RU2375449C2 - CODING DNA (cDNA) FOR MAKING VIRUS-LIKE PARTICLES OF HEPATITIS C VIRUS, METHOD FOR MAKING VIRUS-LIKE PARTICLES AND BASED PHARMACEUTICAL COMPOSITION - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine and biotechnology. There is disclosed application of coding DNA for making virus-like particles of hepatitis C virus, as well as the method for making the specified particles and a pharmaceutical composition using the same. The virus-like particles cause induction of interferon system in vivo.

EFFECT: invention can be used for making a preparation for prevention and treatment of the HCV-related conditions, as well as diagnostics thereof.

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Description

The invention relates to the field of medicine and biotechnology and relates to means for preventing and treating conditions associated with hepatitis C virus (Hepatitis C virus = HCV), as well as their diagnosis.

Hepatitis C virus (Hepatitis C virus = HCV) belongs to the Flaviviridae family and causes an infectious process in people, the most common complication of which is hepatitis, which develops into liver cirrhosis and hepatocarcinoma. This disease has affected more than 170 million people on Earth and the number of infected continues to increase. Health analysts estimate the loss of the disease in the United States alone at $ 200 million annually.

In the vast majority of cases, HCV-infected people do not experience health problems or have very mild symptoms, while long-term infection often results in hepatitis, characterized by pain and impaired liver function. People who have been infected have anti-HCV antibodies and, after some time, viral RNA in the blood. In a significant percentage of cases, viral RNA in the blood is not detected. There are currently no commercially available HCV vaccines.

In addition to its infectious characteristics, HCV infects lymphocytes, dendritic skin cells, myocytes, glia cells, and therefore can cause extrahepatic pathology: psoriasis, myocarditis, various neuropathies, vasculitis. HCV was also found in tissue samples from deceased to 1987 (date of virus description) people. In addition, around the world there are about 1.5 million cases of hepatocarcinoma caused by HCV infection of genotype 1b. The treatment methods for HCV have been and remain based on the simultaneous use of genetically engineered interferon preparations and one or two inhibitors of virus propagation (virus specific protease inhibitors - helicase or (and) RNA polymerase). Clinicians try to inject Toll-7 or Toll-9 receptor inhibitors into the treatment cocktail, which use the virus to infect the cells of the immune system. There are known developments aimed at using other approaches to inhibit the multiplication of the virus in an infected cell. However, due to the significant number of various complications caused by the introduction of drugs of genetically engineered interferons, researchers continue to search for drugs whose use will reduce the percentage of complications from the introduction of such drugs.

In this strategic direction, the most promising are the searches for various inductors of the interferon system. The first was the Toll-7 antagonist of the Imiquimod receptor, which is part of the Aldara ointment (3M Pharmaceuticals), the cutaneous use of which has been approved by the FDA since 1999. Its interferon-inducing activity makes it possible to achieve a 50-fold, as compared to normal, increase in the concentration of interferon in the culture of human peripheral blood monocytes (MPPC). The most powerful of the known type 1 interferon inducers is the ODN2216 phosphotioate oligonucleotide, which is a strong activator of Toll-9 signaling (10,000-fold activation of the formation of interferon MPCC, which occurs after the inducer binds to the Toll-9 receptor). However, the large-scale chemical synthesis of ODN2216 is more expensive than the production of genetically engineered interferons.

The natural activators of the synthesis of MIPC interferons of the 1st type are inactivated virions of various viruses. However, the widespread use of inactivated virions is constrained by the requirements for the biological safety of the technology and the preparations obtained, as well as the need to use exclusively the BHK cell line for the purpose of obtaining the virus, for which industrial cultivation regulations have been developed in Russia, but which so far have not allowed obtaining virions with the desired properties. The technology of obtaining pseudovirions, recombinantly inactivated virus-like particles that do not contain nucleic acid inside themselves, allows overcoming the above contradictions.

Recombinantly obtained, partially purified preparations of non-infectious HCV-like virions are known in the literature. In most cases, the purification process of HCV virions does not lead to the desired results, since serum virions are part of non-specific immune complexes and low and very low density lipoproteins. The process of virus isolation after propagation in cell cultures is not free from such problems, since the virus enters the medium with 10% serum containing immunoglobulins, as well as low and very low density lipoproteins. HCV-like ones are isolated from a population of infectious virions by centrifugation in a CsCl gradient (“band” with a floating density <1.36 g / cm). RNA in HCV-like virions is found in relatively smaller amounts, which is due to deletion of shortening of genomic RNA. Deletions affect sequences not only in the translation coding frame — ORF, but also in untranslated terminal sequences — UTR's. The former can lead to the loss of about 50% of all RNA, which is almost 4 kb. Genomic RNA truncated as a result of ORF deletions loses its infectious properties. However, DNA sequences have not yet been constructed that provide efficient expression of HCV structural proteins in cells cultured in serum-free medium, which ensured the assembly of pure virions that do not exhibit interferon-inducing activity. Also, recombinant plasmids were constructed that would program cells to produce large numbers of pseudovirion HCVs. The methodical approach, according to which internal IRES sites are used for the translation of double-cyst RNA, does not allow the cells to form a large number of HCV pseudovirions. Further reading includes the following papers and reviews:

The disease is generally described in Surveillance. Hepatitis. CDC Report No. 61. 2006. Extrahepatic manifestations of HCV infection are described in M. Ramos-Casals et al. Sjogren's Syndrome and Hepatitis C Virus. Clin RheumaToll. 1999, v. 18, p. 93-100.

A description of the HCV features of an open translation framework is provided by Satoshi Ogata et al. Comparative Sequence Analysis of the Core Protein and Its Frameshift Product, the F Protein, of Hepatitis C Virus Subtype 1b Strains Obtained from Patients with and without Hepatocellular Carcinomaio J. Clin. Mi-crob. 2002, v. 40, No. 10, p. 3625-3630.

A description of the DNA and amino acid sequences of HCV is given in an article by K. Nagayama et al. Time-related changes in full-length hepatitis C virus and hepatitis activity. Virology 1999, v.263, No. 1, p.244-253 and in patent application US 20060035335.

Features of HCV RNA replication are disclosed in ZHANG G. et al. Conformational changes involved in initiation of minus-strand synthesis of a virus-associated RNA, RNA2006, v. 12, p. 147-162.

Features of the cultivation of foot and mouth disease virus in the BHK cell line21 are disclosed in C. Escarmis et al. Rapid Selection in Modified BHK-21 Cells of a Foot-and-Mouth Disease Virus Variant Showing Alterations in Cell Tropism, J Virol, 1998, v. 72, No. 12, p. 10171-10179.

Features of reproduction and replication of the virus of foot and mouth disease are disclosed in M. J. Grubman and B. Baxt. Foot-and-Mouth Disease, Clin. Microb. Reviews 2004, v.17, No. 2, p. 465-493.

A description of the various genomes and their comparative characteristics are given by C. Carrillo et al. Comparative Genomics of Foot-and-Mouth Disease Virus, J Virol, 2005, v. 79, No. 10, p.6487-6504.

The following sources may be indicated as prior art:

A known method for producing pseudovirions and Isolation of a cDNA clone derived from a blood-bome non-A, non-B viral hepatitis genome, QL Choo, G Kuo, AJ Weiner, LR Overby, DW Bradley, and M Houghton (this is the basis on which built most of the methods for producing pseudovirus particles).

Mizuno M. et al. Virion-like structures in HeLa G cells transfected with the full-length sequence of the hepatitis C virus genome. Gastroenterology 1995, v. 109, p. 1933-1940.

Kolykhalov A. A. et al. Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA // Science 1997, v.277, p. 570-574.

Baumert T.F. et al. The hepatitis C virus structural proteins assembled into viruslike particles in in-sect cells. J Virol. 1998; 72: 3827-3836.

Beard M. et al. An infectious clone of a Japanese genotype 1b hepatitis C virus // HepaTollogy 1999, v.30, p. 316-324.

Baumert T.F. et al. Hepatitis C Virus-like Particles Synthesized in Insect Cells as a Potential Vacine Cine Candidate // GASTROENTEROLOGY 1999; 117: 1397-1407.

Owsianka A. et al. Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2 // J. Gen. Virol. 2001, v. 82, p. 1877-1883.

Wellnitz S. et al. Binding of hepatitis C virus-like particles derived from infectious clone H77C to defined human cell lines // J. Virol. 2002, v. 76, p. 1181-1193.

Xiang J et al. Recombinant hepatitis C virus-like particles expressed by baculovirus: utility in cell-binding and antibody detection assays // J. Med. Virol. 2002, v. 68, p. 537-543.

Miriam Triyatni et al. Interaction of Hepatitis With Virus-Like Particles and Cells: a Model System for Studying Viral Binding and Entry // JOURNAL OF VIROLOGY, Sept. 2002, v. 76, No. 18, p. 9355-9344.

Sivov I.G. et al. Genetic antivirus against hepatitis C virus // Doklady Akademii Nauk 2003, vol. 382, No. 5, pp. 1-4.

Zhao Wei et al. Expression and self-assembly of HCV structural proteins into virus-like particles and their immunogenicity // Chin Med J 2004, v.117, No. 8, p.1217-1222.

Tazio Storni et al. Nonmethylated CG Motifs Packaged into Virus-Like Particles Induce Protective Cytotoxic T Cell Responses in the Absence of Systemic Side Effects // The Journal of Immunology, 2004, v.172, p. 1777-1785.

Heidi Barth et al. Uptake and presentation of hepatitis C virus-like particles by human dendritic cells // Blood, 1 May 2005, v. 105, No. 9, p. 3605-3614.

As the closest analogue, for example, US Pat. No. 6,387,662, which describes a method for the preparation and purification of hepatitis C virus-like particles, can be indicated.

The introduction of hepatitis C virus (HCV) cDNA into human, animal and insect cells makes it possible to program the formation and release of infectious viral particles. To obtain pseudo-HVC (pseudo-HCV) particles, each research group uses its own modified cDNA. There may be many such modifications.

However, only the present invention solved the problem of efficiently obtaining purified non-infectious virus-like particles of hepatitis C virus in large quantities and developed compositions based on them that induce the formation of interferons and other cytokines in the human body.

The problem was solved using two RNAs with structures that contained modifications compared to natural genomic sequences.

A system of plasmid DNAs has been proposed that program the cells of the BHK21 line to produce virus-like particles of hepatitis C virus that do not contain nucleic acid. Hepatitis C virus-like particles are used in a pharmaceutical composition to induce an in vivo interferon system. Plasmid DNAs are composed of a vector part that allows them to be produced in a bacterial host cell, and after isolation and transfection, they cause the formation of structural proteins for the hepatitis C virus and their assembly into virus-like particles. The DNA in the vectors is transcribed only after transfection, as a result of which two types of RNA are formed. One of them encodes non-structural proteins of the replicative complex of foot and mouth disease virus, which are able to replicate another type of RNA that encodes the structural proteins of hepatitis C virus.

Thus, in one aspect of the present invention, cDNA variants of the HCV subtype 1b sequence are provided that encode the formation of protein-like virus-like particles. DNA sequences of the present invention may include an isolated cDNA sequence that encodes the expression of homogeneous proteins of a virus-like particle.

The coding DNA (cDNA) for obtaining virus-like particles of hepatitis C virus, characterized by the nucleotide sequence corresponding to SEQ ID NO: 1, is shown in figure 1.

One cDNA sequence obtained from a fragment of the HCV genome includes 51 non-coding sequence (5'-UTR) flanking the coding sequence. So that RNA transcribed from such cDNA can be used by the replicative mechanism of foot and mouth disease virus, the necessary motifs are included in its composition. The structure of the franking ORF non-coding sequences in such DNA is shown in FIG. DNA sequences of the variants of the present invention are obtained by introducing mutations and new motifs into the DNA sequence shown in FIG. 1 that encodes the formation of structural proteins of virus-like particles. This eliminates the production of defective protein, so that only

"C-E1-E2-p7-β-gal _αpep " polyprotein.

Accordingly, another aspect of the invention includes proteins of the replicative complex of foot and mouth disease virus and methods for producing HCV proteins from an RNA replicon encoded by a second cDNA sequence in eukaryotic cells. We note that proteins recombinantly obtained in mammalian cells using a second cDNA obtained from genomic RNA of foot and mouth disease virus, as far as we know, have never been mentioned in the scientific literature in this context.

A binary recombinant DNA system is proposed that includes the vector part and sequences encoding truncated

“C-E1-E2-p7-β-gal _αpep ” and “P2-P3” are polyproteins. The RNA molecule transcribed from the second cDNA provides the formation of a “P2-P3” polyprotein capable of post-translational modification, after which the protein complex is able to cause RNA replication of RNA coding for the formation of “C-E1-E2-p7-β-gal _αpep ". Modified host cells transformed with the binary recombinant DNA system express “C-E1-E2-p7-P-gal _αpep ” and “P2-P3” polyproteins are also provided in this invention.

DNA molecules and transformed host cells in the present invention are used in another aspect of the invention - in a new method for producing recombinant homogeneous virus-like particles, which, after purification, can induce the synthesis of interferons and other cytokines in MCPC in vitro and in vivo. In this method, a cell line transformed with a binary recombinant DNA system is cultured under appropriate conditions for expression of recombinant virus-like particles. The resulting virus-like particles are collected from the culture medium using affinity magnetic microparticles.

As the host cells in this method, cells of the BHK21 line were used, which are currently the preferred object for creating laboratory and industrial regulations in Russian biotechnology.

The DNA sequences and proteins of the present invention can be used in the preparation of therapeutic and immunogenic compounds containing HCV and FMDV antigenic determinants. Such compounds are used for the prevention, treatment and diagnosis of diseases that are associated with HCV and hepatocarcinoma. Accordingly, in another aspect of the present invention, there are provided diagnostic compositions for performing RT-PCR diagnosis of HCV-related conditions and diseases in humans. Such diagnostic compositions include virus-like particles containing RNA encoding structural protein genes, saline or saline prepared in phosphate buffer, as is known to those skilled in the art. By "diagnostic compositions" is meant the amount of virus-like particles that are sufficient for detection in human serum samples with or without anti-HCV antibodies. In other embodiments, the active ingredient may be incorporated into other diagnostic preparations.

In a further aspect of the invention, there is provided a method of inducing a complex of interferons (cytokines) associated with HCV diseases and immunodeficiency conditions by administering to a patient, in particular a human, an immunogenically inducing therapeutically effective amount of virus-like particles in a suitable pharmaceutical formulation. Another method of the invention is a method of stimulating a non-specific defense reaction against certain other viruses by administering to a patient an immunogenically inducing therapeutically effective amount of virus-like particles in a suitable pharmaceutical formulation.

Figure 1 illustrates the structure of plasmids pHCV and pFMDV, namely: SEQ ID NO: 1 and SEQ ID NO: 2. The sequences encoding the formation of functionally essential proteins are given in a one-letter code below the corresponding nucleotide codes, separated by the proteins indicated in the text and underlined.

Figure 2 shows the scheme for obtaining a pharmaceutical composition based on virus-like particles.

Figure 3 illustrates the results of determining the induction activity of the drug in vitro. A. Plaques of the virus of vesicular stomatitis in the culture of BHK21, not treated with an inducer; B. Plaques of the virus of vesicular stomatitis on the culture of BHK21 treated with an inducer.

Figure 4 presents the results of determining the induction activity of the drug in vivo.

cDNA sequences provide for the formation of virus-like particles completely devoid of infectious nucleic acid. The system of introducing cDNA into cells allows the introduction of two or three sequences that are different in structure, which makes it possible to program cells not only for the formation of virus-like particles, but also to obtain virus-like particles with RNA molecules of various structures. This is achieved using the Tat transduction protocol described by Carsten R. et al. Oligomers of the Arginine-rich Motif of the HIV-1 TAT Protein Are Capable of Transferring Plasmid DNA into Cells // J. Biol. Chem. 2003, v. 278, No. 13, p. 11411-11418.

The present invention includes modifications of several or all of the cDNA signals obtained from genomic RNA sequences to be expressed in BHK21 cell lines. Mutations were introduced to prevent the shift of the “C” frame of the protein when it expresses in the cells of the BHK21 line. As a result, mRNA transcripts encoding only the proteins of the virus-like particle are obtained. The possible production of the “F” protein is not essential for the production of virus-like particles, since the “F” protein does not contain potentially antigenic sites located on the virus-like particle.

Therefore, the present invention uses a binary recombinant DNA system encoding almost the same non-structural proteins as foot-and-mouth disease virus, as well as virus-like particle proteins, except that codons for virus-specific proteolytic sites encoding various amino acids are replaced by a unique PYEGP site due to modifications, carried out by replacing nucleotides. Preferably, the native nucleotides are replaced with non-native nucleotides in such a way as to maintain a “translation frame”. So, for example, the six-membered nucleotide TCAAGA is replaced with a triplet of GGG nucleotides, respectively. As a result of such a replacement, the PYEGP site and the like were changed to PWTP. As a result of this modification, the flanking proline residues in the site remain the same. Substitutions other than those given may be carried out by specialists.

The present invention provides a simpler method for isolating virus-like particles, since they can serve as a “label” of floating density for the isolation and purification of pharmaceutical compositions. Cellular expression of cDNA constructs facilitates the isolation of proteins of virus-like particles. This reduces the cost of obtaining them. The present invention also makes it easier to obtain starting material for isolating virus-like particles. Due to the presence of only one type of particles, analysis of the protein content and analysis of their amino acid sequence can be carried out without taking into account the presence of other particles.

The present invention further offers the advantage of enhanced production of virus-like particles. In cells of the BHK21 line, it was found that the concentration of the virus of a specific protein is up to 3-10% of the total concentration.

Proteins of virus-like particles encoded by a single reading frame are programmed with a sequence of 3900 bases encoding 1300 amino acids and defining a primary translation product of about 145 kD. The difference between the predicted and real values is associated with intense glycosylation of the E1 and E2 proteins. The apparent molecular weight of the gene product may also vary depending on the utilization of glycosylation sites and various types of cells of the BHK21 line. The term "polyprotein" refers to a pre-modified product being translated having a molecular weight of about 127 kD. The consensus sequences for glycosylation, experimentally identified on various model systems and on other proteins, have the form: ASP-X- (SER) -THR.

The present invention describes compositions prepared using constitutive proteolysis sites of HCV strain 1b for the translation product of genomic RNA from 342 bases from the 5'-end. The cDNA sequence that was used in the examples has a shortened 5'-UTR sequence. It lacks the 60-membered Cap sequence that the “C” protein recognizes in the formation of ribonucleoprotein. In other embodiments, cDNA that can be used to create virus-like particles containing RNA contains a Cap sequence in accordance with the guidelines here that will produce virus-like particles with RNA.

Virus-like particles have low antigenicity, but have significant immunogenic properties that are not specific to a particular HCV strain, and are therefore suitable for use in immunogenic and / or therapeutic pharmaceutical compositions for treating HCV-related diseases. Table 1 shows the result of the induction of INF-α / β with the introduction of a pharmaceutical composition based on virus-like particles into a surviving culture of human peripheral blood mononuclear cells (MPPC).

To induce the formation of cytokines by a pharmaceutical composition based on virus-like particles, up to 100 μg protein / ml of the composition is introduced into the surviving MPPC culture and the mixture is incubated in RPMI1640 medium with 5% fetal bovine serum. As a positive control, the phosphorothioate oligonucleotide ODN2216 described by Krieg A.M. is used. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 2002. v.20, p.709-760. As a negative control, a warmed-up composition based on virus-like particles (100 μg protein / ml) is used. As shown earlier, to make the composition based on virus-like particles non-functional, it should be heated for 92 minutes at 92 ° C. Lower heating temperatures do not allow inactivation of the composition.

Treatment with various enzymes of a composition based on virus-like particles gave irreproducible results.

Although one aspect of the present invention includes a non-specific variant of induction of INF-α / β and INF-γ, it may be desirable to further describe the use of coding sequences. To obtain functionally active proteins, namely, “ER proteins” associated with the membrane of the endoplasmic reticulum, in order to avoid the problems of assembly of a virus-like particle arising from insufficient expression of the membrane apparatus, cell exchange is modified by pretreatment of cells with one of the P450 cytochrome inducers. Preferably, at least spectrophotometrically, the presence of cytochrome P450 is fixed at OD ₄₅₀ > 0.15. Accordingly, in another aspect of the present invention, conditions are provided that can achieve at least a significant increase in the mass of ER membranes.

These cDNA sequence variants can be constructed using methods well known in the art. Modified cDNA sequences of the present invention can be expressed recombinantly using not only known methods for isolating plasmid DNA from bacterial host cells, but also using preparative high-precision PCR technology. Such recombinant plasmids can be isolated and included in pharmaceutical compositions for the prophylactic treatment and prevention of diseases associated with hepatitis C.

Transformation of BHK21 cells is carried out using a method with an 11-membered PTD fragment of a Tat protein with a YGRKKRRQRRR structure, as described in Jong-Sub by Yoon et al. J. Microb. 328, 42 (4), (2004). The construction of plasmids containing the sequence of hepatitis C virus and foot and mouth disease, with or without additional modifications, is carried out using conventional ligation and restriction techniques well known to those skilled in the art. Site-specific DNA cleavage is carried out by treating with a suitable restriction enzyme or enzymes under standard conditions, in particular those normally indicated by the manufacturers of restriction enzymes. For size separation of the cleaved fragments, gel electrophoresis was used using standard techniques to repair the single-stranded ends of the fragments using the Maple DNA fragment of E. coli polymerase I. Treatment with S1 nuclease under conditions known to those skilled in the art allows hydrolysis of any single-stranded DNA. Synthetic oligonucleotides were obtained by diethylphosphoamidite method known in the art. Ligation was carried out using T4 bacteriophage DNA ligase under standard conditions and temperatures, and ligation was confirmed by transforming E. coli with a ligation mixture. Transformants were selected for resistance to ampicillin, tetracycline or other antibiotics, or using other markers known to those skilled in the art. Polymerase chain reaction (PCR) and its modifications were carried out under conditions that were calculated by methods and using programs built into Internet sites known to specialists. The determination of enzymatic activities was carried out by methods known in the art. The determination of the toxicity of virus-like particles was carried out on gilts and cultures of human stem cells by methods known in the art. Virus-like particles of hepatitis C were obtained by lactoperoxidase iodination, determination of J ¹³¹ in the urine, interferon-α / β, -γ in the samples, was carried out by methods known to specialists. A group of volunteers was formed in accordance with the recommendations of the FDA (United States Food and Drug Administration) collected in the Belmont Report 04/18/1979. “Quality of life” was determined according to the QL-36 protocol.

The techniques are fully disclosed in the relevant literature. See: Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED, (1989); DNA CLONING, Vol 1 and 11 (DN Glover ed 1985); OLIGONUCLEOTIDE SYNTHESIS (MG Gait ed 1984); NUCLEIC ACID HYBRIDIZATION (BD Hames ed 1984); TRANSCRIPTION AND TRANSLATION (BD Hames 1984); ANIMAL CELL CULTURE (Rl Freshney ed 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (JH Miller ed 1987 Cold Spring Harbor Laboratory); Scopes PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, 2nd ed, (1987 Springer-Verlag NY) and HANDBOOK OF EXPERIMENTAL IMMUNOLOGY Vols 1-IV (DM Weired 1986); KRUG A. ET AL. IDENTIFICATION OF CPG OLIGONUCLEOTIDE SEQUENCES WITH HIGH INDUCTION OF IFN-α / β IN PLASMACYTOID DENDRITIC CELLS. EUR J. IMMUNOL. 2001, V.31, No. 5: 2154-2163; JOHN E. W. JR. SF-36® HEALTH SURVEY UPDATE // http://www.sf-36.com; JACOBS J. SEROLOGICAL STUDIES ON IODINATED SERA. 1 PRECIPITINES AND PRERCIPITINOGENS // J. IMMUNOL. 1932, V. 23, PP. 361-374; SIVOV I.G. AND ETC. GENETIC ANTIVIRUS AGAINST HEPATITIS C VIRUS // REPORTS OF THE RAS 2003, Vol. 392, No. 5, PAGE. 1-4. All of these publications are incorporated herein by reference.

Accordingly, in a further aspect, the invention includes vectors containing cDNA sequences and prepared host cells. Further, the method involves obtaining virus-like particles by transfecting BHK21 cells with plasmids isolated from bacteria or obtained by preparative PCR that carry cDNA sequences operatively associated with the expression of the replicative complex of foot-and-mouth disease virus and proteins of virus-like particles, as well as controlling RNA replication encoding the formation of the latter in cultivation conditions that ensure the formation of a mass of membranes of the endoplasmic reticulum (ER-membranes). Formed virus-like particles are released from the disintegrating cells and enter the culture medium. Using the method of deposition of virus-like particles on lactoferin mobilized on magnetic microparticles, virus-like particles were extracted from a culture medium containing disrupted cells, which is a magneto-affinity sorption method. After KCl elution, dialysis and sterilizing filtration, a composition based on virus-like particles was obtained. For large-scale production of commercially significant quantities of virus-like particles when used in vaccine compositions, combinations of differential NH ₄ SO ₄ precipitation and ultrafiltration or lectin columns are preferred. Small amounts of pharmaceutical compositions are most readily isolated using differential PEG-6000 / NaCl precipitation. Small amounts of pharmaceutical compositions were used in experiments on the immune response and toxicity. The purified virus-like particles obtained according to the present invention can be used in therapeutic and / or immunogenic pharmaceutical compositions for the prevention and treatment of conditions associated with hepatitis C and diseases such as tumors and hepatocarcinoma. Such pharmaceutical compositions comprise an immunogenic inducing amount of virus-like particles prepared according to the present invention, in admixture with a pharmaceutically acceptable carrier that is an adjuvant / antigen system. The virus-like particle system, which is an adjuvant similar to drugs such as MF59 (Chiron Corp), QS-21 (Cambridge Biotech Corp), 3-DMPL (3-Deacyl-monophosphoryl lipid A 3-Deacyl-Monophosphoryl Lipid A) (Ribi- lmmuno-Chem Research, Inc.), clinical grade Freund adjuvant (IFA), fusogenic liposomes, water soluble polymers or Iscoms (immunostimulatory complexes). The pharmaceutical compositions can be administered systemically, preferably subcutaneously or intramuscularly in the form of acceptable solutions for subcutaneous or intramuscular administration. Inoculation can also be accomplished by scratching the surface or by inoculation in the body cavity. The preparation of such solutions, due to the selection of pH isotonicity, stability, etc., is known to specialists. The dose regimen is determined by the attending physician taking into account factors such as physical condition, weight, gender, nutrition, severity of the condition, time of administration and other clinical factors that may affect the effect of the drug. Examples of doses range from about 1 μg to about 100 mg of virus-like particles. In the practice of the method of treatment of the present invention, an immunologically inducing effective amount of virus-like substances is administered to a patient in need of therapeutic or prophylactic treatment. An immunogen-inducing effective amount of a pharmaceutical composition of the present invention is in the range of about 1 μg to about 100 mg per introductory dose. The number of doses administered may vary depending on the above factors. The invention is further illustrated by the following examples without limiting their scope.

Example 1

Obtaining cDNA sequences and virus-like particles.

1. Construction of plasmids with HCV and FMDV specific sequences.

Sources of full-sized cDNA (ace. No. AB031663 and ace. No. Х74812 GenBank) of the HCV genomes of subtype 1b (strain VAT96) and FMDV of subtype “O” (strain A22550) were plasmids pHCV and pFMVD, respectively. To create the desired constructs, the PCR fragments obtained from the desired cDNA sites were cloned into two plasmids: 1) 8.3kb pNSFMDV containing the ClaI-EcoRV “fusion” PCR fragment, and 2) pHCV, 10.7kb EcoRI-KpnI “fusion” PCR fragment. These fragments are cloned in pUC vectors so that deletions of a number of motifs and / or proteasensitive domains can be made. For this, synthetic, partially complementary, 70-membered oligodeoxyribonucleotides were obtained. Used primers, the structure of which was calculated using Oligo 5.0 and on the Blast site. Primers and oligodeoxyribonucleotides were synthesized at Syntol. Automatic control sequencing was performed on a 373A Automatic Sequencer (Applied Biosysthems, USA) using the reagent kit Dye Deoxy Terminator ABI sequencing Kit with Taq-polymerase FS (Perkin-Elmer, USA).

The pUC of the vector is pUC18 (ace. No. VB0025 GenBank) and pUC19 (ace. No. VB0026 GenBank).

2. Tat transfection of competent cells of the BHK21 line.

Preliminarily, a peptide was synthesized with the structure of the PTD domain of Tat protein of HIV-1 protein. The peptide was prepared by the solid phase method on an NPS-4000 semi-automatic synthesizer (Neosystem Lc.x. oratoires [France]) using the protocol with tert-butyloxycarbonyl / benzyl on covalently labeled 4-methylbenzhydryl amine (0.55 mM / g; 0.25 mM) resin. To introduce a fluorescent network, 4 (5) -carboxyfluorescein was attached to the N-terminus of the Tat domain of the protein in N, N'-dimethylformamide, with preliminary activation in the system of N, N'-para-diisopropylcarbodiimide / 1-hydrobenzotrioazole. Simultaneous removal of the obtained peptides from the carrier and deprotection were performed with trifluoromethanesulfonic acid (according to the standard protocol of Applied Biosystems). The resulting peptides were dissolved in 50% acetic acid and purified by chromatography on Sephadex G15 in 50% acetic acid, and then HPLC chromatography on a Delta Cancer C 18 column (Waters, Milford, MA) with an aqueous 0.01% acetonitrile trifluoroacetic acid solution . The purity of the obtained peptide (YGRKKRRQRRR) was not less than 97%. Ampoules with 10 mg (± 2 mg) of the peptide were dried under nitrogen, sealed and stored at -70 ° C.

Preparative amounts of plasmid DNA were generated by PCR and, after alcohol reprecipitation and quantification (at least 100 μg), were transferred to phosphate-buffered saline (PBS, pH 7.4), to which a peptide dissolved in 1 ml of deionized water was added dropwise. To quantify the conversion efficiency of deoxynucleoprotein [DNP], linear DNA was stained with ethidium bromide. The mixture was stirred and incubated at room temperature for 30 minutes, and then DNP was added to the previously prepared competent cells.

Competent cells were prepared as follows. Cells were grown on RPMI1640 medium from 10% PBS (fetal calf serum) to 10 ⁷ / ml. After a day, the cells were transferred to DMEM medium with 10% PBS, 0.1% sodium phenobarbital, 1 mM hemin and incubated for another 60 hours. The cytochrome content was measured on a Hitachi-557 spectrophotometer according to a two-wave scheme in 1 ml of 100 mM sodium phosphate buffer, pH 7.5, using the molar extinction coefficient ε ₄₅₀ = 91 mM / cm for OD ₄₅₀ , or by recording the differential absorption spectrum of the CO-reduced hemoprotein complex (OD ₄₅₀ = 0.4). Then the cells were washed thoroughly from the medium and transferred for 2 hours in 1 L of fresh DMEM medium with 0.1% sodium phenobarbital, 1 mM hemin and 0.5 M lysophosphatidylcholine. At the end of time, DNP was added to the competent cells.

3. Getting the mass of virus-like particles.

Competent cells treated with DNP were kept at 37 ° C for 6 hours. Then CaNRO _{4 was} added to the medium to a final concentration of 85 mM and after 3 hours PBS to 10%. Cells were incubated for 48-72 hours until the medium was enlightened. At the end of the incubation period, Triton X100 was added to the cells to a final concentration of 0.45% (lysis of cell cytoplasmic membranes). The effectiveness of the current transfection process was monitored after reconstruction of β-galactosidase activity by α-compliment using X-gal [5-bromo-4-chloro-3-indoyl- | 3-O-galactopyranoside] (OD ₆₀₀ > 1.5). A lysate with the indicated characteristics and containing fragments of destroyed cells was clarified either by centrifugation at 5000 g for 20 minutes, or by filtration through filters with a pore diameter of 0.22 μm. 1/2 volume of a solution containing 1.5 M NaCl with 50% PEG6000 was added to the volume of clarified lysate. The mixture containing PEG6000 was held for 12-18 hours at + 4 ° C. The flocculent precipitate was precipitated by centrifugation at 6000 g for 30 minutes. The precipitate obtained after centrifugation was dissolved in 100 ml of PBS buffer (pH 7.4) containing 25 mM NaCl, 1 mM MgSO ₄ , 0.1 mM CaCl ₂ . The protein concentration in the resulting buffer was at least 60 mg / ml.

Example 2

Properties of virus-like particles.

Preliminarily, the sorption parameters of the virulent HCV strain were determined on cells of the Hu-7 and 293T lines, on the monolayer of which the virus strain forms visible negative colonies. Cells of the indicated lines (1-2 × 10 ^5/100 ul) was incubated with VLPs formulation (2 mg) for 1 hour at 4 ° C and then challenged with a virulent strain of HCV (August ¹⁰ pfu). It was found that with the indicated ratio, the composition completely blocks the formation of visible negative colonies. A 50% inhibition of PFU titer was observed at a drug concentration of 0.4 μg / ml. If the cells were kept for 18 hours after treatment with the drug, but before infection with the virus, the cells regained their ability to maintain the reproduction of the virulent virus. Anti-HCV serum (Hoffman La Rosch, USA) also protected cells from infection with a virulent HCV strain, and the titration curve of the protective effect of serum (curve "dose-effect") allowed us to calculate the serum titer, which reduces the activity of the composition of virus-like particles by 50%.

Cell lines HU-7 and 293T (1-2 × 10 ^5/10 ul) infected with a virulent strain of HCV, include biotinylated uridine in HCV-specific RNA. In the case of preliminary incubation of cells with a composition of virus-like particles (2 μg for 1 hour at 4 ° C), there is no inclusion of biotinylated uridine in HCV-specific RNA for 18 hours.

2. ELISA analysis of the resulting pharmaceutical composition.

Flat-bottom well plates were washed twice with 200 μl of distilled water, and then twice with 200 μl PBS buffer. Carefully remove the liquid from the wells and dry the plates. The composition of virus-like particles was dissolved in PBS buffer to the desired concentration, and then 50 μl of the suspension was dispensed into dry wells with a multichannel pipette. The plates were left at 4 ° C. overnight, and then 2% skim milk was added to each well and the plates were incubated at 37 ° C. for another 1.5 hours. After removing liquid from the wells, they were washed with twice distilled water, 200 μl each. The tablets were dried.

The reference sample was taken from the Orion (England) diagnostic kit, which, according to the data obtained by the diagnostic center of the 1st Moscow City Infectious Diseases Hospital, determines the presence of anti-HCV IgG in 98% of cases in the absence of false-positive responses. Using this kit, the values of the limiting dilutions of control sera (healthy and hepatitis C patient, designations K- and K +, respectively) in PBS buffer with 0.5% skim milk were determined and compared with the values obtained on the isolated composition of virus-like particles. It was found that the diagnostic amount of the drug is 250 ng of the introduced protein / well. Wash the dry plate by adding 200 μl of PBS buffer with 2% Tween-20 to each well, and then 200 μl of distilled water. 50 μl of PBS buffer with 0.5% skim milk was added to each well and 10 μl of samples or controls were added. A plate with diluted samples was incubated in the dark for 1 hour at 37 ° C under continuous shaking. After incubation, the contents of the wells were poured, the wells were washed 2 times with PBS with 2% Tween-20 buffer and distilled water (200 μl per well), after which the plate was blotted overturned onto a sheet of filter paper. 50 μl of rabbit anti-human IgG antibody conjugate solution were added to all wells and the plate was incubated for 40 min at 37 ° C with constant shaking. 15-20 min before the end of the incubation time, a working substrate solution was prepared by mixing 1 volume of TMB substrate solution with 4 volumes of substrate buffer containing H ₂ O ₂ .

After the second incubation, the contents were poured from the wells of the plate and washed as indicated after the first incubation. Then, 100 μl of the working solution of the substrate were added to all wells and incubated for 5 minutes at room temperature in the dark. The enzymatic reaction was stopped by the addition of 0.1 M

H ₂ SO ₄ 100 μl per well. The optical density at a wavelength of 450 nm was measured on a Bio-Rad automatic photometer (OD ₄₅₀ , the time between stopping the reaction and the measurement should not exceed 30 minutes). Controls in the reaction were human serum non-reactive for HBsAg, anti-HCV and anti-HIV, as well as inactivated positive for anti-HCV in a buffer with protein stabilizers. The entire array included 395 serum samples that were obtained from multiple studies of the 181st “positive” serum containing anti-HCV IgG and 114 “negative” in which there was no anti-HCV IgG. There were no cases of false-positive ELISA results. In 16 "positive" ELISA sera with the obtained drug did not give the correct result

3. Binding to low density lipoproteins.

Centrifugation in a sucrose density gradient was carried out for 18 hours at 50,000 g in a sucrose gradient of 50-10% w / v (Spinco L2 65 V, Beckman, Ti-rotor SW55) at 4 ° C. Complexes of lipoprotein with virus-like particles were detected after digging the gradient.

100 μl of antilipoprotein antisera (goat anti-HDL, anti-LDL, lipid-free; Sigma) was diluted 1: 2 with PBS and kept at 4 ° C overnight. The obtained precipitates were precipitated by centrifugation (10 minutes at 5000 g) and the presence of HCV antigen was detected in them, as well as in the supernatant, with HCV-positive sera.

4. Binding to lactoferin immobilized on magnetic particles.

In this experiment, lactoferin-biotin complex was coupled using MagneSphere Magnetic Separation Products (Promega protocol). Avidin-labeled magnetic microparticles require BSA for stabilization, which is present when stored in PBS buffer with 1 mg / ml BSA and 0.02% sodium azide and is removed as soon as the particles are washed. The working concentration of particles is 1 mg / ml. Particles should be washed three times with an equal volume of 0.5X SSC and used within 30 minutes after washing, which is required for optimal performance. Finished magnetic microparticles were added to a mixture of lactoferin-biotin and a composition of virus-like particles (2 μg). As an experimental control, the finished magnetic microparticles were added to CV.CH of lactoferin-biotin and the virulent HCV strain (10 ⁸ PFU). After washing the precipitate three times on the walls of the tube when placed on a magnet, the precipitate was washed with 0.4 M KCl and the amount of HCV-specific antigen was determined by ELISA. When comparing the results obtained by diluting the composition of virus-like particles and particles of hepatitis C virus, we estimated the value of 50% inhibition by the drug (about 0.3 μg / ml) of binding between lactoferin and the virus.

Example 3

Induction of virus-like particles of hepatitis C virus interferon and other cytokines.

1. The induction of virus-like particles of hepatitis C virus interferon and other cytokines in human peripheral blood

PBMCs were isolated after centrifugation of fresh heparinized peripheral blood in a density gradient of ficol-hypak. Blood was received “incognito” from healthy volunteers based on complete information and after obtaining consent. A group of volunteers was selected and formed according to the recommendations of the FDA (United States Food and Drug Administration) collected in the Belmont Report 04/18/1979.

PBMCs were reprecipitated twice in MACS buffer (PBS with BSA at 0.5% and EDTA at 2 mm (Sigma)) or in RPMI 1640 medium containing 10% inactivated at high temperature, fetal bovine serum (Hyclone), La-amide- aminoglutaric acid per 2 mm, 100 units / ml of penicillin and streptomycin and HEPES per 25 mm.

Determination of interferon-α / β was performed using an ELISA kit (Nunc Maxisorp ™ USA). Data from a typical experiment using FITZ-labeled antibodies are presented in table 1.

TABLE 1 in pg / ml The control Experience Control (after 3 days) Experience (in 3 days) TNF-ά / β 12.76 316057.3 21.45 111566.5

2. Induction of virus-like particles of interferon and other cytokines in the body of volunteers.

The experiment consisted in the determination of interferon-α / β in the blood serum of five volunteers before and after a single rectal administration of a pharmaceutical composition based on virus-like particles in an amount of 20 μg to each volunteer. After a single rectal administration of a pharmaceutical composition based on virus-like particles in an amount of 20 μg, the level of interferon α / β in the blood serum briefly increased on average 10,000 times. Subjective deviations in the indicators of “quality of life” by the QL-36 coefficient in five volunteers during the experiment and 6 months after it was not observed.

A joint analysis of the results obtained in examples 2 and 3 indisputably indicates the applicability of the pharmaceutical composition described in this invention for the treatment of hepatitis C. As example 2 shows, the virus-like particles described in this invention are so similar to the particles of hepatitis C virus that they compete for the same cellular receptors and are bound by the same antibodies and lipoproteins. At the same time, Example 3 demonstrates that virus-like particles cause a sharp immunostimulation, which is known to be the key mechanism for curing hepatitis C. Thus, it is obvious that when a patient is introduced into the patient’s body, the virus-like particles will interact with cells infected with hepatitis virus C, and cause immunostimulation in them, which will lead to a cure.

Claims (5)

1. The use of coding DNA (cDNA), characterized by a nucleotide sequence corresponding to SEQ ID NO: 1, to obtain virus-like particles of hepatitis C virus. 2. The use of coding DNA (cDNA), characterized by a nucleotide sequence corresponding to SEQ ID NO: 2, to obtain virus-like particles of hepatitis C virus. 3. A method for producing virus-like particles of hepatitis C virus, characterized in that it includes preparatory cultivation of BHK cells 21 transformed with sequences according to claims 1, 2 in a suitable culture medium and isolation of virus-like particles from these cells by chromatography on lactoferrin immobilized on magnetic particles . 4. A pharmaceutical composition for the induction of interferons and other cytokines, characterized in that it contains virus-like particles obtained by the method according to claim 3, in an effective amount and a pharmaceutically acceptable carrier. 5. The pharmaceutical composition according to claim 4, characterized in that it is intended to prevent and treat hepatitis C conditions for which interferon therapy is recommended.

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