Title:  Vaccine composition for preventing or treating hepatitis C

United States Patent:  6,538,123

Issued:  March 25, 2003

Inventors:  Barban; Veronique (Lyons, FR)

Assignee:  Aventis Pasteur (Lyons, FR)

Appl. No.:  916359

Filed:  July 26, 2001

The invention concerns a pharmaceutical composition for treating or preventing C hepatitis (HCV), induced infections, which in a preferred embodiment, comprises a main active principle, (i) a fusion polypeptide, including the HCV capsid polypeptide (C191) and polypeptide coat (E1) and in which at least one cleavage site 173/174 and 191/192 has been made inoperative by mutation; (ii) an equimolar mixture of the C191 polypeptide of which the cleavage site 173/174 has been made inoperative and of the E1 polypeptide (mixture equivalent to the fusion polypeptide); or (iii) a DNA molecule coding for this fusion polypeptide. Products (i) to (iii) 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.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a pharmaceutical composition intended for the treatment or prophylaxis of infections induced by the hepatitis C virus (HCV).

The hepatitis C virus (HCV) is a positive single-stranded RNA virus. On the basis of structural resemblance, HCV has been linked to the flavivirus and pestivirus families.

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 FIG. 1, 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 hereafter, 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 test, 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 a 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 acitvity 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.

Accordingly, the subject of the invention is a pharmaceutical composition comprising:

(i) A polypeptide which contains:

(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;

(ii) A mixture (preferably) in substantially equimolar quantity,

(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 a pharmaceutically acceptable carrier or diluent.

(iv) 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.

(v) "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-translation modifications which may take place such as, for example, a lipidation. It is also possible to use the term protein interchangeable.

(vi) "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. Hepatoloty, 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 an 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 thr 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 positions 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. h 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 capabiity 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 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 inone 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 of 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 makine 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's Pharmaceutical Sciences, a standard reference text in this field and in USP/NP.

Claim 1 of 21 Claims

What is claimed is:

1. A nucleic acid molecule that encodes a fusion polypeptide that comprises:

(a) a first region consisting essentially of the C polypeptide of a hepatitis C virus (HCV) or a portion thereof that comprises a polypeptide region responsible for gene regulatory activity; and

(b) a second region consisting essentially of the E1 envelope polypeptide of the virus or a portion thereof that comprises a site for cytoplasmic anchorage of the E1 polypeptide;

wherein the first region is fused by a peptide bond to the second region, and the fusion polypeptide is not cleaved by a mammalian protease.
 

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