Polypeptide and a method thereof, a reagent for immunoassay, the method of determining the presence of antibodies and method of induction of an immune response

 

(57) Abstract:

The invention relates to polypeptides, used as immunological reagents for the identification, prevention, and treatment of infections caused by HCV. Describes a new polypeptide comprising a truncated sequence of HCV (hepatitis C virus) containing the epitope of HCV with the formula AAx-AAyable to induce immune response in the subject, where AA denotes an amino acid, x and y are integers such that x 14, AAx-AAydesignates the portion of the amino acid sequence of Fig. 1 and x is chosen from the group: 66, 413, 465, 540, 1218, 1940, 2244, 2281, moreover, the specified polypeptide can have an optional sequence of N-terminal amino acids corresponding to the sequence of the HCV polypeptide adjacent to the N-end amino acids AAxto which it is attached, or an optional sequence C-terminal amino acids corresponding to the sequence of the HCV polypeptide adjacent to the C-end amino acids AAyto which it is attached, and the total number of additional amino acids does not exceed 15 for AA413-AA-AA4274 for AA465-AA480, 85 for AA540-AA55410 for AA

The invention relates to materials and methodologies to eliminate the spread of infection caused by hepatitis C virus (HCV). More specifically it relates to polypeptides, used as immunological reagents for the identification, prevention, and treatment of infections caused by HCV.

Background of the invention

HCV was first identified and characterized as a cause of hepatitis neither A nor B (NANBH) Houghton et al. This led to the discovery of a number of basic and specific polypeptides, applicable as of immunological reagents. See, for example, Houghton et al., N 318,216; Houghton et al., EPO Pub. N 388,232; Choo et al., Science (1989) 244: 359-362; Kuo et al., Science (1989) 244: 362-364; Houghton et al., Hepatology (1991) 14: 381-388. These publications provide information with a wide range of sources mainly for HCV, as well as the production and use of the polypeptide of the immunological reagents of HCV. For brevity, therefore, the description of these publications, in particular, incorporated herein by reference.

Other readily applied and expanded development Houghton et al. See, for example, Highfield et al., UK Pat.App.2.239245 (The Welcome Foundation Ltd); by wahg, EPO Pub. N 442394 (United Biomedical Inc.); Leung et al. , EPO Pub. N 445423 (Ab69.348 (Shionogi Seiyaku K. K.).

Sensitive, specific methods for verification and determination of native HCV and HCV infected blood and blood products from the blood are important achievement in medicine. Hepatitis after blood transfusion (PTH) occurs in approximately 10% of patients who have received a blood transfusion and HCV was up to 90% of these cases. The main problem with this disease is a common disease progression to chronic liver disease (25-55%). The treatment of patients, as well as the prevention of HCV transmission by blood and blood products or by close personal contact, requires reliable diagnostic and prognostic tools, such as the HCV polypeptides to detect antibodies associated with HCV. Such polypeptides also are used as vaccines and immunotherapy and therapeutic agents for the prevention and/or treatment of disease.

Because HCV is a relatively new pathogen, there is a continuing need to identify additional immunological reagents that will enable further study of the clinical course of the disease and the epidemiology of HCV among the population.

Description of the invention

The invention relates to the characterization of new HCV epitopes. The defining characteristic is irout with antibodies to HCV and/or cause the formation of antibodies against HCV in vivo. These polypeptide products are suitable as standards or reagents in diagnostic tests and/or as components of vaccines. Antibodies, including, for example, both polyclonal and monoclonal, directed against HCV epitopes contained within these polypeptide sequences are also applicable as reagents, for example, for diagnostic tests, as therapeutic agents, the selection of antiviral drugs for the separation/purification of the HCV polypeptides or particles.

In its broadest sense, the present invention is directed to obtaining polypeptides containing the newly characterized epitopes of HCV described here, the development of methods for such polypeptides (e.g., chemical synthesis), the development of methods of using such polypeptides (e.g., diagnostics, vaccines and therapy). Immunological study for the detection of antibodies against HCV, comprising incubating a sample suspected the content of antibodies against HCV, with the polypeptide described above, under conditions that allow formation of a complex of antibody-antigen: the definition of a complex of antibody-antigen containing polypeptide, is also in the scope of the claims izaberete the th introduction to the individual selected immunogenic polypeptide, containing an HCV epitope described herein, in an amount sufficient to induce an immune response.

The above objects of the present invention can be carried out using HCV epitopes with the formula

aax-aay,

where aa denotes an amino acid;

x and y are integers such that y-x 14;

aax-aayshows a portion of the amino acid sequence of figure 1;

and x is chosen from the group: 66, 413, 540, 1218, 1940, 2244, 2281, moreover, the specified polypeptide can have an optional sequence of N-terminal amino acids corresponding to the sequence of the HCV polypeptide adjacent to the N-end amino acids aaxto which it is attached, or an optional sequence C-terminal amino acids corresponding to the sequence of the HCV polypeptide, adjacent to the C-end amino acids aayto which it is attached, and the total number of additional amino acids does not exceed 15 for aa413-aa4274 for aa465-aa480, 85 for aa540-aa554, 10 aa2244-aa2258and for 30 aa2281-aa2300.

Brief description of drawings

Fig.1 shows polyprotein prototype isolate HCV-HCV1.

Fig.2 is lnost human isolate 23, sequences are shown below the line sequence. Also shown are the amino acids encoded by the consensus sequence.

Fig. 4 shows the nucleotide consensus sequence of the human isolate 27, variant sequences are shown below the line of the sequence.

Fig.5 shows elongated in a number of the nucleotide sequence of the human isolates, 23 and 27 and HCV1. Homologous sequences are indicated with an asterisk (*). Non-homologous sequence represented by small letters.

Fig. 6 shows elongated in the number of amino acid sequences of the human isolates, 23 and 27 and HCV1. Homologous sequences are indicated with an asterisk (*). Non-homologous sequence represented by small letters.

Fig. 7 shows a comparison of the ranked compound of the nucleotide sequences of isolates Thorn, EC1, HCT 18 and HCV1.

Fig. 8 shows a comparison of the nucleotide sequences EU10 and composite HCV1 sequence; the sequence EU10 is on the line over the points, and the HCV1 sequence is on the line below the point.

Fig. 9 shows comparison of the amino acid poceski isolates PCT#18, JH, 2J, JH27, Thorne, EC1 and HCV1.

Fig. 10 shows a comparison of amino acid sequences 330-360 (relative to the HCV1) encoded in the areas of EnvL" consensus sequences of human isolates nst 18, JA23, JH27, Thorne, ECT, HCV1.

Ways of carrying out the invention

A complete listing of the publications referenced here can be found in the sections "Background of invention" or "Bibliography".

1. Definition

"Hepatitis C virus" or "HCV" refers to nanopiano viral mind pathogenic strains which cause NANBH and from them received attenuated strains or defective interfering particles. See mainly the publications cited in the section entitled "Background of invention". The genome of HCV contains PHK. It is known that the content of PHK viruses have a relatively high degree of spontaneous mutations, i.e., by message, of the order of 10-3up to 10-4at the nucleotide (Fields and Knipl (1986)). Therefore, due to the heterogeneity and fluidity of genotype, characteristic of RNA viruses, there are many strains/isolates, which may be virulent or avirulent, inside the form of HCV. Distribution, identification, detection and selection razlichayushchiesya drugs and vaccines for different strains/isolates, as well as the composition and also to develop methods that are applicable when the selection procedure for antiviral agents for pharmacological purposes, such as tools that inhibit the replication of HCV.

It reveals information on several different strains/isolates of HCV, in particular strain or isolate CDC/HCV/ (also called). The information obtained from one strain or isolate, such as parts of the genome or the amino acid sequence sufficient to enable experienced professionals, using standard techniques, to identify new strains/ isolates and to determine whether such new strains/isolates of HCV. For example, several different strains/isolates described below. These strains, which were obtained from a number of human sera from different geographical areas) were selected using the information on the genomic sequence of HCV1.

The information provided here shows that HCV may be distantly related flaviviruses. The Flavivirus family contains a large number of viruses, which are small, oblechennymi pathogens of man. The morphology and structure flavivirus particles known and discussed by Brinton (1986). In the core and layers. Virions are spherical and have a diameter of about 40-50 nm. Their nucleoid have about 25-30 nm in diameter. On the outer surface of the shell of the virion are outgrowths, which have a length of about 5-10 nm with terminal swellings with a diameter of approximately 2 nm. Typical representatives of this family are the yellow fever virus, West Nile virus, Dengue viruses. They have positively-chain RNA genomes (~ 11,000 nucleotides), which are slightly larger than those of HCV, and encode polyproteins predecessor approximately 3500 amino acids. Individual viral proteins are separated from the predecessor of the polypeptide.

A summary of the structure of the genome and nucleotide sequence of the genomic RNA of HCV. The genome appears to be a single-stranded RNA containing ~ 10,000 nucleotides. The genome is positive-chain and has a constant translational open reading frame (ORF), which encodes polyprotein of approximately 3000 amino acids. In ORF structural protein(s), apparently, are encoded in approximately the first quarter of the N-terminal region, with most of polyprotein responsible for non-structural proteins. When compared with all known viral Poza of flaviviruses and pestiviruses (which, as now considered, are part of the Flavivirus family).

Based on inferred amino acids encoded in the nucleotide sequence of HCV1 and other data may be protein domains encoded polyprotein HCV, as well as the approximate boundaries are the following:

Estimated domain Approximate boundaries (number of amino acids)

C (nucleocapsid protein) - 1-131

E1(envelope protein of the virus) - 192-383

E2/NS1/ (shell?) - 384-800

(unknown function) - 800-1050

(PR?) - 1050-1650

(unknown function) - 1651-2100

(polymerase) - 2100-3011 (end)

These domains are, however, preliminary. For example, the boundary EI-NS2 is in the area 750-810, and NS3-NS4 border is about 1640-1650. There is also evidence that the variant C - 191 is not a precursor, which is then processed (e.g., to a length of about 170 AA), and that proteins NS2, NS4 and NS5 each, then processed into two Mature protein.

As expected, different strains, isolates or subtypes of HCV include variants of the amino acids and nucleic acids compared with HCV1. Suggest that many isolates show a greater degree (i.e., more that about 40%) homology in on what others are less homologous isolates of HCV. These isolates can be defined as HCV according to different criteria, such as, for example, the ORF in the range from about 9000 nucleotides to about 12,000 nucleotides encoding polyprotein, similar in size to that of HCV1, coded polyprotein with hydrophobic and/or antigenic characteristics similar to those of HCV1, and the presence of kolyanych peptide sequences that are saved from HCV1. In addition, would be positively-chain RNA.

HCV encodes at least one epitope which is immunologically comparable to the epitope in polyprotein HCV1. The epitope is unique to HCV in comparison with the previously known flaviviruses. The uniqueness of the epitope may be identified by its immunological reactivity with antibodies to known types of flaviviruses. Methods for determining the immunological reactivity known in the field of science, for example, here is radioimmunoassay, the determination by ELISA haemagglutination and even some examples of suitable methods for the study.

In addition to the above can be used for identification of the strain/isolate as HCV following parameters homology of nucleic and amino acids alone or in combination. Since m level can be at the level of 10% or more, it may be about 40% or more, possible, about 60% or more, and even more probably about 80% or more; and in addition, there will be corresponding adjacent sequence of at least about 13 nucleotides. It should be noted that variable and hypervariable region present in the HCV genome; therefore, it is expected that the homology in these areas will be significantly less than the homology in the whole genome. The correspondence between the intended genomic sequence of HCV strain and, for example, the cDNA sequence of SDS/HCV1 can be identified by methods known in this field of science. For example, it can be determined by direct comparison of data sequence polynucleotide the estimated HCV, and cDNA sequences described herein. For example, it can also be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions (for example, those that would be used to splitting of S1), followed by cleavage with specific nuclease single-stranded nucleic acids with subsequent determination of the amount of cleavage fragments.

The benefit of their homology to the polypeptide level. Mainly it is expected that the strains and isolates of HCV are homologous to at least 10%, more than about 40%, possibly more than about 70%, and even more likely more than about 80%, and some may be homologous more than about 90% at the polypeptide level. Methods for determining homology of amino acid sequences known in the art. For example, it is possible to directly determine the amino acid sequence and compared with the sequences presented here. Or as otherwise may be determined nucleotide sequence of the material of the genome of the intended HCV (usually via a cDNA intermediate), can be defined encoded therein amino acid sequence and comparison of the respective areas.

As used here, polynucleotide "derived from" a specified sequence, refers to a polynucleotide sequence that contains a sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding to the region of the x sequence. Preferably the sequence of the region from which the received polynucleotide homologous or complementary sequences, which are unique to the genome of HCV. Unique or not, the sequence for the HCV genome can be determined by methods known in the art. For example, the sequence can be compared with sequences in databases (priority date), for example, Genebank, to determine whether she uninfected hosts or other microorganisms. The sequence can also be compared with known (priority date) sequences of other viral agents, including those which are known to cause hepatitis, for example NAU, NWO and HDV, and representatives of the family Flaviviridae. Compliance or noncompliance with the received sequence to other sequences can also be identified by hybridization to meet strict conditions. Methods of hybridization to determine the complementary sequences of nucleic acids are known in the industry. See, for example, Maniatis et al. (1982). In addition, inappropriate pair duplex polynucleotides formed by hybridization, can be determined by known methods is minute in duplex polynucleotide. Areas from which can be "obtained" a typical DNA sequences include, but are not limited to, for example, regions encoding specific epitopes, as well as naschityvaetsya and/or noncoding region.

The resulting polynucleotide not necessarily physically derived from shows the nucleotide sequence, but can be done by other means, including, for example, chemical synthesis or replication or reverse transcription or transcription of DNA. In addition, combinations of fields corresponding to this sequence can be modified by well-known specialists of ways to fit the intended application.

Similarly, polypeptide or amino acid sequence "derived from" a specified amino acid or nucleic acid sequence corresponds to a polypeptide having the amino acid sequence identical to the sequence of the polypeptide encoded in the sequence, or portion thereof, and this portion consists of at least 3-5 amino acids and more preferably at least at least 8-10 amino acids, and even more preferably at least 10-15 of aminocellulose. This terminology also includes the polypeptide expressed by the specified nucleic acid sequence.

A recombinant or derived polypeptide is not necessarily translated from a specified nucleic acid sequence; it can be produced by other means, including, for example, chemical synthesis or expression of a recombinant expression system, or isolation from HCV, including mutated HCV. Recombinant or produced polypeptide may include one or more analogs of amino acids or unnatural amino acids in its sequence. Methods of building analogs of amino acids in the sequence is known in this field of science. It may also include one or more labels, which are known to skilled practitioners.

The term "recombinant polynucleotide", as used here, implies polynucleotide genomic ndnc, semi-synthetic or synthetic origin, which due to its origin or manipulation: (1) is not associated with all or part of polynucleotide with which it is associated in nature, (2) connects with polynucleotides than the one with which it is associated in nature, Kili (3) does not occur in nature.

"Purified" polypeptide is the which contains at least about 50% by weight, the desired polypeptide/total number of polypeptides in the composition), preferably, at least about 70%, and even more preferably at least about 90% of the desired polypeptide, without considering deproteinize materials in the composition. Methods purification of viral polypeptides are known in this field of science. Purified antibodies gives a similar definition.

"Recombinant cell hosts", "cell host", "cell", "cell line", "cell culture" and other such terms denoting microorganisms or higher line eukaryotically cells, cultured as unicellular beings are cells that can be used or were used as recipients for recombinant vector or other distortion of the DNA, and include the progeny of the original cell, which was transliterowany. It is clear that the offspring of a single parent cell does not need to be completely identical in morphology or in genomic or total DNA chromosomes of the original parent, due to natural, accidental, or deliberate mutation.

"Replicon" is any genetic element, such as a plasmid, chromosome, virus, kosmidou and so on, which behaves as an Autonomous unit of polynucleotide replication vnutri which is attached to another polynucleotide segment, so to invoke the replication and/or expression of the attached segment.

"Control sequence" refers to polynucleotide sequences which are necessary to affect the expression of coding sequences with which they are associated. The nature of such control sequences differs depending on the host body; in prokaryotes also control sequences generally include promoter, the binding site with the ribosome and terminators; eukaryota basically also control sequences include promoters, terminators and, in some cases, enhancery. The term "control sequences" is intended to include, as a minimum, all components whose presence is necessary for expressions, and may also include additional components whose presence is expedient, for example, a leader sequence.

"Operatively linked" refers to a location at which the components, so-called, are in connection, allowing them to function in the intended way, the control sequence is linked in such a way that expression of the coding posledovatelya frame" (ORF) is a region of the polynucleotide sequence, which encodes the polypeptide; this area may be part of a coding sequence or a total coding sequence.

"Coding sequence" is a polynucleotide sequence which is read at the mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' - end and a translation stop codon at the 3'-end. The coding sequence can include, but is not limited to mRNA, cDNA, and recombinant polynucleotide sequences.

"Immunologically identifiable with/as" refers to the presence of epitope(s) and polypeptide(s) that are also present in the specified(s), polypeptide(s), usually proteins of HCV. Immunological identity can be determined by linking with antibodies and/or competition when linking; these techniques are known to skilled practitioners.

Used here, the term "epitope" refers to an antigenic determinant of a polypeptide. The epitope can include 3 or more amino acids that define the binding site of the antibody. In about the types of epitope mapping are known in this field of science.

The polypeptide is immunologically reactive with an antibody when it binds with the antibody due to the detection antibody specific epitope contained in the polypeptide. Immunological reactivity may be determined by binding of antibodies, more specifically, using the kinetics of antibody binding and/or by competition binding using a competition(s) component(s) known polypeptide(s) containing the epitope against which the antibody directed. Methods of determining whether a polypeptide immunologically reactive with an antibody known in this field of science.

Used here, the term "antibody" refers to a polypeptide or group of polypeptides that contain at least one connecting antibodies website. "Connecting customers antibodies" or "binding domain" is formed as a result of folding the various domains of the molecule(s) of the antibody(a) for the formation of three-dimensional spatial units with an internal surface shape and charge distribution complementary to this characteristic epitope antibodies, which gives the possibility of immunological reaction with the antigen. The binding site of the antibody may be formed on the aphid, providing a binding to the antigen. The term "antibody" includes, for example, vertebral antibodies, hybrid antibodies, chimeric antibodies, altered antibodies, incomplete antibodies, Fab proteins and antibodies with a single domain.

Used here, the term "odnogolosoe antibody" (d:b) is an antibody that includes Vndomain immunologically reactive with a specified antigen, dAb does not contain a domain V1but may contain other antigen binding domains, which are known to exist in the antibodies, for example, Kappa and lambda domains. Methods of obtaining dAb known in this area. See for example Ward et. al. (1989).

Antibodies may also contain Vnand V1domains, as well as other well-known antigen binding domains. An example of these types of antibodies and methods for their preparation are known in this field of science (see, for example, U.S. patent N 4816467, which are hereby incorporated by reference) and include the following. For example, "vertebral antibodies" refer to antibodies that are tetramine or their complexes, including light and heavy chains, which are usually collected in a "y" configuration and which may or may not have covalent bonds between the chains. In vertebral antibodies, amimi one antibody, produced by lymphocyte that produces the antibody in situ or in vitro (e.g., hybridomas). Vertebral antibodies typically include natural antibodies, such as purified polyclonal antibodies and monoclonal antibodies. Examples of methods of producing these antibodies are described below.

"Hybrid antibodies are antibodies in which one pair of heavy and light chains is homologous to those in the first antibody, while the other pair of heavy and light chains are homologous to those of the other second antibody. Typically, each of these two pairs will be contacted different epitopes, in particular, on different antigens. This results in a property of divalently", i.e. the ability to bind two antigens simultaneously. Such hybrids may also be formed using chimeric chains, as set forth below.

"Chimeric antibodies" are antibodies in which the heavy and/or light chain is fused proteins. Usually the constant domain of a chain going from one particular type and/or class, and variabelny domains originate from other species and/or class. This also includes any antibody in which one or both of the heavy or light chains are composed of combinations of peaks representatives of different classes, or species of different origin, and anyway the point of confluence is located on the border variable/constant regions. It is thus possible to obtain antibodies, in which neither a constant nor a variable region does not mimic the known sequence of the antibody. Then it becomes possible, for example, to build an antibody variable region which has high specific affinity for a particular antigen, or a constant region which can cause increased fixation of complement, or to obtain other improvements of properties, which has a constant area.

Another example is the "modified antibodies, which are antibodies in which a naturally occurring amino acid sequence in vertebral modified antibodies. Using techniques of recombinant DNA, antibodies can be rearranged to obtain the desired characteristics. Possible changes are many and they range from changes in one or more amino acids to the complete restructuring of the area, such as a constant region. Changes in the constant region primarily in order to achieve the desired characteristics of cell processing, i.e., the change in fixation of complement, usaste, in order to change the characteristics of binding antigen. Antibodies can also be designed to help specific delivery of molecules or substances to a specific cell or tissue site. The desired changes can be made known molecular biology methods, such as recombinant techniques, siteprovides mutagenesis, etc.

Another example that "incomplete antibodies", which are units that includes a dimer of a heavy chain/light chain bordering the Fc (i.e., constant) a second region of the heavy chain. This type of antibody eliminates antigenic modulation. See, for example, Glennie et.al. (1982).

In the detection of antibodies also include "Fab" fragments of antibodies. Area "Fab" refers to those parts of the heavy and light chains, which are approximately equivalent or similar to the sequences contained in the branched part of the heavy and light chains, which have been shown to exhibit immunological binding to the specific antigen, but do not have the effector part of the Fc. "Fab" includes units of one heavy and one light chain (commonly known as "Fab"), as well as tetramer containing 2H and 2L chain (called F (ab)can be divided into sub -, biological to those described above, i.e., "vertebral Fab, hybrid Fab", "chimeric Fab" and "modified Fab". Methods of obtaining "Fab" fragments of antibodies known in this area of science and include, for example, proteolysis and synthesis using recombinant techniques.

The term "antibody" also includes single-stranded binding antigen (SCA) proteins of this type, which is described in a paper coauthored with Schlom J. the issue of Cancer Research, 15 June 1992, (as well as in the articles cited here).

So, as it is used here, the term "immunogenic polypeptide" refers to a polypeptide that causes cellular and/or humoral immune response, one or associated with a carrier in the presence or absence of adjuvant.

The term "polypeptide" refers to a polymer of amino acids without regard to the specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. This term also does not refer to or are not permitted modifications of the polypeptide after expression, for example, glikozilirovanie, acetylation, phosphorylation and the like. This definition includes, for example, polypeptides containing one or more analogs of an amino acid (including, e.g. the ones in this area, as found in nature, and nature does not exist.

"Transformation", as used here, refers to the insertions of exogenous polynucleotide cells of the host, regardless of the method used for installation, for example, direct uptake, transakcji, f-mating or electroporation. Exogenous polynucleotide can be saved as reintegrating vector such as a plasmid, or alternatively, can be integrated into the host genome.

"Treatment" as used here, refers to the prevention and/or therapy.

"Individual" as used here, refers to a vertebrate, particularly to the representatives of the mammalian species, and includes, but is not limited to animals (e.g. dog, cat, cattle, pigs, sheep, goats, rabbits, mice, rats, Guinea pigs, etc. and primates, including monkeys, chimpanzees, baboons and humans.

Used here, the term "semantic chain nucleic acid contains a sequence that is Ecologia sequence of mRNA. "Antimyeloma chain contains a sequence that is complementary to the sequence of the sense circuit".

As ispolzovat and which encodes a viral(e) the polypeptide(s). Examples of RNA viruses with a positive chain include Jogaviridal, oronaviridal, Retroviridal, Picornaviridal and Caliciviridal.

This also includes Flaviridal that were originally classified as Jogaviridal. See Fields and Knipl (1986).

Thus, this component of the body that contains antibodies" refers to a component of the body of the individual who is the source of the antibodies of interest. Components of the organism that contains antibodies known in this field of knowledge and include (but are not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, external detachable respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells and myeloma.

As used here, a "biological sample" refers to a sample of tissue or fluid isolated from an individual, including, but not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, detachable outer skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples that make up a cell culture in vitro (including, but not limited to, environment, meets the standard, the resulting cell growth in the medium for Kultuk).

II. Description of the invention

In the practice of the present invention will be applied, unless otherwise indicated, conventional techniques of molecular biology, Microbiology, recombinant DNA, and immunology, which constitute the methodological basis of this area. The explanation of such techniques are fully described in the literature. See, for example, Maniatis, Fitsch & Sambrook, "Molecular Cloning; A Laboratory Manual" (1982); "DNA Cloning, Volumes I and II (D. N Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed, 1984); "Nucleic Acid Hybridization" (B. D. Hames &S. J. Higgins eds. 1984); "reduced and Translation" (B. D. Hames &S. J. Higgins eds. 1984); "Animal Cell Culture" (R. I. Freshney ed. 1986); "Immobilized Cells And Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide To Molecular Cloning" (1984); the series "Methods in Enzymology" (Academic Press, Inc.); "Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory), Meth Enzymol Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds. , respectively), Mayer and Walker, eds. (1987), "Immunochemical Methods In Cell And Molecular Biology" (Academic Press, London); Scopes (1987) "Protein Purification: Principles and Practice", Second Edition 10 (Springer-Verlag, N. Y.); and "Handbook of Experimental Immunology", Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

All patents, patent applications and publications referred to here both above and below, thus incorporated herein by reference.

II. A. Truncated polypeptides HCV

Applied polypeptides and processes of the present invention become Vozmi) enables the construction of polypeptides, containing truncated sequence of HCV, which can be used as immunological reagents.

Truncated amino acid sequence of HCV encoding at least one viral epitope, as applicable immunological reagents. For example, polypeptides comprising these truncated sequences can be used as reagents in immunological research. These polypeptides are also candidates for subunit antigens in compositions for the preparation of antisera or vaccines. While these truncated sequences can be obtained by various known treatments natural viral protein, generally it is preferable to produce synthetic or recombinant polypeptides comprising a sequence of HCV. Polypeptides comprising these truncated sequences of HCV may be composed entirely of HCV sequences (one or more epitopes adjacent or neprikacaemyh) or from sequences and heterologous sequences into a single protein. Applicable heterologous sequences include sequences that can be isolated from a recombinant host, which increase the ize or vincinnes carrier. See, for example, EPO Pub N 116 201; US Pat, N 4722840; EPO Pub N 259149; US. Pat. N 4629783, descriptions of which are incorporated herein by reference.

The size of the polypeptide, including truncated HCV sequence, can be widely varied, and the minimum size is determined by the sequence of sufficient size to create an HCV epitope, while the maximum size is not critical. For convenience, the maximum size usually does not substantially exceed that required to provide the desired HCV epitopes and function(s) of a heterologous sequence, if present. Usually truncated amino acid sequence of HCV will vary in the range from about 5 (or 8) to about 100 amino acids in length. More typically, however, that the HCV sequence is maximum at about 50 (or 40) of amino acids, and sometimes a maximum of approximately 20, 25 or 30 amino acids. Usually it is desirable to choose HCV sequence at least about 8, 10, 12 or 15 amino acids.

Examples of truncated HCV amino acid sequences (oktamery), which are suitable, as described herein below in the examples. You must understand that these peptides are not necessarily exactly the apartment and removed from the described sequences. In addition, it can be identified and described additional truncated amino acid sequence of HCV, which include the epitope or are immunogenic.

Polypeptide products containing truncated HCV amino acid sequences disclosed below, can be obtained in the form of discrete peptides or included in the polypeptide of the larger sizes and can find the application here described. In the preferred application of the truncated sequence of domains E1 and/or E2 are used in vaccines for therapeutic products. Although in General any domains may have some use for diagnosis, especially preferred C, N S3, N S4, S5 and N, and combinations C epitopes with epitopes from one or more of the N S3 N S4 or N S5 the most preferred.

P. C. Obtaining polypeptides

The availability of DNA sequences encoding the amino acid sequence of HCV, enables the construction and expression vectors that encode antigenic active region of the polypeptide (See, for example, Fig. 2). These antigenic active area can occur from membrane antigens or antigens kernel or antigens that are not structurally uklocalgov, necessary for replication and/or Assembly of viral particles. Fragments encoding the desired polypeptides are obtained, for example, clones of viral cDNA using conventional restriction cleavage or methods of synthesis and with the subsequent ligation into vectors, which can, for example, contain part of the joined sequences, such as betagalactosidase or superoksidismutazy (SOD), preferably. Methods and vectors that are applicable for the production of polypeptides containing the joined sequence SOD, described in the European Patent Office Publication N 0196056, published October 1, 1986 Vectors encoding joined the peptides and polypeptides of HCV, i.e., NANB5-1-1, NANB81and C100-3, which are encoded in the structure of HCV cDNA are described in sections IV.B.I., IV.B.2 in IV.B.4, respectively. Any desired part of the HCV cDNA containing the hidden reading frame (or its synthetic variant), can be used for expression of recombinant polypeptide, such as Mature or (merged) United protein.

Or otherwise, a polypeptide containing an HCV epitopes, can be obtained by chemical synthesis using standard techniques based on the amino acid sequence represented on the figures and in the containing a series or not containing a signal sequence, enabling selection can be Legerova in the expression vector, acceptable to any suitable host. At the present time for the production of recombinant peptides, use of the host cells as eukaryotic and prokaryotes, and cell line-hosts presented in EPO Pub. N 318216. Then, the polypeptide is released from lysed cells or culture medium and purified to the extent necessary for its intended use. Cleaning can be made known in this region methods, for example, differential extraction, salt fractionation, chromatography on ion-exchange resins, affinal chromatography, centrifugation, etc., See, for example, Methods in Enzimology on various methods of protein purification. Such polypeptides can be used as a diagnostic, or those that give rise neutralizing antibodies, can be made in the form of vaccines. Antibodies generated against these polypeptides, may also be used as diagnostics or for passive immunotherapy. In addition, as discussed below, antibodies to these polypeptides are applicable, for example, for isolation and identification of HCV particles.

The HCV polypeptides can be that avannah HCV cells in tissue culture or in the infected host.

P.F. Obtaining antigenic polypeptides and conjugation with carrier

Antigenic region polypeptides mainly small - usually 8 to 10 amino acids or less in length. Fragments from such a small number, such as 5 amino acids, can determine the antigenic region. These segments may correspond to regions of the HCV antigen. In line with this, using the cDNA of HCV as the basis, the DNA encoding short segments of the HCV polypeptides can be expressed recombinante or in the form of joint proteins, or as separate polypeptides. In addition, short amino acid sequences can be conveniently obtained by means of chemical synthesis. When synthesized polypeptide well-formed, so is the correct epitope, but is too small to be immunogenic, the polypeptide may be associated with a suitable carrier.

To obtain such a binding in this area of science, the number of known techniques, including the formation of disulfide linkages using N-Succinimidyl-3-(2-pyridylthio)propionate (SpDp) and Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SM CC) obtained from the Pirce Company, Rockford, Jllinois (if no peptide sulfhydryl group, such martignani cysteine residues in one protein and an amide bond via the Epsilon-amino group on lysine, or other free amino group in other amino acids. Known a number of such disulfide/ameloblastoma funds. See, for example, Jmmun, Rev (1982), 62:185. Other bifunctional United agents form a thioester, and no disulfide bonds. Many of these thioether-forming agents are commercially available and include reactive esters of 6-multimediaphoto acid, 2-bromoxynil acid, 2-iodixanol acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid, and the like. Carboxyl groups can be activated by connecting them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid sodium salt. Additional methods of communication antigens incorporates rotavirus /"binding peptide", is described in EPO Pub. N 259149, the disclosure of which is included here by reference. The above list is not considered exhaustive, and it is clear that can be used in modifications of the above-mentioned compounds.

You can use any carrier that does not itself cause the formation of antibodies, dangerous for the owner. Convenient media is typically large, slowly metabolisable molecules such as proteins; polysaccharides, such as latex, funktsionalizirovannyi as polyglutamine acid, polylysine and the like; amino acid copolymers; and inactive virus particles, see, for example, the

II. D. Especially suitable protein substrates are serum albumins, hemocyanin mollusk "saucer" of the immunoglobulin molecule, thyroglobulin, ovalbumin, tetanus toxoid and other proteins known to experienced professionals.

II. E. Obtaining particles of hybrid immunogens containing epitopes of HCV

The immunogenicity of HCV epitopes can also be increased by obtaining their systems of mammalian cells or yeast, merged or United with proteins, forming particles, such as, for example, those associated with surface antigen hepatitis B. See, for example, U.S. patent 4622840. Structures in which the epitope of HCV is associated directly with the sequences encoding the protein, forming particles, produce hybrids which are immunogenic in relation to HCV epitope. In addition, all received vectors include epitopes that are specific and have different degree of immunogenicity, such as, for example, pre-S peptide. Thus, particles are built from protein, forming particles that include HCV sequences are immunogen is moved into particles in S. ceverisial P. Valenjuela et al. (1982)), as well as, for example, and in mammalian cells (P. Vallnguela et.al. (1984)). The formation of such particles, as shown, increases the immunogenicity of the Monomeric subunit. Designs can also include immunodominant epitope of HBSAg containing 55 amino acids pripoverkhnostnoi (pre-S) region. Neurath et.al. (1984). Design particle Pre-S-HBSAg expressed in yeast are disclosed in EPO 174444, published March 19, 1966; hybrids comprising heterologous viral sequences for expression in yeast are disclosed in EPO 175261, published on 26 March 1966. These designs can also be expressed in mammalian cells, such as oocytes Chinese hamster (CHO) using SV40-dihydrotetrazolo vector (wichelle et.al. (1984)).

In addition, parts of the sequence that encodes a protein, forming particles may be replaced by columns, encoding the HCV epitope. When this substitution areas that are not needed to mediate the aggregation of units in the formation of immunogenic particles, yeast cells or mammalian cells, can be eliminated, thereby removing additional antigenic sites of HBV competition with HCV epitope.

II. E. Obtaining vaccines

Vaccines can be aromatica in different cells of the host (for example, bacteria, yeast, insect cells and mammalian), or otherwise, can stand out from preparations of viruses or be produced synthetically. One - or polyvalent vaccine against HCV may include one or more epitopes of one or more nonstructural proteins. These vaccines may consist of, for example, recombinant proteins. These vaccines may consist of, for example, recombinant HCV polypeptides and/or polypeptides isolated from virions. In particular, it is assumed that vaccines contain one or more of the following protein or subunit antigens derived from them: E1, E2, C, NS2, NS3, NS4 and NS5. Especially preferred vaccine, including E1 and/or E2 or their subunits.

In addition to the above, it is also possible to receive live vaccines are attenuated microorganisms that Express one or more combinant HCV polypeptides. Suitable attenuated microorganisms known in this field and include, for example, viruses (e.g. vaccinia viruses (see Brown et.al. (1986)), as well as bacteria.

Receive vaccines that contain immunogenic polypeptides as active ingredients known in the art. Typically, such vaccines receive injectable Festa, suitable for solution or suspension in liquid prior to injection. The preparation can also be emulsified, or the protein can be encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable fillers are, for example, water, saline dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as moisturizing or emulsifying means, the buffer means of maintaining pH and/or adjuvants which enhance the effectiveness of vaccines. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetylmuramyl-L-travelsolutions (Tr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanyl-2-(1'-2'-dipalmitoyl-Sn-glycero-3-hydrogenphosphonate)-ethylamine (CGP 19835A, called MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryl-lipid A, limicola triazole and the skeleton of the cellular machinery (MP + TDM + CWS) in a 2% emulsion of squalene/ tweenagainst the immunogenic polypeptide, containing antigenic HCV sequence resulting from the use of this polypeptide in vaccines which are also contained various adjuvants.

Vaccines according to the generally accepted rules introduced parenterally by injection, for example, either subcutaneously or intramuscularly. Additional dosage forms that are suitable for other routes of administration include suppositories and, in some cases, oral dosage forms. For suppositories, traditional binders and bases may include, for example, polyalkylene glycols or triglycerides; such candles can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral dosage forms include also commonly used excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, dosage forms with prolonged selection or powders and contain 10-95% of active ingredient, preferably 25-70%.

Proteins may be incorporated into the vaccine as neutral substance or in the form of salts. Pharmaceutically reception is carried out with inorganic acids, such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, grape, maleic, and the like. Salts formed with the free carboxyl groups can also be obtained with inorganic bases, such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as Isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

II. F. Dosage and administration of vaccines

Vaccines are introduced by that is compatible with the dosage form dose, and in such a quantity that is prophylactically and/or therapeutically effective. The number that you want to enter, and which mainly is in the range from 5 μg to 250 μg of antigen per dose, depends on the subject to be treated, capacity of the immune system of a subject to synthesize antibodies, and the degree of desired protection. The exact amount of the active ingredient required for introduction may depend on the evaluation praktikujuscego doctor and can be individualized for each subject.

The vaccine may be given under the scheme a single injection or preferably according to the scheme of multiple introduction the individual doses, followed by other doses given over successive time intervals required to maintain and enhance the immune response, for example, 1-4 months for a second dose, and if necessary subsequent (s) dose(s) after several months. The dosage regimen will also, at least partly, be determined by the needs of the individual and will depend on the assessment of the practitioner.

In addition, the vaccine containing the immunogenic HCV antigens may be administered together with other immunoregulatory means, such as immunoglobulins.

II. G. Obtaining antibodies against epitopes of HCV

Immunogenic polypeptides described herein are used for the production of antibodies, including polyclonal and monoclonal. If desired polyclonal antibody, a selected mammal (e.g., mouse, rabbit, goat, horse, and so on) immunities immunogenic polypeptide bearing the epitope (s) of HCV. Serum from the immunized animal is collected is processed by the known procedure. If serum containing polyclonal antibodies to an epitope of HCV contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography. Metodi). Or otherwise polyclonal antibodies can be isolated from a mammal, which previously infected HCV. Monoclonal antibodies directed against HCV epitopes,can also be easily obtained by experienced specialists. The basic methodology for the production of monoclonal antibodies using hybrids are well known. A continuous line of antibody-producing cells of other methods, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr. See, for example, M. Schrier et. al. (1980); Hammerling et.al. (1981); Kennet et. al. (1980); see also U.S. patent N 4341761, 4399121, 4427783, 4444887; 4466917; 4472500; 4491632; and 4495890. Panels of monoclonal antibodies produced against HCV epitopes, can be sorted according to various properties; i.e., for isotype, affinity epitope, etc.

Antibodies, both monoclonal and polyclonal, which are directed against epitopes of HCV, particularly useful in diagnosis, and those which are neutralizing, suitable for passive immunotherapy. Monoclonal antibodies, in particular, can be used to induce antibioticsa antibodies.

Antiidiotypic antibodies are immunoglobulins that are "GNC is.al. (1981) and Dieesman et.al. (1985). Methods of inducing antiidiotypic antibodies known in this area. See, for example, Grzych (1985), Mac Namara et.al. (1984) Vytadehaag et. al. (1985). These antiidiotypic antibodies can also be used for treatment, vaccination and/or diagnosis of NANBH, as well as to identify immunogenic regions of the HCV antigens.

B.p. Immunoassay and diagnostic kits

As the polypeptides and antibodies of the present invention is applicable to immunoassays to determine the presence of antibodies to HCV or the presence of the virus and/or HCV polypeptides (or epitopes), for example, in a biological sample. Development of immunoassays is subject to a large number of variations and in this area there are many formats. In the immunoassay will be used at least one viral epitope derived from HCV. When one of the realizations in the immunoassay uses a combination of viral epitopes derived from HCV. These epitopes can be derived from the same or from different viral polypeptides, and may be in separate recombinant or natural polypeptides, or together in the same recombinant polypeptides. In immunostimulant can be used, for example, monoclone is paapam one viral antigen, monoclonal antibodies directed to different epitopes of viral antigens, polyclonal antibodies directed to the same viral antigen or polyclonal antibodies directed to different viral antigens. Procedures can be based, for example, on competition or direct reaction, or on the definitions of the sandwich type. The procedures can also for example be used a solid substrate or may be immunoprecipitate. Most studies include the use of labeled antibodies or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Studies that amplify the signals from the sample, also known; examples of which include studies that use Biotin and avidin, and immunostimulant with enzyme label and posredstvennye immunostimulant, such as ELISA (described below).

Usually immunostimulant for antibodies against HCV will include the selection and preparation of the test sample suspected the content of antibodies, such as a biological sample, and then incubated with antigenic (i.e., containing the epitope) polypeptide (s) of HCV in terms the of kompleksow. Suitable incubation conditions are well known in this area. Immunostimulant can be without limitations in heterogeneous or homogeneous form, and standard or competitive type.

In a heterogeneous form of the polypeptide is usually associated with a solid substrate, to facilitate separation of the sample from the polypeptide after incubation. Examples of solid media (substrates) that can be used are nitrocellulose (e.g., in the form of a membrane or microtiter cell), polyvinyl chloride (e.g., in the form of sheets or microtiter wells), polystyrene latex (e.g., in the form of granules or microtiter cards), polyvinylidenfluoride (known as immulon), diazotized paper, nylon membranes, activated granules and pellets of protein A. for Example, in the heterogeneous form can be used microtiter Board Dynatech Immulon1 or Immulon2, or 0.25 inch polystyrene granules (Precisi on Plastic Bale).

The solid substrate containing the antigenic polypeptide is typically washed after its separation from the test sample and before the definition of related antibodies. Known as standard and competitive forms of research which may occur under conditions in which will be deposited any complexes antigen-antibody, which are formed at the same time. Known both standard and competitive forms of these studies.

When the standard form is directly determined by the number of HCV antibodies, forming a complex of the antibody-antigen. This can be accomplished by determining whether the contact labeled antixenosis (for example, anti-human) antibodies that recognize the epitope of anti-HCV antibodies, due to the formation of the complex. When the competitive form of the number of antibodies to HCV in the sample is deduced by monitoring the competitive effect on the binding of a known quantity of labeled antibody (or other competing ligand) in the complex.

Formed complexes comprising anti-HCV antibodies (or in the case of competitive research number of competing antibody), are determined by any number of known techniques, depending on the form. For example, unlabeled antibodies in the complex may be determined by using the conjugate anticariogenic Ig in combination with a label (e.g. an enzyme label).

In immunostimulant, where the HCV polypeptides are analyzed substances, the subject of obrazovaniya antigen-or antibody-based test systems. You can use various forms of research. For example, you can use the "sandwich study, when the antibody associated with the solid substrate are incubated with test pattern: washed; then incubated with a second labeled antibody to analyze the substance and the substrate basis washed. An analyte is logged by definition, contacted the second antibody to the substrate. In competitive form, which may be either heterogeneous or homogeneous, the test sample is typically incubated with the antibodies and labeled competing antigens or sequentially or simultaneously. These and other forms well known in this field of science.

An effective system for identifying infections caused by HCV may include the use of panels of epitopes, as described above. Epitopes on the panel can be embedded in one or multiple polypeptides. Research on the different epitopes can be sequential or simultaneous.

Enzyme-linked immunosorbent assay (ELISA) can be used to determine the concentrations or antigen, or antibody. This method is based on the coupling of the enzyme to the antigen or antibody, and associated enzymatic asset is I in the solid phase (for example, on microplate or plastic Cup), incubated with subjects with dilutions of serum, washed, incubated with antiimmunoglobulin labeled with enzyme, and again washed. Frumenty suitable for labels known in this area of science and include, for example, horseradish peroxidase, enzyme activity associated with the solid phase, is determined by adding a specific substrate and defining product education or use of the substrate colorimetrically. Associated enzymatic activity is a direct function of the number of related antibodies.

To determine the antigen known specific antibody is fixed on a solid phase, and is added to the test material containing antigen after incubation, the solid phase is washed and added a second, enzyme labeled antibodies. After laundering is added to the substrate and is defined colorimetrically enzymatic activity and is associated with the concentration of antigen. Kits suitable for immunodiagnostic and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the polypeptides of this invention containing HCV epitopes or antibodies directed protene this study, as well as the appropriate set of instructions.

III. The main methods

The main techniques that are used in the practice of the present invention can be found, for example, in the references cited here, in particular, EPO Pub, NN 318216 and 388232, as well as in the references in the bibliography, which are included here for reference.

IV. Examples

Described below are examples of this invention, which are presented for purposes of illustration only and do not limit the scope of this invention. In the light of the present description specialists will be obvious to numerous examples of implementation in the volume formula of this invention.

IV. A. I. Synthesis of overlapping peptides

Plastic pegs are collected in the block in the order of 8 x 12 (Coselco, Mimetopes, Victoria, Australia), prepared by placing the pegs in the tub (20% volume/volume of piperidine in dimethylformamide (DMF) for 30 min at room temperature. Then the pegs were removed, washed for 5 min, then washed in methanol four times (2 min/wash). The pegs were allowed to air dry for at least 10 min and then washed last time (5 min). 1-hydroxybenzotriazole (HOBt, 367 mg) was dissolved in DMF (80 ml) for use in connection Fm Is-OPfp, Fmoc-L-Lys(Boc)-OPfp, Fmoc-L-Leu-OPfp, Fmoc-L-Met-OPfp, Fmoc-L-Asu-OPfp, Fmoc-L-Pro-OPfp, Fmoc-L-Gln-OPfp, Fmoc-L-Arg(Mtr)-OPfp, Fmoc-L-Ser(t-Bu)-ODhbt, Fmoc-L-Thr(t-Bu)-ODhbt, Fmoc-L-Val-OPfp, Fmoc-L-Tyr-OPfp

Protected amino acids were placed in microtiter cell Board with HOBt and the charge was placed block of pegs, immersing the pegs in the cell. Then the kit was sealed in a plastic bag and allowed to react at 25oC for 18 h to the first amino acid to attach to the pegs. Then the block was removed and the pegs were washed in DMF (2 min) to clear and remove protection from linked amino acids. The procedure was repeated for each additional attached amino acids, until, until you have obtained all oktamery.

Free N-ends then epitelium to compensate for the free amide, as most of the epitopes is not at the N-end and thus should not have an associated positive charge. Acetylation was performed by filling the cells in the microtiter Board DMF/acetanhydride/ triethylamine (5: 2: 1 V/V/V), allowing the substances on the pegs to react in the cells for 90 min at 20oC. Pegs then washed DMF (2 min) and MeOH (4 and 2 min) and dried on air for at least 10 minutes

Side chains zasimauskienes pouches for 4 h at room temperature. The pegs are then washed in dichloromethane (2 x 2 min), 5% diisopropylethylamine/dichloromethane (2 x 5 min), dichloromethane 5 min) and dried on air for at least 10 minutes Pegs are then washed in water (2 min), MeOH (18 h), dried under vacuum and kept in sealed plastic bags over silica gel.

IV. A. 2. Research peptides

Bearing oktamery pegs, obtained as described above, are first treated with ultrasound for 30 min in the buffer of disintegration (1% sodium dodecylsulfate, 0.1% of 2-mercaptoethanol, 0.1 M NaH2PO4) at 60oC. Pegs then repeatedly immersed in water (60oC), followed by boiling MeOH (2 min), and allowed to air dry. Then Spanky pre-coated for 1 h at 25oC in microtiter cells containing 200 μl of blocking buffer (1% ovalbumin, 1% BSA, 0.1% tweenand 0.05% NaN3in PB9) with stirring. The pegs were then loaded into microtiter wells containing 175 μl of antisera obtained from patients diagnosed with a disease caused by HCV, and incubated at 4oC during the night. The study was conducted with pegs against antisera from three different Bo the SA to clone C100-3 (dilution 1:1000) and the reaction RIBA > 4 + C100, 5-1-1 t SS (C22 were not). (The names of the antigen/clone presented in EPO Pub. N 318216 and 388232, as described in the literature related to immunostimulant for HCV, available from Ortho Diagnostics Systemes, Inc). Blank plasma was diluted 1:500 in blocking buffer. Sample PAA 33028 ("B") showed a strong reaction with HCV by Western-blotting, HCV competitive ELISA, HCV ELISA with a clone of C100-3 (dilution 1:500) and reaction RIBA > 4 + C100, 5-1-1, C33C and C22. Polyclonal anticigarette was partially purified by passing through a column of protein A and used in a dilution of 1:200 and blocking buffer. Sample PAA's 32931 ("C") showed a moderate response with HCV by Western-blotting (3+), HCV specific with the clone C100-3 (dilution 1: 64), and the reaction RIBA 3+ and 4+ with C100 and 5-1-1 respectively (SS and C22 were not). Polyclonal anticigarette was partially purified by passing through a column of protein A and used in a dilution of 1:500 in blocking buffer.

The pegs were washed in PBS tween20 (4 x 10 min) at room temperature, then incubated in microtiter cells containing labeled with horseradish peroxidase goat against human Ig anticigarette (175 μl, dilution 1:2000 in blocking buffer without NaN3) for 1 h at 25oC when posesiv the AMI, as IgG and IgM. The pegs again washed in PBS tween20 (4 x 10 min) at room temperature. The substrate solution was prepared by diluting NaH2PO4(1 M, 200 ml) and citric acid (1 M, 160 ml) to 2 l of distilled water, bringing the pH to 4.0. Immediately prior to use to 100 ml of buffer was added Azino-diethylenetriaminepenta (50 mg ABTS) and hydrogen peroxide (0,3 µl/ml) to complete the receiving substrate solution. The substrate solution (150 μl) was added to each well of microtiter Board and pegs were loaded into the wells and incubated at 25oC in the dark. After development of the color reaction is stopped by removing the pegs and filmed performance of the absorption of the solution at 405 nm.

Oktamery listed at the end of the description, were immunoreactive with anti-HCV by the antisera. Peptides that react with all three antisera were listed as epitopes, whereas peptides that react only with one or two antisera are listed as weak epitopes (shows " ~ "). Particularly strong epitopes are marked with letters instead of numbers (for example, EpAA).

IV. C. Differential study

The following study was performed to distinguish between early antigens is, caused by HCV, more quickly.

Were obtained serial blood samples from the sick person, having high levels of ALT, but negative for anti-C100-3 antibody. Five blood samples obtained before completion of seroconversion (C100-3 positive), were combined and used in the study at a dilution of 1:2000. The study was carried out as described above in section IV A. However, one double set of pegs were incubated with goat against human IgG specific anticorodal, horseradish peroxidase, while the other set was incubated with goat against human IgM specific anticorodal labeled with peroxidase is horseradish. The epitopes immunoreactive with antibodies IgM, are early epitopes.

The results showed that most of the early epitopes are located in a region located from about amino acids 480 to about 650 amino acids. Especially strong epitopes and IgM were oktamery, starting from amino acid number 506, 510, 523, 553, 562, 580 and the region from 590 to 620. Studies that use antigens carrying the epitope of this area will enable the diagnosis of infections caused by HCV, at an earlier stage than studies using drpaulita NANB in the blood transfusion with further study for 3-12 years. Timing of initial blood samples were less than the interval of one week. Each sample was tested for IgG and IgM against nuclear antigen (G22), two envelope antigens (E1 and E2), and three non-structural antigens region (SS, C100 and N 5) using immunofermentative analysis. We found that IgM response to C22 and C33c exceeded IgG response to these antigens.

NS - 5 also caused an IgM response, but this response did not exceed the IgM response to this antigen. Thus, it is possible to develop tests that allow to determine very early stage of infection, through the use of epitopes derived from areas C22 and C33c and specific binding of IgM . Antibodies to C33c region was present during the greatest period of time and this suggests that the most reliable should be diagnostic studies aimed at C33c.

IV. C. Variants of the sequences in isolates of HCY from different individuals.

In some people, some of whom were serologically positive for anti - C100-3 antibody (EC10 was negative for antibodies) were identified isolates of HCV, which contain sequences with deviations from CDC/HV/. Identification of these new isolates was performed by cloning and sekri this method uses primers and samples based on the cDNA sequences of HCV, as described here. The first step in this method is the synthesis of cDNA or genomic HCY or replication mediator using reverse transcriptase. After the synthesis of the HCV cDNA before amplification of RNA in the sample is cleaved known in this region methods. The specified segment of the HCV cDNA then amplificates through the use of appropriate primers. Amplificatoare sequence are cloned, and the clones containing amplificatoare sequence is determined using a sample that is complementary to the sequence between the primers, but which does not cover the primer.

IV. C. I. HCV Isolates, isolated from people in the United States.

Blood samples that were used as a source of HCV virions, were obtained from the American red cross in Charlotte, North Carolina, and from the civic centre of blood transfusion Kansas. Kansas city, Missouri. The samples were taken for antibodies to the antigen C100-3 HCV, using the study by ELISA, and were subjected to additional Western blot analysis using polyclonal goat protivojinfekiy HRP-antibodies to quantitatively detect anti - HCV antibodies. Two samples 23 and 27 of the American red cross is implementing on HCV in these studies.

Viral particles present in the serum of these samples was separated using ultracentrifugation under the conditions described by Bradley et.al. (1985). RNA was extracted from the particles by cleavage with proteinase K and sodium dodecyl sulfate at final concentrations of proteinase K 10 mg/ml and sodium dodecyl sulfate 0,1%; cleavage was held for 1 h at 37oC. Viral RNA was further purified by extraction with chloroform-phenol.

HCV RNA preparation RNA was subjected to reverse reading on the cDNA. After it was synthesized both strands of cDNA, the resulting cDNA was then amplificadores by PCR. For HCV cDNA of the three clones obtained from each isolate HCY, performed the sequence analysis. The analysis was mainly carried out by the method described in Chen and Seeburg (1985).

Consensus sequences of clones derived from HCV in samples 23 and 27, shown in Fig. 3 and Fig. 4, respectively. The variable sequence is also shown in these figures, as well as amino acids encoded in the consensus sequences.

Figures 5 and 6 show the comparison lined nucleotide sequences of positive circuits (Fig. 5) the alleged arestable amino acids with non-129-467 polyprotein HCV, to encode a large ORF in the genomic HCV RNA. A study of Fig. 5 and 6 shows that in the three sequences of isolated clones there are variations. Variants of the sequences at the nucleotide level and the amino acid level are summarized in the table directly below. In table. 1 polypeptides, designated S and NSI represent amino acid numbers from 130 to~ 380, and c 380 to ~ 470, respectively, since these domains have been previously known. The countdown is on to the intended initiator methionine. Terminology S and NSI is based on determining the location of the sequences encoding the polypeptides, using the model flavivirus. As discussed above, however, recent data suggest that there is no complete correlation between and flaviviruses relative to the viral polypeptide domains, in particular in the alleged domains E/NSI. Of course, polypeptides and caliraya their domains may exhibit significant deviations from flaviviruses model.

Although there are options for the newly selected HCV sequences, cloned sequence of samples 23 and 27 (named 23 HCV and HCV 27) each contains 1019 nucleotides, indicating the absence of deletions and additional mutations outlinesthe not rearranged in this area.

Comparison of the consensus sequences for HCVI and for other HCV isolates is summarized in the table above. Differences in the sequences between isolates from chimpanzees HCV1 and HCV isolated from people about the same as observed between HCV-mi of human origin.

Interestingly, differences in the sequences of two of the proposed domains are not the same. The sequence in the intended, apparently, is relatively constant and randomly distributed over this area. In contrast, the estimated area of the NSI has a higher degree of variability than the entire sequence, and change, apparently, is hypervariable pocket of about 28 amino acids, which is located approximately 70 amino acids in the forward direction from the intended N-end of the proposed polypropylene.

Although it is possible to prove that the defined changes were introduced during the amplification process, it is unlikely that all the changes that have occurred as a result of this. It was found that the Tag polymerizate introduces errors in the sequence about the same based on 10 cilaisnowbuy DNA matrix for the cycle (Saiki et.al. (1988). On the basis of this assessment up to 7 errors could -27 gave 29 and 14 changes bases respectively. The following suggests that these changes occur in nature. About 60% of changes of bases are silent mutations that do not change amino acid sequence. The amendments introduced Ta; polymerase during amplification by PCR, as would be expected, there are chaotic; however, the results show that different sequences are collected in at least one specific area.

IV.C. 2.The HCV isolates from people in Italy and in the USA

Segments of HCV RNA present in different isolates amplified by the method of HCV/cPCP. These segments connect the region from 0.6 To 1.6 K in the forward direction from methionine encoding of the start codon estimated polypropylene HCV. The isolates originate from biological samples obtained from individuals infected with HCV. More specifically, the isolate HCT 18 comes from human plasma from individuals in the United States, ECI and EC10 obtained from biopsy of the liver Italian patient and Th obtained from peripheral blood, mononucleocytes faction, the American patient. Compare segments of HCV RNA were isolated from chimpanzees.

RNA was extracted from samples of human plasma, using extraction with phenol: CHCl3: solidsurface sodium (0.05 M Tris-HCl, pH 8.0, 0.001 M EDTA, 0.1 M NaCl, 1 mg/ml proteinase K and 0.5% sodium dodecyl sulfate) containing from 10 to 40 μg/ml polyadenylic acid, and incubated at 37oC within 60 minutes After cleavage by proteinase K derived from the plasma fraction was freed from protein by extraction with TE (50 nm Tris-HCl, pH 8.0, 1 mm EDTA) saturated phenol, pH 6; 5. Phenolic phase was separated by centrifugation and re-extracted TENB, containing 0.1% sodium dodecyl sulfate. The resulting aqueous phase from each extraction were combined and was extracted twice with equal volumes of phenol/chloroform/isoamyl alcohol(1:1(99: 1)) and then double-volume 99:1 mixture of chloroform/isoamyl alcohol. After phase separation by centrifugation the aqueous phase was brought to a final concentration of 0.2 M Na acetate, and nucleic acids besieged by adding two volumes of ethanol. Precipitated nucleic acids were isolated by ultracentrifugation and SW 41 rotor at 38 K for 60 min at 4oC or microcentrifuge for 10 min at 10 K, 4oC.

RNA extracted from liver biopsy, was received by Dr. F. Bonino, Ospedale Maggiore di Giovanni Battista in Turin, Italy. Mononucleotide fraction was obtained by the manufacturer's instructions. Whole RNA was extracted from fractions using the guanidine-thiocyanate the procedure described by Choo et. al. (1989).

Synthesis of cDNA NCV of the samples was carried out using reverse transcriptase. After precipitation with ethanol precipitated RNA or fraction of nucleic acids were dried and resuspendable in DEPC-treated distilled water. Secondary structure in nucleic acids was destroyed by heating at 65oC for 10 min, and the samples were immediately cooled on ice, cDNA was synthesized using 1 to 3 µg total RNA from the liver or from nucleic acids (or RNA) extracted from 10 to 100 μl of plasma. When the synthesis was using reverse transcriptase and the reaction was carried out in a volume of 25 µl, using the Protocol provided by the manufacturer, BRL. All of the reaction mixture for the synthesis of cDNA consisted of 23 units of RNase inhibitor, Rnasin(Fischer Promepa).

After cDNA synthesis reaction mixture was diluted with water, boiled for 10 min and quickly cooled on ice.

Each choice of the samples was subjected to two cycles of amplification using PCP. The primers for the reaction were chosen to amplify regions marked "EnvL" and "En vR". Area "EnvL" concludes nucleotides 669-1243, and Prelate 300-408 (alleged amino acids are numbered starting from the estimated methioninamide of the initiation codon).

The reaction of PCP was performed essentially according to the manufacturer's instructions (Cetus-Perkin-Elmer), except for the addition of 1 μg RNase A. the Reaction was carried out in a final volume of 100 μl, was performed in 30 cycles using the mode 94oC (1 min), 37oC (2 min), and 72oC (3 min), with the development for 7 min at 72oC for the last cycle. The samples were then extracted with phenol; CHCl2, precipitated with ethanol twice, resuspendable in 10 mm Tris HCl, pH 8.0 and concentrated using a filtering Method - 30 (Amicon). This procedure effectively removes the oligonucleotides that are smaller than 30 nucleotides; thus, the primers of the first cycle of amplification by PCR are removed.

Samples kontsentrirovannye in Method 30, and then subjected to the second cycle of amplification using PCR. Amplification by PCR was performed for 35 cycles, using 94oC (1 min), 60oC ( 1 min) and 72oC (2 min) with the development for 7 min at 72oC for the last cycle. The samples were then extracted with phenol: CHCl, was besieged twice and split cp RI. The PCR reaction products were analyzed by separation of the products by electrophoresis in 6% were polyacrylonitrile or plasmid vector pGEM-4 or gt 11. After the first cycle of amplification is expected products with sizes for EnvL and EnvR 615 683 bp and bp, respectively; after the second cycle of the expected amplification products with sizes for EnvL and EnvR 414 bp 575 bp, respectively. Plasmids containing amplificatoare products were used to transform host cells; plasmid pGEM-4 was used to transform DH5-alpha, and gt 11 was used for transformation C600 Delta HFL. Clones of transformed cells, or which hybridize with the corresponding samples of HCV or those that had inserts of the correct size were chosen. The insert was then cloned into M13 and sequenced. Samples for all products HCY/cPCP consisted of 32P labeled sections HCV cDNA, which was obtained by amplification using PCP.

Information about sequence variants in the field of En obtained from 3 clones from HCT 18, two clones from TN, 3 clones from ECI and clones HCV1. Comparison of the composite nucleotide sequence of each isolate obtained from these clones is shown in Fig. 7. In the figure each sequence is shown from 5' to 3' for sense circuit EnvL region, and sequences were stretched out in a line. Vertical lines and capital letters show the homology of the sequence is s, are as follows: line I, Thorn; line 2, EC1; line 3, HCT 18; line 4 HCV 1.

Information about sequence variants in EnVR region derived from two clones EC10 and clones HCV1. Two clones EU10 differed by only one nucleotide. Comparison of the nucleotide sequences EC10 (clone 2) and a compound of HCV sequences shown in Fig. 8; each sequence is shown from 5' to 3' for sense circuit EnvR region, and sequences were stretched out in a line. Double point between sequences were stretched out in a line. Double point between sequences show homology sequence.

Comparison of amino acid sequences encoded in EnvL (amino acids 117-308) and EnvR region (amino acids 300-438) for each of the isolates is shown in Fig. 9 and Fig. 10, respectively. In the figures included sequences for isolates JH 23 and JH 27 described above. Identifies the sequence of the Japanese isolate; these sequences provided by Dr. T. Miyamura, Japan. On the figures of the amino acid sequence for this region is given fully for HCV1, and non-homologous amino acids are indicated in different isolates.

As can be seen in Fig. 9, in EnvL region exists in a the ome 96% and about 95% homology, respectively, with HCV1. Fig. 10 shows that the homology in EnvR area less than EnvL region; moreover, one one, apparently, is hypervariable (i.e. amino acids 383-405). These data are summarized in table. 2.

IV. Industrial applicability

Epitopes identified herein may be used to obtain a polypeptide products, as described above, for applications such as analysis of blood samples for infection caused by HCV, clinical diagnosis of HCV, antibodies, drugs. Another application of the above-described and other methods will be easily visible to the experts.

1. Polypeptide comprising a truncated sequence of HCV (hepatitis C virus) containing the epitope of HCV with the formula

aax- aay,

able to induce immune response in the subject, where aa denotes an amino acid, x and y are integers such that y - x 14-aax- aaydesignates the portion of the amino acid sequence of figure 1 and x is chosen from the group: 66, 413, 465, 540, 1218, 1940, 2244, 2281, moreover, the specified polypeptide can have an optional sequence of N-terminal amino acids corresponding to the sequence of the HCV polypeptide adjacent to the N-end of the AOT, the corresponding sequence of the HCV polypeptide, adjacent to the C-end amino acids aayin which it is attached, and the total number of additional amino acids does not exceed 15 for aa413- aa4274 for aa465- aa480, 85 for aa540- aa554, 10 aa2244- aa2258and for 30 aa2281- aa2300.

2. The polypeptide under item 1, where aax- aaynot have additional amino acid sequence with either N-Terminus or C-end.

3. The method of producing the polypeptide under item 1 or 2, consisting in that the polypeptide is produced by multi-stage solid-phase chemical synthesis on polyethylene needles.

4. Reagent for immunoassay for detection of antibodies to epitopes of hepatitis C virus, including synthetic HCV polypeptides, characterized in that the said synthetic HCV polypeptides are polypeptide under item 1 or 2.

5. The method of determining the presence of antibodies, immunoregulin with hepatitis C virus (HCV) in a sample, comprising the incubation of the test sample with immunoreagents under conditions that allow the antibody to bind to the reagent, and detection of antibodies associated with the specified reagent, characterized kiuchumi introduction to the subject of immunologically active polypeptide of HCV in combination with a pharmaceutically acceptable excipient, characterized in that said immunological active HCV polypeptide is a polypeptide described in paragraph 1 or 2.

 

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