US20080138894A1 - Purified hepatitis c virus envelope proteins for diagnostic and therapeutic use - Google Patents

Purified hepatitis c virus envelope proteins for diagnostic and therapeutic use Download PDF

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US20080138894A1
US20080138894A1 US11/678,513 US67851307A US2008138894A1 US 20080138894 A1 US20080138894 A1 US 20080138894A1 US 67851307 A US67851307 A US 67851307A US 2008138894 A1 US2008138894 A1 US 2008138894A1
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hcv
protein
proteins
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Geert Maertens
Fons Bosman
Guy De Martynoff
Marie-Ange Buyse
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Fujirebio Europe NV SA
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Innogenetics NV SA
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
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    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
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    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
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    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24241—Use of virus, viral particle or viral elements as a vector
    • C12N2770/24243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y10S435/803—Physical recovery methods, e.g. chromatography, grinding
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    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/911—Microorganisms using fungi
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/975—Kit

Definitions

  • the present invention relates to the general fields of recombinant protein expression, purification of recombinant proteins, synthetic peptides, diagnosis of HCV infection, prophylactic treatment against HCV infection and to the prognosis, monitoring of the clinical efficiency of treatment of an individual with chronic hepatitis, or the prognosis/monitoring of natural disease.
  • the present invention relates to purification methods for hepatitis C virus envelope proteins, the use in diagnosis, prophylaxis or therapy of HCV envelope proteins purified according to the methods described in the present invention, the use of single or specific oligomeric E1 and/or E2 and/or E1/E2 envelope proteins in assays for monitoring disease, and/or diagnosis of disease, and/or treatment of disease.
  • the invention also relates to epitopes of the E1 and/or E2 envelope proteins and monoclonal antibodies thereto, as well their use in diagnosis, prophylaxis or treatment.
  • the E2 protein purified from cell lysates according to the methods described in the present invention reacts with approximately 95% of patient sera. This reactivity is similar to the reactivity obtained with E2 secreted from CHO cells (Spaete et al., 1992). However, the intracellularly expressed form of E2 may more closely resemble the native viral envelope protein because it contains high mannose carbohydrate motifs, whereas the E2 protein secreted from CHO cells is further modified with galactose and sialic acid sugar moieties. When the aminoterminal half of E2 is expressed in the baculovirus system, only about 13 to 21% of sera from several patient groups can be detected (Inoue et al., 1992). After expression of E2 from E. coli , the reactivity of HCV sera was even lower and ranged from 14 (Yokosuka et al., 1992) to 17% (Mita et al., 1992).
  • HCV sera About 75% of HCV sera (and 95% of chronic patients) are anti-s positive using the purified, vaccinia-expressed recombinant E1 protein of the present invention, in sharp contrast with the results of Kohara et al. (1992) and Hsu et al. (1993).
  • Kohara et al. used a vaccinia-virus expressed E1 protein and detected anti-E1 antibodies in 7 to 23% of patients, while Hsu et al. only detected 14150 (28%) sera using baculovirus-expressed E1.
  • the vaccinia virus system may be used for selecting the best expression constructs and for limited upscaling, and large-scale expression and purification of single or specific oligomeric envelope proteins containing high-mannose carbohydrates may be achieved when expressed from several yeast strains.
  • hepatitis B for example, manufacturing of HBsAg from mammalian cells was much more costly compared with yeast-derived hepatitis B vaccines.
  • compositions comprising purified (single or specific oligomeric) recombinant E1 and/or E2 and/or E1/E2 glycoproteins comprising conformational epitopes from the E1 and/or E2 domains of HCV.
  • E1 and/or E2 peptides which can be used for diagnosis of HCV infection and for raising antibodies.
  • Such peptides may also be used to isolate human monoclonal antibodies.
  • kits for monitoring/prognosing the response to treatment e.g. with interferon
  • monitoring/prognosing the outcome of the disease e.g. with interferon
  • hepatitis C virus single envelope protein refers to a polypeptide or an analogue thereof (e.g. mimotopes) comprising an amino acid sequence (and/or amino acid analogues) defining at least one HCV epitope of either the E1 or the E2 region.
  • These single envelope proteins in the broad sense of the word may be both monomeric or homo-oligomeric forms of recombinantly expressed envelope proteins.
  • the sequences defining the epitope correspond to the amino acid sequence of either the E1 or the E2 region of HCV (either identically or via substitution of analogues of the native amino acid residue that do not destroy the epitope).
  • the epitope-defining sequence will be 3 or more amino acids in length, more typically, 5 or more amino acids in length, more topically 8 or more 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 homooligomer or heterooligomer.
  • the HCV antigens of the present invention comprise conformational epitopes from the E1 and/or E2 (envelope) domains of HCV.
  • the E1 domain which is believed to correspond to the viral envelope protein, is currently estimated to span amino acids 192-383 of the HCV polyprotein (Hijikata et al., 1991).
  • the E2 protein previously called NS1, is believed to span amino acids 384-809 or 384-746 (Grakoui et al. 1993) of the HCV polyprotein and to also be an envelope protein.
  • the E2 protein may also be expressed together with the E1, P7 (aa 747-809), NS2 (aa 810-1025), NS4A (aa 1658-1711) or NS4B (aa 1712-1972). Expression together with these other HCV proteins may be important for obtaining the correct protein folding.
  • the E1 and E2 antigens used in the present invention may be full-length viral proteins, substantially full-length versions thereof, or functional fragments thereof (e.g. fragments which are not missing sequence essential to the formation or retention of an epitope).
  • 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 though screening the antigen of interest with an antibody (polygonal serum or monoclonal to the conformational epitope) and comparing its reactivity to that of a denatured version of the antigen which retains only linear epitopes (if any). In such screening using polyclonal antibodies, it may be advantageous to adsorb the polyclonal serum first with the denatured antigen and see if it retains antibodies to the antigen of interest.
  • the HCV antigens of the present invention can be made by any recombinant method that provides the epitope of interest.
  • recombinant intracellular expression in mammalian or insect cells is a preferred method to provide glycosylated E1 and/or E2 antigens in ‘native’ conformation as is the case for the natural HCV antigens.
  • Yeast cells and mutant yeast strains e.g.
  • mnn 9 mutant (Kniskern et al., 1194) or glycosylation mutants derived by means of vanadate resistance selection (Ballou et al., 1991)) may be ideally suited for production of secreted high-mannose-type sugars; whereas proteins secreted from mammalian cells may contain modifications including galactose or sialic acids which may be undesirable for certain diagnostic or vaccine applications. However, it may also be possible and sufficient for certain applications, as it is known for proteins, to express the antigen in other recombinant hosts (such as E. coli ) and renature the protein after recovery.
  • other recombinant hosts such as E. coli
  • fusion polypeptide intends a polypeptide in which the HCV antigen(s) are part of a single continuous chain of amino acids, which chain does not occur in nature.
  • the HCV antigens may be connected directly to each other by peptide bonds or be separated by intervening amino acid sequences.
  • the fusion polypeptides may also contain amino acid sequences exogenous to HCV.
  • solid phase intends a solid body to which the individual HCV antigens or the fusion polypeptide comprised of HCV antigens are bound covalently or by noncovalent means such as hydrophobic adsorption.
  • biological sample intends a fluid or tissue of a mammalian individual (e.g. an anthropoid, a human) that commonly contains antibodies produced by the individual, more particularly antibodies against HCV.
  • the fluid or tissue may also contain HCV antigen.
  • HCV antigen include, without limitation, blood, plasma, serum, urine, spinal fluid, lymph fluid, secretions of the respiratory, intestinal or genitourinary tracts, tears, saliva, milk, white blood cells and myelomas.
  • Body components include biological liquids.
  • biological liquid refers to a fluid obtained from an organism. Some biological fluids are used as a source of other products, such as clotting factors (e.g. Factor VIII:C), serum albumin, growth hormone and the like. In such cases, it is important that the source of biological fluid be free of contamination by virus such as HCV.
  • immunologically reactive means that the antigen in question will react specifically with anti-HCV antibodies present in a body component from an HCV infected individual.
  • immune complex intends the combination formed when an antibody binds to an epitope on an antigen.
  • E1 refers to a protein or polypeptide expressed within the first 400 amino acids of an HCV polyprotein, sometimes referred to as the E, ENV or S protein. In its natural form it is a 35 kDa glycoprotein which is found in strong association with membranes. In most natural HCV strains, the E1 protein is encoded in the viral polyprotein following the C (core) protein. The E1 protein extends from approximately amino acid (aa) 192 to about aa 383 of the full-length polyprotein.
  • E1 as used herein also includes analogs and truncated forms that are immunologically cross-reactive with natural E1, and includes E1 proteins of genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other newly identified HCV type or subtype.
  • E2 refers to a protein or polypeptide expressed within the first 900 amino acids of an HCV polyprotein, sometimes referred to as the NS1 protein. In its natural form it is a 72 kDa glycoprotein that is found in strong association with membranes. In most natural HCV strains, the E2 protein is encoded in the viral polyprotein following the E1 protein. The E2 protein extends from approximately amino acid position 384 to amino acid position 746, another form of E2 extends to amino acid position 809.
  • the term ‘E2’ as used herein also includes analogs and truncated forms that are immunologically cross-reactive with natural E2. 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).
  • E1/E2 refers to an oligomeric form of envelope proteins containing at least one E1 component and at least one E2 component.
  • E1 and/or E2 and/or E1/E2 envelope proteins refers to all possible oligomeric forms of recombinantly expressed E1 and/or E2 envelope proteins which are not aggregates.
  • E1 and/or E2 specific oligomeric envelope proteins are also referred to as homo-oligomeric E1 or E2 envelope proteins (see below).
  • E1 and/or E2 and/or E1/E2 envelope proteins refers to single monomeric E1 or E2 proteins (single in the strict sense of the word) as well as specific oligomeric E1 and/or E2 and/or E1/E2 recombinantly expressed proteins.
  • homo-oligomer refers to a complex of E1 and/or E2 containing more than one E1 or E2 monomer, e.g. E1/E1 dimers, E1/E1E1 trimers or E1/E1/E1/E1, tetramers and E2/E2 dimers, E2/E2/E2 trimers or E2/E2/E2/E2 tetramers, E1 pentamers and hexamers, E2 pentamers and hexamers or any higher-order homo-oligomers of E1 or E2 are all ‘homo-oligomers’ within the scope of this definition.
  • the oligomers may contain one, two, or several different monomers of E1 or E2 obtained from different types or subtypes of hepatitis C virus including for example those described in an international application published under WO 94/25601 and European application No. 94870166.9 both by the present applicants.
  • Such mixed oligomers are still homo-oligomers within the scope of this invention, and may allow more universal diagnosis, prophylaxis or treatment of HCV.
  • the term ‘purified’ as applied to proteins herein refers to a composition wherein the desired protein comprises at least 35% of the total protein component in the composition.
  • the desired protein preferably comprises at least 40%, more preferably at least about 50%, more preferably at least about 60%, still more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% of the total protein component.
  • the composition may contain other compounds such as carbohydrates, salts, lipids, solvents, and the like, without affecting the determination of the percentage purity as used herein.
  • An ‘isolated’ HCV protein intends an HCV protein composition that is at least 35% pure.
  • essentially purified proteins refers to proteins purified such that they can be used for in vitro diagnostic methods and as a therapeutic compound. These proteins are substantially free from cellular proteins, vector-derived proteins or other HCV viral components. Usually these proteins are purified to homogeneity (at least 80 to pure, preferably, 90%, more preferably 95%, more preferably 97%, more preferably 98%, more preferably 99%, even more preferably 99%, and most preferably the contaminating proteins should be undetectable by conventional methods like SDS-PAGE and silver staining.
  • recombinantly expressed used within the context of the present invention refers to the fact that the proteins of the present invention are produced by recombinant expression methods be it in prokaryotes, or lower or higher eukaryotes as discussed in detail below.
  • lower eukarycote refers to host cells such as yeast, fungi and the like.
  • Lower eukaryotes are generally (but not necessarily) unicellular.
  • Preferred lower eukaryotes are yeasts, particularly species within Saccharomyces. Schizosaccharomyces, Kluveromyces, Pichia (e.g. Pichia pastoris ). Hansenula (e.g. Hansenula polymorpha ), Yarowia, Schwaniomyces, Schizosaccharomyces. Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts.
  • prokaryotes refers to hosts such as E. coli, Lactobacillus. Lactococcus, Salmonella, Streptococcus, Bacillus subtilis or Streptomyces . Also these hosts are contemplated within the present invention.
  • higher eukaryote refers to host cells derived from higher animals, such as mammals, reptiles, insects, and the like.
  • Presently preferred higher eukaryote host cells are derived from Chinese hamster (e.g. CHO), monkey (e.g. COS and Vero cells), baby hamster kidney (BHK), pig kidney (PK15), rabbit kidney 13 cells (RK13), the human osteosarcoma cell line 143 B, the human cell line HeLa and human hepatoma cell lines like Hep G2, and insect cell lines (e.g. Spodoptera frugiperda ).
  • the host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively the host cells may also be transgenic animals.
  • polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product, thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogues of an amino 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.
  • polynucleotide or nucleic acid intends a polynucleotide or nucleic acid of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.
  • recombinant host cells refer to cells which can be or have been, used as recipients for a recombinant vector or other transfer polynucleotide, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • replicon is any genetic element, e.g., a plasmid, a chromosome, a virus, a cosmid, etc., that behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control.
  • vector is a replicon further comprising sequences providing replication and/or expression of a desired open reading frame.
  • control sequence refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism: in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and terminators; in eukaryotes, generally, such control sequences include promoters, terminators and, in some instances, enhancers.
  • control sequences is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences which govern secretion.
  • promoter is a nucleotide sequence which is comprised of consensus sequences which allow the binding of RNA polymerase to the DNA template in a manner such that mRNA production initiates a; the normal transcription initiation site for the adjacent structural gene.
  • control sequence ‘operably linked’ refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence ‘operably linked’ to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • ORF ‘open reading frame’ is a region of a polynucleotide sequence which encodes a polypeptide and does not contain stop codons, this region may represent a portion of a coding sequence or a total coding sequence.
  • a ‘coding sequence’ is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries or the coding sequence are determined by a translation star codon at the terminus and a translation stop codon at the 3′-terminus.
  • a coding sequence can include but is not limited to mRNA, DNA (including cDNA), and recombinant polynucleotide sequences.
  • epitope or ‘antigenic determinant’ means an amino acid sequence that is immunoreactive. Generally an epitope consists of at least 3 to 4 amino acids, and more usually, consists of at least 5 or 6 amino acids, sometimes the epitope consists of about 7 to 8, or even about 10 amino acids.
  • immunogenic refers to the ability of a substance to cause a humoral and/or cellular response, whether alone or when linked to a carrier, in the presence or absence of an adjuvant.
  • Negtralization refers to an immune response that blocks the infectivity, either partially or fully, of an infectious agent.
  • a ‘vaccine’ is an immunogenic composition capable of eliciting protection against HCV, whether partial or complete.
  • a vaccine may also be useful for treatment of an individual, in which case it is called a therapeutic vaccine.
  • terapéutica refers to a composition capable of treating HCV infection.
  • an effective amount refers to an amount of epitope-bearing polypeptide sufficient to induce an immunogenic response in the individual to which it is administered, or to otherwise detectably immunoreact in its intended system (e.g., immunoassay).
  • the effective amount is sufficient to effect treatment, as defined above.
  • the exact amount necessary will vary according to the application.
  • the effective amount may vary depending on the species, age, and general condition of the individual, the severity of the condition being treated, the particular polypeptide selected and its mode of administration, etc. It is also believed that effective amounts will be found within a relatively large, non-critical range. An appropriate effective amount can be readily determined using only routine experimentation.
  • E1 and/or E2 and/or E1/E2 single or specific oligomeric envelope proteins for prophylaxis of HCV disease are 0.01 to 100 ⁇ g/dose, preferably 0.1 to 50 ⁇ g/dose. Several doses may be needed per individual in order to achieve a sufficient immune response and subsequent protection against HCV disease.
  • the present invention contemplates a method for isolating or purifying recombinant HCV single or specific oligomeric envelope protein selected from the group consisting of E1 and/or E2 and/or E1/E2, characterized in that upon lysing the transformed host cells to isolate the recombinantly expressed protein a disulphide bond cleavage or reduction step is carried out with a disulphide bond cleaving agent.
  • the proteins according to the present invention are recombinantly expressed in lower or higher eukaryotic cells or in prokaryotes.
  • the recombinant proteins of the present invention are preferably glycosylated and may contain high-mannose-type, hybrid, or complex glycosylations, preferentially said proteins are expressed from mammalian cell lines as discussed in detail in the Examples section, or in yeast such as in mutant yeast strains also as de-ailed in the Examples section.
  • the proteins according to the present invention may be secreted or expressed within components of the cell, such as the ER or the Golgi Apparatus.
  • the proteins of the present invention bear high-mannose-type glycosylations and are retained in the ER or Golgi Apparatus of mammalian cells or are retained in or secreted from yeast cells, preferably secreted from yeast mutant strains such as the mnn9 mutant (Kniskern et al., 1994), or from mutants that have been selected by means of vanadate resistance (Ballou et al., 1991).
  • HCV envelope proteins Upon expression of HCV envelope proteins, the present inventors could show that some of the free thiol groups of cysteines not involved in intra- or inter-molecular disulphide bridges, react with cysteines of host or expression-system-derived (e.g. vaccinia) proteins or of other HCV envelope proteins (single or oligomeric), and form aspecific intermolecular bridges. This results in the formation of ‘aggregates’ of HCV envelope proteins together with contaminating proteins. It was also shown in WO92/08734 that ‘aggregates’ were obtained after purification, but it was not described which protein interactions were involved. In patent application WO 92/08734, recombinant E1/E2 protein expressed with the vaccinia virus system were partially purified as aggregates and only found to be 70% pure, rendering the purified aggregates not useful for diagnostic, prophylactic or therapeutic purposes.
  • host or expression-system-derived proteins e.g. vaccinia
  • other HCV envelope proteins single or oli
  • a major aim of the present invention resides in the separation of single or specific-oligomeric HCV envelope proteins from contaminating proteins, and to use the purified proteins (>959% pure) for diagnostic, prophylactic and therapeutic purposes.
  • the present inventors have been able to provide evidence that aggregated protein complexes (‘aggregates’) are formed on the basis of disulphide bridges and non-covalent protein-protein interactions.
  • the present invention thus provides a means for selectively cleaving the disulphide bonds under specific conditions and for separating the cleaved proteins from contaminating proteins which greatly interfere with diagnostic, prophylactic and therapeutic applications.
  • the free thiol groups may be blocked (reversibly or irreversibly) in order to prevent the reformation of disulphide bridges, or may be left to oxidize and oligomerize with other envelope proteins (see definition homo-oligomer). It is to be understood that such protein oligomers are essentially different from the ‘aggregates’ described in WO 92/08734 and WO 94/01778, since the level of contaminating proteins is undetectable.
  • Said disulfide bond cleavage may also be achieved by:
  • Said disulphide bond cleavage (or reducing) step of the present invention is preferably a partial disulphide bend cleavage (reducing) step (carried out under partial cleavage or reducing conditions).
  • a preferred disulphide bond cleavage or reducing agent according to the present invention is dithiothreitol (DTT). Partial reduction is obtained by using a low concentration of said reducing agent, i.e. for DTT for example in the concentration range of about 0.1 to about 50 mM, preferably about 0.1 to about 20 mM, preferably about 0.5 to about 10 mM, preferably more than 1 mM, more than 2 mM or more than 5 mM, more preferably about 1.5 mM, about 2.0 mM, about 2.5 mM, about 5 mM or about 7.5 mM.
  • DTT dithiothreitol
  • Said disulphide bond cleavage step may also be carried out in the presence or a suitable detergent (as an example of a means for cleaving disulphide bonds or in combination with a cleaving agent) able to dissociate the expressed proteins, such as DecylPEG, EMPIGEN-BB, NP-40, sodium cholate, Triton X-100.
  • a suitable detergent as an example of a means for cleaving disulphide bonds or in combination with a cleaving agent
  • Said reduction or cleavage step (preferably a partial reduction or cleavage step) is carried out preferably in in the presence of (with) a detergent.
  • a preferred detergent according to the present invention is Empigen-BB.
  • the amount of detergent used is preferably in the range of 1 to 10%, preferably more than 3%, more preferably about 3.5% of a detergent such as Empigen-BB.
  • a particularly preferred method for obtaining disulphide bond cleavage employs a combination of a classical disulphide bond cleavage agent as detailed above and a detergent (also as detailed above).
  • a detergent also as detailed above.
  • the particular combination of a low concentration of DTT (1.5 to 7.5 mM) and about 3.5% of Empigen-BB is proven to be a particularly preferred combination of reducing agent and detergent for the purification of recombinantly expressed E1 and E2 proteins.
  • said partial reduction is shown to result in the production of possibly dimeric E1 protein and separation of this E1 protein from contaminating proteins that cause false reactivity upon use in immunoassays.
  • a disulphide bond cleaving means may also include any disulphide bridge exchanging agents (competitive agent being either organic or proteinaeous, see for instance Creighton, 1988) known in the art which allows the following type of reaction to occur: R1S ⁇ S R2 ⁇ R3SH ⁇ R1S ⁇ S R3 ⁇ R2SH
  • R1, R2 compounds of protein aggregates
  • R3 SH competitive agent (organic, proteinaeous)
  • disulphide bridge exchanging agent is to be interpretated as including disulphide bond reforming as well as disulphide bond blocking agents.
  • the present invention also relates to methods for purifying or isolating HCV single or specific oligomeric envelope proteins as set out above further including the use of any SH group blocking or binding reagent known in the art such as chosen from the following list:
  • conditions such as low pH (preferably lower than pH 6) for preventing free SH groups from oxidizing and thus preventing the formation of large intermolecular aggregates upon recombinant expression and purification of E1 and E2 (envelope) proteins are also within the scope of the present invention.
  • a preferred SH group blocking reagent according to the present invention is N-ethylmaleimide (NEM).
  • NEM N-ethylmaleimide
  • Said SH croup blocking reagent may be administrated during lysis of the recombinant host cells and after the above-mentioned partial reduction process or after any other process for cleaving disulphide bridges.
  • Said SH group blocking reagent may also be modified with any group capable of providing a detectable label and/or any group aiding in the immobilization of said recombinant protein to a solid substrate, e.g. biotinylated NEM.
  • a method to purify single or specific oligomeric recombinant E1 and/or E2 and/or E1/E2 proteins according to the present invention as defined above is further characterized as comprising the following steps:
  • Preferred lectin-chromatography systems include Galanthus nivalis agglutinin (GNA)-chromatography, or Lens culinaris agglutinin (LCA) (lentil) lectin chromatography as illustrated in the Examples section.
  • Other useful lectins include those recognizing high-mannose type sugars, such as Narcissus pseudonarcissus agglutinin (NPA), Pisum sativum agglutinin (PSA), or Allium ursinum agglutinin (AUA).
  • Preferably said method is usable to purify single or specific oligomeric HCV envelope protein produced intracellularly as detailed above.
  • lectins binding complex sugars such as Ricinus communis agglutinin I (RCA I) are preferred lectins.
  • the present invention more particularly contemplates essentially purified recombinant HCV single or specific oligomeric envelope proteins, selected from the group consisting of E1 and/or E2 and/or E1/E2, characterized as being isolated or purified by a method as defined above.
  • the present invention more particularly relates to the purification or isolation of recombinant envelope proteins which are expressed from recombinant mammalian cells such as vaccinia.
  • the present invention also relates to the purification or isolation of recombinant envelope proteins which are expressed from recombinant yeast cells.
  • the present invention equally relates to the purification or isolation of recombinant envelope proteins which are expressed from recombinant bacterial (prokaryotic) cells.
  • the present invention also contemplates a recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic promoter sequence followed by a nucleotide sequence allowing the expression of the single or specific oligomeric E1 and/or E2 and/or E1/E2 of the invention.
  • the present invention contemplates a recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic promoter sequence followed by a nucleotide sequence allowing the expression of the single E1 or E1 of the invention.
  • the present invention contemplates a recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic promoter sequence followed by a nucleotide sequence allowing the expression of the single E1 or E2 of the invention.
  • the segment of the HCV cDNA encoding the desired E1 and/or E2 sequence inserted into the vector sequence may be attached to a signal sequence.
  • Said signal sequence may be that from a non-HCV source, e.g. the IgG or tissue plasminogen activator (tpa) leader sequence for expression in mammalian cells, or the a-mating factor sequence for expression into yeast cells, but particularly preferred constructs according to the present invention contain signal sequences appearing in the HCV genome before the respective start points of the E1 and E2 proteins.
  • the segment of the HCV cDNA encoding the desired E1 and/or E2 sequence inserted into the vector may also include deletions e.g. of the hydrophobic domain(s) as illustrated in the examples section, or of the E2 hypervariable region 1.
  • the recombinant vectors according to the present invention encompass a nucleic acid having an HCV cDNA segment encoding the polyprotein starting in the region between amino acid positions 1 and 192 and ending in the region between positions 250 and 400 of the HCV polyprotein, more preferably ending in the region between positions 250 and 341, even more preferably ending in the region between positions 290 and 341 for expression of the HCV single E1 protein.
  • the present recombinant vector encompasses a recombinant nucleic acid having a HCV cDNA segment encoding part of the HCV polyprotein starting in the region between positions 117 and 192, and ending at any position in the region between positions 263 and 326, for expression of HCV single E1 protein.
  • forms that have the first hydrophobic domain deleted positions 264 to 293 plus or minus 8 amino acids
  • forms to which a 5′-terminal ATC codon and a 3′-terminal stop codon has been added or forms which have a factor Xa cleavage site and/or 3 to 10, preferably 6 Histidine codons have been added.
  • the recombinant vectors according to the present invention encompass a nucleic acid having an HCV cDNA segment encoding the polyprotein starting in the region between amino acid positions 290 and 406 and ending in the region between positions 600 and 320 of the HCV polyprotein, more preferably starting in the region between positions 322 and 406, even more preferably starting in the region between positions 347 and 406 even still more preferably starting in the region between positions 364 and 406 for expression of the HCV single E2 protein.
  • the present recombinant vector encompasses a recombinant nucleic acid having a HCV cDNA segment encoding the polyprotein starting in the region between positions 290 and 406, and ending at any position of positions 623, 650, 661, 673, 710, 715, 720, 746 or 809, for expression of HCV single E2 protein.
  • forms to which a 5′-terminal ATG codon and a 3′-terminal stop codon has been added, or forms which have a factor Xa cleavage site and/or 3 to 10, preferably 6 Histidine codons have been added.
  • a variety of vectors may be used to obtain recombinant expression of HCV single or specific oligomeric envelope proteins of the present invention.
  • Lower eukaryotes such as yeasts and glycosylation mutant strains are typically transformed with plasmids, or are transformed with a recombinant virus.
  • the vectors may replicate within the host independently, or may integrate into the host cell genome.
  • Higher eukaryotes may be transformed with vectors, or may be infected with a recombinant virus, for example a recombinant vaccinia virus.
  • a recombinant virus for example a recombinant vaccinia virus.
  • Techniques and vectors for the insertion of foreign DNA into vaccinia virus are well known in the art, and utilize, for example homologous recombination.
  • viral promoter sequences, possibly terminator sequences and poly(A)-addition sequences, possibly enhancer sequences and possibly amplification sequences, all required for the mammalian expression, are available in the art.
  • Vaccinia is particularly preferred since vaccinia halts the expression of host cell proteins.
  • Vaccinia is also very much preferred since it allows the expression of E1 and E2 proteins of HCV in cells or individuals which are immunized with the live recombinant vaccinia virus.
  • AMV Ankara Modified Virus
  • insect expression transfer vectors derived from baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), which is a helper-independent viral expression vector.
  • AcNPV Autographa californica nuclear polyhedrosis virus
  • Expression vectors derived from this system usually use the strong viral polyhedrin gene promoter to drive the expression of heterologous genes.
  • Different vectors as well as methods for the introduction of heterologous DNA into the desired site of baculovirus are available to the man skilled in the art for baculovirus expression.
  • signals for posttranslational modification recognized by insect cells are known in the art.
  • Also included within the scope of the present invention is a method for producing purified recombinant single or specific oligomeric HCV E1 or E2 or E1/E2 proteins, wherein the cysteine residues involved in aggregates formation are replaced at the level of the nucleic acid sequence by other residues such that aggregate formation is prevented.
  • the recombinant proteins expressed by recombinant vectors carrying such a mutated E1 and/or E2 protein encoding nucleic acid are also within the scope of the present invention.
  • the present invention also relates to recombinant E1 and/or E2 and/or E1/E2 proteins characterized in that at least one of their glycosylation sites has been removed and are consequently termed glycosylation mutants.
  • different glycosylation mutants may be desired to diagnose (screening, confirmation, prognosis, etc.) and prevent HCV disease according to the patient in question.
  • An E2 protein glycosylation mutant lacking the GLY4 has for instance been found to improve the reactivity of certain sera in diagnosis.
  • These glycosylation mutants are preferably purified according to the method disclosed in the present invention.
  • Also contemplated within the present invention are recombinant vectors carrying the nucleic acid insert encoding such a E1 and/or E2 and/or E1/E2 glycosylation mutant as well as host cells transformed with such a recombinant vector.
  • the present invention also relates to recombinant vectors including a polynucleotide which also forms part of the present invention.
  • the present invention relates more particularly to the recombinant nucleic acids as represented in SEQ ID NO 3, 5, 7, 9, 11, 13, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 and 49, or parts thereof.
  • the present invention also contemplates host cells transformed with a recombinant vector as defined above, wherein said vector comprises a nucleotide sequence encoding HCV E1 and/or E2 and/or E1/E2 protein as defined above in addition to a regulatory sequence operably linked to said HCV E1 and/or E2 and/or E1/E2 sequence and capable of regulating the expression of said HCV E1 and/or E2 and/or E1/E2 protein.
  • Eukaryotic hosts include lower and higher eukaryotic hosts as described in the definitions section.
  • Lower eukaryotic hosts include yeast cells well known in the art.
  • Higher eukaryotic hosts mainly include mammalian cell lines known in the art and include many immortalized cell lines available from the ATCC, including HeLa cells, Chinese hamster ovary (COC) cells. Baby hamster kidney (BHK) cells. PK15, RK13 and a number of other cell lines.
  • the present invention relates particularly to a recombinant E1 and/or E2 and/or E1/E2 protein expressed by a host cell as defined above containing a recombinant vector as defined above. These recombinant proteins are particularly purified according to the method of the present invention.
  • E1 and/or 2 and, or E1/E2 proteins may be produced in a form which elute differently from the large aggregates containing vector-derived components and, or cell components in the avoid volume of the gelfiltration column or the IMAC column as illustrated in the Examples section.
  • the disulphide bridge cleavage step advantageously also eliminates the false reactivity due to the presence of host and/or expression-system-derived proteins.
  • the presence of NEM and a suitable detergent during lysis of the cells may already partly or even completely prevent the aggregation between the HCV envelope proteins and contaminants.
  • Ni 2 ⁇ -IMAC chromatography followed by a desalting step is preferably used for constructs bearing a (His) 5 as described by Janknecht et al. 1991, and Hochuli et al., 1988.
  • the present invention also relates to a method for producing monoclonal antibodies in small animals such as mice or rats, as well as a method for screening and isolating human B-cells that recognize anti-HCV antibodies, using the HCV single or specific oligomeric envelope proteins of the present invention.
  • the present invention further relates to a composition comprising at least one of the following E1 peptides as listed in Table 3:
  • the present invention also relates to a composition comprising at least one of the following E2 peptides as listed in Table 3:
  • the present invention also relates to a composition comprising at least one of the following E2 conformational epitopes:
  • the present invention also relates to an E1 or E2 specific antibody raised upon immunization with a peptide or protein composition, with said antibody being specifically reactive with any of the polypeptides or peptides as defined above, and with said antibody being preferably a monoclonal antibody.
  • the present invention also relates to an E1 or E2 specific antibody screened from a variable chain library in plasmids or phages or from a population of human B-cells by means of a process known in the art, with said antibody being reactive with any of the polypeptides or peptides as defined above, and with said antibody being preferably a monoclonal antibody.
  • the E1 or E2 specific monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly from a mouse or rat, immunizes against the HCV polypeptides or peptides according to the invention, as defined above on the one hand, and of cells of a myeloma cell line on the other hard, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the polypeptides which has been initially used for the immunization of the animals.
  • the antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
  • the monoclonal antibodies according to this preferred embodiment of the invention may be humanized versions of mouse monoclonal antibodies made by means of recombinant DNA technology, departing from parts of mouse and/or human genomic DNA sequences coding for H and L chains from cDNA or genomic clones coding for H and L chains.
  • the monoclonal antibodies according to this preferred embodiment of the invention may be human monoclonal antibodies.
  • These antibodies according to the present embodiment of the invention can also be derived from human peripheral blood lymphocytes of patients infected with HCV, or vaccinated against HCV.
  • Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice (for recent review, see Duchosal et al., 1992).
  • PBL peripheral blood lymphocytes
  • SCID severe combined immune deficiency
  • the invention also relates to the use of the proteins or peptides of the invention, for the selection of recombinant antibodies by the process of repertoire cloning (Persson et al., 1991).
  • Antibodies directed to peptides or single or specific oligomeric envelope proteins derived from a certain genotype may be used as a medicament, more particularly for incorporation into an immunoassay for the detection of HCV genotypes (for detecting the presence of HCV E1 or E2 antigen), for prognosing/monitoring of HCV disease, or as therapeutic agents.
  • the present invention also relates to the use of any of the above-specified E1 or E2 specific monoclonal antibodies for the preparation of an immunoassay kit for detecting the presence of E1 or E2 antigen in a biological sample, for the preparation of a kit for prognosing/monitoring of HCV disease or for the preparation of a HCV medicament.
  • the present invention also relates to the a method for in vitro diagnosis or detection of HCV antigen present in a biological sample, comprising at least the following steps
  • the present invention also relates to a kit for in vitro diagnosis of HCV antigen present in a biological sample, comprising:
  • the present invention also relates to a composition comprising E1 and/or E2 and/or E1, E2 recombinant HCV proteins purified according to the method of the present invention or a composition comprising at least one peptides as specified above for use as a medicament.
  • the present invention more particularly relates to a composition comprising at least one of the above-specified envelope peptides or a recombinant envelope protein composition as defined above, for use as a vaccine for immunizing a mammal, preferably humans, against HCV, comprising administering a sufficient amount of the composition possibly accompanied by pharmaceutically acceptable adjuvant(s), to produce an immune response.
  • the present invention relates to the use of any of the compositions as described here above for the preparation of a vaccine as described above.
  • the present invention relates to a vaccine composition for immunizing a mammal, preferably humans, against HCV, comprising HCV single or specific oligomeric proteins or peptides derived from the E1 and/or the E2 region as described above.
  • Immunogenic compositions can be prepared according to methods known in the art.
  • the present compositions comprise an immunogenic amount of a recombinant E1 and/or E2 and/or E1/E2 single or specific oligomeric proteins as defined above or E1 or E2 peptides as defined above, usually combined with a pharmaceutically acceptable carrier, preferably further comprising an adjuvant.
  • the single or specific oligomeric envelope proteins of the present invention are expected to provide a particularly useful vaccine antigen, since the formation of antibodies to either E1 or E2 may be more desirable than to the other envelope protein, and since the E2 protein is cross-reactive between HCV types and the E1 protein is type-specific.
  • Cocktails including type 1 E2 protein and E1 proteins derived from several genotypes may be particularly advantageous.
  • Cocktails containing a molar excess of E1 versus E2 or E2 versus E1 may also be particularly useful.
  • Immunogenic compositions may be administered to animals to induce production of antibodies, either to provide a source of antibodies or to induce protective immunity in the animal.
  • Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • 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 of ordinary skill in the art.
  • Preferred adjuvants to enhance effectiveness of the composition include, but are nor limited to: aluminim hydroxide (alum), N-acetyl-muramyl-L-threonyl-O-isoglutamine (thr-MOP) as found in U.S. Pat. No.
  • any of the 3 components MPL, TDM or CWS may also be used alone or combined 2 by 2. Additionally, adjuvants such as Stimulon (Cambridge Bioscience, Worcester, Mass.) or SAF-1 (Syntex) may be used. Further, Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may be user for non-human applications and research purposes.
  • adjuvants such as Stimulon (Cambridge Bioscience, Worcester, Mass.) or SAF-1 (Syntex) may be used. Further, Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may be user for non-human applications and research purposes.
  • CFA Complete Freund's Adjuvant
  • IFA Incomplete Freund's Adjuvant
  • the immunogenic compositions typically will contain pharmaceutically acceptable vehicles, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, preservatives, and the like, may be included in such vehicles.
  • pharmaceutically acceptable vehicles such as water, saline, glycerol, ethanol, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives, and the like, may be included in such vehicles.
  • the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect.
  • the E1 and E2 proteins may also be incorporated into Immune Stimulating Complexes together with saponins, for example Quil A (ISCOMS).
  • Immunogenic compositions used as vaccines comprise a ‘sufficient amount’ or ‘an immunologically effective amount’ of the envelope proteins of the present invention, as well as any other of the above mentioned components, as needed.
  • ‘Immunologically effective amount’ means that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment, as defined above. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, the strain of infecting HCV, and other relevant factors. It is expected that the amount will tall in a relatively broad range that can be determined through routine trials. Usually, the amount will vary from 0.01 to 1000 ⁇ g/dose, more particularly from 0.1 to 100 ⁇ g/dose.
  • the single or specific oligomeric envelope proteins may also serve as vaccine carriers to present homologous (e.g. T cell epitopes or B cell epitopes from the core, NS2, NS3, NS4 or NS5 regions) or heterologous (non-HCV) haptens, in the same manner as Hepatitis 3 surface antigen (see European Patent Application 174, 444).
  • envelope proteins provide an immunogenic carrier capable of stimulating an immune response to haptens or antigens conjugated to the aggregate.
  • the antigen may be conjugated either by conventional chemical methods, or may be cloned into the gene encoding E1 and/or E2 at a location corresponding to a hydrophilic region of the protein.
  • Such hydrophylic regions include the V1 region (encompassing amino acid positions 191 to 202), the V2 region (encompassing amino acid positions 213 to 223), the V3 region (encompassing amino acid positions 230 to 242), the V4 region (encompassing amino acid positions 230 to 242), the V1 region (encompassing amino acid positions 294 to 303) and the V6 region (encompassing amino acid positions 329 to 336).
  • Another useful location for insertion of haptens is the hydrophobic region (encompassing approximately amino acid positions 264 to 293). It is shown in the present invention that this region can be deleted without affecting the reactivity of the deleted E1 protein with antisera. Therefore, haptens may be inserted at the site of the deletion.
  • the immunogenic compositions are conventionally administered parenterally, typically by injection, for example, subcutaneously or intramuscularly. Additional formulations suitable for other methods of administration include oral formulations and suppositories. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.
  • the present invention also relates to a composition comprising peptides or polypeptides as described above, for in vitro detection of HCV antibodies present in a biological sample.
  • the present invention also relates to the use of a composition as described above for the preparation of an immunoassay kit for detecting HCV antibodies present in a biological sample.
  • the present invention also relates to a method for in vitro diagnosis of HCV antibodies present in a biological sample, comprising at least the following steps
  • the present invention also relates to competition immunoassay formats in which recombinantly produced purified single or specific oligomeric protein E1 and/or E2 and/or E1/E2 proteins as disclosed above are used in combination with E1 and/or E2 peptides in order to compete for HCV antibodies present in a biological sample.
  • the present invention also relates to a kit for determining the presence of HCV antibodies, in a biological sample, comprising
  • the immunoassay methods according to the present invention utilize single or specific oligomeric antigens from the E1 and/or E2 domains that maintain linear (in case of peptides) and conformational epitopes (single or specific oligomeric proteins) recognized by antibodies in the sera from individuals infected with HCV. It is within the scope of the invention to use for instance single or specific oligomeric antigens, dimeric antigens, as well as combinations of single or specific oligomeric antigens.
  • the HCV E1 and E2 antigens of the present invention may be employed in virtually any assay format that employs a known antigen to detect antibodies. Of course, a format that denatures the HCV conformational epitope should be avoided or adapted.
  • a common feature of all of these assays is that 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 strength 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, chemiluminescent, radioactive, or dye molecules.
  • Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin or streptavidin, and enzyme-labeled and mediated immunoassays, such as LISA assays.
  • the 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 ImmunolonTM), diazotized paper, nylon membranes, activated beads, and Protein A beads.
  • Dynatech ImmunolonTM1 or ImmunlonTM2 microtiter plates or 0.25 inch polystyrene beads 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 rest sample is incubated with the combination of antigens in solution.
  • the combination of antigens in solution.
  • 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 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 anti-HCV antibodies will bind due to complex formation.
  • labeled anti-xenogeneic e.g. anti-human
  • the amount of HCV antibodies in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labeled antibody (or other competing ligand) in the complex.
  • Complexes formed comprising anti-HCV antibody are detected by any of a number of Known techniques, depending on the format.
  • unlabeled HCV antibodies in the complex may be detected using a conjugate of anti-xenogeneic Ig complexed with a label (e.g. an enzyme label).
  • the reaction between the HCV antigens and the antibody forms a network that precipitates from the solution or suspension and forms a visible layer or film of precipitate. If no anti-HCV antibody is present in the test specimen, no visible precipitate is formed.
  • PA particle agglutination
  • two artificial carriers may be used instead of RBC in the PA.
  • the most common of these are latex particles.
  • gelatin particles may also be used.
  • the assays utilizing either of these carriers are based on passive agglutination of the particles coated with purified antigens.
  • the HCV single or specific oligomeric E1 and/or E2 and/or E1/E2 antigens 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.
  • Immunoassays that utilize the native HCV antigen are useful in screening blood for the preparation of a supply from which potentially infective HCV is lacking.
  • the method for the preparation of the blood supply comprises the following steps. Reacting a body component, preferably blood or a blood component, from the individual donating blood with HCV E1 and/or E2 proteins of the present invention to allow an immunological reaction between HCV antibodies, if any, and the HCV antigen. Detecting whether anti-HCV antibody—HCV antigen complexes are formed as a result of the reacting. Blood contributed to the blood supply is from donors that do no: exhibit antibodies to the native HCV antigens, E1 or E2.
  • the ‘confirmation’ tests are typically formatted to increase specificity (to insure that no false-positive samples are confirmed) at the expense of sensitivity. Therefore the purification method described in the present invention for E1 and E2 will be very advantageous for including single or specific oligomeric envelope proteins into HCV diagnostic assays.
  • 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 chemiluminescent 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 further contemplates the use of E1 proteins, or parts thereof, more particularly HCV single or specific oligomeric E1 proteins as defined above, for in vitro monitoring HCV disease or prognosing the response to treatment (for instance with Interferon) of patients suffering from HCV infection comprising:
  • an anti-E1 assay may be very useful for prognosing long-term response to IFN treatment, or to treatment of Hepatitis C virus disease in general.
  • E1 peptides as listed in Table 3 were found to be useful for in vitro monitoring HCV disease or prognosing the response to interferon treatment of patients suffering from HCV infection:
  • the present invention also relates to a kit for monitoring HCV disease or prognosing the response to treatment (for instance to interferon) of patients suffering from HCV infection comprising:
  • E2 protein and peptides according to the present invention can be used to a certain degree to monitor/prognose HCV treatment as indicated above for the E1 proteins or peptides because also the anti-E2 levels decrease in comparison to antibodies to the other HCV antigens. It is to be understood, however, that it might be possible to determine certain epitopes in the E2 region which would also be suited for use in an test for monitoring/prognosing HCV disease.
  • the present invention also relates to a stereotyping assay for detecting one or more serological types of HCV present in a biological sample, more particularly for detecting antibodies of the different types of HCV to be detected combined in one assay format, comprising at least the following steps
  • compositions of proteins or peptides used in this method are recombinantly expressed type-specific envelope proteins or type-specific peptides.
  • the present invention further relates to a kit for serotyping one or more serological types of HCV present in a biological sample, more particularly for detecting the antibodies to these serological types of HCV comprising:
  • the present invention also relates to the use of a peptide or protein composition as defined above, for immobilization on a solid substrate and incorporation into a reversed phase hybridization assay, preferably for immobilization as parallel lines onto a solid support such as a membrane strip, for determining the presence or the genotype of HCV according to a method as defined above. Combination with other type-specific antigens from other HCV polyprotein regions also lies within the scope of the present invention.
  • FIG. 1 Restriction map of plasmid pgpt ATA 18
  • FIG. 2 Restriction map of plasmid pgs ATA 18
  • FIG. 3 Restriction map of plasmid pMS 66
  • FIG. 4 Restriction map of plasmid pv HCV-11A
  • FIG. 5 Anti-1 levels in non-responders to IFN treatment
  • FIG. 6 Anti-E1 levels in responders to IFN treatment
  • FIG. 7 Anti-E1 levels in patients with complete response to IFN treatment
  • FIG. 8 Anti-E1 levels in incomplete responders to IFN treatment
  • FIG. 9 Anti-E2 levels in non-responders to IFN treatment
  • FIG. 10 Anti-E2 levels in responders to IFN treatment
  • FIG. 11 Anti-E2 levels in incomplete responders to IFN treatment
  • FIG. 12 Anti-E2 levels in complete responders to IFN treatment
  • FIG. 13 Human anti-E1 reactivity competed with peptides
  • FIG. 14 Competition of reactivity of anti-E1 monoclonal antibodies with peptides
  • FIG. 15 Anti-E1 (epitope 1) levels in non-responders to IFN treatment
  • FIG. 16 Anti-E1 (epitope 1) levels in responders to IFN treatment
  • FIG. 17 Anti-E1 (epitope 2) levels in non-responders to IFN treatment
  • FIG. 18 Anti-E1 (epitope 2) levels in responders to IFN treatment
  • FIG. 19 Competition of reactivity of anti-E2 monoclonal antibodies with peptides
  • FIG. 20 Human anti-E2 reactivity competed with peptides
  • FIG. 21 FIGS. 21 A-L provide nucleic acid sequences of the present invention.
  • the nucleic acid sequences encoding an E1 or E2 protein according to the present invention may be translated (SEQ ID NO 3 to 13, 21-31, 35 and 41-49 are translated in a reading frame starting from residue number 1, SEQ ID NO:37-39 are translated in a reading frame starting from residue number 2), into the amino acid sequences of the respective E1 or E2 proteins as shown in the sequence listing.
  • FIG. 22 ELISA results obtained from lentil lectin chromatography eluate fractions of 4 different E1 purifications of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b), vvHCV62 type 3a), and vvHCV63 (type 5a).
  • FIG. 23 Elution profiles obtained from the lentil lectin chromatography of the 4 different E1 constructs on the basis of the values as shown in FIG. 22 .
  • FIG. 24 ELISA results obtained from fractions obtained after gelfiltration chromatography of 4 different E1 purifications of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b), vvHCCV62 (type 3a), and vvHCV63 (type 5a).
  • FIG. 25 Profiles obtained from purifications of E1 proteins of type 1b (1), type 3a (2), and type 5a (3) (from RK13 cells infected with vvHCV39, vvHCV62, and vvHCV63, respectively; purified on lentil lectin and reduced as in example 5.2-5.3) and a standard ( 4 ).
  • FIG. 26 Silver staining of an SDS-PACE as described in example 4 of a raw lysate of E1 vvHCV40 (type 1b) (lane 1), pool 1 of the gelfiltration of vvHCV40 representing fractions 10 to 17 as shown in FIG. 25 (lane 2), pool 2 of the gelfiltration of vvHCV40 representing fractions 13 to 25 as shown in FIG. 25 (lane 3), and E1 pool (fractions 25 to 30) (lane 4).
  • FIG. 27 Streptavidine-alkaline phosphatase blot of the fractions of the gelfiltration of E1 constructs 39 (type 1b) and 62 (type 3a). The proteins were labelled with NEM-biotin. Lane 1: start gelfiltration construct 39, lane 2: fraction 26 construct 39, lane 3: fraction 27 construct 39, lane 4: fraction 28 construct 39, lane 5: fraction 29 construct 39, lane 6: fraction 30 construct 39, lane 7 fraction 31 construct 39, lane 8: molecular weight marker, lane 9: start gelfiltration construct 62, lane 10: fraction 26 construct 62, lane 11: fraction 27 construct 62, lane 12: fraction 28 construct 62, lane 13: fraction 29 construct 62, lane 14: fraction 30 construct 62, lane 15: fraction 31 construct 62.
  • FIG. 28 Siver staining of an SOS-PAGE gel of the gelfiltration fractions of vvHCV-39 (E1s, type 1b) and vvHCV-62 (E1s, type 3a) run under identical conditions as FIG. 26 .
  • Lane 1 start gelfiltration construct 39
  • lane 2 fraction 26 construct 39
  • lane 3 fraction 27 construct 39
  • lane 4 fraction 28 construct 39
  • lane 5 fraction 29 construct 39
  • lane 6 fraction 30 construct 39
  • lane 7 fraction 31 construct 39
  • lane 8 molecular weight marker
  • lane 9 start gelfiltration construct 62
  • lane 10 fraction 26 construct 62
  • lane 11 fraction 27 construct 62
  • lane 12 fraction 28 construct 62
  • lane 13 fraction 29 construct 62
  • lane 14 fraction 30 construct 62
  • lane 15 fraction 31 construct 62.
  • FIG. 29 Western Blot analysis with anti-cl mouse monoclonal antibody 5E1A10 giving a complete overview of the purification procedure.
  • Lane 1 crude lysate
  • Lane 2 flow through of lentil chromagtography
  • Lane 3 wash with Empigen BB after lentil chromatography
  • Lane 4 Eluate of lentil chromatography
  • Lane 5 Flow through during concentration of the lentil eluate
  • Lane 6 Pool of E1 after Size Exclusion Chromatography (gelfiltration).
  • FIG. 30 OD 280 profile (continuous line) of the lentil lectin chromatography of E2 protein from RK13 cells infected with vvHCV44.
  • the dotted line represents the 0.7 reactivity as detected by ELISA (as in example 6).
  • FIG. 31A OD 290 profile (continuous line) of the lentil-lectin gelfiltration chromatography E2 protein pool from RK13 cells infected with vvHCV44 in which the 2 pool is applied immediately on the gelfiltration column (non-reduced conditions).
  • the dotted line represents the E2 reactivity as detected by ELISA (as in example 6).
  • FIG. 31B OD 280 profile (continuous line) of the lentil-lectin gelfiltration chromatography E2 protein pool from RK13 cells infected with vvpCV44 in which the 2 pool was reduced and blocked according to Example 5.3 (reduced conditions).
  • the dotted line represents the 2 reactivity as detected by ELISA (as in example 6).
  • FIG. 32 Ni 2 ⁇ -IMAC chromatography and ELISA reactivity of the E2 protein as expressed from vvHCV44 after gelfiltration under reducing conditions as shown in FIG. 31B .
  • FIG. 33 Silver staining of an SOS-PAGE of 0.5 ⁇ g of purified E2 protein recovered by a 200 mM imidazole elution step (lane 2) and a 30 mM imidazole wash (lane 1) of the Ni 2 ⁇ -IMAC chromatography as shown in FIG. 32 .
  • FIG. 34 OD profiles of a desalting step of the purified E2 protein recovered by 200 mM immidazole as shown in FIG. 33 , intended to remove imidazole.
  • FIGS. 35A-1 to 35 A- 8 Antibody levels to the different HCV antigens (Core 1 , Core 2 , E2HCVR, NS3) for NR and LTR followed during treatment and over a period of 6 to 12 months after treatment determined by means of the LIAscan method. The average values are indicated by the curves with the open squares.
  • FIGS. 35B-1 to 35 B- 8 Antibody levels to the different HCV antigens (NS4, NS5, E1 and E2) for NR and LTR followed during treatment and over a period of 6 to 12 months after treatment determined by means of the LIAscan method. The average values are indicated by the curve with the open squares.
  • FIGS. 36 A and 36 B Average E1 antibody (E1Ab) and E2 antibody (E2Ab) levels in the LTR and NR groups.
  • FIGS. 36 A-D Averages E1 antibody (E1Ab) levels for non-responders (NR) and long term responders (LTR) for type 1b and type 3a.
  • E1Ab E1 antibody
  • NR non-responders
  • LTR long term responders
  • FIG. 37 Averages E1 antibody (E1Ab) levels for non-responders (NR) and long term responders (LTR) for type 1b and type 3a.
  • FIG. 38 Relative map positions of the anti-E2 monoclonal antibodies.
  • FIG. 39 Partial deglycosylation of HCV E1 envelope protein.
  • the lysate of vvHCV10A-infected RK13 cells were incubated with different concentrations of glycosidases according to the manutacturer's instructions.
  • Right panel Glycopepidase F (PNC-ase F).
  • Left panel Endoclycosidase H (Endo H).
  • FIG. 40 Partial deglycosylation of HCV E2 envelope proteins.
  • the lysate of vvHCV64-infected (E2) and vvHCV41-infected (E2s)RK13 cells were incubated with different concentrations of Glycopeptidase F (PNGase F) according to the manufacturer's instructions.
  • PNGase F Glycopeptidase F
  • FIG. 41 in vitro mutagenesis of HCV E1 glycoproteins. Map of the mutated sequences and the creation of new restriction sites.
  • FIG. 42A In vitro mutagenesis of HCV E1 glycoprotein (part 1). First step of PCR amplification.
  • FIG. 42B In vitro mutagensis of HCV E1 glycoprotein (part 2). Overlap extension and nested PCR.
  • FIG. 43 In vitro mutagenesis of HCV E1 glycoproteins. Map of the PCR mutated fragments (GLY-# and OVR-#) synthesized during the first step of amplification.
  • FIG. 44A Analysis of E1 glycoprotein mutants by Western blot expressed in HeLa (left) and RK13 (right) cells.
  • Lane 1 wild type VV (vaccinia virus), Lane 2:original E1 protein (vvHCV-10A).
  • Lane 3 E1 mutant Gly-1 (vvHCV-81).
  • Lane 4 E1 mutant Gly-2 (vvHCV-82), Lane 5: E1 mutant Gly-3 (vvHCV-83), Lane 6: E1 mutant Gly-4 (vvHCV-g8).
  • Lane 7 E1 mutant Gly-5 (vvHCV-85).
  • Lane 8 E1 mutant Gly-6 (vvHCV-86).
  • FIG. 44B Analysis of E1 glycosylation mutant vaccinia viruses by PCR amplification/restriction.
  • Lane 1 E1 (vvHCV-10A), BspE I.
  • Lane 2 E1.GLY-1 (vvHCV-81).
  • Lane 4 E1 (vvHCV-10A), Sac Lane 5: E1.GLY-2 (vvHCV-82), Sac I, Lane 7: E1 (vvHCV-10A), Sac I, Lane 8: E1.GLY-3 (vvHCV-82), Sac Lane 10: E1 (vvHCV-10A), Stu I, Lane 11: E1.GLY-4 (vvHCV-84).
  • FIG. 45 SDS polyacrylamide gel electrophoresis of recombinant E2 expressed in S. cerevisiae . Innoculates were crown in leucine selective medium for 72 hrs. and diluted 1/15 in complete medium. After 10 days of culture at 28° C., medium samples were taken. The equivalent of 200 ⁇ l of culture supernatant concentrated by speedHvc was loaded on the gel. Two independent transformants were analysed.
  • FIG. 46 SDS polyacrylamide gel electrophoresis of recombinant E2 expressed in a glycosylation deficient S. cerevisiae mutant. Innoculate were grown in leucine selective medium for 72 hrs. and diluted 1/15 in complete medium. After 10 days of culture at 28° C. medium samples were taken. The equivalent of 350 ⁇ l of culture supernatant, concentrated by ion exchange chromatography, was loaded on the gel.
  • Table 1 Features of the respective clones and primers used for amplification for constructing the different forms of the E1 protein as despected in Example 1.
  • Table 4 Changes of envelope antibody levels over time.
  • the pgptATA18 vaccinia recombination plasmid is a modified version of pATA18 (Stunnenberg et al, 1988) with an additional insertion containing the E. coli xanthine guanine phosphoribosyl transferase gene under the control of the vaccinia virus 13 intermediate promoter ( FIG. 1 ).
  • the plasmid pgsATA18 was constructed by inserting an oligonucleotide linker with SEQ ID NO 1/94, containing stop codons in the three reading frames, into the Pst I and HindIII-cut pATA18 vector. This created an extra Pac I restriction site ( FIG. 2 ). The original HindIII site was not restored.
  • Oligonucleotide linker with SEQ ID NO 1/94 5′ G GCATGC AAGCTT AATTAATT 3′ 3′ ACGTC CGTACG TTCGAA TTAATTAA TCGA 5′ PstI SphI HindIII Pac I (HindIII)
  • the vaccinia recombination vector pMSc6 was designed to express secreted proteins with an additional carboxy-terminal histidine tag.
  • An oligonucleotide linker with SEQ ID NO 2/95, containing unique sites for 3 restriction enzymes generating blunt ends (Sma I, Stu I and Pml I/Bbr PI) was synthesized in such a way that the carboxy-terminal end of any cDNA could be inserted in frame with a sequence encoding the protease factor Xa cleavage site followed by a nucleotide sequence encoding 6 histidines and 2 stop codons (a new Pac I restriction site was also created downstream the 3′ end).
  • This oligonucleotide with SEC ID NO 2/95 was introduced between the Xma I and Pst I sites of pgptATA18 ( FIG. 3 ).
  • Oligonucleotide linker with SEQ ID NO 2/95 ′5′ CCGGG GAGGCCTGCACGTGATCGAGGGCAGACACCATCACCACCATCACTAATAGTTAATTAA CTGCA3 3′ C CTCCGGACGTGCACTAGCTCCCGTCTGTGGTAGTGGTGGTAGTGATTATCAATTAATT G XmaI PstI
  • PCB Polymerase Chain Reaction
  • HCCl12A (SEQ ID NO 9), HCCl13A (SEQ ID NO 11), and HCCl17A (SEQ ID NO 13) as depicted in FIG. 21 .
  • cDNA fragments containing the E1-coding regions were cleaved by EcoI and HindIII restriction from the respective pS-q72 plasmids and inserted into the EcoRI/HindIII-cut pgptATA-18 vaccinia recombination vector (described in example 1), downstream of the 11 K vaccinia virus late promoter.
  • the respective plasmids were designated pvHCV-9A, pvHCV-10A, pvHCV-11A, pVHCV-12A, pvHCV-13A and pvHCV-17A, of which PvHCV-11A is shown in FIG. A,
  • Clone HCCl-37 containing a deletion of codons Asp264 to Val2S7 (nucleotides 790 to 861, region encoding hydrophobic domain 1) was generated as follows: 2 PCR fragments were generated from clone HCCl10A with primer sets HCPr52 (SEQ ID NO 16)/HCPr107 (SEQ ID NO 19) and HCPr108 (SEQ ID NO 20)/HCPR54 (SEQ ID NO 12). These primers are shown in FIG. 21 . The two PCR fragments were purified from agarose gel after electrophoresis and 1 ng of each fragment was used together as template for PCR by means of primers HCPr52 (SEQ ID NO 16) and HCPr54 (SEC ID NO 18).
  • Plasmid pSP72HCCl37 containing clone HCCl37 (SEQ ID 15) was selected.
  • a recombinant vaccinia plasmid containing the full-length E1 cDNA lacking hydrophobic domain I was constructed by inserting the HCV sequence surrounding the deletion (fragment cleaved by Xma I and SamH I from the vector pSP72—HCCl37) into the Xma I-Bam H I sites of the vaccinia plasmid PVHCV-10A.
  • the resulting plasmid was named pvHCV-37.
  • the amino-terminal region containing the internal deletion was isolated from this vector pvHCV-37 (cleavage by EcoR I and BstE II) and reinserted into the Eco R1 and Bst Ell-cut pvHCV-11A plasmid.
  • This construct was expected to express an E1 protein with both hydrophobic domains deleted and was named pvHCV-38.
  • the E1-coding region of clone HCCl38 is represented by SEQ ID NO 23.
  • HCCl62 (SEQ ID NO 29) was derived from a type 3a-infected patient with chronic hepatitis C (serum BR6, clone BR36-9-13, SEQ ID NO 19 in WO 94/25601, and see also Stuyver et al. 1993a) and HCCl63 (SEQ ID NO 31) was derived from a type 5a-infected child with post-transfusion hepatitis (serum BE95, clone PC-4-1, SEQ ID NO 45 in WO 94125601).
  • the HCV E2 PCR fragment 22 was obtained from serum BE11 (genotype 1b) by means of primers HCPr109 (SEQ ID NO 33) and HCPr72 (SEQ ID NO 34) using techniques of RNA preparation, reverse-transcription and PCR, as described in Stuyver et al., 1993b, and the fragment was cloned into the Sma I-cut pSP72 vector.
  • Clone HCCl22A (SEQ ID NO 35) was cut with NcoI/AlwNI or by BamHI/AlwNI and the sticky ends of the fragments were blunted (NcoI and BamHI sites with Klenow DNA Polymerase I (Boehringer), and AlwNI with T4 DNA polymerase (Boehringer)).
  • the BamHI/AlwNI cDNA fragment was then inserted into the vaccinia pgsATA-18 vector that had been linearized by EcoR I and Hind III cleavage and of which the cohesive ends had been filled with Klenow DNA Polymerase (Boehringer).
  • the resulting plasmid was named pvHCV-41 and encoded the E2 region from amino acids Met347 to Gln673, including 37 amino acids (from Met347 to Gly383) of the E1 protein that can serve as signal sequence.
  • the same HCV cDNA was inserted into the EcoR I and Sbr PI-cut vector pMS66, that had subsequently been blunt ended with Klenow DNA Polymerase.
  • the resulting plasmid was named pvHCV-42 and also encoded amino acids 347 to 683.
  • the NcoI/AlwNI fragment was inserted in a similar way into the same sites of pgsA7A-18 (pvHCV-43) or pMS-66 vaccinia vectors (pvHCV-14).
  • pvHCV-43 and pvHCV-44 encoded amino acids 364 to 673 of the HCV polyprotein, of which amino acids 364 to 383 were derived from the natural carboxyterminal region of the E1 protein encoding the signal sequence for E2, and amino acids 384 to c73 of the mature E2 protein.
  • Rabbit kidney RK13 cells (ATCC CCL 37), human osteosarcoma 143B thymidine kinase deficient (TK) (ATC CRL 8303), HeLa (ATCC CCL 2), and Hep G2 (ATCC HB 8065) cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, Md., USA). The cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% foetal calf serum, and with Earle's salts (EMEM) for RK13 and 143 B (TK-), and with glucose (4 g/l) for Hep G2.
  • DMEM Dulbecco's modified Eagle medium
  • EMEM Earle's salts
  • m.o.i. multiplicity of infection
  • PFU plaque forming unit
  • the vaccinia recombination plasmid was transfected into the infected cells in the form of a calcium phosphate coprecipitate containing 500 ng of the plasmid DNA to allow homologous recombination (Graham & van der Eb, 1973; Mackett et al., 1985).
  • Recombinant viruses expressing the Escherichia coli xanthine-guanine phosphoribosyl transferase (gpt) protein were selected on rabbit kidney RK13 cells incubated in selection medium (EMEM containing 25 ⁇ g/ml mycophenolic acid (MPA), 250 ⁇ g/ml xanthine, and 15 ⁇ g/ml hypoxanthine: Falkner and Moss, 1988; Janknecht et al, 1991). Single recombinant viruses were purified on fresh monolayers of RK13 cells under a 0.9% agarose overlay in selection medium.
  • selection medium EMEM containing 25 ⁇ g/ml mycophenolic acid (MPA), 250 ⁇ g/ml xanthine, and 15 ⁇ g/ml hypoxanthine: Falkner and Moss, 1988; Janknecht et al, 1991.
  • Thymidine kinase deficient (TK-) recombinant viruses were selected and then plaque purified on fresh monolayers of human 1432 cells (TK-) in the presence of 25 ⁇ g/ml 5-bromo-2′-deoxyuridine.
  • Stocks of purified recombinant HCV-vaccinia viruses were prepared by infecting either human 1433 or rabbit RK13 cells at an m.o.i. of 0.05 (Mackett et al, 1988). The insertion of the HCV cDNA fragment in the recombinant vaccinia viruses was confirmed on an aliquot (50 all) of the cell lysate after the MPA selection by means of PCR with the primers used to clone the respective HCV fragments (see Table 1).
  • the recombinant vaccinia-HCV viruses were named according to the vaccinia recombination plasmid number, e.g. the recombinant vaccinia virus vvHCV-10A was derived from recombining the wild type WR strain with the pvHCV-10A plasmid.
  • a confluent monolayer of RK13 cells was infected at a m.o.i. of 3 with the recombinant HCV-vaccinia viruses as described in example 2.
  • the cell monolayer was washed twice with phosphate-buffered saline pH 7.4 (P—S) and the recombinant vaccinia virus stock was diluted in MEM medium.
  • Two hundred ⁇ l of the virus solution was added per 10 5 cells such that the m.o.i. was 3, and incubated for A5 min at 24° C.
  • the virus solution was aspirated and 2 ml of complete growth medium (see example 2) was added per 10 5 cells.
  • the cells were incubated for 24 hr at 37° C. during which expression of the HCV proteins took place.
  • the infected cells were washed two times with PBS, directly lysed with lysis buffer (50 mM Tris.HCl pH 7.5, 150 mM NaCl. 1% Triton X-100, 5 mM MgCl 2 , 1 ⁇ g/ml aprotinin (Sigma, Bornem, Belgium)) or detached from the flasks by incubation in 50 mM Tris.HCL pH 7.5/10 mM EDTA/150 mM NaCl for 5 min, and collected by centrifugation (5 min at 1000 g).
  • lysis buffer 50 mM Tris.HCl pH 7.5, 150 mM NaCl. 1% Triton X-100, 5 mM MgCl 2 , 1 ⁇ g/ml aprotinin (Sigma, Bornem, Belgium)
  • the cell pellet was then resuspended in 200 ⁇ l lysis buffer (50 mM Tris.HCL pH 8.0, 2 mM EDTA, 150 mM NaCl, 5 mM MgCl 2 aprotinin; 1% Triton X-100) per 10 8 cells.
  • the cell lysates were cleared for 5 min at 14,000 rpm in an Eppendorf centrifuge to remove the insoluble debris. Proteins of 20 ⁇ l lysate were separated by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • the proteins were then electro-transferred from the gel to a nitrocellulose sheet (Amersham) using a Hoefer HSI transfer unit cooled to 4° C. for 2 hr at 100 V constant voltage, in transfer buffer (25 mM Tris.HCl pH 8.0, 192 mM glycine, 20% (v/v) methanol). Nitrocellulose filters were blocked with Blotto (5% (w/v) fat-free instant milk powder in PBS; Johnson et al., 1981) and incubated with primary antibodies diluted in Blotto/0.1% Tween 20.
  • a human negative control serum or serum of a patient infected with HCV were 200 times diluted and preincubated for 1 hour at room temperature with 200 times diluted wild type vaccinia virus-infected cell lysate in order to decrease the non-specific binding.
  • the nitrocellulose filters were incubated with alkaline phosphatase substrate solution diluted in Blotto/0.1% Tween 20.
  • the filters were incubated with alkaline phosphates substrate solution (100 mM Tris.HCl pH 9.5, 100 mM NaCl, 5 mM MgCl 2 , 0.38 ⁇ g/ml nitroblue tetrazolium. 0.165 ⁇ g/ml 5-bromo-4-chloro-3-indolylphosphate). All steps, except the electrotransfer, were performed at room temperature.
  • alkaline phosphates substrate solution 100 mM Tris.HCl pH 9.5, 100 mM NaCl, 5 mM MgCl 2 , 0.38 ⁇ g/ml nitroblue tetrazolium. 0.165 ⁇ g/ml 5-bromo-4-chloro-3-indolylphosphate. All steps, except the electrotransfer, were performed at room temperature.
  • Infected RK13 cells (carrying E1 or E2 constructs) were washed 2 times with phosphate-buffered saline (PBS) and detached from the culture recipients by incubation in PBS containing 10 mM EDTA. The detached cells were washed twice with PBS and 1 ml of lysis buffer (50 mM Tris.HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM MgCl 2 , 1 ⁇ g/ml aprotinin (Sigma, Bornem, Belgium) containing 2 mM biotinylated N-ethylmaleimide (biotin-NEM) (Sigma) was added per 10 5 cells at 4 C.
  • PBS phosphate-buffered saline
  • This lysate was homogenized with a type B douncer and left at room temperature for 0.5 hours. Another 5 volumes of lysis buffer containing 10 mM N-ethylmaleimide (NEM. Aldrich, Bornem. Belgium) was added to the primary lysate and the mixture was left at room temperature for 15 min. Insoluble cell debris was cleared from the solution by centrifugation in a Beckman JA-14 rotor at 14,000 rpm (30100 g at r max ) for 1 hour at 4° C.
  • NEM N-ethylmaleimide
  • the cleared cell lysate was loaded at a rate of 1 ml/min on a 0.8 by 10 cm Lentil-lectin Sepharose 48 column (Pharmacia) that had been equilibrated with 5 column volumes of lysis buffer at a rate of 1 ml/min.
  • the lentil-lectin column was washed with 5 to 10 column volumes of buffer 1 (0.1M potassium phosphate pH 7.3, 500 mM KCl, 5% glycerol, 1 mM 6—NH 2 -hexanoic acid, 1 mM MgCl 2 , and 1% DecylPEG (KWANT, Bedum, The Netherlands).
  • the column was subsequently washed with 10 column volumes of buffer 1 containing 0.5% Empigen-BB (Calbiochem, San Diego, Calif., USA) instead of 1% DecylPEGG.
  • the bound material was eluted by applying elution buffer (10 mM potassium phosphate pH 7.3, 59 glycerol, 1 mM hexanoic acid; 1 mM MgCl 2 , 0.5% Empigen-BB, and 0.5 M ⁇ -methyl-mannopyranoside.
  • the eluted material was fractionated and fractions were screened for the presence of E1 or 2 protein by means of ELISA as described in example 6.
  • FIG. 22 shows ELISA results obtained from lentil lectin eluate fractions of 4 different E1 purifications of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b), vvHCV62 (type 3a), and vvHCV53 (type 5a).
  • FIG. 23 shows the profiles obtained from the values shown in FIG. 22 . These results show that the lectin affinity column can be employed for envelope proteins of the different types of HCV.
  • the E1- or E2-positive fractions were pooled and concentrated on a Centricon 30 kDa (Amicon) by centrifugation for 3 hours at 5,000 rpm in a Beckman JA-20 rotor at 4° C. In some experiments the E1- or E2-positive fractions were pooled and concentrated by nitrogen evaporation. An equivalent of 3.10 8 cells was concentrated to approximately 200 ⁇ l. For partial reduction, 30% Empigen-BB (Calbiochem, San Diego, Calif. USA) was added to this 200 ⁇ l to a final concentration of 3.5%, and 1M DTT in H 2 O was subsequently added to a final concentration of 1.5 to 7.5 mM and incubated for 30 min at 37° C. NEM (1M in dimethylsulphoxide) was subsequently added to a final concentration of 50 mM and left to react for another 30 min at 37° C. to block the free sulfhydryl groups.
  • FIG. 24 shows ELISA results obtained from fractions obtained after gelfiltration chromatography of 4 different E1 purifications of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b), vvHCV62 (type 3a), and vvHCVS6 (type 5a).
  • FIG. 25 shows the profiles obtained from purifications of E1 proteins of types 1b, 3, and 5a (from RK13 cells infected with vvHCV39, vvHCVS2, and vvHCV63, respectively; purified on lentil lectin and reduced as in the previous examples).
  • the peaks indicated with ‘1’, ‘2’, and ‘3’, represent sure E1 protein peaks (E1 reactivity mainly in fractions 26 to 30). These peaks show very similar molecular weights of approximately 70 kDa, corresponding to dimeric E1 protein. Other peaks in the three profiles represent vaccinia virus and/or cellular proteins which could be separated from E1 only because of the reduction step as outlined in example 5.3, and because of the subsequent gelfiltration step in the presence of the proper detergent. As shown in FIG. 25 pool 1 (representing fractions 10 to 17) and pool 2 (representing fractions 18 to 25) contain contaminating proteins not present in the E1 pool (fractions 26 to 30).
  • the E1 peak fractions were ran on SDS/PAGE and blotted as described in example 4. Proteins labelled with NEM-biotin were detected by streptavidin-alkaline phosphatase as shown in FIG. 27 . It can be readily observed that, amongst others, the 29 kDa and 45 kDa contaminating proteins present before the gelfiltration chromatography (lane 1) are only present at very low levels in the fractions 26 to 30. The band at approximately 65 kDa represents the E1 dimeric form that could not be entirely disrupted into the monomeric E1 form. Similar results were obtained for the type 3a E1 protein (lanes 10 to 15), which shows a faster mobility on SDS/PAGE because of the presence of only 5 carbohydrates instead of 6. FIG. 23 shows a silver stain of an SDS/PAGE gel run in identical conditions as in FIG. 26 . A complete overview of the purification procedure is given in FIG. 29 .
  • the presence of purified E1 protein was further confirmed by means of western blotting as described in example 4.
  • the dimeric E1 protein appeared to be non-aggregated and free of contaminants.
  • the subtype 1b E1 protein purified from vvHCV40-infected cells according to the above scheme was aminoterminally sequenced on an 477 Perkins-Elmer sequencer and appeared to contain a tyrosine as first residue. This confirmed that the E1 protein had been cleaved by the signal peptidase at the correct position (between A191 and Y192) from its signal sequence. This confirms the finding of Hijikata et al. (1991) that the aminoterminus of the mature E1 protein starts at amino acid position 192.
  • FIG. 30 shows the CCl 2 profile (continuous line) of the lentil lectin chromatography.
  • the dotted line represents the E2 reactivity as detected by ELISA (see example 6).
  • FIG. 31 shows the same profiles part of obtained from gelfiltration chromatography of the lentil-lectin E2 pool (see FIG. 30 ), part of which was reduced and blocked according to the methods as set out in example 5.3, and part of which was immediately applied to the column. Both parts of the E2 pool were run on separate gelfiltration columns.
  • FIG. 32 shows an additional Ni 2 ⁇ -IMAC purification step carried out for the E2 protein purification. This affinity purification step employs the 6 histidine residues added to the E2 protein as expressed from vvHCV44. Contaminating proteins either run through the column or can be removed by a 30 mM imidazole wash.
  • FIG. 32 shows an additional Ni 2 ⁇ -IMAC purification step carried out for the E2 protein purification. This affinity purification step employs the 6 histidine residues added to the E2 protein as expressed from vvHCV44. Contaminating proteins either run through the column or can be removed by a 30 mM imidazole wash.
  • FIG. 33 shows a silver-stained SDS/PAGE of 0.5 ⁇ g of purified E2 protein and a 30 mM imidazole wash.
  • the pure E2 protein could be easily recovered by a 200 mM imidazole elution step.
  • FIG. 34 shows an additional desalting step intended to remove imidazole and to be able to switch to the desired buffer, e.g. PES, carbonate buffer, saline.
  • secreted E2 protein (constituting approximately 30-40%, 60-70% being in the intracellular form) is characterized by aggregate formation (contrary to expectations). The same problem is thus posed to purify secreted E2.
  • the secreted E2 can be purified as disclosed above.
  • Serum samples were diluted 20 times or monoclonal anti-E1 or anti-E2 antibodies were diluted to a concentration of 20 ng/ml in Sample Diluent of the Innotest HCV Ab III kit and 1 volume of the solution was left to react with the E1 or E2 protein for 1 hour at 37° C.
  • the microwells were washed 5 times with 400 ⁇ l of Washing Solution of the Innotest HCV Ab III kit (Innogenetics. Zwijndrecht, Belgium). The bound antibodies were detected by incubating each well for 1 hour at 37° C.
  • HCV hepatitis C virus
  • Type 1b vvHCV-39, see example 2.5.
  • 3a E1 vvHCV-62, see example 2.5. proteins were expressed by the vaccinia virus system (see examples 3 and 4) and purified to homogeneity (example 5).
  • FIG. 5 shows the anti-E1 signal-to-noise ratios of these patients followed during the course of interferon treatment and during the follow-up period after treatment.
  • Anti-Et antibody titers had decreased 6.9 times in LTR but only 1.5 times in NR.
  • the anti-E1 titers had declined by a factor of 22.5 in the patients with sustained response and even slightly increased in NR. Therefore, based on these data, decrease of anti-E1 antibody levels during monitoring of IFN- ⁇ therapy correlates with long-term, sustainer response to treatment.
  • the anti-E1 assay may be very useful for prognosis of long-term response to IFN treatment, or to treatment of the hepatitis C disease in general.
  • the anti-E1 titers were on average at least 2 times higher at the start of treatment in long term responders compared with incomplete responders to treatment. Therefore, measuring the titer of anti-E1 antibodies at the start of treatment, or monitoring the patient during the course of infection and measuring the anti-E1 titer, may become a useful marker for clinical diagnosis of hepatitis C. Furthermore, the use of more defined regions of the E1 or E2 proteins may become desirable, as shown in example 7.3.
  • FIG. 36 depicts average E1 antibody (E1 Ab) and E2 antibody (E2Ab) levels in the LTR and NR groups and Tables 4 and 5 show the statistical analyses.
  • E1 Ab E1 antibody
  • E2Ab E2 antibody
  • RR did not show any decreasing E1 antibody levels, indicating the presence of occult HCV infection that could neither be demonstrated by PCR or other classical techniques for detection of HCV-RNA, nor by raised ALT levels.
  • the anti-E1 ELISA as described in example 6 was modified by mixing 50 ⁇ g/ml of E1 peptide with the 1120 diluted human serum in sample diluent.
  • FIG. 13 shows the results of reactivity of human sera to the recombinant E1 (expressed from vvHCV-40) protein, in the presence of single or of a mixture of E1 peptides.
  • Anti-E2 antibodies clearly followed a different pattern than the anti-E1 antibodies, especially in patients with a long-term response to treatment. Therefore, it is clear that the decrease in anti-envelope antibodies could not be measured as efficiently with an assay employing a recombinant E1/E2 protein as with a single anti-E1 or anti-E2 protein.
  • the anti-E2 response would clearly blur the anti-E1 response in an assay measuring both kinds of antibodies at the same time. Therefore, the ability to test anti-envelope antibodies to the single E1 and E2 proteins, was shown to be useful.
  • conformational epitopes could be grouped according to their relative positions as follows: the IgG antibodies in the supernatant of hybridomas 15C8C1, 12D11F1, 9G3E6, 8G10D1H9, 10D3C4, 4H6B2, 17F2C2, 5H6A7, 15B7A2 recognizing conformational epitopes were purified by means of protein A affinity chromatography and 1 mg/ml of the resulting IgG's were biotinylated in borate buffer in the presence of biotin. Biotinylated antibodies were separated from free biotin by means of gelfiltration chromatography. Pooled biotinylated antibody fractions were diluted 100 to 10,000 times. E2 protein bound to the solid phase was detected by the biotinylated IgG in the presence of 100 times the amount of non-biotinylated competing antibody and subsequently detected by alkaline phosphatase labeled streptavidin.
  • the E1 protein encoded by vvHCV10A, and the E2 protein encoded by vvHCV41 to 44 expressed from mammalian cells contain 6 and 11 carbohydrate moieties, respectively. This could be shown by incubating the lysate of vvHCV10A-infected or vvHCV44-infected RK13 cells with decreasing concentrations of glycosidases (PNGase F or Endoglycosidase H, (Boehringer Mannheim Biochemical according to the manufacturer's instructions), such that the proteins in the lysate (including E1) are partially deglycosylated ( FIGS. 39 and 40 , respectively).
  • PNGase F or Endoglycosidase H glycosidases
  • Mutants devoid of some of their glycosylation sires could allow the selection of envelope proteins with improved immunological reactivity.
  • gp120 proteins lacking certain selected sugar-addition motifs, have been found to be particularly useful for diagnostic or vaccine purpose.
  • the addition of a new oligosaccharide side chain in the hemagglutinin protein of an escape mutant of the A/Hong Kong/3/68 (H 3 N 2 ) influenza virus prevents reactivity with a neutralizing monoclonal antibody (Skehel et al, 1984).
  • N-linked carbohydrate chains is important for stabilization of folding intermediates and thus for efficient folding, prevention of malfolding and degradation in the endoplasmic reticulum, oligomerization, biological activity, and transport of glycoproteins (see reviews by Rose at al., 1988; Doms et al., 1993; Helenius, 1994).
  • the isolate S83 belonging to genotype 2c, even lacks the first carbohydrate motif in the V1 region (on Asn), while it is present on all other isolates (Stuyver et al. 1994)
  • the presence of the carbohydrate may not be required for folding, but may have a role in evasion of immune surveillance. Therefore, identification of the carbohydrate addition motifs which are not required for proper folding (and reactivity) is not obvious, and each mutant has to be analyzed and tested for reactivity.
  • Mutagenesis of a glycosylation motif can be achieved by either mutating the codons for N, S, or T, in such a way that these codons encode amino acids different from N in the case of N, and/or amino acids different from S or T in the case of S and in the case of T.
  • the X position may be mutated into PI since it is known that NPS or NPT are not frequently modified with carbohydrates. After establishing which carbohydrate-addition motifs are required for folding and/or reactivity and which are not, combinations of such mutations may be made.
  • the ‘mispriming’ or ‘overlap extension’ was used for site-directed mutagenesis.
  • the concept is illustrated in FIGS. 42 and 43 .
  • Two separate fragments were amplified from the target gene for each mutated site.
  • the PCR product obtained from the 5′ end (product GLYP) was amplified with the 5′ sense GPT primer (see Table 7) and with the respective 3‘antisense GLYM’ primers.
  • the second fragment (product OVR#) was amplified with the 3′ antisense TK R primer and the respective 5′ sense primers (OVR# primers, see Table 7, FIG. 43 ).
  • the OVR# primers target part of the GLY# primer sequence. Therefore, the two groups of PCR products share an overlap region of identical sequence.
  • these intermediate products are mixed (GLY-1 with OVR-1, GLY-2 with OVR-2, etc.), melted at high temperature, and reannealed, the top sense strand of product GLY# can anneal to the antisense strand of product OVR# (and vice versa) in such a way that the two strands act as primers for one another (see FIG. 42 .B.).
  • Extension of the annealed overlap by Taq polymerase during two FCR cycles created the full-length mutant molecule E1 Gly#, which carries the mutation destroying the glycosylation site number #.
  • the selected clones were analyzed for length of insert by EcoRI/BamHI cleavage and for the presence of each new restriction site. The sequences overlapping the mutated sites were confirmed by double-stranded sequencing.
  • recombinant vaccinia viruses were generated by recombination with wt vaccinia virus as described in example 2.5. Briefly, 175 cm 2 -flasks of subconfluent RK13 cells were infected with the 6 recombinant vaccinia viruses carrying the mutant E1 sequences, as well as with the vvHCV-10A (carrying the non-mutated E1 sequence) and wt vaccinia viruses. Cells were lysed after 24 hours of infection and analyzed on western blot as described in example 4 (see FIG. 444A ).
  • All mutants showed a faster mobility (corresponding to a smaller molecular weight of approximately 2 to 3 kDa) on SDS-PAC-E than the original E1 protein; confirming that one carbohydrate moiety was not added.
  • Recombinant viruses were also analyzed by PCR and restriction enzyme analysis to confirm the identity of the different mutants.
  • FIG. 448 shows that all mutants (as shown in FIG. 41 ) contained the expected additional restriction sites.
  • Another part of the cell lysate was used to test the reactivity of the different mutant by ELISA. The lysates were diluted 20 times and added to microwell plates coated with the lectin GNA as described in example 6.
  • the E2 sequence corresponding to clone HCCL41 was provided with the ⁇ -mating factor pre/pro signal sequence, inserted in a yeast expression vector and S. cerevisiae cells transformed with this construct secreted E2 protein into the growth medium. It was observed that most glycosylation sites were modified with high-mannose type glycosylations upon expression of such a construct in S. cerevisiae strains ( FIG. 45 ). This resulted in a too high level of heterogeneity and in shielding of reactivity, which is not desirable for either vaccine or diagnostic purposes. To overcome this problem, S. cerevisiae mutants with modified glycosylation pathways were generated by means of selection of vanadate-resistant clones.
  • Such clones were analyzed for modified glycosylation pathways by analysis of the molecular weight and heterogeneity of the glycoprotein invertase. This allowed us to identify different glycosylation deficient S. cerevisiae mutants.
  • the E2 protein was subsequently expressed in some of the selected mutants and left to react with a monoclonal antibody as described in example 7, on western blot as described in example 4 ( FIG. 46 ).
  • the present results show that not only a good expression system but also a good purification protocol are required to reach a high reactivity of the HCV envelope proteins with human patient sera.
  • This can be obtained using the proper HCV envelope protein expression system and/or purification protocols of the present invention which guarantee the conservation of the natural folding of the protein and the purification protocols of the present invention which guarantee the elimination of contaminating proteins and which preserve the conformation, and thus the reactivity of the HCV envelope proteins.
  • the amounts of purified HCV envelope protein needed for diagnostic screening assays are in the range of grams per year. For vaccine purposes, even higher amounts of envelope protein would be needed.
  • the vaccinia virus system may be used for selecting the best expression constructs and for limited upscaling, and large-scale expression and purification of single or specific oligomeric envelope proteins containing high-mannose carbohydrates may be achieved when expressed from several yeast strains.
  • hepatitis B for example, manufacturing of HBsAg from mammalian cells was much more costly compared with yeast-derived hepatitis B vaccines.
  • the purification method disclosed in the present invention may also be used for ‘viral envelope proteins’ in general.
  • examples are those derived from Flaviviruses, the newly discovered GB-A.
  • GB-B and GB-C Hepatitis viruses, Pestiviruses such as Bovine viral Diarrhea Virus (BVDV). Hog Cholera Virus (HCV). Border Disease Virus (BDV)), but also less related viruses such as Hepatitis B Virus (mainly for the purification of HBsAg).
  • the envelope protein purification method of the present invention may be used for intra- as well as extracellularly expressed proteins in lower or higher eukaryotic cells or in prokaryotes as set out in the detailed description section.
  • TABLE 1 Recombinant vaccinia plasmids and viruses cDNA subclone Vector used Plasmid name Name construction Length (nt/aa) for insertion pvHCV-13A E1s EcoR I-Hind III 472/157 pgptATA-18 pvHCV-12A E1s EcoR I-Hind III 472/158 pgptATA-18 pvHCV-9A E1 EcoR I-Hind III 631/211 pgptATA-18 pvHCV-11A E1s EcoR I-Hind III 625/207 pgptATA-18 pvHCV-17A E1s EcoR I-Hind III 625/208 pgptATA-18 pvHCV-10A E1 EcoR I-Hind III 783/2

Abstract

The present invention relates to a method for purifying recombinant HCV single or specific oligomeric envelope proteins selected from the group consisting of E1 and/or E1/E2 characterized in that upon lysing the transformed host cells to isolate the recombinantly expressed protein a disulphide bond cleavage or reduction step is carried out with a disulphide bond cleavage agent. The present invention also relates to a composition isolated by such a method. The present invention also relates to the diagnostic and therapeutic application of these compositions. Furthermore, the invention relates to the use of HCV E1 protein and peptides for prognosing and monitoring the clinical effectiveness and/or clinical outcome of HCV treatment.

Description

  • The present application is a divisional of Ser. No. 09/899,303, filed Jul. 6, 2001 (pending), which is a divisional of Ser. No. 08/928,017, filed Sep. 11, 1997 (abandoned), which is a divisional of Ser. No. 08/612,973, filed Mar. 11, 1996 (now U.S. Pat. No. 6,150,134), which is a 371 U.S. national phase of PCT/EP95/03031, filed Jul. 31, 1995 and claims benefit of EP 94870132.1 filed Jul. 29, 1994, the entire contents of each of which is incorporated herein by reference in their entirety.
    • FIELD OF THE INVENTION
  • The present invention relates to the general fields of recombinant protein expression, purification of recombinant proteins, synthetic peptides, diagnosis of HCV infection, prophylactic treatment against HCV infection and to the prognosis, monitoring of the clinical efficiency of treatment of an individual with chronic hepatitis, or the prognosis/monitoring of natural disease.
  • More particularly the present invention relates to purification methods for hepatitis C virus envelope proteins, the use in diagnosis, prophylaxis or therapy of HCV envelope proteins purified according to the methods described in the present invention, the use of single or specific oligomeric E1 and/or E2 and/or E1/E2 envelope proteins in assays for monitoring disease, and/or diagnosis of disease, and/or treatment of disease. The invention also relates to epitopes of the E1 and/or E2 envelope proteins and monoclonal antibodies thereto, as well their use in diagnosis, prophylaxis or treatment.
    • BACKGROUND OF THE INVENTION
  • The E2 protein purified from cell lysates according to the methods described in the present invention reacts with approximately 95% of patient sera. This reactivity is similar to the reactivity obtained with E2 secreted from CHO cells (Spaete et al., 1992). However, the intracellularly expressed form of E2 may more closely resemble the native viral envelope protein because it contains high mannose carbohydrate motifs, whereas the E2 protein secreted from CHO cells is further modified with galactose and sialic acid sugar moieties. When the aminoterminal half of E2 is expressed in the baculovirus system, only about 13 to 21% of sera from several patient groups can be detected (Inoue et al., 1992). After expression of E2 from E. coli, the reactivity of HCV sera was even lower and ranged from 14 (Yokosuka et al., 1992) to 17% (Mita et al., 1992).
  • About 75% of HCV sera (and 95% of chronic patients) are anti-s positive using the purified, vaccinia-expressed recombinant E1 protein of the present invention, in sharp contrast with the results of Kohara et al. (1992) and Hsu et al. (1993). Kohara et al. used a vaccinia-virus expressed E1 protein and detected anti-E1 antibodies in 7 to 23% of patients, while Hsu et al. only detected 14150 (28%) sera using baculovirus-expressed E1.
  • These results show that nor only a good expression system but also a good purification protocol are required to reach a high reactivity of the envelope proteins with human patient sera. This can be obtained using the proper expression system and/or purification protocols of the present invention which guarantee the conservation of the natural folding of the protein and the purification protocols of the present invention which guarantee the elimination of contaminating proteins and which preserve the conformation, and thus the reactivity of the HCV envelope proteins. The amounts of purified HCV envelope protein needed for diagnostic screening assays are in the range of grams per year. For vaccine purposes, even higher amounts of envelope protein would be needed. Therefore, the vaccinia virus system may be used for selecting the best expression constructs and for limited upscaling, and large-scale expression and purification of single or specific oligomeric envelope proteins containing high-mannose carbohydrates may be achieved when expressed from several yeast strains. In the case of hepatitis B for example, manufacturing of HBsAg from mammalian cells was much more costly compared with yeast-derived hepatitis B vaccines.
    • AIMS OF THE INVENTION
  • It is an aim of the present invention to provide a new purification method for recombinantly expressed E1 and/or E2 and/or E1/E2 proteins such that said recombinant proteins are directly usable for diagnostic and vaccine purposes as single or specific oligomeric recombinant proteins free from contaminants instead of aggregates.
  • It is another aim of the present invention to provide compositions comprising purified (single or specific oligomeric) recombinant E1 and/or E2 and/or E1/E2 glycoproteins comprising conformational epitopes from the E1 and/or E2 domains of HCV.
  • It is yet another aim of the present invention to provide novel recombinant vector constructs for recombinantly expressing E1 and/or E2 and/or E1/E2 proteins, as well as host cells transformed with said vector constructs.
  • It is also an aim of the present invention to provide a method for producing and purifying recombinant HCV E1 and/or E2 and/or E1/E2 proteins.
  • It is also an aim of the present invention to provide diagnostic and immunogenic uses of the recombinant HCV. E1 and/or E2 and/or E1/E2 proteins of the present invention, as well as to provide kits for diagnostic use, vaccines or therapeutics comprising any of the recombinant HCV E1 and/or E2 and/or E1/E2 proteins of the present invention.
  • It is further an aim of the present invention to provide for a new use of E1, E2, and/or E1/E2 proteins, or suitable parts thereof, for monitoring/prognosing the response to treatment of patients (e.g. with interferon) suffering from HCV infection.
  • It is also an aim of the present invention to provide for the use of the recombinant E1, E2, and/or E1/E2 proteins of the present invention in HCV screening and confirmatory antibody tests.
  • It is also an aim of the present invention to provide E1 and/or E2 peptides which can be used for diagnosis of HCV infection and for raising antibodies. Such peptides may also be used to isolate human monoclonal antibodies.
  • It is also an aim of the present invention to provide monoclonal antibodies, more particularly human monoclonal antibodies or mouse monoclonal antibodies which are humanized, which react specifically with E1 and/or E2 epitopes, either comprised in peptides or conformational epitopes comprised in recombinant proteins.
  • It is also an aim of the present invention to provide possible uses of anti-E1 or anti-E2 monoclonal antibodies for HCV antigen detection or for therapy of chronic HCV infection.
  • It is also an aim of the present invention to provide kits for monitoring/prognosing the response to treatment (e.g. with interferon) of patients suffering from HCV infection or monitoring/prognosing the outcome of the disease.
  • All the aims of the present invention are considered to have been met by the embodiments as set out below.
    • DEFINITIONS
  • The following definitions serve to illustrate the different terms and expressions used in the present invention.
  • The term ‘hepatitis C virus single envelope protein’ refers to a polypeptide or an analogue thereof (e.g. mimotopes) comprising an amino acid sequence (and/or amino acid analogues) defining at least one HCV epitope of either the E1 or the E2 region. These single envelope proteins in the broad sense of the word may be both monomeric or homo-oligomeric forms of recombinantly expressed envelope proteins. Typically, the sequences defining the epitope correspond to the amino acid sequence of either the E1 or the E2 region of HCV (either identically or via substitution of analogues of the native amino acid residue that do not destroy the epitope). In general, the epitope-defining sequence will be 3 or more amino acids in length, more typically, 5 or more amino acids in length, more topically 8 or more amino acids in length, and even more typically 10 or more amino acids in length. With respect to conformational epitopes, 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). Thus, 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. For example, deletion or substitution of these intervening sequences may not affect the conformational epitope provided sequences critical to epitope conformation are maintained (e.g. cysteines involved in disulfide bonding, glycosylation sites, etc.). A conformational epitope may also be formed by 2 or more essential regions of subunits of a homooligomer or heterooligomer.
  • The HCV antigens of the present invention comprise conformational epitopes from the E1 and/or E2 (envelope) domains of HCV. The E1 domain, which is believed to correspond to the viral envelope protein, is currently estimated to span amino acids 192-383 of the HCV polyprotein (Hijikata et al., 1991). Upon expression in a mammalian system (glycosylated), it is believed to have an approximate molecular weight of 35 kDa as determined via SDS-PAGE. The E2 protein, previously called NS1, is believed to span amino acids 384-809 or 384-746 (Grakoui et al. 1993) of the HCV polyprotein and to also be an envelope protein. Upon expression in a vaccinia system (glycosylated), it is believed to have an apparent gel molecular weight of about 72 kDa. It is understood that these protein endpoints are approximations (e.g. the carboxy terminal end of E2 could lie somewhere in the 730-820 amino acid region, e.g. ending at amino acid 730, 735, 740, 742, 744, 745, preferably 748, 747, 748, 750, 760, 770, 780, 790, 800, 809, 810, 820). The E2 protein may also be expressed together with the E1, P7 (aa 747-809), NS2 (aa 810-1025), NS4A (aa 1658-1711) or NS4B (aa 1712-1972). Expression together with these other HCV proteins may be important for obtaining the correct protein folding.
  • It is also understood that the isolates used in the examples section of the present invention were not intended to limit the scope of the invention and that any HCV isolate from type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or any other new genotype of HCV is a suitable source of E1 and/or E2 sequence for the practice of the present invention.
  • The E1 and E2 antigens used in the present invention may be full-length viral proteins, substantially full-length versions thereof, or functional fragments thereof (e.g. fragments which are not missing sequence essential to the formation or retention of an epitope). Furthermore, the HCV antigens of the present invention can also include other sequences that do not block or prevent the formation of the conformational epitope of interest. The presence or absence of a conformational epitope can be readily determined though screening the antigen of interest with an antibody (polygonal serum or monoclonal to the conformational epitope) and comparing its reactivity to that of a denatured version of the antigen which retains only linear epitopes (if any). In such screening using polyclonal antibodies, it may be advantageous to adsorb the polyclonal serum first with the denatured antigen and see if it retains antibodies to the antigen of interest.
  • The HCV antigens of the present invention can be made by any recombinant method that provides the epitope of interest. For example, recombinant intracellular expression in mammalian or insect cells is a preferred method to provide glycosylated E1 and/or E2 antigens in ‘native’ conformation as is the case for the natural HCV antigens. Yeast cells and mutant yeast strains (e.g. mnn 9 mutant (Kniskern et al., 1194) or glycosylation mutants derived by means of vanadate resistance selection (Ballou et al., 1991)) may be ideally suited for production of secreted high-mannose-type sugars; whereas proteins secreted from mammalian cells may contain modifications including galactose or sialic acids which may be undesirable for certain diagnostic or vaccine applications. However, it may also be possible and sufficient for certain applications, as it is known for proteins, to express the antigen in other recombinant hosts (such as E. coli) and renature the protein after recovery.
  • The term ‘fusion polypeptide’ intends a polypeptide in which the HCV antigen(s) are part of a single continuous chain of amino acids, which chain does not occur in nature. The HCV antigens may be connected directly to each other by peptide bonds or be separated by intervening amino acid sequences. The fusion polypeptides may also contain amino acid sequences exogenous to HCV.
  • The term ‘solid phase’ intends a solid body to which the individual HCV antigens or the fusion polypeptide comprised of HCV antigens are bound covalently or by noncovalent means such as hydrophobic adsorption.
  • The term ‘biological sample’ intends a fluid or tissue of a mammalian individual (e.g. an anthropoid, a human) that commonly contains antibodies produced by the individual, more particularly antibodies against HCV. The fluid or tissue may also contain HCV antigen. Such components are known in the art and include, without limitation, blood, plasma, serum, urine, spinal fluid, lymph fluid, secretions of the respiratory, intestinal or genitourinary tracts, tears, saliva, milk, white blood cells and myelomas. Body components include biological liquids. The term ‘biological liquid’ refers to a fluid obtained from an organism. Some biological fluids are used as a source of other products, such as clotting factors (e.g. Factor VIII:C), serum albumin, growth hormone and the like. In such cases, it is important that the source of biological fluid be free of contamination by virus such as HCV.
  • The term ‘immunologically reactive’ means that the antigen in question will react specifically with anti-HCV antibodies present in a body component from an HCV infected individual.
  • The term ‘immune complex’ intends the combination formed when an antibody binds to an epitope on an antigen.
  • ‘E1’ as used herein refers to a protein or polypeptide expressed within the first 400 amino acids of an HCV polyprotein, sometimes referred to as the E, ENV or S protein. In its natural form it is a 35 kDa glycoprotein which is found in strong association with membranes. In most natural HCV strains, the E1 protein is encoded in the viral polyprotein following the C (core) protein. The E1 protein extends from approximately amino acid (aa) 192 to about aa 383 of the full-length polyprotein.
  • The term ‘E1’ as used herein also includes analogs and truncated forms that are immunologically cross-reactive with natural E1, and includes E1 proteins of genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other newly identified HCV type or subtype.
  • ‘E2’ as used herein refers to a protein or polypeptide expressed within the first 900 amino acids of an HCV polyprotein, sometimes referred to as the NS1 protein. In its natural form it is a 72 kDa glycoprotein that is found in strong association with membranes. In most natural HCV strains, the E2 protein is encoded in the viral polyprotein following the E1 protein. The E2 protein extends from approximately amino acid position 384 to amino acid position 746, another form of E2 extends to amino acid position 809. The term ‘E2’ as used herein also includes analogs and truncated forms that are immunologically cross-reactive with natural E2. 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).
  • ‘E1/E2’ as used herein refers to an oligomeric form of envelope proteins containing at least one E1 component and at least one E2 component.
  • The term ‘specific oligomeric’ E1 and/or E2 and/or E1/E2 envelope proteins refers to all possible oligomeric forms of recombinantly expressed E1 and/or E2 envelope proteins which are not aggregates. E1 and/or E2 specific oligomeric envelope proteins are also referred to as homo-oligomeric E1 or E2 envelope proteins (see below).
  • The term ‘single or specific oligomeric’ E1 and/or E2 and/or E1/E2 envelope proteins refers to single monomeric E1 or E2 proteins (single in the strict sense of the word) as well as specific oligomeric E1 and/or E2 and/or E1/E2 recombinantly expressed proteins. These single or specific oligomeric envelope proteins according to the present invention can be further defined by the following formula (E1) (E2), wherein x can be a number between 0 and 100, and y can be a number between 0 and 100, provided that x and y are not both 0. With x=1 and y=0 said envelope proteins include monomeric E1.
  • The term ‘homo-oligomer’ as used herein refers to a complex of E1 and/or E2 containing more than one E1 or E2 monomer, e.g. E1/E1 dimers, E1/E1E1 trimers or E1/E1/E1/E1, tetramers and E2/E2 dimers, E2/E2/E2 trimers or E2/E2/E2/E2 tetramers, E1 pentamers and hexamers, E2 pentamers and hexamers or any higher-order homo-oligomers of E1 or E2 are all ‘homo-oligomers’ within the scope of this definition. The oligomers may contain one, two, or several different monomers of E1 or E2 obtained from different types or subtypes of hepatitis C virus including for example those described in an international application published under WO 94/25601 and European application No. 94870166.9 both by the present applicants. Such mixed oligomers are still homo-oligomers within the scope of this invention, and may allow more universal diagnosis, prophylaxis or treatment of HCV.
  • The term ‘purified’ as applied to proteins herein refers to a composition wherein the desired protein comprises at least 35% of the total protein component in the composition. The desired protein preferably comprises at least 40%, more preferably at least about 50%, more preferably at least about 60%, still more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% of the total protein component. The composition may contain other compounds such as carbohydrates, salts, lipids, solvents, and the like, without affecting the determination of the percentage purity as used herein. An ‘isolated’ HCV protein intends an HCV protein composition that is at least 35% pure.
  • The term ‘essentially purified proteins’ refers to proteins purified such that they can be used for in vitro diagnostic methods and as a therapeutic compound. These proteins are substantially free from cellular proteins, vector-derived proteins or other HCV viral components. Usually these proteins are purified to homogeneity (at least 80 to pure, preferably, 90%, more preferably 95%, more preferably 97%, more preferably 98%, more preferably 99%, even more preferably 99%, and most preferably the contaminating proteins should be undetectable by conventional methods like SDS-PAGE and silver staining.
  • The term ‘recombinantly expressed’ used within the context of the present invention refers to the fact that the proteins of the present invention are produced by recombinant expression methods be it in prokaryotes, or lower or higher eukaryotes as discussed in detail below.
  • The term ‘lower eukarycote’ refers to host cells such as yeast, fungi and the like. Lower eukaryotes are generally (but not necessarily) unicellular. Preferred lower eukaryotes are yeasts, particularly species within Saccharomyces. Schizosaccharomyces, Kluveromyces, Pichia (e.g. Pichia pastoris). Hansenula (e.g. Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces. Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts.
  • The term ‘prokaryotes’ refers to hosts such as E. coli, Lactobacillus. Lactococcus, Salmonella, Streptococcus, Bacillus subtilis or Streptomyces. Also these hosts are contemplated within the present invention.
  • The term ‘higher eukaryote’ refers to host cells derived from higher animals, such as mammals, reptiles, insects, and the like. Presently preferred higher eukaryote host cells are derived from Chinese hamster (e.g. CHO), monkey (e.g. COS and Vero cells), baby hamster kidney (BHK), pig kidney (PK15), rabbit kidney 13 cells (RK13), the human osteosarcoma cell line 143 B, the human cell line HeLa and human hepatoma cell lines like Hep G2, and insect cell lines (e.g. Spodoptera frugiperda). The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively the host cells may also be transgenic animals.
  • The term ‘polypeptide’ refers to a polymer of amino acids and does not refer to a specific length of the product, thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogues of an amino 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.
  • The term ‘recombinant polynucleotide or nucleic acid’ intends a polynucleotide or nucleic acid of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.
  • The term ‘recombinant host cells’, ‘host cells’, ‘cells’, ‘cell lines’, ‘cell cultures’, and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be or have been, used as recipients for a recombinant vector or other transfer polynucleotide, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.
  • The term ‘replicon’ is any genetic element, e.g., a plasmid, a chromosome, a virus, a cosmid, etc., that behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control.
  • The term ‘vector’ is a replicon further comprising sequences providing replication and/or expression of a desired open reading frame.
  • The term ‘control sequence’ refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism: in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and terminators; in eukaryotes, generally, such control sequences include promoters, terminators and, in some instances, enhancers. The term ‘control sequences’ is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences which govern secretion.
  • The term ‘promoter’ is a nucleotide sequence which is comprised of consensus sequences which allow the binding of RNA polymerase to the DNA template in a manner such that mRNA production initiates a; the normal transcription initiation site for the adjacent structural gene.
  • The expression ‘operably linked’ refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence ‘operably linked’ to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • An ‘open reading frame’ (ORF) is a region of a polynucleotide sequence which encodes a polypeptide and does not contain stop codons, this region may represent a portion of a coding sequence or a total coding sequence.
  • A ‘coding sequence’ is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries or the coding sequence are determined by a translation star codon at the terminus and a translation stop codon at the 3′-terminus. A coding sequence can include but is not limited to mRNA, DNA (including cDNA), and recombinant polynucleotide sequences.
  • As used herein, ‘epitope’ or ‘antigenic determinant’ means an amino acid sequence that is immunoreactive. Generally an epitope consists of at least 3 to 4 amino acids, and more usually, consists of at least 5 or 6 amino acids, sometimes the epitope consists of about 7 to 8, or even about 10 amino acids. As used herein, 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. Such equivalents also include strain, subtype (=genotype), or type(group)-specific variants, e.g. of the currently known sequences or strains belonging to genotypes 1a, 1b, 1c, 1d, 1e, 1f, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 3a, 3b, 3c, 3d, 3e, 3f, 3g, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k, 4l, 5a, 5b, 6a, 6b, 6c, 7a, 7b, 7c, 8a, 8b, 9a, 9b, 10a, or any other newly defined HCV (sub)type. It is to be understood that the amino acids constituting the epitope need not be par, of a linear sequence, but may be interspersed by any number of amino acids, thus forming a conformational epitope.
  • The term ‘immunogenic’ refers to the ability of a substance to cause a humoral and/or cellular response, whether alone or when linked to a carrier, in the presence or absence of an adjuvant. ‘Neutralization’ refers to an immune response that blocks the infectivity, either partially or fully, of an infectious agent. A ‘vaccine’ is an immunogenic composition capable of eliciting protection against HCV, whether partial or complete. A vaccine may also be useful for treatment of an individual, in which case it is called a therapeutic vaccine.
  • The term ‘therapeutic’ refers to a composition capable of treating HCV infection.
  • The term ‘effective amount’ refers to an amount of epitope-bearing polypeptide sufficient to induce an immunogenic response in the individual to which it is administered, or to otherwise detectably immunoreact in its intended system (e.g., immunoassay). Preferably, the effective amount is sufficient to effect treatment, as defined above. The exact amount necessary will vary according to the application. For vaccine applications or for the generation of polyclonal antiserum/antibodies, for example, the effective amount may vary depending on the species, age, and general condition of the individual, the severity of the condition being treated, the particular polypeptide selected and its mode of administration, etc. It is also believed that effective amounts will be found within a relatively large, non-critical range. An appropriate effective amount can be readily determined using only routine experimentation. Preferred ranges of E1 and/or E2 and/or E1/E2 single or specific oligomeric envelope proteins for prophylaxis of HCV disease are 0.01 to 100 μg/dose, preferably 0.1 to 50 μg/dose. Several doses may be needed per individual in order to achieve a sufficient immune response and subsequent protection against HCV disease.
    • DETAILED DESCRIPTION OF THE INVENTION
  • More particularly, the present invention contemplates a method for isolating or purifying recombinant HCV single or specific oligomeric envelope protein selected from the group consisting of E1 and/or E2 and/or E1/E2, characterized in that upon lysing the transformed host cells to isolate the recombinantly expressed protein a disulphide bond cleavage or reduction step is carried out with a disulphide bond cleaving agent.
  • The essence of these ‘single or specific oligomeric’ envelope proteins of the invention is that they are free from contaminating proteins and that they are not disulphide bond linked with contaminants.
  • The proteins according to the present invention are recombinantly expressed in lower or higher eukaryotic cells or in prokaryotes. The recombinant proteins of the present invention are preferably glycosylated and may contain high-mannose-type, hybrid, or complex glycosylations, preferentially said proteins are expressed from mammalian cell lines as discussed in detail in the Examples section, or in yeast such as in mutant yeast strains also as de-ailed in the Examples section.
  • The proteins according to the present invention may be secreted or expressed within components of the cell, such as the ER or the Golgi Apparatus. Preferably, however, the proteins of the present invention bear high-mannose-type glycosylations and are retained in the ER or Golgi Apparatus of mammalian cells or are retained in or secreted from yeast cells, preferably secreted from yeast mutant strains such as the mnn9 mutant (Kniskern et al., 1994), or from mutants that have been selected by means of vanadate resistance (Ballou et al., 1991).
  • Upon expression of HCV envelope proteins, the present inventors could show that some of the free thiol groups of cysteines not involved in intra- or inter-molecular disulphide bridges, react with cysteines of host or expression-system-derived (e.g. vaccinia) proteins or of other HCV envelope proteins (single or oligomeric), and form aspecific intermolecular bridges. This results in the formation of ‘aggregates’ of HCV envelope proteins together with contaminating proteins. It was also shown in WO92/08734 that ‘aggregates’ were obtained after purification, but it was not described which protein interactions were involved. In patent application WO 92/08734, recombinant E1/E2 protein expressed with the vaccinia virus system were partially purified as aggregates and only found to be 70% pure, rendering the purified aggregates not useful for diagnostic, prophylactic or therapeutic purposes.
  • Therefore, a major aim of the present invention resides in the separation of single or specific-oligomeric HCV envelope proteins from contaminating proteins, and to use the purified proteins (>959% pure) for diagnostic, prophylactic and therapeutic purposes. To those purposes, the present inventors have been able to provide evidence that aggregated protein complexes (‘aggregates’) are formed on the basis of disulphide bridges and non-covalent protein-protein interactions. The present invention thus provides a means for selectively cleaving the disulphide bonds under specific conditions and for separating the cleaved proteins from contaminating proteins which greatly interfere with diagnostic, prophylactic and therapeutic applications. The free thiol groups may be blocked (reversibly or irreversibly) in order to prevent the reformation of disulphide bridges, or may be left to oxidize and oligomerize with other envelope proteins (see definition homo-oligomer). It is to be understood that such protein oligomers are essentially different from the ‘aggregates’ described in WO 92/08734 and WO 94/01778, since the level of contaminating proteins is undetectable.
  • Said disulfide bond cleavage may also be achieved by:
  • (1) performic acid oxidation by means of cysteic acid in which case the cysteine residues are modified into cysteic acid (Moore et al., 1963).
  • (2) Sulfitolysis (R—S—S—R →2 R—SOC2− 3) for example by means of sulphite (SO2− 3) together with a proper oxidant such as Cu2− in which case the cysteine is modified into S-sulpho-cysteine (Bailey and Cole, 1959).
  • (3) Reduction by means of mercaptans, such as dithiothreitol (DDT), β-mercapto-ethanol, cysteine, glutathione Red, ε-mercapto-ethylamine, or thioglycolic acid, of which DTT and β-mercapto-ethanol are commonly used (Cleland, 1964), is the preferred method of this invention because the method can be performed in a water environment and because the cysteine remains unmodified.
  • (4) Reduction by means of a phosphine (e.g. Bu3P) (Ruegg and Rudinger, 1977).
  • All these compounds are thus to be regarded as agents or means for cleaving disulphide bonds according to the present invention.
  • Said disulphide bond cleavage (or reducing) step of the present invention is preferably a partial disulphide bend cleavage (reducing) step (carried out under partial cleavage or reducing conditions).
  • A preferred disulphide bond cleavage or reducing agent according to the present invention is dithiothreitol (DTT). Partial reduction is obtained by using a low concentration of said reducing agent, i.e. for DTT for example in the concentration range of about 0.1 to about 50 mM, preferably about 0.1 to about 20 mM, preferably about 0.5 to about 10 mM, preferably more than 1 mM, more than 2 mM or more than 5 mM, more preferably about 1.5 mM, about 2.0 mM, about 2.5 mM, about 5 mM or about 7.5 mM.
  • Said disulphide bond cleavage step may also be carried out in the presence or a suitable detergent (as an example of a means for cleaving disulphide bonds or in combination with a cleaving agent) able to dissociate the expressed proteins, such as DecylPEG, EMPIGEN-BB, NP-40, sodium cholate, Triton X-100.
  • Said reduction or cleavage step (preferably a partial reduction or cleavage step) is carried out preferably in in the presence of (with) a detergent. A preferred detergent according to the present invention is Empigen-BB. The amount of detergent used is preferably in the range of 1 to 10%, preferably more than 3%, more preferably about 3.5% of a detergent such as Empigen-BB.
  • A particularly preferred method for obtaining disulphide bond cleavage employs a combination of a classical disulphide bond cleavage agent as detailed above and a detergent (also as detailed above). As contemplated in the Examples section, the particular combination of a low concentration of DTT (1.5 to 7.5 mM) and about 3.5% of Empigen-BB is proven to be a particularly preferred combination of reducing agent and detergent for the purification of recombinantly expressed E1 and E2 proteins. Upon gelfiltration chromatography, said partial reduction is shown to result in the production of possibly dimeric E1 protein and separation of this E1 protein from contaminating proteins that cause false reactivity upon use in immunoassays.
  • It is, however, to be understood that also any other combination of any reducing agent known in the art with any detergent or other means known in the art to make the cysteines better accessible is also within the scope of the present invention, insofar as said combination reaches the same goal of disulphide bridge cleavage as the preferred combination exemplified in the present invention.
  • Apart from reducing the disulphide bonds, a disulphide bond cleaving means according to the present invention may also include any disulphide bridge exchanging agents (competitive agent being either organic or proteinaeous, see for instance Creighton, 1988) known in the art which allows the following type of reaction to occur:
    R1S−S R2÷R3SH→R1S−S R3÷R2SH
  • R1, R2: compounds of protein aggregates
  • R3 SH: competitive agent (organic, proteinaeous)
  • The term ‘disulphide bridge exchanging agent’ is to be interpretated as including disulphide bond reforming as well as disulphide bond blocking agents.
  • The present invention also relates to methods for purifying or isolating HCV single or specific oligomeric envelope proteins as set out above further including the use of any SH group blocking or binding reagent known in the art such as chosen from the following list:
      • Glutathion
      • 5,5′-dithiobis-(2-nitrobenzoic acid) or bis-(3-carboxy-4-nitrophenyl)-disulphide (DTNB or Ellman's reagent) (Elmann. 1959)
      • N-ethylmaleimide (NEM; Benesch et al., 1956)
      • N-(4-dimethylamino-3,5-dinitrophenyl) maleimide or Tuppy's maleimide which provides a color to the protein
      • P-chloromercuribenzoate (Grassetti et al., 1969)
      • 4-vinylpyridine (Friedman and Krull, 1969) can be liberated after reaction by acid hydrolysis
      • acrylonitrile, can be liberated after reaction by acid hydrolysis (Weil and Seibles, 1961)
      • NEM-biotin (e.g. obtained from Sigma B1267)
      • 2,2′-dithiopyridine (Grassetti and Murray, 1967)
      • 4,4′-dithiopyridine (Grassetti and Murray, 1967)
      • 6,6′-dithiodinicontinic acid (DTDNA; Brown and Cunnigham, 1970)
      • 2,2′-dithiobis-(5′-nitropyridine) (DTNP: U.S. Pat. No. 3,597,160) or other dithiobis (heterocyclic derivative) compounds (Grassetti and Murray, 1969)
  • A survey of the publications cited shows that often different reagents for sulfhydryl groups will react with varying numbers of thiol groups of the same protein or enzyme molecule. One may conclude that this variation in reactivity of the thiol groups is due to the steric environment of these groups, such as the shape of the molecule and the surrounding groups of atoms and their charges, as well as to the size, shape and charge of the reagent molecule or ion. Frequently the presence of adequate concentrations of denaturants such as sodium dodecylsulfate, urea or guanidine hydrochloride will cause sufficient unfolding of the protein molecule to permit equal access to all of the reagents for thiol groups. By varying the concentration of denaturant, the degree of unfolding can be controlled and in this way thiol groups with different degrees of reactivity may be revealed. Although up to date most of the work reported has been done with p-chloromercuribenzoate, N-ethylmaleimide and DTNE, it is likely that the other more recently developed reagents may prove equally useful. Because of their varying structures, it seems likely, in fact, that they may respond differently to changes in the steric environment of the thiol groups.
  • Alternatively, conditions such as low pH (preferably lower than pH 6) for preventing free SH groups from oxidizing and thus preventing the formation of large intermolecular aggregates upon recombinant expression and purification of E1 and E2 (envelope) proteins are also within the scope of the present invention.
  • A preferred SH group blocking reagent according to the present invention is N-ethylmaleimide (NEM). Said SH croup blocking reagent may be administrated during lysis of the recombinant host cells and after the above-mentioned partial reduction process or after any other process for cleaving disulphide bridges. Said SH group blocking reagent may also be modified with any group capable of providing a detectable label and/or any group aiding in the immobilization of said recombinant protein to a solid substrate, e.g. biotinylated NEM.
  • Methods for cleaving cysteine bridges and blocking free cysteines have also been described in Darbre (1987), Mears and Feeney (1971), and by Wong (1993).
  • A method to purify single or specific oligomeric recombinant E1 and/or E2 and/or E1/E2 proteins according to the present invention as defined above is further characterized as comprising the following steps:
      • lysing recombinant E1 and/or E2 and/or E1/E2 expressing host cells, preferably in the presence of an SH group blocking agent, such as N-ethylmaleimide (NEM), and possibly a suitable detergent, preferably Empigen-BB.
      • recovering said HCV envelope protein by affinity purification for instance by means lectin-chromatography, such as lentil-lectin chromatography, or immunoaffinity chromatography using anti-E1 and/or anti-E2 specific monoclonal antibodies, followed by,
      • reduction or cleavage of disulphide bonds with a disulphide bond cleaving agent, such as DTT, preferably also in the presence of an SH group blocking agent, such as NEM or Biotin-NEM, and,
      • recovering the reduced HCV E1 and/or E2 and/or E1/E2 envelope proteins for instance by gelfiltration (size exclusion chromatography or molecular sieving) and possibly also by an additional Ni2−-IMAC chromatography and desalting step.
  • It is to be understood that the above-mentioned recovery steps may also be carried out using any other suitable technique known by the person skilled in the art.
  • Preferred lectin-chromatography systems include Galanthus nivalis agglutinin (GNA)-chromatography, or Lens culinaris agglutinin (LCA) (lentil) lectin chromatography as illustrated in the Examples section. Other useful lectins include those recognizing high-mannose type sugars, such as Narcissus pseudonarcissus agglutinin (NPA), Pisum sativum agglutinin (PSA), or Allium ursinum agglutinin (AUA).
  • Preferably said method is usable to purify single or specific oligomeric HCV envelope protein produced intracellularly as detailed above.
  • For secreted E1 or E2 or E1/E2 oligomers, lectins binding complex sugars such as Ricinus communis agglutinin I (RCA I), are preferred lectins.
  • The present invention more particularly contemplates essentially purified recombinant HCV single or specific oligomeric envelope proteins, selected from the group consisting of E1 and/or E2 and/or E1/E2, characterized as being isolated or purified by a method as defined above.
  • The present invention more particularly relates to the purification or isolation of recombinant envelope proteins which are expressed from recombinant mammalian cells such as vaccinia.
  • The present invention also relates to the purification or isolation of recombinant envelope proteins which are expressed from recombinant yeast cells.
  • The present invention equally relates to the purification or isolation of recombinant envelope proteins which are expressed from recombinant bacterial (prokaryotic) cells.
  • The present invention also contemplates a recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic promoter sequence followed by a nucleotide sequence allowing the expression of the single or specific oligomeric E1 and/or E2 and/or E1/E2 of the invention.
  • Particularly, the present invention contemplates a recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic promoter sequence followed by a nucleotide sequence allowing the expression of the single E1 or E1 of the invention.
  • Particularly, the present invention contemplates a recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic promoter sequence followed by a nucleotide sequence allowing the expression of the single E1 or E2 of the invention.
  • The segment of the HCV cDNA encoding the desired E1 and/or E2 sequence inserted into the vector sequence may be attached to a signal sequence. Said signal sequence may be that from a non-HCV source, e.g. the IgG or tissue plasminogen activator (tpa) leader sequence for expression in mammalian cells, or the a-mating factor sequence for expression into yeast cells, but particularly preferred constructs according to the present invention contain signal sequences appearing in the HCV genome before the respective start points of the E1 and E2 proteins. The segment of the HCV cDNA encoding the desired E1 and/or E2 sequence inserted into the vector may also include deletions e.g. of the hydrophobic domain(s) as illustrated in the examples section, or of the E2 hypervariable region 1.
  • More particularly, the recombinant vectors according to the present invention encompass a nucleic acid having an HCV cDNA segment encoding the polyprotein starting in the region between amino acid positions 1 and 192 and ending in the region between positions 250 and 400 of the HCV polyprotein, more preferably ending in the region between positions 250 and 341, even more preferably ending in the region between positions 290 and 341 for expression of the HCV single E1 protein. Most preferably, the present recombinant vector encompasses a recombinant nucleic acid having a HCV cDNA segment encoding part of the HCV polyprotein starting in the region between positions 117 and 192, and ending at any position in the region between positions 263 and 326, for expression of HCV single E1 protein. Also within the scope of the present invention are forms that have the first hydrophobic domain deleted (positions 264 to 293 plus or minus 8 amino acids), or forms to which a 5′-terminal ATC codon and a 3′-terminal stop codon has been added, or forms which have a factor Xa cleavage site and/or 3 to 10, preferably 6 Histidine codons have been added.
  • More particularly, the recombinant vectors according to the present invention encompass a nucleic acid having an HCV cDNA segment encoding the polyprotein starting in the region between amino acid positions 290 and 406 and ending in the region between positions 600 and 320 of the HCV polyprotein, more preferably starting in the region between positions 322 and 406, even more preferably starting in the region between positions 347 and 406 even still more preferably starting in the region between positions 364 and 406 for expression of the HCV single E2 protein. Most preferably, the present recombinant vector encompasses a recombinant nucleic acid having a HCV cDNA segment encoding the polyprotein starting in the region between positions 290 and 406, and ending at any position of positions 623, 650, 661, 673, 710, 715, 720, 746 or 809, for expression of HCV single E2 protein. Also within the scope of the present invention are forms to which a 5′-terminal ATG codon and a 3′-terminal stop codon has been added, or forms which have a factor Xa cleavage site and/or 3 to 10, preferably 6 Histidine codons have been added.
  • A variety of vectors may be used to obtain recombinant expression of HCV single or specific oligomeric envelope proteins of the present invention. Lower eukaryotes such as yeasts and glycosylation mutant strains are typically transformed with plasmids, or are transformed with a recombinant virus. The vectors may replicate within the host independently, or may integrate into the host cell genome.
  • Higher eukaryotes may be transformed with vectors, or may be infected with a recombinant virus, for example a recombinant vaccinia virus. Techniques and vectors for the insertion of foreign DNA into vaccinia virus are well known in the art, and utilize, for example homologous recombination. A wide variety of viral promoter sequences, possibly terminator sequences and poly(A)-addition sequences, possibly enhancer sequences and possibly amplification sequences, all required for the mammalian expression, are available in the art. Vaccinia is particularly preferred since vaccinia halts the expression of host cell proteins. Vaccinia is also very much preferred since it allows the expression of E1 and E2 proteins of HCV in cells or individuals which are immunized with the live recombinant vaccinia virus. For vaccination of humans the avipox and Ankara Modified Virus (AMV) are particularly useful vectors.
  • Also known are insect expression transfer vectors derived from baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), which is a helper-independent viral expression vector. Expression vectors derived from this system usually use the strong viral polyhedrin gene promoter to drive the expression of heterologous genes. Different vectors as well as methods for the introduction of heterologous DNA into the desired site of baculovirus are available to the man skilled in the art for baculovirus expression. Also different signals for posttranslational modification recognized by insect cells are known in the art.
  • Also included within the scope of the present invention is a method for producing purified recombinant single or specific oligomeric HCV E1 or E2 or E1/E2 proteins, wherein the cysteine residues involved in aggregates formation are replaced at the level of the nucleic acid sequence by other residues such that aggregate formation is prevented. The recombinant proteins expressed by recombinant vectors carrying such a mutated E1 and/or E2 protein encoding nucleic acid are also within the scope of the present invention.
  • The present invention also relates to recombinant E1 and/or E2 and/or E1/E2 proteins characterized in that at least one of their glycosylation sites has been removed and are consequently termed glycosylation mutants. As explained in the Examples section, different glycosylation mutants may be desired to diagnose (screening, confirmation, prognosis, etc.) and prevent HCV disease according to the patient in question. An E2 protein glycosylation mutant lacking the GLY4 has for instance been found to improve the reactivity of certain sera in diagnosis. These glycosylation mutants are preferably purified according to the method disclosed in the present invention. Also contemplated within the present invention are recombinant vectors carrying the nucleic acid insert encoding such a E1 and/or E2 and/or E1/E2 glycosylation mutant as well as host cells transformed with such a recombinant vector.
  • The present invention also relates to recombinant vectors including a polynucleotide which also forms part of the present invention. The present invention relates more particularly to the recombinant nucleic acids as represented in SEQ ID NO 3, 5, 7, 9, 11, 13, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 and 49, or parts thereof.
  • The present invention also contemplates host cells transformed with a recombinant vector as defined above, wherein said vector comprises a nucleotide sequence encoding HCV E1 and/or E2 and/or E1/E2 protein as defined above in addition to a regulatory sequence operably linked to said HCV E1 and/or E2 and/or E1/E2 sequence and capable of regulating the expression of said HCV E1 and/or E2 and/or E1/E2 protein.
  • Eukaryotic hosts include lower and higher eukaryotic hosts as described in the definitions section. Lower eukaryotic hosts include yeast cells well known in the art. Higher eukaryotic hosts mainly include mammalian cell lines known in the art and include many immortalized cell lines available from the ATCC, including HeLa cells, Chinese hamster ovary (COC) cells. Baby hamster kidney (BHK) cells. PK15, RK13 and a number of other cell lines.
  • The present invention relates particularly to a recombinant E1 and/or E2 and/or E1/E2 protein expressed by a host cell as defined above containing a recombinant vector as defined above. These recombinant proteins are particularly purified according to the method of the present invention.
  • A preferred method for isolating or purifying HCV envelope proteins as defined above is further characterized as comprising at least the following steps:
      • growing a host cell as defined above transformed with a recombinant vector according to the present invention or with a known recombinant vector expressing E1 and/or E2 and/or E1/E2 HCV envelope proteins in a suitable culture medium,
      • causing expression of said vector sequence as defined above under suitable conditions, and,
      • lysing said transformed host cells, preferably in the presence of a SH group blocking agent, such as N-ethylmaleimide (NEM), and possibly a suitable detergent, preferably Empigen-BE,
      • recovering said HCV envelope protein by affinity purification such as by means of lectin-chromatography or immunoaffinity chromatography using anti-E1 and/or anti-E2 specific monoclonal antibodies, with said lectin being preferably lentil-lectin or GNA, followed by,
      • incubation of the eluate of the previous step with a disulphide bond cleavage means, such as DTT, preferably followed by incubation with an SH group blocking agent, such as NEM or Biotin-NECM, and,
      • isolating the HCV single or specific oligomeric E1 and/or E2 and/or E1/E2 proteins such as by means of gelfiltration and possibly also by a subsequent Ni2−-IMAC chromatography followed by a desalting step.
  • As a result of the above-mentioned process, E1 and/or 2 and, or E1/E2 proteins may be produced in a form which elute differently from the large aggregates containing vector-derived components and, or cell components in the avoid volume of the gelfiltration column or the IMAC column as illustrated in the Examples section. The disulphide bridge cleavage step advantageously also eliminates the false reactivity due to the presence of host and/or expression-system-derived proteins. The presence of NEM and a suitable detergent during lysis of the cells may already partly or even completely prevent the aggregation between the HCV envelope proteins and contaminants.
  • Ni2−-IMAC chromatography followed by a desalting step is preferably used for constructs bearing a (His)5 as described by Janknecht et al. 1991, and Hochuli et al., 1988.
  • The present invention also relates to a method for producing monoclonal antibodies in small animals such as mice or rats, as well as a method for screening and isolating human B-cells that recognize anti-HCV antibodies, using the HCV single or specific oligomeric envelope proteins of the present invention.
  • The present invention further relates to a composition comprising at least one of the following E1 peptides as listed in Table 3:
      • E1-31 (SEQ ID NO 56) spanning amino acids 181 to 200 of the Core/E1 V1 region,
      • E1-33 (SEQ ID NO 57) spanning amino acids 193 to 212 of the E1 region.
      • E1-35 (SEQ ID NO 58) spanning amino acids 205 to 224 of the E1 V2 region (epitope B),
      • E1-35A (SEQ ID NO 59) spanning amino acids 208 to 227 of the E1 V2 region (epitope B).
      • 1bE1 (SEQ ID NO 53) spanning amino acids 192 to 228 of E1 regions (V1, C1, and V2 regions (containing epitope B)),
      • E1-51 (SEC ID NO 66) spanning amino acids 301 to 320 of the E1 region,
      • E1-53 (SEQ ID NO 67) spanning amino acids 313 to 332 of the E1 C4 region (epitope A),
      • E1-55 (SEC ID NO 68) spanning amino acids 325 to 344 of the E1 region.
  • The present invention also relates to a composition comprising at least one of the following E2 peptides as listed in Table 3:
      • Env 67 or E2-67 (SEQ ID NO 72) spanning amino acid positions 397 to 416 of
      • the E2 region (epitope A, recognized by monoclonal antibody 2-10H10. see FIG. 19),
      • Env 69 or E2-69 (SEQ ID NO 73) spanning amino acid positions 409 to 423 of the E2 region (epitope A),
      • Env 23 or 2-23 (SEQ ID NO 86) spanning positions 583 to 602 of the E2 region (epitope E).
      • Env 25 or E2-25 (SEQ ID NO 87) spanning positions 595 to 614 of the 2 region (epitope E).
      • Env 27 or E2-27 (SEQ ID NO 88) spanning positions 607 to 626 of the E2 region (epitope E),
      • Env 17B or E2-17B (SEQ ID NO 83) spanning positions 5417 to 566 of the E2 region (epitope D),
      • Env 13B or E2-13B (SEQ ID NO 82) spanning positions 523 to 542 of the E2 region (epitope C; recognized by monoclonal antibody 16A6E7, see FIG. 19).
  • The present invention also relates to a composition comprising at least one of the following E2 conformational epitopes:
      • epitope F recognized by monoclonal antibodies 15C8C1, 12D11F1 and 8G10D1H9,
      • epitope G recognized by monoclonal antibody 9G3E6.
      • epitope H (or C) recognized by monoclonal antibody 10D3C4 and 4H6B2, or,
      • epitope I recognized by monoclonal antibody 17F2C2.
  • The present invention also relates to an E1 or E2 specific antibody raised upon immunization with a peptide or protein composition, with said antibody being specifically reactive with any of the polypeptides or peptides as defined above, and with said antibody being preferably a monoclonal antibody.
  • The present invention also relates to an E1 or E2 specific antibody screened from a variable chain library in plasmids or phages or from a population of human B-cells by means of a process known in the art, with said antibody being reactive with any of the polypeptides or peptides as defined above, and with said antibody being preferably a monoclonal antibody.
  • The E1 or E2 specific monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly from a mouse or rat, immunizes against the HCV polypeptides or peptides according to the invention, as defined above on the one hand, and of cells of a myeloma cell line on the other hard, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the polypeptides which has been initially used for the immunization of the animals.
  • The antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
  • The monoclonal antibodies according to this preferred embodiment of the invention may be humanized versions of mouse monoclonal antibodies made by means of recombinant DNA technology, departing from parts of mouse and/or human genomic DNA sequences coding for H and L chains from cDNA or genomic clones coding for H and L chains.
  • Alternatively the monoclonal antibodies according to this preferred embodiment of the invention may be human monoclonal antibodies. These antibodies according to the present embodiment of the invention can also be derived from human peripheral blood lymphocytes of patients infected with HCV, or vaccinated against HCV. Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice (for recent review, see Duchosal et al., 1992).
  • The invention also relates to the use of the proteins or peptides of the invention, for the selection of recombinant antibodies by the process of repertoire cloning (Persson et al., 1991).
  • Antibodies directed to peptides or single or specific oligomeric envelope proteins derived from a certain genotype may be used as a medicament, more particularly for incorporation into an immunoassay for the detection of HCV genotypes (for detecting the presence of HCV E1 or E2 antigen), for prognosing/monitoring of HCV disease, or as therapeutic agents.
  • Alternatively, the present invention also relates to the use of any of the above-specified E1 or E2 specific monoclonal antibodies for the preparation of an immunoassay kit for detecting the presence of E1 or E2 antigen in a biological sample, for the preparation of a kit for prognosing/monitoring of HCV disease or for the preparation of a HCV medicament.
  • The present invention also relates to the a method for in vitro diagnosis or detection of HCV antigen present in a biological sample, comprising at least the following steps
      • (i) contacting said biological sample with any of the E1 and/or E2 specific monoclonal antibodies as defined above, preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex,
      • (ii) removing unbound components.
      • (iii) incubating the immune complexes formed with heterologous antibodies, which specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions,
      • (iv) detecting the presence of said immune complexes visually or mechanically (e.g. by means of densitometry, fluorimetry, colorimetry).
  • The present invention also relates to a kit for in vitro diagnosis of HCV antigen present in a biological sample, comprising:
      • at least one monoclonal antibody as defined above, with said antibody being preferentially immobilized on a solid substrate.
      • a buffer or components necessary for producing the buffer enabling binding reaction between these antibodies and the HCV antigens present in the biological sample,
      • a means for detecting the immune complexes formed in the preceding binding reaction,
      • possibly also including an automated scanning and interpretation device for inferring the HCV antigens present in the sample from the observed binding pattern.
  • The present invention also relates to a composition comprising E1 and/or E2 and/or E1, E2 recombinant HCV proteins purified according to the method of the present invention or a composition comprising at least one peptides as specified above for use as a medicament.
  • The present invention more particularly relates to a composition comprising at least one of the above-specified envelope peptides or a recombinant envelope protein composition as defined above, for use as a vaccine for immunizing a mammal, preferably humans, against HCV, comprising administering a sufficient amount of the composition possibly accompanied by pharmaceutically acceptable adjuvant(s), to produce an immune response.
  • More particularly, the present invention relates to the use of any of the compositions as described here above for the preparation of a vaccine as described above.
  • Also, the present invention relates to a vaccine composition for immunizing a mammal, preferably humans, against HCV, comprising HCV single or specific oligomeric proteins or peptides derived from the E1 and/or the E2 region as described above.
  • Immunogenic compositions can be prepared according to methods known in the art. The present compositions comprise an immunogenic amount of a recombinant E1 and/or E2 and/or E1/E2 single or specific oligomeric proteins as defined above or E1 or E2 peptides as defined above, usually combined with a pharmaceutically acceptable carrier, preferably further comprising an adjuvant.
  • The single or specific oligomeric envelope proteins of the present invention, either E1 and/or E2 and/or E1/E2, are expected to provide a particularly useful vaccine antigen, since the formation of antibodies to either E1 or E2 may be more desirable than to the other envelope protein, and since the E2 protein is cross-reactive between HCV types and the E1 protein is type-specific. Cocktails including type 1 E2 protein and E1 proteins derived from several genotypes may be particularly advantageous. Cocktails containing a molar excess of E1 versus E2 or E2 versus E1 may also be particularly useful. Immunogenic compositions may be administered to animals to induce production of antibodies, either to provide a source of antibodies or to induce protective immunity in the animal.
  • Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. 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 of ordinary skill in the art.
  • Preferred adjuvants to enhance effectiveness of the composition include, but are nor limited to: aluminim hydroxide (alum), N-acetyl-muramyl-L-threonyl-O-isoglutamine (thr-MOP) as found in U.S. Pat. No. 4,606,918, N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-aceylmuramyl-L-alanyl-O-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE) and R1E1, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL÷TDM÷CWS) in a 2% squalene. Tween 80 emulsion. Any of the 3 components MPL, TDM or CWS may also be used alone or combined 2 by 2. Additionally, adjuvants such as Stimulon (Cambridge Bioscience, Worcester, Mass.) or SAF-1 (Syntex) may be used. Further, Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) may be user for non-human applications and research purposes.
  • The immunogenic compositions typically will contain pharmaceutically acceptable vehicles, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, preservatives, and the like, may be included in such vehicles.
  • Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect. The E1 and E2 proteins may also be incorporated into Immune Stimulating Complexes together with saponins, for example Quil A (ISCOMS).
  • Immunogenic compositions used as vaccines comprise a ‘sufficient amount’ or ‘an immunologically effective amount’ of the envelope proteins of the present invention, as well as any other of the above mentioned components, as needed. ‘Immunologically effective amount’, means that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment, as defined above. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (e.g. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, the strain of infecting HCV, and other relevant factors. It is expected that the amount will tall in a relatively broad range that can be determined through routine trials. Usually, the amount will vary from 0.01 to 1000 μg/dose, more particularly from 0.1 to 100 μg/dose.
  • The single or specific oligomeric envelope proteins may also serve as vaccine carriers to present homologous (e.g. T cell epitopes or B cell epitopes from the core, NS2, NS3, NS4 or NS5 regions) or heterologous (non-HCV) haptens, in the same manner as Hepatitis 3 surface antigen (see European Patent Application 174, 444). In this use, envelope proteins provide an immunogenic carrier capable of stimulating an immune response to haptens or antigens conjugated to the aggregate. The antigen may be conjugated either by conventional chemical methods, or may be cloned into the gene encoding E1 and/or E2 at a location corresponding to a hydrophilic region of the protein. Such hydrophylic regions include the V1 region (encompassing amino acid positions 191 to 202), the V2 region (encompassing amino acid positions 213 to 223), the V3 region (encompassing amino acid positions 230 to 242), the V4 region (encompassing amino acid positions 230 to 242), the V1 region (encompassing amino acid positions 294 to 303) and the V6 region (encompassing amino acid positions 329 to 336). Another useful location for insertion of haptens is the hydrophobic region (encompassing approximately amino acid positions 264 to 293). It is shown in the present invention that this region can be deleted without affecting the reactivity of the deleted E1 protein with antisera. Therefore, haptens may be inserted at the site of the deletion.
  • The immunogenic compositions are conventionally administered parenterally, typically by injection, for example, subcutaneously or intramuscularly. Additional formulations suitable for other methods of administration include oral formulations and suppositories. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.
  • The present invention also relates to a composition comprising peptides or polypeptides as described above, for in vitro detection of HCV antibodies present in a biological sample.
  • The present invention also relates to the use of a composition as described above for the preparation of an immunoassay kit for detecting HCV antibodies present in a biological sample.
  • The present invention also relates to a method for in vitro diagnosis of HCV antibodies present in a biological sample, comprising at least the following steps
      • (i) contacting said biological sample with a composition comprising any of the envelope peptide or proteins as defined above, preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex, wherein said peptide or protein can be a biotinylated peptide or protein which is covalently bound to a solid substrate by means of streptavidin or avidin complexes.
      • (ii) removing unbound components,
      • (iii) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions,
      • (iv) detecting the presence of said immune complexes visually or mechanically (e.g. by means of densitometry, fluorimetry, colorimetry).
  • Alternatively, the present invention also relates to competition immunoassay formats in which recombinantly produced purified single or specific oligomeric protein E1 and/or E2 and/or E1/E2 proteins as disclosed above are used in combination with E1 and/or E2 peptides in order to compete for HCV antibodies present in a biological sample.
  • The present invention also relates to a kit for determining the presence of HCV antibodies, in a biological sample, comprising
      • at least one peptide or protein composition as defined above, possibly in combination with other polypeptides or peptides from HCV or other types of HCV, with said peptides or proteins being preferentially immobilized on a solid substrate, more preferably on different microwells of the same ELISA plate, and even more preferentially on one and the same membrane strip,
      • a buffer or components necessary for producing the buffer enabling binding reaction between these polypeptides or peptides and the antibodies against HCV present in the biological sample,
      • means for detecting the immune complexes formed in the preceding binding reaction,
      • possibly also including an automated scanning and interpretation device for inferring the HCV genotypes present in the sample from the observed binding pattern.
  • The immunoassay methods according to the present invention utilize single or specific oligomeric antigens from the E1 and/or E2 domains that maintain linear (in case of peptides) and conformational epitopes (single or specific oligomeric proteins) recognized by antibodies in the sera from individuals infected with HCV. It is within the scope of the invention to use for instance single or specific oligomeric antigens, dimeric antigens, as well as combinations of single or specific oligomeric antigens. The HCV E1 and E2 antigens of the present invention may be employed in virtually any assay format that employs a known antigen to detect antibodies. Of course, a format that denatures the HCV conformational epitope should be avoided or adapted. A common feature of all of these assays is that 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 strength using an excess of antigen. The incubation of the antigen with the specimen is followed by detection of immune complexes comprised of the antigen.
  • Design of the immunoassays is subject to a great deal of variation, and many formats are known in the art. 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, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin or streptavidin, and enzyme-labeled and mediated immunoassays, such as LISA assays.
  • The immunoassay may be, without limitation, in a heterogeneous or in a homogeneous format, and of a standard or competitive type. In a heterogeneous format, the polypeptide is typically bound to a solid matrix or support to facilitate separation of the sample from the polypeptide after incubation. Examples of 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 Immunolon™), diazotized paper, nylon membranes, activated beads, and Protein A beads. For example, Dynatech Immunolon™1 or Immunlon™2 microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball) 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.
  • In a homogeneous format, the rest 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.
  • In a standard format, the amount of 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 anti-HCV antibodies will bind due to complex formation. In a competitive format, the amount of HCV antibodies in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labeled antibody (or other competing ligand) in the complex.
  • Complexes formed comprising anti-HCV antibody (or in the case of competitive assays, the amount of competing antibody) are detected by any of a number of Known techniques, depending on the format. For example, unlabeled HCV antibodies in the complex may be detected using a conjugate of anti-xenogeneic Ig complexed with a label (e.g. an enzyme label).
  • In an immunoprecipitation or agglutination assay format the reaction between the HCV antigens and the antibody forms a network that precipitates from the solution or suspension and forms a visible layer or film of precipitate. If no anti-HCV antibody is present in the test specimen, no visible precipitate is formed.
  • There currently exist three specific types of particle agglutination (PA) assays. These assays are used for the detection of antibodies to various antigens when coated to a support. One type of this assay is the hemagglutination assay using red blood cells (RBCs) that are sensitized by passively adsorbing antigen (or antibody) to the REC. The addition of specific antigen antibodies present in the body component, if any, causes the RSCs coated with the purified antigen to agglutinate.
  • To eliminate potential non-specific reactions in the hemagglutination assay, two artificial carriers may be used instead of RBC in the PA. The most common of these are latex particles. However, gelatin particles may also be used. The assays utilizing either of these carriers are based on passive agglutination of the particles coated with purified antigens.
  • The HCV single or specific oligomeric E1 and/or E2 and/or E1/E2 antigens 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.
  • Immunoassays that utilize the native HCV antigen are useful in screening blood for the preparation of a supply from which potentially infective HCV is lacking. The method for the preparation of the blood supply comprises the following steps. Reacting a body component, preferably blood or a blood component, from the individual donating blood with HCV E1 and/or E2 proteins of the present invention to allow an immunological reaction between HCV antibodies, if any, and the HCV antigen. Detecting whether anti-HCV antibody—HCV antigen complexes are formed as a result of the reacting. Blood contributed to the blood supply is from donors that do no: exhibit antibodies to the native HCV antigens, E1 or E2.
  • In cases of a positive reactivity to the HCV antigen, it is preferable to repeat the immunoassay to lessen the possibility of false positives. For example, in the large scale screening of blood for the production of blood products (e.g. blood transfusion, plasma, Factor VII, immunoglobulin, etc.) ‘screening’ tests are typically formatted to increase sensitivity (to insure no contaminated blood passes) at the expense of specificity: i.e. the false-positive rare is increased. Thus, it is typical to only defer for further resting those donors who are ‘repeatedly reactive’; i.e. positive in two or more runs of the immunoassay on the donated sample. However, for confirmation of HCV-positivity, the ‘confirmation’ tests are typically formatted to increase specificity (to insure that no false-positive samples are confirmed) at the expense of sensitivity. Therefore the purification method described in the present invention for E1 and E2 will be very advantageous for including single or specific oligomeric envelope proteins into HCV diagnostic assays.
  • 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 chemiluminescent compound. Examples of enzymes include alkaline phosphatase, horseradish peroxidase and beta-galactosidase. Examples of enhancer compounds include biotin, anti-biotin and avidin. Examples of enhancer compounds binding members include biotin, anti-biotin and avidin. In order to block the effects of rheumatoid factor-like substances, the test sample is subjected to conditions sufficient to block the effect of rheumatoid factor-like substances. These 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 further contemplates the use of E1 proteins, or parts thereof, more particularly HCV single or specific oligomeric E1 proteins as defined above, for in vitro monitoring HCV disease or prognosing the response to treatment (for instance with Interferon) of patients suffering from HCV infection comprising:
      • incubating a biological sample from a patient with hepatitis C infection with an E1 protein or a suitable part thereof under conditions allowing the formation of an immunological complex,
      • removing unbound components.
      • calculating the anti-E1 titers present in said sample (for example at the start of and/or during the course of (interferon) therapy),
      • monitoring the natural course of HCV disease, or prognosing the response to treatment of said patient on the basis of the amount anti-E1 titers found in said sample at the start of treatment and/or during the course of treatment.
  • Patients who show a decrease of 2, 3, 4, 5, 7, 10, 15, or preferably more than 20 times of the initial anti-E1 titers could be concluded to be long-term, sustained responders to HCV therapy, more particularly to interferon therapy. It is illustrated in the Examples section, that an anti-E1 assay may be very useful for prognosing long-term response to IFN treatment, or to treatment of Hepatitis C virus disease in general.
  • More particularly the following E1 peptides as listed in Table 3 were found to be useful for in vitro monitoring HCV disease or prognosing the response to interferon treatment of patients suffering from HCV infection:
      • E1-31 (SEQ ID NO 56) spanning amino acids 181 to 200 of the Core/E1 V1 region,
      • E1-33 (SEC ID NO 57) spanning amino acids 193 to 212 of the E1 region.
      • E1-35 (SEQ ID NO 58) spanning amino acids 205 to 224 of the E1 V2 region (epitope B).
      • E1-35A (SEQ ID NO 59) spanning amino acids 208 to 227 of the E1 V2 region (epitope B).
      • 1bE1 (SEQ ID NC 53) spanning amino acids 192 to 228 of E1 regions (V1, C1, and V2 regions (containing epitope B)),
      • E1-51 (SEQ ID NO 66) spanning amino acids 307 to 320 of the E1 region,
      • E1-53 (SEQ ID NO 67) spanning amino acids 313 to 332 of the E1 C4 region (epitope A),
      • E1-55 (SEQ ID NO 68) spanning amino acids 325 to 344 of the E1 region.
  • It is to be understood that smaller fragments of the above-mentioned peptides also fall within the scope of the present invention. Said smaller fragments can be easily prepared by chemical synthesis and can be rested for their ability to be used in an assay as detailed above and in the Examples section.
  • The present invention also relates to a kit for monitoring HCV disease or prognosing the response to treatment (for instance to interferon) of patients suffering from HCV infection comprising:
      • at least one E1 protein or E1 peptide, more particularly an E1 protein or E1 peptide as defined above,
      • a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins or peptides and the anti-E1 antibodies present in a biological sample,
      • means for detecting the immune complexes formed in the preceding binding reaction,
      • possibly also an automated scanning and interpretation device for inferring a decrease of anti-E1 titers during the progression of treatment.
  • It is to be understood that also E2 protein and peptides according to the present invention can be used to a certain degree to monitor/prognose HCV treatment as indicated above for the E1 proteins or peptides because also the anti-E2 levels decrease in comparison to antibodies to the other HCV antigens. It is to be understood, however, that it might be possible to determine certain epitopes in the E2 region which would also be suited for use in an test for monitoring/prognosing HCV disease.
  • The present invention also relates to a stereotyping assay for detecting one or more serological types of HCV present in a biological sample, more particularly for detecting antibodies of the different types of HCV to be detected combined in one assay format, comprising at least the following steps
      • (i) contacting the biological sample to be analyzed for the presence of HCV antibodies of one or more serological types, with at least one of the E1 and/or E2 and/of E1/E2 protein compositions or at least one of the E1 or E2 peptide compositions as defined above, preferentially in an immobilized form under appropriate conditions which allow the formation of an immune complex,
      • (ii) removing unbound components,
      • (iii) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies being conjugated to a detectable label under appropriate conditions,
      • (iv) detecting the presence of said immune complexes visually or mechanically (e.g. by means of densitometry, fluorimetry, colorimetry) and inferring the presence of one or more HCV serological types present from the observed binding pattern.
  • It is to be understood that the compositions of proteins or peptides used in this method are recombinantly expressed type-specific envelope proteins or type-specific peptides.
  • The present invention further relates to a kit for serotyping one or more serological types of HCV present in a biological sample, more particularly for detecting the antibodies to these serological types of HCV comprising:
      • at least one E1 and/or E2 and/or E1/E2 protein or E1 or E2 peptide, as defined above,
      • a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins or peptides and the anti-E1 antibodies present in a biological sample,
      • means for detecting the immune complexes formed in the preceding binding reaction,
      • possibly also an automated scanning and interpretation device for detecting the presence of one or more serological types present from the observed binding pattern.
  • The present invention also relates to the use of a peptide or protein composition as defined above, for immobilization on a solid substrate and incorporation into a reversed phase hybridization assay, preferably for immobilization as parallel lines onto a solid support such as a membrane strip, for determining the presence or the genotype of HCV according to a method as defined above. Combination with other type-specific antigens from other HCV polyprotein regions also lies within the scope of the present invention.
    • FIGURE AND TABLE LEGENDS
  • FIG. 1: Restriction map of plasmid pgpt ATA 18
  • FIG. 2: Restriction map of plasmid pgs ATA 18
  • FIG. 3: Restriction map of plasmid pMS 66
  • FIG. 4: Restriction map of plasmid pv HCV-11A
  • FIG. 5: Anti-1 levels in non-responders to IFN treatment
  • FIG. 6: Anti-E1 levels in responders to IFN treatment
  • FIG. 7: Anti-E1 levels in patients with complete response to IFN treatment
  • FIG. 8: Anti-E1 levels in incomplete responders to IFN treatment
  • FIG. 9: Anti-E2 levels in non-responders to IFN treatment
  • FIG. 10 Anti-E2 levels in responders to IFN treatment
  • FIG. 11: Anti-E2 levels in incomplete responders to IFN treatment
  • FIG. 12: Anti-E2 levels in complete responders to IFN treatment
  • FIG. 13: Human anti-E1 reactivity competed with peptides
  • FIG. 14: Competition of reactivity of anti-E1 monoclonal antibodies with peptides
  • FIG. 15: Anti-E1 (epitope 1) levels in non-responders to IFN treatment
  • FIG. 16: Anti-E1 (epitope 1) levels in responders to IFN treatment
  • FIG. 17: Anti-E1 (epitope 2) levels in non-responders to IFN treatment
  • FIG. 18: Anti-E1 (epitope 2) levels in responders to IFN treatment
  • FIG. 19: Competition of reactivity of anti-E2 monoclonal antibodies with peptides
  • FIG. 20: Human anti-E2 reactivity competed with peptides
  • FIG. 21: FIGS. 21A-L provide nucleic acid sequences of the present invention. The nucleic acid sequences encoding an E1 or E2 protein according to the present invention may be translated (SEQ ID NO 3 to 13, 21-31, 35 and 41-49 are translated in a reading frame starting from residue number 1, SEQ ID NO:37-39 are translated in a reading frame starting from residue number 2), into the amino acid sequences of the respective E1 or E2 proteins as shown in the sequence listing.
  • FIG. 22: ELISA results obtained from lentil lectin chromatography eluate fractions of 4 different E1 purifications of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b), vvHCV62 type 3a), and vvHCV63 (type 5a).
  • FIG. 23: Elution profiles obtained from the lentil lectin chromatography of the 4 different E1 constructs on the basis of the values as shown in FIG. 22.
  • FIG. 24: ELISA results obtained from fractions obtained after gelfiltration chromatography of 4 different E1 purifications of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b), vvHCCV62 (type 3a), and vvHCV63 (type 5a).
  • FIG. 25: Profiles obtained from purifications of E1 proteins of type 1b (1), type 3a (2), and type 5a (3) (from RK13 cells infected with vvHCV39, vvHCV62, and vvHCV63, respectively; purified on lentil lectin and reduced as in example 5.2-5.3) and a standard (4). The peaks indicated with ‘1’, ‘2’, and ‘3’, represent pure E1 protein peaks (see FIG. 24, E1 reactivity mainly in fractions 26 to 30).
  • FIG. 26: Silver staining of an SDS-PACE as described in example 4 of a raw lysate of E1 vvHCV40 (type 1b) (lane 1), pool 1 of the gelfiltration of vvHCV40 representing fractions 10 to 17 as shown in FIG. 25 (lane 2), pool 2 of the gelfiltration of vvHCV40 representing fractions 13 to 25 as shown in FIG. 25 (lane 3), and E1 pool (fractions 25 to 30) (lane 4).
  • FIG. 27: Streptavidine-alkaline phosphatase blot of the fractions of the gelfiltration of E1 constructs 39 (type 1b) and 62 (type 3a). The proteins were labelled with NEM-biotin. Lane 1: start gelfiltration construct 39, lane 2: fraction 26 construct 39, lane 3: fraction 27 construct 39, lane 4: fraction 28 construct 39, lane 5: fraction 29 construct 39, lane 6: fraction 30 construct 39, lane 7 fraction 31 construct 39, lane 8: molecular weight marker, lane 9: start gelfiltration construct 62, lane 10: fraction 26 construct 62, lane 11: fraction 27 construct 62, lane 12: fraction 28 construct 62, lane 13: fraction 29 construct 62, lane 14: fraction 30 construct 62, lane 15: fraction 31 construct 62.
  • FIG. 28: Siver staining of an SOS-PAGE gel of the gelfiltration fractions of vvHCV-39 (E1s, type 1b) and vvHCV-62 (E1s, type 3a) run under identical conditions as FIG. 26. Lane 1: start gelfiltration construct 39, lane 2: fraction 26 construct 39, lane 3: fraction 27 construct 39, lane 4: fraction 28 construct 39, lane 5: fraction 29 construct 39, lane 6: fraction 30 construct 39, lane 7 fraction 31 construct 39, lane 8: molecular weight marker, lane 9: start gelfiltration construct 62, lane 10: fraction 26 construct 62, lane 11: fraction 27 construct 62, lane 12: fraction 28 construct 62, lane 13: fraction 29 construct 62, lane 14: fraction 30 construct 62, lane 15: fraction 31 construct 62.
  • FIG. 29: Western Blot analysis with anti-cl mouse monoclonal antibody 5E1A10 giving a complete overview of the purification procedure. Lane 1: crude lysate, Lane 2: flow through of lentil chromagtography, Lane 3: wash with Empigen BB after lentil chromatography. Lane 4: Eluate of lentil chromatography, Lane 5: Flow through during concentration of the lentil eluate, Lane 6: Pool of E1 after Size Exclusion Chromatography (gelfiltration).
  • FIG. 30: OD280 profile (continuous line) of the lentil lectin chromatography of E2 protein from RK13 cells infected with vvHCV44. The dotted line represents the 0.7 reactivity as detected by ELISA (as in example 6).
  • FIG. 31A: OD290 profile (continuous line) of the lentil-lectin gelfiltration chromatography E2 protein pool from RK13 cells infected with vvHCV44 in which the 2 pool is applied immediately on the gelfiltration column (non-reduced conditions). The dotted line represents the E2 reactivity as detected by ELISA (as in example 6).
  • FIG. 31B: OD280 profile (continuous line) of the lentil-lectin gelfiltration chromatography E2 protein pool from RK13 cells infected with vvpCV44 in which the 2 pool was reduced and blocked according to Example 5.3 (reduced conditions). The dotted line represents the 2 reactivity as detected by ELISA (as in example 6).
  • FIG. 32: Ni2−-IMAC chromatography and ELISA reactivity of the E2 protein as expressed from vvHCV44 after gelfiltration under reducing conditions as shown in FIG. 31B.
  • FIG. 33: Silver staining of an SOS-PAGE of 0.5 μg of purified E2 protein recovered by a 200 mM imidazole elution step (lane 2) and a 30 mM imidazole wash (lane 1) of the Ni2−-IMAC chromatography as shown in FIG. 32.
  • FIG. 34: OD profiles of a desalting step of the purified E2 protein recovered by 200 mM immidazole as shown in FIG. 33, intended to remove imidazole.
  • FIGS. 35A-1 to 35A-8: Antibody levels to the different HCV antigens (Core 1, Core 2, E2HCVR, NS3) for NR and LTR followed during treatment and over a period of 6 to 12 months after treatment determined by means of the LIAscan method. The average values are indicated by the curves with the open squares.
  • FIGS. 35B-1 to 35B-8: Antibody levels to the different HCV antigens (NS4, NS5, E1 and E2) for NR and LTR followed during treatment and over a period of 6 to 12 months after treatment determined by means of the LIAscan method. The average values are indicated by the curve with the open squares.
  • FIGS. 36A and 36B: Average E1 antibody (E1Ab) and E2 antibody (E2Ab) levels in the LTR and NR groups.
  • FIGS. 36A-D: Averages E1 antibody (E1Ab) levels for non-responders (NR) and long term responders (LTR) for type 1b and type 3a.
  • FIG. 37: Averages E1 antibody (E1Ab) levels for non-responders (NR) and long term responders (LTR) for type 1b and type 3a.
  • FIG. 38: Relative map positions of the anti-E2 monoclonal antibodies.
  • FIG. 39: Partial deglycosylation of HCV E1 envelope protein. The lysate of vvHCV10A-infected RK13 cells were incubated with different concentrations of glycosidases according to the manutacturer's instructions. Right panel: Glycopepidase F (PNC-ase F). Left panel: Endoclycosidase H (Endo H).
  • FIG. 40: Partial deglycosylation of HCV E2 envelope proteins. The lysate of vvHCV64-infected (E2) and vvHCV41-infected (E2s)RK13 cells were incubated with different concentrations of Glycopeptidase F (PNGase F) according to the manufacturer's instructions.
  • FIG. 41: in vitro mutagenesis of HCV E1 glycoproteins. Map of the mutated sequences and the creation of new restriction sites.
  • FIG. 42A: In vitro mutagenesis of HCV E1 glycoprotein (part 1). First step of PCR amplification.
  • FIG. 42B: In vitro mutagensis of HCV E1 glycoprotein (part 2). Overlap extension and nested PCR.
  • FIG. 43: In vitro mutagenesis of HCV E1 glycoproteins. Map of the PCR mutated fragments (GLY-# and OVR-#) synthesized during the first step of amplification.
  • FIG. 44A: Analysis of E1 glycoprotein mutants by Western blot expressed in HeLa (left) and RK13 (right) cells. Lane 1: wild type VV (vaccinia virus), Lane 2:original E1 protein (vvHCV-10A). Lane 3: E1 mutant Gly-1 (vvHCV-81). Lane 4: E1 mutant Gly-2 (vvHCV-82), Lane 5: E1 mutant Gly-3 (vvHCV-83), Lane 6: E1 mutant Gly-4 (vvHCV-g8). Lane 7: E1 mutant Gly-5 (vvHCV-85). Lane 8: E1 mutant Gly-6 (vvHCV-86).
  • FIG. 44B: Analysis of E1 glycosylation mutant vaccinia viruses by PCR amplification/restriction. Lane 1: E1 (vvHCV-10A), BspE I. Lane 2: E1.GLY-1 (vvHCV-81). BspE1. Lane 4: E1 (vvHCV-10A), Sac Lane 5: E1.GLY-2 (vvHCV-82), Sac I, Lane 7: E1 (vvHCV-10A), Sac I, Lane 8: E1.GLY-3 (vvHCV-82), Sac Lane 10: E1 (vvHCV-10A), Stu I, Lane 11: E1.GLY-4 (vvHCV-84). Stu I, Lane 13: E1 (vvHCV-10A), Sma I, Lane 14: E1.GLY-5 (vvHCV-8E). Sma I, Lane 16: E1 (vvHCV-10A), Stu I, Lane 17: E1.GLY-6 (vvHCV-86), Stu I, Lane 3-6-9-12-15: Low Molecular Weight Marker, pBluescript SK÷Msp 1
  • FIG. 45: SDS polyacrylamide gel electrophoresis of recombinant E2 expressed in S. cerevisiae. Innoculates were crown in leucine selective medium for 72 hrs. and diluted 1/15 in complete medium. After 10 days of culture at 28° C., medium samples were taken. The equivalent of 200 μl of culture supernatant concentrated by speedHvc was loaded on the gel. Two independent transformants were analysed.
  • FIG. 46: SDS polyacrylamide gel electrophoresis of recombinant E2 expressed in a glycosylation deficient S. cerevisiae mutant. Innoculate were grown in leucine selective medium for 72 hrs. and diluted 1/15 in complete medium. After 10 days of culture at 28° C. medium samples were taken. The equivalent of 350 μl of culture supernatant, concentrated by ion exchange chromatography, was loaded on the gel.
  • Table 1: Features of the respective clones and primers used for amplification for constructing the different forms of the E1 protein as despected in Example 1.
  • Table 2: Summary of Anti-E1 tests
  • Table 3: Synthetic peptides for competition studies
  • Table 4: Changes of envelope antibody levels over time.
  • Table 5: Difference between LTR and NR
  • Table 6: Competition experiments between murine E2 monoclonal antibodies
  • Table 7: Primers for construction of E1 glycosylation mutants
  • Table 8: Analysis of E1 glycosylation mutants by ELISA
    • EXAMPLE 1 Cloning and Expression of the Hepatitis C Virus E1 Protein
  • 1. Construction of Vaccinia Virus Recombination Vectors
  • The pgptATA18 vaccinia recombination plasmid is a modified version of pATA18 (Stunnenberg et al, 1988) with an additional insertion containing the E. coli xanthine guanine phosphoribosyl transferase gene under the control of the vaccinia virus 13 intermediate promoter (FIG. 1). The plasmid pgsATA18 was constructed by inserting an oligonucleotide linker with SEQ ID NO 1/94, containing stop codons in the three reading frames, into the Pst I and HindIII-cut pATA18 vector. This created an extra Pac I restriction site (FIG. 2). The original HindIII site was not restored.
    Oligonucleotide linker with SEQ ID NO 1/94:
    5′     G GCATGC AAGCTT AATTAATT      3′
    3′ ACGTC CGTACG TTCGAA TTAATTAA TCGA 5′
       PstI SphI HindIII Pac I (HindIII)
  • In order to facilitate rapid and efficient purification by means of Ni2− chelation of engineered histidine stretches fused to the recombinant proteins, the vaccinia recombination vector pMSc6 was designed to express secreted proteins with an additional carboxy-terminal histidine tag. An oligonucleotide linker with SEQ ID NO 2/95, containing unique sites for 3 restriction enzymes generating blunt ends (Sma I, Stu I and Pml I/Bbr PI) was synthesized in such a way that the carboxy-terminal end of any cDNA could be inserted in frame with a sequence encoding the protease factor Xa cleavage site followed by a nucleotide sequence encoding 6 histidines and 2 stop codons (a new Pac I restriction site was also created downstream the 3′ end). This oligonucleotide with SEC ID NO 2/95 was introduced between the Xma I and Pst I sites of pgptATA18 (FIG. 3).
    Oligonucleotide linker with SEQ ID NO 2/95:
    ′5′ CCGGG GAGGCCTGCACGTGATCGAGGGCAGACACCATCACCACCATCACTAATAGTTAATTAA CTGCA3
    3′     C CTCCGGACGTGCACTAGCTCCCGTCTGTGGTAGTGGTGGTAGTGATTATCAATTAATT G
       XmaI                                                              PstI
    • EXAMPLE 2 Construction of HCV Recombinant Plasmids
  • 2.1. Constructs Encoding Different Forms of the E1 Protein
  • Polymerase Chain Reaction (PCB) products were derived from the serum samples by RNA preparation and subsequent reverse-transcription and PCR as described previously (Stuyver et al., 1993b). Table 1 shows the features of the respective clones and the primers used for amplification. The PCR fragments were cloned into the Sma I-cut pSP72 (Promega) plasmids. The following clones were selected for insertion into vaccinia recombination vectors: HCCl9A (SEQ ID NO 31. HCCl10A (SEQ ID NO 5), HCCl11A (SEQ ID NO 7). HCCl12A (SEQ ID NO 9), HCCl13A (SEQ ID NO 11), and HCCl17A (SEQ ID NO 13) as depicted in FIG. 21. cDNA fragments containing the E1-coding regions were cleaved by EcoI and HindIII restriction from the respective pS-q72 plasmids and inserted into the EcoRI/HindIII-cut pgptATA-18 vaccinia recombination vector (described in example 1), downstream of the 11 K vaccinia virus late promoter. The respective plasmids were designated pvHCV-9A, pvHCV-10A, pvHCV-11A, pVHCV-12A, pvHCV-13A and pvHCV-17A, of which PvHCV-11A is shown in FIG. A,
  • 2.2. Hydrophobic Region E1 Deletion Mutants
  • Clone HCCl-37, containing a deletion of codons Asp264 to Val2S7 (nucleotides 790 to 861, region encoding hydrophobic domain 1) was generated as follows: 2 PCR fragments were generated from clone HCCl10A with primer sets HCPr52 (SEQ ID NO 16)/HCPr107 (SEQ ID NO 19) and HCPr108 (SEQ ID NO 20)/HCPR54 (SEQ ID NO 12). These primers are shown in FIG. 21. The two PCR fragments were purified from agarose gel after electrophoresis and 1 ng of each fragment was used together as template for PCR by means of primers HCPr52 (SEQ ID NO 16) and HCPr54 (SEC ID NO 18). The resulting fragment was cloned into the Sma I-cut pSP72 vector and clones containing the deletion were readily identified because of the deletion of 24 codons (72 base pairs). Plasmid pSP72HCCl37 containing clone HCCl37 (SEQ ID 15) was selected. A recombinant vaccinia plasmid containing the full-length E1 cDNA lacking hydrophobic domain I was constructed by inserting the HCV sequence surrounding the deletion (fragment cleaved by Xma I and SamH I from the vector pSP72—HCCl37) into the Xma I-Bam H I sites of the vaccinia plasmid PVHCV-10A. The resulting plasmid was named pvHCV-37. After confirmatory sequencing, the amino-terminal region containing the internal deletion was isolated from this vector pvHCV-37 (cleavage by EcoR I and BstE II) and reinserted into the Eco R1 and Bst Ell-cut pvHCV-11A plasmid. This construct was expected to express an E1 protein with both hydrophobic domains deleted and was named pvHCV-38. The E1-coding region of clone HCCl38 is represented by SEQ ID NO 23.
  • As the hydrophilic region at the E1 carboxyterminus (theoretically extending to around amino acids 337-340) was not completely included in construct pvHCV-38, a larger E1 region lacking hydrophobic domain I was isolated from the pvHCV-37 plasmid by EcoR I/Bam HI cleavage and cloned into an EcoRI/BamHI-cut pgsATA-18 vector. The resulting plasmid was named pvHCV-39 and contained clone HCCl39 (SEQ ID NO 21). The same fragment was cleaved from the pvHCV-37 vector by BamH I (of which the sticky ends were filled with Klenow DNA Polymerase I (Boehringer)) and subsequently by Ecor I (5′ cohesive end). This sequence was inserted into the EcoRI and Bbr PI-cut vector pMS-66. This resulted in clone HCCl40 (SEQ ID NO 27) in plasmid pvHCV-40, containing a 6 histidine tail at its carboxy-terminal end.
  • 2.3. E1 of Other Genotypes
  • Clone HCCl62 (SEQ ID NO 29) was derived from a type 3a-infected patient with chronic hepatitis C (serum BR6, clone BR36-9-13, SEQ ID NO 19 in WO 94/25601, and see also Stuyver et al. 1993a) and HCCl63 (SEQ ID NO 31) was derived from a type 5a-infected child with post-transfusion hepatitis (serum BE95, clone PC-4-1, SEQ ID NO 45 in WO 94125601).
  • 2.4. E2 Constructs
  • The HCV E2 PCR fragment 22 was obtained from serum BE11 (genotype 1b) by means of primers HCPr109 (SEQ ID NO 33) and HCPr72 (SEQ ID NO 34) using techniques of RNA preparation, reverse-transcription and PCR, as described in Stuyver et al., 1993b, and the fragment was cloned into the Sma I-cut pSP72 vector. Clone HCCl22A (SEQ ID NO 35) was cut with NcoI/AlwNI or by BamHI/AlwNI and the sticky ends of the fragments were blunted (NcoI and BamHI sites with Klenow DNA Polymerase I (Boehringer), and AlwNI with T4 DNA polymerase (Boehringer)). The BamHI/AlwNI cDNA fragment was then inserted into the vaccinia pgsATA-18 vector that had been linearized by EcoR I and Hind III cleavage and of which the cohesive ends had been filled with Klenow DNA Polymerase (Boehringer). The resulting plasmid was named pvHCV-41 and encoded the E2 region from amino acids Met347 to Gln673, including 37 amino acids (from Met347 to Gly383) of the E1 protein that can serve as signal sequence. The same HCV cDNA was inserted into the EcoR I and Sbr PI-cut vector pMS66, that had subsequently been blunt ended with Klenow DNA Polymerase. The resulting plasmid was named pvHCV-42 and also encoded amino acids 347 to 683. The NcoI/AlwNI fragment was inserted in a similar way into the same sites of pgsA7A-18 (pvHCV-43) or pMS-66 vaccinia vectors (pvHCV-14). pvHCV-43 and pvHCV-44 encoded amino acids 364 to 673 of the HCV polyprotein, of which amino acids 364 to 383 were derived from the natural carboxyterminal region of the E1 protein encoding the signal sequence for E2, and amino acids 384 to c73 of the mature E2 protein.
  • 2.5. Generation of Recombinant HCV-Vaccinia Viruses
  • Rabbit kidney RK13 cells (ATCC CCL 37), human osteosarcoma 143B thymidine kinase deficient (TK) (ATC CRL 8303), HeLa (ATCC CCL 2), and Hep G2 (ATCC HB 8065) cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, Md., USA). The cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% foetal calf serum, and with Earle's salts (EMEM) for RK13 and 143 B (TK-), and with glucose (4 g/l) for Hep G2. The vaccinia virus WR strain (Western Reserve, ATTC VR119) was routinely propagated in either 143B or RK13 cells, as described previously (Panicali & Paoletti. 1982; Piccini et al., 1987; Mackett et al., 1982, 1984, and 1986). A confluent monolayer of 1432 cells was infected with wild type vaccinia virus at a multiplicity of infection (m.o.i.) of 0.1 (=0.1 plaque forming unit (PFU) per cell). Two hours later, the vaccinia recombination plasmid was transfected into the infected cells in the form of a calcium phosphate coprecipitate containing 500 ng of the plasmid DNA to allow homologous recombination (Graham & van der Eb, 1973; Mackett et al., 1985). Recombinant viruses expressing the Escherichia coli xanthine-guanine phosphoribosyl transferase (gpt) protein were selected on rabbit kidney RK13 cells incubated in selection medium (EMEM containing 25 μg/ml mycophenolic acid (MPA), 250 μg/ml xanthine, and 15 μg/ml hypoxanthine: Falkner and Moss, 1988; Janknecht et al, 1991). Single recombinant viruses were purified on fresh monolayers of RK13 cells under a 0.9% agarose overlay in selection medium. Thymidine kinase deficient (TK-) recombinant viruses were selected and then plaque purified on fresh monolayers of human 1432 cells (TK-) in the presence of 25 μg/ml 5-bromo-2′-deoxyuridine. Stocks of purified recombinant HCV-vaccinia viruses were prepared by infecting either human 1433 or rabbit RK13 cells at an m.o.i. of 0.05 (Mackett et al, 1988). The insertion of the HCV cDNA fragment in the recombinant vaccinia viruses was confirmed on an aliquot (50 all) of the cell lysate after the MPA selection by means of PCR with the primers used to clone the respective HCV fragments (see Table 1). The recombinant vaccinia-HCV viruses were named according to the vaccinia recombination plasmid number, e.g. the recombinant vaccinia virus vvHCV-10A was derived from recombining the wild type WR strain with the pvHCV-10A plasmid.
    • EXAMPLE 3 Infection of Cells with Recombinant Vaccinia Viruses
  • A confluent monolayer of RK13 cells was infected at a m.o.i. of 3 with the recombinant HCV-vaccinia viruses as described in example 2. For infection, the cell monolayer was washed twice with phosphate-buffered saline pH 7.4 (P—S) and the recombinant vaccinia virus stock was diluted in MEM medium. Two hundred μl of the virus solution was added per 105 cells such that the m.o.i. was 3, and incubated for A5 min at 24° C. The virus solution was aspirated and 2 ml of complete growth medium (see example 2) was added per 105 cells. The cells were incubated for 24 hr at 37° C. during which expression of the HCV proteins took place.
    • EXAMPLE 4 Analysis of Recombinant Proteins by Means of Western Blotting
  • The infected cells were washed two times with PBS, directly lysed with lysis buffer (50 mM Tris.HCl pH 7.5, 150 mM NaCl. 1% Triton X-100, 5 mM MgCl2, 1 μg/ml aprotinin (Sigma, Bornem, Belgium)) or detached from the flasks by incubation in 50 mM Tris.HCL pH 7.5/10 mM EDTA/150 mM NaCl for 5 min, and collected by centrifugation (5 min at 1000 g). The cell pellet was then resuspended in 200 μl lysis buffer (50 mM Tris.HCL pH 8.0, 2 mM EDTA, 150 mM NaCl, 5 mM MgCl2 aprotinin; 1% Triton X-100) per 108 cells. The cell lysates were cleared for 5 min at 14,000 rpm in an Eppendorf centrifuge to remove the insoluble debris. Proteins of 20 μl lysate were separated by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then electro-transferred from the gel to a nitrocellulose sheet (Amersham) using a Hoefer HSI transfer unit cooled to 4° C. for 2 hr at 100 V constant voltage, in transfer buffer (25 mM Tris.HCl pH 8.0, 192 mM glycine, 20% (v/v) methanol). Nitrocellulose filters were blocked with Blotto (5% (w/v) fat-free instant milk powder in PBS; Johnson et al., 1981) and incubated with primary antibodies diluted in Blotto/0.1% Tween 20. Usually, a human negative control serum or serum of a patient infected with HCV were 200 times diluted and preincubated for 1 hour at room temperature with 200 times diluted wild type vaccinia virus-infected cell lysate in order to decrease the non-specific binding. After washing with Blotto/0.1% Tween 20, the nitrocellulose filters were incubated with alkaline phosphatase substrate solution diluted in Blotto/0.1% Tween 20. After washing with 0.1% Tween 20 in PBS, the filters were incubated with alkaline phosphates substrate solution (100 mM Tris.HCl pH 9.5, 100 mM NaCl, 5 mM MgCl2, 0.38 μg/ml nitroblue tetrazolium. 0.165 μg/ml 5-bromo-4-chloro-3-indolylphosphate). All steps, except the electrotransfer, were performed at room temperature.
    • EXAMPLE 5 Purification of Recombinant E1 or E2 Protein
  • 5.1. Lysis
  • Infected RK13 cells (carrying E1 or E2 constructs) were washed 2 times with phosphate-buffered saline (PBS) and detached from the culture recipients by incubation in PBS containing 10 mM EDTA. The detached cells were washed twice with PBS and 1 ml of lysis buffer (50 mM Tris.HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM MgCl2, 1 μg/ml aprotinin (Sigma, Bornem, Belgium) containing 2 mM biotinylated N-ethylmaleimide (biotin-NEM) (Sigma) was added per 105 cells at 4 C. This lysate was homogenized with a type B douncer and left at room temperature for 0.5 hours. Another 5 volumes of lysis buffer containing 10 mM N-ethylmaleimide (NEM. Aldrich, Bornem. Belgium) was added to the primary lysate and the mixture was left at room temperature for 15 min. Insoluble cell debris was cleared from the solution by centrifugation in a Beckman JA-14 rotor at 14,000 rpm (30100 g at rmax) for 1 hour at 4° C.
  • 5.2. Lectin Chromatography
  • The cleared cell lysate was loaded at a rate of 1 ml/min on a 0.8 by 10 cm Lentil-lectin Sepharose 48 column (Pharmacia) that had been equilibrated with 5 column volumes of lysis buffer at a rate of 1 ml/min. The lentil-lectin column was washed with 5 to 10 column volumes of buffer 1 (0.1M potassium phosphate pH 7.3, 500 mM KCl, 5% glycerol, 1 mM 6—NH2-hexanoic acid, 1 mM MgCl2, and 1% DecylPEG (KWANT, Bedum, The Netherlands). In some experiments, the column was subsequently washed with 10 column volumes of buffer 1 containing 0.5% Empigen-BB (Calbiochem, San Diego, Calif., USA) instead of 1% DecylPEGG. The bound material was eluted by applying elution buffer (10 mM potassium phosphate pH 7.3, 59 glycerol, 1 mM hexanoic acid; 1 mM MgCl2, 0.5% Empigen-BB, and 0.5 M α-methyl-mannopyranoside. The eluted material was fractionated and fractions were screened for the presence of E1 or 2 protein by means of ELISA as described in example 6. FIG. 22 shows ELISA results obtained from lentil lectin eluate fractions of 4 different E1 purifications of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b), vvHCV62 (type 3a), and vvHCV53 (type 5a). FIG. 23 shows the profiles obtained from the values shown in FIG. 22. These results show that the lectin affinity column can be employed for envelope proteins of the different types of HCV.
  • 5.3. Concentration and Partial Reduction
  • The E1- or E2-positive fractions were pooled and concentrated on a Centricon 30 kDa (Amicon) by centrifugation for 3 hours at 5,000 rpm in a Beckman JA-20 rotor at 4° C. In some experiments the E1- or E2-positive fractions were pooled and concentrated by nitrogen evaporation. An equivalent of 3.108 cells was concentrated to approximately 200 μl. For partial reduction, 30% Empigen-BB (Calbiochem, San Diego, Calif. USA) was added to this 200 μl to a final concentration of 3.5%, and 1M DTT in H2O was subsequently added to a final concentration of 1.5 to 7.5 mM and incubated for 30 min at 37° C. NEM (1M in dimethylsulphoxide) was subsequently added to a final concentration of 50 mM and left to react for another 30 min at 37° C. to block the free sulfhydryl groups.
  • 5.4 Gel Filtration Chromatography
  • A Superdex-200 HR 10/20 column (Pharmacia) was equilibrated with 3 column volumes PSS/3% Empigen-BB. The reduced mixture was injected in a 500 μl sample loop of the Smart System (Pharmacia) and PBS/3% Empigen-BB buffer was added for gelfiltration. Fractions of 250 μl were collected from V0 to V1. The fractions were screened for the presence of E1 or E2 protein as described in example 6.
  • FIG. 24 shows ELISA results obtained from fractions obtained after gelfiltration chromatography of 4 different E1 purifications of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b), vvHCV62 (type 3a), and vvHCVS6 (type 5a). FIG. 25 shows the profiles obtained from purifications of E1 proteins of types 1b, 3, and 5a (from RK13 cells infected with vvHCV39, vvHCVS2, and vvHCV63, respectively; purified on lentil lectin and reduced as in the previous examples). The peaks indicated with ‘1’, ‘2’, and ‘3’, represent sure E1 protein peaks (E1 reactivity mainly in fractions 26 to 30). These peaks show very similar molecular weights of approximately 70 kDa, corresponding to dimeric E1 protein. Other peaks in the three profiles represent vaccinia virus and/or cellular proteins which could be separated from E1 only because of the reduction step as outlined in example 5.3, and because of the subsequent gelfiltration step in the presence of the proper detergent. As shown in FIG. 25 pool 1 (representing fractions 10 to 17) and pool 2 (representing fractions 18 to 25) contain contaminating proteins not present in the E1 pool (fractions 26 to 30). The E1 peak fractions were ran on SDS/PAGE and blotted as described in example 4. Proteins labelled with NEM-biotin were detected by streptavidin-alkaline phosphatase as shown in FIG. 27. It can be readily observed that, amongst others, the 29 kDa and 45 kDa contaminating proteins present before the gelfiltration chromatography (lane 1) are only present at very low levels in the fractions 26 to 30. The band at approximately 65 kDa represents the E1 dimeric form that could not be entirely disrupted into the monomeric E1 form. Similar results were obtained for the type 3a E1 protein (lanes 10 to 15), which shows a faster mobility on SDS/PAGE because of the presence of only 5 carbohydrates instead of 6. FIG. 23 shows a silver stain of an SDS/PAGE gel run in identical conditions as in FIG. 26. A complete overview of the purification procedure is given in FIG. 29.
  • The presence of purified E1 protein was further confirmed by means of western blotting as described in example 4. The dimeric E1 protein appeared to be non-aggregated and free of contaminants. The subtype 1b E1 protein purified from vvHCV40-infected cells according to the above scheme was aminoterminally sequenced on an 477 Perkins-Elmer sequencer and appeared to contain a tyrosine as first residue. This confirmed that the E1 protein had been cleaved by the signal peptidase at the correct position (between A191 and Y192) from its signal sequence. This confirms the finding of Hijikata et al. (1991) that the aminoterminus of the mature E1 protein starts at amino acid position 192.
  • 5.5. Purification of the E2 Protein
  • The E2 protein (amino acids 384 to 673) was purified from RK13 cells infected with vvHCVA4 as indicated in Examples 5.1 to 5.4. FIG. 30 shows the CCl2 profile (continuous line) of the lentil lectin chromatography. The dotted line represents the E2 reactivity as detected by ELISA (see example 6). FIG. 31 shows the same profiles part of obtained from gelfiltration chromatography of the lentil-lectin E2 pool (see FIG. 30), part of which was reduced and blocked according to the methods as set out in example 5.3, and part of which was immediately applied to the column. Both parts of the E2 pool were run on separate gelfiltration columns. It could be demonstrated that E2 forms covalently-linked aggregates with contaminating proteins if no reduction has been performed. After reduction and blocking, the majority of contaminating proteins segregated into the Vo fraction. Other contaminating proteins copurified with the E2 protein, were not covalently linked to the E2 protein any more because these contaminants could be removed in a subsequent step. FIG. 32 shows an additional Ni2−-IMAC purification step carried out for the E2 protein purification. This affinity purification step employs the 6 histidine residues added to the E2 protein as expressed from vvHCV44. Contaminating proteins either run through the column or can be removed by a 30 mM imidazole wash. FIG. 33 shows a silver-stained SDS/PAGE of 0.5 μg of purified E2 protein and a 30 mM imidazole wash. The pure E2 protein could be easily recovered by a 200 mM imidazole elution step. FIG. 34 shows an additional desalting step intended to remove imidazole and to be able to switch to the desired buffer, e.g. PES, carbonate buffer, saline.
  • Starting from about 50,000 cm2 of RK13 cells infected with vvHCV11A (or vvHCV40) for the production of E1 or vvHCV41, vvHCV42, vvHCV 43, or vvHCV44 for production of E2 protein, the procedures described in examples 5.1 to 5.5 allow the purification of approximately 1.3 mg of E1 protein and 0.6 mg of 2 protein.
  • It should also be remarked that secreted E2 protein (constituting approximately 30-40%, 60-70% being in the intracellular form) is characterized by aggregate formation (contrary to expectations). The same problem is thus posed to purify secreted E2. The secreted E2 can be purified as disclosed above.
    • EXAMPLE 6 ELISA for the Detection of anti-E1 or anti-E2 Antibodies or for the Detection of E1 or E2 Proteins
  • Maxisorb microwell plates (Nunc. Roskilde, Denmark) were coated with 1 volume (e.g. 50 μl or 100 μl or 200 μl) per well of a 5 μg/ml solution of Streptavidin (Boehringer Mannheim) in PBS for 10 hours at 4° C. or for 1 hour at 37° C. Alternatively, the wells were coated with 1 volume of 5 μg/ml of Galanthus nivalis agglutinin (GNA) in 50 mM sodium carbonate buffer pH 9.6 for 16 hours at 4° C. or for 1 hour at 37° C. In the case of coating with GNA, the plates were washed 2 times with 400 μl of Washing Solution of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium). Unbound coating surfaces were blocked with 1.5 to 2 volumes of blocking solution (0.1% casein and 0.1% NaN, in PBS) for 1 hour at 37° C. or for 16 hours at 4° C. Blocking solution was aspirated. Purified E1 or E was diluted to 100-1000 ng/ml (concentration measured at A=280 nm) or column fractions to be screened for E1 or E2 (see example 5), or E1 or E2 in non-purified cell lysates (example 5.1.) were diluted 20 times in blocking solution, and 1 volume of the E1 or E2 solution was added to each well and incubated for 1 hour at 37° C. on the Streptavidin- or GNA-coated plates. The microwells were washed 3 times with 1 volume of Washing Solution of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium). Serum samples were diluted 20 times or monoclonal anti-E1 or anti-E2 antibodies were diluted to a concentration of 20 ng/ml in Sample Diluent of the Innotest HCV Ab III kit and 1 volume of the solution was left to react with the E1 or E2 protein for 1 hour at 37° C. The microwells were washed 5 times with 400 μl of Washing Solution of the Innotest HCV Ab III kit (Innogenetics. Zwijndrecht, Belgium). The bound antibodies were detected by incubating each well for 1 hour at 37° C. with a goat anti-human or anti-mouse IgG, peroxidase-conjugated secondary antibody (DAKO, Glostrup, Denmark) diluted 1/80,000 in 1 volume of Conjugate Diluent of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium), and color development was obtained by addition of substrate of the Innotesz HCV Ab III kit (Innogenetics. Zwijndrecht, Belgium) diluted 100 times in 1 volume of Substrate Solution of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium) for 30 min at 24° C. after washing of the plates 3 times with 400 μl of Washing Solution of the Innotest HCV Ab III kit (Innogenerics, Zwijndrecht, Belgium).
    • EXAMPLE 7 Follow Up of Patient Groups with Different Clinical Profiles
  • 7.1. Monitoring of Anti-E1 and Anti-E2 Antibodies
  • The current hepatitis C virus (HCV) diagnostic assays have seen developed for screening and confirmation of the presence of HCV antibodies. Such assays do not seem to provide information useful for monitoring of treatment or for prognosis of the outcome of disease. However, as is the case for hepatitis 2, detection and quantification of anti-envelope antibodies may prove more useful in a clinical setting. To investigate the possibility of the use of anti-E1 antibody titer and anti-E2 antibody titer as prognostic markers for outcome of hepatitis C disease, a series of IFN-α treated patients with long-term sustained response (defined as patients with normal transaminase levels and negative HCV-RNA test (PCR in the a non-coding region) in the blood for a period of at least 1 year after treatment) was compared with patients showing no response or showing biochemical response with relapse at the end of treatment.
  • A group of 8 IFN-α treated patients with long-term sustained response (LTR, follow up 1 to 3.5 years, 3 type 3a and 5 type 1b) was compared with 9 patients showing non-complete responses to treatment (NR, follow up 1 to 4 years, 6 type 1b and 3 type 3a). Type 1b (vvHCV-39, see example 2.5.) and 3a E1 (vvHCV-62, see example 2.5.) proteins were expressed by the vaccinia virus system (see examples 3 and 4) and purified to homogeneity (example 5). The samples derived from patients infected with a type 1b hepatitis C virus were tested for reactivity with purified type 1b E1 protein, while samples of a type 3a infection were tested for reactivity of anti-type 3a E1 antibodies in an ELISA as described in example 6. The genotypes of hepatitis C viruses infecting the different patients were determined by means of the Inno-LiPA genotyping assay (Innogenetics, Zwijndrecht, Belgium). FIG. 5 shows the anti-E1 signal-to-noise ratios of these patients followed during the course of interferon treatment and during the follow-up period after treatment. LTR cases consistently showed rapidly declining anti-E1 levels (with complete negativation in 3 cases), while anti-E1 levels of NR cases remained approximately constant. Some of the obtained anti-E1 data are shown in Table 2 as average S/N ratios±SD (mean anti-E1 titer). The anti-E1 titer could be deduced from the signal to noise ratio as show in FIGS. 5, 6, 7, and 8.
  • Already at the end of treatment, marked differences could be observed between the 2 groups. Anti-Et antibody titers had decreased 6.9 times in LTR but only 1.5 times in NR. At the end of follow up, the anti-E1 titers had declined by a factor of 22.5 in the patients with sustained response and even slightly increased in NR. Therefore, based on these data, decrease of anti-E1 antibody levels during monitoring of IFN-α therapy correlates with long-term, sustainer response to treatment. The anti-E1 assay may be very useful for prognosis of long-term response to IFN treatment, or to treatment of the hepatitis C disease in general.
  • This finding was not expected. On the contrary, the inventors had expected the anti-E1 antibody levels to increase during the course of IFN treatment in patients with long term response. As is the case for hepatitis B, the virus is cleared as a consequence of the seroconversion for anti-HBsAg antibodies. Also in many other virus infections, the virus is eliminated when anti-envelope antibodies are raised. However, in the experiments of the present invention, anti-E1 antibodies clearly decreased in patients with a long-term response to treatment, while the antibody-level remained approximately at the same level in non-responding patients. Although the outcome of these experiments was not expected, this non-obvious finding may be very important and useful for clinical diagnosis of HCV infections. As shown in FIGS. 9, 10, 11, and 12, anti-E2 levels behaved very differently in the same patients studied and no obvious decline in titers was observed as for anti-E1 antibodies. FIG. 35 gives a complete overview of the pilot study.
  • As can be deduced from Table 2, the anti-E1 titers were on average at least 2 times higher at the start of treatment in long term responders compared with incomplete responders to treatment. Therefore, measuring the titer of anti-E1 antibodies at the start of treatment, or monitoring the patient during the course of infection and measuring the anti-E1 titer, may become a useful marker for clinical diagnosis of hepatitis C. Furthermore, the use of more defined regions of the E1 or E2 proteins may become desirable, as shown in example 7.3.
  • 7.2. Analysis of E1 and E2 Antibodies in a Larger Patient Cohort
  • The pilot study lead the inventors to conclude that, in case infection was completely cleared, antibodies to the HCV envelope proteins changed more rapidly than antibodies to the more conventionally studied HCV antigens, with E1 antibodies changing most vigorously. We therefore included more type 1b and 3a-infected LTR and further supplemented the cohort with a matched series of NR, such that both groups included 14 patients each. Some partial responders (PR) and responders with relapse (RR) were also analyzed.
  • FIG. 36 depicts average E1 antibody (E1 Ab) and E2 antibody (E2Ab) levels in the LTR and NR groups and Tables 4 and 5 show the statistical analyses. In this larger cohort, higher E1 antibody levels before IFN-a therapy were associated with LTR (P<0.03). Since much higher E1 antibody levels were observed in type 3a-infected patients compared with type 1b-infected patients (FIG. 37), the genotype was taken into account (Table 4). Within the type 1b-infected group, LTR also had higher E1 antibody levels than NR at the initiation of treatment (P<0.0-1; the limited number of type 3a-infected NR did not allow statistical analysis.
  • Of antibody levels monitored in LTR during the 1.5-year follow up period, only E1 antibodies cleared rapidly compared with levels measured at initiation of treatment [P=0.0058, end of therapy; P=0.00-7 and P=0.0051 at 6 and 12 months after therapy, respectively]. This clearance remained significant within type 1- or type 3-infected LTR (average P values<0.05). These data confirmed the initial finding that E1Ab levels decrease rapidly in the early phase of resolvement. This feature seems to be independent of viral genotype. In NR, PR, or RR, no changes in any of the antibodies measured were observed throughout the follow up period. In patients who responded favourably to treatment with normalization of ALT levels and HCV-RNA negative during treatment, there was a marked difference between sustained responders (LTR) and responders with a relapse (RR). In contrast to LTR, RR did not show any decreasing E1 antibody levels, indicating the presence of occult HCV infection that could neither be demonstrated by PCR or other classical techniques for detection of HCV-RNA, nor by raised ALT levels. The minute quantities of viral RNA, still present in the RR group during treatment, seemed to be capable of anti-E1 B cell stimulation. Anti-E1 monitoring may therefore not only be able to discriminate LTR from NR, but also from RR.
  • 7.3. Monitoring of Antibodies of Defined Regions of the E1 Protein
  • Although the molecular biological approach of identifying HCV antigens resulted in unprecedented breakthrough in the development of viral diagnostics, the method of immune screening of λgt11 libraries predominantly yielded linear epitopes dispersed throughout the core and non-structural regions, and analysis of the envelope regions had to await cloning and expression of the E1/E2 region in mammalian cells. This approach sharply contrasts with many other viral infections of which epitopes to the envelope regions had already been mapped long before the deciphering of the genomic structure. Such epitopes and corresponding antibodies often had neutralizing activity useful for vaccine development and/or allowed the development of diagnostic assays with clinical or prognostic significance (e.g. antibodies to hepatitis B surface antigen). As no HCV vaccines or tests allowing clinical diagnosis and prognosis of hepatitis C disease are available today, the characterization of viral envelope regions exposed to immune surveillance may significantly contribute to new directions in HCV diagnosis and prophylaxis.
  • Several 20-mer peptides (Table 3) that overlapped each other by 8 amino acids, were synthesized according to a previously described method (EP-A-0 439 968) based on the HC-J1 sequence (Okamoto et al., 1990). None of these, except peptide env35 (also referred to as E1-35), was able to detect antibodies in sera of approximately 200 HCV cases. Only 2 sera reacted slightly with the env35 peptide. However, by means of the anti-E1 ELISA as described in example 6, it was possible to discover additional epitopes as follows: The anti-E1 ELISA as described in example 6 was modified by mixing 50 μg/ml of E1 peptide with the 1120 diluted human serum in sample diluent. FIG. 13 shows the results of reactivity of human sera to the recombinant E1 (expressed from vvHCV-40) protein, in the presence of single or of a mixture of E1 peptides. While only 2% of the sera could be detected by means of E1 peptides coated on strips in a Line Immunoassay format, over half of the sera contained anti-E1 antibodies which could be competed by means of the same peptides, when tested on the recombinant E1 protein. Some of the murine monoclonal antibodies obtained from Balb/C mice after injection with purified E1 protein were subsequently competed for reactivity to E1 with the single peptides (FIG. 14). Clearly, the region of env53 contained the predominant epitope, as the addition of env53 could substantially compete reactivity of several sera with E1, and antibodies to the env31 region were also detected. This finding was surprising, since the env53 and env31 peptides had not shown any reactivity when coated directly to the solid phase.
  • Therefore peptides were synthesized using technology described by applicant previously (in WO 93/18054). The following peptides were synthesized:
  • peptide env35A-biotin
      • NH2—SNSSEAADMIMHTPGCV-GKbiotin (SEQ ID NO 51)
      • spanning amino acids 208 to 227 of the HCV polyprotein in the E1 region peptide biotin-env53 (‘epitope A’)
      • biotin-GG-ITCHRMAWDMMNWSPTTAL-COOH (SEQ ID NO 52)
      • spanning amino acids to 313 of 332 of the HCV polyprotein in the E1 region
  • peptide 1bE1 (‘epitope B’)
      • H2N-YEVRNVSGIYHVTNDCSNSSIVYEAADMHTPGCGK-biotin (SEQ ID NO 53)
      • spanning amino acids 192 to 228 of the HCV polyprotein in the E1 region and compared with the reactivities of peptides E1a-BB (biotin-GG-TPTVATRDGKLPATQLRRHIDLL, SEQ ID NO 54) and E1b-BB (biotin-GG-TPTLAARDASVPTTTIRRHVDLL, SEQ ID NO 55) which are derived from the same region of sequences of genotype 1a and 1b respectively and which have been described at the IXth international virology meeting in Glasgow, 1993 (‘epitope C’). Reactivity of a panel of HCV sera was tested on epitopes A, B and C and epitope B was also compared with env35A (of 47 HCV-positive sera, 8 were positive on epitope 6 and none reacted with env35A). Reactivity towards epitopes A, B, and C was tested directly to the biotinylated peptides (50 μg/ml) bound to streptavidin-coated plates as described in example 6. Clearly, epitopes A and B were most reactive while epitopes C and env35A-biotin were much less reactive. The same series of patients that had been monitored for their reactivity towards the complete E1 protein (example 7.1.) was tested for reactivity towards epitopes A, B, and C. Little reactivity was seen to epitope C, while as shown in FIGS. 15, 16, 17, and 18, epitopes A and B reacted with the majority of sera. However, antibodies to the most reactive epitope (epitope A) did not seem to predict remission of disease, while the anti-1bE1 antibodies (epitope B) were present almost exclusively in long term responders at the start of IFN treatment. Therefore, anti-1bE1 (epitope B) antibodies and anti-env-53 (epitope A) antibodies could be shown to be useful markers for prognosis of hepatitis C disease. The env53 epitope may be advantageously used for the defection of cross-reactive antibodies (antibodies that cross-react between major genotypes) and antibodies to the env53 region may be very useful for universal E1 antigen detection in serum or liver tissue. Monoclonal antibodies that recognized the env53 region were reacted with a random epitope library. In 4 clones that reacted upon immunoscreening with the monoclonal antibody 5E1A10, the sequence -GWD- was present. Because of its analogy with the universal HCV sequence present in all HCV variants in the env53 region, the sequence AWD is thought to contain the essential sequence of the env53 cross-reactive murine epitope. The env31 clearly also contains a variable region which may contain an epitope in the amino terminal sequence —YQVRNSTGL— (SEQ ID NO 93) and may be useful for diagnosis. Env31 or E1-31 as shown in Table 3, is a part of the peptide 1bE1. Peptides 1-33 and E1-51 also reacted to some extent with the murine antibodies, and peptide E1-55 (containing the variable region 6 (V6); spanning amino acid positions 329-336) also reacted with some of the patient sera.
  • Anti-E2 antibodies clearly followed a different pattern than the anti-E1 antibodies, especially in patients with a long-term response to treatment. Therefore, it is clear that the decrease in anti-envelope antibodies could not be measured as efficiently with an assay employing a recombinant E1/E2 protein as with a single anti-E1 or anti-E2 protein. The anti-E2 response would clearly blur the anti-E1 response in an assay measuring both kinds of antibodies at the same time. Therefore, the ability to test anti-envelope antibodies to the single E1 and E2 proteins, was shown to be useful.
  • 7.4. Mapping of Anti-E2 Antibodies
  • Of the 24 anti-12 Mabs only three could be competed for reactivity to recombinant E2 by peptides, two of which reacted with the HVRI region (peptides E2-67 and E2-69, designated as epitope A) and one which recognized an epitope competed by peptide E2-13B (epitope C). The majority of murine antibodies recognized conformational anti-E2 epitopes (FIG. 19). A human response to HVRI (epitope A), and to a lesser extent HVRII (epitope B) and a third linear epitope region (competed by peptides E2-23. E2-25 or E2-27, designated epitope c) and a fourth linear epitope region (competed by peptide E2-17B, epitope D) could also frequently be observed, but the majority of sera reacted with conformational epitopes (FIG. 20). These conformational epitopes could be grouped according to their relative positions as follows: the IgG antibodies in the supernatant of hybridomas 15C8C1, 12D11F1, 9G3E6, 8G10D1H9, 10D3C4, 4H6B2, 17F2C2, 5H6A7, 15B7A2 recognizing conformational epitopes were purified by means of protein A affinity chromatography and 1 mg/ml of the resulting IgG's were biotinylated in borate buffer in the presence of biotin. Biotinylated antibodies were separated from free biotin by means of gelfiltration chromatography. Pooled biotinylated antibody fractions were diluted 100 to 10,000 times. E2 protein bound to the solid phase was detected by the biotinylated IgG in the presence of 100 times the amount of non-biotinylated competing antibody and subsequently detected by alkaline phosphatase labeled streptavidin.
  • Percentages of competition are given in Table 6. Based on these results, 4 conformational anti-E2 epitope regions (epitopes F, C, H and I) could be delineated (FIG. 38). Alternatively, these Mabs may recognize mutant linear epitopes not represented by the peptides used in this study. Mabs 4H6B2 and 10D3C4 competed reactivity of 16A6E7, but unlike 16A6E7, they did not recognize peptide E2-13B. These Mabs may recognize variants of the same linear epitope (epitope C) or recognize a conformational epitope which is sterically hindered or chances conformation after binding of 16A6E7 to the E2-13B region (epitope H).
    • EXAMPLE 8 E1 Glycosylation Mutants
  • 8.1. Introduction
  • The E1 protein encoded by vvHCV10A, and the E2 protein encoded by vvHCV41 to 44 expressed from mammalian cells contain 6 and 11 carbohydrate moieties, respectively. This could be shown by incubating the lysate of vvHCV10A-infected or vvHCV44-infected RK13 cells with decreasing concentrations of glycosidases (PNGase F or Endoglycosidase H, (Boehringer Mannheim Biochemical according to the manufacturer's instructions), such that the proteins in the lysate (including E1) are partially deglycosylated (FIGS. 39 and 40, respectively).
  • Mutants devoid of some of their glycosylation sires could allow the selection of envelope proteins with improved immunological reactivity. For HIV for example, gp120 proteins lacking certain selected sugar-addition motifs, have been found to be particularly useful for diagnostic or vaccine purpose. The addition of a new oligosaccharide side chain in the hemagglutinin protein of an escape mutant of the A/Hong Kong/3/68 (H3N2) influenza virus prevents reactivity with a neutralizing monoclonal antibody (Skehel et al, 1984). When novel glycosylation sites were introduced into the influenza hemaglutinin protein by site-specific mutagenesis, dramatic antigenic changes were observed, suggesting that the carbohydrates serve as a modulator of antigenicity (Gallagher et al., 1988). In another analysis, the 8 carbohydrate-addition motifs of the surface protein gp70 of the Friend Murine Leukemia Virus were deleted. Although seven of the mutations did not affect virus infectivity, mutation of the fourth glycosylation signal with respect to the amino terminus resulted in a non-infectious phenotype (Kayman et al., 1991). Furthermore, it is known in the art that addition of N-linked carbohydrate chains is important for stabilization of folding intermediates and thus for efficient folding, prevention of malfolding and degradation in the endoplasmic reticulum, oligomerization, biological activity, and transport of glycoproteins (see reviews by Rose at al., 1988; Doms et al., 1993; Helenius, 1994).
  • After alignment of the different envelope protein sequences of HCV genotypes, it may be inferred that not all 6 glycosylation sites on the HCV subtype 1b E1 protein are required for proper folding and reactivity, since some are absent in certain (sub)types. The fourth carbohydrate motif (on Asn251), present in types 1b, 6a, 7, 8, and 9, is absent in all other types know today. This sugar-addition motif may be mutated to yield a type 1b E1 protein with improved reactivity. Also the type 2b sequences show an extra glycosylation site in the V5 region (on Asn299). The isolate S83, belonging to genotype 2c, even lacks the first carbohydrate motif in the V1 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. Therefore, identification of the carbohydrate addition motifs which are not required for proper folding (and reactivity) is not obvious, and each mutant has to be analyzed and tested for reactivity. Mutagenesis of a glycosylation motif (NXS or NXT sequences) can be achieved by either mutating the codons for N, S, or T, in such a way that these codons encode amino acids different from N in the case of N, and/or amino acids different from S or T in the case of S and in the case of T. Alternatively, the X position may be mutated into PI since it is known that NPS or NPT are not frequently modified with carbohydrates. After establishing which carbohydrate-addition motifs are required for folding and/or reactivity and which are not, combinations of such mutations may be made.
  • 8.2. Mutagenesis of the E1 Protein
  • All mutations were performed on the E1 sequence of clone HCCl10A (SEQ ID NO. 5). The first round of PCR was performed using sense primer ‘GPT’ (see Table 7) targetting the CPT sequence located upstream of the vaccinia 11 K late promoter, and an antisense primer (designated GLY#, with # representing the number of the glycosylation size, see FIG. 41) containing the desired base change to obtain the mutagenesis. The six GLYM# primers (each specific for a given glycosylacion site) were designed such that:
      • Modification of the codon encoding for the N-glycosylated Asn (AAC or AAT) to a Gln codon (CAA or CAG). Glutamine was chosen because it is very similar to asparagine (both amino acids are neutral and contain non-polar residues, glutamine has a longer side chain (one more —CH2— group).
      • The introduction of silent mutations in one or several of the codons downstream of the glycosylation site, in order to create a new unique or rare (e.g. a second SmaI site for E1Gly5) restriction enzyme site. Without modifying the amino acid sequence, this mutation will provide a way to distinguish the mutated sequences from the original E1 sequence (pvHCV-10A) or from each other (FIG. 41). This additional restriction site may also be useful for the construction of new hybrid (double, triple, etc.) glycosylation mutants.
      • 18 nucleotides extend 5′ of the first mismatched nucleotide and 12 to 16 nucleotides extend to the 3′ end. Table 7 depicts the sequences of the six GLY# primers overlapping the sequence of N-linked glycosylation sites.
  • For site-directed mutagenesis, the ‘mispriming’ or ‘overlap extension’ (Horton. 1993) was used. The concept is illustrated in FIGS. 42 and 43. First, two separate fragments were amplified from the target gene for each mutated site. The PCR product obtained from the 5′ end (product GLYP) was amplified with the 5′ sense GPT primer (see Table 7) and with the respective 3‘antisense GLYM’ primers. The second fragment (product OVR#) was amplified with the 3′ antisense TKR primer and the respective 5′ sense primers (OVR# primers, see Table 7, FIG. 43).
  • The OVR# primers target part of the GLY# primer sequence. Therefore, the two groups of PCR products share an overlap region of identical sequence. When these intermediate products are mixed (GLY-1 with OVR-1, GLY-2 with OVR-2, etc.), melted at high temperature, and reannealed, the top sense strand of product GLY# can anneal to the antisense strand of product OVR# (and vice versa) in such a way that the two strands act as primers for one another (see FIG. 42.B.). Extension of the annealed overlap by Taq polymerase during two FCR cycles created the full-length mutant molecule E1 Gly#, which carries the mutation destroying the glycosylation site number #. Sufficient quantities of the E1GLY# products for cloning were generated in a third PCR by means of a common set of two internal nested primers. These two new primers are respectively overlapping the 3′ end of the vaccinia 11 K promoter (sense GPT-2-primer) and the 5′ end of the vaccinia thymidine kinase locus (antisense TKR-2 primer, see Table 7). All PCR conditions were performed as described in Stuyver et al. (1993).
  • Each of these PCR products was cloned by EcoRI/BamHI cleavage into the EcoRI/BamHI-cut vaccinia vector containing the original E1 sequence (pvHCV-10A).
  • The selected clones were analyzed for length of insert by EcoRI/BamHI cleavage and for the presence of each new restriction site. The sequences overlapping the mutated sites were confirmed by double-stranded sequencing.
  • 8.3. Analysis of E1 Glycosylation Mutants
  • Starting from the 6 plasmids containing the mutant E1 sequences as described in example 8.2, recombinant vaccinia viruses were generated by recombination with wt vaccinia virus as described in example 2.5. Briefly, 175 cm2-flasks of subconfluent RK13 cells were infected with the 6 recombinant vaccinia viruses carrying the mutant E1 sequences, as well as with the vvHCV-10A (carrying the non-mutated E1 sequence) and wt vaccinia viruses. Cells were lysed after 24 hours of infection and analyzed on western blot as described in example 4 (see FIG. 444A). All mutants showed a faster mobility (corresponding to a smaller molecular weight of approximately 2 to 3 kDa) on SDS-PAC-E than the original E1 protein; confirming that one carbohydrate moiety was not added. Recombinant viruses were also analyzed by PCR and restriction enzyme analysis to confirm the identity of the different mutants. FIG. 448 shows that all mutants (as shown in FIG. 41) contained the expected additional restriction sites. Another part of the cell lysate was used to test the reactivity of the different mutant by ELISA. The lysates were diluted 20 times and added to microwell plates coated with the lectin GNA as described in example 6. Captured (mutant) E1 glycoproteins were left to react with 20-times diluted sera of 24 HCV-infected patients as described in example 6. Signal to noise (S/N) values (CD of GLY#/OD of wt) for the six mutants and E1 are shown in Table 8. The table also shows the ratios between S/N values of GLY# and E1 proteins. It should be understood that the approach to use cell lysates of the different mutants for comparison of reactivity with patient sera may result in observations that are the consequence of different expression levels rather then reactivity levels. Such difficulties can be overcome by purification of the different mutants as described in example 5, and by testing identical quantities of all the different E1 proteins. However, the results shown in table 5 already indicate that removal of the 1st (GLY1), 3rd (GLY3), and 6th (GLY6) glycosylation motifs reduces reactivity of some sera, while removal of the 2nd and 5th site does not. Removal of GLY4 seems to improve the reactivity of certain sera. These data indicate that different patients react differently to the glycosylation mutants of the present invention. Thus, such mutant E1 proteins may be useful for the diagnosis (screening, confirmation, prognosis, etc.) and prevention of HCV disease.
    • EXAMPLE 9 Expression of HCV E2 Protein in Glycosylation-Deficient Yeasts
  • The E2 sequence corresponding to clone HCCL41 was provided with the α-mating factor pre/pro signal sequence, inserted in a yeast expression vector and S. cerevisiae cells transformed with this construct secreted E2 protein into the growth medium. It was observed that most glycosylation sites were modified with high-mannose type glycosylations upon expression of such a construct in S. cerevisiae strains (FIG. 45). This resulted in a too high level of heterogeneity and in shielding of reactivity, which is not desirable for either vaccine or diagnostic purposes. To overcome this problem, S. cerevisiae mutants with modified glycosylation pathways were generated by means of selection of vanadate-resistant clones. Such clones were analyzed for modified glycosylation pathways by analysis of the molecular weight and heterogeneity of the glycoprotein invertase. This allowed us to identify different glycosylation deficient S. cerevisiae mutants. The E2 protein was subsequently expressed in some of the selected mutants and left to react with a monoclonal antibody as described in example 7, on western blot as described in example 4 (FIG. 46).
    • EXAMPLE 10 General Utility
  • The present results show that not only a good expression system but also a good purification protocol are required to reach a high reactivity of the HCV envelope proteins with human patient sera. This can be obtained using the proper HCV envelope protein expression system and/or purification protocols of the present invention which guarantee the conservation of the natural folding of the protein and the purification protocols of the present invention which guarantee the elimination of contaminating proteins and which preserve the conformation, and thus the reactivity of the HCV envelope proteins. The amounts of purified HCV envelope protein needed for diagnostic screening assays are in the range of grams per year. For vaccine purposes, even higher amounts of envelope protein would be needed. Therefore, the vaccinia virus system may be used for selecting the best expression constructs and for limited upscaling, and large-scale expression and purification of single or specific oligomeric envelope proteins containing high-mannose carbohydrates may be achieved when expressed from several yeast strains. In the case of hepatitis B for example, manufacturing of HBsAg from mammalian cells was much more costly compared with yeast-derived hepatitis B vaccines.
  • The purification method disclosed in the present invention may also be used for ‘viral envelope proteins’ in general. Examples are those derived from Flaviviruses, the newly discovered GB-A. GB-B and GB-C Hepatitis viruses, Pestiviruses (such as Bovine viral Diarrhea Virus (BVDV). Hog Cholera Virus (HCV). Border Disease Virus (BDV)), but also less related viruses such as Hepatitis B Virus (mainly for the purification of HBsAg).
  • The envelope protein purification method of the present invention may be used for intra- as well as extracellularly expressed proteins in lower or higher eukaryotic cells or in prokaryotes as set out in the detailed description section.
    TABLE 1
    Recombinant vaccinia plasmids and viruses
    cDNA subclone Vector used
    Plasmid name Name construction Length (nt/aa) for insertion
    pvHCV-13A E1s EcoR I-Hind III 472/157 pgptATA-18
    pvHCV-12A E1s EcoR I-Hind III 472/158 pgptATA-18
    pvHCV-9A E1 EcoR I-Hind III 631/211 pgptATA-18
    pvHCV-11A E1s EcoR I-Hind III 625/207 pgptATA-18
    pvHCV-17A E1s EcoR I-Hind III 625/208 pgptATA-18
    pvHCV-10A E1 EcoR I-Hind III 783/262 pgptATA-18
    pvHCV-18A COREs Acc I (Kl)-EcoR I (Kl) 403/130 pgptATA-18
    pvHCV-34 CORE Acc I (Kl)-Fso I 595/197 pgptATA-18
    pvHCV-33 CORE-E1 Acc I (Kl) 1150/380  pgptATA-18
    pvHCV-35 CORE-E1b.his EcoR I-BamH I (Kl) 1032/352  pMS-66
    pvHCV-36 CORE-E1n.his EcoR I-Nco I (Kl) 1106/376  pMS-66
    pvHCV-37 E1Δ Xma I-BamH I 711/239 pvHCV-10A
    pvHCV-38 E1Δs EcoR I-BstE II 553/183 pvHCV-11A
    pvHCV-39 E1Δb EcoR I-BamH I 960/313 pgsATA-18
    pvHCV-40 E1Δb.his EcoR I-BamH I (Kl) 960/323 pMS-66
    pvHCV-41 E2bs BamH I (Kl)-AlwN I (T4) 1005/331  pgsATA-18
    pvHCV-42 E2bs.his BamH I (Kl)-AlwN I (T4) 1005/341  pMS-66
    pvHCV-43 E2ns Nco I (Kl)-AlwN I (T4) 932/314 pgsATA-18
    pvHCV-44 E2ns.his Nco I (Kl)-AlwN I (T4) 932/321 pMS-66
    pvHCV-62 E1s (type 3a) EcoR I-Hind III 625/207 pgsATA-18
    pvHCV-63 E1s (type 5) EcoR I-Hind III 625/207 pgsATA-18
    pvHCV-64 E2 BamH I-Hind III 1410/463  pgsATA-18
    pvHCV-65 E1-E2 BamH I-Hind III 2072/691  pvHCV-10A
    pvHCV-66 CORE-E1-E2 BamH I-Hind III 2427/809  pvHCV-33
    HCV cDNA subclone Vector
    Plasmid Length used for
    Name Name Construction (nt/aa) insertion
    pvHCV-81 E1*-GLY 1 EcoRI-BamH I 783/262 pvHCV-10A
    pvHCV-82 E1*-GLY 2 EcoRI-BamH I 783/262 pvHCV-10A
    pvHCV-83 E1*-GLY 3 EcoRI-BamH I 783/262 pvHCV-10A
    pvHCV-84 E1*-GLY 4 EcoRI-BamH I 783/262 pvHCV-10A
    pvHCV-85 E1*-GLY 5 EcoRI-BamH I 783/262 pvHCV-10A
    pvHCV-86 E1*-GLY 6 EcoRI-BamH I 783/262 pvHCV-10A

    nt: nucleotide

    aa: aminoacid

    Kl: Klenow DNA Pol filling

    T4: T4 DNA Pol filling

    Position: aminoacid position in the HCV polyprotein sequence
  • TABLE 2
    Summary of anti-E1 tests
    S/N ± SD (mean anti-E1 titer)
    Start of treatment End of treatment Follow-up
    LTR 6.94 ± 2.29 4.48 ± 2.69 2.99 ± 2.69
    (1:3946) (1:568)  (1:175) 
    NR 5.77 ± 3.77 5.29 ± 3.99 6.08 ± 3.73
    (1:1607) (1:1060) (1:1978)

    LTR: Long-term, sustained response for more than 1 year

    NR: No response, response with relapse, or partial response
  • TABLE 3
    Synthetic peptides for competition studies
    SEQ
    ID
    PROTEIN PEPTIDE AMINO ACID SEQUENCE POSITION NO
    E1 E1-31 LLSCLTVPASAYCVRNSTGL 181-200 56
    E1-33 QVRNSTGLYHVTNDCPNSSI 193-212 57
    E1-35 NDCPNSSIVYEAHDAILHTP 205-224 58
    E1-35A SNSSIVYEAADMIMHTPGCV 208-227 59
    E1-37 HDAILHTPGCVPCVREGNVS 217-236 60
    E1-39 CVREGNVSRCWVAMTPTVAT 229-248 61
    E1-41 AMTPTVATRDGKLPATQLRR 241-260 62
    E1-43 LPATQLRRHIDLLVGSATLC 253-272 63
    E1-45 LVGSATLCSALYVGDLCGSV 265-284 64
    E1-49 QLFTFSPRRHWTTQGCNCSI 289-308 65
    E1-51 TQGCNCSIYPGHITGHRMAW 301-320 66
    E1-53 ITGHRMAWDMMMNWSPTAAL 313-332 67
    E1-55 NWSPTAALVMAQLLRIPQAI 325-344 68
    E1-57 LLRIPQAILDMIAGAHWGVL 337-356 69
    E1-59 AGAHWGVLAGIAYFSMVGNM 349-368 70
    E1-63 VVLLLFAGVDAETIVSGGQA 373-392 71
    E2 E2-67 SGLVSLFTPGAKQNIQLINT 397-416 72
    E2-69 QNIQLINTNGSWHINSTALN 409-428 73
    E2-$3B LNCNESLNTGWWLAGLIYQHK 427-446 74
    E2-$1B AGLIYQHKFNSSGCPERLAS 439-458 75
    E2-1B GCPERLASCRPLTDFDQGWG 451-470 76
    E2-3B TDFDQGWGPISYANGSGPDQ 463-482 77
    E2-5B ANGSGPDQRPYCWHYPPKPC 475-494 78
    E2-7B WHYPPKPCGIVPAKSVCGPV 487-506 79
    E2-9B AKSVCGPVYCFTPSPVVVGT 499-518 80
    E2-11B PSPVVVGTTDRSGAPTYSWG 511-530 81
    E2-13B GAPTYSWGENDTDVFVLNNT 523-542 82
    E2-17B GNWFGCTWMNSTGFTKVCGA 547-566 83
    E2-19B GFTKVCGAPPVCIGGAGNNT 559-578 84
    E2-21 IGGAGNNTLHCPTDCFRKHP 571-590 85
    E2-23 TDCFRKHPDATYSRCGSGPW 583-602 86
    E2-25 SRCGSGPWITPRCLVDYPYR 595-614 87
    E2-27 CLVDYPYRLWHYPCTINYTI 607-626 88
    E2-29 PCTINYTIFKIRMYVGGVEH 619-638 89
    E2-31 MYVGGVEHRLEAACNWTPGE 631-650 90
    E2-33 ACNWTPGERCDLEDRDRSEL 643-662 91
    E2-35 EDRDRSELSPLLLTTTQWQV 655-674 92
  • TABLE 4
    Change of Envelope Antibody levels over time (complete study, 28 patients)
    Wilcoxon Signed
    E1Ab NR E1Ab NR E1Ab NR E1Ab LTR E1Ab LTR E1Ab LTR E2Ab NR E1Ab LTR
    Rank test (P values)
    All type 1b type 3a All type 1b type 3a All All
    End of therapy* 0.1167 0.2604 0.285 0.0058** 0.043** 0.0499** 0.0186** 0.0640
    6 months follow up* 0.86 0.7213 0.5930 0.0047** 0.043** 0.063 0.04326 0.0464**
    12 months follow up* 0.7989 0.3105 1 0.0051** 0.0679 0.0277** 0.0869 0.0058**

    *Data were compared with values obtained at initiation of therapy

    **P values < 0.05
  • TABLE 5
    Difference between LTR and NR (complete study)
    Mann-Withney
    E1Ab E1Ab E1Ab E1Ab E2Ab
    S/N titers S/N S/N S/N
    U test (P values)
    All All type 1b type 3a All
    Initiation of therapy 0.0257* 0.05* 0.68 0.1078
    End of therapy 0.1742 0.1295
    6 months follow up 1 0.6099 0.425 0.3081
    12 months follow up 0.67 0.23 0.4386 0.6629

    *P values < 0.05
  • TABLE 6
    Competition experiments between murine E2 monoclonal antibodies
    Decrease (%) of anti-E2 reactivity of biotinylated anti-E2 mabs
    17H10F4D10 2F10H10 16A6EQ7 10D3C4 4H6B2 17C2F2 9G3E6 12D11F1 15C8C1 8G10D1H9
    competitor
    17H10F4D10 — 62  10 ND 11 ND 5 6 30 ND
    2F10H10 90  —  1 ND 30 ND 0 4 12 ND
    16A6E7 ND ND — ND ND ND ND ND ND ND
    10D3C4
    11  50  92 — 94 26  28  43  53 30 
    4H6B2 ND ND 82 ND — ND ND ND ND ND
    17C2F2
    2 ND 75 ND 56 — 11  10   0 0
    9G3E6 ND ND 68 ND 11 ND — 60  76 ND
    12D11F1 ND ND 26 ND 13 ND ND — 88 ND
    15C8C1 ND ND 18 ND 10 ND ND ND — ND
    8G10D1H9
    2 2 11 ND 15 ND 67  082  81 —
    competitor
    controls
    15B7A2 0 0 9 15 10 9 0 0  0 5
    5H6A7 0 2 0 12  8 0 0 4  0 0
    23C12H9 ND ND 2 12 ND 4 ND ND ND 2

    ND, not done
  • TABLE 7
    Primers
    SEQ ID NO. 96 GPT 5′-GTTTAACCACTGCATGATG-3′
    SEQ ID NO. 97 TK ∥ 5′-GTCCCATCGAGTGCGGCTAC-3′
    SEQ ID NO. 98 GLY1 5′-CGTGACATGGTACATTCCGGACACTT
    GGCGCACTTCATAAGCGGA-3′
    SEQ ID NO. 99 GLY2 5′-TGCCTCATACACAATG GAGCTC TGGG
    ACGAGTCGTTCGTGAC-3′
    SEQ ID NO. 100 GLY3 5′-TACCCAGCAGCGGGAGCTC TGTTGCT
    CCCGAACGCAGGGCAC-3′
    SEQ ID NO. 101 GLY4 5′-TGTCGTGGTGGGGACGG AGGCCT GCC
    TAGCTGCGAGCGTGGG-3′
    SEQ ID NO. 102 GLY5 5′-CGTTATGTGGCCCGGGTAGATTGAGC
    ACTGGCAGTCCTGCACCGTCTC-3′
    SEQ ID NO. 103 GLY6 5′-CAGGGCCGTTGTAGGCCTCCACTGCA
    TCATCATATCCCAAGC-3′
    SEQ ID NO. 104 OVR1 5′-CCGGAATGTACCATGTCACGAACG
    AC-3′
    SEQ ID NO. 105 OVR2 5′- GCTC CATTGTGTATGAGGCAGC
    GG-3′
    SEQ ID NO. 106 OVR3 5′- GAGCTCCCGCTGCTGGGTAGCGC-3′
    SEQ ID NO. 107 OVR4 5′-CCTCCGTCCCCACCACGACAATA
    CG-3′
    SEQ ID NO. 108 OVR5 5′-CTA
    Figure US20080138894A1-20080612-P00801
    CC
    Figure US20080138894A1-20080612-P00802
    GGCCACATAACGGGTCAC
    CG-3′
    SEQ ID NO. 109 OVR6 5′-GG AGGCCTACAACGGCCCTGGT
    GG-3′
    SEQ ID NO. 110 GPT-2 5′-TTCTATCGATTAAATAGAATTC-3′
    SEQ ID NO. 111 TK∥.2 5′-GCCATACGCTCACAGCCGATCCC-3′

    nucleotides underlined represent additional restriction site

    nucleotides in bold represent mutations with respect to the original HCCl10A sequence
  • TABLE 8
    Analysis of E1 glycosylation mutants by ELISA
    SERUM
    1 2 3 4 5 6 7 8 9
    LY1 1.802462 2.120971 1.403871 1.205597 2.120191 2.866913 1.950345 1.866183 1.730193
    LY2 2.400795 1.76818  2.325495 2.639308 2.459019 5.043993 2.146302 1.595477 1.688973
    LY3 1.642718 1.715477 2.261646 2.354748 1.591818 4.833742 1.96692 1.482099 1.602222
    LY4 2.578154 3.824038 3.874605 1.499387 3.15 4.71302  4.198751 3.959542 3.710507
    LY5 2.482051 1.793761 2.409344 2.627358 1.715311 4.964765 2.13912 1.576336 1.708937
    LY6 2.031487 1.495737 2.131613 2.527925 2.494833 4.784027 2.02069 1.496489 1.704976
    I 2.828205 2.227036 2.512792 2.790881 3.131579 4.869128 2.287753 1.954198 1.805556
    10 11 12 13 14 15 16 17 18
    LY1 2.468162 1.220654 1.629403 5.885561 3.233604 3.763498 1.985105 2.317721 6.675179
    LY2 2.482212 1.467582 2.070524 7.556682 2.567613 3.621928 3.055649 2.933792 7.65433
    LY3 2.191558 1.464216 1.721164 7.930538 2.763055 3.016099 2.945628 2.515305 5.775357
    LY4 5.170841 4.250784 3.955153 8.176816 6.561122 5.707668 5.684498 5.604813 6.4125
    LY5 3.021807 1.562092 2.07278  8.883408 2.940334 3.125561 3.338912 2.654224 5.424107
    LY6 2.677757 1.529608 1.744221 8.005561 2.499952 2.621704 2.572385 2.363301 5.194107
    I 2.616822 1.55719  2.593886 8.825112 3.183771 3.067265 3.280335 2.980354 7.191964
    Sum Average
    19 20 21 22 23 24 S/N S/N
    LY1 1.93476  2.47171  4.378633 1.188748 2.158889 1.706992 59.88534 2.495223
    LY2 2.127712 2.921288 4.680101 1.150781 1.661914 1.632785 69.65243 2.902185
    LY3 1.980185 2.557384 4.268633 0.97767  1.336775 1.20376 62.09872 2.587447
    LY4 3.813321 3.002535 4.293038 2.393011 3.68213  2.481585 102.6978 4.279076
    LY5 2.442804 3.126761 4.64557 1.153656 1.817901 1.638211 69.26511 2.886046
    LY6 1.506716 2.665433 2.781063 1.280743 1.475062 1.716423 61.32181 2.555075
    I 2.771218 3.678068 5.35443 1.167286 2.083333 1.78252 76.54068 3.189195
    1 2 3 4 5 6 7 8 9
    Figure US20080138894A1-20080612-P00899
    E1    		 0.637316 0.952374 0.55869  0.431977 0.677036 0.588794 0.852516 0.954961 0.958261
    Figure US20080138894A1-20080612-P00899
    E1    		 0.848876 0.793961 0.925463 0.94569 0.785233 1.035913 0.93817  0.816436 0.935431
    Figure US20080138894A1-20080612-P00899
    E1    		 0.580834 0.770296 0.900053 0.84373 0.508312 0.992733 0.859761 0.758418 0.887385
    Figure US20080138894A1-20080612-P00899
    E1    		 0.911587 1.717097 1.541952 0.537245 1.005882 0.967939 1.835317 2.026172 2.05505 
    Figure US20080138894A1-20080612-P00899
    E1    		 0.877607 0.805447 0.958831 0.941408 0.547746 1.019642 0.935031 0.806641 0.946488
    Figure US20080138894A1-20080612-P00899
    E1    		 0.718296 0.671626 0.848305 0.90578 0.796669 0.982522 0.883264 0.765781 0.944294
    10 11 12 13 14 15 16 17 18
    Figure US20080138894A1-20080612-P00899
    E1    		 0.94319 0.783882 0.628171 0.644248 1.015652 1.226988 0.605153 0.777666 0.928144
    Figure US20080138894A1-20080612-P00899
    E1    		 0.94856 0.942455 0.798232 0.85627 0.806469 1.180833 0.931505 0.984377 1.064289
    Figure US20080138894A1-20080612-P00899
    E1    		 0.837488 0.940294 0.663547 0.898633 0.867856 0.983319 0.897966 0.843962 0.803029
    Figure US20080138894A1-20080612-P00899
    E1    		 1.976 2.72978  1.524798 0.92654 2.060802 1.860833 1.732902 1.880587 0.89162
    Figure US20080138894A1-20080612-P00899
    E1    		 1.154762 1.003148 0.799102 1.006606 0.923538 1.019006 1.017857 0.890574 0.75419
    Figure US20080138894A1-20080612-P00899
    E1    		 1.023286 0.982288 0.672435 0.907134 0.785217 0.854737 0.784184 0.79296  0.72221
    Sum Average
    19 20 21 22 23 24 E1/GLY// E1/GLY//
    Figure US20080138894A1-20080612-P00899
    E1    		 0.698162 0.672013 0.817759 1.018386 1.036267 0.957628 19.36524 0.806885
    Figure US20080138894A1-20080612-P00899
    E1    		 0.76779  0.794245 0.874061 0.98586  0.797719 0.915998 21.67384 0.903077
    Figure US20080138894A1-20080612-P00899
    E1    		 0.714554 0.695306 0.797215 0.837558 0.641652 0.675314 19.19921 0.799967
    Figure US20080138894A1-20080612-P00899
    E1    		 1.376045 0.816335 0.801773 2.050064 1.767422 1.392178 36.38592 1.51608 
    Figure US20080138894A1-20080612-P00899
    E1    		 0.881491 0.850109 0.867612 0.988323 0.872593 0.919042 21.78679 0.907783
    Figure US20080138894A1-20080612-P00899
    E1    		 0.543702 0.724683 0.519395 1.097197 0.70803  0.962919 19.59691 0.816538
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Claims (13)

1. A recombinant vector comprising a vector sequence and an: appropriate eukaryotic or viral promoter sequence operably linked to a nucleotide sequence comprising an HCV signal sequence attached before a sequence encoding an HCV envelope E2 protein starting at amino acid position 384 and ending in the region between amino acid positions 600 and 820 and wherein said HCV envelope E2 protein is produced by recombinant expression in lower eukaryotic or mammalian host cells.
2. The recombinant vector according to claim 1, with said nucleotide sequence being characterized further in that it ends at any of amino acid positions 623, 650, 673, 710, 715, 720, 746 or 809.
3. The recombinant vector according to claim 1, wherein said nucleotide sequence is comprising a sequence encoding an HCV signal sequence starting at amino acid position 347 or 364 and ending at amino acid position 383 and a sequence encoding an HCV envelope E2 protein starting at amino acid position 384.
4. The recombinant vector according to claim 1, with said nucleotide sequence being characterized further in that a 5′-terminal ATG codon and a 3′-terminal stop codon have been added to it.
5. The recombinant vector according to claim 1, with said nucleotide sequence being characterized further in that a factor Xa cleavage site and/or 3 to 10, histidine codons have been added.
6. The recombinant vector according to claim 5, wherein said histidine codons are added 3′-terminally to the coding region.
7. The recombinant vector according to claim 1, with said HCV E2 encoding sequence being chosen from the group as represented in SEQ ID NO 35, 37, 39, 41, 43, and 45.
8. The recombinant vector according to claim 1, with said nucleotide sequence encoding an HCV protein spanning amino acid positions 384-746, 384-809, 384-673, 347-746, 347-809, 347-673, 347-683, 364-673, 364-746 or 384-673.
9. Recombinant nucleic acid comprising any of the HCV E2 encoding sequences as represented in SEQ ID NO 35, 37, 39, 41, 43, 45.
10. Recombinant nucleic acid encoding an HCV E2 protein spanning amino acid positions 384-746, 384-809, 384-673, 347-746, 347-809, 347-673, 347-683, 364-673, 364-746 or 384-673.
11. Recombinant vector according to claim 1, further characterized in that at least one of the glycosylation sites present in said HCV protein has been removed by site-directed mutagenesis of the nucleotide sequence encoding a glycosylation site.
12. An isolated host cell transformed with at least one recombinant vector according to claim 1.
13. A composition comprising a recombinant vector according to claim 1.
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