during an infectious event , the hcv genome is first translated into a precursor polyprotein of about 3000 amino acids . this polyprotein then undergoes post - translational cleavages to give various precursors and mature viral proteins . the structural proteins of hcv are located in the n - terminal region of the polyprotein . as shown in fig1 they are more particularly the capsid or core protein ( c ), and the envelope proteins e1 and e2 , which are present in the following order : nh2 - c - e1 - e2 . this portion is cleaved by the host cell proteases . the numbering of the amino acids of the polyprotein as well as of its derivatives , which is adopted here after , is that commonly used and in particular presented by choo et al ., pnas [ vol . 88 : p . 2451 ( 1991 )]. thus , the c protein corresponds to the amino acids at positions 1 to 191 of the polyprotein and the e1 protein to the amino acids at positions 192 to 380 . in the remainder of the text , it is appropriate to number the amino acids of the sequence of the e1 protein , from position 192 to position 380 , 381 , 382 or 383 . in the remainder of the text , for the sake of simplicity , reference is made solely to the c - terminal position 380 . the c protein derived from the direct cleavage of the polyprotein contains 191 amino acids . this c protein , also called c191 , may itself be truncated toward its c - terminal end by enzymatic cleavage to give a protein of 173 amino acids , called c173 . in the remainder of the text , the term “ c protein or polypeptide ” will preferably designate the c191 form . the c protein is a good vaccine candidate since it is of course a structural protein of the virus and since the region encoding this protein is relatively well conserved by the various hcv strains . it is known that a region of the c protein capable of generating a high antibody response corresponds to the first 120 amino acids ; the first 48 amino acids constituting the major antigenic domain . however , a major obstacle to its use as vaccine lies in the fact that this protein is capable of transactivating genes belonging to the host cell , in particular genes such as oncogenes , which may have , inter alia , the consequence of inducing a carcinogenesis event . indeed , it has in particular been shown that the c173 form was capable of translocation in the nucleus of the host cell and of transactivation . the region of the c protein responsible for the translocation in the nucleus and for the regulatory activity appears to be located in the n - terminal portion ( first 123 amino acids ). thus , the region of the c protein which is of interest from a vaccine point of view is , on the other hand , responsible for a toxic effect toward the host cell . to overcome this difficulty , a solution commonly envisaged in the scientific community would be to use a c191 protein whose cleavage site at position 173 / 174 would have been made inoperative by mutation . as will be seen below , such a protein nevertheless proves capable of regulatory activity , even if it is to a lesser degree . surprisingly , it has now been demonstrated that it was possible to abolish the regulatory activity of the c protein by modifying it and by combining it , under certain conditions , with the e1 protein . the present invention provides means for abolishing the regulatory activity by preventing the migration of the c protein into the nucleus . this migration no longer takes place in the presence of the e1 protein which possesses , inter alia , the property of becoming anchored in the cytoplasm , at the level of the endoplasmic reticulum , and which , unexpectedly , has the capacity to retain the c protein therein when certain conditions are met . the migration may be abolished by producing , for example , a fusion of the two proteins , cleavable or otherwise ; in the case where it is cleavable , the products generated should be capable of interacting with each other so that there is no leakage of one of them into the nucleus ; the complex formed by the product of cleavage being capable of becoming anchored in the cytoplasm . the equivalent of a cleavable peptide fusion is a mixture , in equimolar quantity , of the components constituting the fusion . ( a ) a first region corresponding to all or part of the c polypeptide of the hepatitis c virus ; and ( b ) a second region corresponding to all or part of the e1 polypeptide of said virus and , proves , as such or via its products of cleavage , incapable of regulatory activity toward one or more genes ; ( a ) of a first polypeptide containing a region which corresponds to all or part of the c polypeptide of hcv and ( b ) of a second polypeptide containing a region corresponding to all or part of the e1 polypeptide of hcv ; and which proves incapable of regulatory activity toward one or more genes ; or ( iii ) a dna molecule comprising a sequence encoding the polypeptide as described in ( i ) of the present claim , placed under the control of elements necessary for its expression in a mammalian cell ; and according to another aspect of the invention , the subject of the invention is also a method for the treatment or prevention of an infection induced by hcv according to which a pharmaceutical composition according to the invention is administered to a mammal , preferably a human , requiring such a treatment . “ polypeptide ” is understood to mean any chain of amino acids covalently linked to each other , regardless of the length of the chain and regardless of the post - translational modifications which may take place such as , for example , a lipidation . it is also possible to use the term protein interchangeably . “ c polypeptide of hcv ” is understood to mean in particular a c polypeptide which possesses the amino acid sequence as disclosed by choo et al . as well as any other c polypeptide obtained from any other strain and whose sequence could differ from that of choo et al . for example , it may represent the c polypeptides described by takeuchi k et al . [ nucleic acids research 18 : 4626 ( 1990 )], houghton m et al . [ hepatology 14 : 381 ( 1991 )], delisse et al . [ j . hepatology , 13 , suppl . 4 ( 1991 )], bukh j et al . [ pnas 91 : 8239 ( 1994 )] and hiroaki o et al . [ intervirology 37 : 68 ( 1994 )]. “ e1 polypeptide of hcv ” is understood to mean in particular an e1 polypeptide which possesses the amino acid sequence as disclosed by choo et al . as well as any other e1 polypeptide obtained from any other strain and whose sequence could differ from that of choo et al . for example , it may represent the e1 polypeptides described in hiroaki o et al . [ intervirology 37 : 68 ( 1994 )], grakoui et al . [ j . virol . 67 : 1385 ( 1993 )] and spaete et al . [ virology 188 : 819 ( 1992 )], matsumia et al . [ j . virol . 66 : 1425 ] or in kohara et al . [ j . gen . virol . 73 : 2313 ( 1992 )]. thus , the amino acid sequence of the c polypeptide and that of the e1 polypeptide of hcv may vary according to the viral strain , reflecting the phenomenon of allelic variance . for example , a virus is usually represented by a set of strains which differ from each other in minor allelic characteristics . a polypeptide which fulfills the same biological function in different strains may have an amino acid sequence which is not the same for all the strains . such an allelic variation is also found at the level of the dna . at the level of the amino acid sequence , the allelic differences may consist of one or more amino acid substitutions , deletions or additions which do not alter the biological function . as regards the polypeptide included in the pharmaceutical composition according to the invention , two cases must be envisaged : either the polypeptide is incapable of being cleaved by a protease in a mammalian cell , or it is susceptible to such a cleavage . when the polypeptide is incapable of being cleaved by a protease in a mammalian cell , it advantageously contains : ( a ) a first region corresponding at least to the portion of the c polypeptide of the hcv virus responsible for the regulatory activity of said c polypeptide toward one or more genes ; and ( b ) a second region corresponding at least to a portion of the e1 polypeptide of said virus responsible for the cytoplasmic anchorage of the e1 polypeptide . when the polypeptide is capable of being cleaved by a protease in a mammalian cell , it advantageously contains : ( a ) a first region corresponding at least to a portion of the c polypeptide of hcv responsible for the regulatory activity of said c polypeptide toward one or more genes and to the portion of said c polypeptide responsible for the interaction of said c polypeptide with the e1 polypeptide of said virus ; and ( b ) a second region corresponding at least to a portion of the e1 polypeptide of said virus responsible for the interaction of the e1 polypeptide with the c polypeptide of said virus and to a portion of the e1 polypeptide of said virus responsible for the cytoplasmic anchorage of the e1 polypeptide . “ portion of the c polypeptide of hcv responsible for the regulatory activity of said c polypeptide toward one or more genes ” is understood to mean in particular any portion of the c polypeptide of hcv capable of activating , transactivating or suppressing the transcription or the expression of any gene , according to any mechanism . this gene may be a eukaryotic gene , a viral gene , an oncogene or a protooncogene . a portion of the c polypeptide of hcv responsible for the regulatory activity of said polypeptide may in particular correspond to the amino acids at positions 38 to 43 , 58 to 64 , 66 to 71 , 6 to 23 , 39 to 74 , 99 to 102 , 101 to 121 , 101 to 122 , 58 to 121 , 1 to 120 , 1 to 121 , 1 to 122 , 1 to 123 or 1 to 173 . preferably , a portion of the c polypeptide of hcv responsible for the regulatory activity may be a portion of the c polypeptide ranging from the amino acid at position 1 to the amino acid in one of positions 48 to 191 . for this purpose , one of positions 48 to 191 may be for example position 119 , 120 , 121 , 123 and 173 . a portion of the c polypeptide of hcv responsible for the interaction of said c polypeptide with the e1 protein of hcv may in particular correspond to the amino acids at positions 151 to 173 or at positions 173 to 191 of the c polypeptide of hcv . a portion of the e1 polypeptide of hcv responsible for the cytoplasmic anchorage of the e1 polypeptide may be a hydrophobic domain of the e1 polypeptide . such hydrophobic domains are for example located at positions 262 to 291 , 370 to 380 and 330 to 380 of the e1 polypeptide . a portion of the e1 polypeptide of hcv responsible for the interaction of the c polypeptide with the e1 protein may be in particular the c - terminal domain of the e1 polypeptide , preferably the domain at positions 330 to 380 or at positions 370 to 380 . in a polypeptide useful for the purposes of the present invention , the first region may be located on the n - or c - terminal side of the polypeptide , advantageously on the n - terminal side ; likewise , the second region may be located on the c - or n - terminal side , advantageously on the c - terminal side . according to a preferred mode , the c - terminal end of the first region may be fused by peptide bonding to the n - terminal end of the second region . when the polypeptide contained in the pharmaceutical composition according to the invention comprises the region corresponding at least to the amino acids at positions 172 to 175 of the c polypeptide of hcv , this polypeptide advantageously contains a mutation making the cleavage site at position 173 / 174 inoperative . according to a preferred mode , such a mutation is a point mutation , carried out in one of positions 172 to 175 . it may be obtained , for example , by deletion , addition or substitution of one or more amino acids , in particular by deletion , addition or substitution of one or more amino acid at positions 172 to 175 . preferably the mutation will be produced by substitution of one or two amino acids ; a double mutation by substitution being most particularly preferred . according to a particular example , the residue naturally existing at position 173 ( serine ) may be in particular substituted by the methionine residue and the residue naturally existing at position 173 ( phenylalanine ) may be substituted in particular by the leucine residue . in general , it is within the capability of persons skilled in the art to produce one or more mutations capable of making inoperative the cleavage site at position 173 / 174 of the c polypeptide of hcv . when the polypeptide contained in the pharmaceutical composition according to the invention comprises the region corresponding at least to the amino acids at positions 190 to 193 of the hcv polyprotein , this polypeptide advantageously contains a mutation making inoperative the cleavage site at position 191 / 192 of the hcv polyprotein . according to a preferred mode , such a mutation is a point mutation produced in one of positions 190 to 193 . it may be obtained , for example , by deletion , addition or substitution of one or more amino acids , in particular by deletion , addition or substitution of one or more amino acids at positions 190 to 193 . preferably , the mutation will be produced by substitution of one or two amino acids , a double mutation by substitution being most particularly preferred . according to a specific example , the residue naturally existing at position 191 ( alanine ) may in particular be substituted by the valine residue and the residue naturally existing at position 192 ( tyrosine ) may in particular be substituted by the asparagine residue . in general , it is within the capability of persons skilled in the art to produce one or more mutations capable of making inoperative the cleavage site at position 191 / 192 . when the polypeptide useful for the purposes of the present invention comprises both the region corresponding at least to amino acids 190 to 193 of the hcv polyprotein and the region corresponding at least to amino acids 172 to 175 of the c polypeptide of hcv , only one of the two cleavage sites 191 / 192 and 173 / 174 can be made inoperative , preferably both will be made inoperative . when only the site 173 / 174 is made inoperative , the polypeptide is capable of being cleaved and in this particular case , it is necessary that this polypeptide possesses a first region which corresponds , inter alia , to the portion of the c polypeptide responsible for the interaction of said polypeptide with the e1 polypeptide and a second region which corresponds , inter alia , to the portion of the e1 polypeptide responsible for the interaction of said polypeptide with the c polypeptide . when a polypeptide useful for the purposes of the present invention is incapable of being cleaved by a protease , it may contain a cleavage site on the condition , however , that this cleavage site is not functional . for example , in the particular case of a polypeptide consisting of the c191 polypeptide fused with the e1 polypeptide and containing a mutation making inoperative the cleavage site at position 191 / 192 , the cleavage site at position 173 / 174 may not be mutated ; however , it will not be , or will be only slightly , functional , insofar as the cleavage at position 191 / 192 is no longer possible . indeed , it is known that the cleavage at position 191 / 192 must be carried out for the cleavage at position 173 / 174 to take place . according to a specific mode , a polypeptide useful for the purposes of the present invention is incapable of being cleaved by a protease and contains : ( a ) a first region which substantially corresponds to the domain of the c polypeptide ranging from the amino acid at position 1 to the amino acid in one of positions 120 to 173 , and ( b ) a second region which substantially corresponds to a domain of the e1 polypeptide containing at least one hydrophobic region , for example to the domain of the e1 polypeptide ranging from the amino acid at position 192 to the amino acid at position 380 , or from the amino acid at position 330 to the amino acid at position 380 , or from the amino acid at position 260 to the amino acid at position 290 , or from the amino acid at position 260 to the amino acid at position 380 . according to another particular mode , a polypeptide useful for the purposes of the present invention is incapable of being cleaved by a protease and contains : ( a ) a first region which substantially corresponds to the domain of the c polypeptide ranging from the amino acid at position 1 to the amino acid in one of positions 120 to 191 , and ( b ) a second region which substantially corresponds to a domain of the e1 polypeptide containing at least one hydrophobic region , for example to the domain of the e1 polypeptide ranging from the amino acid at position 192 to the amino acid at position 380 , or from the amino acid at position 330 to the amino acid at position 380 , or from the amino acid at position 260 to the amino acid at position 290 , or from the amino acid at position 260 to the amino acid at position 380 ; on the condition that said polypeptide does not contain a cleavage site 191 / 192 or alternatively when the cleavage site is reconstituted , then a mutation is introduced in order to make it inoperative . advantageously , the first region of the polypeptide useful for the purposes of the present invention corresponds to the amino acids at positions 1 to 191 of the c polypeptide of hcv and / or the second region of this polypeptide corresponds at least to the amino acids at positions 192 to 380 of the e1 polypeptide or hcv . in a particularly preferred manner , the first and second regions are as defined above in this same paragraph , the amino acid at position 191 being fused by peptide bonding to the amino acid at position 192 . according to a particular mode , the polypeptide consists of the first and second regions as defined above in this same paragraph . when the polypeptide useful for the purposes of the present invention is as described in the preceding paragraph , it imperatively contains a mutation making inoperative the cleavage site at position 191 / 192 or at position 173 / 174 . preferably , the two cleavage sites are made inoperative . a mixture useful for the purposes of the present invention advantageously comprises : ( a ) a first polypeptide containing a region which corresponds at least to the portion of the c polypeptide of the hcv virus responsible for the regulatory activity of said c polypeptide toward one or more genes and to the portion of said c polypeptide responsible for the interaction of said c polypeptide with the e1 polypeptide of said virus , and ( b ) a second polypeptide containing a region corresponding to a portion of the e1 polypeptide of said virus responsible for the interaction of the e1 polypeptide with the c polypeptide of said virus and to a portion of the e1 polypeptide of said virus responsible for the cytoplasmic anchorage of the e1 polypeptide . the portions of the c and e1 polypeptides responsible for the properties listed in points ( a ) and ( b ) of the preceding paragraph may be as described above for the fusion polypeptide . preferably , the first polypeptide of the mixture contains and in a most particularly preferred manner consists of a region corresponding to the amino acids at positions 1 to 191 of the c polypeptide ( c191 ) of hcv . in the latter case , the cleavage site at position 173 / 174 must be made inoperative by mutation . this mutation may be produced as described above for the fusion polypeptide . preferably , the second polypeptide of the mixture contains and in a most particularly preferred manner consists of a region corresponding to the amino acids at positions 192 to 380 of the e1 polypeptide of hcv . for the purposes of the present invention , a dna molecule may be a simple linear dna fragment , or alternatively a plasmid or alternatively a viral vector such as a pox vector . a polypeptide , a mixture or a molecule of dna as described in the present application are of a most special interest when they are used for the manufacture of a medicament intended for the treatment or prevention of infections induced by hcv . they are in particular useful in the immunotherapy of infections induced by hcv , most particularly a dna molecule . finally , the invention relates to a method for inducing an immune response toward hcv in a mammal , according to which an immunologically effective quantity of a composition according to the invention is administered to said mammal in order to develop an immune response . the invention also relates to a method for the prevention or treatment of an infection induced by hcv , according to which a prophylactically or therapeutically effective quantity of a composition according to the invention is administered to an individual . the methods and the pharmaceutical compositions according to the invention can treat or prevent hcv infections and consequently hepatic diseases associated with such infections . they are in particular persistent chronic hepatitis , active chronic hepatitis , cirrhosis of the liver and hepatocarcinomas . a composition according to the invention may be administered by any conventional route used in the field of vaccines , in particular by the parenteral ( e . g . subcutaneous , intradermal , intramuscular , intravenous or intraperitoneal ) route . the choice of the route of administration depends on a number of parameters such as the nature of the active principle , polypeptide or dna molecule , the adjuvant combined with the polypeptide or with the dna molecule . a composition according to the invention may comprise , in addition to a polypeptide or a mixture of polypeptides useful for the purposes of the present invention , at least one other hcv antigen such as the e2 protein or alternatively such as a nonstructural protein ns1 , ns2 , ns3 , ns4 or ns5 , or a subunit , fragment , homolog , mutant or derivative of these antigens . a polypeptide , a mixture or a molecule of dna useful for the purposes of the present invention may be formulated in or with liposomes , preferably neutral or anionic liposomes , microspheres iscoms or virus - like particles ( vlps ), in order to promote the screening of the protein or of the polypeptide or to increase the immune response . persons skilled in the art have these compounds available without difficulty ; for example see liposomes : a practical approach . rrc new ed , irl press ( 1990 ). adjuvants other than liposomes may also be used . a large number are known to persons skilled in the art . such adjuvants are identified by references below : for parenteral administration , there may be mentioned in particular aluminum compounds such as aluminum hydroxide , aluminum phosphate and aluminum hydroxyphosphate . the antigen may be absorbed or precipitated on an aluminum compound according to standard methods . other adjuvants useful for parenteral administration include in particular polyphosphazene ( wo 95 / 2415 ), dc - chol ( 3 - beta -[ n -( n ′, n ′- dimethylaminomethane ) carbamoyl ] cholesterol ) ( u . s . pat . no . 5 , 283 , 185 and wo 96 / 14831 ), qs - 21 ( wo 88 / 9336 ) and ribi from immunochem ( hamilton , mont .). the administration may take place in a single dose or in a dose repeated once or several times after a certain period . the appropriate dosage varies according to various parameters , for example the individual treated ( adult or child ), the vaccinal antigen itself , the mode and frequency of administration , the presence or absence of adjuvant and , if present , the type of adjuvant and the desired effect ( e . g . protection or treatment ), as will be determined by persons skilled in the art . a composition according to the invention may be manufactured conventionally . in particular , a polypeptide , a mixture or a molecule of dna contained in the composition according to the invention is combined with a pharmaceutically acceptable diluent or carrier , e . g . water or a saline solution such as phosphate - buffered saline ( pbs ). in general , the diluent or the carrier is selected on the basis of the mode and route of administration and of standard pharmaceutical practices . pharmaceutically acceptable diluents and carriers as well as all that is necessary for their use in pharmaceutical formulations are described in remington &# 39 ; s pharmaceutical sciences , a standard reference text in this field and in usp / np . the constructs called prc / e1 , prc / ce1m1 , prc / ce1m2 and prc / ce1m1m2 ( also call prc / ce1dm ), prc / c191m1 and prc / c173 , which are used below in example 2 , have been described in : liu q et al ., j . virol . 71 : 657 ( 1997 ). the inserts used are represented in fig2 . all the constructs are produced in the vector prc which is obtained from invitrogen ( ref : v780 - 20 ). the vector prc carries the ampicillin gene and allows the expression of inserts under the control of a cmv promoter . mutations called m1 and m2 are present in the constructs prc / c191m1 , prc / ce1m1 , prc / ce1m2 and prc / ce1m1m2 . they were generated by site - directed mutagenesis performed by pcr . the mutation called m1 corresponds to the replacement of the amino acids serine 173 and phe 174 of the c protein with the amino acids methionine and leucine , respectively . the mutation called m2 corresponds to the replacement of the amino acids alanine 191 and tyrosine 192 of the ce1 protein with the amino acids valine and asparagine , respectively . the plasmids expressing the reporter genes for luciferase and for β - galactosidase were constructed by modifying the vector puc 18 ( appligene ; ref : 161131 ). the expression of the genes is under the control of the immediate - early promoter 1 ( ie1 ) of the human cmv . sequences derived from the 3 ′ region of the bovine gene for the growth hormone were moreover added in the 3 ′ of the genes in order to stabilize the mrnas . these plasmids carry more than one ampicillin gene . cho - k1 cells ( atcc ccl 61 ) were stored in α - mem medium [ nature 230 : 310 ( 1971 )], supplemented with 10 % foetal calf serum ( fcs ) ( hyclone , ref : a1115 - l ) and 20 % dimethyl sulfoxide ( dmso ) in liquid nitrogen . these cells are cultured under humid atmosphere at 37 ° c . with 5 % co 2 and 95 % air . to carry out subcultures , the medium removed and the cellular lawn is rinsed with 5 ml of phosphate buffer ( pbs ). the supernatant is then removed before addition of 1 . 5 ml of trypsine per 75 cm 2 flask ( trypsine at 0 . 025 %). after incubating for 10 min in an incubator at 37 ° c ., the reaction is stopped by addition of 10 ml of α - mem medium containing 10 % fcs . the cells are counted on a malassez cell after a one - half dilution in 0 . 02 % trypan blue . 5 × 10 5 cells are then inoculated into dishes 6 cm in diameter with complete medium . the cho cells are then cotransfected with one of the recombinant plasmids ( phcv ) described above and a reporter plasmid ( pcmv ) which contains either the β - galactosidase gene under the control of the cmv promoter ( pcmv β - gal ) or the luciferase gene under the control of the cmv promoter ( pcmv luc ). for that , 5 μg of dna ( 4 . 5 μg of plasmid phcv / 0 . 5 μg of plasmid pcmv ) are diluted in 500 μl of opti - mem medium ( gibco ), and mixed with 14 μl of lipofectamine diluted in 500 μl of the same medium . the two solutions are mixed and incubated for 20 min at room temperature in order to allow the formation of the dna - liposome complexes . the dna liposome mixture diluted with 2 ml of opti - mem is then added to the cells after removing the culture medium and rinsing in pbs . after incubating for 5 hours , the medium is again changed and 48 h after the transfection , it is then possible to test for the transient expression of the recombinant genes . the transfected cells are lysed with the aid of the reagent “ luciferase cell culture lysis reagent ” ( promega , luciferase assay system ). 100 μl of substrate are added to 100 μl of cell supernatant , directly by the bioluminometer injector ( lumat lb / 9501 / 16 from berthold ) which measures the quantity of light emitted ( relative light units ) for 10 seconds . the quantity of light emitted is then converted to nanograms of proteins per ml of cell lysate , by comparing with a standard curve established with the aid of purified luciferase . the results , which are presented in fig2 show that a point mutation at the amino acid 191 in the construct ce1m2 abolishes the transactivating effect . a point mutation at the amino acid 173 ( in the construct ce1m1 ) abolishes the transactivating effect only in the case where c is fused with e1 . a double mutation at the amino acids 173 and 191 abolishes the transactivating effect . the teachings of all the articles , patents , and patent applications cited herein are incorporated by reference in their entirety . while this invention has been particularly shown and described with references to preferred embodiments thereof , it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims .
the invention concerns a pharmaceutical composition for treating or preventing c hepatitis , induced infections , which in a preferred embodiment , comprises a main active principle , a fusion polypeptide , including the hcv capsid polypeptide and polypeptide coat and in which at least one cleavage site 173 / 174 and 191 / 192 has been made inoperative by mutation ; an equimolar mixture of the c191 polypeptide of which the cleavage site 173 / 174 has been made inoperative and of the e1 polypeptide ; or a dna molecule coding for this fusion polypeptide . products to are characterized in that the c191 element is incapable of regulating the functioning of the genes , in particular of causing them to interact . such a composition can also include any form equivalent to the products described above .