invention is an immunogenic composition comprising the recombinant asialoglycoprotein selected from the group consisting of E1 and E2 HCV in combination with a pharmaceutically acceptable carrier. If desired (optional) you can include immunological adjuvant.

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

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

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

Another aspect of the invention is the method of propagation of HCV in cell culture, comprising: (a) providing cells expressing a receptor selected from the group consisting of mannose receptor and receptor asialoglycoprotein; (b) infection of cells by HCV; (b) growing the infected cells. Preferably, the cell expressed recombinant receptor.

Ways of carrying out the invention

A. Definition

The term "asialoglycoprotein" refers to glycosylated protein, which is substantially freed from residues of sialic acid. Asialoglycoprotein can be obtained by recombinant means, or by purification from cell culture or natural sources.

In the currently preferred asialoglycoprotein, resulting from HCV, preferably, the glycoproteins E1 and E2, the most preferably, the recombinant E1 and E2 (pE1 and pE2). Protein, largely exempt" from sialic acid, falls under this definition, if the number of sialic acid residues has little effect on the binding glycoprotein with proteins, mannose-binding, such as GNA. This degree of sililirovanie, in General, dostigaetsya 30%, more preferably less than 20%, more preferably less than 10, more preferably less than 5% and most preferred less than 2%.

The term "E1" is used here to refer to a protein or polypeptide expressed within the first 400 amino acids of polyprotein HCV, plot, sometimes referred to as protein E or S. In natural form it is a glycoprotein 35 KD, strictly related with the shell. Most natural strains of HCV protein E1 is encoded in the viral polyprotein after protein C (core).

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

The term "E2" is used here to refer to a protein or polypeptide expressed within the first 900 amino acids polyprotein HCV, plot, sometimes referred to as protein NSI. In its natural form it is a glycoprotein 72 KD, strictly related with the shell. Most of the natural strains of HCV protein E2 follows E1 protein.

The E2 protein is located approximately between 384 and 820 amino acids. In the concept of the natural E2.

The term "unit" is used here to denote a complex of E1 and/or E2, containing more than one monomer E1 or E2. Under this definition dimers E1:E1 and E2:E2 and heterodimeric E1:E2 are the "units". Compositions of the invention may also include larger aggregates with a molecular weight in excess of 800 KD.

The term "particle" is used here to denote a unit E1, E2 or E1/E2, visible under electron microscopy, and having dimensions of not less than 20 nm. The preferred particles are those that have an approximately spherical shape and a diameter of about 40 nm electron microscopy.

The term "purified" is used here in relation to the proteins in the composition, where a given protein is not less than 35% of the total protein component of the composition. Preferably, the predetermined 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 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 definition about the HCV protein, cleaned not less than 35%.

"Protein, mannose-binding" here means lectin or other proteins that specifically bind to proteins with glycosylation, ending with mannose (for example, asialoglycoprotein), such as lectins, mannose-binding, antibody-specific glycosylation, ending with mannose, a protein receptor mannose (R. A. B. Ezekowitz et al., J. Exp. Med. (1990) 176-1785-94), proteins receptor asialoglycoprotein (H. Kurata et al. , J. Biol Chem. (1990) 265: 11295-98), whey protein, mannose-binding, (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., Lectins, mannose-binding, include, for example, GNA, concanavalin a (CONA) and other lectins with similar binding properties.

The term "lectin GNA" refers to agglutinin plant Galanthus nivalus, sales lectin that binds glycoproteins with mannose.

"Recombinant glycoprotein here is called a glycoprotein expressed from recombinant polynucleotide, in which the structural gene that encodes a glycoprotein that is expressed under the control of regulatory sequences that do not belong in the natural is about to create a vector in which structural E1 gene placed under the control of the functional fragment of the promoter of glyceraldehyde-3-phosphate dehydrogenase of yeast (GAPDH). Currently, the preferred promoters for use in yeast is a hybrid promoter ADG/GAF described in U.S. patent N 4.880.73, which is a fragment of the promoter GAPDH in combination with the previous activation sequence derived from alcohol dehydrogenase 2. Modification of the structural gene may include the substitution of different codons are degenerate codons (for example, to use the preferred host codons, to eliminate or create restriction sites, to control the formation of hairpins and so on) and the substitution, insertion or deletion of a limited number of codons coding for different amino acids (preferably not 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, "recombinant receptor called protein receptor, expressed with recombinant polynucleotide, in which the structural gene that encodes a receptor that is expressed under the control of regulatory sequences not adjacent in its natural is hydrated polypeptide" refers to a polypeptide, substantially free from other components of the HCV virus, especially from polynucleotides. Polypeptide composition is "substantially free from other components, if the weight of the polypeptide in the composition is at least 70% of the weight of the polypeptide and another component, United with him, more preferably at least 80%, even more preferably about 90% and most preferred is 95% or more.

For example, a composition containing 100 μg/ml E1 and only 3 μg/ml of the other components of the GHS (e.g., DNA, lipids, and so on), is substantially free from other components of the GHS, and so is the composition of the isolated polypeptide within this definition.

The term "leader secretion" refers to a polypeptide that, when encoding aminocore protein causes after the broadcast, the secretion of the protein into the culture medium host cells. The leader of the secretion, usually obtained from the used host cell.

For example, among the leaders of the secretion, suitable for use in yeast is the leader of the alpha-factor of Saccharomyces cerevisial (see U.S. patent N 4.870.008).

The term "lower eukaryote" refers to cells of the host, such as yeast, fungi, etc., the Bottom of the yeast, especially species belonging to Saccharomyces, Schizosaccharomyces, Kluveromyces, Pichia, Hansenuba, etc., Saccharomyces cerevisiae, S. Carisbergensis and K. lactis are the most commonly used yeast hosts, and common fungal hosts.

The term "higher eukaryotes" refers to cells-owners, received from higher animals such as mammals, reptiles, insects, etc.

In the currently preferred higher eukaryotic cell hosts, obtained from Chinese hamster (e.g., CHO-cells Chinese hamster ovary), monkeys (e.g., COS cells), human and insect (e.g., spodoptera frugiperda). Cell host can be represented in the form of a suspension or culture in the mattress, tissue culture, cultures, bodies, etc.

The term "modulator of calcium" refers to a compound capable of sequestrati or bind calcium ions in the endoplasmic reticulum or to change the concentration of calcium ions in the ESR by action on proteins that regulate the metabolism of calcium (e.g. calcium channel proteins, calcium pumps, and so on ). In a number of suitable modulators of calcium include, for example, thapsigargin, EGTA (ethylene glycol bis/beta aminoacylase ether/ N, N, N', N' - tetraoxane acid). Currently predpochtitel "immunogenic" refers to the ability of a substance to induce humoral and/or cellular immune response either alone or associated with a carrier state in the presence of adjuvant or without him. "Neutralization" refers to an immune response that blocks the infectivity, either partially or completely, an infectious pathogen. "The vaccine is immunogenic composition capable of protection against HCV, partial or complete, applicable to the treatment of the individual.

The term "biological fluid" refers to liquid derived from an organism, such as serum, plasma, saliva, gastric juice, mucus, etc., In General, the biological fluid to be screened for the presence of HCV particles. Some biological fluids are used as a source of other products such as coagulation factors (e.g. factor VIII:C), serum albumin, growth hormone, etc., In such cases it is important that the source of biological fluid was free of contamination by the virus, such as HCV.

B. General way

Region E1 of the HCV genome is described in EP 388.232 as a region "E", whereas E2 is described as "NS1". Region E1 comprises approximately amino acids 192-383 full-viral polyprotein. Region E2 comprises approximately amino acids 384-820. The full sequence of prototypes of these proteins (shtam the RC. As E1 and E2, can be expressed with polynucleotide encoding the first 850-900 amino acids polyprotein HCV: post-translational processing in most eukaryotic cells masters breaks down the initial polyprotein C, E1 and H2. You can truncate the 5' end of the coding region in order to reduce the amount of produced protein C.

The expression of asialoglycoprotein can be obtained in a variety of ways. For example, you can obtain the expression in lower eukaryotes (such as yeast) that normally do not add residues of sialic acid to glycosylated proteins. In yeast expression systems currently preferred leader secretion, such as the leader of the alpha - factor S. cerevisial, so that the protein expressively after the broadcast in a nutrient medium. Also in the currently preferred mutants lack of glycosylation, such as pmr1, as these mutants provide only glycosylation core and often secrete heterologous proteins with high efficiency (N. To. Pudolph et al., Cell (1989) 58:133-45). Alternatively, you can use other types of yeast, such as Pichia pastaris that Express glycoproteins containing capitalsim in S. cerevisia.

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

The yeast in the future it is necessary to stimulate calcium modulator, in order to obtain secretion of the protein in the endoplasmic reticulum. In a number of suitable modulators include thapsigargin and A (see, for example: o Thastup et al., Proc. Nat. Accd. Sci. USA (1990) 87:2466-70). For example, you can Express a lot E1 or E2 in intracellular mammalian cells (such as CHO cells, COS, HeLa and Kapitsa in the endoplasmic reticulum, cells treated with calcium modulator in concentrations large enough to cause the release of ER content. Then the protein is recovered from the culture medium which is replaced for the next cycle.

Additionally it may be advantageous to Express a truncated form of the protein shell. As E1 and E2, apparently, have vysokoriskovannym domain, which obviously keeps the protein within the endoplasmic reticulum and prevents the efficient allocation. So you wish to remove parts of the sequence found in one or more areas of amino acids 170-190, 260-290 or 330-380 El (numbering from the start of polyprotein) and 660-830 in E2 (see, for example, figure 20-1 in EP 388.232). It seems that at least one of these hydrophobic domains forms a transmembrane region, which is not essential for the antigenicity of the protein and therefore can be removed without damage. What better to delete, you can define a few experiments on the destruction carried out in the usual way. Remove hydrophobic 3'-end of the E2 leads to the secretion of part downregulation of E2 with sililirovanie secreted protein.

To obtain the expression Moni by the method of precipitation of calcium phosphate or subjected to transfection with the recombinant virus. Vectors can independently reproduce in the cell host or can integrate into the genome of the host cell. Higher eukaryotes can be transformed plasmids, but usually they are infected with the recombinant virus, for example by recombinant vaccinia virus. Vaccinia virus is particularly preferred, since infection with vaccinia virus stops the protein expression host cell. Currently, the number of preferred host cells include cell lines HeLa and cancer. In the present system, this means that E1 and E2 are accumulated as the major glycosylated variants in ER owner.

As pE1 and pE2 are the predominant glycoproteins with mannosyl the end, they are easy to isolate in pure form from cells by lectins such as agglutinin Galanthus nivalus (GNA), which binds to terminal mannose residues

Proteins that are naturally expressed as glycoproteins with mannosyl end in the physiology of mammals are relatively rare. In most cases glycoprotein mammals has mannose end in transient intermediate form generated in the process of glycosylation. The fact that membrane proteins In degrees) N-acetylglucosamine, means that HCV proteins and whole virani possible to separate and partly purify from endogenous proteins using lectins specific for terminal mannose or N-acetylglucosamine. Recombinant proteins seem authentic and are essentially identical membrane proteins found in Mature free virion or protein associated with the membrane of the cells. So you can use lectins, such as GNA for proteins with mannosyl end and WCA (agglutinin of wheat seeds) and their equivalents for proteins with N-acetylglucosaminyl the end. You can use lectins, fixed on a solid phase (for example, column lectin-sepharose) to separate E1 and E2 from the supernatant of cell culture or from other liquids, for example, for cleaning during retrieval of antigens for vaccines or for use in the immunoassay.

Alternatively, you can provide suitable lectin to highlight E1, E2 or HCV virions from fluid or tissue of persons with suspected HCV infection. As glycoproteins with mannosyl the end of the relatively rare, such a procedure should serve as the purification of proteins present in the sample, significantly weakening the intensity of the background. After the corded Verona, possible alternative to detect nucleic acid of HCV using techniques of polymerase chain reaction (PCR) or other methods of accumulation of nucleic acids, preservation of areas of the HCV genome (for example, non-coding region 5'). This method allows to isolate and characterize different strains of HCV, regardless of disability or variants of antigenicity, for example, when a new strain does not cross-immune reaction from the strain used to generate antibodies. There are many other ways to take advantage of the unique recognition of glycoproteins with mannosyl end using specific lectins.

For example, it is possible to incubate the samples suspected of containing Verona or HCV proteins with Biotin - or avidinbiotin the lectins, and to precipitate the protein-leckenby complex using avidin or Biotin. You can also use the affinity of the lectin to the HCV proteins to direct connection to Verona for therapeutic purposes, for example, conjugial antivirus connection with GNA. Alternative you can use the appropriate lectins in order to remove glycoproteins with mannosyl completion of serum or plasma, reducing or acclimatation E1 and/or E2 from crude cell lysates by incubation with immobilized protein, mannose-binding, especially with lectins such as CONA or GNA. Cells lyse, for example, mechanical destruction in hypotonic buffer, then the lysate centrifuged to prepare postnuclear lysate, and again centrifuged to obtain the crude fraction microsomal membranes. The crude membrane fraction consistently solubilizated in buffer containing a detergent, such as Triton X-100, Np40, or the like. This extract with detergent further purified from insoluble particles by centrifugation and the resulting OSVETLENIE lysate incubated in a chromatographic column containing immobilizovannyi protein, mannose-binding, preferably GNA, fixed on a solid substrate, such as agarose or sepharose, in a period of time sufficient for binding, usually 16-20 hours. Then the suspension is passed through the column until such time as E1/E2 begin to appear in eluent, then incubated in the column for a time sufficient for binding, usually about 12 to 24 hours. Associated material is then washed with additional buffer with detergent (such as Triton X-100, NP40 or similar) and eluted with mannose to get cleaned will begin to appear protein, at this point, the elution is stopped and the column equilibrated for 2-3 hours, before the elution of the protein. I believe that this gives sufficient time to react the remaining large protein aggregates. In cases where E1 and E2 are expressed together in a natural way (i.e. without processing domain communication with the shell), a substantial portion of asialoglycoprotein appears in the form of aggregates E1:E2. In the study under the electronic microscope, a significant portion of these units looks like the particles are almost spherical shape with a diameter of about 40 nm, which corresponds to the expected size of intact virus. These particles seem to be self-organizing viral subunits.

It is expected that these units must form a Quaternary structure, very similar to the authentic structure of the particles of HCV virion, and it is therefore expected that they will serve as a highly immunogenic vaccine.

Complexes E1/E2 can be further purified by gel-chromatography on a basic environment, such as fractogel-DEAE or DEAE-sepharose. Using fractogel-deaa gel chromatography can be obtained complexes E1/E2, peeled, roughly, up to 60-80%. E1 can then clean the AET E2 and allows easy separation of E1.

Tissue specificity HCV together with the observation that the membrane glycoproteins of HCV have mannone the end, suggests that the virus uses the mannose receptor or asialoglycoprotein receptor (ASGR) in order to penetrate into the cell host. Mannose receptor found on macrophages and cells of sinusoidal liver, whereas ASGR detected in parenchymal hepatocytes. Therefore, it should be possible to grow HCV in cells of the host expressing one or both of these receptors. You can either use the primary culture cells, which naturally Express this receptor, under conditions that preserve the receptor, or you can enter in another cell line, such as HeLa, Cho, COS, etc., vector conducive to the expression of this receptor.

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

Cloning and sequencing ASGR described K. Drickamer et al., J. Biol. Chem (1984) 259; 770-78 and M. Spiess, Proc. Nat. Acad. Sci., USA (1985) 82:6465-69; transfection and expression of functional ACHR in NTS homothetic teruya cells of rats described M. McPhaul and R. Wegg, Pros. Nat Acad. Sci. USA (1986) 83:8863-67 and M. McPhaul and R. Berg, Mc1. Cell Biol. (1987) 7: 1841-47.

T the studied cells as a host for propagation of HCV in culture. Serial passages of HCV in such a culture should lead to weakened strains suitable for use as a live vaccine. You can either use the primary culture cells, which naturally Express this receptor, creating conditions in which the receptor is saved, or you can transfection of other cell lines such as HeLa, CHO, COS, etc., vector, providing receptor expression.

Cloning of the mannose receptor and its transfection and expressiona in fibroblasts have been shown by Taylor et al. (see above), whereas transfection and expression of functional ACHR in homozygous teruya cells of rats described McPhaul et al. (see above).

At present, it is preferable to use an immortal cell line, transfected with one or both of recombinant receptors.

Immunogenic compositions can be prepared by known methods. These compositions comprise immunogenic amount of a polypeptide, for example the composition of the particles E1, E2 or E1/E2, usually connected with a pharmaceutically acceptable carrier, preferably further comprising adjuvant.

If desirable "cocktail" - a combination of polypeptides CI E1/E2 will as expected, especially useful vaccine antigen. Immunogenic compositions can enter an animal to induce formation of antibodies, or get the source of the antibodies, or to create a protective immunity in the animal.

Pharmaceutically acceptable carriers include any carrier that does not itself cause the formation of antibodies, dangerous for the individual receiving the composition. Suitable carriers are typically large, slowly decaying macromolecules, such as proteins, polysaccharides, polyethoxylate, polyglycolide acid, polymers of amino acids, copolymers of amino acids and inactive virus particles. Such carriers are well known to specialists.

In a 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 U.S. patent N 4.606.918, N-acetyl - norbornyl-L-alanyl-D-isoglutamine (hor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanyl-2-(1'-2'-dipalmitoyl-Sn-glycero-3-hydroxyrisperidone)-ethylamine (MTP-PE) and R1B1, which contains three components extracted from bacteria, monophosphorylated, dimycolate trehalose and cell wall skeleton (MPL + TDM + CWS) in order Bioscience, Morcester, MA). Further, drugs that are not designed for people, and for research purposes, you can use the full adjuvant's adjuvant(PAF) and incomplete adjuvant's adjuvant (NAF).

Immunogenic compositions will typically contain pharmaceutically acceptable excipients, such as water, saline, glycerol, ethanol, etc. Additionally, the number of such fillers may be included auxiliary substances such as wetting or emulsifying means, buffering agents to maintain pH, etc.

Typical immunogenic compositions are prepared in a form suitable for injection, or as a liquid solution or as an emulsion, it is also possible to prepare dry forms suitable for dissolution in the liquid filler with the formation of a solution or suspension before injection. The preparations can also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.

Immunogenic compositions used as vaccines comprise immunologically effective amount of the polypeptide of the GHS, as well as the other aforementioned components on demand.

"Immunologically effective amount" means that the introduction of such a large number of individuum number varies, depending on the health and physical condition of the individual, which it is introduced, the taxonomic group of individuals exposed to (e.g., nonhuman primates, primates, and so on), the ability of the immune system of an individual to synthesize antibodies, the degree of desired protection, vaccine composition, assessment of the medical situation doctor, HCV strain that caused the infection and other relevant factors.

It is expected that this number will fluctuate within wide limits, which can be determined by conventional tests.

These self-assembling aggregates E1/E2 can also serve as carriers of vaccines for heterologous (not SSC) haptens in the same way as the surface antigen of hepatitis b (see European patent application 174.444). In such applications, the units E1/E2 represent immunogenic carrier, capable of stimulating the immune response to haptens or antigens, conjugated with the unit.

The antigen can be konjugierte or conventional chemical methods, or you can clone it in the gene encoding the E1 and/or E2, with localization corresponding to the hydrophilic region of the protein.

Immunogenic compositions decided to enter a pair of the x way of introduction, include recipe for oral administration and suppositories. Therapeutic dosage can be entered once or in several stages in the scheme. The vaccine can be administered in conjunction with other immunoregulatory agents.

Century Examples

The following examples are provided as further guidance for practical specialist and should not be construed as any limitation of the invention.

EXAMPLE 1 Cloning and expression)


Vectors constructed from plasmids containing the 5'part of the genome of HCV, as described in EP 318.216 and EP 388.232. Cassette HCV (S/B) contains a DNA fragment StuI - Bgt II encoding the 5'-end of polyprotein from Meth1to Lei906starting from the nucleotide-63 related to Meth1. This includes protein core (C) protein E1 (also sometimes referred to as S), the E2 protein (also referred to as NSI) and 5'-part of the region NS2a. After the expression of this construct separated by proteolysis on separate proteins, E1 and E2.

Cassette HCV (a/b) contains a DNA fragment ApaLI-BgIII encoding the 5'-end of polyprotein from Meth1to Lei906starting from the nucleotide-6, related to Meth1. This includes protein core (C) protein E1 (also sometimes referred to cacheline proteins, E1 and E2 receive proteolytic processing.

The cassette C-E1 (S/B) (part StuI - BamHI) contains the 5'-end from Meth1to Ile340(BamHI site in the gene). The expression of this cassette leads to expression With and, to some extent, a truncated E1 (E1'). The part cut from the 3'-end is hydrophobic region, which is serving as the broadcast signal.

Cassette NSI (In/In) (part BamHI - BgIII contains a small portion of 3' from E1 (Meth364all E2 and part NSA (up to MDL906). This constructor fragment E1 serves as a translocation signal.

Cassette TRA NSI uses the leader of the activator tissue plasminogen person (tPA) as a translocation signal instead of the 3'-part E1. This tape contains a truncated form of E2 from Gli406to the Chap661, which lost hydrophobic end 3'.

Each cassette is embedded in the vector pGEM3Z (Promega) with or without synthetic beta-Globino non-coding sequences for transcription and translation in vitro using the T7 expression and reticulocyte rabbit.

Vectors recombinant vaccinia virus (rVV) were prepared by embedding cassette in plasmid pSC11 (obtained from Dr. C. Moss, NIH), followed by recombination with vaccinia virus as described Charkrabar HCV (A/B) pS59 (obtained from Dr. C. Moss NIH) between sites StaI and SpeI, followed by recombination with vaccinia virus as described Charkrabarty et al., Mol. Cell Biol. (1985) 5:3403-09.


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

Precipitation resuspendable additional culture medium (Joklik - modified MEM Spenner environment + 5% horse serum and gentamicin) ("centrifuge environment) to a final concentration of 2107cells/ml. Destroyed by ultrasound crude mass of the virus vv/SC59 - HCV was added to many infection in 8 plaque-forming units/cell, and the mixture was stirred for 30 minutes at 37oC. Then infected cells was transferred into a centrifuge bottle containing 8 l of the centrifuge environment, and incubated for 3 days at 37oC.

Then, the cultured cells were collected by centrifugation and precipitation resuspendable buffer (10 mm Tris-HCL, pH 9,0, 152 ml). The cells are then homogenized by the homogenizer 40 ml Dounce (50 strokes) and kernel besieged by centrifugation (5 minutes, 1600 rpm, 4oC, rotor JA-20). Nuclear fallout resuspendable in Tris-buffer (24 ml) was rehomogenization and again Osaka 3 x 30 minutes in sonicator in the form of wine horns at medium power. The treated lysate (15 ml) was layered on 17 ml cushion of sucrose (36%) in centrifuge tubes SW 28 and centrifuged for 80 minutes at 13500 rpm at 4oC in order to precipitate the virus. Precipitate the virus resuspendable in 1 ml of Tris buffer (1 mm Tris-HCL, pH 9,0) and frozen at -80oC.

EXAMPLE 2 (Comparison of products in vitro and in vivo)


E1 and E2 expressed both in vitro and in vivo and mately35S-Met used the vectors described above in example 1. Cells BSC-40 and HeLa were infected with rVV vectors for expression in vivo. Culture media and cell lysates were tested recombinant proteins. Conducted immunoprecipitation products of human HCV immune serum, whereas proteins in vitro was analyzed by the direct method. The resulting proteins were analyzed by chromatography SDS-PAGE.

Reticulocytopenia expression system (pGFM3Z with HCV (S/B) or HCV (a/b) was produced proteins, E1 and E2 with a molecular mass of approximately 18 KD, 35 KD and 72 KD, respectively. Lysates of cells BSC-40 and HeLa, after transfection rVV containing HCV (S/B), HCV (a/b) or C-E1 (S/B) gave the same proteins. Due to the fact that reticulocytopenia system does not provide efficient processing in the Golgi apparatus and is therefore not the mobility, suggests that the proteins are not cialisyou in vitro. Only vector rVV containing TPA-NSI causes some extracellular secretion of E2 with altered mobility corresponding to sililirovanie.


HCV (S/B) 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, suirable buffer, Lemli for sample and analyzed by chromatography SDS-PAGE. The results showed that E1 and E2 are fixed on GNA and con, which indicates the presence of mannose. GNA binds to terminal Mennonite groups, while Kona is associated with any alpha-linked mannose. The lack of binding to SNA, PNA and WGA States that none of the proteins does not contain sialic acid, galactose-N-atsetilgalaktozamin or N-acetylglucosamine.


E1 and E2 with a radioactive label was obtained in cells BSC - 40 by infection with rVV containing HCV (S/B) (vv/SC11-HCV) and produced immunoprecipitate human HCV+immune serum. Half of precipitated material was treated with neuraminidase during the night to remove any sialic acid.

After processing the processed and neopentecostal no cialisonline in vivo.


E1 and E2 with a radioactive label was obtained in cells BSC-40 by infection with rVV containing HCV (a/b) (vv/S 59-HCV), and produced either immunoprecipitation HCV+serum human or besieged the biotinylated lectin GNA associated with acrylic beads, using vv/'s 11 free of HCV sequences as control. The precipitates were examined by chromatography SDS-PAGE. The data showed that E1 and E2 were the main varieties of proteins with mannosyl ending in vv/SC 59-HCV infected cells. GNA as effectively as anticavity man, besieged E1 and E2 of the environment of cell culture. Observed component 25 KD, but he seemed to be specific for cells infected with vaccinia virus.

EXAMPLE 3 (Cleaning using lectin)


HeLa cells were inoculable mass of purified vv/SC 59-HCV virus to high titer in the multitude of infection of 5 plaque-forming units/cell, and the mixture was stirred for 30 minutes at 37oC. Then infected cells was transferred into a centrifuge bottle containing 8 l centrifuge medium and incubated for 3 days at 37oC. the Cells were again collected by centrifugation and resuspendable in hypotonic buffer (20 mm HEPES, 10 mm NaCI, 1 mm MgCl2that 120 is 5 minutes 1600 rpm, 4oC, rotor JA-20). Sediments were collected, resuspendable in 48 ml of hypotonic buffer, re-rehomogenization, centrifuged again collected and frozen at -80oC.

Then the frozen supernatant was unfrozen and the fraction of microsomal membranes from post-nuclear lysate was isolated by centrifugation for 20 minutes in the rotor JA-20 at 13500 rpm at 4oC. the Supernatant was removed by suction.

Precipitation which was in 96 ml of detergent buffer (20 mm Tris-HCI, 100 mm NaCl, 1 mm EDTA, 1 mm DDT, 0.5% of Triton-X-100, pH 7.5) and homogenized (50 strokes). The product was osvetleni by centrifugation for 20 minutes at 13500 rpm 4oC and collected supernatant.

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

Purified proteins E1/E2 was suirable alpha-D-mannoside (0.9 M in detergent buffer) at a speed of current of 0.5 ml/min, the Elution was stopped when E1/E2 in eluent and gave the column again to balance the morals together and was irradiated with UV to inactivate any residual vaccinia virus.


Asialoglycoprotein E1 and E2, purified GNA-agarose, were subjected to sedimentation in 20-60% glycerol. The gradients were divided and analyzed the proteins by methods SDS-PAGE and Western batirovna. Spots probed GNA for the identification of E1 and E2.

The results indicate the presence of heterodimer E1:E2, deposited with the expected rate (i.e., the position characteristic of the protein of 110 KD). Also appear larger aggregates of the protein shell of the GHS. Also appeared E2: E2 homodimer. It seemed that E2 prevailed over E1 in the larger species, although some varieties E1:E2 was also detected.

Larger aggregates were deposited significantly more likely than thyreoglobulin token.


E1 and E2, purified GNA-agarose, was besieged in the gradient of 20-60% glycerol and 1 mm EDTA. Fractions were analyzed by SDS-PAGE with and without beta-mercaptoethanol (ME). The number of E1 and E2 is almost not dependent on the presence of ME, pointing at the absence of disulfide bonds between heterodimers.


Complexes E1/E2 (approximately 40% purity) were analyzed on a Coulter DM-4 analyzer sub-micron particles. Material found in the range of 20-60 nm.


Complexes E1/E2 (peeled approximately 40%) ...">

E micrograms revealed the presence of spherical particles with a diameter of about 40 nm. Complexes E1/E2 were incubated with HCV + immune human sera, then examined under EM with negative contrast. Observed complexes of antibodies containing large aggregates and smaller particles.

EXAMPLE 4 (Chromatographic purification)


Purified by lectin GNA material, prepared as described in example 3 (0.5 to 0.8 ml), was diluted 10 times with buffer A (20 mm Tris-Cl buffer, pH 8.0; 1 mm EDTA) and applied to a column of Fractogel EMD DEAE-650 1.8 x 1.5 cm (EM Separations, Gibbstown, New Jersey, catalogue N 16883), equilibrated in buffer A.

The fraction of protein that contains E1/E2, suirable with the same buffer at a rate of 0.2 ml/min and collecting 1 ml fractions. The fractions containing E1 and E2 (identified by SDS-PAGE), were combined and stored at -80oC.


Material purified by the above method (A) according to the results of SDS-PAGE, was purified by 60-80%. Identification bands alleged E1 and E2 was confirmed by analysis of the N-terminal sequence after the implementation of the methods of transfer. For this material E1/E2, peeled Fractogel-DEAE, restored by the addition of a buffer Laemmli (pH 6,8, is 0.06 M Tris-Cl, and 2.3% SDS, 10% glycerol, 0,72 M beta-merchavim SDS-PAGE proteins were transferred to 0.2 μm membrane of polyvinylidene-diferida (PVDF) (Bio-Rad Laboratories, Richmond, CA). Strips corresponding to putative proteins E1 and E2, were cut out of the spot and subjected to N-terminal amino acid analysis, although in the preparation of the material did not take special measures to prevent aminoterminal blocking. The first 15 cycles showed that the sample E1 has the sequence: Tyr-GLn-Val - Arg-X-Ser-Tra-Gli-X-Tyr-GIS-Val-X-ASN-ASP, whereas the sequence of E2 was: Tre-GIS-Val-Tre-Gli-x-X-Ala-Gli-GIS-X - Val-X-Gli-Hairdryer. These data determine the amino acid sequence consistent with that expected for the corresponding DNA sequences.

Product E1/E2, purified as described above, Fractogel chromatography-DEDE, is considered to be aggregated on the basis that a large number of E1 and E2 together elute in the region of the free volume of the chromatographic column gel filtration Bio-Sil TSK-4000 SW. This indicates that under natural conditions a significant number of complex E1/E2 has a molecular weight of at least 800 KD. It was also observed the presence of the material E1/E2 with a molecular weight of about 650 BC.

1. Asialoglycoprotein hepatitis C virus (HCV) selected from the group comprising truncated glycoprotein expressed from El-district HCV, Kotora, numbered from the beginning of polyprotein HCV, and truncated glycoprotein expressed from the E2 region of HCV containing a deletion in part of the sequence, is found in a region spanning amino acids 660-830 E2-district, numbered from the beginning of polyprotein HCV.

2. Asialoglycoprotein under item 1, characterized in that the said truncated glycoprotein expressed from the E1 region of HCV and contains a deletion in part of the sequence, is found in a region spanning amino acids 330-380 E1-district, numbered from the beginning of polyprotein HCV.

3. Asialoglycoprotein under item 1, characterized in that the said truncated glycoprotein is expressed from the E2 region of HCV and contains a deletion in part of the sequence, is found in a region spanning amino acids 660-830 E2-district, numbered from the beginning of polyprotein HCV.

4. Immunogenic composition for the diagnosis, treatment or prevention of infection caused by the hepatitis C virus containing asialoglycoprotein hepatitis C virus (HCV) according to any one of paragraphs.1-3 and a pharmaceutically acceptable carrier.

5. Method of induction of immune responses by antigen in an animal, comprising the stage of: obtaining a composition containing an effective coomarasamy animal wherein the immunogenic substance contains asialoglycoprotein HCV according to any one of paragraphs.1-3.

6. The immunoassay method for detecting in a biological sample, of antibodies to HCV comprising obtaining a biological sample, obtaining a polypeptide reagent, the reaction of the biological sample with a polypeptide reagent under conditions ensuring formation of complexes of antigen-antibody and determining the formation of complex antigen-antibody containing the aforementioned polypeptide reagent, characterized in that the polypeptide reagent use composition under item 4.


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