WO2002086101A2 - Core-glycosylated hcv envelope proteins - Google Patents
Core-glycosylated hcv envelope proteins Download PDFInfo
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- WO2002086101A2 WO2002086101A2 PCT/BE2002/000064 BE0200064W WO02086101A2 WO 2002086101 A2 WO2002086101 A2 WO 2002086101A2 BE 0200064 W BE0200064 W BE 0200064W WO 02086101 A2 WO02086101 A2 WO 02086101A2
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- Prior art keywords
- hcv
- protein
- envelope protein
- hcv envelope
- fragment
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/18—Togaviridae; Flaviviridae
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5258—Virus-like particles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24223—Virus like particles [VLP]
Definitions
- the present invention relates to the general field of recombinant protein expression, to diagnosis of HCV infection, to treatment or prevention of HCV infection and to the prognosing/monitoring of the clinical efficiency of treatment of an individual with chronic hepatitis, or the prognosing/ monitoring of the natural disease. More particularly, the present mvention relates to the expression of hepatitis C virus envelope proteins in yeast, yeast strains for the expression of core-glycosylated viral envelope proteins, and the use in diagnosis, prophylaxis or therapy of HCV envelope proteins according to the present invention,
- HCV infection is a major health problem in both developed and developing countries. It is estimated that about 1 to 5 % of the world population is affected by the virus. HCV infection appears to be the most important cause of transfusion-associated hepatitis and frequently progresses, to chronic liver damage. Moreover, evidence exists implicating HCV in induction of hepatocellular carcinoma. Consequently, the demand for reliable diagnostic methods and effective therapeutic agents is high. Also sensitive and specific screening methods of HCN-contaminated blood-products and improved methods to culture HCN are needed.
- HCN is a positive stranded R ⁇ A virus of approximately 9,600 bases which encode a single polyprotein precursor of about 3000 amino acids. Proteolytic cleavage of the precursor coupled to co- and posttranslational modifications has been shown to result in at least three structural and six non-structural proteins. Based on sequence homology, the structural proteins have been functionally assigned as one single core protein and two envelope glycoproteins: El and E2. The El protein consists of 192 amino acids and contains 4 to 5 ⁇ - glycosylation sites, depending on the HCN genotype.
- the E2 protein consists of 363 to 370 amino acids and contains 9 to 11 ⁇ -glycosylation sites, depending on the HCN genotype (for reviews see: Major and Feinstone, 1997; Maertens and Stuyver, 1997).
- the El protein contains various variable domains (Maertens and Stuyver, 1997).
- the E2 protein contains three hypervariable domains, of which the major domain is located at the N-terminus of the protein (Maertens and Stuyver, 1997).
- the HCV glycoproteins localize predominantly in the ER where they are modified and assembled into oligomeric complexes.
- sugar residues are commonly linked to four different amino acid residues. These amino acid residues are classified as O-linked (serine, threonine, and hydroxylysine) and N-linked (asparagine).
- O-linked sugars are synthesized in the Golgi or rough Endoplasmic Reticulum (ER) from nucleotide sugars.
- ER Endoplasmic Reticulum
- the N-linked sugars are synthesized from a common precursor, and subsequently processed. It is believed that HCN envelope proteins are ⁇ - glycosylated.
- the oligosaccharide is further processed into the complex type (containing ⁇ -acetylglucosamine, mannose, fucose, galactose and sialic acid) or the high-mannose type (containing ⁇ -acetylglucosamine and mannose).
- HCN envelope proteins are believed to be of the high-mannose type.
- ⁇ -linked oligosaccharide processing in yeast is very different from mammalian Golgi processing. In yeast the oligosaccharide chains are elongated in the Golgi through stepwise addition of mannose, leading to elaborate high mannose structures, referred to as hyperglycosylation.
- the methylotrophic yeast Pichia pastoris was reported to attach an average of 8 to 14 mannose units, i.e. Man(8-14)Glc ⁇ Ac(2) per glycosylation site (Tschopp in EP0256421) and approximately 85% of the N-linked oligosaccharides are in the size range Man(8- 14)GlcNAc(2) (Grinna and Tschopp 1989).
- Other researchers have published slightly different oligosaccharide structures attached to heterologous proteins expressed in P. pastoris: Man(8-9)GlcNAc(2) (Montesino et al. 1998), Man(9-14)GlcNAc(2) or Man(9-15)GlcNAc(2) (Kalidas et al.
- Saccharomyces cerevisiae glycosylation deficient mutant mnn9 differs from wild- type S. cerevisiae in that mm ⁇ 9 cells produce glycosylated proteins with a modified oligosaccharide consisting of Man(9-13)GlcNAc(2) instead of hyperglycosylated proteins (Mackay et al. in US5,135,854 and Kniskern et al. in WO94/01132).
- Another S. cerevisiae mutant, ochlmnn9 was reported to add Man(8)GlcNAc(2) to N-glycosylation sites in proteins (Yoshifumi et al. JP06277086).
- Characteristic for S. cerevisiae (wild-type and mnn9 mutant) core oligosaccharides is the presence of terminal ⁇ l,3-linked mannose residues (Montesino et al. 1998). Oligosaccharides attached to N-glycosylation sites of proteins expressed in P. pastoris or S. cerevisiae ochlmnnl are devoid of such terminal ⁇ l,3-linked mannoses (Gellissen et al. 2000). Terminal ⁇ l,3-linked mannoses are considered to be allergenic (Jenkins et al. 1996). Therefor, proteins carrying on their oligosaccharides terminal ⁇ l,3-linked mannose residues are not suitable for diagnostic or therapeutic purposes.
- Hansenula polymorpha have, despite the use of this yeast for production of a considerable number of heterologous proteins (see Table 3 in Gellissen et al. 2000), not been studied in great detail. From the experiments of Janowicz et al. (1991) and Diminsky et al. (1997), it seems that H. polymorpha is not or only poorly glycosylating the large or small hepatitis B viral surface antigen (HBsAg). Most likely this is due to the fact that the HBsAg was expressed without signal peptide, thus preventing the produced HBsAg to enter the lumen of the endoplasmic reticulum (ER) and glycosylation.
- HBsAg hepatitis B viral surface antigen
- G-CSF granulocyte colony stimulating factor
- HCN antigenic determinants can be divided in at least two forms, i.e. lineair and conformational epitopes. Conformational epitopes result from the folding of a molecule in a three- dimensional space, including co- and posttranslational modifications, such as glycosylation.
- HCN envelope proteins have been produced by recombinant techniques in
- a first aspect of the current invention relates to an isolated HCV envelope protein or a fragment thereof comprising at least one N-glycosylation site, said protein or fragment thereof characterized in that it is the product of expression in a eukaryotic cell and further characterized in that on average up to 80 % of the N-glycosylation sites are core-glycosylated.
- more than 70 % of said core-glycosylated sites are glycosylated with an 20 oligomannose with a structure defined by Man(8 to 10)-GlcNAc(2).
- the ratio of the oligomannose with structure Man(7)-GlcNAc(2) over the oligomannose with structure Man(8)-GlcNAc(2) is less than or equal to 0.45. More specifically said oligomannoses contain less than 10 % terminal ⁇ l,3 mannose.
- the eukaryotic cell expressing said isolated HCN envelope protein or part thereof can be a yeast cell such as a Hansenula cell. 25.
- a further aspect of the current invention relates to an isolated HCN envelope protein or part thereof according to the invention which is derived from a protein comprising an avian lysozyme leader peptide or a functional variant thereof joined to said HCN envelope protein or fragment thereof. More specifically, said isolated HCN envelope protein or part thereof is
- CL is an avian lysozyme leader peptide or a functional equivalent thereof
- Al, A2, A3 and A4 are adaptor peptides which can be different or the same
- PSI and PS2 are processing sites which can be the different or the same
- HCNENV is a HCN envelope protein or a part thereof, a, b, c, d, e and fare 0 or 1, and wherein, optionally, Al and/or A2 are part of PSI and or wherein A3 and/or A4 are part of PS2.
- Another aspect of the invention covers an isolated HCN envelope protein or fragment thereof according to the invention which is comprised in a structure chosen from the group consisting of monomers, homodimers, heterodimers, homo-oligomers and hetero-oligomers.
- said isolated HCN envelope protein or fragment thereof according to the invention which is comprised in a virus-like particle.
- any of the isolated HCN envelope protein or fragment thereof according to the invention may comprise cysteines of which the cysteine thiol-groups are chemically modified.
- HCN envelope proteins or fragment thereofs according to the invention which are antigenic or immunogenic and/or which comprise a T-cell stimulating epitope.
- a further aspect relates to a composition
- a composition comprising an isolated HCN 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.
- the invention also relates to a method for producing the isolated HCN envelope protein or fragment thereof according to the invention.
- Another method of the invention is a method for the detection of the presence of anti- HCN antibodies in a sample suspected to comprise anti-HCN antibodies, said method comprising: (i) contacting a HCN envelope protein or part thereof according to any of claims 1 to
- said method may comprise step (i) in which said contacting is occurring under competitive conditions.
- said methods may utilize a solid support to which said HCN envelope protein or part thereof is attached.
- the invention further relates to a diagnostic kit for the detection of the presence of anti-HCN antibodies in a sample suspected to comprise anti-HCN antibodies, said kit comprising a HCN envelope protein or part thereof according to the invention. More specifically, said kit may comprise said HCN envelope protein or part thereof attached to a solid support.
- the invention also relates to a medicament or a vaccine comprising a HCN envelope protein or part thereof according to the invention.
- a pharmaceutical composition for inducing a HCN- specific immune response in a mammal comprising an effective amount of a HCN envelope protein or part thereof according to the invention and, optionally, a pharmaceutically acceptable adjuvant.
- Said pharmaceutical composition may alternatively be capable of inducing HCN-specific antibodies in a mammal or of inducing a T-cell function in a mammal.
- said pharmaceutical composition may be a prophylactic composition or a therapeutic composition.
- said mammal is a human.
- Figure 1 Schematic map of the vector pGEMT-ElsH6RB which has the sequence as defined in SEQ ID NO:6.
- Figure 2 Schematic map of the vector pCHH-Hir which has the sequence as defined in SEQ ID NO:9.
- Figure 3 Schematic map of the vector pFPMT121 which has the sequence as defined in SEQ ID NO-.12.
- Figure 4 Schematic map of the vector pFPMT-CHH-El-H6 which has the sequence as defined in SEQ ID NO: 13.
- Figure 5 Schematic map of the vector pFPMT-MFa-El-H6 which has the sequence as defined in SEQ ID NO:16.
- Figure 6 Schematic map of the vector pUC18-FMD-MFa-El-H6 which has the sequence as defined in SEQ ID NO:17.
- Figure 7 Schematic map of the vector pUC18-FMD-CL-El-H6 which has the sequence as defined in SEQ ID NO:20.
- Figure 8 Schematic map of the vector pFPMT-CL-El-H6 which has the sequence as defined i SEQ ID NO:21.
- Figure 9 Schematic map of the vector pSP72E2H6 which has the sequence as defined in SEQ ID NO:22.
- Figure 10 Schematic map of the vector pMPT121 which has the sequence as defined in SEQ ID NO:23.
- Figure 11 Schematic map of the vector pFPMT-MFa-E2-H6 which has the sequence as defined in SEQ ID NO:24.
- Figure 12 Schematic map of the vector pMPT-MFa-E2-H6 which has the sequence as defined in SEQ ID NO:25.
- Figure 13 Schematic map of the vector pMF30 which has the sequence as defined in SEQ ID NO:28.
- Figure 14 Schematic map of the vector pFPMT-CL-E2-H6 which has the sequence as defined in SEQ ID NO:32.
- Figure 15. Schematic map of the vector pUC18-FMD-CL-El which has the sequence as defined in SEQ ID NO:35.
- Figure 16 Schematic map of the vector pFPMT-CL-El which has the sequence as defined in SEQ ID NO:36.
- FIG. 17 Schematic map of the vector pUC18-FMD-CL-H6-El-K-H6 wliich has the sequence as defined in SEQ ID NO:39.
- Figure 18 Schematic map of the vector pFPMT-CL-H6-K-El which has the sequence as defined in SEQ ID NO:40.
- Figure 19 Schematic map of the vector pYIG5 which has the sequence as defined in SEQ ID NO:41.
- Figure 20 Schematic map of the vector pYIG5ElH6 which has the sequence as defined in SEQ ID NO:42.
- Figure 21 Schematic map of the vector pSYl which has the sequence as defined in SEQ ID NO:43.
- Figure 22 Schematic map of the vector pSYlaMFElsH6a wliich has the sequence as defined in SEQ ID NO:44.
- Figure 23 Schematic map of the vector pBSK-E2sH6 wliich has the sequence as defined in SEQ ID NO:45.
- Figure 24 Schematic map of the vector pYIG5HCCL-22aH6 which has the sequence as defined in SEQ ID NO:46.
- Figure 25 Schematic map of the vector pYYIGSE2H6 which has the sequence as defined in SEQ ID NO:47.
- Figure 26 Schematic map of the vector pYIG7 which has the sequence as defined in SEQ ID NO:48.
- Figure 27 Schematic map of the vector pYIG7El which has the sequence as defined in SEQ ID NO:49.
- Figure 28 Schematic map of the vector pSYl YIG7Els wliich has the sequence as defined in SEQ ID NO:50.
- Figure 29 Schematic map of the vector pPICZalphaA which has the sequence as defined in SEQ ID NO.51.
- Figure 30 Schematic map of the vector pPICZalphaD' which has the sequence as defined in SEQ ID NO:52.
- Figure 31 Schematic map of the vector pPICZalphaE' which has the sequence as defined in SEQ ID NO:53.
- Figure 32 Schematic map of the vector pPICZalphaD 'El sH6 which has the sequence as defined in SEQ ID NO:58.
- Figure 33 Schematic map of the vector pPICZalphaE 'ElsH6 which has the sequence as defined in SEQ ID NO:59.
- Figure 34 Schematic map of the vector pPICZalphaD 'E2sH6 which has the sequence as defined in SEQ ID NO:60.
- Figure 35 Schematic map of the vector pPICZalphaE'E2sH6 which has the sequence as defined in SEQ ID NO:61.
- Figure 36 Schematic map of the vector pUC18MFa wliich has the sequence as defined in SEQ ID NO:62.
- Figure 37 Elution profile of size exclusion chromatography of IMAC-purified E2-H6 protein expressed from the MF ⁇ -E2-H6-expressing Hansenula polymorpha (see Example 15).
- the X- axis indicates the elution volume (in mL).
- the vertical lines through the elution profile indicate the fractions collected.
- "PI' - pooled fractions 4 to 9
- P3 pooled fractions 37 to 44.
- the Y-axis indicates absorbance given in mAU ( illi absorbance units).
- the X-axis indicates the elution volume in mL.
- FIG 38 The different pools and fractions collected after size exclusion chromatography (see Figure 37) were analyzed by non-reducing SDS-PAGE followed by silver staining of the polyacrylamide gel.
- the analyzed pools (“PI", "P2”, and “P3") and fractions (16 to 26) are indicated on top of the picture of the silver-stained gel.
- At the left (lane "M") are indicated the sizes of the molecular mass markers.
- Figure 39 Fractions 17 to 23 of the size exclusion chromatographic step as shown in Figure 37 were pooled and alkylated. Thereafter, the protein material was subjected to Endo H treatment for deglycosylation. Untreated material and Endo H-treated material were separated on an SDS-PAGE gel and blotted to a PVDF membrane. The blot was stained with amido black.
- Lane 1 Alkylated E2-H6 before Endo H-treatment
- Lane 2 Alkylated E2-H6 after Endo H-treatment.
- Figure 40 Western-blot analysis of cell lysates of El expressed in Saccharomyces cerevisiae.
- the Western-blot was developed using the El-specific monoclonal antibody IGH 201.
- Lanes 1-4 expression product after 2, 3, 5 or 7 days expression, respectively, in a Saccharomyces clone transformed with pSYlYIG7Els (SEQ ID NO:50, Figure 28) comprising the nucleotide sequence encoding the chicken lysozyme leader peptide joined to
- Lanes 5-7 expression product after 2, 3 or 5 days expression, respectively, in a
- Saccharomyces clone transformed with pSYlaMFElsH ⁇ aYIGl (SEQ ID NO:44, Figure 22) comprising the nucleotide sequence encoding the ⁇ -mating factor leader peptide joined to El-
- Lane 8 molecular weight markers with sizes as indicated.
- Lane 9 purified Els produced by HCV-recombinant vaccinia virus-infected mammalian cells.
- Figure 41 Analysis of the immobilized metal ion affinity chromatography (IMAC)-purified E2-H6 protein expressed by and processed from CL-E2-H6 to E2-H6 by H. polymorpha (see Example 17). Proteins in different wash fractions (lanes 2 to 4) and elution fractions (lanes 5 to 7) were analyzed by reducing SDS-PAGE followed by silver staining of the gel (A, top picture) or by western blot using using a specific monoclonal antibody directed against E2 (B, bottom picture). The sizes of the molecular mass markers are indicated at the left.
- IMAC immobilized metal ion affinity chromatography
- Figure 42 Elution profile of the first IMAC chromatography step on a Ni-IDA column (Chelating Sepharose FF loaded with Ni 2+ , Pharmacia) for the purification of the sulfonated
- H6-K-E1 protein produced by H. polymorpha (see Example 18).
- the column was equilibrated with buffer A (50 mM phosphate, 6 M GuHCl, 1 % Empigen BB (v/v), pH 7.2) supplemented with 20 mM imidazole. After sample application, the column was washed sequentially with buffer A containing 20 mM and 50 mM imidazole, respectively (as indicated on chromatogram). A further washing and elution step of the His-tagged products was performed by the sequential application of buffer B (PBS, 1% empigen BB, pH 7.2) supplemented with
- the wash pool 1 fractions 8 to 11, wash with 50 mM imidazole.
- the eluted material was collected as separate fractions 63 to 72 or an elution pool (fractions 63 to 69) was made.
- the Y-axis indicates absorbance given in mAU (milli absorbance units).
- the X-axis indicates the elution volume in L
- FIG 43 Analysis of the IMAC-purified H6-K-E1 protein (see Figure 42) expressed by and processed from CL-H6-K-E1 to H6-K-E1 by H. polymorpha. Proteins in the wash pool 1 (lane 12) and elution fractions 63 to 72 (lanes 2 to 11) were analyzed by reducing SDS-PAGE followed by silver staining of the gel (A, top picture). Proteins present in the sample before IMAC (lane 2), in the flow-through pool (lane 4), in wash pool 1 (lane 5) and in the elution pool (lane 6) were analyzed by western blot using a specific monoclonal antibody directed against El (B, bottom picture; no sample was loaded in lane 3). The sizes of the molecular mass markers (lanes M) are indicated at the left.
- FIG 44 Elution profile of the second IMAC chromatography step on a Ni-IDA column (Chelating Sepharose FF loaded with Ni 2+ , Pharmacia) for the purification of El resulting from the in vitro processing of H6-K-E1 (purification: see Figure 42) with Endo Lys-C.
- the flow through was collected in different fractions (1 to 40) that were screened for the presence of Els-products.
- the fractions (7 to 28), containing intact El processed from H6-K-E1 were pooled.
- the Y-axis indicates absorbance given in mAU (milli absorbance units).
- the X-axis mdicates the elution volume in mL
- FIG 45 Western-blot analysis indicating specific Els proteins bands reacting with biotinylated heparin (see also Example 19).
- Els preparations purified from HCN-recombinant vaccinia virus-infected mammalian cell culture or expressed by H. polymojpha were analyzed.
- the panel right from the vertical line shows a Western-blot developed with the biotinylated El specific monoclonal IGH 200.
- the panel left from the vertical line shows a Western-blot developed with biotinylated heparin. From these results it is concluded that mainly the lower-glycosylated Els has high affinity for heparin.
- Lanes M molecular weight marker (molecular weights indicated at the left).
- Lanes 1 Els from mammalian cells and alkylated during isolation. Lanes 2: El s-H6 expressed by H. polymorpha and sulphonated during isolation. Lanes 3: Els-H6 expressed by H. polymorpha and alkylated during isolation. Lanes 4: same material as loaded in lane 2 but treated with dithiotreitol to convert the sulphonated Cys-thiol groups to Cys-thiol.
- FIG. 46 Size exclusion chromatography (SEC) profile of the purified H. polymorpha- expressed E2-H6 in its sulphonated form, submitted to a run in PBS, 3% betain to force viruslike particle formation by exchange of Empigen BB for betain.
- the pooled fractions containing the NLPs used for further study are indicated by " ⁇ -»".
- the Y-axis indicates absorbance given in mAU (milli absorbance units).
- the X-axis indicates the elution volume in mL. See also Example 20.
- FIG 47 Size exclusion chromatography (SEC) profile of the purified H. polymorpha- expressed E2-H6 in its alkylated form, submitted to a run in PBS, 3% betain to force viruslike particle formation by exchange of Empigen BB for betain.
- the pooled fractions containing the NLPs are indicated by " ⁇ -»".
- the Y-axis indicates absorbance given in mAU (milli absorbance units).
- the X-axis indicates the elution volume in mL. See also Example 20.
- Figure 48 Size exclusion chromatography (SEC) profile of the purified H.
- polymorpha- expressed El in its sulphonated form submitted to a run in PBS, 3% betain to force virus-like particle formation by exchange of Empigen BB for betain.
- the pooled fractions containing the NLPs are indicated by " ⁇ ⁇ - ".
- the Y-axis indicates absorbance given in mAU (milli absorbance units).
- the X-axis indicates the elution volume in mL. See also Example 20.
- FIG. 49 Size exclusion chromatography (SEC) profile of the purified H. polymorpha- expressed El in its alkylated form, submitted to a run in PBS, 3% betain to force virus-like particle formation by exchange of Empigen BB for betain.
- the pooled fractions containing the NLPs are indicated by " ⁇ -»".
- the Y-axis indicates absorbance given in mAU (milli absorbance units).
- the X-axis indicates the elution volume in mL. See also Example 20.
- Figure 50 SDS-PAGE (under reducing conditions) and western blot analysis of NLPs as isolated after size exclusion chromatography (SEC) as described in Figures 48 and 49.
- Left panel silver-stained SDS-PAGE gel.
- Right panel western blot using a specific monoclonal antibody directed against El (IG ⁇ 201).
- Lanes 1 molecular weight markers (molecular weights indicated at the left);
- lanes 2 pool of NLPs containing sulphonated El (cfr. Figure 48); lanes 3: pool of NLPs containing alkylated El (cfr. Figure 49). See also Example 20.
- FIG 51 El produced in mammalian cells ("M”) or Hansenula-p ⁇ oduced El (“H”) were coated on a ELISA solid support to determine the end point titer of antibodies present in sera after vaccination of mice with El produced in mammalian cells (top panel), or after vaccination of mice with Hansenula-produced El (bottom panel).
- the horizontal bar represents the mean antibody titer.
- the end-point titers (fold-dilution) are indicated on the Y- axis. See also Example 22.
- Figure 52 Hansenula-produced El was alkylated ("A") or sulphonated ("S") and coated on a ELISA solid support to determine the end point titer of antibodies present in sera after vaccination of mice with Hansenula-produced El that was alkylated (top panel), or after vaccination of mice with Hansenula-produced El that was sulphonated (bottom panel).
- the horizontal bar represents the mean antibody titer.
- the end-point titers (fold-dilution) are indicated on the Y-axis. See also Example 23.
- Figure 53 HCN El produced by HCN-recombinant vaccinia virus-infected mammalian cells and HCN El produced by H. polymorpha were coated directly to ELISA plates.
- End point titers of antibodies were deteremined in sera of chimpanzees vaccinated with El produced by mammalian cells (top panel) and of murine monoclonal antibodies raised against El produced by mammalian cells (bottom panel).
- Chimpanzees Yoran and Marti were prophylactically vaccinated.
- Chimpanzees Ton, Phil, Marcel, Peggy and Femma were therapeutically vaccinated.
- Black filled bars ELISA plate coated with El produced by mammalian cells.
- Open bars ELISA plate coated with El produced by Hansenula. The end-point titers (fold- dilution) are indicated on the Y-axis. See also Example 24.
- Figure 54 Fluorophore-assisted carbohydrate gelelectrophoresis of oligosaccharides released from El produced by recombinant vaccinia virus-infected mammalian cells and from E1-H6 protein produced by Hansenula.
- Lane 1 Glucose ladder standard with indication at the left of the number of monosaccharides (3 to 10, indicated by G3 to G10).
- Lane 2 25 ⁇ g ⁇ -linked oligosaccharides released from (alkylated) El produced by mammalian cells.
- Lane 3 25 ⁇ g ⁇ - linked oligosaccharides released from (alkylated) E1-H6 produced by Hansenula.
- Lane 4 lOO pmoles maltotetraose.
- Figure 55 This figure shows the simplified structures of the reference oligomannoses Man-9 (Figure 55.A), Man-8 (Figure 55.B), Man-7 (Figure 55.C), Man-6 ( Figure 55.D) and Man-5 ( Figure 55.E).
- Man mannose;
- GlcNAc ⁇ ⁇ -acetylglucosamine;
- ⁇ ⁇ -linkage between 2 mannoses;
- ⁇ ⁇ -linkage between 2 mannoses;
- (1-3)", “(1-4)” and “(1-6)” (1- 3), (1-4) and (1-6) linkage between 2 mannoses, respectively.
- the thin upward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by 1-2 Mannosidase (none for this oligomannose)
- the thick upward or leftward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ 1-2-linked mannoses (not applicable to this oligomannose)
- the empty downward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ -linked mannoses. See also Example 26.
- Figure 57 This figure shows a higher oligomannose consisting of 9 mannose moieties coupled to chitobiose. In this oligomannose, one terminal mannose residue is linked by an ⁇ 1-2 bond to the non-terminal mannose residue.
- the thin upward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ 1-2 Mannosidase
- the thick upward or leftward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ 1-2-linked mannoses
- the empty downward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ -linked mannoses. See also Example 26.
- Figure 58 This figure shows the reference higher oligomannose Man-9 consisting of 9 mannose moieties coupled to chitobiose. In this oligomannose, all terminal mannose residues are linked by an ⁇ 1-2 bond to a non-terminal mannose residue.
- the thin upward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ 1-2 Mannosidase
- the thick upward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ 1-2-linked mannoses
- the empty downward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ -linked mannoses. See also Example 26.
- FIG 59 This figure shows the higher oligomannose Man-8 consisting of 8 mannose moieties coupled to chitobiose.
- Man-8 consisting of 8 mannose moieties coupled to chitobiose.
- all terminal mannose residues are linked by an ⁇ 1-3 or an ⁇ 1-6 bond to a non-terminal mannose residue which renders this structure fully resistant to cleavage by ⁇ 1-2 Mannosidase.
- the thick upward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase and the empty downward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ -linked mannoses. See also Example 26.
- FIG. 60 This figure shows the higher oligomannose Man-7 consisting of 7 mannose moieties coupled to chitobiose.
- Man-7 consisting of 7 mannose moieties coupled to chitobiose.
- all terminal mannose residues are linked by an ⁇ 1-3 bond to a non-terminal mannose residue which renders this structure fully resistant to cleavage by ⁇ 1-2 Mannosidase.
- the thick upward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase and the empty downward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ -linked mannoses. See also Example 26.
- Figure 61 This figure shows a higher oligomannose consisting of 9 mannose moieties coupled to chitobiose. In this oligomannose, one terminal mannose residue is linked by an ⁇ 1-2 bond to the non-terminal mannose residue.
- the thin upward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ 1-2 Mannosidase
- the thick upward or leftward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ 1-2-linked mannoses
- the empty downward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ -linked mannoses. See also Example 26.
- FIG 62 This figure shows the putative glucose-containing oligosaccharide consisting of 1 or 2 glucose moieties and 8 mannose moieties coupled to chitobiose.
- this oligosaccharide either one of the terminal ⁇ 1-2-linked mannose residues of the A- or B-branch ("A - " and "B - * " in the Figure) is carrying one or two glucose residues, as indicated by (Glc)Glc to the left of the of the bracket.
- the thin upward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ 1-2 Mannosidase given that no glucose is attached to the terminal mannose residue.
- the thick upward or leftward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ 1-2-linked mannoses and the empty downward pointing arrow indicates the oligosaccharide bonds which are prone to cleavage by ⁇ Mannosidase after removal of the ⁇ -linked mannoses.
- An overview of the possible reaction products is given in Table 10 of Example 26.
- the reaction products of Man-9 after overnight incubation with or without exoglycosidases were separated on a TSK gel-Amide-80 (0.46 x 25 cm, Tosoh Biosep) column coupled to a Waters Alliance HPLC station.
- Separation of the oligosaccharides was carried out at ambient temperature at 1.0 mL/min.
- Solvent A consisted of 0.1% acetic acid in acetonitrile and solvent B consisted of 0.2 % acetic acid-0.2 % triethylamine in water.
- Separation of 2-AB labeled oligosaccharides was carried out using 28 % B isocratic for 5 column volumes followed by a linear increase to 45 % B over fifteen column volumes.
- the composition of the elution solvent is indicate on the right Y-axis as % solvent B in solvent A (v/v).
- the elution time is indicated in minutes on the X-axis.
- the left Y-axis indicates fluorescence of eluting 2-aminobenzamide (2-AB)-labeled oligosaccharides.
- the excitation wavelength of 2-AB is 330 nm, the emission wavelength 420 nm.
- Trace 1 of the chromatogram shows the elution of Man-9 incubated overnight without exoglycosidases.
- Trace 2 shows the elution of a mixture of Man-5 and Man-6 after overnight incubation of Man-9 with ⁇ 1-2 Mannosidase.
- Traces 3 and 4 (“3” and "4") show the elution of 4'- ⁇ -mannosyl chitobiose after 1 h and overnight incubation, respectively, of Man-9 with ⁇ -Mannosidase.
- Trace 5 (“5") shows the elution of chitobiose after overnight incubation of Man-9 with ⁇ - and ⁇ -Mannosidase.
- Traces 1-5 are represented as overlays, as such their respective baselines are not all on the zero level.
- Trace 6 (“6") indicates the applied solvent gradient.
- the peaks, when present, indicated by the letters A to K on top of the Figure represent: A, chitobiose; B, 4'- ⁇ -mannosyl-chitobiose; C, Man-2; D, Man-3; E, Man-4; F, Man-5; G, Man- 6; H, Man-7; I, Man-7; J, Man-8; and K, Man-10. See also Example 26.
- Figure 64 The reaction products of the oligosaccharides derived from Saccharomyces- produced Els after overnight incubation with or without exoglycosidases were separated on a TSK gel-Amide-80 (0.46 x 25 cm, Tosoh Biosep) column coupled to a Waters Alliance HPLC station. Separation of the oligosaccharides was carried out at ambient temperature at 1.0 mL/min. Solvent A consisted of 0.1 % acetic acid in acetonitrile and solvent B consisted of 0.2 % acetic acid-0.2 % triethylamine in water.
- Trace 1 of the chromatogram shows the elution of oligosaccharides derived from S ⁇ ccA ⁇ rorayces-produced Els incubated overnight without exoglycosidases.
- Trace 2 shows the elution of oligosaccharides derived from Saccharomyces-produced Els after overnight incubation of Man-9 with ⁇ 1-2 Mannosidase.
- Traces 3 and 4 (“3" and "4") show the elution of oligosaccharides derived from Saccharomyces-produced Els after 1 h and overnight incubation, respectively, with ⁇ -Mannosidase.
- Trace 5 (“5") shows the elution of oligosaccharides derived from Saccharomyces-produced Els after overnight incubation with ⁇ - and ⁇ -Mannosidase. Traces 1-5 are represented as overlays, as such their respective baselines are not all on the zero level. Trace 6 (“6") indicates the applied solvent gradient. The peaks, when present, indicated by the letters A to K on top of the Figure represent: A, chitobiose; B, 4'- ⁇ -mannosyl-chitobiose; C, Man-2; D, Man-3; E, Man-4; F, Man-5; G, Man- 6; H, Man-7; I, Man-7; J, Man-8; and K, Man-10. See also Example 26.
- Figure 65 The reaction products of the oligosaccharides derived from Els produced in vaccinia-transfected mammalian cells after overnight incubation with or without exoglycosidases were separated on a TSK gel-Amide-80 (0.46 x 25 cm, Tosoh Biosep) column coupled to a Waters Alliance HPLC station. Separation of the oligosaccharides was carried out at ambient temperature at 1.0 mL/min. Solvent A consisted of 0.1% acetic acid in acetonitrile and solvent B consisted of . 0.2 % acetic acid-0.2 % triethylamine in water.
- Trace 1 of the chromatogram shows the elution of oligosaccharides derived from Els produced in vaccinia-transfected mammalian cells incubated overnight without exoglycosidases.
- Trace 2 shows the elution of oligosaccharides derived from Els produced in vaccinia-transfected mammalian cells after overnight incubation of Man-9 with ⁇ 1-2 Mannosidase.
- Traces 3 and 4 (“3" and "4") show the elution of oligosaccharides derived from Els produced in vaccinia-transfected mammalian cells after 1 h and overnight incubation, respectively, with ⁇ -Mannosidase.
- Trace 5 (“5") shows the elution of oligosaccharides derived from Els produced in vaccinia-transfected mammalian cells after overnight incubation with ⁇ - and ⁇ -Mannosidase. Traces 1-5 are represented as overlays, as such their respective baselines are not all on the zero level. Trace 6 (“6") indicates the applied solvent gradient.
- peaks, when present, indicated by the letters A to K on top of the Figure represent: A, chitobiose; B, 4'- ⁇ -mannosyl-chitobiose; C, Man-2; D, Man-3; E, Man-4; F, Man-5; G, Man- 6; H, Man-7; I, Man-7; J, Man-8; and K, Man-10. See also Example 26.
- Figure 66 The reaction products of the oligosaccharides derived from Hansenula-produced Els after overnight incubation with or without exoglycosidases were separated on a TSK gel- Amide-80 (0.46 x 25 cm, Tosoh Biosep) column coupled to a Waters Alliance HPLC station. Separation of the oligosaccharides was carried out at ambient temperature at 1.0 mL/min. Solvent A consisted of 0.1% acetic acid in acetonitrile and solvent B consisted of 0.2 % acetic acid-0.2 % triethylamine in water.
- Trace 1 of the chromatogram shows the elution of oligosaccharides derived from Hansenula-produced Els incubated overnight without exoglycosidases.
- Trace 2 shows the elution of oligosaccharides derived from Hansenula-produced Els after overnight incubation of Man-9 with ⁇ 1-2 Mannosidase.
- Traces 3 and 4 (“3" and "4") show the elution of oligosaccharides derived from Hansenula-produced Els after overnight incubation with ⁇ - Mannosidase.
- Trace 5 (“5") shows the elution of oligosaccharides derived from Hansenula - produced Els after overnight incubation with ⁇ - and ⁇ -Mannosidase. Traces 1-5 are represented as overlays, as such their respective baselines are not all on the zero level. Trace 6 (“6") indicates the applied solvent gradient. The peaks, when present, indicated by the letters A to K on top of the Figure represent: A, chitobiose; B, 4'- ⁇ -mannosyl-chitobiose; C, Man-2; D, Man-3; E, Man-4; F, Man-5; G, Man- 6; H, Man-7; I, Man-7; J, Man-8; and K, Man-10. See also Example 26.
- Figure 67 SDS-PAGE analysis and Coomassie Brilliant Blue staining of El proteins produced by Hansenula and by a HCN-recombinant vaccinia virus-infected mammalian cells.
- Lane 1 molecular weight markers with molecular weights indicated at the left;
- Lane 2 alkylated Els produced by Hansenula polymorpha (10 ⁇ g);
- Lane 3 alkylated Els produced by Hansenula polymorpha (5 ⁇ g);
- Lane 4 alkylated Els produced by Hansenula polymorpha (2.5 ⁇ g);
- Lane 5 alkylated Els produced by HCN-recombinant vaccinia virus-infected vero cells (10 ⁇ g);
- Lane 6 alkylated Els produced by HCN-recombinant vaccinia virus-infected vero cells (5 ⁇ g);
- Lane 7 alkylated Els produced by HCN-recombinant vaccinia virus- infected vero cells (2.5 ⁇
- Figure 68 Sequence of HCN E2-H6 protein (SEQ ID ⁇ O:5) with indication of tryptic fragments (boxed sequences) of the deglycosylated protein.
- the glycosylated Asn-residues are converted into Asp-residues by the PNGase F enzyme and are indicated with a "*" under the sequence.
- the Asn-residues are prone to proteolytic cleavage by the Asp-N endoproteinase.
- the possible N-glycosylation sites in E2-H6 are N 17 , N 423 , N 430 , N 8 , N 78 , N 532 , N 540 , N 556 , N 576 , N 623 and N 645 according to the numbering in the HCN polyprotein; these sites are numbered ⁇ 3 , N 0 , N , N 65 , N 95 , N 149 , N 157 , N 1 , N 193 , N 2 0 and N 262 in this figure. See also Example 28.
- glycosylated HCN envelope proteins in Saccharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha was possible by expression of said HCN envelope proteins as proteins comprising a signal peptide sequence joined to said HCN envelope proteins.
- the glycosylation patterns of the HCN envelope proteins expressed in these three yeast species were, however, very different (see Examples 6, 10, 13 and 25). More specifically the S. cerevisiae (glycosylation deficient mutant)- and H. polymorpha-expressed HCN envelope proteins were glycosylated in a manner resembling core-glycosylation.
- the HCN envelope proteins expressed in Pichia pastoris were hyperglycosylated despite earlier reports that proteins expressed in this yeast are normally not hyperglycosylated (Gellissen et al. 2000, Sugrue et al. 1997).
- HCN proteins produced in S. cerevisiae glycosylation deficient strain
- H polymorpha glycosylation deficient strain
- H-recombinant vaccinia virus-infected mammalian cells it was surprisingly found that the Hansenula-produced HCN envelope proteins displayed a glycosylation pattern which is very advantageous for diagnostic, prophylactic and therapeutic application of these HCN envelope proteins (see Examples 21-24 and 26-29). This unexpected finding is reflected in the different aspects and embodiments of the present invention as presented below.
- a first aspect of the invention is related to an isolated HCN envelope protein or a fragment thereof comprising at least one ⁇ -glycosylation site, said protein or fragment thereof characterized in that it is the product of expression in a eukaryotic cell and further characterized in that on average up to 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% of the ⁇ -glycosylated sites are core-glycosylated.
- the ratio of sites core- glycosylated with an oligomannose with structure Man(7)-GlcNAc(2) over the sites core- glycosylated with an oligomannose with structure Man(8)-GlcNAc(2) is less than or equal to 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.44, 0.45, or 0.50.
- said oligomannoses contain less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% terminal ⁇ l,3 mannose.
- N-glycosylated sites glycosylated with an oligomannose with a structure defined by Man(8 to 10)-GlcNAc(2) is meant that said N-glycosylated sites are glycosylated with either one of Man(8)-GlcNAc(2), Man(9)-GlcNAc(2), or Man(10)-GlcNAc(2).
- protein refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and polypeptides are included within the definition of protein. This term also does not refer to or exclude post-expression modifications of the protein, for example, glycosylations, acetylations, phosphorylations and the like.
- polypeptides containing one or more analogues of an arnino acid including, for example, unnatural amino acids, PNA, etc.
- polypeptides with substituted linkages as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
- pre-pro-protein or "pre-protein” is, when used herein, meant a protein comprising a pre-pro-sequence joined to a protein of interest or a protein comprising a pro- sequence joined to a protein of interest, respectively.
- pre-sequence the terms “signal sequence”, “signal peptide”, “leader peptide”, or “leader sequence” are used; all refer to an amino acid sequence that targets a pre-protein to the rough endoplasmic reticulum (ER) which is a prerequisite for (N-)glycosylation.
- the "signal sequence”, “signal peptide”, “leader peptide”, or “leader sequence” is cleaved off, i.e.
- pre-pro-protein is converted to a pro-protein upon translocation to the lumen of the ER.
- pro amino acid sequence
- a well known pre-pro-amino acid sequence is the ⁇ mating factor pre-pro- sequence of the S. cerevisiae a mating factor.
- HCV envelope protein is meant a HCV El or HCV E2 envelope protein or a part thereof whereby said proteins may be derived from a HCN strain of any genotype. More specifically, HCNE ⁇ N is chosen from the group of amino acid sequences consisting of SEQ ID NOs:85 to 98, amino acid sequences wliich are at least 90% identical to SEQ ID NOs:85 to98, and fragments of any thereof. As “identical” amino acids are considered the groups of conserved amino acids as described above, i.e.
- HCN envelope proteins relates to a polypeptide or an analogue thereof (e.g. mimotopes) comprising an amino acid sequence (and/or amino acid analogues) defining at least one HCN epitope of either the El or the E2 region, in addition to a glycosylation site.
- These envelope proteins may be both monomeric, hetero-oligomeric or homo-oligomeric forms of recombinantly expressed envelope proteins.
- sequences defining the epitope correspond to the amino acid sequences of either the El or the E2 region of HCN (either identically or via substitutions of analogues of the native amino acid residue that do not destroy the epitope). It will be understood that the HCN epitope may co-locate with the glycosylation site.
- the epitope-defining sequence will be 3 or 4 amino acids in length, more typically, 5, 6, or 7 amino acids in length, more typically 8 or 9 amino acids in length, and even more typically 10 or more amino acids in length.
- the length of the epitope-defining sequence can be subject to wide variations, since it is believed that these epitopes are formed by the three-dimensional shape of the antigen (e.g. folding).
- the amino acids defining the epitope can be relatively few in number, but widely dispersed along the length of the molecule being brought into the correct epitope conformation via folding.
- the portions of the antigen between the residues defining the epitope may not be critical to the conformational structure of the epitope.
- a conformational epitope may also be formed by 2 or more essential regions of subunits of a homo-oligomer or hetero-oligomer.
- an epitope of a designated polypeptide denotes epitopes with the same amino acid sequence as the epitope in the designated polypeptide, and immunologic equivalents thereof.
- the amino acids constituting the epitope need not be part of a linear sequence, but may be interspersed by any number of amino acids, thus forming a conformational epitope.
- the HCN antigens of the present invention comprise conformational epitopes from the
- El and/or E2 (envelope) domains of HCN are believed to correspond to the viral envelope protein.
- the El domain which is believed to correspond to the viral envelope protein, is currently estimated to span amino acids 192-383 of the HCN polyprotein (Hijikata et al., 1991).
- the E2 protein previously called ⁇ S1, is believed to span amino acids 384-809 or 384-746 (Grakoui et al., 1993) of the HCN polyprotein and also to be an envelope protein.
- the E2 protein may also be expressed together with El, and/or core (aa 1-191), and/or P7 (aa 747-809), and or ⁇ S2 (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 HCN proteins different from E2 .
- the El protein may also be expressed together with the E2, and/or core (aa 1-191), and/or P7 (aa 747-809), and/or ⁇ S2 (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 El. Expression together with these other HCN proteins may be important for obtaining the correct protein folding.
- El as used herein also includes analogs and truncated forms that are immunologically cross-reactive with natural El, and includes El proteins of genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 or any other newly identified HCN type or subtype.
- '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 HCN 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 by Kato et al. (1992).
- HCN proteins that are co-expressed with the HCN envelope proteins of the present invention can be derived from any HCN type, thus also from the same type as the HCN envelope proteins of the present invention.
- E1/E2 refers to an oligomeric form of envelope proteins containing at least one El component and at least one E2 component.
- specific oligomeric El and/or E2 and/or E1/E2 envelope proteins refers to all possible oligomeric forms of recombinantly expressed El and/or E2 envelope proteins wliich are not aggregates. El and/or E2 specific oligomeric envelope proteins are also referred to as homo-oligomeric El or E2 envelope proteins (see below).
- the term 'single or specific oligomeric' El and/or E2 and/or E1/E2 envelope proteins refers to single monomeric El or E2 proteins (single in the strict sense of the word) as well as specific oligomeric El and/or E2 and/or E1/E2 recombinantly expressed proteins.
- homo-oligomer refers to a complex of El or E2 containing more than one El or E2 monomer, e.g. El/El dimers, El/El/El trimers or El/El/El/El tetramers and E2/E2 dimers, E2/E2/E2 trimers or E2/E2/E2/E2 tetramers, El pentamers and hexamers, E2 pentamers and hexamers or any higher-order homo.oligomers of El or E2 are all 'homo-oligomers' within the scope of this definition.
- the oligomers may contain one, two, or several different monomers of El or E2 obtained from different types or subtypes of hepatitis C virus including for example those described by Maertens et al. in WO94/25601 and WO96/13590, 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 HCN.
- the El 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).
- 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).
- the HCN 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 HCN genotypes, it may be inferred that not all 6 glycosylation sites on the HCN El protein are required for proper folding and reactivity.
- glycosylation site at position 325 is not modified by ⁇ -glycosylation (Fournillier- Jacob et al. 1996, Meunier et al. 1999).
- HCN subtype lb El 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 lb, 6a, 7, 8, and 9, is absent in all other types know today. This sugar-addition motif may be mutated to yield a type lb El protein with improved reactivity.
- 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 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 can be achieved by either mutating the codons for ⁇ , S, or T, in such a way that these codons encode amino acids different from ⁇ in the case of ⁇ , and/or amino acids different from S or T in the case of S and in the case of T.
- the X position may be mutated into P, since it is known that ⁇ PS or ⁇ PT are not frequently modified with carbohydrates.
- the present invention relates to HCV envelope proteins, or parts thereof that are core-glycosylated.
- core-glycosylation refers to a structure "similar” to the structure as depicted in the boxed structure in Figure 3 of Herscovics and Orlean (1993).
- the carbohydrate structure referred to contains 10 to 11 monosaccharides.
- said disclosure is herein incorporated by reference.
- similar intends that not more than 4 additional monosaccharide has been added to the structure or that not more than about 3 monosaccharides have been removed from the structure.
- the core-glycosylation carbohydrate structure referred to in the current invention consists minimally of 7 and maximally of 15 monosaccharides and can consist of 8, 9, 10, 11, 12, 13 or 14 monosaccharides.
- the monosaccharides connoted are preferentially glucose, mannose or N-acetyl glucosamine.
- An alternative aspect of the current invention relates to an isolated HCV envelope protein or a fragment thereof comprising at least one N-glycosylation site, said protein or fragment thereof characterized in that it is the product of expression in a eukaryotic cell and further characterized in that more than 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95 % of the N-glycosylated sites are glycosylated with an oligomannose with a structure defined by Man(8 to 10)-GlcNAc(2).
- the ratio of sites core-glycosylated with an oligomannose with structure Man(7)-GlcNAc(2) over the sites core-glycosylated with an oligomannose with structure Man(8)-GlcNAc(2) is less than or equal to 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.44, 0.45, or 0.50.
- said oligomannoses contain less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% terminal ⁇ l,3 mannose.
- Another alternative aspect of the invention is related to an isolated HCV envelope protein or a fragment thereof comprising at least one N-glycosylation site, said protein or fragment thereof characterized in that it is the product of expression in a eukaryotic cell and further characterized in that N-glycosylated sites are occupied by oligomannoses wherein the ratio of the oligomannoses with structure Man(7)-GlcNAc(2) over the oligomannoses with structure Man(8)-GlcNAc(2) is less than or equal to 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.44, 0.45, or 0.50.
- said oligomannoses contain less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% terminal ⁇ l,3 mannose.
- an isolated HCV envelope protein or a fragment thereof comprising at least one N-glycosylation site said protein or fragment thereof characterized in that it is the product of expression in a non- mammalian eukaryotic cell and further characterized in that the number of N-glycosylated sites is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, less than the number of N-glycosylated sites in the same protein or fragment thereof expressed from a vaccinia virus in a eukaryotic cell liable of being infected with said vaccinia virus. More particular to the above N-glycosylation characteristic, up to 50%, 51%, 52%), 53%, 54%, 55%,
- N-glycosylated sites are core- glycosylated.
- the ratio of sites core-glycosylated with an oligomannose with structure Man(7)-GlcNAc(2) over the sites core-glycosylated with an oligomannose with structure Man(8)-GlcNAc(2) is less than or equal to 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50.
- said oligomannoses contain less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% terminal ⁇ l,3 mannose.
- the isolated HCV envelope protein or part thereof according to the invention is the product of expression in a yeast cell. More particularly, the isolated HCV envelope protein or part thereof according to the invention is the product of expression in 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.
- Saccharomyces such as Saccharomyces cerevisiae, Saccharo
- the isolated HCV envelope protein or part thereof according to the invention is the product of expression in a Hansenula cell. Even more specifically, the isolated HCV envelope protein or part thereof according to the invention is the product of expression in a yeast, e.g. Hansenula, cell in the absence of glycosylation inhibitors such as tunicamycin.
- the isolated HCV envelope protein or part thereof according to the invention is derived from a protein comprising an avian lysozyme leader peptide or a functional variant thereof joined to said HCV envelope protein or fragment thereof. More specifically, the isolated HCV envelope protein or part thereof according to the invention is derived from a protein characterized by the structure CL-[(Al) a - (PSl) b - (A2) c ]-HCVENV-[(A3) d - (PS2) e - (A4) f ] wherein:
- CL is an avian lysozyme leader peptide or a functional equivalent thereof
- Al, A2, A3 and A4 are adaptor peptides wliich can be different or the same
- PSI 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
- Al and/or A2 are part of PSI and/or wherein A3 and/or A4 are part ofPS2.
- an avian leader peptide or a functional equivalent thereof joined to a HCV envelope protein or a part thereof is meant that the C-terminal amino acid of said leader peptide is covalently linked via a peptide bond to the N-terminal amino acid of said HCV envelope protein or part thereof.
- the C-terminal amino acid of said leader peptide is separated from the N-terminal amino acid of said HCV envelope protein or part thereof by a peptide or protein.
- Said peptide or protein may have the structure -[(Al) a - (PSl) b - (A2) c ] as defined above.
- the derivation of the HCV envelope protein of interest from the protein comprising an avian lysozyme leader peptide or a functional equivalent thereof joined to an HCV envelope protein or a part thereof or of the protein characterized by the structure CL-[(Al) a - (PSl) b - (A2) c ]-HCVENV-[(A3) d - (PS2) e - (A4) f ] can be performed in vivo by the proteolytic machinery of the cells in which the pre-protein protein is expressed. More specifically, the step consisting of removal of the avian leader peptide is preferably performed in vivo by the proteolytic machinery of the cells in which the pre-protein is expressed.
- Derivation may, however, also be performed solely in vitro after and/or during isolation and/or purification of the pre-protein and/or protein from the cells expressing the pre-protein and/or from the culture fluid in which the cells expressing the pre-protein are grown.
- said in vivo derivation is performed in combination with said in vitro derivation.
- Derivation of the HCV protein of interest from a recombinantly expressed pre-protein can further comprise the use of (an) proteolytic enzyme(s) in a polishing step wherein all or most of the contaminating proteins co-present with the protein of interest are degraded and wherein the protein of interest is resistant to the polishing proteolytic enzyme(s).
- HCV Els protein of HCV genotype lb SEQ ID NO:2
- wliich is devoid of Lys-residues.
- endoproteinase Lys-C Endo-lys C
- the El proteins will not be degraded whereas contaminating proteins containing one or more Lys-residues are degraded.
- Such a process may significantly simplify or enhance isolation and/or purification of the HCV El proteins.
- an additional Lys-residue e.g.
- HCV El proteins may comprise a Lys-residue at either one or more of the positions 4, 40, 42, 44, 61, 65 or 179 (wherein position 1 is the first, N-terminal natural amino acid of the El protein, i.e. position 192 in the HCV polyprotein).
- said Lys-residues may be mutated into another amino acid residue, preferably into an Arg-residue.
- leader peptide is removed from the protein comprising the signal sequence joined to a protein of interest with high efficiency, i.e. a large number of pre-(pro-)proteins is converted to (pro-)proteins, and with high fidelity, i.e. only the pre-amino acid sequence is removed and not any amino acids of the protein of interest joined to said pre-amino acid sequence.
- removal of a leader peptide with high efficiency is meant that at least about 40%, but more preferentially about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or even 99% of the pre- proteins is converted to the protein from which the pre-sequence is removed.
- these pre-proteins may still be purified or be removed during purification.
- avian lysozyme (CL) leader peptide a CL leader peptide wherein one or more amino acids have been substituted for another amino acid and whereby said substitution is a conservative amino acid substitution.
- conservative amino acid substitution is meant a substitution of an amino acid belonging to a group of conserved amino acids with another amino acid belonging to the same group of conserved amino acids.
- a or "adaptor peptide” is meant a peptide (e.g. 1 to 30 amino acids) or a protein which may serve as a linker between e.g. a leader peptide and a processing site (PS), a leader peptide and a protein of interest, a PS and a protein of interest, and/or a protein of interest and a PS; and/or may serve as a linker N- or C-terminal of e.g. a leader peptide, a PS or a protein of interest.
- the adaptor peptide "A” may have a certain three-dimensional structure, e.g. an ⁇ -helical or ⁇ -sheet structure or a combination thereof.
- the three-dimensional structure of A is not well defined, e.g. a coiled-coil structure.
- the adaptor A may be part of e.g. a pre-sequence, a pro-sequence, a protein of interest sequence or a processing site.
- the adaptor A may serve as a tag enhancing or enabling detection and/or purification and/or processing of the protein of which A is a part.
- An A peptide is the his-tag peptide (HHHHHH; SEQ ID NO:63) Hn wherein n usually is six, but may be 7, 8, 9, 10, 11, or 12.
- Other examples of A-peptides include the peptides EEGEPK (Kjeldsen et al.
- Adaptor peptides are given in SEQ ID NOs:63-65, 70-72 and 74-82.
- Another example of an adaptor peptide is the G4S immunosilent linker.
- Other examples of adaptor peptides or adaptor proteins are listed in Table 2 of Stevens (Stevens et al. 2000).
- PS protein processing or processable site. Said processing may occur enzymatically or chemically.
- processing sites prone to specific enzymatic processing include IEGR- X (SEQ ID NO:66), IDGR-iX (SEQ ID NO:67), AEGR-iX (SEQ ID NO:68), all recognized by and cleaved between the Arg and Xaa (any amino acid) residues as indicated by the "*! by the bovine factor Xa protease (Nagai, K. and Thogersen, H. C. 1984).
- Another example of a PS site is a dibasic site, e.g.
- the PS site may also be a monobasic Lys-site. Said monobasic Lys-PS-site may also be included at the C-terminus of an A peptide. Examples of A adaptor peptides comprising a C-terminal monobasic Lys-PS-site are given by SEQ ID NOs:64-65 and 74-76.
- Exoproteolytic removal of a His-tag is possible by using the dipeptidyl aminopeptidase I (DAPase) alone or in combination with glutamine cyclotransferase (Qcyclase) and pyroglutamic aminopeptidase (pGAPase) (Pedersen, J. et al. 1999).
- DAPase dipeptidyl aminopeptidase I
- Qcyclase glutamine cyclotransferase
- pGAPase pyroglutamic aminopeptidase
- Said exopeptidases comprising a recombinant His-tag (allowing removal of the peptidase from the reaction mixture by immobilize metal-affinity chromatography, IMAC) are commercially available, e.g. as the TAGZyme System of Unizyme Laboratories (H ⁇ rsholm, DK).
- processing is thus generally meant any method or procedure whereby a protein is specifically cleaved or cleavable at at least one processing site when said processing site is present in said protein.
- a PS may be prone to endoproteolytic cleavage or may be prone to exproteolytic cleavage, in any case the cleavage is specific, i.e. does not extend to sites other than the sites recognized by the processing proteolytic enzyme.
- a number of PS sites are given in SEQ ID NOs:66-68 and 83-84.
- the versatility of the [(Al/3) a/d - (PSl/2)b/ e - (A2/4) c/f ] structure as outlined above is demonstrated by means of some examples.
- the HCV protein of interest can (optionally) be purified by IMAC.
- the (optionally purified) HCV protein of interest will carry at its C- terminus a processed PS site which is "IEGR" (SEQ ID NO:70).
- Variant processed factor Xa processing site can be IDGR (SEQ ID NO:71) or AEGR (SEQ ID NO.J2).
- the [(Al/3) a/d - (PSl/2) b/e - (A2/4) c /f] structure is present at the N-terminus of the HCV protein of interest.
- Al is the histidine-tag (SEQ ID NO:63)
- the resulting HCV protein of interest can be purified by IMAC (optional).
- IMAC optionally used to purify the protein of interest.
- the protein of interest will be devoid of the [(Al) a - (PSl) b - (A2) c ] structure. It will furthermore be clear that any of Al, A2, A3, A4, PSI and PS2, when present, may be present in a repeat structure.
- Such a repeat structure when present, is in this context still counted as 1, i.e. a, b, c, d, e, or f are 1 even if e.g. Al is occurring as e.g. 2 repeats (Al- Al).
- Yet another aspect of the current invention relates to any of the isolated HCV envelope protein or fragment thereof according to the invention in which the cysteine thiol- groups are chemically modified.
- Yet another aspect of the current invention relates to any of the isolated HCV envelope protein or fragment thereof according to the invention which is antigenic. Yet another aspect of the current invention relates to any of the isolated HCV envelope protein or fragment thereof according to the invention which is immunogenic.
- Yet another aspect of the current invention relates to any of the isolated HCV envelope protein or fragment thereof according to the invention which comprises a T-cell stimulating epitope.
- Another aspect of the current invention relates to any of the isolated HCV envelope protein or fragment thereof according to the invention which is comprised in a structure chosen from the group consisting of monomers, homodimers, heterodimers, homo-oligomers and hetero-oligomers.
- Yet another aspect of the current invention relates to any of the isolated HCV envelope protein or fragment thereof according to the invention wliich is comprised in a viruslike particle.
- the cysteine thiol-groups can be irreversibly protected by chemical or enzymatic means.
- “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.
- Alkylation can be performed by any method known in the art, such as, for example, active halogens X(CH 2 ) n R in which X is a halogen such as I, Br, CI or F.
- active halogens are methyliodide, iodoacetic acid, iodoacetamide, and 2- bromoethylamine.
- alkylation examples 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 2 -CH 2 -NH 2 (Hermanson, G. T. 1996).
- alkylating agents refers to compounds which are able to perform alkylation as described herein. Such alkylations finally result in a modified cysteine, which can mimic other aminoacids.
- 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).
- the usage of methyliodide results in an amino acid resembling methionine
- the usage of iodoacetate and iodoacetamide results in amino acids resembling glutamic acid and glutamine, respectively.
- these amino acids are preferably used in direct mutation of cysteine.
- the present mvention 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.
- mutated refers to site-directed mutagenesis of nucleic acids encoding these amino acids, ie to the well kown 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.
- 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.
- the sulfur-containing functional group eg thiols and disulfides
- the sulfur-containing functional group 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 disulfi.de bonds, such as, for example
- reversible protection or "reversible blocking” as used herein contemplates covalently binding of modification agents to the cysteine thiol-groups, as well as manipulating the enviromnent 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.
- reversible protection by enzymatical means contemplates reversible protection mediated by enzymes, such as for example acyl-transferases, e.g. acyl- transferases that are involved in catalysing thio-esterification, such as palmitoyl acyltransf erase (see below).
- enzymes such as for example acyl-transferases, e.g. acyl- transferases that are involved in catalysing thio-esterification, such as palmitoyl acyltransf erase (see below).
- reversible protection by chemical means 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 - RS-SO 3 " (Darrow, A. 1986) or RS-SR-> 2 RS-SO 3 " (sulfitolysis; (Kumar, N. et al. 1986)).
- Reagents for sulfonation are e.g. Na 2 SO 3 , or sodium tetrathionate. The latter reagents for sulfonation are used in a concentration of 10-
- sulfonation can be performed in the presence of a catalysator such as, for example Cu 2+ (100 ⁇ M-1 mM) or cysteine (1-10 mM).
- a catalysator such as, for example Cu 2+ (100 ⁇ 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:
- modification agents that reversibly modify the cysteinyls of the present invention such as, for example, by heavy metals, in particular Zn 2+ ', Cd 2+ , mono-, dithio- and disulfide- compounds (e.g. aryl- and alkylmethanethiosulfonate, dithiopyridine, dithiomorpholine, dihydrolipoamide, Ellmann reagent, aldrothiolTM (Aldrich) (Rein, A. et al.
- Dithiocarbamate comprise a broad class of molecules possessing an R- .
- R 2 NC(S)SR 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;
- modification agents that preserve the thiol status 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 ⁇ M-10 mM, more preferentially in a concentration of 1-10 mM;
- thiol stabilising conditions such as, for example, (i) cofactors as metal ions (Zn 2+ ,
- pH control e.g. for proteins in most cases pH ⁇ 5 or pH is preferentially thiol pK a -2; e.g. for peptides purified by Reversed Phase Chromatography at pH ⁇ 2.
- combination compounds can be used, such as, for example Z103 (Zn carnosine), preferentially in a concentration of 1-10 mM.
- 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.
- 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.
- 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 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.
- enzymes in particular thioesterases, glutaredoxine, thioredoxine, in particular in a concentration of 0.01-5 ⁇ M, even more particular in a concentration range of 0.1-5 ⁇ 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 3 " - * RSH).
- An antioxidant is any reagent which preserves the thiol status or rmnimises "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 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), DitMoerythritol (DTE), ⁇ -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 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.
- immunogenic refers to the ability of a protein or a substance to produce an immune response.
- the immune response is the total response of a body to the introduction of an antigen, including antibody formation, cellular immunity, hypersensitivity, or immunological tolerance.
- Cellular immunity refers to a T-helper cell- and/or CTL-response.
- antigenic refers to the ability of a protein or a substance to causes the formation of an antibody or to elicit a cellular response.
- T-cell stimulating epitope refers to an epitope capable of stimulating T-cells or CTL-cells, respectively.
- a T-helper cell stimulating epitope may be selected by monitoring the lymphoproliferative response towards polypeptides containing in their amino acid sequence a (putative) T-cell stimulating epitope. Said lymphoproliferative response may be measured by either a T-helper assay comprising in vitro stimulation of peripheral blood mononuclear cells (PMBCs) from patient sera with varying concentrations of peptides to be tested for T-cell stimulating activity and counting the amount of radiolabelled thymidine uptake.
- PMBCs peripheral blood mononuclear cells
- a CTL-stimulating epitope may be selected by means of a cytotoxic T-cell (CTL) assay measuring the lytic activity of cytotoxic cells using 51 Cr release. Proliferation is considered positive when the stimulation index (mean cpm of antigen-stimulated cultures/mean cpm of controle cultures) is more than 1, preferably more than 2, most preferably more than 3.
- CTL cytotoxic T-cell
- 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 mvention may be prophylactic composition or a therapeutic composition.
- 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.
- 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.
- 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, OR, USA), avidin or streptavidin.
- 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.
- RIBI ImmunoChem Research Inc., Hamilton, MT, USA
- MPL 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.
- adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA, 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 inulin-based adjuvants, such as Algammulin and Gammalnulin (Anutech), In
- 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.
- 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.
- a vaccine composition is prepared as an injectable, either as a liquid solution or suspension. Injection may be subcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal, intradermal.
- admimstration 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 Hposomes 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.
- the amount will vary from 0.01 to 1000 ⁇ g/dose, more particularly from 0.1 to 100 ⁇ 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.
- Another aspect of the current invention relates to a method for producing the isolated HCV envelope protein or fragment thereof according to the invention.
- Said method for producing an HCV envelope protein or part thereof is e.g. comprising transformation of a host cell with a recombinant nucleic acid or vector comprising an open reading frame encoding said HCV envelope protein or part thereof, and wherein said host cell is capable of expressing said HCV envelope protein or part thereof.
- Said method 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.
- chaotropic agents are guanidinium chloride and urea.
- 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
- recombinant nucleic acid is intended a nucleic acid of natural or synthetic origin which has been subjected to at least one recombinant DNA technical manipulation such as restriction enzyme digestion, PCR, ligation, dephosphorylation, phosphorylation, mutagenesis, adaptation of codons for expression in a heterologous cell etc.
- a recombinant nucleic acid is a fragment of a naturally occurring nucleic acid or comprises at least two nucleic acid fragments not naturally associated or is a fully synthetic nucleic acid.
- polynucleotide refers to nucleotides, either ribonucleotides, deoxyribonucleotides, peptide nucleotides or locked nucleotides, or a combination thereof, in a polymeric form of any length or any shape (e.g. branched DNA). Said terms furthermore include double-stranded (ds) and single- stranded (ss) polynucleotides as well as triple-stranded polynucleotides.
- ds double-stranded
- ss single- stranded
- Said terms also include known nucleotide modifications such as methylation, cyclization and 'caps' and substitution of one or more of the naturally occurring nucleotides with an analog such as inosine or with non-amplifiable monomers such as HEG (hexethylene glycol).
- Ribonucleotides are denoted as NTPs, deoxyribonucleotides as dNTPs and dideoxyribonucleotides as ddNTPs.
- Nucleotides can generally be labeled radioactively, chemiluminescently, fluorescently, phosphorescently or with infrared dyes or with a surface-enhanced Raman label or plasmon resonant particle (PRP).
- PRP surface-enhanced Raman label or plasmon resonant particle
- polynucleotide also encompass peptide nucleic acids (PNAs), a DNA analogue in which the backbone is a pseudopeptide consisting of N-(2-aminoethyl)-glycine units rather than a sugar.
- PNAs peptide nucleic acids
- the neutral backbone of PNA results in stronger binding and greater specificity than normally achieved.
- PNA probes can generally be shorter than DNA probes and are generally from 6 to 20 bases in length and more optimally from 12 to 18 bases in length (Nielsen, P. E. 2001). Said terms further encompass locked nucleic acids (LNAs) which are RNA derivatives in which the ribose ring is constrained by a methylene linkage between the 2'-oxygen and the 4'-carbon. LNAs display unprecedented binding affinity towards DNA or RNA target sequences.
- LNAs locked nucleic acids
- LNA nucleotides can be oligomerized and can be incorporated in chimeric or mix-meric LNA/D A or LNA/RNA molecules. LNAs seem to be nontoxic for cultured cells (Orum, H. and Wengel, J. 2001, Wahlestedt, C. et al. 2000). In general, chimeras or mix-mers of any of DNA, RNA, PNA and LNA are considered as well as any of these wherein thymine is replaced by uracil. It is clear from the above that the present invention also relates to the use of a core- glycosylated HCV envelope proteins according to the invention or a composition according to the invention for the manufacture of an HCV vaccine composition.
- the present invention relates to the use of a core-glycosylated HCV envelope protein according to the invention for inducing immunity against HCV in chronic HCV carriers. More in particular, the present invention relates to the use of a core-glycosylated HCV envelope protein as defined herein for inducing immunity against HCV in chronic HCV carriers prior to, simultaneously to or after any other therapy, such as, for example, the well-known interferon therapy either or not in combination with the administration of small drugs treating HCV, such as, for example, ribavirin. Such composition may also be employed before or after liver transplantation, or after presumed infection, such as, for example, needle-stick injury.
- Another aspect of the invention relates to a method for the detection of the presence of anti-HCV antibodies in a sample suspected to comprise anti-HCV antibodies, said method comprising:
- step (iii) inferring from (ii) the presence of said anti-HCV antibodies in said sample.
- the contacting in step (i) of said method is occurring under competitive conditions.
- said HCV envelope protein or part thereof is attached to a solid support.
- said sample suspected to comprise anti-HCV antibodies is a biological sample.
- 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.
- said HCV envelope protein or part thereof is attached to a solid support.
- said sample suspected to comprise anti-HCV antibodies is a biological sample.
- biological sample 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.
- the HCV envelope proteins of the present invention are particularly suited for incorporation into methods, such as immunoassay methods, for the detection of HCV, and/or genotyping of HCV, for prognosing/monitoring of HCV disease, or as a therapeutic agent.
- the methods, such as immunoassay methods, according to the present invention utilize the HCV envelope proteins of the present invention that maintain linear (in case of peptides) and conformational epitopes, recognized by antibodies in the sera from individuals infected with HCV.
- the HCV El and E2 antigens of the present invention may be employed in virtually any assay format that employs a known antigen to detect antibodies.
- the antigen is contacted with the body component suspected of containing HCV antibodies under conditions that permit the antigen to bind to any such antibody present in the component. Such conditions will typically be physiologic temperature, pH and ionic strenght using an excess of antigen. The incubation of the antigen with the specimen is followed by detection of immune complexes comprised of the antigen.
- Protocols may, for example, use solid supports, or immunoprecipitation.
- Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemiliiminescent, radioactive, or dye molecules.
- Assays which amplify the signals from the immxine complex are also known; examples of which are assays which utilize biotin and avidin or streptavidin, and enzyme-labeled and mediated immunoassays, such as ELISA and RIA assays.
- An immunoassay may be, without limitation, in a heterogeneous or in a homogeneous format, and of a standard or competitive type.
- the polypeptide is typically bound to a solid matrix or support to facilitate separation of the sample from the polypeptide after incubation.
- solid supports that can be used are nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., in sheets or microtiter wells), polystyrene latex (e.g., in beads or microtiter plates, polyvinylidine fluoride (known as HnmunolonTM), diazotized paper, nylon membranes, activated beads, and Protein A beads.
- Dynatech Immunolon 1 or Immunlon 2 microtiter plates can be used in the heterogeneous format.
- the solid support containing the antigenic polypeptides is typically washed after separating it from the test sample, and prior to detection of bound antibodies.
- Both standard and competitive formats are know in the art.
- the test sample is incubated with the combination of antigens in solution. For example, it may be under conditions that will precipitate any antigen-antibody complexes which are formed. Both standard and competitive formats for these assays are known in the art.
- the amount of antibodies, such as anti-HCV antibodies, in the antibody-antigen complexes is directly monitored. This may be accomplished by determining whether labeled anti-xenogeneic (e.g. anti-human) antibodies which recognize an epitope on said antibodies, such as said anti-HCV antibodies, will bind due to complex formation.
- labeled anti-xenogeneic e.g. anti-human
- the amount of said antibodies, such as said anti-HCV antibodies, in a sample is deduced by monitoring the competitive effect on the binding of a known amount of (labeled) antibody (or other competing ligand) or antigen in the complex.
- Antigen-antibody complexes can be detected by any of a number of known techniques, depending on the format.
- unlabeled antibodies such as anti-HCV antibodies in the complex may be detected using a conjugate of anti-xenogeneic lg complexed with a label (e.g. an enzyme label).
- a label e.g. an enzyme label.
- Hi an immunoprecipitation or agglutination assay format the reaction between an antigen and an antibody forms a protein cluster that precipitates from the solution or suspension and forms a visible layer or film of precipitate. If no antibody is present in the test specimen or sample, no such precipitate is formed.
- the HCV envelope proteins, or specific parts thereof of the present invention comprised of conformational epitopes will typically be packaged in the form of a kit for use in these immunoassays.
- the kit will normally contain in separate containers the native HCV antigen, control antibody formulations (positive and/or negative), labeled antibody when the assay format requires the same and signal generating reagents (e.g. enzyme substrate) if the label does not generate a signal directly.
- the native HCV antigen may be already bound to a solid matrix or separate with reagents for binding it to the matrix. Instructions (e.g. written, tape, CD-ROM, etc.) for carrying out the assay usually will be included in the kit.
- the solid phase selected can include polymeric or glass beads, nitrocellulose, microparticles, microwells of a reaction tray, test tubes and magnetic beads.
- the signal generating compound can include an enzyme, a luminescent compound, a chromogen, a radioactive element and a chemilxxminescent compound.
- enzymes include alkaline phosphatase, horseradish peroxidase and beta-galactosidase.
- enhancer compounds include biotin, anti-biotin and avidin.
- enhancer compounds binding members include biotin, anti-biotin and avidin.
- the test sample is subjected to conditions sufficient to block the effect of rheumatoid factor-like substances.
- conditions comprise contacting the test sample with a quantity of anti-human IgG to form a mixture, and incubating the mixture for a time and under conditions sufficient to form a reaction mixture product substantially free of rheumatoid factor-like substance.
- the present invention relates to the use of an HCV envelope protein or part thereof according to the invention for the preparation of a diagnostic kit.
- the present invention further relates also to a kit for detecting HCV related T cell response, comprising the oligomeric particle or the purified single HCV envelope protein of the instant invention.
- HCV T cell response can for example be measured as described by Leroux-Roels et al. in WO95/12677.
- a further aspect of the mvention relates to a method of inducing a HCV-specific immune response in a mammal, said method comprising administering to said mammal an effective amount of a HCV envelope protein or part thereof according to the invention optionally comprising a pharmaceutically acceptable adjuvant.
- Said method comprising administering to said mammal an effective amount of a HCV envelope protein or part thereof according to the invention may also be used for inducing HCV-specific antibodies in a mammal or for inducing a specific T-cell function in a mammal.
- said administering may be for prophylactic purposes, i.e. prophylactic administering or for therapeutic purposes, i.e. therapeutic administering.
- Yet another aspect of the invention relates to a method of immunizing a mammal, said method comprising administering to said mammal an effective amount of a HCV envelope protein or part thereof according to the invention optionally comprising a pharmaceutically acceptable adjuvant.
- the current invention also relates to a method of treating a mammal infected with HCV, said method comprising administering to said mammal an effective amount of a HCV envelope protein or part thereof according to the invention optionally comprising a pharmaceutically acceptable adjuvant.
- any of the above described aspects of the invention or the embodiments specific to said aspects is also applicable generally to proteins of interest which are the product of expression in a eukaryotic cells and which are further characterized by the same glycosylation properties as described above for the two different HCV envelope proteins.
- the invention thus relates to an isolated protein of interest or a fragment thereof comprising at least one N-glycosylation site, said protein or fragment thereof characterized in that it is the product of expression in a eukaryotic cell and further characterized in that on average up to 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% of the N-glycosylated sites are core-glycosylated.
- the ratio of sites core- glycosylated with an oligomannose with structure Man(7)-GlcNAc(2) over the sites core- glycosylated with an oligomannose with structure Man(8)-GlcNAc(2) is less than or equal to 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.45, or 0.50.
- said oligomannoses contain less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% terminal ⁇ l,3 mannose.
- Another alternative aspect of the invention is related to an isolated protein of interest or a fragment thereof comprising at least one N-glycosylation site, said protein or fragment thereof characterized in that it is the product of expression in a eukaryotic cell and further characterized in that N-glycosylated sites are occupied by oligomannoses wherein the ratio of the oligomannoses with structure Man(7)-GlcNAc(2) over the oligomannoses with structure Man(8)-GlcNAc(2) is less than or equal to 0.15, 0.2, 0.25, 0.30, 0.35, 0.40, 0.44, 0.45, or 0.50.
- said oligomannoses contain less than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% terminal ⁇ l,3 mannose.
- said isolated protein of interest or fragment thereof is the product of expression in a yeast cell such as a Hansenula cell.
- Said isolated 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.
- viral envelope proteins include the HIV (human immunodeficiency virus) envelope protein g ⁇ l20 and viral envelope proteins of a virus belonging to the Flavirideae.
- HIV human immunodeficiency virus
- said isolated protein of interest or fragment thereof can be any protein needing the N- glycosylation characteristics of the current invention.
- HCV-recombinant vaccinia virus is meant a vaccinia virus comprising a nucleic acid sequence encoding a HCV protein or part thereof.
- 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 El and/or E2 envelope proteins, which on their own are thought to consist of one or two El 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 wliich can be empty, consisting of a shell of envelope proteins in which Hpids, detergents, the HCV core protein, or adjuvant molecules can be incorporated.
- the latter particles can also be encapsulated by Hposomes 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.
- 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.
- empty spherical particles are often referred to as "virus-like particles" or VLPs.
- the higher-order particles can be solid spherical structures, in which the complete sphere consists of HCV El or E2 envelope protein oligomers, in which Hpids, detergents, the HCV core protein, or adjuvant molecules can be additionally incorporated, or which in turn may be themselves encapsulated by Hposomes 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 Els in 0.2% CHAPS as exemplified herein which apparently contain 8-10 monomers of Els.
- Such rosette-like structures are usually organized in a plane and are round-shaped, e.g. in the form of a wheel.
- Hpids, detergents, the HCV core protein, or adjuvant molecules can be additionally incorporated, or the smaller particles may be encapsulated by Hposomes 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.
- Hposomes 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 El or E2 envelope proteins in which Hpids, detergents, the HCV core protein, or adjuvant molecules could be additionally incorporated, or which in turn may be encapsulated by Hposomes 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.
- Hposomes 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.
- apolipoproteins such as, for example, apolipoprotein B or low density lipoproteins
- the diameter) of the above-defined particles 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.
- the present invention relates to a method for purifying core glycosylated 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 El and/or HCV E2 protein, or any part thereof, in a suitable culture medium; (ii) causing expression of said HCV El and or HCV E2 gene, or any part thereof; and (iii) purifying said core-glycosylated HCV El and/or HCV E2 protein, or any part thereof, from said cell culture.
- HCV hepatitis C virus
- the invention further pertains to a method for purifying core-glycosylated 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 El and/or HCV E2 protein, or any part thereof, in a suitable culture medium; (ii) causing expression of said HCV El and/or HCV E2 gene, or any part thereof; and (iii) purifying said intraceUularly expressed core-glycosylated HCV El and/or HCV E2 protein, or any part thereof, upon lysing the transformed host cell.
- HCV hepatitis C virus
- the invention further pertains to a method for purifying core-glycosylated hepatitis C virus (HCV) envelope proteins, or any part thereof, suitable for use in an immunoassay or vaccine, which method comprising:
- the invention further pertains to a method for purifying core-glycosylated hepatitis C virus (HCV) envelope proteins, or any part thereof, suitable for use in an immunoassay or vaccine, which method comprising:
- the present invention specifically relates to a method for purifying recombinant core- glycosylated HCV yeast proteins, or any part thereof, as described herein, in wliich said purification includes heparin affinity chromatography.
- the present invention also relates to a method for purifying recombinant core- glycosylated HCV yeast proteins, or any part thereof, as described above, in which said chemical means is sulfonation.
- the present invention also relates to a method for purifying recombinant core- glycosylated 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.
- the present invention also relates to a method for purifying recombinant core- glycosylated HCV yeast proteins, or any part thereof, as described above, in which said irreversible protection by chemical means is iodo-acetamide.
- the present invention also relates to a method for purifying recombinant core- glycosylated 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, El, E2, P7, NS2, NS3, NS4A, NS4B, NS5A and/or NS5B protein, or parts thereof.
- the core-glycosylated proteins El, 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.
- the present invention relates to the use of a core-glycosylated envelope protein as described herein for inducing immunity against HCV, characterized in that said core- glycosylated envelope protein is used as part of a series of time and compounds.
- 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 core-glycosylated envelope protein, HCV DNA vaccine composition, HCV polypeptides.
- a series comprises administering, either: (i) an HCV antigen, such as, for example, a core-glycosylated envelope protein, with time intervals, or (ii) an HCV antigen, such as, for example, a core-glycosylated envelope protein in combination with a HCV DNA vaccine composition, in which said core-glycosylated envelope protein oligomeric particles and said HCV DNAvaccine composition, can be administered simultaneously, or at different time intervals, including at alternating time intervals, or
- a HCV DNA vaccine composition comprises nucleic acids encoding HCV envelope peptide, including E1-, E2-, El/E2-peptides, NS3 peptide, other HCV peptides, or parts of said peptides.
- said HCV peptides comprises HCV envelope peptides, including E1-, E2-, El/E2-peptides, other HCV peptides, or parts thereof.
- the term "other HCV peptides" refers to any HCV peptide or fragment thereof.
- the HCV DNA vaccine composition comprises preferentially nucleic acids encoding HCV envelope peptides.
- an 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.
- 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.
- a "plasmid vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to wliich it has been linked.
- Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they have been linked.
- plasmid vectors are circular double stranded DNA loops which, in their vector form, are not bound to the chromosome.
- a "polynucleotide sequence" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- 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.
- 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.
- operably linked refers to a juxtaposition wherein the components are configured so as to perform their usual function.
- transcription regulatory elements operably linked to a nucleotide sequence are capable of effecting the expression of said nucleotide sequence.
- the DNA vaccine may be delivered through a live vector such as adenovirus, canary pox virus, MNA, and the like.
- Plasmids for Hansenula polymorpha transformation were constructed as follows.
- E1-H6 DNA fragment (coding for HCV type lb Els protein consisting of the amino acids 192 to 326 of Els elongated with 6 His-residues; SEQ ID NO:5) was isolated by PCR from the plasmid pGEMTElsH6 (SEQ ID NO:6; Figure 1). The following primers were used thereto:
- the Eaml ⁇ 04l site is underlined, the dot marks the cleavage site.
- the bold printed bases are complementary to those of primer CHH-links.
- the non-marked bases anneal within the start region of El (192-326) in sense direction; and - CHHE1-R: 5'- agttactcttca . cagj_7atcctccttaatggtgatggtggtggtgcc-3'
- the Earn 11041 site is underlined, the dot marks the cleavage site.
- the bold printed bases are complementary to those of primer MF30-rechts.
- the bases forming the BaniRl site usefull for later cloning procedures are printed in italics.
- the non-marked bases anneal in antisense direction within the end of the E1-H6 unit, including the stop codon and three additional bases between the stop codon and the Bam I site.
- the reaction mixture was constituted as follows: total volume of 50 ⁇ L containing 20 ng of Eco311-linearized pG ⁇ MT ⁇ lsH6, each 0.2 ⁇ M of primers CHHEl-F and CHHEl-R, dNTP's
- Program 1 was used, said program consisting of the following steps: 1. denaturation: 5min 95°C;
- the acceptor fragment was made by PCR from the pCHH-Hir plasmid (SEQ ID NO:9; Figure 2) and consists of almost the complete pCHH-Hir plasmid, except that the Hir-coding sequence is not present in the PCR product. Following primers were used for this PCR:
- CHH-links 5'-agttactcttca . cctctttccaacgggtgtgtag-3' (SEQ ID NO: 10);
- the Earn 11041 site is underlined, the dot marks the cleavage site.
- the bold printed bases are complementary to those of primer CHH ⁇ 1-F.
- the non-marked bases anneal within the end of the CHH sequence in antisense direction;
- the Eaml 1041 site is underlined, the dot marks the cleavage site.
- the bold printed bases are complementary to those of primer CHHEl-R.
- the non-marked bases anneal within the pUC18 sequences behind the cloned CHH-Hirudin HL20 of pCHH-Hir, pointing away from the insert.
- the reaction mixture was constituted as follows: total volume of 50 ⁇ L containing 20 ng of _4_ ⁇ ?718I-linearized pCHH-Hir, each 0.2 ⁇ M of primers CHH-links and MF30-rechts, dNTP's (each at 0.2 ⁇ M), 1 x buffer 2 (Expand Long Template PCR System; Boeringer; Cat No 1681 834), 2.5 U polymerase mix (Expand Long Template PCR System; Boeringer; Cat No 1681 834).
- E. coli XL-Gold cells were transformed with the ligation mixture and the plasmid DNA of several ampicillin-resistant colonies were analyzed by digestion with EcoRI and BamHI. A positive clone was selected and denominated as pCHH ⁇ l.
- shuttle vector pFPMT-MF ⁇ -El-H6 was generated by ligation of three fragments, said fragments being: 1. the 6.961 kb EcoWIBamHL digested pFPMT121 (SEQ ID NO:12; Figure 3),
- the 0.454 kb PCR product giving rise to fragment No.3 was obtained by PCR using the following primers:
- primer MFa-El f-Hi 5'-aggggtaagcttggataaaaggtatgaggtgcgcaacgtgtccgggatgt-3' (SEQ ID NO: 14); and 2. primer El back-Bam:
- reaction mixture was constituted as follows: Reaction mixture volume 50 ⁇ L, ⁇ FPMT-CHH-El-H6 (EcoRI-linearized; 15 ng/ ⁇ L), 0.5 ⁇ L; primer MFa- ⁇ l f-Hi (50 ⁇ M), 0.25 ⁇ L; primer ⁇ l back-Bam (50 ⁇ M), 0.25 ⁇ L; dNTP's (all at 2mM), 5 ⁇ L; DMSO, 5 ⁇ L; H 2 O, 33.5 ⁇ L; Expand Long Template PCR System (Boeringer Mannheim; Cat No 1681 834) Buffer 2 (10 x concentrated), 5 ⁇ L; Expand Long Template PCR System Polymerase mixture (1 U/ ⁇ L), 0.5 ⁇ L.
- the PCR program consisting of the following steps was used: 1. denaturation: 5 min at 95 °C
- the resulting 0.454kb PCR product contained the codons of El(192-326) followed by six histidine codons and a "taa" stop codon, upstream flanked by the 22 3 '-terminal base pairs of the MF ⁇ prepro sequence (including the cloning relevant HmdIII site plus a six base pairs overhang) and downstream flanked by a (cloning relevant) BamHI site and a six base pairs overhang.
- T4 DNA ligase Boehringer Mannheim
- example volume 20 ⁇ L sample volume 20 ⁇ L.
- E.coli HB101 cells were transformed with the ligation mixture and positive clones withheld after restriction analysis of the plasmids isolated from several transformants.
- a positive plasmid was selected and denominated as pFPMT-MF ⁇ -El-H6 (SEQ ID NO:16; Figure 5).
- Plasmids for Hansenula polymorpha transformation were constructed as follows.
- the pFPMT-CL-El-H6 shuttle vector was constructed in three steps starting from pFPMT-MF ⁇ - E1-H6 (SEQ ID NO: 16, Figure 5).
- the MF ⁇ -El-H6 reading frame of pFPMT-MF ⁇ -El-H6 was subcloned into the pUC18 vector. Therefore a 1.798kb SaWBamHl fragment of pFPMT-MF ⁇ -El-H6 (containing the FMD promotor plus MF ⁇ -El-H6) was ligated to the SaWBamHl vector fragment of pUC18 with T4 ligase (Boehringer) accordig to the supplier's conditions.
- a correct clone was used for PCR directed mutagenesis to replace the MF ⁇ pre-pro-sequence with the codons of the avian lysozyme pre-sequence ("CL"; corresponding to amino acids 1 to 18 of avian lysozyme; SEQ ID NO:l).
- CL codons of the avian lysozyme pre-sequence
- SEQ ID NO:l codons of the avian lysozyme pre-sequence
- the principle of the applied PCR-directed mutagenesis method is based on the amplification of an entire plasmid with the desired alterations located at the 5 '-ends of the primers. In downstream steps, the ends of the linear PCR product are modified prior to self-ligation resulting in the desired altered plasmid.
- the following primers were used for the PCR reaction:
- primer CL hin 5 '-tgcttcctaccactagcagcactaggatatgaggtgcgcaacgtgtccggg-3 ' (SEQ ID NO: 18); 2. primer CL her neu: 5 '-tagt ⁇ ct ⁇ fftattagtaggcttcgcatgaattcccgatgaaggcagagagcg-3 ' (SEQ ID NO: 18).
- the underlined 5' regions of the primers contain the codons of about half of the avian lysozyme pre-sequence.
- Primer CL her neu includes a Spel restriction site (italic).
- the non- underlined regions of the primers anneal with the codons for amino acid residues 192 to 199 of El (CL hin) or the with the "atg" start codon over the EcoRI site up to position -19 (counted from the EcoRI site) of FMD promoter.
- the primers are designed to amplify the complete pMal2-l thereby replacing the codons of the MF ⁇ pre-pro-sequence with the codons of the avian lysozyme pre sequence.
- the reaction mixture was constituted as follows: pUC18-FMD-Mf ⁇ - ⁇ l-H6 (pMal2-l; 1.3 ng/ ⁇ L), 1 ⁇ L; primer CL hin (100 ⁇ M), 2 ⁇ L; primer CL her neu (100 ⁇ M), 2 ⁇ L; dNTP's (all at 2.5mM), 8 ⁇ L; H 2 O, 76 ⁇ L; Expand Long Template PCR System (Boeringer; Cat No 1681 834) Buffer 2 (10 x concentrated), lO ⁇ L; Expand Long Template PCR System Polymerase mixture (1 U/ ⁇ L), 0.75 ⁇ L.
- the PCR program consisting of the following steps was applied:
- PCR product was checked by agarose gel electrophoresis for its correct size (3.5 kb). Thereafter the 3'-A overhangs form the PCR product were removed by a T4 polymerase reaction resulting in blunt ends with 3'- and 5'-OH-groups. Therefore, the PCR product was treated with T4 polymerase (Boehringer; 1 U/ ⁇ L): to the remaining 95 ⁇ L of PCR reaction mix were added 1 ⁇ L T4 polymerase and 4 ⁇ L dNTP's (all at 2.5 mM). The sample was incubated for 20 min at 37°C. Subsequently, the DNA was precipitated with ethanol and taken up in 16 ⁇ L H O.
- T4 polymerase Boehringer; 1 U/ ⁇ L
- the DNA was applied onto a 1% agarose gel and the correct product band was isolated by means of the gel extraction kit (Qiagen) according to the supplier's conditions. Fifty (50) ng of the purified product was then self-ligated by use of T4 ligase (BQebringer) according to the supplier's conditions. After 72 h incubation at 16°C, the DNA in the ligation mix was precipitated with ethanol and dissolved in 20 ⁇ L water.
- E.coli DH5 ⁇ -F' cells were subsequently transformed with 10 ⁇ L of the ligation sample.
- the plasmid DNA of several ampicillin-resistant clones was checked by means of restriction enzyme digestion.
- a positive clone was withheld and denominated p27d-3 (pUCl 8-FMD-CL- E1-H6, SEQ ID NO:20, Figure 7).
- the pFPMT-CL-El-H6 shuttle vector was constructed as described below.
- the 0.486kb EcoRUBamHl fragment of p27d-3 (harboring CL- ⁇ l(192-326)-H6) was ligated with EcoRI/_5 ⁇ HI-digested pFPMT121 (S ⁇ Q ID NO: 12, Figure 3).
- T4 ligase (Boehringer) has been used according to the supplier's recommendations.
- the DNA in the ligation sample was precipitated with ethanol and dissolved in 10 ⁇ L H 2 O.
- Plasmid clone p37-5 (pFPMT-CL- ⁇ l-H6; SEQ ID NO:21, Figure 8) showed the desired fragment sizes of 0.486kb and 6.961kb. The correct sequence of CL-E1-H6 of p37-5 was verified by sequencing.
- Plasmids for Hansenula polymorpha transformation were constructed as follows. The DNA sequence encoding the MF ⁇ -E2s (amino acids 384-673 of HCV E2)-VIEGR-His6 (SEQ ID NO:5) was isolated as a 1.33 lkb Ec ⁇ SUBglH. fragment from plasmid pSP72 ⁇ 2H6 (SEQ ID NO:22, Figure 9).This fragment was ligated with either the EcoRI Bg/II-digested vectors pFPMT121 (S ⁇ Q ID NO.T2, Figure C+2) or pMPT121 (S ⁇ Q ID NO:23, Figure 10) using T4 DNA ligase (Boehringer Mannheim) according to the supplier's recommendations. After transformation of E.
- the shuttle vector pFPMT-CL-E2-H6 was assembled in a three-step procedure.
- An intermediate construct was prepared in which the E2 coding sequence was cloned behind the signal sequence of ⁇ -amylase of Schwanniomyces accidentalis. This was done by the seamless cloning method (Padgett, K. A. and Sorge, J. A. 1996).
- E2s-H6 encoding DNA fragment At first the DNA sequence encoding E2-H6 (amino acids 384 to 673 of HCV E2 extended with the linker peptide "VIEGR" and with 6 His residues, SEQ ID NO: 5) was amplified from the pSP72E2H6 plasmid (SEQ ID NO:24, Figure 11) by PCR. The used primers were denoted MF30E2/F and MF30E2/R and have the following sequences:
- caqggatccttagtgatggtgg-tqa.tq-3' (SEQ ID NO:27; the Eaml l04l site is underlined, the dot marks the enzyme's cleavage site; the bold printed bases are complementary to the bold printed bases of primer MF30-Rechts (see below); a
- BamHI site to be introduced into the construct is printed in italic; the non- marked sequence anneals with the stop codon and the six terminal His codons of E2 (384-673)-
- VIEGR-H6 (SEQ ID NO:5).
- the reaction mixture was constituted as follows: total volume of 50 ⁇ L containing 20 ng of the 1.33kb Eco I/Bglll fragment of pSP72 ⁇ 2H6, each 0.2 ⁇ M of primers MF30E2/F and
- MF30E2/R dNTP's (each 0.2 ⁇ M), lx buffer 2 (Expand Long Template PCR System; Boeringer; Cat No 1681 834), 2.5 U polymerase mix (Expand Long Template PCR System; Boeringer; Cat No 1681 834).
- the PCR program 3 consisting of the following steps was used: 1. denaturation: 5 min at 95°C 2. 10 cycles of 30 sec denaturation at 95°C, 30 sec annealing at 65°C, and 1 min elongation at 68°C 3. termination at 4°C.
- the second fragment originated from the plasmid pMF30 (SEQ ID NO:28, Figure 13), the amplicon was almost the complete pMF30 plasmid excluding the codons of the mature ⁇ - amylase of S.occidentalis, modifications relevant for cloning were introduced by primer design.
- the following set of primers was used:
- Earn 11041 site is underlined, the dot marks the enzyme's cleavage site; the bold printed "cct” is complementary to the bold printed "agg" of primer MF30 ⁇ 2/F (see above); the non-marked and the bold printed bases anneal with the 26 terminal bases of the codons of the ⁇ - Amylase of S. occidentalis in pMF30);
- primer MF30-Rechts 5'-agtcac cttca . ctgcaggcatgcaagcttggcg-3 ' (SEQ ID NO:l l; the E ⁇ mll04I site is underlined, the dot marks the enzyme's cleavage site; the bold printed "ctg” is complementary to the bold printed "cag” of primer MF30 ⁇ 2/R (see above); the non-marked bases anneal with pUC18 sequences downstream of the stop codon of the ⁇ - Amylase of S. occidentalis in pMF30).
- the reaction mixture was constituted as follows: total volume of 50 ⁇ L containing 20 ng of the Rg/II-linearized pMF30, each 0.2 ⁇ M of primers MF30-Links and MF30-Rechts, dNTP's (each 0.2 ⁇ M), lx buffer 1 (Expand Long Template PCR System; Boeringer; Cat No 1681 834), 2.5 U polymerase mix (Expand Long Template PCR System; Boeringer; Cat No 1681 834).
- the same PCR programs programs 3 and 4) as described above were used, except for the elongation times wliich were extended from 1 minute to 4 minutes in both programs.
- the E2s-H6 encoding DNA fragment and pMF30-derived acceptor plasmid obtained by PCR were controlled on their respective size by gel electrophoresis on a 1 % agarose gel.
- the PCR products were purified with a PCR product purification kit (Qiagen) according to the supplier's instructions. Subsequently the purified fragments were digested separately with E ⁇ l 10041. Ligation of the ⁇ 2s-H6 fragment with the pMF30-derived acceptor plasmid was performed by using T4 ligase (Boehringer) according to the supplier's recommendations.
- the ligation mixture was used to transform E.coli DH5 ⁇ F'cells and the plasmid DNA of several clones was analyzed by EcoRI/R ⁇ mHI digestion. A positive clone was selected, its plasmid further denominated as pAMY- ⁇ 2, and utilized for further modifications as described below.
- primer CL2 her 5'-taqtactaqtattaqtaqqcttcqcatq raattcactqqccqtcqtttta- caacgtc-3' (SEQ ID NO:31).
- the underlined 5 '-regions of the primers contain the DNA sequence of about half of the avian lysozyme pre sequence.
- Primer CL2 her includes Spel (italic) and EcoRI (italic, double underlined) restriction sites.
- the non-underlined regions of the primers anneal with the codons of amino acid residues 384 to 392 of ⁇ 2 (CL2 hin) or the with the "atg" start codon over the EcoRI site up to position -19 (counted from the EcoRI site) of FMD promoter.
- the primers are designed to amplify the complete pAMY- ⁇ 2 vector thereby replacing the codons of the ⁇ -amylase signal sequence with the codons of the avian lysozyme pre-sequence.
- the PCR reaction was performed according to the following program: 1. denaturation: 15 min at 95°C
- reaction mixture was used: pAMY-E2 (1 ng/ ⁇ L), l ⁇ L; primer CL2 hin (100 ⁇ M), 2 ⁇ L; primer CL2 her (100 ⁇ M), 2 ⁇ L; dNTP's (2.5mM each), 8 ⁇ L; H 2 O, 76 ⁇ L; Expand Long Template PCR System (Boeringer; Cat No 1681 834) Buffer 2 (10 x concentrated), 10 ⁇ L; Expand Long Template PCR System Polymerase mixture (lU/ ⁇ L), 0J5 ⁇ L. The resulting PCR product was checked by gel electrophoresis on a 1% agarose gel. Prior to ligation the PCR fragment was modified as follows.
- T4 polymerase resulting in blunt ends with 3'- and 5'-OH-groups.
- 1 ⁇ L T4 polymerase (Boehringer, lU/ ⁇ L) was added to the residual 95 ⁇ L PCR reaction mixture along with 4 ⁇ L dNTP's (2.5 mM each). The sample was incubated for 20 min at 37°c. Subsequently the DNA was precipitated with ethanol and dissolved in 16 ⁇ L deionized water. This was followed by a kinase treatment to add 5 '-phosphates to the blunt-ended PCR product.
- a 0.966 kb EcoRI/R ⁇ mHI fragment was isolated from pUC18-CL- ⁇ 2-H6 (harboring CL- E2(384 - 673)-VIEGR-H6) and was ligated into the EcoRI/R ⁇ mHI-digested pFPMT121 (S ⁇ Q ID NO: 12, Figure 3).
- T4 ligase (Boehringer) was used according to the supplier's conditions. The ligation sample was precipitated with ethanol and dissolved in 10 ⁇ L water. This was used to transform E.coli DH5 ⁇ F' cells, a positive clone was withheld after restriction analysis and the respective plasmid is denominated pFPMT-CL- ⁇ 2-H6 (SEQ ID NO:32, Figure 14).
- the construction of the shuttle vector was comprised of two steps.
- the pUC18-FMD-CL-H6-K-El-H6 construct was constructed by site-directed mutagenesis.
- the pUC18-FMD-CL-El-H6 was used as template (SEQ ID NO:20; Figure 7).
- the PCR reaction mixture was constituted as follows: pUC18-FMD-CL-El-H6 (2 ng/ ⁇ L), l ⁇ L; primer H6K hin neu (100 ⁇ M), 2 ⁇ L; primer H6KRK her neu (100 ⁇ M), 2 ⁇ L; dNTP's
- the sample was applied onto a 1% agarose gel and the correct product band was isolated, by means of the gel extraction kit (Qiagen) according to the supplier's conditions.
- DNA in the ligation sample was precipitated with ethanol and dissolved in 10 ⁇ L water.
- E.coli DH5 ⁇ F' cells were transformed with 5 ⁇ L of the ligation sample.
- the plasmid DNA of several ampicillin-resitant colonies was analyzed by restriction enzyme digestion, a positive clone was withheld and the corresponding plasmid denominated: pUC18-FMD-CL-H6-El-K-
- the transfer vector was constructed by a two-fragment ligation. In the following construction fragments with Bell cohesive ends were involved. Since BcFl can cleave its site only on unmethylated DNA, an E. coli dam " strain was transformed with the involved plasmids pUC18-FMD-CL-H6-K-El-H6 (SEQ ID NO:39, Figure 17) and pFPMT- CL-E1 (SEQ ID NO: 36, Figure 16). From each transformation, an ampicillin-resistant colony was picked, grown in a liquid culture and the unmethylated plasmid DNAs were prepared for the further use.
- the 1.273kb BcW Hindlll fragment of the unmethylated plasmid pUC18- FMD-CL-H6-K-E1-H6 (harbouring the FMD promoter, the codons of the CL-H6-K unit, and the start of El) and the 6.057kb BclllHindlll fragment of plasmid pFPMT-CL-El (harbouring the missing part of the El reading frame starting from the Bc ⁇ site, without C-terminal His tag, as well as the pFPMTl 21 -located elements except for the FMD promoter) were prepared and ligated together for 72 h at 16°C by use of T4 ligase (Boehringer) in a total volume of 20 ⁇ L according to the supplier's specifications.
- T4 ligase Boehringer
- the ligation mixture was placed on a piece of nitrocellulose membrane floating on sterile deionized water in order to desalt the ligation mixture (incubation for 30 min at room temperature).
- E. coli TOP10 cells were transformed by electroporation with 5 ⁇ L of the desalted sample.
- the plasmid DNA of several resulting ampicillin-resistant colonies was analyzed by restriction enzyme digestion.
- a positive clone was withheld and denominated pFPMT-CL-H6-K-El (SEQ ID NO:40, Figure 18).
- H.polymorpha strain RB11 was been transformed (PEG-mediated DNA uptake protocol essentially as described by (Klebe, R. J. et al. 1983) with the modification of (Roggenkamp, R. et al. 1986) with the different parental shuttle vectors as described in Examples 1 to 5.
- 72 uracil-prototrophic colonies were selected and used for strain generation by the following procedure.
- a 2 mL liquid culture was inoculated and grown in test tubes for 48h (37°C; 160 rpm; angle 45°) in selective medium (YNB/glucose, Difco). This step is defined as the first passaging step.
- a 150 ⁇ L aliquot of the cultures of the first passaging step were used to inoculate 2 mL fresh YNB/glucose medium. Again, the cultures have been incubated as described above (second passaging step). Together, eight of such passaging steps were carried out. Aliquots of the cultures after the third and the eighth passaging steps were used to inoculate 2 mL of non-selective YPD medium (Difco). After 48 h of incubation at 37°C (160 rpm; angle 45°; the so-called first stabilization step), 150 ⁇ L aliquots of these YPD cultures have been used to inoculate fresh 2 mL YPD cultures which were incubated as described above (second stabilization step).
- Rabbit- Anti- Mouse- AP was used as second antibody. Staining was performed with NBT-BCIP. Positive strains were withheld for further investigation. Five of these positive clones were used in a shake flask expression experiment. A colony of the respective strain was picked from YNB plate and used to inoculate 2 mL YPD. These cultures were incubated as described above. This cell suspension was used to inoculate a second seed culture of 100 mL YPD medium in a 500 mL shake flask. This shake flask was incubated on a rotary shaker for 48 h at 37°C and 200 rpm.
- H. polymorpha a yeast strain closely related to Pichia pastoris (Gellissen, G. 2000), is able to express HCV proteins essentially without hyperglycosylation and thus with sugar moieties comparable in size to the HCV envelope proteins expressed by HCV- recombinant vaccinia virus-infected mammalian cells.
- the Hansenula polymorpha strain RB11 was deposited on April 19, 2002 under the conditions of the Budapest Treaty at the Mycotheque de l'UCL (MUCL), Universite Catholique de Louvain, Laboratoire de mycologie, Place Croix du Sud 3 bte 6, B-1348 Louvain-la-Neuve, Belgium and has the MUCL accession number MUCL43805.
- MUCL Mycotheque de l'UCL
- the S. cerevisiae expression plasmid was constructed as follows. An El -coding sequence was isolated as a NsRIEcoSll fragment from pG ⁇ MT- ⁇ lsH6 (SEQ ID NO:6, Figure 1) which was made blunt-ended (using T4 DNA polymerase) and cloned in the pYIG5 vector (SEQ ID NO:41, Figure 19) using T4 DNA ligase (Boehringer) according to the supplier's specifications. The cloning was such that the Els-H6 encoding fragment was joined directly and in frame to the ⁇ MF-coding sequence. The ligation mixture was transformed in E.coli DH5 ⁇ F'cells.
- pYIG5ElH6 pYIG5ElH6
- the expression cassette (containing the ⁇ MF-sequence and the Els-coding region with a His- tag) was transferred as a BamHI fragment (2790 bp) of pYIG5ElH6 into the _3 ⁇ HI-digested E .colilS. cerevisiae pSYl shuttle vector (SEQ ID NO:21, Figure 43).
- the ligation was performed with T4 DNA ligase (Boehringer) according to supplier's conditions.
- the ligation mix was transformed to E .coli D ⁇ 5 ⁇ F' cells, and the plasmid DNA of several ampicilin resistant colonies was analyzed by restriction enzyme digestion.
- a positive clone was withheld and denominated pSYlaMFElsH6a (ICCG3479; SEQ ID NO:44, Figure 22).
- the S. cerevisiae expression plasmid pSYYIGSE2H6 was constructed as follows. An E2 coding sequence was isolated as a SaWKpnl fragment from pBSK-E2sH6 (SEQ ID NO:45, Figure 23) which was made blunt-ended (using T4 DNA polymerase) and subsequently cloned in the pYIG5 vector (SEQ ID NO:41, Figure 19) using T4 DNA ligase (Boehringer) according to the supplier's specifications. The cloning was such that the E2-H6 encoding fragment was joined directly and in frame to the ⁇ MF-coding sequence. The ligation mixture was then transformed to E.
- pYIG5HCCL-22aH6 plasmid DNA of several ampicilin resistant clones was analyzed by restriction digestion and a positive clone withheld and denominated as pYIG5HCCL-22aH6 (ICCG2424; SEQ ID NO:46, Figure 24).
- the expression cassette (containing the ⁇ MF-sequence and the E2 (384 - 673) coding region with a His-tag) was transferred as a BamHI fragment (3281 bp) of pYIG5HCCL-22aH6 into the BamHI opened E. colilS. cerevisiae pSYl shuttle vector (SEQ ID NO:43, Figure 21).
- the ligation was performed with T4 DNA ligase (Boehringer) according to supplier's conditions.
- the ligation mix was transformed to E. coli DH5 ⁇ F' cells and the plasmid DNA of several ampicilin resistant colonies was analyzed by restriction enzyme digestion. A restriction positive clone was withheld and denominated pSYYIGSE2H6 (ICCG2466; SEQ ID NO:47, Figure 25).
- the S. cerevisiae expression plasmid pSYlYIG7Els was constructed as follows. An El coding sequence was isolated as a NsR/Eco52l fragment from pGEMT-Els (SEQ ID NO:6, Figure 1) wliich was made blunt-ended and cloned into the pYIG7 vector (SEQ ID NO:48, Figure 26) using T4 DNA ligase (Boehringer) according to the supplier's specifications. The cloning was such that the El -encoding fragment was joined directly and in frame to the ⁇ MF- coding sequence. The ligation mixture was transformed to E.
- coli DH5 ⁇ F' cells the plasmid DNA of several ampicilin resistant clones analyzed by restriction digestion and a positive clone withheld and denominated as pYIG7El (SEQ ID NO:49, Figure 27).
- the expression cassette (containing the CL leader sequence and the El (192-326) coding region) was transferred as a BamHI fragment (2790 bp) of pYIG7El into the R ⁇ mHI-digested E. colilS. cerevisiae pSYl shuttle vector (SEQ ID NO:43, Figure 21).
- the ligation was performed with T4 DNA ligase (Boehringer) according to supplier's conditions.
- the ligation mix was transformed to E.
- Said glycosylation-deficient strain IYCC155 was transformed with the plasmids as described in Examples 7 to 9 essentially by to the lithium acetate method as described by Elble (Elble, R. 1992).
- Several Ura complemented strains were picked from a selective YNB + 2 % agar plate (Difco) and used to inoculate 2ml YNB+2%glucose. These cultures were incubated for 72 h, 37°C, 200 rpm on orbital shaker, and the culture supernatant and intracellular fractions were analysed for expression of El by western blot developed with a El specific murine monoclonal antibody (IGH 201). A high producing clone was withheld for further experiments.
- IGH 201 El specific murine monoclonal antibody
- the shuttle vector pPICZalphaE 'ElsH6 was constructed starting from the pPICZalphaA vector (Invitrogen; SEQ ID NO:51, Figure 29). In a first step said vector was adapted in order to enable cloning of the El coding sequence directly behind the cleavage site of the KEX2 or
- pPICZalphaA was digested with Xhol and Notl.
- the digest was separated on a 1% agarose gel and the 3519 kb fragment (major part of vector) was isolated and purified by means of a gel extraction kit (Qiagen).
- This fragment was then ligated using T4 polymerase (Boehringer) according to the supplier's conditions in presence of specific oligonucleotides yielding pPICZalphaD' (SEQ ID NO:52, Figure 30) or pPICZalphaE' (SEQ ID NO:53, Figure 31).
- linker oligonucleotide 5'-GGCCGCATGCGAATTCGGGCCCCTTTTC-3' (SEQ ID NO:55) which yield, after annealing, the linker oligonucleotide:
- linker oligonucleotide 5'-TCGAGAAAAGAGAGGCTGAAGCCTGCAGCATATGC-3' (SEQ ID NO: 56) 8650: 5'-GGCCGCATATGCTGCAGGCTTCAGCCTCTCTTTTC-3' (SEQ ID NO:57) which yield, after annealing, the linker oligonucleotide:
- CTTTTCTCTCCGACTTCGGACGTCGTATACGCCGG (SEQ ID NO : 57 )
- shuttle vectors pPICZalphaD' and pPICZalphaE' have newly introduced cloning sites directly behind the cleavage site of the respective processing proteases, KEX2 and STE13.
- the E1-H6 coding sequence was isolated as a NsIl/Eco52l fragment from pGEMT-ElsH6 (SEQ ID NO:6, Figure 1). The fragment was purified using a gel extraction kit (Qiagen) after separation of the digest on a 1% agarose gel. The resulting fragment was made blunt-ended (using T4 DNA polymerase) and ligated into either pPICZalphaD' or pPICZalphaE' directly behind the respective processing protease cleavage site.
- the ligation mixtures were transformed to E. coli TOPI OF' cells and plasmid DNA of several zeocin resistant colonies analyzed by restriction enzyme digestion. Positive clones were withheld and denominated pPICZalphaD 'ElsH6 (ICCG3694; SEQ ID NO:58, Figure 32) and pPICZalphaE'ElsH6 (ICCG3475; SEQ ID NO:59, Figure 33), respectively.
- the shuttle vectors pPICZalphaD' and pPICZalphaE' were constructed as described in Example 11.
- the E2-H6 coding sequence was isolated as a Sall/Kpnl fragment from pBSK-E2sH6 (SEQ ID NO: 45, Figure 23).
- the fragment was purified with a gel extraction kit (Qiagen) after separation of the digest on a 1% agarose gel.
- the resulting fragment was made blunt-ended (using T4 DNA polymerase) and ligated into either pPICZalphaD' or pPICZalphaE' directly behind the respective processing protease cleavage site.
- the ligation mixture was transformed to E.
- the P. pastoris shuttle plasmids as described in Examples 11 and 12 were transformed to P. pastoris cells according to the supplier's conditions (Invitrogen). An El- and an E2 -producing strain were withheld for further characterization.
- the HCV envelope proteins were expressed in P. pastoris, a yeast strain well known for the fact that hyperglycosylation is normally absent (Gellissen, G. 2000) and previously used to express dengue virus E protein as GST fusion (Sugrue, R. J. et al. 1997).
- the resulting P. pastoris-expressed HCV envelope proteins displayed a comparable glycosylation as is observed in wild-type Saccharomyces strains. More specifically, the HCV envelope proteins produced by P. pastoris are hyperglycosylated (based on the molecular weight of the expression products detected in western-blots of proteins isolated from transformed P. pastoris cells).
- a master cell bank and working cell bank were prepared.
- Cryo-vials were prepared from a mid-exponentially grown shake flask culture (incubation conditions as for fermentation seed cultures, see below). Glycerol was added (50 % final cone.) as a cryoprotectant.
- Seed cultures were started from a cryo-preserved working cell bank vial and grown in 500 mL medium (YNB supplemented with 2 % sucrose, Difco) in a 2 L Erlenmeyer shake flasks at 37°C, 200 rpm for 48h.
- Fermentations were typically performed in Biostat C fermentors with a working volume of 15 L (B.Braun Int., Melsungen, Germany).
- the fermentation medium contained 1% Yeast Extract, 2% Peptone and 2 % sucrose as carbon source. Poly-ethylene glycol was used as anti- foam agent.
- the fermentation was started by the addition of 10 % seed-culture. During the growth phase the sucrose concentration was monitored off-line by HPLC analysis (Polysphere Column OAKC Merck).
- the dissolved oxygen was controlled by cascade control (agitation/aeration). After complete metabolisation of sucrose the heterologous protein production was driven by the endogenous produced ethanol supplemented with stepwise addition of EtOH in order to maintain the concentration at approximately 0.5 % (off-line HPLC analysis, polyspher OAKC column) During this induction phase the dissolved oxygen was controlled below 5% air-saturation, by manual adjustment of airflow rate and agitator speed.
- the fermentation was harvested 48 to 72 h post induction by concentration via tangential flow filtration followed by centrifugation of the concentrated cell suspension to obtain cell pellets. If not analyzed immediately, cell pellets were stored at -70°C.
- a master cell bank and working cell bank were prepared.
- Cryo-vials were prepared from a mid-exponentially grown shake flask culture (incubation conditions as for fermentation seed cultures, see below). Glycerol was added (50 % final cone.) as a cryoprotectant.
- Seed cultures were started from a cryo-preserved (-70°C) working cell bank vial and grown in 500 mL medium (YPD, Difco) in a 2 L Erlenmeyer shake flasks at 37°C, 200 rpm for 48h. Fermentations were typically performed in Biostat C fermentors with a working volume of 15 L (B.Braun Int., Melsungen, Germany).
- the fermentation medium contained 1% Yeast Extract, 2% Peptone and 1% glycerol as carbon source. Poly-ethylene glycol was used as anti- foam agent.
- the fermentation was started by the addition of 10 % seed-culture. During the growth phase the glycerol concentration was monitored off-line (Polysphere Column OAKC Merck) and 24 h after complete glycerol consumption 1% methanol was added in order to induce the heterologous protein expression. The fermentation was harvested 24 h post induction by concentration via tangential flow filtration followed by centrifugation of the concentrated cell suspension to obtain cell pellets. If not analyzed immediately, cell pellets were stored at - 70°C. Pichia pastoris
- the small scale production was typically performed at 500 mL scale in 2 L shake flasks and were started with a 10 % inoculation in expression medium, containing 1% Yeast extract, 2 %
- Peptone both Difco
- 2 % glycerol as carbon source. Incubation conditions were as for the seed culture. Induction was started by addition of 1 % MeOH approximately 72 h after inoculation. The cells were collected 24 h post induction by centrifugation. If not analyzed immediately, cell pellets were stored at -70°C.
- cell pellets were resuspended in 50 mM phosphate, 6M GuHCl, pH 7.4 (9 vol/g cells). Proteins were sulfonated overnight at room temperature (RT) in the presence of 320 mM (4% w/v) sodium sulfite and 65 mM (2% w/v) sodium tetrathionate. The lysate was cleared after a freeze-thaw cycle by centrifugation (10.000 g, 30 min, 4°C) and Empigen (Albright &Wilson, UK) and imidazole were added to the supernatant to final concentrations of 1% (w/v) and 20 mM, respectively.
- the sample was filtrated (0.22 ⁇ M) and loaded on a Ni-IDA Sepharose FF column, which was equilibrated with 50 mM phosphate, 6M GuHCl, 1% Empigen (buffer A) supplemented with 20 mM imidazole.
- the column was washed sequentially with buffer A containing 20 mM and 50 mM imidazole, respectively, till absorbance at 280 nm reached baseline level.
- the his-tagged products were eluted by applying buffer D, 50 mM phosphate, 6M GuHCl, 0.2 % (for El) or 1 % (for E2) Empigen, 200 mM imidazole.
- the eluted materials were analyzed by SDS-PAGE and western-blot using a specific monoclonal antibodies directed against El (IGH201), or E2 (IGH212).
- Several of the degradation products may be a result of a Kex-2 like cleavage (e.g. the cleavage observed after aa 196 of El which is a cleavage after an arginine), which is also required for the cleavage of the ⁇ -mating factor leader and which can thus not be blocked without disturbing this essential process.
- a high El producing clone derived from transformation of S. cerevisiae IYCC155 with pSYlYIG7Els (SEQ ID NO:50; Figure 28) was compared with a high producing clone derived from transformation of S. cerevisiae IYCC155 with pSYlaMFElsH6aYIGlEls (SEQ ID NO:44; Figure 22).
- the intracellular expression of the El protein was evaluated after 2 up to 7 days after induction, and this by means of Western-blot using the El specific monoclonal antibody (IGH 201). As can be judged from Figure 40, maximal expression was observed after 2 days for both strains but the expression patterns for both strains are completely different. Expression with the ⁇ -mating factor leader results in a very complex pattern of bands, which is a consequence from the fact that the processing of the leader is not efficient. This leads to several expression products with a different ammo-terminus and of which some are modified by 1 to 5 N-glycosylations.
- the hybridoma cell line producting the monoclonal antibody directed against El (IGH201) was deposited on March 12, 1998 under the conditions of the Budapest Treaty at the European Collection of Cell Cultures, Centre for Applied Microbiology & Research, Salisbury, Wiltshire SP4 0JG, UK, and has the accession number ECACC 98031216).
- the monoclonal antibody directed against E2 (IGH212) has been described as antibody 12D11F2 in Example 7.4 by Maertens et al. in WO96/04385.
- leader sequences were used to replace the S. cerevisiae ⁇ MF leader peptide including CHH (leader sequence of Carcinus maenas hyperglycemic hormone), Amyl (leader sequence of amylase from S. occidentalis), Gaml (leader sequence of glucoamylase from S. occidentalis), Phy5 (leader sequence from fungal phytase), phol (leader sequence from acid phosphatase from Pichia pastoris) and CL (leader of avian lysozyme C, 1,4-beta-N- acetylmuramidase C) and linked to E1-H6 (i.e. El with C-terminal his-tag).
- CHH leader sequence of Carcinus maenas hyperglycemic hormone
- Amyl leader sequence of amylase from S. occidentalis
- Gaml leader sequence of glucoamylase from S. occidentalis
- Phy5 leader sequence from fungal phytase
- phol leader sequence from acid phosphata
- the higher hydrophobic character of the non-glycosylated material may be used to select and optimize other enrichment procedures.
- the correct removal of the CL leader peptide from the CL-E1-H6 protein was further confirmed by mass spectrometry wliich also confirmed that up to 4 out of the 5 N-glycosylation sites of genotype lb Els can be occupied, whereby the sequence NNSS (amino acids 233 to 236; SEQ ID NO:73) are considered to be a single N- glycosylation site.
- CL-E2-H6 The efficiency of removal of the CL leader peptide from CL-E2-VIEGR-H6 (furtheron in this Example denoted as "CL-E2-H6") protein expressed in Hansenula polymorpha was analyzed. Since the HCV E2s (aa 383-673) was expressed as a bis-tagged protein, a rapid and efficient purification of the expressed protein after GuHCl-solubilization of collected cells was performed on Ni-IDA. In brief, cell pellets were resuspended in 30 mM phosphate, 6 M GuHCl, pH 7.2 (9 mL buffer/g cells).
- the protein was sulfonated overnight at room temperature in the presence of 320 mM (4% w/v) sodium sulfite and 65 mM (2% w/v) sodium tetrathionate.
- the lysate was cleared after a freeze-thaw cycle by centrifugation (10.000 g, 30 min, 4°C).
- Empigen BB Albright & Wilson
- imidazole were added to a final concentration of 1% (w/v) and 20 mM, respectively. All further chromatographic steps were executed on an Akta FPLC workstation (Pharmacia).
- the sample was filtrated through a 0.22 ⁇ m pore size membrane (cellulose acetate) and loaded on a Ni-IDA column (Chelating Sepharose FF loaded with Ni 2+ , Pharmacia), which was equilibrated with 50 mM phosphate, 6 M GuHCl, 1 % Empigen BB, pH 7.2 (buffer A) supplemented with 20 mM imidazole.
- the column was washed sequentially with buffer A containing 20 mM and 50 mM imidazole, respectively, till the absorbance at 280 nm reached the baseline level.
- the his-tagged products were eluted by applying buffer D, 50 mM phosphate, 6 M GuHCl, 0.2 % Empigen BB (pH 7.2), 200 mM imidazole.
- the purified materials were analysed by SDS-PAGE and western- blot using a specific monoclonal antibody directed against E2 (IGH212) ( Figure 41).
- the IMAC-purified E2-H6 protein was also subjected to N-terminal sequencing by Edman degradation. Thereto proteins were treated with N-glycosidase F (Roche) (0.2 U/ ⁇ g E2, 1 h incubation at 37°C in PBS/3% empigen BB) or left untreated.
- the glycosylated and deglycosylated E2-H6 proteins were subjected to SDS-PAGE and blotted on a PVDF- membrane for amino acid sequencing (analysis was performed on a PROCISETM 492 protein sequencer, Applied Biosystems). Since at this stage, SDS-PAGE revealed some degradation products, a further fractionation by size exclusion chromatography was performed.
- the Ni-IDA eluate was concentrated by ultrafiltration (MWCO 10 kDa, centriplus, Amicon, Millipore) and loaded on a Superdex G200 (Pharmacia) in PBS, 1% Empigen BB.
- Elution fractions containing mainly intact E2s related products with a Mr between ⁇ 30 kDa and ⁇ 70 kDa based on the migration on SDS-PAGE, were pooled and eventually alkylated (incubation with 5 mM DTT for 30 minutes at 37°C, followed by incubation with 20 mM iodoacetamide for 30 minutes at 37°C).
- the possible presence of degradation products after IMAC purification can thus be overcome by a further fractionation of the intact product by means of size exclusion chromatography. An unexpectedly good result was obtained.
- Based on the N- terminal sequencing the amount of E2 product from which the CL leader peptide is removed could be estimated.
- the total amount of protein products is calculated as pmol of protein based on the intensity of the peaks recovered by Edman degradation. Subsequently, for each specific protein (i.e. for each 'detected N-terminus') the mol %> versus the total is estimated. In the current experiment, only the correct N-terminus of E2-H6 was detected and other variants of E2-H6 lacking amino acid of the E2 protein or containing N-terminal amino acids not comprised in the E2 protein were absent. In conclusion, the E2-H6 protein expressed by H. polymorpha as CL-E2-H6 protein was isolated without any further in vitro processing as a > 95 % correctly cleaved protein. This is in sharp contrast with the fidelity of leader peptide removal by H polymorpha of the ⁇ MF-E2-H6 protein to the E2-H6 protein, which was estimated to occur in 25 % of the isolated proteins (see Table 3).
- Endoproteinase Lys-C digested protein sample was applied on a Ni-IDA column after a 10-fold dilution with 10 mM NaH 2 PO .3H 2 O, 1 % (v/v) Empigen B, pH 7.2 (buffer B) followed by washing with buffer B till the absorbance at 280 nm reached the baseline level.
- the flow through was collected in different fractions (1-40) that were screened for the presence of Els-products ( Figure 44).
- the fractions (7-28), containing intact El from wliich the N-terminal H6-K (and possibly residual CL-H6- K) tail is removed (with a Mr between ⁇ 15 kDa and -30 kDa based on the migration on SDS- PAGE followed by silver staining or western blot analysis using a specific monoclonal antibody directed against El (IGH201), were pooled and eventually alkylated (incubation with 5 mM DTT for 30 minutes at 37°C, followed by incubation with 20 mM iodoacetamide for 30 minutes at 37°C).
- the total amount of protein products is calculated as pmol of protein based on the intensity of the peaks recovered by Edman degradation. Subsequently, for each specific protein (i.e. for each 'detected N-terminus') the mol % versus the total is estimated. Hi the current experiment, only the correct N-terminus of El was detected and not the N-termini of other processing variants of H6-K-E1. Based thereon, in vitro processing by Endo Lys-C of the H6-K-E1 El (and possibly residual CL-H6-K-E1) protein to the El protein was estimated to occur with a fidelity of more than 95 %.
- HCV envelope proteins In order to find specific purification steps for HCV envelope proteins from yeast cells binding with heparin was evaluated. 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 order to analyze this potential binding, heparin was biotinylated and interaction with HCV El analyzed in microtiterplates coated with either sulfonated HCV El from H. polymorpha, alkylated HCV El from H. polymorpha (both produced as described in Example 16) and alkylated HCV El from a culture of mammalian cells transfected with a vaccinia expression vector.
- HCV El and E2 envelope proteins expressed in H. polymorpha were converted to VLPs was done essentially as described by Depla et al. in WO99/67285 and by Bosman et al. in WO01/30815. Briefly, after cultivation of the transformed H polymorpha cells during which the HCV envelope proteins were expressed, cells were harvested, lysed in GuHCl and sulphonated as described in Example 17. His-tagged proteins were subsequently purified by IMAC and concentrated by ultrafiltration as described in Example 17.
- the concentrated HCV envelope proteins sulphonated during the isolation procedure were not subjected to a reducing treatment and loaded on a size-exclusion chromatograpy column (Superdex G200, Pharmacia) equilibrated with PBS, 1 % (v/v) Empigen.
- the eluted fractions were analyzed by SDS-PAGE and western blotting.
- the fractions with a relative Mr -29 — 15 kD (based on SDS-PAGE migration) were pooled, concentrated and loaded on Superdex G200, equilibrated with PBS, 3%> (w/v) betain, to enforce virus like particle formation (VLP).
- the fractions were pooled, concentrated and desalted to PBS, 0.5% (w/v) betain.
- the concentrated HCV envelope proteins sulphonated during the isolation procedure were subjected to a reducing treatment (incubation in the presence of 5 mM DTT in PBS) to convert the sulphonated Cys-thiol groups to free Cys-thiol groups.
- Irreversible Cys-thiol modification was performed by (i) incubation for 30 min in the presence of 20 mM iodoacetamide, or by (ii) incubation for 30 min in the presence of 5 mM N-ethylmaleimide (NEM) and 15 mM biotin-N-ethylmaleimide.
- the proteins were subsequently loaded on a size-exclusion chromatograpy column (Superdex G200, Pharmacia) equilibrated with PBS, 1 % (v/v) Empigen in case of iodoacetamide-blocking, or with PBS, 0.2 % CHAPS in case of blocking with NEM and biotin-NEM.
- the eluted fractions were analyzed by SDS-PAGE and Western blotting.
- the fractions with a relative Mr -29—15 kD were pooled, concentrated and, to force virus-like particle formation, loaded on a Superdex G200 column equilibrated with PBS, 3% (w/v) betain.
- the concentrated HCV envelope proteins sulphonated during the isolation procedure were subjected to a reducing treatment (incubation in the presence of 5 mM DTT in PBS) to convert the sulphonated Cys-thiol groups to free Cys-thiol groups.
- Reversible Cys-thiol modification was performed by incubation for 30 min in the presence of dithiodipyridine (DTDP), dithiocarbamate (DTC) or cysteine.
- DTDP dithiodipyridine
- DTC dithiocarbamate
- cysteine cysteine.
- the proteins were subsequently loaded on a size-exclusion chromatograpy column (Superdex G200, Pharmacia) equilibrated with PBS, 1 % (v/v) Empigen. The eluted fractions were analyzed by SDS-PAGE and Western blotting.
- the fractions with a relative Mr -29 — 15 kD were pooled, concentrated and loaded on Superdex G200, equilibrated with PBS, 3% (w/v) betain, to enforce virus like particle formation (VLP).
- the fractions were pooled, concentrated and desalted to PBS, 0.5% (w/v) betain.
- the VLP particle size was determined by Dynamic Light Scattering.
- a particle-size analyzer Model Zetasizer 1000 HS, Malvern Instruments Ltd., Malvern, Worcester UK
- PCS photon correlation spectroscopy
- Photon correlation spectroscopy or dynamic light scattering is an optical method that measures brownian motion and relates this to the size of particles.
- Light from a continuous, visible laser beam is directed through an ensemble of macromolecules or particles in suspension and moving under brownian motion. Some of the laser light is scattered by the particles and this scattered light is measured by a photomultiplier.
- Fluctuations in the intensity of scattered light are converted into electrical pulses which are fed into a correlator. This generates the autocorrelation function which is passed to a computer where the appropriate data analysis is performed.
- the laser used was a 10 W monochromatic coherent He-Ne laser with a fixed wavelength of 633 nm. For each sample, three to six consecutive measurements were taken.
- HCV El The reactivity of Hansenula-produced HCV E1-H6 with sera from HCV chronic carriers was compared to the reactivity of HCV El produced by HCV-recombinant vaccinia virus-infected mammalian cells as described by Depla et al. in WO 99/67285. Both HCV-El preparations tested consisted of VLP's wherein the HCV El proteins were alkylated with NEM and biotin- NEM. The reactivities of both HCV El VLP-preparations with sera from HCV chronic carriers was determined by ELISA. The results are summarized in Table 6. As can be derived from Table 6, no differences in reactivity were noted between HCV El expressed in HCV- recombinant vaccinia virus-infected mammalian cells and HCV El expressed in H. polymorpha.
- HCV E1-H6 The immunogenecity of Hansenula-produced HCV E1-H6 was compared to the immunogenecity of HCV El produced by HCV-recombinant vaccinia virus-infected mammalian cells as described by Depla et al. in WO99/67285.
- Both HCV-El preparations tested consisted of VLP's wherein the HCV El proteins were alkylated with iodoacetamide. Both VLP preparations were formulated with alum and injected in Balb/c mice (3 intramuscular/subcutaneous injections with a three week interval between each and each consisting of 5 ⁇ g El in 125 ⁇ l containing 0.13% Alhydrogel, Superfos, Denmark). Mice were bled ten days after the third immunization.
- Antibody titers were determined by ELISA (see Example 21) wherein either El produced in mammalian cells ("M”) or Hansenula-produced El (“H”) were coated directly on the ELISA solid support whereafter the ELISA plates were blocked with casein.
- the antibody titers determined were end point titers.
- the end point titer is determined as the dilution of serum resulting in an OD (as determined by ELISA) equal to two times the mean of the background of the assay.
- Figure 51 shows that no significant differences were observed between the immunogenic properties of both El -compositions and that the determined antibody titers are independent of the antigen used in the ELISA to perform the end point titration.
- the yeast-derived HCV El induced upon vaccination a protective response similar to the protective response obtained upon vaccination with alkylated HCV El derived from mammalian cell culture. The latter response was able to prevent chronic evolution of HCV after an acute infection.
- EXAMPLE 23 ANTIGENIC AND IMMUNOGENIC PROFILE OF HANSENULA-PROOVC ⁇ D HCV
- HCV-El The reactivity of Hansenula-produced HCV E1-H6 with sera from HCV chronic carriers was compared to the reactivity of HCV El produced by HCV-recombinant vaccinia virus-infected mammalian cells as described by Depla et al. in WO99/67285. Both HCV-El preparations tested consisted of VLP's wherein the Hansenula-produced HCV El proteins were sulphonated and the HCV El produced by mammalian cells was alkylated. The results are given in Table 7. Although the overall (average) reactivity was identical, some major differences were noted for individual sera. This implies that the sulphonated material presents at least some of its epitopes in a way different from alkylated HCV El.
- HCV-El preparations tested consisted of VLP's. Both VLP preparations . were formulated with alum and injected in Balb/c mice (3 intramuscular/subcutaneous injections with a three week interval between each and each consisting of 5 ⁇ g El in 125 ⁇ l containing 0.13% Alhydrogel, Superfos, Denmark). Mice were bled ten days after the third immunization.
- Antibody titers were determined similarly as described in Example 22. Surprisingly, immxinization with sulphonated material resulted in higher antibody titers, regardless of the antigen used in ELISA to assess these titers ( Figure 51; top panel: titration of antibodies raised against alkylated El; bottom panel: titration of antibodies raised against sulphonated El; "A”: alkylated El coated on ELISA plate; "S”: sulphonated El coated on ELISA plate). However, in this experiment individual titers are different dependent on the antigen used for analysis which confirms the observation noted with sera from HCV patients.
- HCV El wherein the cysteine thiol-gorups are modified in a reversible way may be more immunogenic and thus have an increased potency as a vaccine protecting against HCV (chronic infection). In addition thereto, induction of a response to neo-epitopes induced by irrreversible blocking is less likely to occur.
- Table 7 Antigenicity of alkylated El (produced in mammalian cell culture) or sulphonated E1-H6 (produced in H. polymorpha) was evaluated on a panel of sera from human HCV chronic carriers ("patient sera") and a panel of control sera ("blood donor sera”). To this purpose El was bound to ELISA plates, after which the plates were further saturated with casein.
- the glycosylation profiles were compared of Hansenula-produced HCV El and HCV El produced by HCV-recombinant vaccinia virus-infected mammalian cells as described by Depla et al. in WO99/67285. This was done by means of fluorophore-assisted carbohydrate electrophoresis (FACE). Thereto, oligosaccharides were released from Els produced by mammalian cells or Hansenula by peptide-N-glycosidase (PNGase F) and labelled with ANTS (the El proteins were alkylated with iodoacetamide prior to PNGase F digestion).
- FACE fluorophore-assisted carbohydrate electrophoresis
- ANTS-labeled oligosaccharides were separated by PAGE on a 21% polyacrylamide gel at a current of 15 mA at 4°C for 2-3 h. From Figure 54, it was concluded that the oligosaccharides on El produced by mammalian cells and E1-H6 produced by Hansenula migrate like oligomaltose with a degree of polymerization between 7 and 11 monosaccharides. This indicates that the Hansenula expression system surprisingly leads to an El protein which is not hyperglycosylated and which has sugar chains with a length similar to the sugar chains added to El proteins produced in mammalian cells.
- deglycosilation incubation buffer 50 mM Na 2 HPO 4, 0J5 % Nonidet P-40
- PNGase F Peptide-N 4 -(acetyl-bet ⁇ -glucosaminyl)-asparagine amidase of Flavobacterium meningosepticum; EC 3.5.1.52; obtained from Roche
- the sample was incubated overnight at 37°C.
- the pH of the solution was adjusted to pH 5.5 with concentrated H 3 PO .
- Proteins and oligosaccharides were subsequently precipitated by adding 4 volumes of acetone (at -20°C), and the mixtures were incubated at -20°C for 15 min. Samples were centrifuged at 4°C at 13000 rpm during 5 min.
- the acetone supernatant was discarded and the pellet was incubated at -20°C for 1 hour after adding 150 ⁇ L ice-cold 60% methanol.
- the methanol supernatant containing the released oligosaccharides was collected and dried by rotary evaporation (SpeedVac).
- the sample was mixed by vortexing. After conjugation, the excess of 2-AB was removed as follows.
- the sample was diluted with 16- ⁇ L purified water (MilliQ) and applied to a Sephadex G-10 column (diameter of 1 cm, height of 1.2 cm, Amersham Biosciences; coupled to a VacElut system, Varian) after the column was pulled dry.
- the labeled oligosaccharides were eluted by applying 2 x 100- ⁇ L purified water (MilliQ) to the column.
- the eluates of the reference carbohydrates Man-9, Man-8, Man-7 and Man-6) were dried and stored at -70°C until HPLC analysis.
- the eluates of the Els samples as well as the Man-9 reference glycan were distributed over 4 numbered PCR tubes and dried.
- the reactions as outlined in Table 8 were performed, all reactions were allowed to proceed overnight at 37°C, except for the reaction in tube 3 which was terminated after 1 h.
- the final concentration of the exoglycosidase enzymes (all obtained from Glyko, Bicester, UK) used were: for ⁇ l-2 Mannosidase (Aspergillus saitoi): 2 mU/mL; for ⁇ -Mannosidase (Jack Bean): 50 U/mL; and for ⁇ -Mannosidase (Helix pomatia): 4 U/mL.
- Figure 56 shows a higher oligomannose consisting of 10 mannose moieties coupled to chitobiose. Each terminal mannose residue is linked by an ⁇ 1-3 bond to a non-terminal mannose residue.
- the oligomannose of Figure 56 is fully resistant to cleavage by the exoglycosidase ⁇ 1-2 Mannosidase. Long-term (overnight) incubation of the oligomannose of Figure 56 with the exoglycosidase ⁇ -Mannosidase will result in cleavage of all ⁇ -linkages ( ⁇ 1-2, ⁇ 1-3, ⁇ 1-6), but not of the ⁇ -linkages.
- the resulting oligosaccharide will thus be 4'- ⁇ - mannosyl chitobiose.
- This 4'- ⁇ -mannosyl chitobiose moiety can be converted to mannose and chitobiose through the action of the exoglycosidase ⁇ -Mannosidase.
- ⁇ -Mannosidase converts the reference oligosaccharide Man-6 (see Figure 55.D) to 4'- ⁇ -mannosyl chitobiose completely and the further conversion thereof to mannose and chitobiose by ⁇ -Mannosidase is also reported to be complete.
- Figure 57 shows a higher oligomannose consisting of 9 mannose moieties coupled to chitobiose.
- this oligomannose one terminal mannose residue is linked by an ⁇ 1-2 bond to the non-terminal mannose residue.
- said ⁇ 1-2-linked mannose will be removed.
- the reaction products as described for the oligomannose of Figure 56 will be obtained.
- ⁇ 1-2 Mannosidase is capable of converting the reference oligosaccharides Man-9 and Man-6 to Man-5 (see Figure 55) with an efficiency of >90 %.
- Figure 58 shows the reference higher oligomannose Man-9 consisting of 9 mannose moieties coupled to chitobiose. In this oligomannose, all terminal mannose residue is linked by an ⁇ 1- 2 bond to a non-terminal mannose residue.
- Man-9 will thus be converted to Man-5, according to the specification of the supplier by > 90%).
- Solvent A consisted of 0.1% acetic acid in acetonitrile and solvent B consisted of 0.2 %> acetic acid-0.2 % triethylamine in water. Separation of 2-AB labeled oligosaccharides was carried out using 28 % B isocratic for 5 column volumes followed by a linear increase to 45 % B over fifteen column volumes.
- Man-6 is eluting at 53+1 min
- Man-7 is eluting at 59+1 min
- Man-8 at 67+2 min
- Man-9 at 70+1 min
- 4'- ⁇ -mannosyl chitobiose is eluting at 10+1 min and chitobiose at 6 ⁇ 1 min (not shown).
- Oligomannoses with terminal glucoses have been marked with a "*", for some reference is made to "from 62*", meaning that the structure can be derived from the structure given in Figure 62.
- “1” is the 'reaction' without exoglycosidases
- "2” is the reaction with ⁇ 1-2 Mannosidase.
- the oligomannose with retention time 45 ⁇ 1 min is supposedly an oligomannose comprising 5 mannose residues linked to chitobiose.
- the oligomannose with retention time 75+1 min is supposedly an oligomannose comprising 10 mannose residues linked to chitobiose.
- Possible glycosylation sites in E2s are N 417 , N 423 , N 430 , N 448 , N 478 , N 532 , N 540 , N 556 , N 5 6 , N 623 and N 6 (see Figure 68). Maldi-MS analysis showed for each of these glycosylation sites that N-glycosylation was not complete, because after deglycosylation with PNGase F, peptides were found with either N or D at the glycosylation site (mass difference 1 Da).
- Both proteins were directly adsorbed on a microtiterplate at 0.5 ⁇ g/mL (lh, 37°C) and after blocking of the plate (PBS-0.1% casein, lh, 37°C) sera from HCV-screened and negative blood donors were incubated at a dilution of 1/20 (PBS-0.5%> casein, 10% (w/v) sucrose, 0.2% (v/v) Triton X-705, lh, 37°C). Finally binding was detected using a secondary rabbit anti-human IgG-F c specific antiserum coupled to peroxidase (Dako, Denmark) at a dilution of 1/50000 (PBS-0.1%> casein, lh, RT) followed by color development.
- the cut-off for this ELISA was set at 2 times the mean of the background (i.e. the reactivity of all sera in an identical set-up but with streptavidin adsorbed to the wells). From Table 15 it can be judged that many (75 %>) sera show reactivity above the cut-off towards the Saccharomyces-produced El while only a few sera (6 %) show some reactivity above cut-off with the Hansenula-produced El. This difference in reactivity was attributed to the presence of terminal ⁇ 1-3 mannoses linked to ⁇ 1-2 mannoses on the Saccharomyces- produced El as evidenced in Example 26.
- Nanadate-resistant yeast mutants are defective in protein glycosylation. Proc. ⁇ atl.Acad.Sci.U.S.A 88:3209-3212.
- hepatitis B surface antigen HBsAg particles derived from mammalian cells (CHO) and yeast cells (Hansenula polymorpha): composition, structure and immunogenicity.
- Vingerhoeds,M.£ Haisma,H.J., Belliot,S.O., Smit,R.H., Crommelin,D.J., and Storm,G.
- trimera mouse system a mouse model for hepatitis C infection and evaluation of therapeutic agents. 6th International Symposium on hepatitis C and related viruses. Bethesda 6-9 June, 1999.
Abstract
Description
Claims
Priority Applications (12)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ529324A NZ529324A (en) | 2001-04-24 | 2002-04-24 | Core-glycosylated HCV envelope proteins made in yeast cells |
AU2002257392A AU2002257392B2 (en) | 2001-04-24 | 2002-04-24 | Core-glycosylated hcv envelope proteins |
BR0209034-1A BR0209034A (en) | 2001-04-24 | 2002-04-24 | Glycosylated hcv wrap proteins in the nucleus |
MXPA03009632A MXPA03009632A (en) | 2001-04-24 | 2002-04-24 | Core-glycosylated hcv envelope proteins. |
CA002443781A CA2443781A1 (en) | 2001-04-24 | 2002-04-24 | Core-glycosylated hcv envelope proteins |
JP2002583616A JP4173741B2 (en) | 2001-04-24 | 2002-04-24 | Core glycosylated HCV envelope protein |
EP02727059A EP1417298A2 (en) | 2001-04-24 | 2002-04-24 | Core-glycosylated hcv envelope proteins |
HU0303924A HUP0303924A2 (en) | 2001-04-24 | 2002-04-24 | Core-glycosylated hcv envelope proteins |
SK1314-2003A SK13142003A3 (en) | 2001-04-24 | 2002-04-24 | Core-glycosylated HCV envelope proteins |
KR1020037013778A KR100950104B1 (en) | 2001-04-24 | 2002-04-24 | Core-glycosylated HCV envelope proteins |
IL15842202A IL158422A0 (en) | 2001-04-24 | 2002-04-24 | Core-glycosylated hcv envelope proteins |
HR20030946A HRP20030946A2 (en) | 2001-04-24 | 2003-11-20 | Core-glycosylated hcv envelope proteins |
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PCT/BE2002/000063 WO2002086100A2 (en) | 2001-04-24 | 2002-04-24 | Expression of core-glycosylated hcv envelope proteins in yeast |
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US (3) | US7048930B2 (en) |
EP (3) | EP1414942A2 (en) |
JP (2) | JP4173741B2 (en) |
KR (1) | KR100950104B1 (en) |
CN (1) | CN1636050A (en) |
AR (3) | AR035869A1 (en) |
AU (3) | AU2002308449B2 (en) |
BR (2) | BR0209034A (en) |
CA (3) | CA2443781A1 (en) |
CZ (1) | CZ20032853A3 (en) |
HU (1) | HUP0303924A2 (en) |
MX (2) | MXPA03009626A (en) |
NZ (2) | NZ529324A (en) |
OA (1) | OA13092A (en) |
PL (1) | PL366621A1 (en) |
RU (1) | RU2274643C2 (en) |
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