KR100457790B1 - Preparation of infectious HCV-like particles - Google Patents

Abstract

The present invention relates to hepatitis C virus like particles having infectivity. Specifically, a recombinant alphavirus expression vector expressing core, E1, and E2, which are structural proteins of hepatitis C virus, and a helper vector, which expresses core, E1, and E2, which are structural proteins of hepatitis C virus, are used. It relates to an infectious hepatitis C virus analogous particles prepared by The virus like particle of the present invention may be used as an antigen for detecting an HCV vaccine or an antibody of HCV, or may be useful for assaying an infection inhibitor.

Description

Preparation of infectious HCV-like particles {Preparation of infectious HCV-like particles}

The present invention relates to hepatitis C virus like particles having infectivity. Specifically, the present invention relates to an infectious hepatitis C virus-like particle generated by using a recombinant alpha-virus expression vector expressing a structural protein of hepatitis C virus and a helper vector expressing a structural protein of hepatitis C virus.

Hepatitis C virus (hereinafter referred to as "HCV") is a virus that is a major cause of non-A or non-B hepatitis. HCV is known as a pathogen of acute or chronic hepatitis, cirrhosis and liver cancer. HCV, which belongs to Flaviviridae of Hepacivirus, has been identified at least six subtypes worldwide since the first genetic clone was identified in 1989 by researchers from Chiron, USA.

HCV is an enveloped virus that has a positive-stranded single linear RNA genome of about 9.5 kb. The genome of HCV has one open reading frame (ORF) and encodes a polyprotein consisting of about 3000 amino acids. The HCV genome consists of structural genes of core, E1, and E2, and nonstructural genes of NS2, NS3, NS4A, NS4B, NS5A, NS5B, and non-translated regions at 5 'and 3' ends. (untranslated region: UTR) exists. UTRs in the HCV genome are essential parts for initiating transcription and replication (Tanaka et. Al., J. Virol. 70, 3307-3312 (1996); Choo, QL et al., Science, 244, 359-362 (1989) Choo, QL et al., Science, 88, 2451-2455 (1991)). The genomic structure of HCV is shown in FIG. 1.

HCV vaccine research has been attempted in many directions. Examples include isolated recombinant antigens, recombinant viruses, DNA vaccines, and the like. Recently, research to make virus analogous particles has been conducted. Mizuno et al. Reported the formation of virus pseudoparticles by transfecting HeLa G cells with Vaccinia virus, which cloned the full length genome of HCV (Mizuno et al., Gastroenterology, 109). , 1933-1940 (1995). Falcon et al. Confirmed that virus-like particles were produced in cells by expressing only core and E1 of HCV structural proteins in Pichia pastoris yeast (Falcon et al., Tissue and Cell, 31, 117-). 125 (1999)). However, the above results only confirm the presence of the virus in the host cell, but did not obtain the virus particles.

The literature also reported the production of virus analogs by expressing structural proteins of HCV in insect cells using baculovirus (Baumert et al., J. of Virology, 72, 3827-3836 (1998). Baumert et al., Gastroenterology, 117, 1397-1407 (1999); Baumert et al., Hepatology, 32, 610-617 (2000). However, it was confirmed that the cell culture system used in the above case was an insect cell, and that the protein produced in the insect cell had a different glycosylation pattern from that expressed in mammalian cells, and the virus-like particles thus produced were not infectious. Was.

The present invention provides an alpha viral expression vector expressing the structural protein of HCV, RNA transcripts thereof, and a method of preparing the same.

The present invention provides a helper vector, an RNA transcript thereof, and a method for producing the same, which expresses HCV structural proteins.

The present invention provides a cell or cell line transiently or stably transformed with an alpha viral expression vector or an RNA transcript thereof expressing the structural protein gene of HCV and a method for producing the structural protein of HCV using the transformant. do.

The present invention provides a method for producing an HCV virus analogous particle and a virus analogous particle produced by the method using an alpha viral expression vector expressing an HCV structural protein gene and a helper vector expressing the HCV structural protein. To provide.

The present invention provides a vaccine composition comprising the expression vector or RNA transcript thereof, or the RNA transcript of the expression vector and the helper vector or two of them.

The present invention provides a vaccine composition comprising HCV virus analogous particles.

The present invention provides a method for preventing a disease by administering the vaccine composition to a patient.

The present invention provides a method for producing an antibody using the HCV structural protein or HCV virus analog particles as an antigen.

The present invention provides a method for detecting HCV antibody using HCV structural protein and / or HCV virus analogous particles prepared by the above method, and HCV antibody, characterized by using the structural protein and / or viral analogous particle of HCV as a capture antigen. It provides a kit for detection.

1 is a schematic diagram showing the genomic structure of HCV.

2 is a schematic diagram illustrating a process of preparing a pSFHCV / c740 expression vector.

3 is a schematic diagram illustrating a process of preparing a pHelper / c740 vector.

Figure 4 is a photograph confirming the expression of E2 protein by immunostaining with monoclonal antibody against mouse E2 after coatffecting pSFHCV / c740 and pSFV-helper or pSFHCV / c740 and pHelper / c740 RNA transcripts to BHK-21 cells (A: negative control; B: cells transfected with pSFHCV / c740 and pSFV-helper; C: cells transfected with pSFHCV / c740 and pHelper / c740).

5 is a photograph showing the results of Western blotting using mouse E2 monoclonal antibody after transfection of pSFHCV / c740 and pSFV-helper or pSFHCV / c740 and pHelper / c740 RNA transcripts to BHK-21 cells (FIG. Lane 1: negative control group; lane 2: cells transfected with pSFHCV / c740 and pSFV-helper; lane 3: cells transfected with pSFHCV / c740 and pHelper / c740).

FIG. 6 shows the results of RT-PCR analysis of RNA packaged in these particles after coating and transfecting pSFHCV / c740 and pSFV-helper or pSFHCV / c740 and pHelper / c740 RNA transcripts to BHK-21 cells. (M: marker; lane 1: coatfection of pSFHCV / c740 and pSFV-helper RNA (sense: primer 1, antisense: primer 3); lane 2: coat of pSFHCV / c740 and pSFV-helper RNA Lancefection (Sense: Primer 4, Antisense: Primer 3); Lane 3: Coatfection of pSFHCV / c740 and pHelper / c740 (Sense: Primer 1, Antisense: Primer 3); Lane 4: pSFHCV / c740 and pHelper Coatfection of / c740 (Sense: Primer 4, Antisense: Primer 3).

7 is a photograph taken by TEM of VLP in cells. After 40 hours of transfection, the cells were collected to identify the virus present in the cells. Panel A is a cell transfected with pSFHCV / c740 and pSFV-helper, and panel B is a cell transfected with pSFHCV / c740 and pHelper / c740. Bar is 100 nm.

FIG. 8 is a photograph taken by TEM of VLPs, and is a photograph obtained by TEM by shade staining of VLP obtained by ultracentrifugation in a cell culture medium after 48 hours of transfection. Panel A is a virus like particle from cells transfected with pSFHCV / c740 and pSFV-helper, and panel B is a virus like particle from cells transfected with pSFHCV / c740 and pHelper / c740. Bar is 167 nm.

Figure 9 is a photograph showing the immunostaining results for confirming the infectivity of the virus-like particle of the present invention. Panel A activates VLPs with chymotrypsin and infects BHK-21 cells, panel B infects BHK-21 cells with VLPs that have not been treated with chymotrypsin, and Panel C shows VLPs activated with chymotrypsin Was infected with COS-1 cells, Panel D was infected with Hep3B cells with VLP activated with chymotrypsin, and Panel E was infected with HepG2 cells with VLP activated with chymotrypsin (column 1: negative control; Column 2: VLP by coatfection of pSFHCV / c740 and pSFV-helper; column 3: VLP by coatfection of pSFHCV / c740 and pHelper / c740).

10 shows the results of RT-PCR following infection with BLP in BHK-21 cells. (Lane 1: negative control group; lane 2: cells transfected with pSFHCV / c740 and pSFV-helper; lane 3: cells transfected with pSFHCV / c740 and pHelper / c740).

The present invention provides alpha viral expression vectors and RNA transcripts thereof that express structural proteins of HCV.

The "HCV structural protein" means the core, E1 and E2 proteins of HCV. The "core" protein is the nucleocapsid protein that surrounds the RNA of HCV on the outside. It has been reported that the sequence difference is the smallest among several genotypes of HCV while showing high antigenicity (Houghton, M. et al., Hepatology, 14, 381-388 (1991)). The core as defined in the present invention is an analog or truncated form of a native core protein, which includes immunologically cross-reacting with a native core.

"E1 and E2" are the outermost envelope glycoproteins of HCV and are transmembrane proteins with N-terminal ectodomains and C-terminal hydrophobic anchors (Dubussion, J., Curr. Top. Microbio.Imunmunol., 242, 135-148 (2000)). E1 and E2 proteins are produced by the host's signal peptidase cleaving the polyprotein of HCV and N-linked glycosylation with high intensity. Administration of purified HCV El and E2 proteins together as an antigen has been reported to induce protective immunity against HCV infection of the same genotype in chimpanzees (Choo, QL, et al., Proc. Natl. Acad. Sci. , USA 91, 1294-1298 (1994)). E1 and E2 proteins of the present invention include analogues of the native E1 or E2 proteins or their truncated forms and include immunologically cross-reaction with the native E1 or E2.

"Alphavirus-based" refers to an alpha virus. "Alphavirus" is a virus belonging to Togaviridae, a positive-strand RNA virus with a coating, and can infect a wide range of cells (insects, birds, mammals). It is an expression system in which RNA replication and transcription are efficient because the RNA genome of the virus itself encodes an RNA replicaase (Liljestrom, P. and H. Garoff, Biotechnology, 9, 1356-1361 (1991)).

Alpha viruses of the invention refer to viral species and subtypes commonly classified in the art as alpha viruses, for example Eastern Equine Encephalitisvirus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Encephalitis virus (WEE), Sindbis virus, South African Arbovirus No. 86, Girdwood S.A. virus, Ockelbo virus, Semliki Forest virus, Middleburg virus, Chikungunya virus, O'Nyong-Nyong virus, Ross River virus, Barmah Forest virus, Mayaro virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus This includes what is classified as an alpha virus by the International Committee on Taxonomy of Viruses, Whataroa virus, Babanki virus, Kyaylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, Buggy Creek virus and other virus scientific names. .

The alpha virus of the present invention is preferably "Semliki Forest Virus (SFV)" or "Sindbis Virus" or "Ross River Virus".

"Alpha viral expression vector" refers to an expression vector that has been engineered so that a foreign gene can be inserted in place of an alpha virus structural gene and the inserted foreign gene can be expressed in a suitable host cell. An "expression vector" is a nucleic acid construct that has been genetically engineered to express a foreign gene that is not naturally encoded by a plasmid or virus, etc. used as a vector, and its basic characteristics are already known in the art. Therefore, the alpha viral expression vector of the present invention is an expression vector engineered so that a foreign gene is inserted in place of the alpha virus structural gene and the inserted foreign gene can be expressed in a suitable host cell. Enabling sequences (e.g. SP6 promoter), non-structural proteins of alpha virus (e.g. nsP1, nsP2, nsP3, nsP4) genes, subgenomic promoters (e.g. 26S promoter) and HCV structural protein genes for SFV It is an expression vector characterized in that it is connected to be operable sequentially. Expression vectors of the present invention may include conventional structural modifications to those skilled in the art to optimize the expression of the desired foreign genes.

The present invention provides an alpha viral expression vector expressing the structural protein of HCV and a method for producing the RNA transcript thereof. In the expression vector of the present invention, HCV structural protein genes are inserted into an alpha viral expression vector. Alpha virus is an RNA virus, which is usually converted into DNA form, and is used as an expression vector. In the embodiment of the present invention was used SFV expression vector. PSFV, the SFV-based basic expression vector exemplified in the embodiment, is inserted with the genes of the non-structural proteins nsP1, nsP2, nsP3, and nsP4, which are the non-structural proteins of SFV under the SP6 promoter, and the foreign gene is inserted under the 26S subgenomic promoter. The expression vector is transformed to be. In the present invention, pSFV, a SFV-based expression vector sold by Gibco BRL, was used. The structure of pSFV is shown in FIG.

The present inventors made pSFHCV / c740, an expression vector expressing the structural protein of HCV, by inserting the core, E1, and E2 protein genes of HCV into pSFV as an example of an embodiment of the present invention. Amino acid sequence comprising the structural protein of HCV using the whole genome of J strain of HCV (H. Marusawa, M. Hijikata, T. Chiba, K. Shimotohno. Journal of virology 73, 4713-4720 (1999)) as a template (Nos. 1 to 740) were amplified by PCR. The amplified gene was cut out by Bgl II treatment with a restriction enzyme and cloned into the Bam H1 region of the vector pSFV. A manufacturing process of pSFHCV / c740 is shown in FIG. 2. The pSFHCV / c740 thus prepared was deposited in the Korean Culture Center of Microorganisms, an international depository under the Budapest Treaty, under accession number KCCM-10287. The alpha viral expression vector of the present invention can be transcribed in vitro in RNA form using SP6 polymerase.

The present invention provides an alpha viral helper vector, an RNA transcript thereof, and a method of producing the same, which expresses HCV structural proteins. The alpha viral helper vector of the present invention is a helper vector for the purpose of providing a virus coat protein for HCV virus particle formation. The helper vector of the present invention preferably lacks the sequence necessary for replicating its own nucleic acid sequence, but is linked to operate the gene of the HCV structural protein under a subgenomic promoter to provide an HCV structural protein, and the HCV structural protein. Refers to a vector capable of packaging a nucleic acid construct having a nucleic acid sequence necessary for packaging in a coat composed of the viral coat.

In an embodiment of the present invention, the SFV-helper SFV structural protein is coded for the purpose of preparing HCV analogous particles containing HCV recombinant gene in HCV viral shell, not SFV analogous particles containing HCV recombinant gene in viral shell of SFV. A new pHelper / c740 vector was prepared by substituting the entire coding site with a gene encoding structural proteins (core, E1, E2) of HCV (Example 2). pSFV-helper contains information capable of expressing SFV structural genes, but is commercially available from Gibco BRL as a helper vector that lacks the genomic RNA packaging signal.

First, pSFHCV / c740 prepared in Example 1 was treated with Xba I and Spe I to obtain DNA fragments containing cores, E1 and E2, which are structural proteins of 26S subgenomic promoter and HCV, and then pSFV-helper (Gibco BRL). Was treated with Xba I and Spe I to remove C, p62, 6K, E1 sites, which are structural protein parts of SFV. A pHelper / c740 was constructed by inserting a DNA fragment cut out from the pSFHCV / c740 containing the structural protein gene of HCV in the portion (FIG. 3). When the helper vector of the present invention is introduced into a host cell at the same time as the vector expressing the structural gene of HCV, a virus like particle in which the HCV structural gene genome is packaged in the coat protein of HCV is produced. The pHelper / c740 vector of the present invention was deposited with the Korean Culture Center of Microorganisms, an international depository under the Budapest Treaty, under accession number KCCM-10286.

In the present invention, for the sake of convenience, the SFV system has been described as an expression vector expressing HCV structural protein and a helper vector expressing structural protein of HCV. However, the above concept may be applied to other alpha viruses. Vectors of the present invention can be prepared by those skilled in the art based on genetic recombination techniques known in the art, with modifications appropriate to the teachings of the invention (Maniatis et al., Molecula Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982). Sambrook et al., Supra; Gene Expression Technology, Methodin Enzymology, Genetics and Molecular Biology, Methods in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif. (1991); Hitzeman et al., J. Biol. Chem., 255, 12073-12080 (1980); US Pat. No. 4,935,349).

The present invention provides a cell or cell line transiently or stably transformed with an alpha viral expression vector or an RNA transcript thereof expressing the structural protein gene of HCV and a method for producing the structural protein of HCV using the transformant. do.

Since the structural protein of HCV is a highly antigenic part, it can be used as a vaccine for inducing antibodies of HCV by separating the structural protein of recombinantly expressed HCV. Alpha viral expression vector expressing the structural protein of HCV provided in the present invention has the advantage that can express the protein in a wide range of host cells. The present invention provides a method for producing core, E1, E2 structural proteins of HCV by expressing the above expression vector or its RNA transcript in a host cell, preferably an animal cell. The animal cell of the invention is preferably a bird, mammal, reptile, amphibian, insect or fish cell. Examples of mammalian cells include human, monkey, hamster, rat and pig cells. In particular, BHK cells, COS cells and Hep3B cells may be used as preferred host and vector systems, but are not limited thereto.

Methods for preparing the expression vectors of the present invention are known in the art (Maniatis et al., Molecula Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982); Sambrook et al., Supra; Gene Expression Technology, Methodin Enzymology, Genetics and Molecular Biology, Methods in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif. (1991); Hitzeman et al., J. Biol. Chem., 255, 12073-12080 (1980 US Patent Publication No. 4,935,349).

Alpha viral expression vectors or RNA transcripts thereof can be introduced into cells using conventional transfection methods such as DEAE-dextran, calcium phosphate, electroporation. Preferably it can be introduced into animal cells by electroporation. In the case of the vector of the present invention, it is possible to introduce RNA transcripts into the host cell in a very high yield by electroporation (eg, Example 3). The above methods can also be used when coatring one or more vectors. Techniques for transforming host cells, including electroporation and the like, and methods for expressing cloned foreign DNA sequences within them are well known in the art (Maniatis et al., Molecula Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982); Sambrook et al., Supra; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Methods in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif. (1991) Hitzeman et al., J. Biol. Chem., 255, 12073-12080 (1980); US Pat. No. 4,935,349).

Next, the vector construct of the present invention or a host cell containing the RNA is cultured to express the HCV structural protein. The cells are cultured in culture medium containing the nutrients necessary for the growth of the host cell. Suitable media are known in the art and generally include carbon sources, nitrogen sources, essential amino acids, vitamins and minerals, as well as other components, such as growth factors or serum that may be required by certain host cells.

HCV proteins produced by infection of viral particles prepared using the transcripts of the expression vectors and helper vectors of the present invention can be isolated by various methods. The supernatant from the cell line, or whole cells that are broken down can be treated by various methods for separating and purifying proteins. For example, the supernatant may be first concentrated using commercial protein enrichment filters such as Amicon or Millpore ultrafiltration devices. Various methods known in the art can be used to separate HCV proteins from concentrates or centrifuged cell lysates, such as ion exchange chromatography, affinity column chromatography, gel permeation chromatography, and the like.

HCV, although cloned, is still a very difficult virus to culture. However, the transformation of cells increases their expression efficiency when infected with viral particles rather than RNA. Therefore, the provision of HCV-like particles by genetic recombination technology is expected to contribute greatly to the research of virus infectivity, intracellular reactivity, pathogenicity, and virus evasion mechanism.

Attempts have been made to make virus analogs of HCV. For example, Mizuno et al transfected HeLa G cells with vaccinia virus, which cloned the full length genome of HCV, to form virus mimics (Mizuno et al., Gastroenterology, 109). , 1933-1940 (1995)), and Falcon et al. Have confirmed that virus-like particles were produced in cells by expressing only core and E1 of the structural protein of HCV in Pichia pastoris yeast (Falcon et al. ., Tissue and Cell, 31, 117-125 (1999)). Nevertheless, the above findings only confirm the presence of the virus in the host cell, and in fact the virus particles can be used for future research or development as a product. It was not prepared and obtained separately.

The present inventors succeeded in making and recovering HCV virus analogous particles using an alpha viral expression vector expressing an HCV structural protein gene and an alpha viral helper vector characterized by expressing HCV structural protein. By definition, "viral pseudoparticles" (VLPs), although not exactly identical to wild-type viruses, refer to the formation of particles that are morphologically similar in size to the wild-type virus when viewed under an electron microscope.

Such VLPs can be achieved using a binary vector system of expression vectors expressing HCV structural protein genes and helper vectors expressing HCV structural proteins. Specifically, the expression vector constituting the binary vector system of the present invention is an alpha virus engineered so that a foreign gene is inserted in place of an alpha virus structural gene and the inserted foreign gene is expressed in an appropriate host cell. Refers to a systemic expression vector, preferably a sequence (e.g., SP6 promoter) which enables transcription of the vector, a nonstructural protein of an alpha virus (e.g., nsP1, nsP2, nsP3, nsP4 in the case of SFV), subgenomic A promoter (eg, a 26S promoter) and an HCV structural protein gene are sequentially linked to each other so as to be operable in sequence; A helper vector is a helper vector aimed at providing a viral coat protein for HCV particle formation. Preferably, the helper vector lacks the sequence necessary to replicate its nucleic acid sequence, but the gene of the HCV structural protein operates under a subgenomic promoter. It refers to a helper vector capable of packaging a nucleic acid structure having a nucleic acid sequence necessary for providing the HCV structure protein and having the nucleic acid sequence required for packaging in a coat composed of the HCV structure protein.

Specifically, an RNA transcript of an alpha viral system expression vector expressing the HCV structural protein gene and a helper vector expressing the HCV structural protein is introduced into one host cell, or a sequence enabling the transcription of the vector is provided. The containing DNA vectors are introduced into a host cell to express these two vectors (or their RNA transcripts) simultaneously. When the expression vector and the helper vector are expressed in one host cell, a VLP consisting of the structural protein of HCV is obtained. This fact indicates that the host cell (BHK-21) coated with the expression vector and the helper vector (BHK-21) is applied to the mouse. It can be seen from the result of immunostaining with monoclonal antibody and Western blot with the same antibody by dissolving the host cell (FIGS. 4 and 5). The binary vector system is also used in the experimental results (Example 7) of observing the virus analog particles present in the cells transfected in Examples 3 and 4 and the virus analog particles recovered in Example 5 with an electron microscope (Example 7). This confirms that the VLP has been created.

Baumert et al. Have used Baculovirus to express structural proteins of HCV in insect cells to produce virus analogs (Baumert et al., J. of Virology, 72, 3827-3836). (1998); Baumert et al., Gastroenterology, 117, 1397-1407 (1999); Baumert et al., Hepatology, 32, 610-617 (2000)). However, the virus-like particles produced in this case were not infectious. When a virus particle is infectious, it is usually infected in a target cell to express the antigenic protein of the virus and thus a more effective immune response, for example, a humoral immune response or a cell mediated immune response. There is an advantage that can cause).

It was found by the RT-PCR that the HCV VLP prepared in the present invention contained RNA corresponding to a part of the HCV genome. That is, when amplifying a sequence corresponding to the core portion of HCV, a constant nucleic acid sequence was amplified in both VLP having a coat of HCV or VLP having a coat of SFV (FIG. 6). Furthermore, even if the gene was amplified using a primer prepared above 200bp from the foreign gene insertion part, the signal was detected in both the SFV VLP and the HCV VLP, and it is assumed that the SFV replicator was included in the VLP. The replicator sequence comes from the expression vector, demonstrating that the expression vector is replicable in cells.

It was also confirmed by the infectious test results of Example 6 that the RNA present in the gastric VLP is infectious. When the VLPs prepared in Examples 3 to 5 were infected with liver cell lines including BHK-21, COS-1 and Hep3B cell lines, it can be seen that E2 protein was expressed from infected cells (FIG. 9). Furthermore, when the total RNA was extracted from the cells infected with the VLP and amplified the core gene region of the HCV, the nucleic acid sequence was amplified. Thus, the recombinant RNA containing the HCV structural gene was packaged in the VLP to become infectious. . The VLP of HCV prepared in the present invention is expected to be useful for identifying infection of hepatitis C virus and interaction with intracellular proteins, virus assembly process and immune system evasion mechanism.

The present invention further provides a vaccine composition for preventing hepatitis comprising the expression vector, an RNA transcript of the vector, an expressed HCV structural protein or the VLP in an amount sufficient to cause an immune response, and a method for preventing hepatitis by administering the same. do. The use of VLPs as a vaccine can induce an excellent immune response because it can provide the conformation of HCV. The vaccine may be administered to humans to provide immune defense mechanisms or to animals to induce antibodies.

The vaccine composition may comprise a pharmaceutically acceptable carrier. The carrier may be used as long as it does not produce an undesirable antibody in the subject to which the vaccine composition is administered. Many suitable carriers or diluents can be used in the present invention, particularly saline, buffered saline, and saline mixed with nonspecific serum albumin. The pharmaceutical composition may contain water, saline, glycerol, ethanol, or the like or an emulsifier, wetting agent, or the like, including adjuvant, buffer, antioxidant, carbohydrates such as glucose, sucrose or dextrin, and chelating agents such as EDTA. Substances that adjust pH may be included. The vaccine composition may comprise an adjuvant to enhance the immune response. Examples of the adjuvant include aluminum hydroxide (alum), thr-MDP, nor-MDP, MPT-PE and the like.

The vaccine composition of the present invention is administered to an individual in an amount sufficient to have efficacy as a vaccine. The amount may vary depending on the degree of protection required, the age or weight of the subject, the formulation of the vaccine composition, the method of administration and the like. In a preferred embodiment, the immunization will comprise oral administration. In addition, the vaccine may be administered parenterally, either via the subcutaneous route or by other routes. Booster immunization can be braked after 4-6 weeks.

The present invention provides a method of inducing an immune response against HCV by administering the vaccine composition to an animal or human.

Monoclonal and polyclonal antibodies specific for HCV can be prepared using the HCV structural protein or VLP of the present invention as an antigen. Antibodies are prepared through standard methods of preparing hybridomas using the polypeptides or VLPs of the invention as immunogens. The resulting antibodies are particularly useful for detecting HCV in samples, samples from humans. Polyclonal antibodies can be readily produced by one of ordinary skill in the art from several warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats. Briefly, the protein or peptide of interest is used to immunize an animal, typically via intraperitoneal, intramuscular, intraocular or subcutaneous injection. Immunity of the protein or peptide of interest can be increased using an adjuvant, for example Freund's complete or incomplete adjuvant. Following various booster immunizations, small samples of serum are collected and tested for reactivity to the desired protein or peptide. Once the titer of the animal has reached a standstill in terms of its reactivity to the protein, large amounts of polyclonal immune serum can be readily obtained by bleeding weekly or by bleeding the animal.

Monoclonal antibodies can also be readily produced using well known techniques (Monoclonal Antibodies, Hybridomas; A New Dimension in Biological Analyses, Plenum Press, Kennettm McKearn, and Bechtol (eds.) (1980)). Briefly, in one embodiment, a subject animal, such as a rat or mouse, is injected with the desired protein or peptide. If desired, several techniques can be used to increase the resulting immune response generated by the protein and to develop greater antibody reactivity. For example, the protein or peptide of interest can be bound to another protein, such as ovalbumin or keyholelimpet hemocyanin (KLH), using an adjuvant such as Freund's complete or incomplete adjuvant. have. Initial induction of the immune response can be via the intraperitoneal, intramuscular, intraocular or subcutaneous route. Between one and three weeks after initial immunization, animals can be reimmunized with booster immunization. Animals are then tested for bleeding and plasma is tested for binding to untreated polypeptide using the assay as described above. Further immunotreatment may be performed until the animal has reached a state of stagnation in reactivity to the desired protein or peptide. Animals can then be given a final increase in the desired protein or peptide and sacrificed after 3-4 days. Antibodies of the invention are particularly useful for the detection and diagnosis of HCV. The fluorescent antibody test (FA-test) uses fluorescently labeled antibodies capable of binding to the VLPs of the present invention. This method can detect the result using a fluorescence microscope. This assay is also used for the study of tissue samples or histological parts. In latex bead aggregation assays, antibodies of the invention bind to latex beads. The HCV binding to the antibody after contact with the sample in which the HCV is present can be analyzed. Methods such as enzyme immunoassay (EIA), radioimmunoassay (RIA) and the like can be used.

For example, HCV-antibodies bound to the VLPs of the present invention may be measured by immunochemical methods after the VLPs are immobilized on the stationary phase and then contacted with the test sample under conditions suitable for generating antigen-antibody binding. Alternatively, the test sample is coated on a stationary phase and then reacted with the VLP of the present invention, and the HCV-antibody in the test sample is detected again by using the HCV-antibody again detecting the VLP of the present invention bound to the HCV-antibody in the test sample. Can be detected. Methods of immunochemical analysis based on antigen-antibody responses are known in the art (Short protocols in molecular biology, 3rd edition). Although the detection method of HCV antibody which this invention intends is preferably a stationary phase method, the detection method which is not a stationary phase can also be used.

The present invention provides a method for detecting an HCV antibody from a test sample using the VLP and a kit for detecting an HCV antibody. Like HIV, HCV is transmitted through blood transfusions and post-transfusion hepatitis (PTH) occurs in about 10% of transfusion patients, with HCV accounting for 90% of these cases. Therefore, there is an urgent need for sensitive and specific methods for detecting HCV in contaminated blood or blood products. In this way, many methods for detecting antibodies against HCV have been used.

Many studies have been conducted on antibodies specific for HCV. Kyron EP 0 318 216 Al describes a synthesized polypeptide, C100-3, which can be used for the detection of one type of HCV antibody. Currently, kits for detecting HCV antibodies based on the C100-3 antigen are commercially available from Abbott Laboratories. In addition to studies on nonstructural C100-3 antigens, serological diagnostic methods of hepatitis C virus (HCV) infection using HCV core protein (p22) have been developed. Chiba used unglycosylated core proteins to provide specific and sensitive methods for diagnosing infections of HCV (Chiva, et al, Proc. Natl. Acad. Sci. USA 88: 4641-4645 (1991)). However, a satisfactory HCV antibody detection kit has not been developed.

The present invention comprises the steps of: a) contacting a VLP of the invention with a sample comprising an HCV antibody under a time and condition sufficient for the HCV antibody to bind to the VLP of the invention; And b) detecting the bound antibody to determine whether the sample comprises an antibody against HCV. In a preferred embodiment, the sample is a crude sample obtained from an animal or human. In another preferred embodiment, the assay is reverse current immuno-electrophoresis (CIEP) analysis, radioimmunoassay, radioimmunoprecipitation, ELISA, dot blot analysis, inhibition 'or competition analysis, sandwich analysis, immunostick analysis (diff-stick It is selected from the group consisting of analysis, simultaneous analysis, immunochromatographic analysis, immunofiltration analysis, latex bead analysis, immunofluorescence analysis, biosensor analysis and low light detection analysis (Antibodies: A Laboratory Manual, Harlow and Lane (eds). Cold Spring Harbor Laboratory Press (1988); U.S. Patent No. 4,376,110; U.S. Patent No. 4,486,530; International Patent Publication WO 94/25598.

The following examples are intended to illustrate the invention. Therefore, the scope of the present invention should not be limited by the following examples.

<Example 1>

Preparation of pSFHCV / c740 Vectors

In order to prepare infectious hepatitis C virus-like particles, an expression vector expressing core, E1 and E2 proteins was prepared by inserting HCV structural protein gene into alpha viral replicon.

The entire genome of the J strain of HCV was used as a template (provided by Professor Shimotono, Kyoto University; H. Marusawa, M. Hijikata, T. Chiba, K. Shimotohno. Journal of virology, 73, 4713-4720 (1999) )). The amino acid sequence containing the structural protein of HCV (No. 1-740) was amplified by PCR using the following primers 1 (sense; c1) and primer 2 (antisense; c740). Primer was added with Bgl II site (underlined) to facilitate cloning.

PCR was performed using 1 μl of HCV template (10 ng / μl), 8 μl of dNTP (2.5 mM each, TaKaRa), 5 μl of Primer 1 (10 pmol / μl), 5 μl of Primer 2 (10 pmol / μl), 10 × buffer 10 of Taq polymerase. 2 μl of Taq polymerase (1.5 U / μl) and 79 μl of distilled water were mixed, pretreated with 94 ° C. for 5 minutes, denatured at 94 ° C. for 1 minute, and annealed at 50 ° C. for 2 minutes. (annealing), a series of steps of stretching for 2 minutes at 72 ° C was repeated 35 times.

Primer 1: Sense (SEQ ID NO: 1)

c1; 5'-CGGAATCC AGATCT ATGAGCACAAATCCTAAACCT-3 '

Primer 2: Antisense (SEQ ID NO: 2)

c740; 5'-GGG AGATCT GAATTCCATCAGTAGCATCAT-3 '

The amplified gene was treated with Bgl II and cloned into the Bam HI site of pSFV (Gibco BRL), an SFV expression vector, to prepare pSFHCV / c740. The pSFHCV / c740 vector is linked to the nonstructural proteins of SFV under the SP6 promoter, nsP1, nsP2, nsP3, nsP4, and the core, E1, and E2 genes of HCV under the subgenomic promoter 26S. (ampicillin resistance gene) (FIG. 2).

The pSFHCV / c740 vector of the present invention was deposited with the Korean Culture Center of Microorganisms, an international depository under the Budapest Treaty, under Accession No. KCCM-10287.

<Example 2>

Preparation of the pHelper / c740 Vector

PHelper / c740 vectors expressing the structural proteins of HCV were prepared using the pSFV-helper vector. SFV-helper plasmids were purchased from Gibco BRL. The helper vector is linked to genes such as C (capsid), p62, 6K, and E1 (envelop) which function as structural proteins of SFV under the SP6 promoter, and have a replication start point and an ampicillin resistance gene. In the present invention, the structural protein portion of SFV was replaced with cores, E1 and E2, which are structural proteins of HCV. First, pSFHCV / c740 was treated with Xba I and Spe I to obtain DNA fragments containing cores, E1 and E2, which are the structural proteins of HCV. Then, pSFV-helper was treated with Xba I and Spe I to treat C, p62. PHelper / c740 was constructed by removing the, 6K, El region, and inserting the DNA fragment having the HCV structural protein gene in the region (FIG. 3).

The pHelper / c740 vector of the present invention was deposited with the Korean Culture Center of Microorganisms, an international depository under the Budapest Treaty, under accession number KCCM-10286.

<Example 3>

Expression of HCV Structural Protein Using pSFHCV / c740 and pSFV-helper

pSFHCV / c740 and pSFV-helper were simultaneously expressed in host cells.

pSFHCV / c740 and pSFV-helper were digested with restriction enzyme Spe I. After linearizing the vector, in vitro transcription was performed using SP6 polymerase. Specifically, 5 μl of 10 × SP6 polymerase buffer, 10 μm m ⁷ G (5 ′) ppp (5 ′) G (Roche), 10 μl of DNA linearized with restriction enzymes, rNTP mixture (10 mM ATP, 10 mM CTP, 10 mM) UTP, 5 mM GTP, Roche) 5 µl, 100 mM DTT 2.5 µl, 3 µl RNasin (TaKaRa), 1.5 µl SP6 polymerase, 18 µl of distilled water were prepared and reacted at 37 ° C. for 1 hour 30 minutes. The RNA transcript (50 μl pSFV-helper RNA transcript and 50 μl pSFHCV / c740 transcript) was suspended in PBS buffer (Phosphate buffered saline, 1.37 mM sodium chloride, 0.02 mM potassium chloride, 0.1 mM phosphate buffer / ml). 800 μl of BHK-21 cells (10 ⁷ cells / ml) and pulsed at 0.83 V / 25 μF in 0.4 cm electroporation cuvettes (Bio-Rad Gene Pulser).

Immunostaining and western blotting were performed to confirm the expression of HCV structural proteins in the transfected cells. 48 hours after transfection, the cells were fixed with methanol and immunostained. Cytoplasmic expression of E2 protein using monoclonal antibodies against E2 in mice (Lee JW, Kim Km, Jung SH, Lee KJ, Choi EC, Sung YC, Kang CY. Journal of virology, 73, 11-18 (1999)) It was confirmed. In the negative control group, the expression of the E2 protein could not be confirmed, but the expression of the E2 protein was confirmed when the pSFHCV / c740 and the pSFV-helper were coated (FIG. 4) (A: negative control group; B: pSFHCV / c740 and pSFV-helper cells transfected).

In addition, the transfected cells were lysed with 1% NP-40 (1% NP-40, 50 mM Tris-HCl (pH7.6), 150 mM NaCl, 2 mM EDTA, 1 μg / μl PMSF) and then Western blotting was performed using mouse E2 monoclonal antibody. As a result of detection by ECL (enhance chemiluminescence), it was confirmed that E2 protein was expressed (FIG. 5) (lane 1: negative control group, lane 2: cells transfected with pSFHCV / c740 and pSFV-helper).

<Example 4>

Expression of HCV Structural Protein Using pSFHCV / c740 and pHelper / c740

In this example, pHelper / c740 together with pSFHCV / c740 were expressed in host cells. The experiment was performed in the same manner as in Example 3 using pHelper / c740 prepared in Example 2 instead of pSFV-helper. The results are shown in FIGS. 4 and 5. As in Example 3 in which pSFHCV / c740 and pSFV-helper were coat-fected, E2 protein was expressed even when pSFHCV / c740 and pHelper / c740 were transfected into BHK-21 cells.

Example 5

Recovery of VLP

After 48 hours after transfection of the cells according to the method of Examples 3 and 4, the cell culture solution was collected and ultracentrifuge for 3 hours at 100,000 × g to recover virus-like particles. The recovered virus like particles were suspended in 100 μl of TNE buffer (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.5 mM EDTA) and stored at -70 ° C.

<Example 6>

Analysis of RNA Packaged in VLPs

RT-PCR of the VLP was performed to see if the VLP prepared in Examples 3 to 5 contains the RNA of HCV. After dissolving the VLPs recovered from the cell culture in 100 μl of TNE buffer solution, 3 μl of RNase (1 mg / ml) was added and reacted for 30 minutes at room temperature in order to remove RNA and DNA remaining in the cell culture. After that, 10 μl of 10 × DNase buffer (1 M CH ₃ COONa, 50 mM NgSO ₄ , pH 5.0) and 1 μl of DNase (70 U / μl) were added and reacted at room temperature for 1 hour. After removing RNA and DNA remaining outside the VLP through the above process, RNA was extracted using the RNA extraction kit (QIAGEN) using the method described in the manufacturer's manual and stored at -70 ℃.

RT-PCR was performed using the extracted RNA. Denature 2 min at 98 ° C by adding 5 μl of extracted RNA, 4 μl of 4 × buffer, 2 μl of 100 mM DTT, 3 μl of dNTP mixture (2.5 mM each, TaKaRa), 3 μl of oligodT ₁₀ primer, and 1 μl of RNasin. After 2 minutes at 42 ° C., 1 μl of Superscript II (Gibco BRL) was added to react at 50 ° C. for 50 minutes, and the enzyme was inactivated at 72 ° C. for 15 minutes to prepare cDNA. .

PCR was performed with the cDNA as a template. PCR was performed with 5 μl of cDNA, 5 μl of 10 × Z-Taq polymerase buffer, 3 μl of primer 1 (10 pmol, c1), 3 μl of primer 3 (10 pmol, c191), 4 μl of dNTP (2.5 mM each), Z-taq polymer 0.5 μl of TaKaRa and 30 μl of distilled water were added thereto.

Primer 3: Antisense (SEQ ID NO: 3)

c191: 5'-CGGGATCCAGCGGAAGCTGGGATGGT-3 '

Z-taq was a speedy taq polymerase (speedy taq polymerase) was repeated 30 times at 98 ℃ 2 seconds, 68 ℃ 20 seconds 30 times, PCR was performed with an elongation time of 72 ℃ 7 minutes.

The amplified DNA band amplified the core portion of HCV, and it was found that this partial gene was amplified both when using pSFV-helper (lane 1) and when using pHCV-helper (lane 3) (FIG. 6). ). In addition, RT-PCR was performed using primers 4 designed to amplify all core portions linked to 200bp of the 3 'end of the pSFV expression vector, when using pSFV-helper (lane 2) and using pHelper / c740 (lane 2). Lane 4) All these genes were found to be amplified (Fig. 6). It can be assumed that the packaged genes are all from pSFHCV.

Primer 4; Sense (SEQ ID NO: 4)

SFV7200: 5'-AAAGGCAGAAGCTTGATCCAGAAGCACAAATCCTAAA-3 '

<Example 7>

Identification of VLPs by Electron Microscopy

As in Examples 3 and 4, virus-like particles made by expressing proteins were identified using electron microscopy. First, 40 hours after transfection, the cells were collected, washed twice with PBS, first fixed with 2.5% phosphate buffered glutaldehyde, and then secondary with 1% osmium acid (OsO ₄ ). Fixed. The fixed cells were mixed in the same amount of 1% low-melting agarose (hard-melting agarose) and then hardened at low temperature and cut to 1 ㎣. Then perform a dehydration process. Dehydration was performed by stepwise treatment of 25%, 50%, 75%, 95%, 100% ethanol. The dehydrated sample is embedded in complete Spurr'sresin and solidified at 60 ° C. The block thus made was sectioned and stained with uranyl acetate and red cyanate and observed by electron microscopy. As a result, the virus-like particle of 60-70 nm was confirmed (FIG. 7).

The VLP recovered in Example 5 was confirmed using the electron microscope. First, the virus dissolved in TNE was ultracentrifuged with 10% sucrose cushion to concentrate the VLP, followed by ultracentrifugation using PBS, and the obtained VLP was dissolved in distilled water and concentrated. The concentrated VLPs were negatively stained using uranyl acetate, and then observed at an electron microscope magnification of 60,000 ×. As a result, the VLP of about 60-70 nm could be confirmed (FIG. 8).

<Example 8>

Infectivity Testing of VLPs

Each VLP was infected with a cell line to determine whether the VLPs prepared in Examples 3 to 5 were infectious. The VLP suspended in TNE is taken out of the deep freezer and immediately melted, and then treated with 500 µg / ml chymotrypsin and 0.5 mM CaCl ₂ for 30 minutes on ice to protect the infectivity of the virus. Activated. Chymotrypsin was inactivated by the addition of aprotinin. Activated VLPs were infected with hepatoma cell lines including BHK-21 cells, COS-1 cells and Hep3B cells, respectively (FIG. 9) (A: BHK-21 cells; B: BHK-21 cells (chymotrypsin). Untreated); C: COS-1 cells; D: Hep3B cells).

In the case of SFV, the virus is activated only when the chymotrypsin is treated by giving mutations to the p62 region. However, the hepatitis C virus-like particles we developed do not contain the capsid portion of SFV and were expected to be infected with or without chymotrypsin. However, experiments have shown that the treatment of chymotrypsin enhances the infectivity of HCV VLP (FIGS. 9A and 9B). This suggests that the N-terminal 1/3 of the NS3 protein, one of the nonstructural proteins of hepatitis C virus, may be related to the chymotrypsin-like serine protease.

After 48 hours of infection with the VLPs, the cells were washed with PBS and fixed with cold methanol for 10 minutes at 4 ° C. Fixed cells were washed with PBS and then blocked with 1% gelatin for 1 hour. The monoclonal antibody against E2 was diluted in gelatin and reacted for 3 hours, and then reacted with biotinylated IgG for one hour, followed by an avidin-biotin reaction solution. The reaction was developed with DAB to confirm the expression level under an optical microscope. As a result, it was confirmed that the protein of HCV was expressed by immunostaining in infected cells.

Example 9

Identification of HCV Nucleic Acids in Cells Infected by VLPs

After infecting the VLPs of the present invention BHK-21 cells, whether the gene of the virus is present in the cells was confirmed using the RT-PCR technique. After 48 hours of infection with virus like particles in a cell culture dish with a diameter of 35 mm, the cells were collected and total RNA was extracted from the cells (QIAGEN). Using the extracted RNA, reverse transcription was performed with primer 3 (up and dT ₁₀ ), and then the core gene containing 200bp of the upstream into which the structural protein of HCV was inserted was amplified by PCR. Primers 4 (sense, SFV7200) and primer 3 (antisense, c191) were used. As a result, the core gene amplified by RT-PCR was confirmed in both VLP-infected cells (lane 2) prepared using pSFV-helper and VLP-infected cells (lane 3) prepared using pHCV-helper ( 10).

Virus-like particles of HCV were prepared using an alpha viral expression vector expressing HCV structural protein and a helper vector system characterized by expressing HCV structural protein. Since the virus analog particles of the present invention provide antigens that are very similar to the original HCV antigens and are also infectious, they can be useful as vaccines, and can be used as HCV antibody diagnostic kits using the virus analog particles of the present invention as capture antigens. And useful in HCV infection inhibition drug assays.

<110> KIM, Chul Joong <120> Preparation of infectious HCV-like particles <160> 4 <170> Kopatent In 1.71 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cggaatccag atctatgagc acaaatccta aacct 35 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gggagatctg aattccatca gtagcatcat 30 <210> 3 <211> 26 < 212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cgggatccag cggaagctgg gatggt 26 <210> 4 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aaaggcagaa gcttgatcca gaagcacaaa tcctaaa 37

Claims (29)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete a) an alphavirus recombinant vector expressing the core, E1 and E2 structural proteins of Hepatitis C Virus (HCV) and (ii) the core, E1 and E2 structural proteins of Hepatitis C virus. Providing an alphavirus-based helper vector; b) coatfecting said (i) and (ii) vectors into mammalian cells; c) culturing the transfected cells; And d) harvesting HCV analogous particles from the culture supernatant, the method of producing infectious hepatitis C virus analogous particles. The method of claim 22, wherein the alphavirus is selected from Semiki forest virus, Sindbis virus, or Ross River virus. The method of claim 23, wherein the alphavirus is a Semiki forest virus. The method of claim 22, wherein the recombinant vector of (i) is a plasmid deposited with accession number KCCM-10287. The method of claim 22, wherein the helper vector of (ii) is a plasmid deposited with accession number KCCM-10286. The method of claim 22, wherein the (i) and (ii) vectors are coatfected in the form of an RNA transcript. The method of claim 22, wherein the mammalian cell is selected from BHK cells, COS cells, and Hep3B cells. An infectious hepatitis C virus like particle prepared by the method of claim 22.