Oligomeric particle inducing immunity against hepatitis c virus, method for preparing oligomeric particle, composition, specific antibody, set (variants), immunological analysis and vaccine against hepatitis c virus

FIELD: biotechnology, vaccines.

SUBSTANCE: invention proposes an oligomeric particle that is able to induce immunity against hepatitis C virus. Particle consists of purified proteins or their parts, hepatitis C virus envelope (HCV) E1, E1s and E2. Proposed particle has diameter from 5 to 100 nm. Also, invention proposes a method for preparing such particle. Method involves purification of proteins HCV in solution, formation of oligomeric particles by replacing purified proteins with detergent solution or salt solution and purification of oligomeric particles. Also, invention proposes a composition for inducing immunity against HCV that comprises the proposed particle, antibody specific to particle, sets for detection of antigens HCV, immunological analysis for detection of antibodies to HCV, and vaccine against hepatitis C virus (HCV) that comprises the oligomeric particle. Proposed inventions provide carrying out the induction of specific humoral and cellular immunity to HCV envelope proteins and to carry out diagnosis of HCV. Invention can be used in medicine.

EFFECT: valuable medicinal properties of particle, improved preparing methods.

71 cl, 27 dwg, 6 tbl, 14 ex

 

The SCOPE of the INVENTION

The present invention is based on the discovery that membrane proteins of HCV induce a beneficial immune response in chimpanzees, which are chronically infected with heterologous strain of HCV subtype subtype 1A or 1b. More specifically the present invention relates to the discovery that membrane proteins are highly immunogenic and lead to stimulation of both the cellular and humoral immune response. In addition, the present invention relates to the discovery that blocking cysteine alkylation results in even more immunogenic proteins. In addition, membrane proteins of the present invention should include particles that exhibit high immunogenicity and immune reactivity. In addition, it was demonstrated that such particles may include other proteins.

BACKGROUND of the INVENTION

Infection With hepatitis C virus (HCV) is the most important health problem in both developed and developing countries. It is estimated that about 1 to 5% of the world population affected by the virus. It turned out that HCV infection is the most important cause of hepatitis associated with blood transfusion, and often progresses to chronic liver injury. In addition, there is evidence of involvement of HCV in the induction of hepatic cell carcinoma. Consequently the nutrient, high need for reliable diagnostic methods and effective therapeutic agents. Also needed are sensitive and specific methods of screening for HCV infected blood products and improved methods of cultivation HCV.

HCV is a virus with single-stranded positive RNA of approximately 9600 bases that encodes at least three structural proteins and six non-structural proteins. On the basis of sequence homology, these structural proteins are functionally defined as one nuclear protein and two envelope protein: E1 and E2. Protein E1 consists of 192 amino acids and contains from 5 to 6 sites of N-glycosylation, depending on the HCV genotype. Protein E2 consists of 363-370 amino acids and contains 9-11 sites of N-glycosylation, depending on the HCV genotype (as reviews see: Major and Feinstone, 1997; Maertens and Stuyver, 1997). Protein E1 contains a different variable domains (Maertens and Stuyver, 1997), whereas the E2 protein contains three hypervariable domain, of which the main domain is localized at the N end of this protein (Maertens and Stuyver, 1997). Last membrane proteins obtained using recombinant techniques in Escherichia Li, insect cells, yeast cells and mammalian cells. The use of expression systems in the culture of cells of higher eukaryotes, and especially in the culture of mammalian cells is the result of the atom protein shell, which is effectively recognized by antibodies in samples from patients (Maertens et al., 1994).

It was postulated that the envelope protein E1 needs in the protein shell of E2 to reach the state of the correct installation of subunits (Deleersnyder et al., 1997). In addition, it was assumed that E1 and E2 form heterodimer that can form the basic unit of the viral envelope (Yi et al., 1997). In WO 98/21338 Liang et al. used these assumptions to construct HCV particles, which consist of E1 and E2, as well as from Core and P7. In other words, in the prior art was not asked to use E1 or E2 separately for immunization and other purposes. However Houghton (1997) reported that repeated immunization with recombinant tree (4×25 mcg) 3 chronically HCV infected chimpanzees did not induce a significant immune response. The authors of this application have found that the induction of an immune response against shell in patients with chronic hepatitis C will indeed be desirable and beneficial for the patient, since high levels of these antibodies, apparently correlated with a good response to treatment with interferon and can, therefore, promote the excretion of the virus in the patient (PCT/EP 95/03031, Maertens et al.). The authors of the present invention, moreover, convinced that, since the levels of antibodies against E1 in chronic carriers of HCV are among Nissi is among all HCV antibodies, the raising of these antibody levels and possibly cell response may be beneficial for the induction of dealing with this infection or even in clearance of the infection in the host. Apparently, high levels of cellular immunity against E1 also correlated with a good response to treatment with interferon (Leroux-Roels et al., 1996).

Besides the importance of immunity against E1 in relation to therapy with interferon, other indicators point to the fact that some other parts of the HCV genome may be important for the induction of specific immune response, which may give the opportunity to deal with this infection. In patients responding to therapy with interferon, also often observed reactivity of T cells against C-terminal region of the nuclear protein (Leroux-Roels et al., 1996). Potentially neutralizing antibodies against the protein NS4B were demonstrated in patients displaying HCV after liver transplantation (Villa et al., 1998). Within NS3 mapped several T-cell epitopes, which, apparently, is correlated with the excretion of HCV during the acute phase (see: PCT/EP 94/03555, Leroux-Roels et al.; Leroux-Roels et al., 1996; Rehermann et al., 1996 and 1997; Diepolder et al., 1995 and 1997). In addition, antibodies to NS5A, like antibodies to E1, demonstrate high levels at a basic level before therapy with interferon-alpha in long-term responsible organisms (long term responders, LTR) compared to unresponsive what rganizati.

Currently, therapeutic vaccination against HCV is not successful. It is also shown that prophylactic vaccination is effective only against the homologous strain of HCV (h et al., 1994). The present invention relates to the surprising discovery that the introduction of the antigen membrane HCV can dramatically improve the state of chronic active hepatitis in an individual infected with a heterologous strain or isolate, as in heterologous infection with subtype 1A and heterologous infection with subtype 1b. Indeed, chronically infected chimpanzees, which was introduced six doses of 50 μg E1s (i.e. amino acids 192-326 from E1), unexpectedly showed a strong humoral and cellular immune responses, which did not increase during the whole period of chronic infection until the last vaccination. Moreover, viral antigen was not detectable in the liver during the period from two to five months and remained detectable for at least 5 months after vaccination. Although titers of HCV RNA in the liver did not decrease the levels of liver enzymes in the serum showed a marked tendency to normalization. Most importantly, when both vaccines dramatically improved liver histology. The present invention further relates to the surprising discovery that a protein E1 used for VA is cinali, which is expressed in the form of individual HCV protein without its hydrophobic anchors, forms a stable particles. It should also be noted that in order to avoid the induction of immune responses against unwanted epitopes, protein E1 is used for vaccination, designed in the form of a coherent sequence of individual clones originating from a single serum sample from one chronic carrier. In addition, the present invention relates to the discovery that the induction of such responses against E1 can be enhanced by using antigens of a different genotype than the genotypes of infection that is present in the host. In addition, the present invention relates to the discovery that, when cysteine coat proteins of HCV is alkylated, for example, N-(Iodate)-trifurcation, ethylenimine or active halogen, such as iodoacetamide, oligomeric particle as described above, demonstrated an even higher immunogenicity. Finally, the present invention relates to the discovery that mutation cysteine coat proteins of HCV in any other natural amino acid, preferably methionine, glutamic acid, glutamine or lysine, oligomeric particles, as described above, also leads to higher immunogenicity compared to the original protein shell.

The OBJECTIVE of the INVENTION

From the literature dealing with the s clear that there is an urgent need to develop reliable vaccines and effective therapeutic agents for HCV. Therefore, the object of the present invention to provide an antigenic preparation which is able to induce specific humoral and cellular immunity to membrane proteins of HCV, even (but not exclusively) in chronic HCV carriers. The same antigens can be used for diagnosis of immune response.

More specifically, the present invention is the development of the angiogenic drug, as defined above, which consists of stable particles separate coat proteins of HCV. It should be clear that at the present time such particles or a method of obtaining such particles in the art is unknown. In addition, in the prior art there are no indications that any antigenic preparation comprising such a stable particles or peeled individual membrane proteins of HCV can be successfully used as a (heterologous) a prophylactic or therapeutic vaccine against HCV. Thus, the present invention also is to develop a method of obtaining a stable particles, which can be successfully used as a prophylactic or therapeutic agent against HCV, and, in addition, the development of DNA vaccines encoding HCV of antig the us. More specifically, the present invention is to develop a method of obtaining the last particles on the basis of particle formation in the presence of detergent (see below). In addition, the present invention is to develop methods for producing particles, consisting of antigens derived from different HCV genotypes.

In addition, the present invention is the development of antigen, which is a coherent sequence of individual clones, which can provide a more correct installation of these proteins. This is done to avoid stimulation of the immune system against unwanted epitopes.

In addition, the present invention is the development of antigenic preparation, in particular for therapeutic vaccination, based on the HCV genotype, which with a chronic carrier. In this respect, the present invention is the development envelope protein or a homologous or a very good genotype or subtype compared with genotype or subtype of this chronic carrier.

The next task of the present invention is to develop a method of treatment or therapeutic vaccination of chronically infected patients using the above antigens or DNA vaccines, possibly in combination with other with what disiniame. The present invention also is developing a method of prophylactic vaccination of people against HCV.

Another objective of the present invention to provide an oligomeric particles that have the best immunogenicity due to mutations in at least one cysteine residue envelope protein of HCV in the natural amino acid, preferably methionine, glutamic acid, glutamine or lysine. Alternatively, you can carry out the alkylation of at least one cysteine residue envelope protein of HCV. In particular, the latter protein can be alkilirovanii using etilenimina, N-(Iodate)trifurcated or active Halogens. In this respect, the present invention is the development of additional applications of oligomeric particles as carriers for efficient representation of non-HCV epitopes.

In the present invention are also included development of a treatment for acutely or chronically infected patients with antibody against the membrane, such as an antibody against E1, for example an antibody against the V2 region E1 or separately, or in combination with other therapies.

In the present invention are also included development of antigen, stimulating T-cells, such as Core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A or NS5B, together with the protein shell according to the invention.

It is believed, is that all aspects of the present invention are found in the embodiments, as indicated below.

A BRIEF description of the TABLES AND GRAPHICS

Table 1 presents the sequences of clones E1, obtained from one chronic carrier, the construction of E1, used for production of vaccines, is a consistent sequence of all these individual clones. V1-V5 variable regions 1-5; C4, const domain 4; HR, hydrophobic area; HCV-B con, consensus sequence at positions that are variable between clones and HCV-J.

Table 2 presents the sequence of the protein E1 vaccines and protein E1, as it is found in infected chimps Phil and Ton. The isolate of subtype 1b differs from Phil 5.92% from the vaccine strain. The difference between the vaccine and the isolate of subtype 1A from the Ton was 20,74%.

Table 3 presents a schematic overview of the changes induced by therapeutic vaccination in two chronically infected chimpanzees (Ton and Phil). The analysis was carried out as described for Fig and 11. In addition, evaluated the histology and inflammation on the basis of biopsies of the liver.

Table 4 presents the sequence of the peptides used for epitope mapping of b-cells. Note that HR overlaps with V4V5.

Table 5 shows the reconstruction NS3 to obtain a shorter protein, which carries all the major epitopes, which correlate with clearance of VIR is CA.

Table 6 shows the reactivity in LIA (immunofluorescently analysis) E1s-ndimethylacetamide against E1s-maleimide with sera of chronic carriers of HCV. Proteins were immobilized on LIA membranes. E1s-ndimethylacetamide itself was sprayed on the strips LIA, whereas E1s-maleimid (containing Biotin-maleimide) before dispersion was subjected to komplektovaniiu with streptavidin. Antigens associated with LIA-membranes and strips were processed essentially as described in Zrein et al. (1998). Human antibodies directed against these antigens were visualized using human anti-IgG conjugated with alkaline phosphatase. NBT (nicrosini tetrazolium) and BCIP (5-bromo-4-chloro-3-indolylacetic) used for color displays of strips. Staining was evaluated from 0.5 to 4, as indicated in Zrein et al. (1998). The number (#POS.) and percentage (%POS.) positive samples are indicated in the lower part of the table using the cutoff for this analysis, equal to 0.5.

Figure 1. Superimposed on other profiles gel chromatography in FR (phosphate buffered saline)/3% Empigen-BB proteins E1s and E2s, expressed and purified according to Maertens et al. (PCT/ER/03031).

Figure 2. Superimposed on other profiles gel chromatography (size exclusion chromatography, SEC) proteins E1s and E2s, expressed and purified according to Maertens et al. (PCT/EP95/03031) and subjected to a second run on the same SEC column in SFR/0,2% perhaps (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate) for specific oligomeric structures installed with an approximate molecular weight of 250-300 kDa. A similar degree of binding can be obtained by using 3%betaine.

Figure 3. Superimposed on other profiles gel chromatography of proteins E1s and E2s, expressed and purified according to Maertens et al. (PCT/EP95/03031), subjected to a second run of 0.2%CHAPS or 3%betaine, to obtain a specific oligomeric structure, as shown in figure 2, and subjected to a third run on the same SEC column in 0.05% CHAPS to obtain specific Homo-oligomeric structures installed with an approximate molecular weight of 250-300 kDa (E2s) and more than 600 kDa (E1s). A similar degree of binding can be obtained by using 0.1 or 0.5%betaine.

Figure 4. Analysis of dynamic light scattering, expressed as a percentage of the number of particles in relation to the observed diameter in nm, for 1s in SFR/A 0.05% CHAPS.

Figure 5. Analysis of dynamic light scattering, expressed as a percentage of the number of particles in relation to the observed diameter in nm, for 1s in SFR/0.1% betaine (top) or 0.5% betaine (below).

6. Electron microscopic staining (A) 1s in SFR/A 0.05% CHAPS, and (B) E1s in SFR/3% betaine.

7. Size distribution of particles E1s in SFR/A 0.05% CHAPS.

Fig. Evolution of antibodies anti-E1 induced six consecutive and 3 repeated immunizations (indicated by small arrows) 1b infected chimpanzees (Phil), and the evolution of ALT (LT, alanine aminotransferase), HCV RNA and anti-E1 antibodies. Anti-E1 antibodies to bind to the solid phase E1 was detected using specific secondary anticigarette against human IgG, conjugate with horseradish peroxidase. As a substrate for color manifestations used TMB (tetramethylbenzidine). The results are expressed as the final title. ALT levels were determined by clinical analyzer and expressed as units/liter of HCV RNA in serum was determined using HCV Monitor (Roche, Basel, Switzerland). Viral load in the liver was determined using a semiquantitative determination of the number of E2 antigen, painted in a liver biopsy, using a specific monoclonal antibody (ESAS, catalog number 98031215, as described in the application EP No. 98870060.5).

Fig.9. Epitope mapping humoral responses induced by immunization E1 chimps Phil. The reactivity of antibodies against various peptides was measured using an indirect ELISA (enzyme-linked immunosorbent assay), which biotinylated peptides (see also Table 4) adsorbing on titration microplate using streptavidin. Specific antibodies were detected using specific secondary anticigarette against human IgG, conjugate with horseradish peroxidase. As a substrate for color manifestations used the TMB.

IG. The results of the analysis of proliferation of lymphocytes before and after vaccination among chimpanzees Phil. Frozen RVMS (mononuclear cells peripheral blood) were thawed and stimulated in three repetitions of different antigens. The negative control was clean environment, while as a positive control was used of concanavalin a at a concentration of 5 µg/ml RVS at a concentration of 2-4×105cells/well in a final volume of 150 μl were cultured in medium RPMI 1640, to which was added 10% V / V heat inactivated FCS (fetal calf serum), in U-shaped 96-well titration microplate together with the controls or 1 μg/ml E1 for 90 h at 37°C in humidified atmosphere containing 5% CO2. During the last 18 h, cells were subjected to pulse-labelling of 0.5 µci (3H) thymidine per well. Then these cultures were collected on filters fiberglass and determined the inclusion of the label.

The results are expressed as stimulation indexes (IP): the average number of counts per minute (CPM) antigen/mean number of counts per minute (CPM) net environment in three definitions.

11. Evolution of antibodies anti-E1 induced six consecutive and 3 repeated immunizations (indicated by small arrows) in the chimpanzee Ton infected with HCV subtype 1A. Shows the evolution of ALT, HCV RNA in serum and determination of HCV antigen is in the liver. Anti-E1 antibodies were determined by indirect ELISA: specific antibodies to bind to the solid phase coated with E1 was detected using specific secondary anticigarette against human IgG, conjugate with horseradish peroxidase. As a substrate for color manifestations used the TMB. The results are expressed as the final credits roll. ALT levels were determined by clinical analyzer and expressed as units/liter of HCV RNA in serum was determined using HCV Monitor (Roche, Basel, Switzerland). E2 antigen was stained in a liver biopsy, using a specific monoclonal antibody (ESAS, catalog number 98031215, as described in the application EP No. 98870060.5). Semiquantitative assessment indicated as black squares for obvious positive staining in most cells, gray squares - for obvious staining in most cells and white squares for biopsies do not reveal detectable staining. HCV RNA are indicated by small black rectangles. Staining E2 can be confirmed by staining the Core and E1 (data not shown).

Fig. Epitope mapping of the antibody response induced by immunization E1 at Ton. The reactivity of antibodies against various peptides was measured using an indirect ELISA in which biotinylated peptides (see also Table 4) adsorbing on titration microplate is by using streptavidin. Specific antibodies were detected using specific secondary anticigarette against human IgG, conjugate with horseradish peroxidase. As a substrate for color manifestations used the TMB.

Fig. Analysis of E1 humoral responses to E1 proteins of subtype 1A and subtype 1b chimp Ton. For this purpose, E1 genotype 1A were obtained from HCV-H sequence; received recombinant vaccinia virus expressing the same area E1, and for genotype 1b (see below). E1 was obtained from crude lysates infected with cowpox virus RK13 cells (obtained as described in Maertens et al. (PCT/EP95/03031)). Reactivity of the antibodies were measured by indirect ELISA in which E1 was adsorbing on titration microplate by high mannose-binding of agglutinin Galathus nivalis (GNA). Specific antibodies were detected using specific secondary anticigarette against human IgG, conjugate with horseradish peroxidase. As a substrate for color manifestations used the TMB. The results are expressed as differential absorbance (optical density) (OD of wells with adsorbed E1 minus the OD of the wells without adsorbed E1).

Fig. The results of the analysis of proliferation of lymphocytes before and after vaccination among chimpanzees Ton. Frozen RVMS thawed and stimulated in three repetitions of different antigens. Negative con the role was clean environment, while as a positive control was used of concanavalin a at a concentration of 5 µg/ml RVS at a concentration of 2-4×105cells/well in a final volume of 150 μl were cultured in medium RPMI 1640, to which was added 10% V / V heat inactivated FCS, in U-shaped 96-well titration microplate together with the controls or 1 μg/ml E1 for 90 h at 37°C in humidified atmosphere containing 5% CO2. During the last 18 h, cells were subjected to pulse-labelling of 0.5 µci (3H) thymidine per well. Then these cultures were collected on filters fiberglass and determined the inclusion of the label. The results are expressed as stimulation indexes (IP): the average number of pulses per minute of the antigen/mean number of pulses per minute clean environment with three definitions.

Fig (a, b, C, d). Card designs used to obtain the expression of E2 protein with delegated N-terminal hypervariable region. Design pvHCV-92 and pvHCV-99 are intermediate structures used to construct deletion mutants pvHCV-100 and pvHCV-101.

Fig (a, b). Sequence (nucleotides: A; broadcasting: B), corresponding to the structures depicted in Fig (see above).

Fig. The antibody titers obtained in mice after immunization with various preparations E1, as described in example 9. Titers determined and with the help of ELISA: mouse serum was diluted 1/20 and forth (0,5 log 10), and incubated on E1s (modified or ndimethylacetamide, or maleimido)deposited on titration microplates. After washing, the bound antibodies were detected using specific secondary anticigarette against mouse IgG, conjugate with horseradish peroxidase. As a substrate for color manifestations used the TMB. The results are expressed as the final title, shows the standard deviation (n=6).

Fig. Epitope mapping of the humoral immune response induced by immunization with various preparations E1s in mice. The reactivity of antibodies against various peptides was measured using an indirect ELISA in which biotinylated peptides (listed in table 4) adsorbing on titration microplate by streptavidin. Mouse serum was diluted 1/20, and specific antibodies were detected using specific secondary anticigarette against human IgG, conjugate with horseradish peroxidase. As a substrate for color manifestations used the TMB.

Fig. Profile ittipiboon immunoglobulin mice immunized with various drugs E1s. Specific antibody class and subclass Ig was adsorbing on titration the microplate. After capturing mouse Ig from immune sera, diluted 1/500, E1s were incubated with 1 µg/ml of Education is avchina the immune complexes then incubated with polyclonal anticorodal rabbit, directed against E1. Finally, the rabbit antibodies were detected using secondary anticigarette Ig goat against rabbit conjugate with horseradish peroxidase. As a substrate for color manifestations used the TMB. The results are normalized by IgG (i.e. IgG signal for each animal separately considered equal to 1 and all the results for the other isotypes were expressed relative to that of the IgG1).

Fig. The antibody titers induced by two immunizations of about 1000 days E1s-ndimethylacetamide chimps Phil. Antibodies anti-E1 was determined by indirect ELISA: specific antibodies to bind to the solid phase E1 was detected using specific secondary anticigarette against human IgG, conjugate with horseradish peroxidase. The titer is expressed in units/ml, these units are related to local standard based on human sera.

Fig. The antibody titers induced by two immunizations of about 900 days E1s-ndimethylacetamide the chimpanzee Ton. Antibodies anti-E1 was determined by indirect ELISA: specific antibody associated with the solid phase of E1 was detected using specific secondary anticigarette against human IgG, conjugate with horseradish peroxidase. The titer is expressed in units/ml, these units are related to local standard based on human SIV is rodah.

Fig (A, B, C). SEC profile of the final stage of reduction of detergent (from 0.2 to 0.5% CHAPS): particle one E1 (A), one particle E2 (B) or equimolar mixture of E1 and E2; mixed particle (). This figure also shows the overlap between the values OP ELISA specifically detects only E1 (top), E2 (middle) and ELISA that detects only mixed particles (bottom).

Fig. SEC profile of the final stage of reduction of detergent (from 0.2 to 0.5% CHAPS): particle one E1 genotype 1b (top), particle one E1 genotype 4 (middle) or equimolar mixture of E1 genotype 1b and 4; mixed particle (bottom). This figure also shows the overlap between the values OP ELISA specifically detects only the mixed particles (see also Fig).

DETAILED description of the INVENTION

The invention described here, based on previously published work and patent applications pending. For example, this work consists of scientific works, patents or patent applications pending. All of these publications and applications cited previously or below, incorporated herein by reference.

The present invention relates to HCV vaccination. First successful immunotherapy chimpanzees with chronic active hepatitis C could be achieved through vaccination HCV antigen. This vaccine not only induced high immunodeficiency is nye answers but also induced clearance of viral antigen in the liver and a significant improvement of histological activity and degree of liver disease. In addition, the present invention relates to purified separate membrane proteins of HCV and, in particular, oligomeric particles. These oligomeric particles consist essentially of the coat proteins of HCV and have a diameter of from 1 to 100 nm when measured using dynamic light scattering or perhaps electronic microscopy. In this regard, it should be emphasized that these particles can be formed only E1 and/or E2 proteins, or their parts (see below). Therefore, the oligomeric particle according to the present invention are fundamentally different from HCV-like particles described in WO 98/21338, which must consist of E1 and E2, as well as Core and P7. The term “oligomeric particles consisting essentially of the coat proteins of HCV”, here defined as the design of a specific nature and form that contains several basic units of HCV E1 and/or E2 protein shell, which themselves think of one or two E1 and/or E2 monomers, respectively. It should be clear that the particles of the present invention is defined as free from infectious HCV RNA genomes. Particles according to the present invention can provide a particle of higher order spherical nature that can billposting, consisting of a shell of the envelope proteins, which can be included lipids, detergents, nuclear HCV protein or molecule adjuvants. Last, the particles can be encapsulated by liposomes or apolipoproteins, such as, for example, apolipoprotein b or the low-density lipoprotein, or through any other means, providing targeted delivery of these particles to a specific organ or tissue. In this case, such an empty spherical particles is often referred to as virus-like particles” or HPV. Alternative these particles are of a higher order can be a solid spherical structures in which the filled sphere consists of oligomers coat proteins E1 and E2 of HCV, which may be optionally included lipids, detergents, nuclear HCV protein or molecule adjuvants, or which in turn can themselves be encapsulated by liposomes or apolipoproteins, such as, for example, apolipoprotein b, low-density lipoprotein, or by other means, providing targeted delivery of these particles to a specific organ or tissue, such as asialoglycoprotein. These particles can consist of smaller structures (compared with a blank or solid spherical structures mentioned above), which are usually rounded (see on the form and which usually contain no more than one layer of coat proteins of HCV. A typical example of such smaller particles are rozetka-like structures that consist of a smaller number of coat proteins of HCV, usually between 4 and 16. A specific example of the latter includes smaller particles, obtained with E1s 0.2%CHAPS, an example of which is given here, which contain about 8-10 monomers E1s. Such rozetka-like structures are planar organization and are characterized by a round shape, for example the shape of a wheel. Again, may be optionally included lipids, detergents, capsid protein of HCV, or adjuvant molecules, or these smaller particles can be encapsulated by liposomes or apolipoproteins, such as, for example, apolipoprotein b or the low-density lipoprotein, or through any other means, providing targeted delivery of these particles to a specific organ or tissue. Smaller particles can also form small spherical or globular structure consisting of such smaller number of coat proteins E1 and E2 of HCV, which may be optionally included lipids, detergents, nuclear protein of HCV, or adjuvant molecules, or which in turn can be encapsulated by liposomes or apolipoproteins, such as, for example, apolipoprotein b or the low-density lipoprotein, or by any other means of providing targeted delivery of these particles to a specific organ or tissue. The size (i.e. diameter) of those identified above, particles, measured by means known in the field of techniques of dynamic light scattering (see later in the examples section), is usually between 1 and 100 nm, more preferably between 2 and 70 nm, even more preferably between 2 and 40 nm, between 3 and 20 nm, between 5 and 16 nm, between 7 and 14 nm, or between 8 and 12 nm.

Further, the invention relates to oligomeric particle as defined above, where these proteins are membrane selected from the group consisting of a HCV E1, HCV E1s, HCV E2 protein, SEQ ID No 13, or SEQ ID No 14, or parts thereof. Proteins of HCV HCV E1 and E2, as well as a detailed description of how to purify the latter proteins are detailed and described in PCT/EP 95/03031, Maertens et al. HCV E1s, SEQ ID No 13, or SEQ ID No 14, or their parts can be cleaned just as described for the HCV E1 or HCV E1s in PCT/EP 95/03031, Maertens et al. It should be emphasized that all content, including all definitions, the last document is incorporated into the present application by reference. Protein HCV E1s refers to the amino acids 192-326 E1 and E1 represents a protein without its C-terminal hydrophobic anchor. The term “or part thereof” refers to any part of these protein that is immunogenic, if it is a part of the particles of the present invention.

The invention further relates to oligomeric particles, as described herein, where at the ore one cysteine residue envelope protein of HCV, as described above, is alkylated, preferably alkylated with alkylating agents such as, for example, active halogen, ethylenimine or N-(Iodate)triptorelin. In this regard, it should be understood that the alkylation cysteine refers to zisteinom, in which the hydrogen atom when the atom is replaced by sulfur (CH2)nR, in which n is 0, 1, 2, 3 or 4, a R=H, COOH, NH2, CONH2, phenyl, or any derivative. The alkylation can be accomplished in any way known in the field, such as, for example, the use of active halogen X(CH2)nR, in which X represents a halogen such as I, Br, Cl or F. Examples of active Halogens are methyliodide, youkana acid, iodoacetamide and 2-bromethalin. Other methods of alkylation include the use of ethylenimine or N-(Iodate)trifurcated, both lead to the replacement of the N-CH2-CH2-NH2(Hermanson, 1996). The term “alkylating agents”, as used here, refers to compounds that are capable of alkylation, as described here. Such alkylation ultimately result in a modified cysteine, which can mimic other amino acids. Alkylation of ethylenimine leads to the structure, which has similarity with lysine, so that entering the camping new cleavage sites for trypsin (HerNanson, 1996). Similarly the use under the conditions leads to the production of amino acids, which is like methionine, whereas the use of iodoacetate and iodoacetamide leads to the production of amino acids with similarity to glutamic acid and glutamine, respectively. By analogy, these amino acids are preferably used in direct mutation of cysteine. Therefore, the present invention relates to oligomeric particles, as described herein, where at least one cysteine residue envelope protein of HCV, as described here, mutated in the natural amino acid, preferably methionine, glutamic acid, glutamine or lysine. The term “mutant” refers to a site-directed mutagenesis of nucleic acids encoding these amino acids, that is well known in the field methods, such as, for example, site-directed mutagenesis through PCR or mediated oligonucleotide mutagenesis as described in Sambrook et al. (1989).

The term “purified”, as used here, refers to a composition where the desired components, such as, for example, membrane proteins of HCV or oligomeric particles comprise at least 35% of the total components in this composition. The required components preferably comprise at least about 40%, more preferably at least about 50% even more preferably at least about 60%, even more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, even more preferably at least about 95% and most preferably at least about 98% fraction of the total components in this composition. This composition may contain other compounds, such as, for example, carbohydrates, salts, lipids, solvents and the like, without affecting the determination of percentage purity, as used here. “Selected” oligomeric particle HCV implies the oligomeric composition of HCV particles, which is at least 35% pure. In this regard, it should be clear that the term “purified single HCV envelope protein”, as used here, refers to the selected membrane proteins of HCV in essentially pure form. The terms “essentially purified oligomeric particles” and “separate membrane proteins of HCV, as they are used here, refers to oligomeric particles of HCV or a separate membrane proteins of HCV, so that you can apply for in vitro diagnostics and therapy. These oligomeric HCV particles are essentially free of cellular proteins, proteins of the vector origin or other HCV viral components. Typically, these particles or proteins purified to homogeneity (at least 8% purity, preferably 85%, more preferably 90%, more preferably 95%, more preferably 97%, more preferably 98%, more preferably 99%, even more preferably 99.5%pure, and most preferably contaminating proteins should be detectable conventional means, such as DDS-SDS page (polyacrylamide gel electrophoresis with sodium dodecyl sulfate) and silver staining).

The present invention also relates to oligomeric particle as defined above, where these membrane proteins comprise any mixture of HCV E1, HCV E1s, HCV E2, SEQ ID No 13 and/or SEQ ID No 14, or portions thereof, for example, the particle of the present invention may essentially consist of a protein of HCV HCV E1 and E2 proteins of HCV HCV E1 and E1s, proteins of HCV E1s and HCV E2 and HCV proteins E1, HCV E1s and HCV E2. In addition, the present invention also relates to oligomeric particle as defined above, where these proteins are derived from different strains, subtypes or genotypes of HCV, for example, these proteins are derived from genotype 1b, henati PA 4 or represent a mixture of coat proteins of HCV from a single strain or genotype of HCV and at least one other strain or genotype of HCV. Different strains or genotypes of HCV are well defined and described in PCT/ER/04155, Naertens et al. We emphasize again that the full contents, including all definitions, the last document is incorporated by reference into this application. Thus, the present invention relates to oligomeric particles containing membrane proteins, originating from any strain or genotype of HCV, known in the field, or to particles containing a mixture of proteins having origins from any strain or genotype of HCV, known in this area. In this respect, the present invention also relates to consensus sequences, originating from individual clones, an example of which is given below and in the examples section (see below).

Further, the present invention relates both to the oligomeric particle as described above, which can be obtained by using the method and the described method of obtaining this oligomeric particles. This method is characterized by the following stages:

(I) Purification of membrane proteins of HCV, possibly including the first use of detergent. Essentially, the method of purification stage (I) is described in detail in PCT EP 95/03031, Naertens et al. It is important that according to the present invention stage block in this method of purification, as described in PCT EP 95/03031, for example NEN ((N-ethylmaleimide)-Biotin), carried out together with the stage alkylation, as described in this application, preferably by use of iodoacetamide. In addition, the method of purification stage (I) may include the use of the agent is, cleave disulfide bonds, and may include the use of an alkylating agent. In the end, the technique of stage (I) results in a purified membrane proteins of HCV in solution.

(II) Substitution of the solution of the indicated purified proteins shell HCV detergent or salt, leading to the formation of oligomeric particles.

(III) Removing or clearing these oligomeric particles, possibly including additional decrease in the concentration of detergent or salt stage (II), which further promotes the formation or stabilization of these oligomeric particles formed after the specified replacement.

More preferably the present invention relates both to the oligomeric particle as defined here, and to a method for the specified particles, where the first detergent is a Empigen-BB. More preferably the present invention relates both to the oligomeric particle as defined here, and to a method for the specified particles, where the detergent phase (II) represents CHAPS, octylglucoside or twin room, more preferably tween-20 or tween-80, or any other detergent. More preferably the present invention relates both to the oligomeric particle as defined here, and to a method for the specified particles, where this salt is a betaine. the school is more preferably present invention relates both to the oligomeric particle as defined here, and to a method for the specified particles, where the specified Empigen-BB is used in a concentration of from 1% to 10% and where the specified CHAPS or twin use at a concentration of from 0.01% to 10%, or specified betaine is used in a concentration of from 0.01% to 10%. Even more preferably the present invention relates both to the oligomeric particle as defined here, and to a method for the specified particles, where the specified Empigen-BB is used at a concentration of 3% and where the specified CHAPS or betaine is used in concentrations of 0.2% or 0.3%, respectively, after which the buffer is changed and specified CHAPS or betaine is used in concentrations of 0.05% or 0.1-0.5% respectively. It should be understood that all percentages used in the above method, are given as wt./about. It should be clear that the above method (see also PCT/EP 95/03031 and examples section of this application) is an example of how to obtain particles of the present invention. In this respect, the present invention also applies to any other method known in this field that you can use to obtain oligomeric particle according to the present invention, such as, for example, excluding a reducing agent, as described in PCT/EP 95/03031 and in the examples section (below), and instead using it to restore cysteine bridges cells households is s, which have optimized the redox state of the endoplasmic reticulum. In addition, it should be clear that you can use a number of alkylbetaine, such as, for example, with Cntail, where n is a positive integer in the range from 1 to 20, and derivatives betaine, such as, for example, sulfobetaine.

Since it was first achieved successful immunotherapy chimpanzees with severe chronic active hepatitis by vaccination With purified HCV antigen, the present invention also relates to purified separate membrane proteins of HCV, in particular E1 and E1s. In addition, the present invention relates to compositions containing these separate membrane proteins of HCV, and to its use as a HCV vaccine or for the production of HCV vaccines.

In order to avoid the induction of immune responses against unwanted epitopes, HCV envelope protein used for immunization, preferably designed in the form of a coherent sequence of individual subtypes, strains or clones. Therefore, the present invention also relates to the use of HCV antigen (in the form of a peptide, protein or polynucleotide) for vaccination or diagnostic. In addition, the present invention also relates to oligomeric particle as defined here, and its application in cat is Roy HCV envelope protein encoded by the agreed sequence, based on quasimidi variability within isolates (coherent sequence of isolate), or based on an agreed sequence of different isolates within a subtype (coherent sequence subtype), the type or types of coordinated sequence type or species), or a kind HCV (coherent sequence type). Therefore, the amino acid sequence of this agreed envelope protein of HCV is a coherent sequence, with the origin of the agreed sequence of the isolate, subtype, strain or genus. For the connotations of the term “consistent”, in particular, refer to Naertens and Stuyver (1997), and references used here.

Oligomeric particle according to the present invention displays epitopes extremely effectively (see below). Therefore, this oligomeric particle is a means of presenting epitopes so that they can cause a clear immune response. In this context, it is clear that there is no need to membrane proteins of HCV, as defined here, contained HCV epitopes exclusively. Proteins shell HCV, which form oligomeric particles may contain epitopes that are originated only from HCV, and may contain epitopes that are derived from the other exogenous is hentov, such as, for example, HBV (hepatitis b virus) or HIV (human immunodeficiency virus). In other words, the oligomeric particle, with the frame of the coat proteins of HCV can be used as a carrier to represent non-HCV epitopes, perhaps in addition to HCV epitopes. Therefore, the present invention also includes oligomeric particle as defined here, but perhaps without HCV epitopes, possibly containing non-HCV epitopes, as well as its use and production. The term “exogenous agent”, as used here, refers to any agent, either living or inanimate, is able to induce an immune response, and this agent is not endogenous to the host and is not HCV. Specifically the latter term refers to the group consisting of pathogenic agents, allergens and haptens. Pathogenic agents include prions, viruses, prokaryotes and eukaryotes. More specifically, the virus includes, in particular, HBV, HIV, or the herpes virus, but not HCV. Common allergens include substances or molecules that are able to induce its own immune response in the host, when the host comes in contact with these allergens. The haptens behave like allergens in terms of their ability to provoke an immune response, but, in contrast to allergens, haptens need in the molecule-carrier.

The present invention also is tositsa to the composition, containing oligomeric particle as defined above. More specifically the present invention relates to vaccine compositions. The term “vaccine composition” refers to an immunogenic composition capable of elizerbeth protection against HCV, either partial or full. It is, therefore, includes peptides, proteins or polynucleotide HCV. Protection against HCV include, in particular, to the people, but also applies to human primates, trimera mouse (Zauberman et al., 1999) or other mammals.

Particles according to the present invention can be applied by themselves, biotinylating form (as shown in WO 93/18054) and/or in combination with Neutrality Avidin (Nolecular Probes Inc., Eugene, OR, USA). It should also be noted that the “vaccine composition” includes, in addition to the active compound, suitable excipient, diluent, carrier and/or adjuvant, which by themselves do not induce the production of antibodies harmful to the individual receiving the composition, and not alsirat protection. Suitable carriers are typically large, slowly metabolisable macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, copolymers of amino acids and inactive virus particles. Such carriers are well known to specialists in this field. Preferred adjuvants to enhance effectively the ti composition include aluminum hydroxide, aluminum in combination with 3-O-diallylamine monophosphorylation And as described in WO 93/19780, aluminum phosphate, as described in WO 93/24148, N-acetyl-muramyl-L-threonyl-D-isoglutamine, as described in U.S. Patent No. 4606918, N-acetyl-norbornyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine 2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)ethylamine and RIBI (ImmunoChem Research Inc., Hamilton, NT, USA), which contains monophosphorylated And detoxified endotoxin, trehalose-6,6-dimycolate and cell wall skeleton (MPL+TDM+CWS) in the emulsion 2% squalene/tween 80, but are not limited to. Any of these three components MPL, TDM or CWS may also be used alone or combined 2 by 2. In addition, you can use adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA, USA) or SAF-1 (Syntex), as well as adjuvants such as combinations between QS21 and 3-de-O-acetylated by monophosphorylation And (WO 94/00153) or MF-59 (Chiron), or adjuvants based on poly[di(carboxymethoxy)phosphazene] (Virus Research Institute), or adjuvants based blakolmer, such as Optivax (Vaxcel, Cythx)or adjuvants based on inulin, such as Algammulin and Gammalnulin (Anutech), incomplete adjuvant's adjuvant (IFA) or drugs Gerbu (Gerbu Biotechnik), it Should be understood that complete adjuvant's adjuvant (CFA) can also be used for applications not for man and for research purposes. “Vaccine composition” additional content is t excipients and diluents, which are intrinsically non-toxic and non-therapeutic, such as water, saline, glycerol, ethanol, moisturizing or emulsifying agents, substances, sautereau pH, preservatives and the like. Typically, the vaccine composition is prepared in injectable form or as a liquid solution, or in suspension. You can also prepare solid forms suitable for dissolution or suspension in liquid media prior to injection. The drug can also emulsify or encapsulated in liposomes to enhance the effect of adjuvant. The polypeptides can also be incorporated into immunostimulating complexes together with saponins, such as Quit A (ISCONS). Vaccine compositions contain immunologically effective amount of the polypeptides of the present invention, as well as any other of the above components. “Immunologically effective amount” means that the introduction of this quantity to the individual, either in a single dose or as part of a series, is effective for prevention or treatment. This number varies depending on the health status and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g., human, Primate, in addition to human, Primate, and so forth), on the ability of immunosystem individual to maintain an effective immune response, to the degree desired protection from drug vaccine, from doctor's appointment, from the strain of the infecting HCV and other relevant factors. Suppose that this number is within a relatively broad range that can be determined by conventional tests. Usually this number varies from 0.01 to 1000 μg/dose, more specifically from 0.1 to 100 μg/dose. Typically, the vaccine composition is administered parenterally, usually by injection, for example subcutaneously or intramuscularly. Additional drugs that are suitable for other routes of administration include oral medications and suppositories. Medical system doses can be a mode of a single dosage or multiple dosages. The vaccine can be administered in conjunction with other immunoregulatory agents. Therefore, the present invention relates to the use of oligomeric particles, as defined here, for prophylactic induction of immunity against HCV. It should be noted that the vaccine may also be useful for the treatment of the individual, as described above, and in this case it is called a “therapeutic vaccine”.

The present invention also relates to compositions, as defined above, which also contains nuclear protein (core) HCV, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A and/or NS5B protein or portion thereof. E1, E2 and/who do EE particles can, for example, to combine with antigens that stimulate T-cells, such as, for example, nuclear protein (core), P7, NS3, NS4A, NS4B, NS5A and/or NS5B. In particular, the present invention relates to compositions, as defined above, where the specified NS3 protein or part thereof have the amino acid sequence represented by SEQ ID 1 or SEQ ID 2 (see later in the examples section).

Cleaning these NS3 proteins will preferably include a reversible modification of cysteine residues, and even more preferably sulfonation cysteines. Methods of obtaining such a reversible modification, including sulfonation described for NS3 protein in Maertens et al. (PCT/EP99/02547). It should be emphasized that the full contents, including all definitions, the last document incorporated by reference into this application.

From the above it is clear that the present invention also relates to the use of oligomeric particles, as defined above, or a composition as defined above, for the production of HCV vaccine composition. In particular, the present invention relates to the use of oligomeric particles, as defined above, for the induction of immunity against HCV in chronic HCV carriers. More specifically the present invention relates to the use of oligomeric particles, as defined above, for the induction of immunity against HCV in chronic HCV carriers before, during, or p is after any other therapy, such as, for example, a well-known interferon therapy, or in combination or not in combination with the introduction of small drugs, treating HCV, such as, for example, ribavirin. Such a composition can also be applied before or after liver transplantation or after the alleged infection, such as, for example, damage to the injection needle. In addition, the present invention relates to a kit containing oligomeric particles or individual membrane proteins of HCV of the present invention, for determining HCV antibodies present in a biological sample. The term “biological sample”, as used here, refers to a sample of tissue or fluid isolated from an individual, including, but not limited to, serum, plasma, lymphatic fluid, the surface sections of the skin, respiratory, intestinal and genitourinary tracts, oocytes, tears, saliva, milk, blood cells, tumors, organs, gastric secrets, mucus, cerebrospinal fluid, exterior secrets, such as, for example, excrement, urine, semen and the like. Because of oligomeric particles and separate membrane proteins of HCV according to the present invention are highly immunogenic and stimulate both humoral and cellular immune response, the present invention relates also to a kit for detection associated with HCV T-cell immune response, containing the oligomeric particle or purified single HCV envelope protein of the present invention. HCV T-cell immune response can, for example, be measured as described in the examples section, or as described in PCT/EP 94/03555, Leroux-Roels et al.

It should be emphasized that the full contents, including all definitions, this document incorporated by reference into this application.

It should be clear that the present invention also relates to the use of specific HCV antibodies for the treatment and prevention of HCV infection. Here for the first time demonstrated that sufficient levels of HCV antibodies, particularly antibodies to HCV envelope, induce a decrease in the intensity of hepatitis C. Also the first to demonstrate that sufficient levels of antibodies can bind circulating virus and the presence of the complex AB virus coincides with disappearance of HCV antigen from the liver and decrease the intensity of liver disease. Antibodies to HCV membrane can be induced by vaccination, or they can be passively transferred by injection once these antibodies were purified from pools of HCV infected blood or from blood obtained from vaccinated HCV. Therefore, the present invention further relates to specific antibodies generated against oligomeric particle as described above, or against the arrangement, as described above, or against a separate envelope protein of HCV. In particular, the crust is ASEE the invention relates to a set, containing these antibodies for the detection of HCV antigens. The term “specific antibody”, as used here, refers to antibodies that are induced against epitopes that are specific for oligomeric particles, as described in the present invention. In other words, specific antibodies are induced against epitopes that are the result of the formation of oligomeric particles and are present only in the oligomeric particles. In addition, there are a variety of techniques are known for obtaining HCV peptides. The implementation of these techniques may be HCV peptides capable of presenting epitopes. Presumably these HCV peptides derived from these different and various techniques are able to view similar epitopes. Similar epitopes are epitopes, resulting from the implementation of different methods of obtaining or cleaning, but recognized by the same antibody. However, the oligomeric particle according to the present invention represent epitopes extremely effectively. Therefore, the epitopes on these oligomeric particles are highly immunogenic. Therefore, the present invention also relates to epitopes on oligomeric particles, and these epitopes are at least 10 times, preferably at least 20 RA is, preferably at least 50 times, preferably at least 100 times, preferably at least 500 times and most preferably at least 1000-fold more immunogenic than the epitopes on HCV peptides, which are not produced according to the present invention, that is not produced through the formation of particles in the presence of detergent. Professionals should be understood that the indicated immunogenicity can, for example, to detect, and then to compare by immunization of mammals by administration of a comparable amounts of peptides obtained by any method. In addition, the term “specific antibody” refers to antibodies that are induced against purified separate envelope protein of HCV. As used here, the term “antibody” refers to polyclonal or monoclonal antibodies. The term “monoclonal antibody” refers to a composition of antibodies with a homogeneous population of antibodies. The term “antibody” is not limited in the type or source of this antibody and is not intended to limit the manner in which it is received. In addition, the term “antibody” also refers to a humanized antibody in which at least part of the areas of the reading frames of immunoglobulin derived from the sequences of human immunoglobulin, and the single-chain antibody, such as, for example, described in U.S. patent No. 4946778, fragments of antibodies, such as FabF(AB)2FV, and other fragments which retain the function of antigen binding and specificity of the parent antibody.

In addition, the characteristic of the present invention is also the use of oligomeric particles as described above or a composition as described above for the detection of antibodies against membrane proteins of HCV. As used here, the term “detecting” refers to any analysis, known in the field, suitable for detection. In particular, this term refers to any immunological analysis, as described in WO 96/13590.

The terms “peptide”, “polypeptide” and “protein” in the present invention are used interchangeably. “Polypeptide” refers to a polymer of amino acids (amino acid sequence) and not for the specific length of the molecule. Thus, oligopeptides included within the definition of polypeptide. It should be understood that the peptidomimetics included in the terms “polypeptide” and “protein”.

The present invention also relates to the use of oligomeric particle as described herein for inducing immunity against HCV, characterized in that the specified oligomeric particle is used as part of a system time and connections. In this regard, it should be understood that Ter is in “the system time and connections” refers to the introduction of the individual time intervals compounds, used to eleirovania immune response. The latter compound can contain any of the following components: oligomeric particle vaccine composition HCV DNA, HCV polypeptides.

In this regard, the system includes the introduction of any of:

(I) an HCV antigen, such as, for example, oligomeric particle, with time intervals, or

(II) an HCV antigen, such as, for example, oligomeric particle, in combination with the vaccine composition of HCV DNA, where these oligomeric particles and specified vaccine composition HCV DNA can be administered simultaneously or at various time intervals, including alternating time intervals, or

(III) either (I)or (II), possibly in combination with other HCV peptides, with time intervals.

In this regard, it should be clear that the vaccine composition of HCV DNA contains a nucleic acid encoding the peptide membrane HCV, including E1-, E2-, E1/E2-peptides, E1s peptide, SEQ ID No 13, SEQ ID No 14, NS3 peptide, other HCV peptides or part of the indicated peptides. In addition, it should be understood that these HCV peptides include peptides shell HCV, including E1-, E2-, E1/E2-peptides, E1s peptide, SEQ ID No 13, SEQ ID No 14, NS3 peptide, other HCV peptides or their cactii. The term “other HCV peptides” refers to any HCV peptide or fragment with the condition that the HCV peptide does not represent E1, E2, E1s, SEQ ID No 13, SEQ ID No 14 and the and NS3. In paragraph II above scheme vaccine composition HCV DNA preferably contains nucleic acids encoding the peptides of the shell HCV. In paragraph II above scheme vaccine composition HCV DNA is even more preferably from nucleic acids encoding the peptides of the shell HCV, possibly in combination with a vaccine composition DNA HCV-NS3. In this regard, it should be clear that the vaccine composition of HCV DNA contains a plasmid vector containing a polynucleotide sequence encoding a HCV peptide, as described above, associated with the possibility of functioning with transcriptional regulatory elements. As used here, the term “plasmid vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it was sewn. Preferred vectors are vector capable of Autonomous replication and/or expression of nucleic acids with which they were sewn. Typically, but not limited to, plasmid vectors represent circular double-loop DNA, which, in their vector form are not bound to the chromosome. As used here, the term “polynucleotide sequence” refers to polynucleotides, such as deoxyribonucleic acid (DNA) and, where this is appropriate, ribonucleic to the slot (RNA). It should also be understood that this term include, as equivalents, analogs of either RNA or DNA, obtained using analogs of nucleotides and single-stranded (sense or antisense) and denitive polynucleotide. As used here, the term “transcriptional regulatory elements” refers to a nucleotide sequence which contains essential regulatory elements, such as those which when introduced into the living cell of the spinal capable of directing cellular mechanisms for the production of products broadcast encoded by this polynucleotide. The term “associated with functioning” refers to the immediate neighborhood, where the components are located in such a configuration, in which they carry out their normal function. Thus, transcriptional regulatory elements associated with functioning with a nucleotide sequence capable of expression of the specified nucleotide sequence. Specialists in this field can understand that it is also possible to use different transcriptional promoters, terminators, vectors-media or specific gene sequences.

Finally, the present invention relates to immunological analysis for the detection of HCV antibodies in which: (1) take Oli is omerou particle or purified separate envelope protein of HCV, as defined here, or its functional equivalent, (2) incubate the biological sample with the specified oligomeric particle or a specified protein shell HCV under conditions which allow the formation of complex antigen-antibody, (3) determine, is formed if the specified complex antigen-antibody containing the specified oligomeric particle or a specified envelope protein of HCV.

Hereinafter the present invention will be illustrated by reference to the following examples, which are set out in a particularly preferred embodiment. However, it should be noted that these embodiments are only illustrative and should not be construed as in any way limiting the invention.

EXAMPLES

Example 1: Expression, purification and homologously in the presence of detergent HCV protein E1

HCV E1s protein (amino acids 192-326) expressed and purified from RK13 cells using recombinant vaccinia virus pvHCV-11A, according to the Protocol described in Maertens et al. (PCT/EP 95/03031). In addition, purified E1 protein in 3%Empigen-BB, which shows the apparent molecular mass corresponding to the E1-glycosilated (approximately 60 kDa; Figure 1), was pulirula, and polerowanie fraction was again applied on the column for gel chromatography (according to PCT/EP 95/03031) and missed in the presence of 0.2% CHAPS or 3% betaine. Surprisingly, although E1s protein deprived when ofhis area of the membrane anchor, with both detergents is possible to obtain a homogeneous population specifically associated E1 of komolgorov with an apparent molecular weight of 260-280 kDa (Figure 2). This homologoumena structure can contain about 9 monomers E1s. It should be clear that the latter is a rough estimate, since the form of this oligomer may fundamentally affect its apparent molecular weight as measured by gel-chromatography. During the transition from 0.2% CHAPS to the 0.05% CHAPS, and repeated the same procedure, the apparent molecular mass was then shifted beyond the resolution column (the blank space of the column, more than 600 kDa, Figure 3), suggesting the formation of particles. The transition from 3% betaine 0.1% betaine gave the population of E1s oligomers with similar behavior (data not shown). You can select other detergents, through which you can achieve a similar oligomerization in the presence of detergent. Oligomerization, leading to the formation of particles is not unique to CHAPS or betaine, since similar results can be obtained by the use of Tween-20 or Tween-80 or octylglucoside. In addition, it may be possible to further remove detergent, which may give the possibility of formation of even larger particles. Therefore, to obtain particles the presence of detergent next may not be required. E. the particles can be obtained by using, for example, SCC without any detergent. Exclusively E1 monomer is approximately 31 kDa, whereas E2 monomer is approximately 70 kDa. These values, however, may differ depending on the state of glycosylation of the protein.

Example 2: Analysis of the oligomeric structures E1s higher order by using dynamic light scattering

To confirm the unexpected result that the formed particles, drugs E1s 0.05%CHAPS and 0.1%betaine, obtained according to example 1, or 0.1%betaine obtained by cultivation of drugs in 0.5%betaine, were analyzed using dynamic light scattering (DLS, dynamic light scattering).

The technique of dynamic light scattering makes it possible to measure the Brownian motion and to correlate it with the particle size. The larger the particle, the slower will be the Brownian motion. The speed of Brownian motion defined on the property, known as the diffusion coefficient (usually denoted by the symbol D). The particle size calculated from the diffusion coefficient using the equation of the Stokes-Einstein (Stokes-Einstein): d(H)=kT/3πηD, in which d(H) is the hydrodynamic diameter, k is a Boltzmann constant, T represents absolute temperature, η is the viscosity. Note that the measured diameter of t is made a value that which refers to how a particle diffuses within a fluid. Therefore, referred to as the hydrodynamic diameter. The diffusion coefficient is deduced from the autocorrelation functions (change of intensity fluctuations of the light time). In this device for the automatic calculation of the intensity of the autocorrelation function used correlator with computer control.

To measure distributions by size above function autocorrelation adjusted to obtain a linear curves, and the device is equipped with a computer program for the analysis of distribution by size. However, this technique has restrictive assumptions, such assumptions methodology called multi-angle laser scattering (MALLS, multi-angle laser light scattering), and no method cannot be considered as giving the absolute data. Results distributions by size according to the DLS should rather be interpreted as semi-quantitative indicators of polydispersity than as a true representation of this distribution.

Samples containing E1s particles (80-400 μg E1s/ml FR (phosphate buffered saline) to 0.05% CHAPS, 0.1% or 0.5% betaine) was transferred by pipette into the measuring cell of the device LSP 3.53 DLS equipped with a 10 mW HeNe laser (PolymerLabs). The results of this analysis are presented in figure 4 (E1s 0.05%CHAPS) and 5 (E1s 0.1%or 0.5%of the th betaine).

These tests really confirmed the unexpected result that the received E1 s patterns are monodisperse spherical particles. E1s particles in SFR/0.1% betaine showed the average size distribution of 21.3±4 nm, SFR/0.5% betaine: 27,9±5 nm, whereas in SFR/A 0.05% CHAPS for E1s received a diameter of 12,5.

Example 3: Analysis of size and shape using electron microscopy

Ten μl of E1s (226 ág/ml in SFR/A 0.05% CHAPS and 143 mg/ml in SFR/3% betaine) visualized the standard negative staining with 1%uranylacetate on stable carbon formvar (formvar)-lattices. The sample was applied for 30 seconds, and then before staining for 5 seconds and photography washed d b2About (6).

Statistical analysis gave the following results: E1s particle in CHAPS has an average diameter of 8.7±0,27 nm (the range of 4.3-29,0; 95% Cl 5,4), and E1s particle in the betaine was less homogeneous with an average diameter of 9.7±0,55 nm (the range of 4.3-40,5; 95% Cl 11,0). Surprisingly, the drug in 3% betaine, which is the source showed the molecular mass (MM) 250-300 kDa when analyzed using SEC, showed even larger particles than the drug in the 0.05% CHAPS, which was originally shown MM over 600 kDa. Therefore, the inventors hypothesized that an intermediate homopolyamide form E1s obtained using a 3%betaine, could form particles of higher parades over time. This unexpected effect indicates other possibilities of obtaining particles of the highest order. The distribution of particle size (Fig.7) shows that the drug in CHAPS is monodisperse, while particles larger observed the formation of the tails (up to 29 nm for the 0.05% CHAPS). Because large structures measured in DLS analyses, the presence of these large particles, although in smaller numbers, you can explain a larger diameter obtained using DLS analysis (example 2). The difference in diameter can also be explained by the fact that DLS measures the particle in motion, whereas electron microscopy measures the static particles. It should be clear that the immunogenicity of these drugs, as shown in the examples below, is the result of total drug and may be the result of secondary, smaller or larger particles or a mixture thereof.

Example 4: Immunization of chimpanzees chronically infected with HCV subtype 1b

Chimpanzees (Phil), already infected for more than 13 years (5015 days before immunization) strain of HCV subtype 1b were vaccinated with E1 (amino acids 192-326), which was derived from another strain of genotype 1b with 95.1% identity at the amino acid level (see also table 2) and who received, as described in examples 1-3. Chimpanzees received in the amount of 6 intramuscular immunization, each at 50 μg E1 in SFR/A 0.05% CHAPS, mixed the CSOs with RIBI R-730 (MPLA+TDM+CWS) according to the manufacturer's Protocol (Ribi Inc. Hamilton, MT). These 6 immunizations were given in two series of three immunizations with a three-week intervals and with a lag period of 6 weeks between the two series. Starting with 150 days before immunization during the period of immunization and up to 1 year after immunization (but see below) was performed continuous monitoring of this chimpanzee on various parameters indicative of the activity induced by HCV disease. These parameters included chemical analysis of blood, GPT, SGOT (aspartate aminotransferase, AST), G, chemical analysis of blood, viral load in the serum viral load in the liver and liver histology. In addition, monitoring of immune response to immunization was performed on the humoral and cellular level. During this period conducted monitoring of the animal to any harmful effects of immunization, such as a change in behavior, clinical signs, body weight, temperature and local reactions (redness, swelling, induration). Such effects were not found.

Obviously, the levels of ALT (and especially the G, data not shown) decreased as the level of antibodies against E1 has reached its maximum (Fig). ALT recover once the antibody levels began to decline, but the G remained at a low level, while anti-E1 remained detectable.

E2 antigen in the liver was decreased up on the tee undetectable levels during the period, during which anti-E1 were watchable, and E2 antigen was recovered shortly after the disappearance of these antibodies. However, as the Core antigens and E2 was not detectable in the liver, inflammation of the liver were significantly decreased (see also table 3). This is the main proof that the vaccine induces a decrease in the liver, probably by removing, at least partially, viral antigens from their main target organ, the liver.

The level of viraemia when measured using the Amplicor HCV Monitor (Roche, Basel, Switzerland) remained approximately unchanged in serum during the entire study period.

A more detailed analysis of the humoral response revealed that the maximum end-titer reached 14,5×103(after the sixth immunization) and that the titer decreased to undetectable levels after 1 year after immunization (Fig). Figure 9 shows that the major epitopes, which can be mimicked by peptides that are recognized by b-cells, located in the N-terminal region of E2 (peptides V1V2 and V2V3, the details of the used peptides, see Table 4). Since the reactivity against recombinant E1 is higher and longer, on the basis of this figure a conclusion can be drawn that antibodies that recognize these peptides represent only part of the total population of antibodies against E1. On the remaining part is directed against epitopes, you cannot mimic peptides, i.e. against discontinuous epitopes. Such epitopes are present only on a full-sized molecule E1 or even only on a structure that is similar to the particle. Such an immune response against E1 is unique, at least compared with what is usually observed in people with chronic HCV carriers (WO 96/13590 Maertens et al.) and chimpanzees (van Doorn et al., 1996), in which the formation of anti-E1 antibodies in the natural course of infection. In these patients, anti-E1 partly directed against discontinuous epitopes, but most part directed against C4 epitope (±50% of patient serum), and a minor proportion against V1V2 (varying from 2 to 70%, depending on genotype), and was celebrated exclusively reactivity against V2V3 (Maertens et al., 1997).

Analysis of T-cell reactivity was indicated that the Department of the immune system also specific way stimulated by the vaccine, because the index of stimulation of these T cells is increased from 1 to 2.5 and still is in some way higher in the next period (Figure 10). This T-cell reactivity is observed only in long-term cell-responders treated with interferon (see: PCT/EP 94/03555, Leroux-Roels et al.; Leroux-Roels et al., 1996).

Example 5: Immunization chronic carrier of HCV with another subtype

Chimpanzees (Ton), already infected for more than 10 years (380 days before immunization) strain of HCV genotypes 1A, were vaccinated with E1 from genotype 1b with only 79.3 percent identity at the amino acid level (see also Table 2), obtained as described in the previous examples. Chimpanzee has received a total of 6 intramuscular immunizations of 50 μg E1 in SFR/A 0.05% CHAPS, in each case mixed with RIBI R-730 according to the Protocol of the manufacturer (Ribi Inc. Hamilton, MT). These 6 immunizations were given in two series of three immunizations with a three-week intervals and with a lag period of 4 weeks between the two series. Starting with the 250 days prior to immunization, during the period of immunization and up to 9 months after immunization (but see below) was performed continuous monitoring of this chimpanzee on various parameters indicative of the activity induced by HCV disease. These parameters included chemical analysis of blood, ALT, AST, G, viral load in the serum viral load in the liver and liver histology. In addition, monitoring of immune response to immunization was performed on the humoral and cellular level. During this period conducted monitoring of the animal to any harmful effects of immunization, such as a change in behavior, clinical signs, body weight, temperature and local reactions (redness, swelling, induration). Such effects were not found.

Obviously, the ALT levels (and levels G, data not shown) decreased as t is like the level of antibodies against E1 has reached its maximum (11). ALT and G recover once the antibody levels began to decline, however, ALT and G remained at a lower level during the entire follow-up period. The levels of ALT even decreased significantly after vaccination (62±6 units/l) compared with the period prior to vaccination (85±11 units/l). Because fewer markers of tissue damage were extracted from the serum, these discoveries were the first indication that vaccination induced improvement in liver disease.

Levels of E2 antigen was undetectable in the period during which the anti-E1 kept the title above 1,0×103but again became detectable at lower levels E1 antibodies. Together with the disappearance of HCV antigens inflammation of the liver were significantly decreased from moderate chronic active hepatitis to the lowest forms of chronic persistent hepatitis (table 3). This is another major proof that the vaccine induces a decrease in the liver, probably by removing, at least partially, viral antigens from their main target organ, the liver.

The level of viraemia when measured using the Amplicor HCV Monitor (Roche, Basel, Switzerland) in serum remained at approximately the same levels throughout the study period. A more detailed analysis of the humoral response showed that max the maximum achieved the final titer was 30× 103(after the sixth immunization) and that this title was dropped to 0.5×1039 months after immunization (11). On Fig seen that the major epitopes, which can mimic peptides and which are recognized by b-cells, located in the N-terminal region of E2 (peptides V1V2 and V2V3, the details of the used peptides, see table 4). Since the reactivity against recombinant E1 is higher and longer, on the basis of this figure a conclusion can be drawn that antibodies that recognize these peptides represent only part of the total population of antibodies against E1. The remaining part is most likely directed against epitopes that cannot be mimicked by peptides, i.e. against discontinuous epitopes. Such epitopes are likely to be present only on a full-sized molecule E1 or even only on a structure that is similar to the particle. Such an immune response against E1 is unique, at least compared with what is usually observed in people with chronic HCV carriers who have detectable anti-E1. In these patients, anti-E1 is also partially broken, but a large proportion directed against C4 epitope (50% serum of the patient), and a minor proportion against V1V2 (varying from 2 to 70%, depending on genotype), and noted the exceptional reactivity against V2V3 (Maertens et al., 1997). Pollicott chimpanzees infected with isolate 1A, humoral immune response was also evaluated for cross reactivity with respect to E1-1A antigen. As you can see in Fig such cross-reactive antibodies indeed formed, although they constitute only part of the total population of antibodies. Significant is the correlation between the disappearance of the viral antigen in the liver and the disappearance of detectable anti-1A E1 antibodies in the serum.

Analysis of T-cell reactivity was indicated that the Department of the immune system is also stimulated by specific vaccine, because the index of stimulation of these T cells is increased from 0.5 to 5 and remains elevated during the subsequent period (Fig).

Example 6: Re-vaccination E1 chronic carriers of HCV

As the title E1 antibodies, as observed in examples 4 and 5 were unstable and decreased over time, even to undetectable levels for chimpanzees infected 1b, researched, can I again reinforce this humoral immune response with additional revaccination. Both chimpanzees were immunized again three consecutive intramuscular immunization with a three-week interval (50 μg E1, mixed with RIBI adjuvant). As can be judged on the basis of Fig and 11, anti-E1 the answer really can be strengthened, viral antigen in the liver again declined below detectable levels. The virus is the first load in the liver remained constant even at Ton (11). For the first time in the next period, the level of viraemia was less than 105genomic equivalents ml.

Important is the discovery that, as already was the case in the first series of immunizations, chimpanzees infected with a strain of HCV subtype 1b (Phil), correspond to lower titers of anti-E1 than chimpanzees infected with a strain of HCV subtype 1A (maximum titer in the first round of 14.5×103against 30×103for Ton and after revaccination only 1.2×103Phil against 40×103for Ton). Although both animals favorable effect is similar, from this experiment we can conclude that immunization chronic carrier protein E1, originating from a different subtype or genotype can be especially beneficial to achieve higher titers may bypass the pre-existing and specific immunosuppression, existing from the owner and induced infecting the subtype or genotype. Alternative lower titers observed in the homologous condition (vaccine 1b + 1b infection), may indicate the binding of the main mass of the antibodies to the virus. Therefore, induced antibodies may possess neutralizing capacity.

Example 7a: construction of the NS3 protein by combining the major epitopes, correlating, known as the but, with infection control

In addition to epitopes in E1, other epitopes may also be associated with the elimination of HCV during the acute phase, or by using an interferon therapy. Several of these epitopes localized in NS3 (Leroux-Roels et al., 1996; Rehermann et al., 1996 and 1997; Diepolder et al., 1995 and 1997). Two of these major epitopes represent a CTL epitope mapped Rehermann and co-authors (amino acids 1073-1081) and T-cell (CD4) epitope mapped Diepolder and co-authors (amino acids 1248-1261). Unfortunately, these epitopes are scattered around the NS3 protein. To have at least these epitopes will require a relatively large protein (amino acids 1073-1454). The production of such a large protein usually comes with low yields and vaccination may lead to the answer, a very small portion of which has a target of these important epitopes.

Therefore, the production of smaller protein would be a more appropriate solution to this problem. To do this, some of these epitopes need preraphaelite within this smaller protein. Given the existence of another CTL epitope (amino acids 1169-1177)that is not associated with clearance of HCV (Rehermann et al., 1996, 1997)have constructed a molecule NS3 so that it starts with amino acids 1166 and ends with the amino acid 1468 (table 5). This design already includes the epitopes described by Weiner and co-authors and Diepoler and co-authors. By mutation engine area 1167-1180 century sequence region 1071-1084 unnecessary CTL epitope was changed to the epitope, which opened Rehermann and co-authors, is associated with clearance of the virus. This design is additionally modified so that it contains methionine at position 1166 to enable initiation of translation. This methionine is chipped off in E. coli, because it is followed by alanine. Thus, the introduction of new epitopes that are not present in natural NS3, limited to a minimum. Alternatively, if the expression of this protein would be difficult, CTL epitope can make with a end on the amino acid 1468, as depicted in detail in Table 5.

The coding sequence of HCV NS-3 fragment was isolated and expressed as described in Naertens et al. (PCT/EP99/02547; clone 19b; HCV amino acids 1188-1468 used as source material). CTL epitope, as described Rehermann, which is not present in the fragment 19b NS-3 was merged with this fragment. Constructed as a C-terminal and N-terminal fusions, because the effects of the merger in relation to the levels of expression, sensitivity to proteolytic destruction and functionality can influence the position of the epitope.

Using plasmid pIGR12NS-3, which is a plasmid expression E. coli expressing the fragment 19b NS-3 under control of the left promoter of phage lambda, the quality is TBE matrix for PCR, coding sequences NS3 19b, merged respectively on the N - and C-end with CTL epitope Rehermann (called NS-3 19bTn and NS-3 19b respectively), were first subcloned into the cloning vector pGEM-T (Promega) with the formation of vectors pGEN-TNS-319bTn and pGEN-TNS-319bTc. PCR amplificatoare sequence was verified by DNA sequence.

In the event of a merger sequence T-cell epitope from the N-terminal region NS-3 PCR was carried out with a long sense primer bearing CTL epitope, and short antimuslim primer homologous to sequences 3' NS-3 19b of the stop codon. Sequences of primers are shown below.

Primer 9038 (semantic)

5'-GCCATGGCGACCTGCATCAACGGTGTTTGCTGGACCGTTTACCACGGTCGTGC GGCTGTTTGCACCCGTGGGGTTGCGAAGGCGGTGG-3' (SEQ ID NO 5)

Primer 1901 (antisense)

5'-TTTTATCAGACCGCTTCTGCG-3' (SEQ ID NO 6)

In the case of the slit on the end of the NS-3 PCR was carried out with a short semantic primers homologous to sequences 5' NS-3 19b of the start codon, and long antimuslim primer bearing CTL epitope, followed by stop codons in the reading frame. Sequences of primers are shown below.

Primer 1052 (semantic)

5'-AGCAAACCACCAAGTGGA-3' (SEQ ID NO 7)

Primer 9039 (antisense)

5'-CTCTAGACTATTAACCGTGGTAAACGGTCCAGCAAACACCGTTGATGCAGGTC GCCAGGCTGAAGTCGACTGTCTGG-3' (SEQ ID NO 8)

Since coding sequences cloned into pGEM-T vectors, the sequence of NS-3 19b embedded in the vector e is cpressey E.coli pIGRI2. For fused at the N-end NS-3 19b the coding sequence of the NS-3 19b was allocated in the form of a 379 BP fragment Ncol/SnaBI and ligated with fragments SnaBI/AllwNI and AlwNI/Ncol from the vector pIGRI2NS-3, which gave the result vector pIGRI2NS-3Tn. For fused at the C-end NS-3 19b the coding sequence of the NS-3 19b was allocated in the form of a 585 BP fragment SnaBI/Spel and was built in outdoor SnaBI/Spel vector pIGRI2NS-3, which gave the result vector pIGRI2NS-3Tc.

Both vector pIGRI2NS-3Tn and pIGRI2NS-3Tc successively transformed in expressing strain of E. coli MC1061 (pAcl) and after temperature induction of the promoter pLa lambda expression levels were analyzed on a DDS-SDS page (polyacrylamide gel electrophoresis with sodium dodecyl sulfate) and Western band, using polyclonal calcu serum against NS-3.

The amino acid sequence of the protein NS-3 19bTn

MATCINGVCWTVYHGRAAVCTRGVAKAVDFVPVESMETTMRSPVFTDNSSPPAVPQTF

QVAHLHAPTGSGKSTKVPAAYAAQGYKVLVLNPSVAATLGFGAYMSKAHGVDPNIRTG

VRTTTTGAPITYSTYGKFLADGGCSGGAYDIIICDECHSIDSTSILGIGTVLDQAETAGARL

VVLATATPPGSVTVPHPNTEEVALSSTGEIPFYGKAIPIEVIKGGRHLIFCHSKKKCDELAA

KLSGFGINAVAYYRGLDVSVTPTSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDF

S(SEQ ID NO 1)

The amino acid sequence of the protein NS-3 19b

MGVAKAVDFVPVESMETTMRSPVFTDNSSPPAVPQTFQVAHLHAPTGSGKSTKVPAAY

AAQGYKVLVLNPSVAATLGFGAYMSKAHGVDPNIRTGVRTTTTGAPITYSTYGKFLADG

GCSGGAYDIIICDECHSIDSTSILGIGTVLDQAETAGARLVVLATATPPGSVTVPHPNIEEV

ALSSTGEIPFYGCAIPIEVIKGGRHLIFCHSKKKCDELAAKLSGFGINAVAYYRGLDVSVIP

TSGDVVVVATDALMTGFTGDFDSVIDCNTCVTQTVDFSLATCINGVCWTVYHG (SEQ ID NO 2)

The nucleotide sequence of the coding region NS-3 19bn

ATGGCGACCTGCATCAAGGTGTTTGCTGGACCGTTTACCACGGTCGTGCGGCTGTT

TGCACCCGTGGGGTTGCGAAGGCGGTGGACTTTGTACCCGTAGAGTCTATGGAAACC

ACCATGCGGTCCCCGGTCTTTACGGATAACTCATCTCCTCCGGCCGTACCGCAGACA

TTCCAAGTGGCCCATCTACACGCCCCCACTGGTAGTGGCAAGAGCACTAAGGTGCCG

GCTGCATATGCAGCCCAAGGGTACAAGGTACTTGTCCTGAACCCATCCGTTGCCGCC

ACCTTAGGATTCGGGGCGTATATGTCTAAAGCACATGGTGTCGACCCTAACATTAGA

ACTGGGGTAAGGACCATCACCACGGGCGCCCCCATTACGTACTCCACCTACGGCAA

GTTTCTTGCCGACGGTGGTTGCTCTGGGGGCGCTTACGACATCATAATATGTGATGA

GTGCCACTCGATTGACTCAACCTCCATCTTGGGCATCGGCACCGTCCTGGATCAGGC

GGAGACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACTGCTACACCTCCGGGGTCGGT

CACCGTGCCACATCCCAACATCGAGGAGGTGGCTCTGTCCAGCACTGGAGAGATCCC

CTTTTATGGCAAAGCCATCCCCATCGAGGTCATCAAAGGGGGGAGGCACCTCATTTT

CTGCCATTCCAAGAAGAAATGTGACGAGCTCGCCGCAAAGCTATCGGGCTTCGGAA

TCAACGCTGTAGCGTATTACCGAGGCCTTGATGTGTCCGTCATACCGACTAGCGGAG

ACGTCGTTGTTGTGGCAACAGACGCTCTAATGACGGGCTTTACCGGCGACTTTGACT

CAGTGATCGACTGTAACACATGCGTCACCCAGACAGTCGACTTCAGCTAA (SEQ ID NO 3)

The nucleotide sequence of the coding region NS-3 19b

ATGGGGGTTGCGAAGGCGGTGGACTTTGTACCCGTAGAGTCTATGGAAACCACC

ATGCGGTCCCCGGTCTTTACGGATAACTCATCTCCTCCGGCCGTACCGCAGACAT

TCCAAGTGGCCCATCTACACGCCCCCACTGGTAGTGGCAAGAGCACTAAGGTGC

CGGCTGCATATGCAGCCCAAGGGTACAAGGTACTTGTCCTGAACCCATCCGTTGC

CGCCACCTTAGGATTCGGGGCGTATATGTCTAAAGCACATGGTGTCGACCCTAAC

ATTAGAACTGGGGTAAGGACCATCACCACGGGCGCCCCCATTACGTACTCCACCT

ACGGCAAGTTTCTTGCCGACGGTGGTTGCTCTGGGGGCGCTTACGACATCATAAT

ATGTGATGAGTGCCACTCGATTGACTCAACCTCCATCTTGGGCATCGGCACCGTC

CTGGATCAGGCGGAGACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACTGCTACA

CCTCCGGGGTCGGTCACCGTGCCACATCCCAACATCGAGGAGGTGGCTCTGTCCA

GCACTGGAGAGATCCCCTTTTATGGCAAAGCCATCCCCATCGAGGTCATCAAAGG

GGGGAGGCACCTCATTTTCTGCCATTCCAAGAAGAAATGTGACGAGCTCGCCGC

AAAGCTATCGGGCTTCGGAATCAACGCTGTAGCGTATTACCGAGGCCTTGATGTG

TCCGTCATACCGACTAGCGGAGACGTCGTTGTTGTGGCAACAGACGCTCTAATGA

CGGGCTTTACCGGCGACTTTGACTCAGTGATCGACTGTAACACATGCGTCACCCA

GACAGTCGACTTCAGCCTGGCGACCTGCATCAACGGTGTTTGCTGGACCGTTTAC

CACGGTTAA (SEQ ID NO 4)

Example 7b: Purification of proteins NS-3 19bTn and NS-3 19b

Cell mass .li culture Erlenmeyer destroyed with the help of cell death is ntegrator (CSL, model) at 1.4 kbar (1,4·105kPa) in 50 mm Tris, pH 8. This lysate was osvetleni by centrifugation (15000 g, 30 min, 4°). The supernatant was discarded, because the N - and C-terminal design NS3 was isolated from the sediment. The residue is made highly stable for N-terminal design, producing a thorough washing (first wash: 2% sarkosyl, 0.5 M guanidinium and 10 mm DTT (dithiothreitol), the second and third washing: 1% Triton X-100, 0.5 M guanidinium and 10 mm EDTA) before solubilization. In case C-terminal constructs did not. Purification of N-terminal construction continued. The washed precipitate in the end was dissolved in 6 M guanidinium/50 mm Na2HPO4at pH 7.2 and was sulfanilamide, as described in Maertens et al. (PCT/EP99/02547). From sulphonated precipitate first vysalivanie on a column of Sephadex G25 to 6 M f/50 mm triethanolamine, pH 7.5, and finally purified using two sequential anion-exchange chromatography in the same part of the buffer. The first anion exchange was performed on a column of Hyper DQ (50 μm) (BioSpra Inc.Marlborough, Ma. USA) and was isolated NS3 between 0.11 and to 0.19 M NaCl. After breeding these fractions were applied to a second column Hyper DQ (20 μm) (BioSpra Inc. Narlborough, Na. USA) and was isolated NS3 in the fractions containing 0.125 M NaCl. These fractions were vysalivanie up to 6 M urea in SFR, pH 7.5. Based on DDS-page with subsequent silver staining was found that the final purity was is above 90%. N-terminal sequencing using EDMAN degradation showed that NS3 is intact N-end, in which the desired epitope is present in the correct sequence. Confirmed that the methionine used to start the broadcast, split-off, as predicted.

Example 8: construction of the E2 protein without hypervariable region I

Was marked immunodominant homologous response to the HVR I region E2. This answer may not find a wide application in the development of a vaccine, because the vaccine is a heterologous state (vaccinal strain is always different from field strains). Therefore, deletion of this region will be necessary to have the E2 protein induces antibodies against more conservative, but less immunogenic regions E2. By careful analysis of the leader sequence of E2 and hypervariable region of E2 has developed the perfect design for the expression of E2 protein without HVR I. This construction enables the expression of E2 peptide, from the amino acids 409 instead of amino acids 384. As a leader sequence used 20 C-terminal amino acids of E1. However, since the description of this HVR is not unambiguous, then received a second version (starting with amino acids 412), which also has a high probability of cleavage in the correct position.

P is amerotica design pvHCV-99 (see also Fig and 16)

In expressing cassette coding sequence E2-715 must be preceded by a leader signal peptide E1, beginning with Met364. Therefore, plasmids pvHCV-92 (Fig), which contains the coding sequence of E2-715 HCV type 1b with a long version of the signal peptide E1 (since Met347), received a deletion by double enzymatic hydrolysis EcoRI and Ncol and subsequent completion of the 5' protruding end of the reaction with T4 DNA polymerase. Ligation obtained blunt ends (recircularisation 6621 BP fragment) in the given plasmid pvHCV-99, which encodes the same protein (E2-715) with a shorter leader signal peptide E1 (since Net364), this plasmid pvHCV-99 was deposited in the list of strains as ICCG 3635. It should be clear that you can use HCV or heterologous signal sequences of varying length.

Plasmids pvHCV-100 and -101 must contain a deletion in the sequence of E2, that is, a deletion of hypervariable region I (HVR-I). Plasmid pvHCV-100 was deleterule amino acids 384 (His)-408 (Ala), whereas plasmid pvHCV-101 was deleterule amino acids 384 (His)-411 (IIe).

Design pvHCV-100

To construct pvHCV-100 has designed two of the oligonucleotide:

HCV-RG 409 [8749]:

5'-CTT TGC CGG CGT CGA CGG GCA GAA AAT CCA GCT CGT AA - 3' (SEQ ID NO 9)

HCV-WG 408 [8750]:

5'-TTA CGA GCT GGA TTT TCT GCC CGT CGA CGC CGG CAA AG - 3' (SEQ ID NO 10)

PCR amplification (denaturate the 5 min 95° C, 30 cycles of amplification consisting of annealing at 55°S, polymerization at 72°s and denaturation at 95°C for 1 min, elongation for 10 min at 72° (C) matrix pvHCV-99 Gpt-WG [3757] and HCV-WG 408 [8750] resulted in 221 BP fragment, whereas amplification of HCV-RG 409 [8749] and Tag-WG [3756] resulted in 1006 BP fragment. Both PCR fragments overlap with each other by 19 nucleotides. These two fragments were combined and amplified by PCR with primers Gpt-WG [3757] and Tag-WG [3756]. Received 1200 BP fragment was subjected to enzymatic hydrolysis by EcoRI and HinDIII and ligated into the vector pgsATA18 [ICCG 1998] (5558 BP), subjected to enzymatic hydrolysis by EcoRI/HinDIII. This design, pvHCV-100, and analyzed using restriction analysis and sequence analysis and deposited in the list of strains as ICCG 3636.

Design pvHCV-101

To construct pvHCV-101 has designed two of the oligonucleotide:

HCV-WG 411 [8747]:

5'-CTT TGC CGG CGT CGA CGG GCA GCT CGT AAA CAC CAA CG - 3' (SEQ ID NO 11)

HCV-WG 410 [8748]:

5'-CGT TGG TGT TTA CGA GCT GCC CGT CGA CGC CGG CAA AG - 3' (SEQ ID NO 12)

PCR amplification matrix pvHCV-99 Gpt-WG [3757] and HCV-WG 410 [8748] resulted in 221 BP fragment, whereas amplification of HCV-RG 411 [8747] and Tag-WG [3756] resulted in 997 BP fragment. Both PCR fragments overlap with each other by 19 nucleotides. These two fragments were combined and amplified by PCR with what ramarama Gpt-WG [3757] and Tag-WG [3756]. Received 1200 BP fragment was subjected to enzymatic hydrolysis by EcoRI and HinDIII and ligated into the vector pgsATA18 [ICCG 1998] (5558 BP), subjected to enzymatic hydrolysis by EcoRI/HinDIII. This design, pvHCV-100, was analyzed using restriction analysis and sequence analysis and deposited in the list of strains as ICCG 3637.

All plasmids were checked by sequence analysis and deposited in the list of strains Innogenetics. For each plasmid was awarded two mini-DNA preparation (PLASmix) under sterile conditions and were pulirula. Determined the concentration of DNA was carried out by QA using restriction analysis. Purified DNA was used to generate recombinant vaccinia virus as described in Maertens et al. (PCT/EP95/03031). Recombinant viruses vvHCV-100 and vvHCV-101 was created, however, proven by the WHO (world health organization) Vero cells. After two rounds of purification plaques product expression was analyzed using Western blot analysis, as described in Naertens et al. (PCT/EP95/03031). Proteins were visualized using specific monoclonal anti-E2 antibodies (IGH 212, which can be obtained from the authors of the invention in Innogenetics N.V., Zwijnaarde, Belgium) installed with a molecular mass of 69 and 37 kDa for vvHCV-100 and 68 and 35 kDa for vvHCV-101. These molecular weights indicate the presence of both glycosylated and replicationmanager E2 protein, which was confirmed using the processing is TCI samples NGF before Western blot analysis. This treatment leads to the detection of only a single protein of 37 kDa and 35 kDa for vvHCV-100 and vvHCV-101, respectively.

Amino acid sequence of the Mature E2. obtained from pvHCV-100

QKIQLVNTNGSWHINRTALNCNDSLQTGFFAALFYKHKFNSSGCPERLASCRSIDKFAQG

WGPLTYTEPNSSDQRPYCWHYAPRPCGIVPASQVCGPVYCFTPSPVVVGTTDRFGVPTY

NWGANDSDVLILNNTRPPRGNWFGCTWMNGTGFTKTCGGPPCNIGGAGNNTLTCPTDC

FRKHPEATYARCGSGPWLTPRCMVHYPYRLWHYPCTVNFTIFKVRMYVGGVEHRFEAA

CNWTRGERCDLEDRDRSELSPLLLSTTEWQILPCSFTTLPALSTGLIHLHQNTVDVQYLYG

VGSAVVSLVIK (SEQ ID NO 13)

Amino acid sequence of the Mature E2 obtained from pvHCV-101

QLVNTNGSWHINRTALNCNDSLQTGFFAALFYKHKFNSSGCPERLASCRSIDKFAQGWG

PLTYTEPNSSDQRPYCWHYAPRPCGIVPASQVCGPVYCFTPSPVWGTTDRFGVPTYNW

GANDSDVLILNNTRPPRGNWFGCTWMNGTGFTKTCGGPPCNIGGAGNNTLTCPTDCFR

KHPEATYARCGS GPWLTPRCMVHYPYRLWHYPCTVNFTIFKVRMYVGGVEHRFEAAC

NWTRGERCDLEDRDRSELSPLLLSTTEWQILPCSFTTLPALSTGLIHLHQNIVDVQYLYGV

GSAWSLVIK(SEQ ID NO 14)

Example 9: Particle E1 with additionally improved immunogenicity

As described in example 1, E1s protein was purified according to the Protocol described in PCT/EP 95/03031 Naertens et al. This Protocol involves covalent modification cysteine (free cysteine and cysteine involved in the formation of intermolecular bridges, in the latter case, after recovery of these cysteine bridges using DTT (dithiothreitol)), using derivatives maleimide (N-ethylmaleimide and Biotin-maleimide, both obtained from Sigma). As an alternative method of locking maleimido also assessed active halogen. These compounds, i.e. active halog the us, block free cysteine by alkylation. As an example, was estimated active halogen (iodoacetamide, Merck). The same Protocol used for the purification of E1, as described in Naertens et al. (PCT/EP 95/03031), but instead maleimide compounds used iodoacetamide. Protein E1s obtained using this technique, during the entire cleaning behaved like proteins, blocked by maleimido. When the final reduction of the concentration of detergent (0.05% CHAPS or when switching to a 0.5%increase betaine as described in example 1 was received similar particles, as determined by DLS. However, immunization of mice E1s modified ndimethylacetamide, was discovered an unexpected effect.

A total of three series of 6 mice each were immunized E1s using three injections with a three-week intervals, with each injection consisted of 5 μg E1s concentration of 100 μg/ml in FR, mixing with an equal volume of RIBI adjuvant (R-700). The first series received E1 maleimide in the form of the drug in the 0.05%CHAPS, second series received E1 ndimethylacetamide also in the form of the drug in the 0.05%CHAPS, while the third series of the received E1 ndimethylacetamide in the medication 0.12%betaine. Then all the mice took the blood within 10 days after the third immunization. End titles (defined as the serum dilution still giving OP 2 times higher than background values) for each individual animal is correctly determined against E1 maleimide and E1-ndimethylacetamide. On Fig shows the end credits are presented as mean with standard deviation. The mice that received E1 maleimide, was only the formation of antibodies that were able to recognize maleimidomethyl epitopes (E1 ndimethylacetamide reactivity no), the mice that received E1 ndimethylacetamide, clearly there was a formation of antibodies against the valid E1 epitopes, since these antibodies are reactive against E1 ndimethylacetamide and against E1 maleimide. This is clearly demonstrated in an additional experiment, in which antibodies specific areas E1 was determined using peptides that have not been modified nor ndimethylacetamide, nor maleimido. These results, as shown in Fig demonstrate that mice immunized with E1-ndimethylacetamide (in the form of drugs in CHAPS and betaine), was the formation of antibodies, which can recognize peptides V1V2, V2V3, V3V4, V5C4, C4V6. Since V6 is not part of E1s, the inventors concluded that the antibodies were producirovanie against C4, V3 (V3V4 is positive, whereas V4V5 is not positive) and V1V2. In mice immunized with E1s-maleimido, developed only very low response against peptides V1V2 and V2V3. This further confirms the fact that moderately high titer measured for these mouse antibodies about the Yves maleimid-E1s, mainly directed against maleimid-dependent epitopes. In addition, the inventors were able to prove that the response induced E1s-ndimethylacetamide, partly represents the response of Th1 type, since a substantial number of induced antibodies belong to the IgG2 subtype(a+b). The number of IgG2 even higher for betainovuyu of the drug compared with drug CHAPS (Fig). From these results concluded that membrane proteins of HCV, in which at least one cysteine (but possibly more than one cysteine) is alkylated, are highly immunogenic proteins.

In the modified ndimethylacetamide E1 in the form of the drug in the betaine is also used for revaccination chimps Phil and Ton. Both chimpanzees were immunized again two consecutive intramuscular immunization with a three-week time interval (50 μg E1, mixed with RIBI adjuvant, as in examples 4 and 5). As can be seen from Fig and 21, anti-E1 response really can again enhance and increase to higher levels than those that were obtained in the previous immunizations after two injections. This titration was performed against a standard, which is a mixture of three human anti-E1 of high titers of sera (obtained from chronic carriers of HCV). The titer of anti-E1 these sera was defined as one unit/ml of China who see Phil (Fig) only after two immunizations were induced titles twice, than people-carriers. The chimpanzee Ton (Fig) were induced titers, higher up to 140-fold increase. This again confirms the high immunogenicity of these E1-particles.

Example 10: Alkilirovanny E1 has excellent qualities for diagnostic applications

E1 s-ndimethylacetamide, as described in example 9, was then estimated as antigen for detection of anti-E1 antibodies in serum samples from people with chronic HCV carriers. As an example, these antigens were associated with LIA-membranes and strips were processed essentially as described in Zrein et al. (1998). Serum samples from 72 blood donors were first evaluated to determine the optimum concentration of E1 antigen, which can be used in the analysis to eliminate false positives. For E1s-maleimide this concentration was equal to 8 micrograms/ml, whereas for E1s-ndimethylacetamide concentrations up to 50 µg/ml did not lead to false positive results (none of the samples showed a relative color staining above 0.5). Using 8 and 50 µg/ml, respectively for E1s-maleimide and 1s-ndimethylacetamide, 24 serum of chronic HCV carriers were subjected to screening for antibodies against E1s. As shown in Table 6, E1s-ndimethylacetamide results in more samples being evaluated positively (67% vs. 38% for E1s-maleimide). Have not found a single specimen, which OC is aligned positively only for E1s-maleimide. For samples that are evaluated positively for E1s-maleimide and E1s-ndimethylacetamide, the reactivity of the latter was higher. From this example we can conclude that the alkylated proteins shell HCV are the best antigens for the detection of human antibodies than modified maleimide protein shell.

Example 11: production of mixed particles containing E1 and E2

E1s and E2s (vvHCV-44) were produced and purified as described in Maertens et al., PCT/EP95/03031, except for the fact that the modification of maleimido replaced by alkylation using iodoacetamide. E1s and E2s 3%Empigen separately or in equimolar mixture was injected into the column Superdex-200 PC 3.2/30, balanced STR/0,2% CHAPS. This column is intended for use with HPLC equipment SNART™ from Pharmacia LKB (Sweden). Fractions were subjected to screening using three different sandwich ELISA (enzyme-linked immunosorbent assays). For these ELISA specific monoclonal antibody E1-(IGH 207) and E2(IGH 223) was applied at a concentration of 2 µg/ml. Fractions from gel filtration were incubated in a dilution of 1/2500. Two other monoclonal antibodies E1 (IGH 200) and E2 (IGH 212)conjugated with Biotin, was used to detect the bound antigen. System streptavidin-HRP (horseradish peroxidase)/TMB (3,3',5,5'-tetramethylbenzidine) was used for the manifestation of the bound Biotin in yellow color, which is measured at 450 nm.

This ELISA system was used for homologous (anti-E1 coating/anti-E1 detection or anti-E2 coating/anti-E2 detection) and heterologous production (anti-E1 coating/anti-E2 detection). Last theoretically detect only particles that include and E1, and E2. Reactive fractions were pulirula, concentrated on a 10 kDa filter and again subjected to chromatography on Superdex-200 SVR/A 0.05% CHAPS. All these fractions were tested for reactivity with the help of different productions ELISA. As can be seen from Fig, adding E2 to E1 does not lead to a large shift in retention time compared with the one of E1, which indicates that the particles are indeed still present. These particles contain both E1 and E2, since only in this production of heterologous ELISA evaluate positively.

Example 12: Purification of mixed particles containing E1 from 2 different genotypes

E1s genotype 1b and genotype 4 (vvHCV-72) was produced and purified as described in Maertens et al., PCT/EP95/03031, except for the fact that the modification of maleimido replaced by alkylation using iodoacetamide for genotype 1b. E1s-1b and E1s-4 3% Empigen separately or in equimolar mixture was injected into the column Superdex-200 PC 3.2/30, balanced STR/0,2% CHAPS. This column is intended for use with HPLC equipment SNART™ from Pharmacia LKB (Sweden). Main fractions, the content is interacting protein was pulirula, concentrated on a 10 kDa filter and again subjected to chromatography on Superdex-200 SVR/A 0.05% CHAPS. All these fractions were tested for reactivity by using staging ELISA, which should detect only particles containing E1 from both genotypes. For this ELISA-streptavidin was applied at a concentration of 2 µg/ml. Fractions from gel filtration were incubated in a dilution of 1/2500. To detect the bound antigen used E1 monoclonal antibody (IGH 200), which recognizes only E1 from genotype 1 and 10. The system of goat-anti-mouse-HRP/TMB was used for the manifestation of this analysis in a yellow color, which was measured at 450 nm. As can be seen from Fig, Appendix E1-4 E1-1b does not lead to a large shift in retention time of proteins compared to the one of E1, which indicates that the particles are indeed still present. These particles contain both protein E1, i.e. E1s genotype 1b and genotype 4, because only in this production of heterologous ELISA evaluate positively.

The LIST of REFERENCES

Deleersnyder V., Pillez A., C. Wychowski, Blight, K., Xu J., Hahn, Y.S., Rice C.M., Dubuisson J. Formation of native hepatitis With virus glycoprotein complexes. J. Virol. 1997: 71:697-704.

Diepolder H.M., Zachoval R., Hoffmann R., Wierenga E.A., Santantonio So, M.C. Jung, Eichenlaub D., Pape G.R. Possible mechanism involving T-lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis With virus infection. Lancet 1995: 346: 1006-1007.

Diepolder H.M., Gerlach J.T., Zachoval R., HoffNann R., M.C. Jung, A. Wierenga, S. Scholz, Santntonio So, Houghton M., Southwood, S., Sette, A., Pape G.R. Immunodominant CD4+ T-cell an epitope within the non-structural protein 3 in acute hepatitis With virus infection. J. Virol., 1997: 71: 6011-6019.

Fancy D.A., K. Melcher, S.T. Johnston and T. Kodadek New chemistry for the study of multiprotein complexes: the six-histidine tag as a receptor for a protein crosslinking reagent. Chem. Biol. (1996) 3: 551-559.

G.T.Hermanson in Bioconjugate Techniques (1996) Part I section 1.43 and section 2.2.1, Academic Press San Diego CA, USA.

Houghton M. Immunity to HCV: The case for vaccine development. 4thInternational meeting on hepatitis With Virus and related viruses. Satellite Symposium: New approach to prevention and therapy of HCV infection. March 7 1997, Kyoto, Japan.

Leroux-Roels G., Esquivel C.A., DeLeys R, Stuyver L, Elewaut A., Philippe J., Desombere I, Paradijs J., Maertens G. Lymphoproliterative responses to hepatitis With virus core, E1, E2, NS3 and in patients with chronic hepatitis With infection treated with interferon alfa. Hepatology 1996: 23: 8-16.

Maertens G. and Stuyver L. Genotypes and genetic variation of hepatitis virus. In: The molecular medicine of viral hepatitis. Ed: T.J. Harrison and Zuckerman A.J. 1997.

Major M.E. and S.M. Feinstone The molecular virology of hepatitis C. Hepatology 1997: 25: 1527-1538.

Maertens G., E. Depla, Ducatteeuw A., Vandeponseele P., Bosman F., Venneman A., de Martynoff, G., Stuyver L, F. Dekeyser, Vandeperre Century, Zrein Buyse M. and M. - A. Hepatology 1997: 26: 186A.

Rehermann Century, C.M. Chang, McHutchinson j, Kokka R, Houghton M., Rice C.M., Chisari F.V. Differential cytotoxic T-lymphocyte responsiveness to the hepatitis In and With virus in chronically infected patients. J. Virol. 1996 70: 7092-7102.

Rehermann Century, Takaki, A., Liebetrau, A., Luda S., Seifert U., Salha K., Manns, M., Wiese M. Characterization of the cytotoxic and helper T cell response in patients 18 years after a single-source outbreak of HCV infection. Hepatology, 1997: 26: 406A.

Sambrook J., Fritsch E.F. and Maniatis T. (1989) Nolecular Cloning, a laboratory manual, second edition. Cold Spring Harbor University Press, Cold Spring Harbor, NY USA.

L.J. Van Doorn, Kleter Century, Pike I., W. Quint Analysis of hepatitis With virus isolates by serotyping and genotyping. J. Clin.Microbiol. 1996: 34: 1784-1787.

Villa E., Buttafoco P., Grottola a, Scarcelli A., Giannini F., Manerti F. Neutralizing antibodies against HCV: liver transplant as an experimental model. J. Hepatol. 1998:28:

Weiner A.J., Erickson A.L, Kansopon J., Crawford, K., E. Muchmore, Houghton m, Walker C.M. Association of cytotoxic T lymphocyte (CTL) escapemutations with persistent hepatitis With virus (HCV) infection. Princess Takamatsu Symp, 1995:25:227-235.

Yi M., Nakamoto y, Kaneko S., Yamashita T., Murakami S. Delineation of regions important for heteromeric association of hepatitis With virus E1 and E2. Virol. 1997a 231: 119-129.

Zauberman, A., Nussbaum o, llan E., Eren R, Ben-Moshe O., Arazi Y., Berre, S., I. Lubin, Shouval D., Galun E., Reisner Y. and Dagan petrol S. The trimera mouse system: a mouse model for hepatitis With infection and evaluation of therapeutic agents. June 6-9, 1999; Oral 4.3. In: 6thInternational Symposium on Hepatitis C & Related Viruses. Bethesda USA.

Zrein M., Louwagie J., Boeykens H., Covers L., Hendrickx G., Bosman f, Sablon, E., Demarquilly C., Boniface, M. and Saman, E. (1998). Assessment of a new immunoassay for serological confirmation and discrimination of human T-cell lymphotropic virus infections. Clin. Diagn. Lab. Imm. 5: 45-49.

Example 13 Obtaining oligomeric particles, consisting of parts of the coat proteins of HCV

E1s HCV protein (amino acids 192-326) was expressed in Vero cells using recombinant virus HCV11B. The protein was purified essentially as described in example 9. After cleaning 3% empigen-BB was replaced by 3% betaine using chromatography with the exception of size, as described in Example 1. As a result of this process get E1s in the form of particles.

E2deltaHVRI (amino acids 412-715) expressed and purified from Vero essentially as described for E1, using recombinant vaccinia virus HCV101, which was on what is learned by recombinant from pvHCV-101, described in Example 8, and the vaccine virus wild type. E2deltaHVRI also behaves as a particle (which is measured dynamically by scattering) after replacing empigen on betaine.

In chimpanzees immunized with such particles, was registered immune response.

There were obtained particles composed of another part of the E2 protein, and amino acids 384-673. Constructed accordingly the plasmid were transformed cells of Hansenula polymorpha. Of expressed proteins using chromatography with the exception of size were obtained particles whose size is defined by the dynamic scattering, was 20-56 nm.

1. Oligomeric particle, inducing immunity against HCV (hepatitis C virus), consisting essentially of purified coat proteins of HCV and having a diameter of 5 to 100 nm.

2. Oligomeric particle according to claim 1, having a diameter of 5 to 40 nm.

3. Oligomeric particle according to claim 1 or 2, in which the amino acid sequence of the envelope protein of HCV is a coordinated sequence of the isolate of HCV, HCV subtype, strain HCV or HCV.

4. Oligomeric particle according to any one of paragraphs.-3, where at least one cysteine residue of the specified envelope protein is alkylated.

5. Oligomeric particle according to claim 4, where at least one cysteine residue of the specified envelope protein is alkylated by an alkylating agent.

6. Oligomeric particle according to claim 5, where the specified alkylating agent is ethylenimine, N-(Iodate)triptorelin or active halogen X-(CH2)n-R, where X represents halogen, R represents H, COOH, NH2, CONH2, phenyl, or any derivative, and n is 0, 1, 2, 3, or 4.

7. Oligomeric particle according to any one of claims 1 to 4, where at least one cysteine residue of the specified envelope protein mutated in the natural amino acid, preferably selected from the group consisting of methionine, glutamic acid, glutamine and lysine.

8. Oligomeric particle according to any one of claims 1 to 7, where these membrane proteins represent the E1 protein of HCV, or part thereof.

9. Oligomeric particle according to any one of claims 1 to 7, where these membrane proteins are E1s HCV proteins or parts thereof.

10. Oligomeric particle according to any one of claims 1 to 7, where these membrane proteins represent the E2 protein of HCV, or part thereof.

11. Oligomeric particle according to any one of claims 1 to 7, where these membrane proteins are SEQ ID No 13 and/or SEQ ID No 14, or parts thereof.

12. Oligomeric particle l is the Boma one of claims 1 to 11, where these membrane proteins are encoded by nucleotide coherent sequence of HCV isolate, nucleotide coherent sequence of subtile HCV, nucleotide coherent sequence like HCV, nucleotide coherent sequence kind HCV or their parts.

13. Oligomeric particle according to any one of claims 1 to 7, where these membrane proteins are a mixture of E1 protein of HCV and/or its parts, E1s protein of HCV and/or its parts, the E2 protein of HCV and/or its parts.

14. Oligomeric particle according to item 13, where these E2 proteins of HCV, or part thereof defined by SEQ ID No 13 and/or its parts, or SEQ ID No 14 and/or its parts.

15. Oligomeric particle according to any one of claims 1 to 14, where these proteins shell or parts obtained from different strains of HCV, the HCV subtypes or genotypes of HCV.

16. Oligomeric particle according to any one of claims 1 to 15, where these membrane proteins or parts thereof are a mixture of coat proteins of HCV from one strain of HCV or HCV genotype and coat proteins of HCV from at least one other strain of HCV or HCV genotype.

17. Oligomeric particle according to any one of claims 1 to 16, obtained by the method characterized by the following stages:

(I) purification of membrane proteins of HCV in solution,

(II) formation of oligomeric particles by replacing the purified coat proteins of HCV from the stage (I) with a solution of detergent or a solution of the m salt,

(III) purification of oligomeric particles formed in stage (II).

18. Oligomeric particle according to 17, where the specified stage (I) of the above method involves the use of a first detergent.

19. Oligomeric particle according to 17 or 18 where the specified stage (I) involves the use of a agent that breaks disulfide bonds.

20. Oligomeric particle according to any one of PP-19 where the specified stage (I) involves the use of an alkylating agent.

21. Oligomeric particle according to any one of p-20, where the specified stage (III) of the above method includes an additional reduction of the concentration of detergent or salt from step (II).

22. Oligomeric particle according to any one of p-21, where said first detergent with stage (I) is a Empigen-BB, where the detergent from the stage (II) or stage (III) is a CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate), octylglucoside, twin or any other detergent, and where this salt is a betaine.

23. Oligomeric particle according to item 22, where the specified Empigen-BB is used in a concentration of from 1 to 10% and where the specified CHAPS or twin use at a concentration of from 0.01 to 10%, or specified betaine is used in a concentration of from 0.01 to 10%.

24. Oligomeric particle according to any one of claims 1 to 23 for the production of HCV vaccine composition.

25. Oligomeric particle according to any one of claims 1 to 23 the La production of medicines for inducing immunity against HCV in chronic HCV carriers.

26. Oligomeric particle according A.25 for the production of medicines for inducing immunity against HCV in chronic HCV carriers prior to, simultaneously with or after any other treatment.

27. Oligomeric particle according A.25 for the production of medicines for inducing immunity against HCV in HCV infected individuals before or after liver transplantation or after the alleged infection.

28. Oligomeric particle according to any one of claims 1 to 23 for the production of medicaments for prophylactic induction of immunity against HCV.

29. Oligomeric particle according to any one of claims 1 to 23 for the production of medicines for inducing immunity against HCV, characterized in that the specified oligomeric particle is used as part of the system time and connections.

30. Oligomeric particle according to any one of claims 1 to 23 for use as a HCV vaccine.

31. Oligomeric particle according to any one of claims 1 to 23 for inducing immunity against HCV in chronic HCV carriers.

32. Oligomeric particle according p for inducing immunity against HCV in chronic HCV carriers prior to, simultaneously with or after any other treatment.

33. Oligomeric particle according to any one of claims 1 to 23 for inducing immunity against HCV in HCV infected individuals before or after liver transplantation or after the alleged infection.

34. Oligomeric particle according to any one of claims 1 to 23 for preventive induction of immunity PR is against HCV.

35. Oligomeric particle according to any one of claims 1 to 23 for the induction of immunity against HCV, characterized in that the oligomeric particle is a part of the system time and connections.

36. The way to obtain oligomeric particle according to any one of claims 1 to 16, characterized by the following stages:

(I) purification of membrane proteins of HCV in solution,

(II) formation of oligomeric particles by replacing the purified coat proteins of HCV from the stage (I) with a solution of detergent or a solution of salt,

(III) purification of oligomeric particles formed in stage (II).

37. The method according to p where the specified stage (I) of the above method involves the use of a first detergent.

38. The method according to p or 37 where the specified stage (I) involves the use of a agent that breaks disulfide bonds.

39. The method according to any of p-38 where the specified stage (I) involves the use of an alkylating agent.

40. The method according to any of p-39 where the specified stage (III) of the above method includes an additional reduction of the concentration of detergent or salt from step (II).

41. The way to obtain oligomeric particle according to any one of p-40, where said first detergent with stage (I) is a Empigen-BB, where the detergent from the stage (II) or stage (III) is a CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonic the t), octylglucoside, twin or any other detergent, and where this salt is a betaine.

42. The way to obtain oligomeric particle according to paragraph 41, where the specified Empigen-BB is used in a concentration of from 1 to 10% and where the specified CHAPS or twin use at a concentration of from 0.01 to 10%, or specified betaine is used in a concentration of from 0.01 to 10%.

43. Composition for inducing immunity against HCV containing oligomeric particle according to any one of claims 1 to 23 and at least one suitable excipient, diluent, carrier or adjuvant.

44. The composition according to item 43, which also contains HCV nuclear protein (core) P7, E1, E2, NS2, NS3, NS4A, NS4B, NS5A and/or NS5B protein or part thereof.

45. The composition according to item 44, where the specified NS3 protein or part thereof have the amino acid sequence represented by SEQ ID No 1 or its parts, or represented by SEQ ID No 2 or its parts.

46. Composition according to any one of p-45 for use as a HCV vaccine.

47. Composition according to any one of p-45 for inducing immunity against HCV in chronic HCV carriers.

48. The composition according to p for inducing immunity against HCV in chronic HCV carriers prior to, simultaneously with or after any other treatment.

49. Composition according to any one of p-45 for inducing immunity against HCV in HCV infected individuals before or after liver transplantation or after the alleged infection.

50. Comp is the position of any of PP-45 for preventive induction of immunity against HCV.

51. Composition according to any one of p-45 for inducing immunity against HCV, characterized in that the oligomeric particle is a part of the system time and connections.

52. Specific antibody against oligomeric particle according to any one of claims 1 to 23, obtained by immunization of an animal with particle according to any one of claims 1 to 23.

53. Specific antibody according to paragraph 52, for the production of drugs for treating or preventing HCV infection.

54. Kit for detection of HCV antigens containing the specific antibody, wherein the specific antibody is an antibody according to paragraph 52.

55. Kit for detection of HCV antibodies present in a biological sample containing the specific antigen in a suitable container, characterized in that the specific antigen is an oligomeric particle according to any one of claims 1 to 23.

56. Kit for detection associated with HCV T-cell response containing the specific antigen, characterized in that the specific antigen is an oligomeric particle according to any one of claims 1 to 23.

57. An immunological assay for detection of antibodies to HCV in a biological sample, in which

(I) take oligomeric particle according to any one of items 1 to 23,

(II) incubated specified biological sample with the oligomeric particle with stage (I) the conditions to which that allow the formation of a complex between a specified oligomeric particle and the specified antibodies to HCV,

(III) determine, after stage (II), there are specified complex and, hence, the presence of antibodies to HCV in the specified biological sample.

58. The vaccine against hepatitis C virus (HCV), containing oligomeric particle according to any one of claims 1 to 23, or a composition according to any one of p-45.

59. The vaccine according to § 58, which represents a therapeutic HCV vaccine.

60. The vaccine according to any one of p and 59 for the induction of an immune response in a mammal, chronically infected with HCV.

61. The vaccine p, where specified, the immune response is a humoral and/or cellular immune response.

62. The vaccine according to any one of p and 59 for the induction of an immune response in a mammal, chronically infected with HCV genotype or subtype, homologous genotype or subtype of HCV, membrane proteins which are contained in the specified vaccine.

63. The vaccine according to any one of p and 59 for the induction of an immune response in a mammal, chronically infected with HCV genotype or subtype, heterologous genotype or subtype of HCV, membrane proteins which are contained in the specified vaccine.

64. The vaccine according to any one of p and 59 for clearance of HCV viral antigens from the liver of a mammal infected with HCV.

65. The vaccine p where these viral antigens are antigens of HCV Core and/or HCV E2.

66. The vaccine according to any one of p and 59 to normalize the equal of liver enzymes in the serum of a mammal, infected with HCV.

67. The vaccine p where these liver enzymes are alanine aminotransferase (ALT, alanineaminotransferase) and/or G.

68. The vaccine according to any one of p and 59 to improve the histology of the liver of a mammal infected with HCV.

69. The vaccine according to any one of p and 59 for improvements in liver disease of a mammal infected with HCV.

70. The vaccine according to any one of p and 59 for improvement in the inflammation of the liver of a mammal infected with HCV.

71. The vaccine according to any one of p-70, where specified, the mammal is a human.

Priority claims

24.06.1998 claims 1 to 3, 8-14, 16 to 26, 28-32, 34-48, 50-53 and 58-71 EP 98870142.1;

22.02.1999 claims 4-7, 15 EP 99870033.0;

23.06.1999 PP, 33, 49, 54-57 PCT/EP 99/04342.



 

Same patents:

FIELD: medicine, biochemistry.

SUBSTANCE: in blood serum one should detect the level of lactoferrin and biliary acids. At their ratio being equal to 5-17 it is necessary to detect chronic hepatitis of high activity.

EFFECT: higher accuracy of detection.

3 ex

Diagnostic analysis // 2228530
The invention relates to medicine, in particular to the analysis on the basis of nucleic acids for detection of hepatitis b virus
The invention relates to medicine, in particular for immunochemical analysis, and can be used to improve analytical and technological characteristics of immunochemical analysis

The invention relates to medicine and concerns НСV immunoreactive polypeptide compositions

The invention relates to Virology, biotechnology and immunology, and can be used to identify various biological samples of antibodies to surface antigen of hepatitis b virus

The invention relates to medicine, namely to medical Virology, and concerns the methods rapid diagnosis of hepatitis C

The invention relates to Virology and immunology, and can be used for typing of hepatitis C virus (НСV)

The invention relates to polypeptides, used as immunological reagents for the identification, prevention, and treatment of infections caused by HCV

The invention relates to medicine, in particular of Pediatrics and pathology of cellular immunity

The invention relates to the production potential of drugs aimed at the destruction of protein antigens, in particular glycoprotein gp 120 of the main surface protein of human immunodeficiency virus

The invention relates to genetic engineering

The invention relates to biotechnology and immunology, and can be used to generate neutralizing antibodies against different strains and clinical isolates of HIV-1

The invention relates to medicine and concerns НСV immunoreactive polypeptide compositions

The invention relates to biotechnology and gene therapy and relates to vaccines against hepatitis

The invention relates to medicine and relates to a method of detecting antibodies, the method of screening of blood components and kit for detection of antibodies НСV

The invention relates to biotechnology, namely genetic engineering
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