The current invention relates to vectors and methods for efficient expression of HCV envelope proteins in eukaryotic cells. More particularly said vectors comprise the coding sequence for an avian lysozyme signal peptide or a functional equivalent thereof joined to a HCV envelope protein or a part thereof. Said avian lysozyme signal peptide is efficiently removed when the protein comprising said avian lysozyme signal peptide joined to a HCV envelope protein or a part thereof is expressed in a eukaryotic cell. Suitable eukaryotic cells include yeast cells such as Saccharomyces or Hansenula cells.

Description of the Invention

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to recombinant nucleic acids comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to an HCV envelope protein or a part thereof. More specifically said protein is characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.e-(A4- ).sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2. The recombinant nucleic acids according to the invention may further comprise regulatory elements allowing expression of said protein in a eukaryotic host cell.

Another aspect of the invention relates to a recombinant nucleic acid according to the invention which are comprised in a vector. Said vector may be an expression vector and/or an autonomously replicating vector or an integrative vector.

A further aspect of the invention relates to a host cell harboring a recombinant nucleic acid according to the invention or a vector according to the invention. More particularly, said host cell is capable of expressing the protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to an HCV envelope protein or a part thereof. More specifically, said protein is characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2)- .sub.e-(A4).sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or a part thereof, a, b, c, d, e and fare 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

The host cell according to the invention may be capable of removing the avian lysozyme leader peptide with high efficiency and fidelity and may be capable of processing the processing sites PS1 and/or PS2 in said protein translocated to the endoplasmic reticulum. Said host cell may further be capable of N-glycosylating said protein translocated to the endoplasmic reticulum or said protein translocated to the endoplasmic reticulum and processed at said sites PS1 and/or PS2. The host cell may be an eukaryotic cell such as a yeast cell.

A next aspect of the invention relates to a method for producing an HCV envelope protein or part thereof in a host cell, said method comprising transforming said host cell with a recombinant nucleic acid according to the invention or with a vector according to the invention, and wherein said host cell is capable of expressing a protein comprising the avian lysozyme leader peptide or a functional equivalent thereof joined to an HCV envelope protein or a part thereof. More particularly, said protein is characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.e-(A4- ).sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2. The method according to the invention may further comprise cultivation of said host cells in a suitable medium to obtain expression of said protein, isolation of the expressed protein from a culture of said host cells, or from said host cells. Said isolation may include one or more of (i) lysis of said host cells in the presence of a chaotropic agent, (ii) chemical modification of the cysteine thiol-groups in the isolated proteins wherein said chemical modification may be reversible or irreversible and (iii) heparin affinity chromatography.

DETAILED DESCRIPTION OF THE INVENTION

In work leading to the present invention, it was observed that expression of HCV envelope proteins as .alpha.MF-HCVENV (.alpha. mating factor-HCV envelope protein) pre-proproteins in Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha was possible but that the extent of removal of the pre-pro- or pre-sequences was unacceptably low and that removal of pre-pro- or pre-sequences is very often not occurring with high fidelity. As a result, many different HCV envelope proteins are produced in these yeasts which do not have a natural amino-terminus (see Example 15). The majority of the HCV envelope proteins expressed in these yeast species were glycosylated (see Examples 6, 10, 13 and 25). More specifically the S. cerevisiae (glycosylation deficient mutant)- and H. polymorpha-expressed HCV envelope proteins were glycosylated in a manner resembling core-glycosylation. The HCV envelope proteins expressed in Pichia pastoris were hyperglycosylated despite earlier reports that proteins expressed in this yeast are normally not hyperglycosylated (Gellissen, G. 2000, Sugrue, R. J. et al. 1997).

Constructs were made for expression of the HCV envelope proteins as pre-pro- or pre-proteins wherein these pre-pro- or pre-sequences were either the Carcinus maenas hyperglycemic hormone leader sequence (pre; CHH), the S. occidentalis amylase leader sequence (pre; Amyl), the S. occidentalis glucoamylase Gam1 leader sequence (pre; Gam1), the fungal phytase leader sequence (pre; Phy5), the Pichia pastoris acid phosphatase leader sequence (pre; phol), the yeast aspartic protease 3 signal peptide (pre; YAP3), the mouse salivary amylase signal peptide (pre) and the chicken lysozyme leader sequence (pre; CL). Only for one of these pre-pro-HCVENV or pre-HCVENV proteins, removal of the pre-pro- or pre-sequence with high frequency and high fidelity was observed. This was surprisingly found for the chicken lysozyme leader sequence (CL) and was confirmed both in S. cerevisiae and H. polymorpha (see Example 16). The CL signal peptide is thus performing very well for expression of glycosylated HCV envelope proteins in eukaryotic cells. This unexpected finding is reflected in the different aspects and embodiments of the present invention as presented below.

A first aspect of the current invention relates to a recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to an HCV envelope protein or a part thereof.

In one embodiment thereto, the recombinant nucleic acid comprising nucleotide sequence encodes characterized by the structure CL-[(A1).sub.a-(PS1).sub.b (A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.e-(A4).sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or a part thereof, a, b, c, d, e and fare 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

In a further embodiment, the recombinant nucleic acids according to the invention further comprise regulatory elements allowing expression in a eukaryotic host cell of said protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to an HCV envelope protein or a part thereof, or of said protein characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2)- .sub.e-(A4).sub.f].

The HCV antigens of the present invention comprise conformational epitopes from the E1 and/or E2 (envelope) domains of HCV. The E1 domain, which is believed to correspond to the viral envelope protein, is currently estimated to span amino acids 192-383 of the HCV polyprotein (Hijikata, M. et al. 1991). Upon expression in a mammalian system (glycosylated), it is believed to have an approximate molecular weight of 35 kDa as determined via SDS-PAGE. The E2 protein, previously called NS1, is believed to span amino acids 384-809 or 384-746 (Grakoui, A. et al. 1993) of the HCV polyprotein and also to be an envelope protein. Upon expression in a vaccinia system (glycosylated), it is believed to have an apparent gel molecular weight of about 72 kDa. It is understood that these protein endpoints are approximations (e.g. the carboxy terminal end of E2 could lie somewhere in the 730-820 amino acid region, e.g. ending at amino acid 730, 735, 740, 742, 744, 745, preferably 746, 747, 748, 750, 760, 770, 780, 790, 800, 809, 810, 820). The E2 protein may also be expressed together with E1, and/or core (aa 1-191), and/or P7 (aa 747-809), and/or NS2 (aa 810-1026), and/or NS3 (aa 1027-1657), and/or NS4A (aa 1658-1711) and/or NS4B (aa 1712-1972) and/or NS5A (aa 1973-2420), and/or NS5B (aa 2421-3011), and/or any part of any of these HCV proteins different from E2. Likewise, the E1 protein may also be expressed together with the E2, and/or core (aa 1-191), and/or P7 (aa 747-809), and/or NS2 (aa 810-1026), and/or NS3 (aa 1027-1657), and/or NS4A (aa 1658-1711) and/or NS4B (aa 1712-1972), and/or NS5A (aa 1973-2420), and/or NS5B (aa 2421-3011), and/or any part of any of these HCV proteins different from E1. Expression together with these other HCV proteins may be important for obtaining the correct protein folding.

The term "E1" as used herein also includes analogs and truncated forms that are immunologically cross-reactive with natural E1, and includes E1 proteins of genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 or any other newly identified HCV type or subtype. The term `E2` as used herein also includes analogs and truncated forms that are immunologically cross-reactive with natural E2, and includes E2 proteins of genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 or any other newly identified HCV type or subtype. For example, insertions of multiple codons between codon 383 and 384, as well as deletions of amino acids 384-387 have been reported (Kato, N. et al. 1992). It is thus also understood that the isolates used in the examples section of the present invention were not intended to limit the scope of the invention and that any HCV isolate from type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 or any other new genotype of HCV is a suitable source of E1 and/or E2 sequence for the practice of the present invention. Similarly, as described above, the HCV proteins that are co-expressed with the HCV envelope proteins of the present invention, can be derived from any HCV type, thus also from the same type as the HCV envelope proteins of the present invention.

"E1/E2" as used herein refers to an oligomeric form of envelope proteins containing at least one E1 component and at least one E2 component.

The term "specific oligomeric" E1 and/or E2 and/or E1/E2 envelope proteins refers to all possible oligomeric forms of recombinantly expressed E1 and/or E2 envelope proteins which are not aggregates. E1 and/or E2 specific oligomeric envelope proteins are also referred to as homo-oligomeric E1 or E2 envelope proteins (see below). The term `single or specific oligomeric` E1 and/or E2 and/or E1/E2 envelope proteins refers to single monomeric E1 or E2 proteins (single in the strict sense of the word) as well as specific oligomeric E1 and/or E2 and/or E1/E2 recombinantly expressed proteins. These single or specific oligomeric envelope proteins according to the present invention can be further defined by the following formula (E1).sub.x(E2).sub.y wherein x can be a number between 0 and 100, and y can be a number between 0 and 100, provided that x and y are not both 0. With x=1 and y=0 said envelope proteins include monomeric E1.

The term "homo-oligomer" as used herein refers to a complex of E1 or E2 containing more than one E1 or E2 monomer, e.g. E1/E1 dimers, E1/E1/E1 trimers or E1/E1/E1/E1 tetramers and E2/E2 dimers, E2/E2/E2 trimers or E2/E2/E2/E2 tetramers, E1 pentamers and hexamers, E2 pentamers and hexamers or any higher-order homo-oligomers of E1 or E2 are all `homo-oligomers` within the scope of this definition. The oligomers may contain one, two, or several different monomers of E1 or E2 obtained from different types or subtypes of hepatitis C virus including for example those described by Maertens et al. in WO 94/25601 and WO 96/13590 both by the present applicants. Such mixed oligomers are still homo-oligomers within the scope of this invention, and may allow more universal diagnosis, prophylaxis or treatment of HCV.

The E1 and E2 antigens used in the present invention may be full-length viral proteins, substantially full-length versions thereof, or functional fragments thereof (e.g. fragments comprising at least one epitope and/or glycosylation site). Furthermore, the HCV antigens of the present invention can also include other sequences that do not block or prevent the formation of the conformational epitope of interest. The presence or absence of a conformational epitope can be readily determined through screening the antigen of interest with an antibody (polyclonal serum or monoclonal to the conformational epitope) and comparing its reactivity to that of a denatured version of the antigen which retains only linear epitopes (if any). In such screening using polyclonal antibodies, it may be advantageous to adsorb the polyclonal serum first with the denatured antigen and see if it retains antibodies to the antigen of interest.

The HCV proteins of the present invention may be glycosylated. Glycosylated proteins intend proteins that contain one or more carbohydrate groups, in particular sugar groups. In general, all eukaryotic cells are able to glycosylate proteins. After alignment of the different envelope protein sequences of HCV genotypes, it may be inferred that not all 6 glycosylation sites on the HCV E1 protein are required for proper folding and reactivity. For instance, HCV subtype 1b E1 protein contains 6 glycosylation sites, but some of these glycosylation sites are absent in certain other (sub)types. The fourth carbohydrate motif (on Asn250), present in types 1b, 6a, 7, 8, and 9, is absent in all other types know today. This sugar-addition motif may be mutated to yield a type 1b E1 protein with improved reactivity. Also, the type 2b sequences show an extra glycosylation site in the V5 region (on Asn299). The isolate S83, belonging to genotype 2c, even lacks the first carbohydrate motif in the VI region (on Asn), while it is present on all other isolates (Stuyver, L. et al. 1994). However, even among the completely conserved sugar-addition motifs, the presence of the carbohydrate may not be required for folding, but may have a role in evasion of immune surveillance. Thus, the identification of the role of glycosylation can be further tested by mutagenesis of the glycosylation motifs. Mutagenesis of a glycosylation motif (NXS or NXT sequences) can be achieved by either mutating the codons for N, S, or T, in such a way that these codons encode amino acids different from N in the case of N, and/or amino acids different from S or T in the case of S and in the case of T. Alternatively, the X position may be mutated into P, since it is known that NPS or NPT are not frequently modified with carbohydrates. After establishing which carbohydrate-addition motifs are required for folding and/or reactivity and which are not, combinations of such mutations may be made. Such experiments have been described extensively by Maertens et al. in WO 96/04385 (Example 8), which is included herein specifically by reference. The term glycosylation as used in the present invention refers to N-glycsoylation unless otherwise specified.

In particular, the present invention relates to HCV envelope proteins, or parts thereof that are core-glycosylated. In this respect, the term "core-glycosylation" refers to a structure "similar" to the structure as depicted in the boxed structure in FIG. 3 (see Original Patent) of Herscovics and Orlean (Herscovics, A. and Orlean, P. 1993). Thus, the carbohydrate structure referred to contains 10 or 11 mono-saccharides. Notably, said disclosure is herein incorporated by reference. The term "similar" intends that not more than about 4 additional mono-saccharides have been added to the structure or that not more than about 3 mono-saccharides have been removed from the structure. Consequently, a carbohydrate structure consists most preferentially of 10 mono-saccharides, but minimally of 7, and more preferentially of 8 or 9 mono-sacchariden, and maximally of 15 mono-saccharides, and more preferentially of 14, 13, 12, or 11 mono-saccharides. The mono-saccharides connoted are preferentially glucose, mannose or N-acetyl glucosamine.

Another aspect of the present invention covers vectors comprising a polynucleic acid, or a part thereof, of the invention. Such vectors comprise universal cloning vectors such as the pUC-series or pEMBL-series vectors and furthermore include other cloning vectors such as cloning vectors requiring a DNA topoisomerase reaction for cloning, TA-cloning vectors and recombination-based cloning vectors such as those used in the Gateway system (InVitrogen). Vectors comprise plasmids, phagemids, cosmids, bacmids (baculovirus vectors) or may be viral or retroviral vectors. A vector can merely function as a cloning tool and/or -vehicle or may additionally comprise regulatory sequences such as promoters, enhancers and terminators or polyadenylation signals. Said regulatory sequences may enable expression of the information contained within the DNA fragment of interest cloned into a vector comprising said regulatory sequences. Expression may be the production of RNA molecules or mRNA molecules and, optionally, the production of protein molecules thereof. Expression may be the production of an RNA molecule by means of a viral polymerase promoter (e.g. SP6, T7 or T3 promoter) introduced to the 5'- or 3'- end of the DNA of interest. Expression may furthermore be transient expression or stable expression or, alternatively, controllable expression. Controllable expression comprises inducible expression, e.g. using a tetracyclin-regulatable promoter, a stress-inducible (e.g. human hsp70 gene promoter), a methallothionine promoter, a glucocorticoid promoter or a progesterone promoter. Expression vectors are known in the art that mediate expression in bacteria (e.g. Escherichia coli, Streptomyces species), insect cells (Spodoptera frugiperda cells, Sf9 cells), plant cells (e.g. potato virus X-based expression vectors, see e.g. Vance et al. 1998 in WO98/44097) and mammalian cells (e.g. CHO or COS cells, Vero cells, cells from the HeLa cell line).

This aspect of the invention thus specifically relates to a vector comprising the recombinant nucleic acids according to the invention encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to an HCV envelope protein or a part thereof, or a protein characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.e-(A4- ).sub.f].

Embodied in the present invention are also said vectors further comprising regulatory sequences allowing expression of said protein.

In a specific embodiment, said vector according to the invention is an expression vector.

In another specific embodiment, said vector according to the invention is an autonomously replicating vector or an integrative vector.

In yet another specific embodiment, said vector according to the invention is chosen from any of SEQ ID NOs: 20, 21, 32, 35, 36, 39, 40.

Suitable vectors or expression vectors of the invention are yeast vectors. A yeast vector may comprise a DNA sequence enabling the vector to replicate autonomously. Examples of such sequences are the yeast plasmid 2.mu. replication genes REP 1-3 and origin of replication. Other vectors are integrating partially or completely in the yeast genome. Such integrative vectors are either targeted to specific genomic loci or integrate randomly. In P. pastoris, foreign DNA is targeted to the AOX1 and the HIS4 genes (Cregg, J. M. 1999), in P. methanolica to the AUG1 gene (Raymond, C. K. 1999). In most recombinant H. polymorpha strains, foreign DNA can be randomly integrated using HARS-sequence-harboring circular plasmids for transformation (Hollenberg, C. P. and Gellissen, G. 1997). Targeted integration can be achieved by homologous recombination using the MOX/TRP3 locus for disruption/integration (Agaphonov, M. O. et al. 1995, Sohn, J. H. et al. 1999), the LEU2 gene (Agaphonov, M. O. et al. 1999) or the rDNA cluster (Cox, H. et al. 2000). Transformations in H. polymorpha typically result in a variety of individual, mitotically stable strains containing single to multiple copies of the expression cassette in a head-to-tail arrangement. Strains with up to 100 copies have been identified (Hollenberg, C. P. and Gellissen, G. 1997). Random multiple-copy integration can be forced in the uracil-auxotroph H. polymorpha strain RB11 by a sequence of passages under selective conditions if a H. polymorpha or S. cerevisiae-derived URA3 gene is present. A HARS sequence can be excluded (Gatzke, R. et al. 1995) or can be present (Hollenberg, C. P. and Gellissen, G. 1997). This passaging furthermore leads to mitotically stable strains. The vector may also comprise a selectable marker, e.g. the Schizosaccharomyces pombe TPI gene as described by Russell (Russell, P. R. 1985), or the yeast URA3 gene. Other marker genes so far used for transformation of Saccharomyces, for example TRP5, LEU2, ADE1, ADE2, HIS3, HIS4, LYS2, may be obtained from e.g. Hansenula, Pichia or Schwanniomyces.

"Regulatory elements (or sequences) allowing expression of a protein in a eukaryotic host" are to be understood to comprise at least a genetic element displaying promoter activity and a genetic element displaying terminator activity whereby said regulatory elements are operably linked to the open reading frame encoding the protein to be expressed.

The term "promoter" is a nucleotide sequence which is comprised of consensus sequences which allow the binding of RNA polymerase to the DNA template in a manner such that mRNA production initiates at the normal transcription initiation site for the adjacent structural gene.

The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

An "open reading frame" (ORF) is a region of a polynucleotide sequence which encodes a polypeptide and does not contain stop codons; this region may represent a portion of a coding sequence or a total coding sequence.

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

Many regulatory elements are known in the art. Examples of suitable yeast promoters are the Saccharomyces cerevisiae MF.alpha.1, TPI, ADH I, ADH II or PGK promoters, or corresponding promoters from other yeast species, e.g. Schizosaccharomyces pombe. Examples of suitable promoters are described by, for instance, (Alber, T. and Kawasaki, G. 1982, Ammerer, G. 1983, Ballou, L. et al. 1991, Hitzeman, R. A. et al. 1980, Kawasaki, G. and Fraenkel, D. G. 1982, Russell, D. W. et al. 1983, Russell, P. R. 1983, Russell, P. R. and Hall, B. D. 1983). A suitable yeast terminator is, e.g. the TPI terminator (Alber, T. and Kawasaki, G. 1982), or the yeast CYC1 terminator. For methylotrophic or facultative methylotrophic yeast species, the strong and regulatable promoters of the enzymes involved in the methanol utilization pathway are good candidate promoters and include the promoters of the alcohol oxidase genes (AOX1 of Pichia pastoris, AUG1 of P. methanolica, AOD1 of Candida boidinii, and MOX of Hansenula polymorpha), the formaldehyde dehydrogenase promoter (FLD1 of P. pastoris), the dihydroxyacetone synthase promoter (DAS1 of C. boidinii) and the formate dehydrogenase promoter (FMD of H. polymorpha). Other promoters include the GAP1 promoter of P. pastoris or H. polymorpha and the PMA1 and TPS1 promoter of H. polymorpha ((Gellissen, G. 2000), and references cited therein). The terminator element derived from any of these genes are examples of suitable terminator elements, more specifically suitable terminator elements include the AOD1, AOX1 and MOX terminator elements.

A further aspect of the current invention covers host cells comprising a recombinant nucleic acid or a vector according to the invention.

In a specific embodiment thereto, said host cells comprising a recombinant nucleic acid or a vector according to the invention are capable of expressing the protein according to the invention comprising the avian leader lysozyme leader peptide or a functional variant thereof joined to an HCV envelope protein or a part thereof.

In an alternative embodiment, said host cells are capable of expressing the protein characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.e-(A4- ).sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

In a further specific embodiment thereto, said host cells comprising a recombinant nucleic acid or a vector according to the invention are capable of translocating the protein comprising the avian lysozyme leader peptide or a functional equivalent thereof joined to an HCV envelope protein or a part thereof to the endoplasmic reticulum upon removal of the avian lysozyme leader peptide.

In a further specific embodiment thereto, said host cells comprising a recombinant nucleic acid or a vector according to the invention are capable of translocating the protein [(A1).sub.x-(PS1).sub.y-(A2).sub.z]-HCVENV-[(A3).sub.x-(PS2).sub.y-(A4).s- ub.z] to the endoplasmic reticulum upon removal of the CL peptide wherein said protein and said CL peptide are derived from the protein characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.e-(A4- ).sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

Also embodied are host cells comprising a recombinant nucleic acid or a vector according to the invention which are capable of processing the processing sites PS1 and/or PS2 in said protein translocated to the endoplasmic reticulum.

Also embodied are host cells comprising a recombinant nucleic acid or a vector according to the invention which are capable of N-glycosylating said protein translocated to the endoplasmic reticulum.

Also embodied are host cells comprising a recombinant nucleic acid or a vector according to the invention which are capable of N-glycosylating said protein translocated to the endoplasmic reticulum and processed at said sites PS1 and/or PS2.

More specifically, the host cells comprising a recombinant nucleic acid or a vector according to the invention are eukaryotic cells and, more particularly, yeast cells such as cells of strains of Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces kluyveri, or Saccharomyces uvarum, Schizosaccharomyces, such as Schizosaccharomyces pombe, Kluyveromyces, such as Kluyveromyces lactis, Yarrowia, such as Yarrowia lipolytica, Hansenula, such as Hansenula polymorpha, Pichia, such as Pichia pastoris, Aspergillus species, Neurospora, such as Neurospora crassa, or Schwanniomyces, such as Schwanniomyces occidentalis, or mutant cells derived from any thereof.

The term "eukaryotic cells" includes lower eukaryotic cells as well as higher eukaryotic cells. Lower eukaryotic cells are cells such as yeast cells, fungal cells and the like. Particularly suited host cells in the context of the present invention are yeast cells or mutant cells derived from any thereof as described above. Mutant cells include yeast glycosylation minus strains, such as Saccharomyces glycosylation minus strains as used in the present invention. Glycosylation minus strains are defined as strains carrying a mutation, in which the nature of the mutation is not necessarily known, but resulting in a glycosylation of glycoproteins comparable to the core-glycosylation In particular, it is contemplated that Saccharomyces glycosylation minus strains carry a mutation resulting in a significant shift in mobility on PAGE of the invertase protein. Invertase is a protein which is normally present in Saccharomyces in a hyperglycosylated form only (Ballou, L. et al. 1991). Glycosylation minus strains include mnn2, and/or och1 and/or mnn9 deficient strains. The mutant host cells of the invention do not include cells which, due to the mutation, have lost their capability to remove the avian lysozyme leader peptide from a protein comprising said leader peptide joined to a protein of interest.

Higher eukaryotic cells include host cells derived from higher animals, such as mammals, reptiles, insects, and the like. Presently preferred higher eukaryote host cells are derived from Chinese hamster (e.g. CHO), monkey (e.g. COS and Vero cells), baby hamster kidney (BHK), pig kidney (PK15), rabbit kidney 13 cells (RK13), the human osteosarcoma cell line 143 B, the human cell line HeLa and human hepatoma cell lines like Hep G2, and insect cell lines (e.g. Spodoptera frugiperda). The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively the host cells may also be transgenic animals or transgenic plants.

Introduction of a vector, or an expression vector, into a host cell may be effectuated by any available transformation or transfection technique applicable to said host cell as known in the art. Such transformation or transfection techniques comprise heat-shock mediated transformation (e.g. of E. coli), conjugative DNA transfer, electroporation, PEG-mediated DNA uptake, liposome-mediated DNA uptake, lipofection, calcium-phosphate DNA coprecipitation, DEAE-dextran mediated transfection, direct introduction by e.g. microinjection or particle bombardment, or introduction by means of a virus, virion or viral particle.

Yet another aspect of the invention relates to methods for producing a HCV envelope protein or part thereof in a host cell, said method comprising transforming said host cell with the recombinant nucleic acid according to the invention or with the vector according to the invention, and wherein said host cell is capable of expressing a protein comprising the avian lysozyme leader peptide or a functional equivalent thereof joined to a HCV envelope protein or a part thereof.

In a specific embodiment thereto, said method for producing a HCV envelope protein or part thereof in a host cell is comprising the step of transforming said host cell with the recombinant nucleic acid according to the invention or with the vector according to the invention, and wherein said host cell is capable of expressing the protein characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.e-(A4- ).sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

In another specific embodiment thereto, the host cell in said method is capable of translocating the protein CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.e-(A4- ).sub.f] to the endoplasmic reticulum upon removal of the CL peptide wherein said protein and said CL peptide are derived from the protein characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.e-(A4- ).sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

Also embodied is the method for producing a HCV envelope protein or part thereof wherein said host cell is capable of N-glycosylating said protein translocated to the endoplasmic reticulum.

Further embodied is the method for producing a HCV envelope protein or part thereof wherein said host cell is capable of N-glycosylating said protein translocated to the endoplasmic reticulum and processed at said sites PS1 and/or PS2.

More specifically, the host cell in any of said methods for producing a HCV envelope protein or part thereof is an eukaryotic cell and, more particularly, a yeast cell such as a cell of strains of Saccharomyces, such as Saccharomyces cerevisiae, Saccharomyces kluyveri, or Saccharomyces uvarum, Schizosaccharomyces, such as Schizosaccharomyces pombe, Kluyveromyces, such as Kluyveromyces lactis, Yarrowia, such as Yarrowia lipolytica, Hansenula, such as Hansenula polymorpha, Pichia, such as Pichia pastoris, Aspergillus species, Neurospora, such as Neurospora crassa, or Schwanniomyces, such as Schwanniomyces occidentalis, or mutant cells derived from any thereof.

Any of the methods according to the invention for producing a HCV envelope protein or part thereof may further comprise cultivation of the host cells comprising a recombinant nucleic acid or a vector according to the invention in a suitable medium to obtain expression of said protein.

A further embodiment thereto comprises isolation of the produced HCV envelope protein or part thereof from a culture of said host cells, or, alternatively, from said host cells. Said isolation step may include one or more of (i) lysis of said host cells in the presence of chaotropic agent, (ii) chemical and/or enzymatic modification of the cysteine thiol-groups in the isolated proteins wherein said modification may be reversible or irreversible, and producing a HCV envelope protein or part thereof (iii) heparin affinity chromatography.

Exemplary "chaotropic agents" are guanidinium chloride and urea. In general, a chaotropic agent is a chemical that can disrupt the hydrogen bonding structure of water. In concentrated solutions they can denature proteins because they reduce the hydrophobic effect

In the HCV envelope proteins or parts thereof as described herein comprising at least one cysteine residue, but preferably 2 or more cysteine residues, the cysteine thiol-groups can be irreversibly protected by chemical or enzymatic means. In particular, "irreversible protection" or "irreversible blocking" by chemical means refers to alkylation, preferably alkylation of the HCV envelope proteins by means of alkylating agents, such as, for example, active halogens, ethylenimine or N-(iodoethyl)trifluoro-acetamide. In this respect, it is to be understood that alkylation of cysteine thiol-groups refers to the replacement of the thiol-hydrogen by (CH.sub.2).sub.nR, in which n is 0, 1, 2, 3 or 4 and R=H, COOH, NH.sub.2, CONH.sub.2, phenyl, or any derivative thereof. Alkylation can be performed by any method known in the art, such as, for example, active halogens X(CH.sub.2).sub.nR in which X is a halogen such as I, Br, Cl or F. Examples of active halogens are methyliodide, iodoacetic acid, iodoacetamide, and 2-bromoethylamine. Other methods of alkylation include the use of NEM (N-ethylmaleimide) or Biotin-NEM, a mixture thereof, or ethylenimine or N-(iodoethyl)trifluoroacetamide both resulting in substitution of --H by --CH.sub.2--CH.sub.2--NH.sub.2 (Hermanson, G. T. 1996). The term "alkylating agents" as used herein refers to compounds which are able to perform alkylation as described herein. Such alkylations finally result in a modified cysteine, which can mimic other amino acids. Alkylation by an ethylenimine results in a structure resembling lysine, in such a way that new cleavage sites for trypsine are introduced (Hermanson, G. T. 1996). Similarly, the usage of methyliodide results in an amino acid resembling methionine, while the usage of iodoacetate and iodoacetamide results in amino acids resembling glutamic acid and glutamine, respectively. In analogy, these amino acids are preferably used in direct mutation of cysteine. Therefore, the present invention pertains to HCV envelope proteins as described herein, wherein at least one cysteine residue of the HCV envelope protein as described herein is mutated to a natural amino acid, preferentially to methionine, glutamic acid, glutamine or lysine. The term "mutated" refers to site-directed mutagenesis of nucleic acids encoding these amino acids, ie to the well know methods in the art, such as, for example, site-directed mutagenesis by means of PCR or via oligonucleotide-mediated mutagenesis as described in (Sambrook, J. et al. 1989). It should be understood that for the Examples section of the present invention, alkylation refers to the use of iodo-acetamide as an alkylating agent unless otherwise specified.

It is further understood that in the purification procedure, the cysteine thiol-groups of the HCV proteins or the parts thereof of the present invention can be reversibly protected. The purpose of reversible protection is to stabilize the HCV protein or part thereof. Especially, after reversible protection the sulfur-containing functional group (eg thiols and disulfides) is retained in a non-reactive condition. The sulfur-containing functional group is thus unable to react with other compounds, e.g. have lost their tendency of forming or exchanging disulfide bonds, such as, for example R.sub.1--SH+R.sub.2--SH--X-->R.sub.1--S--S--R.sub.2; R.sub.1--S--S--R.sub.2+R.sub.3--SH--X-->R.sub.1--S--S--R.sub.3+R.sub.2- --SH; R.sub.1--S--S--R.sub.2+R.sub.3--S--S--R.sub.4--X-->R.sub.1--S--S-- -R.sub.3+R.sub.2--S--S--R.sub.4.

The described reactions between thiols and/or disulphide residues are not limited to intermolecular processes, but may also occur intramolecularly.

The term "reversible protection" or "reversible blocking" as used herein contemplates covalently binding of modification agents to the cysteine thiol-groups, as well as manipulating the environment of the HCV protein such, that the redox state of the cysteine thiol-groups remains unaffected throughout subsequent steps of the purification procedure (shielding). Reversible protection of the cysteine thiol-groups can be carried out chemically or enzymatically.

The term "reversible protection by enzymatical means" as used herein contemplates reversible protection mediated by enzymes, such as for example acyl-transferases, e.g. acyl-tranferases that are involved in catalysing thio-esterification, such as palmitoyl acyltransferase (see below).

The term "reversible protection by chemical means" as used herein contemplates reversible protection: 1. by modification agents that reversibly modify cysteinyls such as for example by sulphonation and thio-esterification;

Sulphonation is a reaction where thiol or cysteines involved in disulfide bridges are modified to S-sulfonate: RSH.fwdarw.RS--SO.sub.3.sup.- (Darbre, A. 1986) or RS--SR.fwdarw.2 RS--SO.sub.3.sup.- (sulfitolysis; (Kumar, N. et al. 1986)). Reagents for sulfonation are e.g. Na.sub.2SO.sub.3, or sodium tetrathionate. The latter reagents for sulfonation are used in a concentration of 10-200 mM, and more preferentially in a concentration of 50-200 mM. Optionally sulfonation can be performed in the presence of a catalysator such as, for example Cu.sup.2+ (100 .mu.M-1 mM) or cysteine (1-10 mM).

The reaction can be performed under protein denaturing as well as native conditions (Kumar, N. et al. 1985, Kumar, N. et al. 1986).

Thioester bond formation, or thio-esterification is characterised by: RSH+R'COX.fwdarw.RS--COR' in which X is preferentially a halogenide in the compound R'CO--X. 2. by modification agents that reversibly modify the cysteinyls of the present invention such as, for example, by heavy metals, in particular Zn.sup.2+, Cd.sup.2+, mono-, dithio- and disulfide-compounds (e.g. aryl- and alkylmethanethiosulfonate, dithiopyridine, dithiomorpholine, dihydrolipoamide, Ellmann reagent, aldrothiol.TM. (Aldrich) (Rein, A. et al. 1996), dithiocarbamates), or thiolation agents (e.g. gluthathion, N-Acetyl cysteine, cysteineamine). Dithiocarbamate comprise a broad class of molecules possessing an R.sub.1R.sub.2NC(S)SR.sub.3 functional group, which gives them the ability to react with sulphydryl groups. Thiol containing compounds are preferentially used in a concentration of 0.1-50 mM, more preferentially in a concentration of 1-50 mM, and even more preferentially in a concentration of 10-50 mM; 3. by the presence of modification agents that preserve the thiol status (stabilise), in particular antioxidantia, such as for example DTT, dihydroascorbate, vitamins and derivates, mannitol, amino acids, peptides and derivates (e.g. histidine, ergothioneine, carnosine, methionine), gallates, hydroxyanisole, hydoxytoluene, hydroquinon, hydroxymethylphenol and their derivates in concentration range of 10 .mu.M-10 mM, more preferentially in a concentration of 1-10 mM; 4. by thiol stabilising conditions such as, for example, (i) cofactors as metal ions (Zn.sup.2+, Mg.sup.2+), ATP, (ii) pH control (e.g. for proteins in most cases pH.about.5 or pH is preferentially thiol pK.sub.a-2; e.g. for peptides purified by Reversed Phase Chromatography at pH.about.2).

Combinations of reversible protection as described in (1), (2), (3) and (4) may result in similarly pure and refolded HCV proteins. In effect, combination compounds can be used, such as, for example Z103 (Zn camosine), preferentially in a concentration of 1-10 mM. It should be clear that reversible protection also refers to, besides the modification groups or shielding described above, any cysteinyl protection method which may be reversed enzymatically or chemically, without disrupting the peptide backbone. In this respect, the present invention specifically refers to peptides prepared by classical chemical synthesis (see above), in which, for example, thioester bounds are cleaved by thioesterase, basic buffer conditions (Beekman, N. J. et al. 1997) or by hydroxylamine treatment (Vingerhoeds, M. H. et al. 1996).

Thiol containing HCV proteins can be purified, for example, on affinity chromatography resins which contain (1) a cleavable connector arm containing a disulfide bond (e.g. immobilised 5,5' dithiobis(2-nitrobenzoic acid) (Jayabaskaran, C. et al. 1987) and covalent chromatography on activated thiol-Sepharose 4B (Pharmacia)) or (2) a aminohexanoyl-4-aminophenylarsine as immobilised ligand. The latter affinity matrix has been used for the purification of proteins, which are subject to redox regulation and dithiol proteins that are targets for oxidative stress (Kalef, E. et al. 1993).

Reversible protection may also be used to increase the solubilisation and extraction of peptides (Pomroy, N. C. and Deber, C. M. 1998).

The reversible protection and thiol stabilizing compounds may be presented under a monomeric, polymeric or liposomic form.

The removal of the reversibly protection state of the cysteine residues can chemically or enzymatically accomplished by e.g.: a reductant, in particular DTT, DTE, 2-mercaptoethanol, dithionite, SnCl.sub.2, sodium borohydride, hydroxylamine, TCEP, in particular in a concentration of 1-200 mM, more preferentially in a concentration of 50-200 mM; removal of the thiol stabilising conditions or agents by e.g. pH increase; enzymes, in particular thioesterases, glutaredoxine, thioredoxine, in particular in a concentration of 0.01-5 .mu.M, even more particular in a concentration range of 0.1-5 .mu.M.; combinations of the above described chemical and/or enzymatical conditions. The removal of the reversibly protection state of the cysteine residues can be carried out in vitro or in vivo, e.g. in a cell or in an individual.

It will be appreciated that in the purification procedure, the cysteine residues may or may not be irreversibly blocked, or replaced by any reversible modification agent, as listed above.

A reductant according to the present invention is any agent which achieves reduction of the sulfur in cysteine residues, e.g. "S--S" disulfide bridges, desulphonation of the cysteine residue (RS--SO.sub.3.sup.-.fwdarw.RSH). An antioxidant is any reagent which preserves the thiol status or minimises "S--S" formation and/or exchanges. Reduction of the "S--S" disulfide bridges is a chemical reaction whereby the disulfides are reduced to thiol (-SH). The disulfide bridge breaking agents and methods disclosed by Maertens et al. in WO 96/04385 are hereby incorporated by reference in the present description. "S--S" Reduction can be obtained by (1) enzymatic cascade pathways or by (2) reducing compounds. Enzymes like thioredoxin, glutaredoxin are known to be involved in the in vivo reduction of disulfides and have also been shown to be effective in reducing "S--S" bridges in vitro. Disulfide bonds are rapidly cleaved by reduced thioredoxin at pH 7.0, with an apparent second order rate that is around 10.sup.4 times larger than the corresponding rate constant for the reaction with DTT. The reduction kinetic can be dramatically increased by preincubation the protein solution with 1 mM DTT or dihydrolipoamide (Holmgren, A. 1979). Thiol compounds able to reduce protein disulfide bridges are for instance Dithiothreitol (DTT), Dithioerythritol (DTE), .beta.-mercaptoethanol, thiocarbamates, bis(2-mercaptoethyl) sulfone and N,N'-bis(mercaptoacetyl)hydrazine, and sodium-dithionite. Reducing agents without thiol groups like ascorbate or stannous chloride (SnCl.sub.2), which have been shown to be very useful in the reduction of disulfide bridges in monoclonal antibodies (Thakur, M. L. et al. 1991), may also be used for the reduction of HCV proteins. In addition, changes in pH values may influence the redox status of HCV proteins. Sodium borohydride treatment has been shown to be effective for the reduction of disulfide bridges in peptides (Gailit, J. 1993). Tris (2-carboxyethyl)phosphine (TCEP) is able to reduce disulfides at low pH (Burns, J. et al. 1991). Selenol catalyses the reduction of disulfide to thiols when DTT or sodium borohydride is used as reductant. Selenocysteamine, a commercially available diselenide, was used as precursor of the catalyst (Singh, R. and Kats, L. 1995).

Heparin is known to bind to several viruses and consequently binding to the HCV envelope has already been suggested (Garson, J. A. et al. 1999). In this respect, in order to analyze potential binding of HCV envelope proteins to heparin, heparin can be biotinylated and subsequently the interaction of heparin with HCV envelope proteins can be analyzed, e.g. on microtiterplates coated with HCV envelope proteins. In this way different expression systems can be scrutinized. For example, a strong binding is observed with part of the HCV E1 expressed in Hansenula, while binding with HCV E1 from mammalian cell culture is absent. In this respect, the term "heparin affinity chromatography" relates to an immobilized heparin, which is able to specifically bind to HCV envelope proteins. Proteins of the high-mannose type bind agglutinins such as Lens culinaris, Galanthus nivalis, Narcissus pseudonarcissus Pisum sativum or Allium ursinum. Moreover, N-acetylglucosamine can be bound by lectins, such as WGA (wheat germ agglutinin) and its equivalents. Therefore, one may employ lectins bound to a solid phase to separate the HCV envelope proteins of the present invention from cell culture supernatants, cell lysates and other fluids, e.g. for purification during the production of antigens for vaccine or immunoassay use.

With "HCV-recombinant vaccinia virus" is meant a vaccinia virus comprising a nucleic acid sequence encoding a HCV protein or part thereof.

A further aspect of the invention relates to an isolated HCV envelope protein or part thereof resulting from the method of production as described herein. In particular, the invention relates to an isolated HCV envelope protein or part thereof resulting from the expression in an eukaryotic cell of a recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to said HCV envelope protein or a part thereof. More specifically, said recombinant nucleic acid is encoding a protein which is characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.3-(A4- ).sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

In a specific embodiment, the isolated HCV envelope protein or part thereof is derived from said protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to said HCV envelope protein or a part thereof. In another specific embodiment, the isolated HCV envelope protein or part thereof is derived from said protein which is characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-HCVENV-[(A3).sub.d-(PS2).sub.e-(A4- ).sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, HCVENV is a HCV envelope protein or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

Another aspect of the current invention relates to the use of the avian lysozyme leader peptide to direct a recombinantly expressed protein to the endoplasmic reticulum of Hansenula polymorpha or any mutant thereof.

Thus, all aspects and embodiments of the current invention as described above and relating to a HCV envelope protein can, specific for H. polymorpha or any mutant thereof as host cell, be read as relating to a protein instead of relating to a HCV envelope protein.

More specifically, the current invention also relates to a recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to a protein of interest or a part thereof.

In one embodiment thereto, the recombinant nucleic acid comprising nucleotide sequence encodes characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-PROT-[(A3).sub.d-(PS2).sub.e-(A4).- sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, PROT is a protein of interest or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

In a further embodiment, the recombinant nucleic acids according to the invention further comprise regulatory elements allowing expression in a H. polymorpha cell or any mutant thereof of said protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to a protein of interest or a part thereof, or of said protein characterized by the structure CL-[(A1).sub.x-(PS1).sub.y-(A2).sub.z]-PROT-[(A3).sub.x-(PS2).sub.y-(A4).- sub.z]. Further included are vectors comprising said recombinant nucleic acids, host cells comprising said recombinant nucleic acids or said vectors, said host cells expressing the protein comprising an avian lysozyme leader peptide or a functional variant thereof joined to a protein of interest and methods for producing said protein of interest in said host cells.

A further aspect of the invention relates to an isolated protein of interest or part thereof resulting from the expression in a Hansenula cell of a recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to said protein of interest or a part thereof. More specifically, said recombinant nucleic acid is encoding a protein which is characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-PROT-[(A3).sub.d-(PS2).sub.e-(A4).- sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, PROT is a protein of interest or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

In a specific embodiment, the isolated protein of interest or part thereof is derived from said protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to said protein of interest or a part thereof. In another specific embodiment, the isolated protein of interest or part thereof is derived from said protein which is characterized by the structure CL-[(A1).sub.a-(PS1).sub.b-(A2).sub.c]-PROT-[(A3).sub.d-(PS2).sub.e-(A4).- sub.f] wherein: CL is an avian lysozyme leader peptide or a functional equivalent thereof, A1, A2, A3 and A4 are adaptor peptides which can be different or the same, PS1 and PS2 are processing sites which can be the different or the same, PROT is a protein of interest or a part thereof, a, b, c, d, e and f are 0 or 1, and wherein, optionally, A1 and/or A2 are part of PS1 and/or wherein A3 and/or A4 are part of PS2.

In a specific embodiment of the invention, said protein of interest or fragment thereof can e.g. be a viral envelope protein or a fragment thereof such as a HCV envelope protein or HBV (hepatitis B) envelope protein, or fragments thereto. In general, said protein of interest or fragment thereof can be any protein needing the N-glycosylation characteristics of the current invention. Other exemplary viral envelope proteins include the HIV (human immunodeficiency virus) envelope protein gp120 and viral envelope proteins of a virus belonging to the Flavirideae.

The terms "HCV virus-like particle formed of a HCV envelope protein" "oligomeric particles formed of HCV envelope proteins" are herein defined as structures of a specific nature and shape containing several basic units of the HCV E1 and/or E2 envelope proteins, which on their own are thought to consist of one or two E1 and/or E2 monomers, respectively. It should be clear that the particles of the present invention are defined to be devoid of infectious HCV RNA genomes. The particles of the present invention can be higher-order particles of spherical nature which can be empty, consisting of a shell of envelope proteins in which lipids, detergents, the HCV core protein, or adjuvant molecules can be incorporated. The latter particles can also be encapsulated by liposomes or apolipoproteins, such as, for example, apolipoprotein B or low density lipoproteins, or by any other means of targeting said particles to a specific organ or tissue. In this case, such empty spherical particles are often referred to as "virus-like particles" or VLPs. Alternatively, the higher-order particles can be solid spherical structures, in which the complete sphere consists of HCV E1 or E2 envelope protein oligomers, in which lipids, detergents, the HCV core protein, or adjuvant molecules can be additionally incorporated, or which in turn may be themselves encapsulated by liposomes or apolipoproteins, such as, for example, apolipoprotein B, low density lipoproteins, or by any other means of targeting said particles to a specific organ or tissue, e.g. asialoglycoproteins. The particles can also consist of smaller structures (compared to the empty or solid spherical structures indicated above) which are usually round (see further)-shaped and which usually do not contain more than a single layer of HCV envelope proteins. A typical example of such smaller particles are rosette-like structures which consist of a lower number of HCV envelope proteins, usually between 4 and 16. A specific example of the latter includes the smaller particles obtained with E1s in 0.2% CHAPS as exemplified herein which apparently contain 8-10 monomers of E1s. Such rosette-like structures are usually organized in a plane and are round-shaped, e.g. in the form of a wheel. Again lipids, detergents, the HCV core protein, or adjuvant molecules can be additionally incorporated, or the smaller particles may be encapsulated by liposomes or apolipoproteins, such as, for example, apolipoprotein B or low density lipoproteins, or by any other means of targeting said particles to a specific organ or tissue. Smaller particles may also form small spherical or globular structures consisting of a similar smaller number of HCV E1 or E2 envelope proteins in which lipids, detergents, the HCV core protein, or adjuvant molecules could be additionally incorporated, or which in turn may be encapsulated by liposomes or apolipoproteins, such as, for example, apolipoprotein B or low density lipoproteins, or by any other means of targeting said particles to a specific organ or tissue. The size (i.e. the diameter) of the above-defined particles, as measured by the well-known-in-the-art dynamic light scattering techniques (see further in examples section), is usually between 1 to 100 nm, more preferentially between 2 to 70 nm, even more preferentially between 2 and 40 nm, between 3 to 20 nm, between 5 to 16 nm, between 7 to 14 nm or between 8 to 12 nm.

In particular, the present invention relates to a method for purifying hepatitis C virus (HCV) envelope proteins, or any part thereof, suitable for use in an immunoassay or vaccine, which method comprising: (i) growing Hansenula or Saccharomyces glycosylation minus strains transformed with an envelope gene encoding an HCV E1 and/or HCV E2 protein, or any part thereof, in a suitable culture medium; (ii) causing expression of said HCV E1 and/or HCV E2 gene, or any part thereof; and (iii) purifying said HCV E1 and/or HCV E2 protein, or any part thereof, from said cell culture.

The invention further pertains to a method for purifying hepatitis C virus (HCV) envelope proteins, or any part thereof, suitable for use in an immunoassay or vaccine, which method comprising: (i) growing Hansenula or Saccharomyces glycosylation minus strains transformed with an envelope gene encoding an HCV E1 and/or HCV E2 protein, or any part thereof, in a suitable culture medium; (ii) causing expression of said HCV E1 and/or HCV E2 gene, or any part thereof; and (iii) purifying said intracellularly expressed HCV E1 and/or HCV E2 protein, or any part thereof, upon lysing the transformed host cell.

The invention further pertains to a method for purifying hepatitis C virus (HCV) envelope proteins, or any part thereof, suitable for use in an immunoassay or vaccine, which method comprising: (i) growing Hansenula or Saccharomyces glycosylation minus strains transformed with an envelope gene encoding an HCV E1 and/or HCV E2 protein, or any part thereof, in a suitable culture medium, in which said HCV E1 and/or HCV E2 protein, or any part thereof, comprises at least two Cys-amino acids; (ii) causing expression of said HCV E1 and/or HCV E2 gene, or any part thereof; and (iii) purifying said HCV E1 and/or HCV E2 protein, or any part thereof, in which said Cys-amino acids are reversibly protected by chemical and/or enzymatic means, from said culture.

The invention further pertains to a method for purifying hepatitis C virus (HCV) envelope proteins, or any part thereof, suitable for use in an immunoassay or vaccine, which method comprising: (i) growing Hansenula or Saccharomyces glycosylation minus strains transformed with an envelope gene encoding an HCV E1 and/or HCV E2 protein, or any part thereof, in a suitable culture medium, in which said HCV E1 and/or HCV E2 protein, or any part thereof, comprises at least two Cys-amino acids; (ii) causing expression of said HCV E1 and/or HCV E2 gene, or any part thereof; and, (iii) purifying said intracellulary expressed HCV E1 and/or HCV E2 protein, or any part thereof, upon lysing the transformed host cell, in which said Cys-amino acids are reversibly protected by chemical and/or enzymatic means.

The present invention specifically relates to a method for purifying recombinant HCV yeast proteins, or any part thereof, as described herein, in which said purification includes heparin affinity chromatography.

Hence, the present invention also relates to a method for purifying recombinant HCV yeast proteins, or any part thereof, as described above, in which said chemical means is sulfonation.

Hence, the present invention also relates to a method for purifying recombinant HCV yeast proteins, or any part thereof, as described above, in which said reversibly protection of Cys-amino acids is exchanged for an irreversible protection by chemical and/or enzymatic means.

Hence, the present invention also relates to a method for purifying recombinant HCV yeast proteins, or any part thereof, as described above, in which said irreversible protection by chemical means is iodo-acetamide.

Hence, the present invention also relates to a method for purifying recombinant HCV yeast proteins, or any part thereof, as described above, in which said irreversible protection by chemical means is NEM or Biotin-NEM or a mixture thereof.

The present invention also relates to a composition as defined above which also comprises HCV core, E1, E2, P7, NS2, NS3, NS4A, NS4B, NS5A and/or NS5B protein, or parts thereof. The core-glycosylated proteins E1, E2, and/or E1/E2 of the present invention may, for example, be combined with other HCV antigens, such as, for example, core, P7, NS3, NS4A, NS4B, NS5A and/or NS5B. The purification of these NS3 proteins will preferentially include a reversible modification of the cysteine residues, and even more preferentially sulfonation of cysteines. Methods to obtain such a reversible modification, including sulfonation have been described for NS3 proteins in Maertens et al. (PCT/EP99/02547). It should be stressed that the whole content, including all the definitions, of the latter document is incorporated by reference in the present application.

Also, the present invention relates to the use of a envelope protein as described herein for inducing immunity against HCV, characterized in that said HCV envelope protein is used as part of a series of time and compounds. In this regard, it is to be understood that the term "a series of time and compounds" refers to administering with time intervals to an individual the compounds used for eliciting an immune response. The latter compounds may comprise any of the following components: a HCV envelope protein according to the invention, HCV DNA vaccine composition, HCV polypeptides.

In this respect, a series comprises administering, either: (i) an HCV antigen, such as, for example, a HCV envelope protein according to the invention, with time intervals, or (ii) an HCV antigen, such as, for example, a HCV envelope protein according to the invention in combination with a HCV DNA vaccine composition, in which said envelope protein and said HCV DNA vaccine composition, can be administered simultaneously, or at different time intervals, including at 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 a HCV DNA vaccine composition comprises nucleic acids encoding HCV envelope peptide, including E1-, E2-, E1/E2-peptides, NS3 peptide, other HCV peptides, or parts of said peptides. Moreover, it is to be understood that said HCV peptides comprises HCV envelope peptides, including E1-, E2-, E1/E2-peptides, other HCV peptides, or parts thereof. The term "other HCV peptides" refers to any HCV peptide or fragment thereof. In item (ii) of the above scheme, the HCV DNA vaccine composition comprises preferentially nucleic acids encoding HCV envelope peptides. In item (ii) of the above scheme, the HCV DNA vaccine composition consists even more preferentially of nucleic acids encoding HCV envelope peptides, possibly in combination with a HCV-NS3 DNA vaccine composition. In this regard, it should be clear that an HCV DNA vaccine composition comprises a plasmid vector comprising a polynucleotide sequence encoding an HCV peptide as described above, operably linked to transcription regulatory elements. As used herein, a "plasmid vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they have been linked. In general, but not limited to those, plasmid vectors are circular double stranded DNA loops which, in their vector form, are not bound to the chromosome. As used herein, a "polynucleotide sequence" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and single (sense or antisense) and double-stranded polynucleotides. As used herein, the term "transcription regulatory elements" refers to a nucleotide sequence which contains essential regulatory elements, such that upon introduction into a living vertebrate cell it is able to direct the cellular machinery to produce translation products encoded by the polynucleotide. The term "operably linked" refers to a juxtaposition wherein the components are configured so as to perform their usual function. Thus, transcription regulatory elements operably linked to a nucleotide sequence are capable of effecting the expression of said nucleotide sequence. Those skilled in the art can appreciate that different transcriptional promoters, terminators, carrier vectors or specific gene sequences may be used successfully. Alternatively, the DNA vaccine may be delivered through a live vector such as adenovirus, canary pox virus, MVA, and the like.

The HCV envelope proteins of the present invention, or the parts thereof, are particularly suited for incorporation into an immunoassay for the detection of anti-HCV antibodies, and/or genotyping of HCV, for prognosing/monitoring of HCV disease, or as a therapeutic agent.

A further aspect of the invention relates to a diagnostic kit for the detection of the presence of anti-HCV antibodies in a sample suspected to comprise anti-HCV antibodies, said kit comprising a HCV envelope protein or part thereof according to the invention. In a specific embodiment thereto, said HCV envelope protein or part thereof is attached to a solid support. In a further embodiment, said sample suspected to comprise anti-HCV antibodies is a biological sample.

The term "biological sample" as used herein, refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example, serum, plasma, lymph fluid, the external sections of the skin, respiratory-, intestinal- or genito-urinary tracts, oocytes, tears, saliva, milk, blood cells, tumors, organs, gastric secretions, mucus, spinal cord fluid, external secretions such as, for example, excrement, urine, sperm, and the like.

Another aspect of the invention refers to a composition comprising an isolated HCV envelope protein or fragment thereof according to the invention. Said composition may further comprise a pharmaceutically acceptable carrier and can be a medicament or a vaccine.

A further aspect of the invention covers a medicament or a vaccine comprising a HCV envelope protein or part thereof according to the invention.

Yet another aspect of the invention comprises a pharmaceutical composition for inducing a HCV-specific immune response in a mammal, said composition comprising an effective amount of a HCV envelope protein or part thereof according to the invention and, optionally, a pharamaceutically acceptable adjuvant. Said pharmaceutical composition comprising an effective amount of a HCV envelope protein or part thereof according to the invention may also be capable of inducing HCV-specific antibodies in a mammal, or capable of inducing a T-cell function in a mammal. Said pharmaceutical compostion comprising an effective amount of a HCV envelope protein or part thereof according to the invention may be prophylactic composition or a therapeutic composition. In a specific embodiment said mammal is a human.

A "mammal" is to be understood as any member of the higher vertebrate class Mammalia, including humans; characterized by live birth, body hair, and mammary glands in the female that secrete milk for feeding the young. Mammals thus also include non-human primates and trimera mice (Zauberman et al. 1999).

A "vaccine" or "medicament" is a composition capable of eliciting protection against a disease, whether partial or complete, whether against acute or chronic disease; in this case the vaccine or medicament is a prophylactic vaccine or medicament. A vaccine or medicament may also be useful for treatment of an already ill individual, in which case it is called a therapeutic vaccine or medicament. Likewise, a pharmaceutical composition can be used for either prophylactic and/or therapeutic purposes in which cases it is a prophylactic and/or therapeutic composition, respectively.

The HCV envelope proteins of the present invention can be used as such, in a biotinylated form (as explained in WO 93/18054) and/or complexed to Neutralite Avidin (Molecular Probes Inc., Eugene, Oreg., USA), avidin or streptavidin. It should also be noted that "a vaccine" or "a medicament" may comprise, in addition to an active substance, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable adjuvant" which may be a suitable excipient, diluent, carrier and/or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. Suitable carriers are typically large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. Such carriers are well known to those skilled in the art. Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: aluminium hydroxide, aluminium in combination with 3-0-deacylated monophosphoryl lipid A as described in WO 93/19780, aluminium phosphate as described in WO 93/24148, N-acetyl-muramyl-L-threonyl-D-isoglutamine as described in U.S. Pat. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine2-(1'2'dipalmitoyl-sn-gly- cero-3-hydroxyphosphoryloxy) ethylamine, RIBI (ImmunoChem Research Inc., Hamilton, Mont., USA) which contains monophosphoryl lipid A, detoxified endotoxin, trehalose-6,6-dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. Any of the three components MPL, TDM or CWS may also be used alone or combined 2 by 2. The MPL may also be replaced by its synthetic analogue referred to as RC-529. Additionally, adjuvants such as Stimulon (Cambridge Bioscience, Worcester, Mass., USA), SAF-1 (Syntex) or bacterial DNA-based adjuvants such as ISS (Dynavax) or CpG (Coley Pharmaceuticals) may be used, as well as adjuvants such as combinations between QS21 and 3-de-O-acetylated monophosphoryl lipid A (WO94/00153), or MF-59 (Chiron), or poly[di(carboxylatophenoxy) phosphazene] based adjuvants (Virus Research Institute), or blockcopolymer based adjuvants such as Optivax (Vaxcel, Cythx) or insulin-based adjuvants, such as Algammulin and Gammalnulin (Anutech), Incomplete Freund's Adjuvant (IFA) or Gerbu preparations (Gerbu Biotechnik). It is to be understood that Complete Freund's Adjuvant (CFA) may be used for non-human applications and research purposes as well. "A vaccine composition" may further contain excipients and diluents, which are inherently non-toxic and non-therapeutic, such as water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, preservatives, and the like. Typically, a vaccine composition is prepared as an injectable, either as a liquid solution or suspension. Injection may be subcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal, intradermal. Other types of administration comprise implantation, suppositories, oral ingestion, enteric application, inhalation, aerosolization or nasal spray or drops. Solid forms, suitable for solution on, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or encapsulated in liposomes for enhancing adjuvant effect. The polypeptides may also be incorporated into Immune Stimulating Complexes together with saponins, for example Quil A (ISCOMS). Vaccine compositions comprise an effective amount of an active substance, as well as any other of the above-mentioned components. "Effective amount" of an active substance means that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for prevention or treatment of a disease or for inducing a desired effect. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated (e.g. human, non-human primate, primate, etc.), the capacity of the individual's immune system to mount an effective immune response, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment, the strain of the infecting pathogen and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Usually, the amount will vary from 0.01 to 1000 .mu.g/dose, more particularly from 0.1 to 100 .mu.g/dose. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.

Claim 1 of 32 Claims

1. A recombinant nucleic acid comprising a nucleotide sequence encoding a protein comprising a leader peptide defined by the amino acid sequence MRSLLILVLCFLPLAALG (SEQ ID NO:99) joined to an HCV envelope protein or to the sequence of SEQ ID NO:2 or a corresponding sequence from another HCV.
 

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