+

WO2003051912A2 - Proteines purifiees du virus de l'hepatite c utilisables en diagnostic et therapie - Google Patents

Proteines purifiees du virus de l'hepatite c utilisables en diagnostic et therapie Download PDF

Info

Publication number
WO2003051912A2
WO2003051912A2 PCT/EP2002/014480 EP0214480W WO03051912A2 WO 2003051912 A2 WO2003051912 A2 WO 2003051912A2 EP 0214480 W EP0214480 W EP 0214480W WO 03051912 A2 WO03051912 A2 WO 03051912A2
Authority
WO
WIPO (PCT)
Prior art keywords
hcv
seq
region
spanning
igp
Prior art date
Application number
PCT/EP2002/014480
Other languages
English (en)
Other versions
WO2003051912A3 (fr
WO2003051912A9 (fr
Inventor
Geert Maertens
Erik Depla
Fons Bosman
Original Assignee
Innogenetics N.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Innogenetics N.V. filed Critical Innogenetics N.V.
Priority to CA002468690A priority Critical patent/CA2468690A1/fr
Priority to EP02796675A priority patent/EP1461080A2/fr
Priority to IL16223602A priority patent/IL162236A0/xx
Priority to KR10-2004-7009720A priority patent/KR20040076869A/ko
Priority to NZ533396A priority patent/NZ533396A/en
Priority to BR0215081-6A priority patent/BR0215081A/pt
Priority to AU2002361160A priority patent/AU2002361160B2/en
Priority to JP2003552792A priority patent/JP2005516939A/ja
Publication of WO2003051912A2 publication Critical patent/WO2003051912A2/fr
Publication of WO2003051912A3 publication Critical patent/WO2003051912A3/fr
Publication of WO2003051912A9 publication Critical patent/WO2003051912A9/fr

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/18—Togaviridae; Flaviviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the general fields of recombinant protein expression, purification of recombinant proteins, synthetic peptides, diagnosis of HCN 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 El 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 El 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 baculo virus 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.
  • HCV sera 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-El positive using the purified, vaccinia-expressed recombinant El protein of the present invention, in sha ⁇ contrast with the results of Kohara et al. (1992) and Hsu et al. (1993).
  • Kohara et al. used a vaccinia- virus expressed El protein and detected anti- El antibodies in 7 to 23% of patients, while Hsu et al. only detected 14/50 (28%) sera using baculovirus-expressed El.
  • 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.
  • liver disease Clinical importance of necro-in ⁇ ammation and ⁇ brosis in HCV infection.
  • the natural history of liver disease after HCV infection does vary significantly from patient to patient. About 20% of the acutely infected persons are able to resolve infection spontaneously, while 80% of infected persons progresses to a chronic infection.
  • This chronic infection increases the risk for development of fibrosis which can lead to development of cirrhosis and ultimately liver carcinoma.
  • HAI Histology Activity Index
  • compositions comprising purified (single or specific oligomeric) recombinant El and/or E2 and/or E1/E2 glycoproteins comprising conformational epitopes from the El and/or E2 domains of HCV.
  • E2 and/or E1/E2 proteins of the present invention in HCV screening and confirmatory antibody tests.
  • 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.
  • monoclonal antibodies more particularly human monoclonal antibodies or mouse monoclonal antibodies which are humanized, which react specifically with El 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 -El or anti-E2 monoclonal antibodies for HCV antigen detection or for therapy of chronic HCV infection.
  • kits for monitoring/prognosing the response to treatment e.g. with interferon
  • 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.
  • '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 El 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 El 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 typically 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 El and/or E2 (envelope) domains of HCV.
  • the El 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 El, P7 (aa 747-809), NS2 (aa 810-1026), 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 El and/or E2 sequence for the practice of the present invention.
  • the El and E2 antigens used in the present invention may be full-length viral proteins, substantially full-length versions thereof, or functional fragments thereof (e.g.
  • 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 (polyclonal serum or monoclonal to the conformational epitope) and comparing its reactivity to that of a denatured version of the antigen which retains only linear epitopes (if any). In such screening using polyclonal antibodies, it may be advantageous to adsorb the polyclonal serum first with the denatured antigen and see if it retains antibodies to the antigen of interest.
  • the HCV antigens of the present invention can be made by any recombinant method that provides the epitope of intrest.
  • recombinant intracellular expression in mammalian or insect cells is a preferred method to provide glycosylated El 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, 1994) or glycosylation mutants derived by means of vanadate resistence 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
  • 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.
  • 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 adso ⁇ tion.
  • 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.
  • 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.
  • 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.
  • 'El' 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 El protein is encoded in the viral polyprotein following the C (core) protein. The El protein extends from approximately amino acid (aa) 192 to about aa 383 of the full-length polyprotein.
  • 'El' as used herein also includes analogs and truncated forms that are immunologically cross-reactive with natural El, and includes El proteins of genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 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 El 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 El component and at least one E2 component.
  • El and/or E2 and/or E1/E2 envelope proteins refers to all possible oligomeric forms of recombinantly expressed El and/or E2 envelope proteins which are not aggregates.
  • El and/or E2 specific oligomeric envelope proteins are also referred to as homo-oligomeric El or E2 envelope proteins (see below).
  • the term 'single or specific oligomeric' El and or E2 and/or E1/E2 envelope proteins refers to single monomeric El or E2 proteins (single in the strict sense of the word) as well as specific oligomeric El and/or E2 and/or E1/E2 recombinantly expressed proteins.
  • 'homo-oligomer' refers to a complex of El and/or E2 containing more than one El or E2 monomer, e.g. El/El dimers, El/El/El trimers or El /El /El /El tetramers and E2/E2 dimers, E2/E2/E2 trimers or E2/E2/E2/E2 teframers, El pentamers and hexamers, E2 pentamers and hexamers or any higher-order homo-oligomers of El or E2 are all 'homo-oligomers' within the scope of this definition.
  • the oligomers may contain one, two, or several different monomers of El or E2 obtained from different types or subtypes of hepatitis C virus including for example those described 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, withouth 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.
  • these proteins are purified to homogeneity (at least 80% pure, preferably, 90%, more preferably 95%, more preferably 97%, more preferably 98%, more preferably 99%, even more preferably 99.5%, 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.
  • lower eukaryote' 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, Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorph ⁇ ), 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.
  • 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.
  • 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.
  • 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', '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 fransfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in mo ⁇ hology 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.
  • the term '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.
  • 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.
  • 'promoter' is a nucleotide sequence which is comprised of consensus sequences which allow the binding of RNA polymerase to the DNA template in a manner such that mRNA production initiates at the normal transcription initiation site for the adjacent structural gene.
  • the 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' is a region of a polynucleotide sequence which encodes a polypeptide and does not contain stop codons; this region may represent a portion of a coding sequence or a total coding sequence.
  • a 'coding sequence' is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus.
  • a coding sequence can include but is not limited to mRNA, DNA (including cDNA), and recombinant polynucleotide sequences.
  • 'epitope' or 'antigenic determinant' means an amino acid sequence that is immunoreactive.
  • 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.
  • '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).
  • 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.
  • the present invention contemplates a method for isolating or purifying recombinant HCV single or specific oligomeric envelope protein selected from the group consisting of El 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 disculphide bond cleaving agent.
  • 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 detailed 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 resistence (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 WO 92/08734 that 'aggregates' were obtained after purification, but it was not described which protein interactions were involved.
  • host or expression-system-derived proteins e.g. vaccinia
  • HCV envelope proteins single or oligomeric
  • 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 disuphide 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).
  • Said disulphide bond cleavage (or reducing) step of the present invention is preferably a partial disulphide bond 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 of 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,
  • 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 El and E2 proteins.
  • said partial reduction is shown to result in the production of possibly dimeric El protein and separation of this El 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:
  • 'disulphide bridge exchanging agent' is to be inte ⁇ retated 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 envelelope 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
  • 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,
  • NEM-biotin e.g. obtained from Sigma B 1267
  • 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; US patent 3597160
  • other dithiobis heterocyclic derivative
  • 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 El 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 group 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 El 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 El 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-El 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 El and/or E2 and/or E1/E2 envelope proteins for
  • 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)
  • RCA I Ricinus communis agglutinin I
  • the present invention more particularly contemplates essentially purified recombinant HCV single or specific oligomeric envelope proteins, selected from the group consisting of El 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 El 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 El or El 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 El or E2 of the invention.
  • the segment of the HCV cDNA encoding the desired El 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 ⁇ -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 El and E2 proteins.
  • the segment of the HCV cDNA encoding the desired El 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 I.
  • 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 El protein.
  • the present recombinant vector encompasses a recombinant nucleic acid having a HCV cDNA seqment 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 El 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 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.
  • 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 820 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 seqment 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 El 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 El 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 caarying such a mutated El and/or E2 protein encoding nucleic acid are also within the scope of the present invention.
  • the present invention also relates to recombinant El 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 El and/or E2 and/or E1/E2 glycosylation mutant as well as host cells tranformed 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
  • 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, inluding HeLa cells, Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, PK15, RK13 and a number of other cell lines.
  • the present invention relates particularly to a recombinant El and/or E2 and/or E1/E2 protein expressed by a host cell as defined above containing a recombinany 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 El 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-BB, recovering said HCV envelope protein by affinity purification such as by means of lectin-chromatography or immunoaffinity chromatography using anti-El 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,
  • El and/or E2 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 void 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 contructs bearing a (His) 6 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 El peptides as listed in Table 3 and described elsewhere herein:
  • E1-35A (SEQ ID NO:59) spanning amino acids 208 to 227 of the El V2 region
  • epitopope B epitopope B
  • IbEl SEQ ID NO:53
  • V2 regions (containing epitope B)
  • epitope A epitope A
  • El-55 SEQ ID NO:68
  • the present invention also relates to a composition comprising at least one of the following E2 peptides as listed in Table 3 :
  • E2 region epitopope A, recognized by monoclonal antibody 2F10H10, see Figure 19
  • Env 69 or E2-69 SEQ ID NO:73
  • Env 23 or E2-23 (SEQ ID NO:86) spanning positions 583 to 602 of the E2 region
  • epitope E epitope E
  • Env 25 or E2-25 SEQ ID NO:87
  • Env 27 or E2-27 (SEQ ID NO:88) spanning positions 607 to 626 of the E2 region
  • epitope E epitope E
  • Env 17B or E2-17B (SEQ ID NO:83) spanning positions 547 to 566 of the E2 region
  • Env 13B or E2-13B (SEQ ID NO:82) spanning positions 523 to 542 of the E2 region
  • epitopope C recognized by monoclonal antibody 16A6E7, see Figure 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 El 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 El 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 El 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, immunized 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 hand, 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 mice
  • 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 inco ⁇ oration into an immunoassay for the detection of HCV genotypes (for detecting the presence of HCV El 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 El or E2 specific monoclonal antibodies for the preparation of an immunoassay kit for detecting the presence of El 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: 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 inte ⁇ retation device for inferring the HCV antigens present in the sample from the observed binding pattern.
  • the present invention also relates to a composition comprising El 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. 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 El and/or the E2 region as described above.
  • Immunogenic compositions can be prepared according to methods known in the art.
  • compositions comprise an immunogenic amount of a recombinant El and/or E2 and/or E1/E2 single or specific oligomeric proteins as defined above or El 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 El 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 El protein is type-specific.
  • Cocktails including type 1 E2 protein and El proteins derived from several genotypes may be particularly advantageous.
  • Cocktails containing a molar excess of El versus E2 or E2 versus El 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 not limited to : aluminim hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP) as found in U.S. Patent No.
  • alum aluminim hydroxide
  • thr- MDP N-acetyl-muramyl-L-threonyl-D-isoglutamine
  • N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP)
  • N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl-sn- glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE)
  • RIBI 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, MA) or SAF-1 (Syntex) may be used. Further, Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IF A) may be used for non-human applications and research pu ⁇ oses.
  • adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA) or SAF-1 (Syntex) may be used. Further, Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IF A) may be used for non-human applications and research pu ⁇ oses.
  • CFA Complete Freund's Adjuvant
  • IF A 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.
  • 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 El and E2 proteins may also be inco ⁇ orated 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 fall 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 B 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 El and/or E2 at a location corresponding to a hydrophilic region of the protein.
  • Such hydrophylic regions include the VI 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 V5 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 El 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 :
  • 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).
  • the present invention also relates to competition immunoassay formats in which recombinantly produced purified single or specific oligomeric protein El and/or E2 and/or E1/E2 proteins as disclosed above are used in combination with El 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 inte ⁇ retation 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 El 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 El 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 strenght using an excess of antigen.
  • the incubation of the antigen with the specimen is followed by detection of immune complexes comprised of the antigen.
  • 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
  • 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 ImmunolonTM 1 or ImmunlonTM 2 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 test sample is incubated with the combination of antigens in solution. For example, it may be under conditions that will precipitate any antigen-antibody complexes which are formed.
  • Both standard and competitive formats for these assays are known in the art.
  • the amount of 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 fo ⁇ ns 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.
  • particle agglutination (PA) assays are used for the detection of antibodies to various antigens when coated to a support.
  • This assay is the hemagglutination assay using red blood cells (RBCs) that are sensitized by passively adsorbing antigen (or antibody) to the RBC.
  • RBCs red blood cells
  • the addition of specific antigen antibodies present in the body component, if any, causes the RBCs coated with the purified antigen to agglutinate.
  • 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 specififc oligomeric El 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 El 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 not exhibit antibodies to the native HCV antigens, El or E2.
  • 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 El proteins, or parts thereof, more particularly HCV single or specific oligomeric El 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 El protein or a suitable part thereof under conditions allowing the formation of an immunological complex, removing unbound components, calculating the anti-El 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-El titers found in said sample at the start of treatment and/or during the course of treatment.
  • E1-35A (SEQ ID NO:59) spanning amino acids 208 to 227 of the El V2 region (epitope B), IbEl (SEQ ID NO:53) spanning amino acids 192 to 228 of El regions (VI, Cl , and
  • V2 regions (containing epitope B)
  • El-51 (SEQ ID NO:66) spanning amino acids 301 to 320 of the El region
  • El -53 (SEQ ID NO:67) spanning amino acids 313 to 332 of the El C4 region (epitope A)
  • El-55 (SEQ ID NO:68) spanning amino acids 325 to 344 of the El region.
  • 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
  • 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 El protein or El peptide, more particularly an El protein or El 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-El antibodies present in a biological sample, means for detecting the immune complexes formed in the preceding binding reaction, - possibly also an automated scanning and inte ⁇ retation device for inferring a decrease of anti-El titers during the progression of treatment.
  • 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 El 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 serotyping 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: at least one El and or E2 and/or E1/E2 protein or El 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-El antibodies present in a biological sample, means for detecting the immune complexes formed in the preceding binding reaction, possibly also an automated scanning and inte ⁇ retation 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 inco ⁇ oration 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.
  • a solid support such as a membrane strip
  • the present invention provides a method for purifying recombinant HCV single or specific oligomeric envelope proteins selected from El and/or E2 and/or E1/E2 proteins which have been produced by a recombinant process comprising contacting said proteins with a disulphide bond cleavage or reducing agent.
  • the contacting of the method of the invention may be carried out under partial cleavage or reducing conditions.
  • the disulphide bond cleavage agent is dithiothreitol (DTT), preferably in a concentration range of 0.1 to 50 mM, preferably 0.1 to 20 mM, more preferably 0.5 to 10 mM.
  • the disulphide bond cleavage agent may be a detergent, such as Empigen-BB (which is a mixture containing N-Docecyl-N,N-dimethylglycine as a major component), preferably at a concentration of 1 to 10%, more preferably at a concentration of 3.5%. Mixtures of detergents, disulphide bond cleavage agents and/or reducing agents may also be used.
  • disulphide bond reformation is prevented with an SH group blocking agent, such as N-ethylmaleimide (NEM) or a derivative thereof. In a preferred embodiment, the disulphide bond reformation is blocked by use of low pH conditions.
  • NEM N-ethylmaleimide
  • the present invention further provides a method as described herein, further involving the following steps: lysing recombinant El and/or E2 and/or E1/E2 expressing host cells, optionally in the presence of an SH blocking agent such as N-ethylmaleimide (NEM); recovering said HCV envelope proteins by affinity purification such as by means of lectin-chromatography, such as lentil-lectin chromatography, or by means of immunoaffinity using anti-El and/or anti-E2 specific monoclonal antibodies; reducing or cleaving of the disulfide bonds with a disulphide bond cleaving agent, such as DTT, preferably also in the presence of an SH blocking agent, such as NEM or Biotin-NEM; and, recovering the reduced El and/or E2 and/or E1/E2 envelope proteins by gelfiltration and optionally additionally by a subsequent Ni 2+ -IMAC chromatography and desalting step.
  • an SH blocking agent such as N-ethylmaleimide (NEM)
  • the present invention provides a composition containing substantially isolated and/or purified, and/or isolated and/or purified recombinant HCV single or specific oligomeric recombinant envelope proteins selected from El and/or E2 and/or E1/E2, which have preferably been isolated from the methods described herein.
  • the recombinant HCV envelope proteins of the invention have been expressed in recombinant mammalian cells, such as vaccinia, recombinant yeast cells.
  • the present invention provides a recombinant vector containing a vector sequence, a prokaryotic, eukaryotic or viral promoter sequence and a nucleotide sequence allowing the expression of a single or specific oligomeric El and/or E2 and/or E1/E2 protein, in operable combination.
  • the nucleotide sequence of the vector encodes a single HCV El protein starting in the region between amino acid positions 1 and 192 and ending in the region between amino acid positions 250 and 400, more particularly ending in the region between positions 250 and 341, even more preferably ending in the region between position 290 and 341.
  • nucleotide sequence of the vector encodes a single HCV El protein starting in the region between amino acid positions 117 and 192 and ending in the region between amino acid positions 263 and 400, more particularly ending in the region between positions 250 and 326.
  • nucleotide sequence of the vector encodes a single HCV El protein bearing a deletion of the first hydrophobic domain between positions 264 to 293, plus or minus 8 amino acids.
  • the nucleotide sequence of the vector encodes a single HCV E2 protein starting in the region between amino acid positions 290 and 406 and ending in the region between amino acid positions 600 and 820, more particularly starting in the region between positions 322 and 406, even more preferably starting in the region between position 347 and 406 and most preferably starting in the region between positions 364 and 406; and preferably ending at any of amino acid positions 623, 650, 661, 673, 710, 715, 720, 746 or 809.
  • the vector of the present invention in one embodiment, contains a 5'-terminal ATG codon and a 3'-terminal stop codon operably linked to the nucleotide sequence.
  • the vector further contains, in one embodiment, a nucleotide sequence further containing at a factor Xa cleavage site and/or 3 to 10, preferably 6, histidine codons added 3'-terminally to the coding region.
  • the vector of the present invention optionally contains a nucleotide sequence wherein at least one of the glycosylation sites present in the El or E2 proteins has been removed at the nucleic acid level.
  • the present invention provides a nucleic acid containing any one of SEQ ID NOs: 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 vector of the invention may preferably contain a nucleotide sequence containing a nucleic acid containing any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 and 49, or parts thereof.
  • composition of the present invention further contains recombinant HCV envelope proteins which have been expressed or are the expression product of a vector described herein.
  • the present invention provides a host cell transformed with at least one recombinant vector as described herein, wherein the vector contains a nucleotide sequence encoding HCV El and/or E2 and/or E1/E2 protein as described herein in addition to a regulatory sequence operable in the host cell and capable of regulating expression of the HCV El and/or E2 and/or E1/E2 protein. Moreover, the present invention provides a ecombinant El and/or E2 and/or E1/E2 protein expressed by a host cell of the invention.
  • the present invention further provides a method as described herein and containing the following steps: growing a host cell as described herein which has been transformed with a recombinant vector as described herein in a suitable culture medium; causing expression of the vector nucleotide sequence of the vector, as described herein under suitable conditions; lysing the transformed host cells, preferably in the presence of an SH group blocking agent, such as N-ethylmaleimide (NEM); recovering the HCV envelope protein by affinity purification by means of for instance lectin-chromatography or immunoaffinity chromatography using anti-El and/or anti-E2 specific monoclonal antibodies, with said lectin being preferably lentil-lectin, followed by, incubation of the eluate of the previous step with a disulphide bond cleavage agent, such as DTT, preferably also in the presence of an SH group blocking agent, such as NEM or Biotin-NEM; and, isolating the HCV single or specific oligomeric El and/or E2 and/or
  • the present invention provides a composition containing at least one of the following El and/or E2 peptides:
  • El-33 (SEQ ID NO:57) spanning amino acids 193 to 212 of the El region
  • El-35 (SEQ ID NO:58) spanning amino acids 205 to 224 of the El V2 region (epitope B)
  • E1-35A (SEQ ID NO:59) spanning amino acids 208 to 227 of the El V2 region (epitope B)
  • IbEl (SEQ ID NO:53) spanning amino acids 192 to 228 of El regions (VI, Cl, and V2 regions (containing epitope B),
  • El-51 (SEQ ID NO:66) spanning amino acids 301 to 320 of the El region
  • El -53 (SEQ ID NO:67) spanning amino acids 313 to 332 of the El C4 region (epitope A)
  • Env 23 or E2-23 (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 E2 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 547 to 566 of the E2 region (epitope D), Env 13B or E2-13B (SEQ ID NO:82) spanning positions 523 to 542 of the
  • the present invention provides a composition containing 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 antibodies 10D3C4 and 4H6B2, epitope I recognized by monoclonal antibody 17F2C2.
  • the present invention provides an El and/or E2 specific monoclonal antibody raised upon immunization with a composition as described herein.
  • the antibodies of the present invention may be used, for example, as a medicament, for inco ⁇ oration into an immunoassay kit for detecting the presence of HCV El or E2 antigen, for prognosis/monitoring of disease or for HCV therapy.
  • the present invention provides for the use of an El and or E2 specific monoclonal antibody as described herein for the preparation of an immunoassay kit for detecting HCV El or E2 antigens, for the preparation of a kit for prognosing/monitoring of HCV disease or for the preparation of a HCV medicament.
  • the present invention provides a method for in vitro diagnosis of HCV antigen present in a biological sample, containing at least the following steps:
  • the present invention provides a kit for determining the presence of HCV antigens present in a biological sample, which includes at least the following: at least one El and/or E2 specific monoclonal antibody as described herein, preferably in an immobilized form on a solid substrate, a buffer or components necessary for producing the buffer enabling binding reaction between these antibdodies and the HCV antigens present in a biological sample, and optionally a means for detecting the immune complexes formed in the preceding binding reaction.
  • composition of the present invention may be provided in the form of a medicament.
  • the present invention provides a composition, as described herein for use as a vaccine for immunizing a mammal, preferably humans, against HCV, comprising administrating an effective amount of said composition being optionally accompanied by pharmaceutically acceptable adjuvants, to produce an immune response.
  • the present invention provides a method of using the composition, as described herein, for the preparation of a vaccine for immunizing a mammal, preferably humans, against HCV, comprising administrating an effective amount of said composition, optionally accompanied by pharmaceutically acceptable adjuvants, to produce an immune response.
  • the present invention provides a vaccine composition for immunzing a mammal, preferably humans, against HCV, which contains an effective amount of a composition containing an El and/or E2 containing composition as described herein, optionally also accompanied by pharmaceutically acceptable adjuvants.
  • the composition of the present invention may be provided in a form for in vitro detection of HCV antibodies present in a biological sample.
  • the present invention also provides a method of preparing an immunoassay kit for detecting HCV antibodies present in a biological sample and a method of detecting HCV antibodies present in a biological sample using the kit of the invention to diagnose HCV antibodies present in a biological sample.
  • Such a method of the present invention includes at least the following steps: (i) contacting said biological sample with a composition as described herein, preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex with HCV antibodies present in the biological sample,
  • the present invention provides a kit for determining the presence of HCV antibodies present in a biological sample, containing: at least one peptide or protein composition as described herein, preferably in an immobilized form on a solid substrate; a buffer or components necessary for producing the buffer enabling binding reaction between these proteins or peptides and the antibodies against HCV present in the biological sample; and, optionally, a means for detecting the immune complexes formed in the preceding binding reaction.
  • the present invention provides a method of in vitro monitoring HCV disease or diagnosing the response of a patient suffering from HCV infection to treatment, preferably with interferon, the method including: incubating a biological sample from the patient with HCV infection with an El protein or a suitable part thereof under conditions allowing the formation of an immunological complex; removing unbound components; calculating the anti-El titers present in the sample at the start of and during the course of treatment; monitoring the natural course of HCV disease, or diagnosing the response to treatment of the patient on the basis of the amount anti-El titers found in the sample at the start of treatment and/or during the course of treatment.
  • the present invention provides a kit for monitoring HCV disease or prognosing the response to treatment, particularly with interferon, of patients suffering from HCV infection, wherein the kit contains: at least one El protein or El peptide, more particularly an El protein or El peptide as described herein; a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins or peptides and the anti-El antibodies present in a biological sample; and optionally, means for detetecting the immune complexes formed in the preceding binding reaction, optionally, also an automated scanning and inte ⁇ retation device for inferring a decrease of anti-El titers during the progression of treatment.
  • the present invention provides a serotyping 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, including 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 El and/or E2 and/or E1/E2 protein compositions as described herein or at least one of the El or E2 peptide compositions described herein, 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 the heterologous antibodies being conjugated to a detectable label under appropriate conditions; and optionally, (iv) detecting the presence of said immune complexes visually or mechanically (e.g. by means of densitometry, fluorimetry, colorimetry) and inferring the presence of
  • the present invention provides 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 containing: at least one El and/or E2 and/or E1/E2 protein as described herein or an El or E2 peptide as described herein; a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins or peptides and the anti-El antibodies present in a biological sample; optionally, means for detecting the immune complexes formed in the preceding binding reaction, optionally, also an automated scanning and inte ⁇ retation device for detecting the presence of one or more serological types present from the observed binding pattern.
  • the present invention provides a peptide or protein composition as described herein, for immobilization on a solid substrate and inco ⁇ oration 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 described herein.
  • the present invention provides a therapeutic vaccine composition, such as a therapeutic HCV vaccine composition, containing or comprising a therapeutically effective amount of: a composition containing at least one purified recombinant HCV single or specific oligomeric recombinant envelope proteins selected from the group consisting of an El protein, an E2 protein, a part of said El and E2 proteins, an E1/E2 protein complex formed from purified HCV single or specific oligomeric recombinant El or E2 proteins or parts thereof; and optionally a pharmaceutically acceptable adjuvant.
  • a composition containing at least one purified recombinant HCV single or specific oligomeric recombinant envelope proteins selected from the group consisting of an El protein, an E2 protein, a part of said El and E2 proteins, an E1/E2 protein complex formed from purified HCV single or specific oligomeric recombinant El or E2 proteins or parts thereof and optionally a pharmaceutically acceptable adjuvant.
  • Another therapeutic vaccine composition such as a therapeutic HCV vaccine composition, of the invention may be comprising a therapeutically effective amount of a combination of at least two purified HCV single or specific oligomeric recombinant envelope proteins selected from the group consisting of El proteins derived from different HCV genotypes or subtypes, E2 proteins derived from different HCV genotypes or subtypes, parts of said El and E2 proteins, and E1/E2 protein complexes formed from purified HCV single or specific oligomeric recombinant El or E2 proteins, or parts thereof, derived from different HCV genotypes or subtypes; and optionally a pharmaceutically acceptable adjuvant.
  • the HCV envelope proteins of the vaccine, or more particularly the HCV vaccine, of the present invention are optionally produced by recombinant mammalian cells, by recombinant yeast cells, or by or via a recombinant virus.
  • the invention provides a therapeutic vaccine composition, such as a therapeutic HCV vaccine composition, containing or comprising a therapeutically effective amount of a composition comprising at least one of the following El and E2 peptides:
  • E1-35A (SEQ ID NO:59) spanning amino acids 208 to 227 of the El V2 region
  • IbEl (SEQ ID NO:53) spanning amino acids 192 to 228 of El regions VI, Cl, and
  • V2 regions (containing epitope B),
  • Env 67 or E2-67 (SEQ ID NO:72) spanning amino acid positions 397 to 418 of the E2 region (epitope A),
  • Env 69 or E2-69 (SEQ ID NO:73) spanning amino acid positions 409 to 428 of the E2 region (epitope A),
  • Env 23 or E2-23 (SEQ ID NO:86) spanning positions 583 to 602 of the E2 region
  • Env 27 or E2-27 (SEQ ID NO:88) spanning positions 607 to 626 of the E2 region
  • Env 17B or E2-17B (SEQ ID NO:83) spanning positions 547 to 586 of the E2 region
  • Env 13B or E2-13B (SEQ ID NO:82) spanning positions 523 to 542 of the E2 region
  • IGP 1626 spanning positions 192 ⁇ 211 of the El region (SEQ ID NO 112), IGP 1627 spanning positions 204 ⁇ 223 of the El region (SEQ ID NO 113), IGP 1628 spanning positions 216 ⁇ 235 of the El region (SEQ ID NO 114), IGP 1629 spanning positions 228 ⁇ 247 of the El region (SEQ ID NO 115), IGP 1630 spanning positions 240 ⁇ 259 of the El region (SEQ ID NO 116), IGP 1631 spanning positions 252 ⁇ 271 of the El region (SEQ ID NO 117), IGP 1632 spanning positions 264 ⁇ 283 of the El region (SEQ ID NO 118), IGP 1633 spanning positions 276 ⁇ 295 of the El region (SEQ ID NO 119), IGP 1634 spanning positions 288 ⁇ 307 of the El region (SEQ ID NO 120), IGP 1635 spanning positions 300 ⁇ 319 of the El region (SEQ ID NO 121) and IGP 1636 spanning positions
  • therapeutic vaccine compositions may also be considered or regarded as therapeutic HCV vaccine compositions, or as therapeutic compositions or therapeutic HCV compositions, or as compositions or HCV compositions.
  • the present invention provides a method of treating a mammal, such as a human, infected with HCV comprising administering an effective amount of a composition as described herein, such as the above described vaccines or therapeutic compositions, and optionally, a pharmaceutically acceptable adjuvant.
  • a composition as described herein such as the above described vaccines or therapeutic compositions, and optionally, a pharmaceutically acceptable adjuvant.
  • the composition of the invention is administered in combination with or at a time in conjunction with antiviral therapy, either soon prior to or subsequent to or with administration of the composition of the invention.
  • any of the compositions of the invention e.g. a therapeutic HCV vaccine composition, can be used for treating a mammal chronically infected with HCV (a "chronic HCV-infected mammal").
  • the present invention provides a composition, such as a therapeutic HCV composition or a HCV composition, containing or comprising at least one purified recombinant HCV recombinant envelope proteins selected from the group consisting of an
  • the composition contains at least one of the following El and E2 peptides:
  • El-31 (SEQ ID NO:56) spanning amino acids 181 to 200 of the Core/El VI region, El-33 (SEQ ID NO:57) spanning amino acids 193 to 212 of the El region, El-35 (SEQ ID NO:58) spanning amino acids 205 to 224 of the El V2 region (epitope B), E1-35A (SEQ ID NO:59) spanning amino acids 208 to 227 of the El V2 region
  • IbEl (SEQ ID NO:53) spanning amino acids 192 to 228 of El regions VI, Cl, and V2 regions (containing epitope B),
  • El-51 (SEQ ID NO:66) spanning amino acids 301 to 320 of the El region
  • El-53 (SEQ ID NO:67) spanning amino acids 313 to 332 of the El C4 region (epitope
  • Env 69 or E2-69 (SEQ ID NO:73) spanning amino acid positions 409 to 428 of the E2 region (epitope A),
  • Env 23 or E2-23 (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 E2 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 547 to 586 of the E2 region
  • Env 13B or E2-13B (SEQ ID NO:82) spanning positions 523 to 542 of the E2 region (epitope C),
  • IGP 1628 spanning positions 216-235 of the El region (SEQ ID NO:l 14), IGP 1629 spanning positions 228-247 of the El region (SEQ ID NO:l 15), IGP 1630 spanning positions 240-259 of the El region (SEQ ID NO:l 16), IGP 1631 spanning positions 252-271 of the El region (SEQ ID NO:l 17), IGP 1632 spanning positions 264-283 of the El region (SEQ ID NO: 118),
  • IGP 1633 spanning positions 276-295 of the El region (SEQ ID NO:l 19), IGP 1634 spanning positions 288-307 of the El region (SEQ ID NO:120), IGP 1635 spanning positions 300-319 of the El region (SEQ ID NO: 121) and IGP 1636 spanning positions 312-331 of the El region (SEQ ID NO:122), and wherein said peptides may be of recombinant or synthetic origin, and optionally combined with a pharmaceutically acceptable adjuvant.
  • compositions such as a therapeutic HCV composition or HCV composition, of the invention may be comprising a therapeutically effective amount of a combination of at least two purified HCV single or specific oligomeric recombinant envelope proteins selected from the group consisting of El proteins derived from different HCV genotypes or subtypes, E2 proteins derived from different HCV genotypes or subtypes, parts of said El and E2 proteins, and E1/E2 protein complexes formed from purified HCV single or specific oligomeric recombinant El or E2 proteins, or parts thereof, derived from different HCV genotypes or subtypes; and optionally a pharmaceutically acceptable adjuvant.
  • the present invention provides a therapeutic composition or therapeutic vaccine composition or composition for inducing HCV-specific antibodies or for stimulating T-cell activity or for stimulating cytokine secretion or cytokine production.
  • the present invention provides a therapeutic HCV composition or therapeutic HCV vaccine composition or HCV composition for inducing HCV-specific antibodies or for stimulating T-cell activity or for stimulating cytokine secretion or cytokine production.
  • the therapeutic composition according to the present invention such as a therapeutic HCV vaccine composition or therapeutic HCV composition, may also be therapeutically effective in a HCV carrier infected with a HCV genotype different from the
  • the recombinant HCV envelope proteins may be produced by recombinant mammalian cells, recombinant HCV envelope proteins are produced by recombinant yeast cells, or recombinant HCV envelope proteins are produced by or via a recombinant virus.
  • the present invention provides a method of treating a mammal, such as a human, infected with HCV including administering an effective amount of a composition described herein, such as a therapeutic HCV vaccine composition, and, optionally, a pharmaceutically acceptable adjuvant. It will be clear that any of the compositions of the invention, such as a therapeutic HCV vaccine composition, can be used for treating a chronic HCV-infected mammal.
  • the present invention provides a therapeutic composition for inducing HCV- specific antibodies, for stimulating T-cell activity or for stimulating cytokine secretion or cytokine production, said composition containing a therapeutic effective amount of a composition containing at least one purified recombinant HCV single or specific oligomeric recombinant envelope protein selected from the group consisting of an El protein and an E2 protein; and optionally a pharmaceutically acceptable adjuvant.
  • any of the compositions according to this invention may comprise recombinant HCV envelope proteins wherein the cysteines of said recombinant HCV envelope proteins are blocked, or may comprise El and/or E2 peptides wherein the cysteines of said El or E2 peptides are blocked.
  • compositions including the vaccine compositions and therapeutic compositions
  • the compositions may comprise recombinant HCV envelope proteins which are added to said compositions as viral-like particles (VLPs).
  • VLPs viral-like particles
  • an immunogenic composition in particular a HCV immunogenic composition, comprising a recombinant virus allowing expression of at least one HCV recombinant envelope protein chosen from an El protein and/or an E2 protein, and parts of said El and E2 proteins; and, optionally, a pharmaceutically acceptable adjuvant.
  • the invention is envisaging a vaccine composition such as a
  • HCV vaccine composition comprising a recombinant virus allowing expression of at least one HCV recombinant envelope protein chosen from an El protein and/or an E2 protein, and parts of said El and E2 proteins; and, optionally, a pharmaceutically acceptable adjuvant.
  • the above recombinant virus compositions may be effective against a HCV genotype or subtype different from the HCV genotype or subytpe from which said El protein and/or E2 protein, or said parts thereof, are derived.
  • the above recombinant virus compositions may be used for inducing HCV-specific antibodies or for stimulating T-cell activity or for stimulating cytokine secretion or cytokine production.
  • said recombinant virus is a vaccinia virus, a recombinant avipox virus or a recombinant Ankara Modified Virus.
  • Another aspect of the invention relates to a method of treating a mammal infected with HCV comprising administering an effective amount of a recombinant vaccine composition as described above.
  • the mammal in any of the above aspects of the invention may in particular be a human.
  • One further aspect of the present invention relates to a method to reduce liver disease in a chronic HCV-infected mammal or human comprising administering a therapeutic vaccine to said mammal or human.
  • a method to reduce liver fibrosis progression in a chronic HCV- infected mammal or human comprising administering a therapeutic vaccine to said mammal or human is covered.
  • a further aspect relates to a method to reduce liver fibrosis in a chronic HCV- infected mammal or human comprising administering a therapeutic vaccine to said mammal or human.
  • a further aspect relates to a method to reduce liver steatosis in a chronic HCV- infected mammal or human comprising administering a therapeutic vaccine to said mammal or human.
  • Yet another aspect of the invention embodies a method to reduce liver disease by at least 2 points according to the overall Ishak score or Ishak activity score in a chronic HCV- infected mammal or human comprising administering a therapeutic vaccine to said mammal or human.
  • Another further aspect relates to a method to reduce liver disease or liver fibrosis by at least 1 point according to the Ishak fibrosis score in a chronic HCV-infected mammal or human comprising administering a therapeutic vaccine to said mammal or human.
  • a further aspect of the invention provides a method to reduce serum ALT levels in a chronic HCV-infected mammal or human comprising administering a therapeutic vaccine to said mammal or human.
  • Yet another aspect of the invention relates to a method to reduce anti-El and/or anti-E2 immunoreactivity in the liver of a chronic HCV-infected mammal or human comprising administering a therapeutic vaccine to said mammal or human.
  • a method for treating a chronic HCV- infected mammal or human comprising administration of multiple doses of any of the compositions of the invention, such as a therapeutic HCV vaccine composition, to said mammal or human and wherein said multiple doses are administrated to said mammal or human separated by a specified time interval.
  • said plurality of administrations of a composition of the invention to treat a chronic HCV-infected carrier may be separated, e.g., by a time-interval of 4 weeks or less.
  • said time intervals could be 1 or 1.5 or 2 or 2.5 or 3 or 3.5 or 4 weeks, or could be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days.
  • said method of treating a chronic HCV-infected mammal or human comprises a plurality of administrations of a composition of the invention, such as a therapeutic HCV vaccine composition, to said mammal or human wherein said administrations are separated by a time interval of 3 weeks.
  • said plurality of administrations consists of a first series of at least 5 administrations followed by an administration-free period of at least 12 weeks followed by a second series of at least 3 administrations.
  • a series of administrations may comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more administrations.
  • the administration- free period may be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more weeks.
  • compositions of the invention such as a therapeutic HCV vaccine composition
  • Said therapeutic vaccine as applied in the methods described above may be any composition of the current invention, such as a therapeutic HCV vaccine composition.
  • HCV E1/E2 proteins or parts thereof or E1/E2 peptides as outlined throughout the above description for the manufacture of a composition, HCV composition, vaccine, HCV vaccine, therapeutic vaccine or therapeutic HCV vaccine for use in any of the methods outlined above or used for obtaining any of the effects aimed at in the various methods outlined above is also envisaged by the current invention.
  • said therapeutic vaccine comprises at least one HCV antigen and, optionally, a pharmaceutically acceptable adjuvant such as alum.
  • said HCV antigen is an El or E2 antigen, or an immunogenic part of an El or E2 antigen.
  • the Els antigen may be defined by SEQ ID NO: 123.
  • liver disease in this context any abnormal liver condition caused by infection with the hepatitis C virus including inflammation, fibrosis, cirrhosis, necrosis, necro-inflammation and hepatocellular carcinoma.
  • steatosis is meant a histological feature of lipid accumulation in the hepatocytes that is indicative of liver involvement in a wide variety of systemic disorders, toxic or drug-induced liver injury, as well as of various specific liver diseases, including hepatitis C infection, Wilson's disease, and galactosemia.
  • reducing liver disease is meant any stabilization or reduction of the liver disease status. Liver disease can be determined, e.g., by the Knodell scoring system or the Knodell scoring system adapted by Ishak. A reduction of this score by two points is accepted as therapeutically beneficial effect in several studies (see, e.g., studies published after 1996 as indicated in Table 2 of Shiffman 1999).
  • reducing liver fibrosis progression is meant any slowing down, halting or reverting of the normally expected progression of liver fibrosis. Liver fibrosis progression can be determined, e.g., by the Metavir scoring system.
  • Fibrosis Normal expected progression of liver fibrosis according to this system was published to be an increase of the Metavir score of an untreated chronic HCV patient of approximately 0.133 per year (Poynard et al. 1997). Fibrosis is considered to include any form of fibrosis, e.g. as scored by the Metavir or Ishak system, including perisinusoidal fibrosis.
  • HCV antigen any HCV protein or fragment thereof comprising at least one T cell epitope or B cell epitope.
  • a further aspect of the current invention provides a method to predict changes in liver disease in a chronic HCV-infected mammal or human, said method comprising:
  • a sustained high level of serum anti-El antibody could be reached trough additional immunizations either by administering a new series of immunizations after an administration free period or by repeating immunizations with a larger time interval, e.g. 6 weeks, after an initial priming series consisting of administrations with a short time interval, e.g. 3 weeks.
  • a new series of immunizations after an administration free period or by repeating immunizations with a larger time interval, e.g. 6 weeks, after an initial priming series consisting of administrations with a short time interval, e.g. 3 weeks.
  • nucleic acid sequences encoding an El 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 El or E2 proteins as shown in the sequence listing.
  • Figure 22 ELISA results obtained from lentil lectin chromatography eluate fractions of
  • Figure 23 Elution profiles obtained from the lentil lectin chromatography of the 4 different El constructs on the basis of the values as shown in Figure 22.
  • Figure 24 ELISA results obtained from fractions obtained after gelfiltration chromatography of 4 different El purifications of cell lysates infected with wHCV39 (type lb), wHCV40 (type lb), wHCV62 (type 3a), and wHCV63 (type 5a).
  • Figure 25 Profiles obtained from purifications of El proteins of type lb (1), type 3a (2), and type 5a (3) (from RK13 cells infected with wHCV39, wHCV62, and wHCV63, respectively; purified on lentil lectin and reduced as in example 5.2 - 5.3) and a standard (4).
  • Figure 26 Silver staining of an SDS-PAGE as described in example 4 of a raw lysate of
  • Figure 27 Streptavidine-alkaline phosphatase blot of the fractions of the gelfiltration of
  • 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
  • Figure 28 Siver staining of an SDS-PAGE gel of the gelfiltration fractions of vvHCV-
  • 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.
  • Figure 29 Western Blot analysis with anti-El 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 El 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 wHCV44.
  • the dotted line represents the E2 reactivity as detected by ELISA (as in example 6).
  • FIG 31 A OD 280 profile (continuous line) of the lentil-lectin gelfiltration chromatography E2 protein pool from RK13 cells infected with wHCV44 in which the E2 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).
  • Figure 3 IB OD 280 profile (continuous line) of the lentil-lectin gelfiltration chromatography E2 protein pool from RK13 cells infected with wHCV44 in which the E2 pool was reduced and blocked according to Example 5.3
  • the dotted line represents the E2 reactivity as detected by ELISA (as in example 6).
  • Figure 32 Ni 2+ -IMAC chromatography and ELISA reactivity of the E2 protein as expressed from wHCV44 after gelfiltration under reducing conditions as shown in Figure 3 IB.
  • Figure 33 Silver staining of an SDS-PAGE of 0.5 ⁇ g of purified E2 protein recovered by a 200 mM imidazole elution step (lane 2) and a 30mM imidazole wash (lane 1) of the Ni 2+ -IMAC chromatography as shown in Figure 32.
  • Figure 34 OD profiles of a desalting step of the purified E2 protein recovered by 200 mM imidazole as shown in Figure 33, intended to remove imidazole.
  • Figure 35A 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.
  • Figure 35B Antibody levels to the different HCV antigens (NS4, NS5, El 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.
  • FIG. 36 Average El antibody (ElAb) and E2 antibody (E2Ab) levels in the LTR and
  • Figure 37 Averages El antibody (ElAb) levels for non-responders (NR) and long term responders (LTR) for type lb and type 3a.
  • Figure 38 Relative map positions of the anti-E2 monoclonal antibodies.
  • Figure 39 Partial deglycosylation of HCV El envelope protein. The lysate of vvHCVl OA-infected RK13 cells were incubated with different concentrations of glycosidases according to the manufacturer's instructions. Right panel: Glycopeptidase F (PNGase F). Left panel: Endoglycosidase H (Endo H).
  • Figure 40 Partial deglycosylation of HCV E2 envelope proteins.
  • FIG. 41 In vitro mutagenesis of HCV El glycoproteins. Map of the mutated sequences and the creation of new restriction sites.
  • Figure 42A In vitro mutagenesis of HCV El glycoprotein (part 1). First step of PCR amplification.
  • Figure 42B In vitro mutagensis of HCV El glycoprotein (part 2). Overlap extension and nested PCR.
  • Figure 43 In vitro mutagesesis of HCV El glycoproteins. Map of the PCR mutated fragments (GLY-# and OVR-#) synthesized during the first step of amplification.
  • Figure 44A Analysis of El glycoprotein mutants by Western blot expressed in HeLa (left) and RK13 (right) cells.
  • Lane 1 wild type W (vaccinia virus)
  • Lane 2 original El protein (wHCV-lOA)
  • Lane 3 El mutant Gly-1 (wHCV-81)
  • Lane 4 El mutant Gly-2 (wHCV-82)
  • Lane 5 El mutant Gly-3 (wHCV- 83)
  • Lane 6 El mutant Gly-4 (wHCV-84)
  • Lane 7 El mutant Gly-5 (vvHCV-85)
  • Lane 8 El mutant Gly-6 (wHCV-86).
  • Figure 44B Analysis of El glycosylation mutant vaccinia viruses by PCR amplification/restriction.
  • Lane 1 El (wHCV-lOA), BspE I
  • Lane 2 Lane 2
  • Figure 46 SDS polyacrylamide gel electrophoresis of recombinant E2 expressed in a glycosylation deficient S. cerevisiae mutant. Innoculae 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.
  • Figure 47 Profile of chimpanzees and immunization schedule.
  • Figure 48 Cellular response after 3 immunizations.
  • Figure 49 Evolution of cellular response upon repeated E 1 immunizations.
  • Figure 50 Cellular response upon NS3 immunizations.
  • FIG 51 Stimulation index through week 28.
  • the stimulation index (SI; cellular immune response) was obtained by culturing PBMC (10 5 cells), drawn from the individuals before immunization (week 0), two weeks after the third immunization (week 8), before the booster immunization (week 26) and two weeks after the booster immunization (week 28), in the presence or absence of 3 ⁇ g of recombinant Els or 2 ⁇ g tetanos toxoid and determining the amount of tritiated thymidine inco ⁇ orated in these cells during a pulse of 18 hours after 5 days of culture.
  • SI cellular immune response
  • the stimulation index is the ratio of thymidine inco ⁇ orated in the cells cultured with envelope antigen versus the ones cultured without antigen.
  • Samples of week 0 and 8 were determined in a first assay (A), while the samples of week 26 and 28 were determined in a second assay (B) in which the samples of week 0 were reanalyzed. Results are expressed as the geometric mean stimulation index of all 20 (A, experiment) or 19 (B, experiment) volunteers.
  • Figure 52 Cytokine production of PBMCs.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • El recombinant Els
  • TT tetanos toxoid
  • Bl no antigen
  • Cytokines were measured in the supernatant taken after 24 hours (interleukin-5) or after 120 hours (interferon-gamma) by means of ELISA.
  • the stimulation index is the ratio of cytokine measured in the supernatants of cells cultured with envelope antigen versus the ones cultured without antigen. Results are expressed as the geometric mean of pg cytokine/ml secreted of all 19 volunteers. Samples with a cytokine amount below detection limit were assigned the value of the detection limit. Similarly samples with extremely high concentrations of cytokine out of the linear range of the assay were assigned the value of the limit of the linear range of the assay.
  • FIG. 53 Thymidine inco ⁇ oration results.
  • the stimulation index (cellular immune response) was obtained by culturing PBMC (3 xlO 5 cells), in the presence or absence of peptides and determining the amount of tritiated thymidine inco ⁇ orated in these cells during a pulse after 5-6 days of culture.
  • the stimulation index is the ratio of thymidine inco ⁇ orated in the cells cultured with peptide versus the ones cultured without peptide. Results are expressed as individual values for vaccinated persons (top panel) or non vaccinated or controls (lower panel).
  • Figure 54 Three-dimensional graph showing for the individual patients (each represented by a dot) the % change in ALT-levels (absolute change from baseline) on the X-axis, the change in serum anti-El antibody levels (in mU/mL) on the Y-axis, and the change in Ishak fibrosis score on the Z-axis.
  • Figure 55 Three-dimensional graph showing for the individual patients (each represented by a dot) the % change in ALT-levels (absolute change from baseline) on the X-axis, the change in serum anti-El antibody levels (in mU/mL) on the Y-axis, and the change in Metavir fibrosis score on the Z- axis.
  • Figure 56 For each individual patient (represented by a dot or a "*"), the Ishak fibrosis score (on the X-axis) and the ALT values (on the Y-axis) are given. In panel A (top), the baseline (pre- vaccination) situation is given whereas panel B (bottom) is illustrating the situation at the time of taking of liver biopsies. The seven patients with the highest increase in serum anti-El antibody level are represented by a "*”.
  • Figure 57 The influence of the age (on the X-axis) of each individual patient (represented by a dot or a "*") on the Ishak fibrosis score (on the Y-axis) is given.
  • panel A the baseline (pre-vaccination) situation is given whereas panel B (bottom) is illustrating the situation at the time of taking of liver biopsies.
  • the seven patients with the highest increase in serum anti-El antibody level are represented by a "*".
  • Table 1 Features of the respective clones and primers used for amplification for constructing the different forms of the El protein as despected in Example 1.
  • Table 2 Summary of Anti-El tests
  • Table 3 Synthetic peptides for competition studies
  • Table 4 Changes of envelope antibody levels over time.
  • Table 8 Analysis of El glycosylation mutants by ELISA
  • Table 9 Profile of adjuvanted E 1 Balb/c mice.
  • Table 10 Humoral responses: No. of immunizations required for different El- antibodies levels.
  • Table 11 Chimpanzee antibody titers.
  • Table 12 Human antibody titers.
  • Table 13 Human antibody titers (8-28 weeks).
  • Table 14 Stimulation index (SI) of cultured PBMC, drawn from the individuals four weeks (W16) after the fourth immunization and two weeks (W26) after the fifth immunization in the presence or absence of 3 ⁇ g of Els. A stimulation index of >3 is considered a positive signal.
  • SI Stimulation index
  • Table 15 Ishak grading of necro-infiammatory intensities for periportal hepatitis, confluent necrosis, focal inflammation, portal inflammation and the overall total inflammation grading. Scores are indicated as the change from baseline (mean and 95 % confidence interval) and the mean baseline- to end- values.
  • Table 16 Overview of frequencies (given as number of patients) of changes of a given baseline Metavir score (given in top row; Baseline 0 to 4) to a given
  • Example 1 Cloning and expression of the hepatitis C virus El protein
  • 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 ( Figure 1).
  • the plasmid pgsATAl ⁇ 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 H dIII-cut pATA18 vector. This created an extra Pac I restriction site ( Figure 2). The original HmdIII site was not restored.
  • Oligonucleotide linker with SEQ ID NO 1/94 Oligonucleotide linker with SEQ ID NO 1/94:
  • the vaccinia recombination vector pMS66 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 (Sm ⁇ I, Stu I and Pml l/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 P ⁇ c I restriction site was also created downstream the 3'end).
  • This oligonucleotide with SEQ ID NO 2/95 was introduced between the Xm ⁇ I and Pst I sites of pgptATAl 8 ( Figure 3).
  • Oligonucleotide linker with SEQ ID NO 2/95 Oligonucleotide linker with SEQ ID NO 2/95:
  • Pstl Plasmid pgptATA-18 contained within Escherichia coli MCI 061 (lambda) has been deposited under the terms of the Budapest Treaty at BCCM/LMBP (Belgian Coordinated Collections of microorganisms/Laboratorium voor Mole Diagram Biologie Plasmidencollectie, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium), and bears accession number LMBP4486. Said deposit was made on January 9, 2002.
  • 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 reombination vectors: HCC19A (SEQ ID NO:3), HCCllOA (SEQ ID NO:5), HCCll lA (SEQ ID NO:7), HCC112A (SEQ ID NO:9), HCC113A (SEQ ID NO:l l), and HCC117A (SEQ ID NO:13) as depicted in Figure 21.
  • cDNA fragments containing the El -coding regions were cleaved by EcoRI and Hindlll restriction from the respective pSP72 plasmids and inserted into the EcoRI/Hindlll-cut pgptATA-18 vaccinia recombination vector (described in example 1), downstream of the UK vaccinia virus late promoter.
  • the respective plasmids were designated pvHCV-9A, pvHCV-lOA, pvHCV-11A, pvHCV-12A, pvHCV-13A and pvHCV- 17A, of which pvHCV-11 A is shown in Figure 4.
  • 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 (SEQ 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 pSP72HCC137 containing clone HCC137 (SEQ ID 15) was selected.
  • a recombinant vaccinia plasmid containing the full-length El cDNA lacking hydrophobic domain I was constructed by inserting the HCV sequence surrounding the deletion (fragment cleaved by Xma I and BamH I from the vector pSP72-HCC137) into the Xma I-Bam H I sites of the vaccinia plasmid pvHCV-lOA.
  • the resulting plasmid was named pvHCV-37.
  • HCC162 (SEQ ID NO:29) was derived from a type 3a-infected patient with chronic hepatitis C (serum BR36, clone BR36-9-13, SEQ ID NO:19 in WO 94/25601, and see also Stuyver et al. 1993a) and HCC163 (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
  • HCV E2 PCR fragment 22 was obtained from serum BE11 (genotype lb) by means of primers HCPrl09 (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 HCC122A (SEQ ID NO:35) was cut with NcoI/AlwNI or by BamHI/AlwNI and the sticky ends of the fragments were blunted (Ncol 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 El protein that can serve as signal sequence.
  • the same HCV cDNA was inserted into the EcoR I and Bbr 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 pgsATA-18 (pvHCV-43) or pMS-66 vaccinia vectors (pvHCV-44).
  • 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 El protein encoding the signal sequence for E2, and amino acids 384 to 673 of the mature E2 protein.
  • Rabbit kidney RK13 cells (ATCC CCL 37), human osteosarcoma 143B thymidine kinase deficient (TK) (ATCC 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/1) for Hep G2.
  • DMEM Dulbecco's modified Eagle medium
  • EMEM Earle's salts
  • the vaccinia virus WR strain (Western Reserve, ATTC VRl 19) 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).
  • the vaccinia recombination plasmid was fransfected 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 E.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 143B 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 143 B 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 ⁇ l) 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 wHCV-lOA was derived from recombining the wild type WR strain with the pvHCV-lOA 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 .
  • the cell monolayer was washed twice with phosphate-buffered saline pH 7.4 (PBS) and the recombinant vaccinia virus stock was diluted in MEM medium.
  • PBS phosphate-buffered saline pH 7.4
  • the virus solution was aspirated and 2 ml of complete growth medium (see example 2) was added per 10 6 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 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 lOOOg).
  • 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)
  • 50 mM Tris.HCL pH 7.5/ 10 mM EDTA/ 150 mM NaCl for 5 min, and
  • 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 6 cells.
  • lysis buffer 50 mM Tris.HCL pH 8.0, 2 mM EDTA, 150 mM NaCl, 5 mM MgCl 2 aprotinin, 1% Triton X-100
  • the cell lysates were cleared for 5 min at 14,000 ⁇ m 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) methanoi).
  • transfer buffer 25 mM Tris.HCl pH 8.0, 192 mM glycine, 20% (v/v) methanoi).
  • 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.
  • Blotto 5 % (w/v) fat-free instant milk powder in PBS; Johnson et al., 1981
  • 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.
  • Blotto/0.1%) Tween 20 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.
  • the filters were incubated with alkaline phosphatase 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 phosphatase 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.
  • Lysis Infected RK13 cells (carrying El 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.
  • PBS phosphate-buffered saline
  • 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. This lysate was homogenized with a type B douncer and left at room temperature for 0.5 hours.
  • 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)
  • the cleared cell lysate was loaded at a rate of lml/min on a 0.8 by 10 cm Lentil-lectin Sepharose 4B column (Pharmacia) that had been equilibrated with 5 column volumes of lysis buffer at a rate of lml/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, CA, USA) instead of 1% DecylPEG.
  • the bound material was eluted by applying elution buffer (10 mM potassium phosphate pH 7.3, 5% glycerol, 1 mM hexanoic acid, ImM 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 El or E2 protein by means of ELISA as described in example 6.
  • Figure 22 shows ELISA results obtained from lentil lectin eluate fractions of 4 different El purifications of cell lysates infected with vvHCV39 (type lb), wHCV40 (type lb), wHCV62 (type 3a), and wHCV63 (type 5a).
  • Figure 23 shows the profiles obtained from the values shown in Figure 22. These results show that the lectin affinity column can be employed for envelope proteins of the different types of HCV.
  • the El- or E2 -positive fractions were pooled and concentrated on a Centricon 30 kDa (Amicon) by centrifugation for 3 hours at 5,000 ⁇ m in a Beckman JA-20 rotor at 4°C. In some experiments the El- 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, CA, 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 sulphydryl groups.
  • Figure 24 shows ELISA results obtained from fractions obtained after gelfiltration chromatography of 4 different El purifications of cell lysates infected with wHCV39 (type lb), wHCV40 (type lb), wHCV62 (type 3a), and wHCV63 (type 5a).
  • Figure 25 shows the profiles obtained from purifications of El proteins of types lb, 3a, and 5a (from RK13 cells infected with wHCV39, wHCV62, and wHCV63, respectively; purified on lentil lectin and reduced as in the previous examples).
  • the peaks indicated with '1', '2', and '3', represent pure El protein peaks (El reactivity mainly in fractions 26 to 30).
  • the dimeric El protein appeared to be non-aggregated and free of contaminants.
  • the subtype lb El protein purified from wHCV40-infected cells according to the above scheme was aminotenninally sequenced on an 477 Perkin-Elmer sequencer and appeared to contain a tyrosine as first residue. This confirmed that the El 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 El 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 wHCV44 as indicated in Examples 5.1 to 5.4.
  • Figure 30 shows the OD 280 profile (continuous line) of the lentil lectin chromatography. The dotted line represents the E2 reactivity as detected by ELISA (see example 6).
  • Figure 31 shows the same profiles obtained from gelfiltration chromatography of the lentil-lectin E2 pool (see Figure 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.
  • 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 wHCV44. Contaminating proteins either run through the column or can be removed by a 30 mM imidazole wash.
  • Figure 33 shows a silver-stained SDS/PAGE of 0.5 ⁇ g of purified E2 protein and a 30 mM imidazole wash.
  • Figure 34 shows an additional desalting step intended to remove imidazole and to be able to switch to the desired buffer, e.g. PBS, carbonate buffer, saline.
  • desired buffer e.g. PBS, carbonate buffer, saline.
  • secreted E2 protein (constituting approximately 30- 40%), 60-70%o 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-El or anti-E2 antibodies or for the detection of El or E2 proteins
  • Serum samples were diluted 20 times or monoclonal anti-El 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 El 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, 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, Belgium), and color development was obtained by addition of substrate of the Innotest HCV Ab III kit (Innogenetics, Belgium) diluted 100 times in 1 volume of Substrate Solution of the Innotest HCV Ab III kit (Innogenetics, Belgium) for 30 min at 24°C after washing of the plates 3 times with 400 ⁇ l of Washing Solution of the Innotest HCV Ab III kit (Innogenetics, Belgium).
  • DAKO peroxidase-conjugated secondary antibody
  • HCV hepatitis C virus
  • Type lb (wHCV-39, see example 2.5.) and 3a El (wHCV-62, see example 2.5.) proteins were expressed by the vaccinia virus system (see examples 3 and 4) and purified to homogeneity (example 5).
  • anti-El data are shown in Table 2 as average S/N ratios ⁇ SD (mean anti-El titer).
  • the anti-El titer could be deduced from the signal to noise ratio as show in Figures 5, 6, 7, and 8.
  • Anti-El antibody titers had decreased 6.9 times in LTR but only 1.5 times in NR.
  • the anti-El 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-El antibody levels during monitoring of IFN- ⁇ therapy correlates with long- term, sustained response to treatment.
  • the anti-El 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-El 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-El antibodies at the start of treatment, or monitoring the patient during the course of infection and measuring the anti-El titer, may become a useful marker for clinical diagnosis of hepatitis C. Furthermore, the use of more defined regions of the El or E2 proteins may become desirable, as shown in example 7.3.
  • LTR and NR groups and Tables 4 and 5 show the statistical analyses.
  • higher El antibody levels before IFN- ⁇ therapy were associated with LTR (P ⁇ 0.03). Since much higher El antibody levels were observed in type 3a-infected patients compared with type lb-infected patients ( Figure 37), the genotype was taken into account (Table 4).
  • LTR also had higher El antibody levels than NR at the initiation of treatment [P ⁇ 0.05]; the limited number of type 3a-infected NR did not allow statistical analysis.
  • the anti-El ELISA as described in example 6 was modified by mixing 50 ⁇ g/ml of El peptide with the 1/20 diluted human serum in sample diluent.
  • Figure 13 shows the results of reactivity of human sera to the recombinant El (expressed from vvHCV-40) protein, in the presence of single or of a mixture of El peptides.
  • peptides were synthesized using technology described by applicant previously (in WO 93/18054).
  • the following peptides were synthesized: peptide env35 A-biotin
  • NH 2 -SNSSEAADMIMHTPGCV-GKbiotin (SEQ ID NO:51) spanning amino acids 208 to 227 of the HCV polyprotein in the El region peptide biotin-env53 ('epitope A') biotin-GG-ITGHRMAWDMMMNWSPTTAL-COOH (SEQ ID NO:52) spanning amino acids to 313 of 332 of the HCV polyprotein in the E 1 region peptide IbEl ('epitope B')
  • H 2 N-YEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGCGK -biotin (SEQ ID NO:53) spanning amino acids 192 to 228 of the HCV polyprotein in the El region and compared with the reactivities of peptides Ela-BB (biotin-GG-
  • TPTVATRDGKLPATQLRRHIDLL SEQ ID NO:54
  • Elb-BB biotin-GG-
  • TPTLAARDASVPTTTIRRHVDLL SEQ ID NO:55
  • 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 B and none reacted with env35A).
  • Reactivity towards epitopes A, B, and C was tested directly to the biotinylated peptides (50 ⁇ g/ml) bound to streptavidin-coated plates as described in example 6.
  • 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 El 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 Figures 15, 16, 17, and 18, epitopes A and B reacted with the majority of sera.
  • antibodies to the most reactive epitope did not seem to predict remission of disease, while the anti- IbEl antibodies (epitope B) were present almost exclusively in long term responders at the start of IFN treatment.
  • anti-lbEl (epitope B) antibodies and anti-env53 (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 detection of cross-reactive antibodies (antibodies that cross-react between major genotypes) and antibodies to the env53 region may be very useful for universal El 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 5E1 A 10, the sequence -GWD- was present.
  • 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 El-31 as shown in Table 3, is a part of the peptide IbEl.
  • Peptides El-33 and El-51 also reacted to some extent with the murine antibodies, and peptide El-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-El 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-El or anti-E2 protein.
  • the anti- E2 response would clearly blur the anti-El response in an assay measuring both kinds of antibodies at the same time. Therefore, the ability to test anti-envelope antibodies to the single El and E2 proteins, was shown to be useful.
  • HVRI epitope A
  • HVRII epitope B
  • epitope E a third linear epitope region
  • epitope D a fourth linear epitope region
  • 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 El protein encoded by wHCVlOA, and the E2 protein encoded by wHCV41 to 44 expressed from mammalian cells contain 6 and 11 carbohydrate moieties, respectively. This could be shown by incubating the lysate of wHCVlOA-infected or wHCV44-infected RK13 cells with decreasing concentrations of glycosidases (PNGase F or Endoglycosidase H, (Boehringer Mannhein Biochemica) according to the manufacturer's instructions), such that the proteins in the lysate (including El) are partially deglycosylated (Fig. 39 and 40, respectively).
  • PNGase F or Endoglycosidase H (Boehringer Mannhein Biochemica) according to the manufacturer's instructions
  • Mutants devoid of some of their glycosylation sites could allow the selection of envelope proteins with improved immunological reactivity.
  • gpl20 proteins lacking certain selected sugar-addition motifs have been found to be particularly useful for diagnostic or vaccine pu ⁇ ose.
  • 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).
  • 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 et al., 1988; Doms et al., 1993; Helenius, 1994).
  • the isolate S83 belonging to genotype 2c, even lacks the first carbohydrate motif in the VI region (on Asn), while it is present on all other isolates (Stuyver et al., 1994).
  • 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 P, since it is known that NPS or NPT are not frequently modified with carbohydrates. After establishing which carbohydrate-addition motifs are required for folding and/or reactivity and which are not, combinations of such mutations may be made.
  • 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 -CH 2 - group).
  • 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.
  • 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 PCR cycles created the full-length mutant molecule ElGly#, which carries the mutation destroying the glycosylation site number #.
  • the selected clones were analyzed for length of insert by EcoRI/BamH I 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 subconfiuent RK13 cells were infected with the 6 recombinant vaccinia viruses carrying the mutant El sequences, as well as with the wHCV-lOA (carrying the non-mutated El sequence) and wt vaccinia viruses. Cells were lysed after 24 hours of infection and analyzed on western blot as described in example 4 (see Figure 44A).
  • All mutants showed a faster mobility (corresponding to a smaller molecular weight of approximately 2 to 3 kDa) on SDS-PAGE than the original El 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.
  • Figure 44B shows that all mutants (as shown in Figure 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 HCC141 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 ( Figure 45). This resulted in a too high level of heterogeneity and in shielding of reactivity, which is not desirable for either vaccine or diagnostic pu ⁇ oses. 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 ( Figure 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 pu ⁇ oses, 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 dislcosed 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
  • BVDV Diarrhoea Virus
  • HCV Hog Cholera Virus
  • BDV Border Disease Virus
  • 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.
  • Liver disease in chimpanzees chronically infected with HCV can be reduced by immunization with El . Multiple immunizations, however, were required in order to reach a significant immune response.
  • immune modulation which is either orchestrated by the virus itself or by the host. In order to analyze if such an immune modulation does exist in HCV, the immune responses against El and NS3 in naive and chronically infected chimpanzees were compared.
  • this group of animals was selected for a more rigorous immunization schedule including the following: use of an adjuvant proven in mice to be more potent for inducing cellular responses (Table 9) compared to alum, which was the adjuvant used for naive animals; and the immunization schedule for chronically infected animals consisted of 12 immunizations compared to 6 for naive animals (Fig. 47).
  • the number of immunized animals does not allow statistical analysis, the following clear tendency can be detected in the humoral responses (Table 10): the number of immunizations for seroconversion is lower in naive animals; and the magnitude of the immune response is substantially greater in the naive animals, 2/3 infected animals do not reach the level of 10 internal units, even after 12 immunizations.
  • Fig. 48a-d The analysis of the cellular responses, after three immunizations, reveals an even larger difference (Fig. 48a-d), including the following: El -specific T-cell proliferation is almost absent in the chronically infected animals, while a clear stimulation can be seen in the naive setting; IL-2 measurements confirmed that the low stimulation of the T-cell compartment in chronic carriers; and, a clear Th2 (IL-4) response in naive animals is induced, as expected for an alum-adjuvant containing vaccine.
  • IL-4 was noted to remain at a low level compared to the level reached after three immunizations in naive animals.
  • RIBI adjuvant
  • nt nucleotide
  • aa amino acid
  • Kl Klenow DNA Pol filling
  • T4 T4 DNA Pol filling
  • Position amino acid position in the HCV polyprotein sequence Table 1 - continued: Recombinant vaccinia plasmids and viruses
  • nt nucleotide
  • aa amino acid
  • Kl Klenow DNA Pol filling
  • T4 T4 DNA Pol filling Position: amino acid position in the HCV polyprotein sequence
  • LTR Long-term, sustained response for more than 1 year NR : No response, response with relapse, or partial response
  • PROTEIN PEPTIDE AMINO ACID SEQUENCE POSITION SEQ ID NO
  • mice 1 after three s.c./i.m. immunizations, 3 randomly selected mice were analyzed individually, the result is expressed as the mean specific cpm obtained after 4 days of El stimulation (1 ⁇ g/ml), the number in brackets refers to the number of mice with specific stimulation above background
  • Example 12 Immunization of a chimpanzee chronically infected with HCV subtype lb
  • a chimpanzee (Phil) already infected for over 13 years (5015 days before immunization) with an HCV subtype lb strain was vaccinated with El (aa 192-326) which was derived from a different strain of genotype 1 b, with a 95.1% identity on the amino acid level (see also Table 2 of WO 99/67285 the whole of which is incorporated herein by reference), and which was prepared as described in examples 1-3 of WO 99/97285.
  • the chimpanzee received in total 6 intramuscular immunizations of each 50 ⁇ g El in PBS/0.05% CHAPS mixed with RIBI R-730 (MPLA+TDM+CWS) according to the manufacturer's protocol (Ribi Inc. Hamilton, MT).
  • the 6 immunizations were given in two series of three shots with a three week interval and with a lag period of 6 weeks between the two series.
  • the chimpanzee was continuously monitored for various parameters indicative for the activity.of the HCV induced disease. These parameters included blood chemistry, ALT ,AST, gammaGT, blood chemistry, viral load in the serum, viral load in the liver and liver histology.
  • the immune answer to the immunization was monitored both on the humoral and cellular level. During this period the animal was also monitored for any adverse effects of the immunization, such as change in behaviour, clinical symptoms, body weight, temperature and local reactions (redness, swelling, indurations). Such effects were not detected.
  • ALT (and especially gammaGT, data not shown) levels decreased as soon as the antibody level against El reached its maximum (see, Figure 8 of WO 99/67285). ALT rebounded rather rapidly as soon as the antibody levels started to decline, but gammaGT remained at a lower level as long as anti-El remained detectable.
  • E2 antigen in the liver decreased to almost undetectable levels during the period in which anti-El was detectable and the E2 antigen rebounded shortly after the disappearance of these antibodies. Together with the Core and E2 antigen becoming undetectable in the liver, the inflammation of the liver markedly decreased (see also Table 3 of WO 99/67285). This is a major proof that the vaccine induces a reduction of the liver damage, probably by clearing, at least partially, the viral antigens from its major target organ, the liver . The viraemia level, as measured by Amplicor HCV Monitor (Roche, Basel, Switzerland), remained approximately unchanged in the serum during the whole study period.
  • anti-El is in part also directed to discontinuous epitopes but a large proportion is directed against the C4 epitope (+50% of the patient sera), a minor proportion against VI V2 (ranging from 2-10% depending on the genotype), and reactivity against V2V3 was only exceptionally recorded (Maertens et al., 1997).
  • Example 13 Immunization of a chronic HCV carrier with different subtype A chimpanzee (Ton) already infected for over 10 years (3809 days before immunization) with HCV from genotype la was vaccinated with El from genotype lb, with only a 79.3 % identity on the amino acid level (see also Table 2 of WO 99/67285), and prepared as described in the previous examples.
  • the chimpanzee received a total of 6 intramuscular immunizations of 50 ⁇ g El in PBS/0.05% CHAPS each mixed with RIBI R-730 according to the manufacturer's protocol (Ribi Inc. Hamilton, MT).
  • the 6 immunizations were given in two series of three shots with a three week interval and with a lag period of 4 weeks between the two series.
  • the chimpanzee was continuously monitored for various parameters indicative for the activity of the HCV induced disease. These parameters included blood chemistry, ALT, AST, gammaGT, viral load in the serum, viral load in the liver and liver histology.
  • the immune answer to the immunization was monitored both on the humoral and cellular level.
  • ALT levels decreased as soon as the antibody level against El reached its maximum ( Figure 11 of WO 99/67285). ALT and gammaGT rebounded as soon as the antibody levels started to decline, but ALT and gammaGT remained at a lower level during the complete follow up period. ALT levels were even significantly reduced after vaccination (62 + 6 U/l) as compared to the period before vaccination (85 ⁇ 11 U/l). Since less markers of tissue damage were recovered in the serum, these findings were a first indication that the vaccination induced an improvement of the liver disease.
  • anti-El is in part also discontinuous, but a large proportion is directed against he C4 epitope (50% of the patient sera), a minor proportion against V1V2 (ranging from 2-10% depending on the genotype) and exceptionally reactivity against V2V3 was recorded (Maertens et al., 1997).
  • V1V2 ranging from 2-10% depending on the genotype
  • V2V3 exceptionally reactivity against V2V3 was recorded
  • the HCV Els protein (amino acids 192-326) was expressed in Vero cells using recombinant vaccinia virus HCV1 IB.
  • This vaccinia virus is essentially identical to vvHCVl lA (as described in U.S. Patent No. 6,150,134, the entire contents of which is hereby incorporated by reference) but has been passaged from RK13 to Vero cells.
  • the protein was purified (by means of lentil chromatography, reduction-alkylation and size exclusion chromatography) essentially as described in example 9 of PCT/E99/04342 (WO 99/67285), making use of iodoacetamide as alkylating agent for the cysteines.
  • HCV E2deltaHVRI (amino acids 412-715) was expressed in and purified from Vero essentially as described for El using recombinant vaccinia virus HCV101 which has been recombined from pvHCV-101 described in Example 8 of PCT/E99/04342 and wild type vaccinia virus. Also E2deltaHVRI behaves as a particle (measured by dynamic light scattering) after exchange of empigen to betain.
  • antibody titers were determined by ELISA two weeks after the 6 th immunization. A serial dilution of the sample was compared to an in house standard (this in house standard defined as having 1000 mU/ml of El or anti-E2deltaHVR I antibody is a mixture of three sera from HCV chronic carriers selected based on a high anti-envelope titer).
  • the stimulation index which reflects the cellular immune response, was obtained by culturing PBMC, drawn from the animals two weeks after the third immunization, in the presence or absence of envelope antigen and determining the amount of tritiated thymidine inco ⁇ orated in these cells during a pulse of 18 hours after 5 days of culture. The stimulation index is the ratio of thymidine inco ⁇ orated in the cells cultured with envelope antigen versus the ones cultured without antigen. A stimulation index of >3 is considered a positive signal.
  • Example 16 Similar El responses which allowed clearing of infection in chimpanzee can be induced in man
  • Table 12 antibody titers were determined by ELISA two weeks after the third immunization. A serial dilution of the sample was compared to an in house standards (this in house standard defined as having 1000 mU/ml of El or anti-E2deltaHVR I antibody is a mixture of three sera from HCV chronic carriers selected based on a high anti-envelope titer).
  • the stimulation index (cellular immune response) was obtained by culturing PBMC, drawn from the individuals two weeks after the third immunization, in the presence or absence of 1 ⁇ g of Els and determining the amount of tritiated thymidine inco ⁇ orated in these cells during a pulse of 18 hours after 5 days of culture. The stimulation index is the ratio of thymidine inco ⁇ orated in the cells cultured with envelope antigen versus the ones cultured without antigen. A stimulation index of >3 is considered a positive signal.
  • Example 17 Boosting of El responses in vaccinated healthy volunteers
  • the T-cell responses were for the majority of individuals still high after the 20 week interval. Taking in account a normalization to the tetanos response, which is present in most individuals as a consequence of previous vaccinations, there is no change in the geomeatric mean of the stimulation index. After the additional boost, taking in account a normalization to the tetanos response, no change is noted (figure 51). This confirms that a strong T-help response was induced after 3 El immunizations and indicates that these immunizations induced already a very good T-help memory which requires, at leeast for a period of 6 months, no further boosting.
  • the stimulation index (cellular immune response) was obtained by culturing PBMC (10 5 cells), drawn from the individuals before immunization (week 0), two weeks after the third immunization (week 8), before the booster immunization (week 26) and two weeks after the booster immunization (week 28), in the presence or absence of 3 ⁇ g of recombinant Els or 2 ⁇ g tetanos toxoid and determining the amount of tritiated thymidine inco ⁇ orated in these cells during a pulse of 18 hours after 5 days of culture.
  • the stimulation index is the ratio of thymidine inco ⁇ orated in the cells cultured with envelope antigen versus the ones cultured without antigen.
  • Samples of week 0 and 8 were determined in a first assay (A), while the samples of week 26 and 28 were determined in a second assay (B) in which the samples of week 0 were reanalyzed. Results are expressed as the geometric mean stimulation index of all 20 (A, experiment) or 19 (B, experiment) volunteers.
  • Thl cytokine interferon-gamma and Th2 cytokine interleukin-5 were measured in the supematants of the PBMC cultures of samples taken at week 26 and 28 and restimulated with El .
  • the predominant cytokine secreted by the El stimulated PBMC is interferon-gamma. It is highly su ⁇ rising to see that a strong Thl biased response is observed with an alum adjuvanted El, since alum is known to be a Th2 inducer. Once more the results confirm that a good T-cell memory response is induced, as prior to the final boost (week 26) already a very strong response is observed.
  • the interferon-gamma secretion was found to be specific as in an additional experiment we saw no difference in interferon-gamma secretion between El stimulated cell cultures and non-stimulated cell cultures of these volunteers using samples drawn at week 0.
  • PBMC (10 5 cells), drawn from the individuals before the booster immunization (week 26) and two weeks after the booster immunization (week 28), were cultured in the presence of 3 ⁇ g of recombinant Els (El) or 2 ⁇ g of tetanos toxoid (TT) or no antigen (Bl). Cytokines were measured in the supernatant taken after 24 hours (interleukin-5) or after 120 hours (interferon-gamma) by means of ELISA. The stimulation index is the ratio of cytokine measured in the supematants of cells cultured with envelope antigen versus the ones cultured without antigen.
  • Results are expressed as the geometric mean of pg cytokine/ml secreted of all 19 volunteers. Samples with a cytokine amount below detection limit were assigned the value of the detection limit. Similarly samples with extremely high concentrations of cytokine out of the linear range of the assay were assigned the value of the limit of the linear range of the assay.
  • Example 18 Fine mapping of cellular response against El in vaccinated healthy volunteers.
  • IGP1626 YEVRNVSGIYHVTNDCSNSS (amino acid 192-211)(SEQ ID NO:112)
  • IGP1627 TNDCSNSSIVYEAADMIMHT (amino acid 204-223)(SEQ ID NO:l 13)
  • IGP1628 AADMIMHTPGCVPCVRENNS (amino acid 216-235)(SEQ ID NO:114)
  • IGP 1629 PCVRENNSSRCWVALTPTLA (amino acid 228-247)(SEQ ID NO:l 15)
  • IGP1631 SVPTTTIRRHVDLLVGAAAF (amino acid 252-271)(SEQ ID NO:117)
  • IGP 1632 LLVGAAAFCSAMYVGDLCGS (amino acid 264-283)(SEQ ID NO:l 18)
  • IGP 1633 YVGDLCG
  • IGP1636 HITGHRMAWDMMMNWSPTTA (amino acid 312-331)(SEQ ID NO : 122 )
  • PBMC from 14 different healthy donors not vaccinated with Els or 10 donors vaccinated with Els were cultured in the presence of 25 ⁇ g/ml (non vaccinated persons) or 10 ⁇ g/ml (vaccinated persons, samples taken after the third or booster injection) of each peptide separately.
  • the peptides IGP 1627, 1629, 1630, 1631, 1633, 1635 and 1635 all induced significantly higher responses in vaccinated persons compared to non-vaccinated persons.
  • a stimulation index of 3 as cut-off the peptides IGP 1627, 1629, 1631 and 1635 were the most frequently recognized (i.e. recognized by at least half of the vaccinated persons tested).
  • the stimulation index (cellular immune response) was obtained by culturing PBMC (3 xlO 5 cells), in the presence or absence of peptides and determining the amount of tritiated thymidine inco ⁇ orated in these cells during a pulse after 5-6 days of culture.
  • the stimulation index is the ratio of thymidine inco ⁇ orated in the cells cultured with peptide versus the ones cultured without peptide. Results are expressed as individual values for vaccinated persons (top panel) or non vaccinated or controls (lower panel).
  • the present invention also provides therefor, the following El peptides, proteins, compisitions and kits containing the same, nucleic acid sequences coding for these peptides and proteins containing the same, and methods of their manufacture and use, as are generally described herein for other El and related peptides of the present invention.
  • IGP 1636 spanning positions 312 ⁇ 331 of the El region (SEQ ID NO 122).
  • the HCV Els protein (amino acids 192-326 (SEQ 10 NO: 123: YEVRNVSGMYHVTNDCSNSSIVYEAADMIMHTPGCVPCVRENNSSRCWVALTPT LAARNASVPTTTIRRHVDLLVGAAAFCSAMYVGDLCGSVFLVSQLFTISPRRHETV QDCNCSIYPGHITGHRMAWDMMMNW)) was expressed in Vero cells using recombinant vaccinia virus HCV11B. This vaccinia virus is essentially identical to vvHCVl l A (described in, for example, PCT/EP95/03031 and U.S. Patent No.
  • the stimulation index (SI; cellular immune response) was obtained by culturing PBMC, drawn from the individuals four weeks (W16) after the fourth immunization and two weeks (W26) after the fifth immunizationt in the presence or absence of 3 ⁇ g of Els and determining the amount of tritiated thymidine inco ⁇ orated in these cells during a pulse of 18 hours after 5 days of culture.
  • the stimulation index is the ratio of thymidine inco ⁇ orated in the cells cultured with envelope antigen versus the ones cultured without antigen. A stimulation index of >3 is considered a positive signal.
  • the antibody titers were determined by ELISA prior to the first immunization (this was done for each patient on three samples taken at different time points before immunization) and after the fifth immunization (this was done for each patient on two samples: W26 and W28).
  • a serial dilution of the sample was compared to an in-house standard (this in-house standard is, as previously described, defined as having 1000 mU/mL of El antibody is a mixture of three sera from HCV chronic carriers selected based on a high anti-envelope titer). From this analysis it was concluded that, on average, the titer doubled from 331 to 715 mU/mL. This result demonstrates that the humoral arm of the immune response has at least been quantitatively altered.
  • An El vaccine formulated on alum significantly changes both the qualitative and quantitative immune response against El .
  • Such an El based vaccine and/or any of the vaccines described herein may be additionally useful if administered in conjunction with antiviral therapy such but not limited to interferon and alternatively with its combination with ribavirin (i.e., prior to, after or with a composition of the present invention).
  • antiviral therapy such but not limited to interferon and alternatively with its combination with ribavirin (i.e., prior to, after or with a composition of the present invention).
  • Example 20 Effects of therapeutic El-vaccination in chronically infected patients.
  • Example 19 In a first course of this study, 26 patients received 5 doses of 20 ⁇ g Els as described in Example 19. In a second course, 25 of said 26 patients received a further 6 intramuscular doses of 20 ⁇ g Els formulated on 0.13% Alhydrogel in 0.5 mL (as in Example 19). The immunizations of this second series were administered with 3 -week intervals and the first injection was given at week 50. Four weeks after the last injection of the second course, a liver biopsy was performed in 24 out of the 25 patients who completed the two courses of therapeutic Els vaccination. A liver biopsy was also performed in all patients prior to the onset of the therapeutic vaccine treatment (i.e. prior to course 1 vaccination scheme).
  • the mean time elapsed between pre-treatment and post-treatment biopsies was 17 months.
  • Liver histology slides thus obtained before and after therapeutic vaccination were scored centrally in a blind way by two expert pathologists for fibrosis and inflammation, this according to the Ishak and Metavir scoring systems (Ishak et al. 1995 which is a modification of the scoring system of Knodell et al. 1981; Bedossa and Poynard 1996), as well as for anti-E2 immunostain using the IGH222 murine anti-E2 HVRI monoclonal antibody as described in International Patent Application WO99/50301. Perisinusoidal fibrosis was assessed based on staining of collagen with Sirius Red (on liver histology slides).
  • the Ishak scores range from 0 to 18 for grading of inflammation and from 0 to 6 for staging of fibrosis/cirrhosis.
  • the sum of the Ishak inflammation and fibrosis scores comes closest to the Histological Activity Index (HAI; Knodell et al. 1981) which has been widely used.
  • HAI Histological Activity Index
  • the change in Ishak fibrosis score in the 24 patients who followed the two courses of therapeutic Els vaccination is -0.04 (with a 95% confidence interval of -0.60 to +0.68) and with a change from baseline- to end-value from 2.54 (baseline) to 2.50 (end).
  • An overview of the different assessed necro-infiammatory intensities (Ishak scoring) is given in Table 15.
  • the Ishak equivalent of the HAI scores for the treated patients showed a mean absolute change from baseline of -0.17 (with a 95% confidence interval of -1.36 to 1.03) and with a change from baseline- to end-value from 8.88 (baseline) to 8.71 (end). Furthermore, nine (9) out of the 24 patients (38%) improved 2 points or more on the sum of Ishak inflammation- and Ishak fibrosis- scores whereas ten (10) out of the 24 patients remained stable (no change or a change of +1 or -1) and five (5) patients evolved to a worse condition (worsening of 2 points or more).
  • the Metavir scores range from 0 to 3 for grading of inflammation and from 0 to 4 for staging of fibrosis/cirrhosis.
  • the overall progression rate of the Metavir score in an untreated patient is estimated to be 0.133 per year (Poynard et al. 1997).
  • the average progression over the 17-month period based on the linear extension of the baseline score and the estimated duration of the infection (which is reported for 19 out of the 24 patients who followed the two courses of therapeutic Els vaccination) would be 0.20. This correlates well with the published overall rate of progression which would be 0.19 for the 17 month period.
  • Metavir score observed for the treated patients is, however, 0.00 (with a 95%o confidence interval of -0.43 to +0.43) and with a mean baseline- an end-score of 1.67.
  • An overview of Metavir scores at baseline and at the end of the second course treatment is given in Table 16. Unblinded comparison of pre- and post treatment slides revealed that 10 patients had diminished perisinusoidal fibrosis based on Sirius Red staining.
  • the anti-E2 immunostain scores for the treated patients showed a mean absolute change from baseline of -0.75 (with a 95% > confidence interval of -1.64 to 0.14) and with a change from baseline- to end- value from 2.54 (baseline) to 1.79 (end).
  • Serum HCV RNA levels were determined using the Amplicor HCV Monitor kit (Roche, Basel, Switzerland). The serum HCV RNA levels remained unchanged or did not change more than one log from baseline except in 1 patient. This concerned a 37-year old treatment-na ' ive female patient, infected with a genotype la virus and with flu-like symptoms at baseline. HCV-RNA dropped 3 logs from week 8 (after two injections with El) to reach levels below 3000 IU/ml at weeks 16 and 20, in parallel ALT dropped from a single peak value of 400 U/ml at week 8 to normal ALT values at weeks 16, 20 and 24. This was accompanied by disappearance of the flu-like symptoms.
  • a further outcome of the current study is the predictive character of the immune response (in terms of increase in serum anti-Els antibody levels) to treatment for improvement in histological scores (fibrosis and overall) as well as serum ALT levels.
  • Antibody titers were determined by ELISA. A serial dilution of a serum sample was compared to an in house standard (this in house standard defined as having 1000 mU/mL of Els antibody is a mixture of three sera from HCV chronic carriers selected based on a high anti-envelope titer). The detection limit for this assay is 5 mU/mL.
  • Figure 54 illustrates the correlation between changes in Ishak fibrosis score, changes in ALT-levels and changes in anti-Els antibody levels.
  • Figure 55 illustrates the correlation between changes in Metavir fibrosis score, changes in ALT-levels and changes in anti-Els antibody levels.
  • Figure 56 illustrates Ishak fibrosis score versus ALT levels both at the start of the first treatment course (panel A) and at the end of the second treatment course (panel B).
  • Figure 57 illustrates patient's age versus Ishak fibrosis score both at the start of the first treatment course (panel A) and at the end of the second treatment course (panel B). Both Figures 56 and 57 further indicate the seven patients with the highest increase in anti-Els antibody levels induced by the therapeutic vaccination treatment.
  • the increase in antibody levels to the Els vaccination significantly predicted improvement in liver fibrosis (Metavir and Ishak scores), sum of inflammation and fibrosis scores (Ishak), and in ALT levels, even after correcting for any other baseline prognostic variables.
  • the increase in anti-El antibodies after vaccination was also significantly correlated with an increase in T cell proliferation index to El. This study is clearly supportive for a El -based therapeutic vaccination strategy to have the potential to halt disease progression towards liver cirrhosis.
  • Example 21 Effects of therapeutic El-vaccination regimen.
  • Knodell RG Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, Kieman TW,
  • WO 96/04385 (PCT/EP95/03031) - Purified Hepatitis C Vims Envelope Proteins for Diagnostic and Therapeutic Use.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Communicable Diseases (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un procédé permettant de purifier des protéines d'enveloppe simple ou d'oligomères spécifiques du VHC (virus de l'hépatite C) recombinant. Ces protéines sont choisies dans le groupe constitué des E1 et/ou E2 et/ou E1/E2. La caractéristique du procédé est qu'au moment de la lyse des cellules transformées, pour isoler la protéine exprimée par recombinaison, on utilise un agent de clivage de la liaison bisulfure de façon à réaliser une réduction ou un clivage de la liaison bisulfure. L'invention concerne également, d'une part une composition isolée par un tel procédé, d'autre part l'application diagnostic et thérapeutique de ces compositions, et enfin l'utilisation des peptides et de la protéine E1 du VHC pour établir un pronostic ou pour surveiller l'efficacité clinique et/ou l'avantage clinique du traitement contre le VHC.
PCT/EP2002/014480 2001-12-18 2002-12-18 Proteines purifiees du virus de l'hepatite c utilisables en diagnostic et therapie WO2003051912A2 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
CA002468690A CA2468690A1 (fr) 2001-12-18 2002-12-18 Proteines purifiees du virus de l'hepatite c utilisables en diagnostic et therapie
EP02796675A EP1461080A2 (fr) 2001-12-18 2002-12-18 Proteines purifiees du virus de l'hepatite c utilisables en diagnostic et therapie
IL16223602A IL162236A0 (en) 2001-12-18 2002-12-18 Purified hepatitis c virus envelopeproteins for dagnostic and therapeutic use
KR10-2004-7009720A KR20040076869A (ko) 2001-12-18 2002-12-18 진단적 및 치료적 용도를 위한 정제된 ｃ형 간염 바이러스외피 단백질
NZ533396A NZ533396A (en) 2001-12-18 2002-12-18 Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
BR0215081-6A BR0215081A (pt) 2001-12-18 2002-12-18 Proteìnas purificadas do envoltório do vìrus de hepatite c para uso diagnóstico e terapêutico
AU2002361160A AU2002361160B2 (en) 2001-12-18 2002-12-18 Purified Hepatitis C virus envelope proteins for diagnostic and therapeutic use
JP2003552792A JP2005516939A (ja) 2001-12-18 2002-12-18 診断及び治療用途の精製ｃ型肝炎ウイルス外被タンパク質

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US2051001A 2001-12-18 2001-12-18
US10/020,510 2001-12-18
US41835802P 2002-10-16 2002-10-16
US60/418,358 2002-10-16

Publications (3)

Publication Number Publication Date
WO2003051912A2 true WO2003051912A2 (fr) 2003-06-26
WO2003051912A3 WO2003051912A3 (fr) 2004-03-04
WO2003051912A9 WO2003051912A9 (fr) 2004-07-15

Family

ID=26693530

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2002/014480 WO2003051912A2 (fr) 2001-12-18 2002-12-18 Proteines purifiees du virus de l'hepatite c utilisables en diagnostic et therapie

Country Status (12)

Country Link
EP (1) EP1461080A2 (fr)
JP (1) JP2005516939A (fr)
KR (1) KR20040076869A (fr)
CN (1) CN1622828A (fr)
AU (1) AU2002361160B2 (fr)
BR (1) BR0215081A (fr)
CA (1) CA2468690A1 (fr)
IL (1) IL162236A0 (fr)
MX (1) MXPA04005891A (fr)
NZ (1) NZ533396A (fr)
RU (1) RU2319505C2 (fr)
WO (1) WO2003051912A2 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2859221A1 (fr) * 2003-08-28 2005-03-04 Centre Nat Rech Scient Vecteur de co-expression de domaines membranaires de proteines d'enveloppe d'un virus et utilisations
US7048930B2 (en) 2001-04-24 2006-05-23 Innogenetics N.V. Expression of core-glycosylated HCV envelope proteins in yeast
US7101561B2 (en) 2000-12-01 2006-09-05 Innogenetics N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US7108855B2 (en) 1998-06-24 2006-09-19 Innogenetics N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US7413741B2 (en) 2004-03-08 2008-08-19 Innogenetics N.V. HCV E1 comprising specific disulfide bridges
WO2009030872A1 (fr) * 2007-09-07 2009-03-12 Mats Axel Atterdag Persson Matériaux et procédés pour le traitement de l'hépatite c
WO2009147248A2 (fr) * 2008-06-05 2009-12-10 Ablynx N.V. Séquences d'acides aminés dirigées contre des protéines d'enveloppe d'un virus, et polypeptides comprenant ces séquences destinés au traitement de maladies virales
CN102614510A (zh) * 2004-10-18 2012-08-01 全球免疫股份有限公司 基于酵母的对慢性丙型肝炎感染的治疗
CN103732250A (zh) * 2011-06-14 2014-04-16 全球免疫股份有限公司 基于酵母的治疗或预防丁型肝炎病毒感染的组合物和方法
US8945567B2 (en) 2009-06-05 2015-02-03 Ablynx N.V. Monovalent, bivalent and trivalent anti human respiratory syncytial virus (HRSV) nanobody constructs for the prevention and/or treatment of respiratory tract infections
RU2570553C2 (ru) * 2013-11-20 2015-12-10 Федеральное бюджетное учреждение "Всероссийский научно-исследовательский институт лесоводства и механизации лесного хозяйства (ФБУ ВНИИЛМ) Способ очистки вирусных полиэдров
US9644022B2 (en) 2009-11-30 2017-05-09 Ablynx N.V. Amino acid sequences directed against human respiratory syncytial virus (HRSV) and polypeptides comprising the same for the prevention and/or treatment of respiratory tract infections
WO2018058177A1 (fr) * 2016-09-29 2018-04-05 Macfarlane Burnet Institute For Medical Research And Public Health Limited Glycoprotéines assemblées
US10493141B2 (en) 2014-09-17 2019-12-03 The University Of Iowa Research Foundation Viral RNA segments as immunomodulatory agents and vaccine components
WO2020117760A1 (fr) * 2018-12-03 2020-06-11 Duke University Procédé de purification de nanoparticules et de protéines recombinantes

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100365124C (zh) * 2005-10-27 2008-01-30 云南大学 一段丙型肝炎病毒特异性cDNA序列的应用
CN104327170A (zh) * 2014-05-16 2015-02-04 中国疾病预防控制中心病毒病预防控制所 丙型肝炎病毒(hcv)细胞进入抑制肽zte1序列及应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995012677A2 (fr) * 1993-11-04 1995-05-11 Innogenetics N.V. Epitopes de lymphocytes t humains immunodominants du virus de l'hepatite c
WO1999067285A1 (fr) * 1998-06-24 1999-12-29 Innogenetics N.V. Particules de proteines d'enveloppe de vhc: utilisation pour la vaccination

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995012677A2 (fr) * 1993-11-04 1995-05-11 Innogenetics N.V. Epitopes de lymphocytes t humains immunodominants du virus de l'hepatite c
WO1999067285A1 (fr) * 1998-06-24 1999-12-29 Innogenetics N.V. Particules de proteines d'enveloppe de vhc: utilisation pour la vaccination

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7108855B2 (en) 1998-06-24 2006-09-19 Innogenetics N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US7101561B2 (en) 2000-12-01 2006-09-05 Innogenetics N.V. Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US7048930B2 (en) 2001-04-24 2006-05-23 Innogenetics N.V. Expression of core-glycosylated HCV envelope proteins in yeast
US7238356B2 (en) 2001-04-24 2007-07-03 Innogenetics N.V. Core-glycosylated HCV envelope proteins
US7314925B2 (en) 2001-04-24 2008-01-01 Innogenetics N.V. Constructs and methods for expression of recombinant HCV envelope proteins
WO2005024031A1 (fr) * 2003-08-28 2005-03-17 Centre National De La Recherche Scientifique Vecteur de co-expression de domaines membranaires de proteines d'enveloppe d'un virus et utilisations
FR2859221A1 (fr) * 2003-08-28 2005-03-04 Centre Nat Rech Scient Vecteur de co-expression de domaines membranaires de proteines d'enveloppe d'un virus et utilisations
US7413741B2 (en) 2004-03-08 2008-08-19 Innogenetics N.V. HCV E1 comprising specific disulfide bridges
CN102614510A (zh) * 2004-10-18 2012-08-01 全球免疫股份有限公司 基于酵母的对慢性丙型肝炎感染的治疗
WO2009030872A1 (fr) * 2007-09-07 2009-03-12 Mats Axel Atterdag Persson Matériaux et procédés pour le traitement de l'hépatite c
US9193780B2 (en) 2008-06-05 2015-11-24 Ablynx N.V. Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases
CN102112155A (zh) * 2008-06-05 2011-06-29 埃博灵克斯股份有限公司 针对病毒包膜蛋白的氨基酸序列和用于治疗病毒疾病的包含其的多肽
WO2009147248A3 (fr) * 2008-06-05 2010-07-15 Ablynx N.V. Séquences d'acides aminés dirigées contre des protéines d'enveloppe d'un virus, et polypeptides comprenant ces séquences destinés au traitement de maladies virales
US20190077847A1 (en) 2008-06-05 2019-03-14 Ablynx N.V. Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases
AU2009254501B2 (en) * 2008-06-05 2014-07-31 Ablynx N.V. Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases
US11518799B2 (en) 2008-06-05 2022-12-06 Ablynx N.V. Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases
WO2009147248A2 (fr) * 2008-06-05 2009-12-10 Ablynx N.V. Séquences d'acides aminés dirigées contre des protéines d'enveloppe d'un virus, et polypeptides comprenant ces séquences destinés au traitement de maladies virales
US10550174B2 (en) 2008-06-05 2020-02-04 Ablynx N.V. Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases
US9834595B2 (en) 2008-06-05 2017-12-05 Ablynx N.V. Amino acid sequences directed against envelope proteins of a virus and polypeptides comprising the same for the treatment of viral diseases
US9803001B2 (en) 2009-06-05 2017-10-31 Ablynx N.V. Monovalent, bivalent and trivalent anti human respiratory syncytial virus (hRSV) nanobody constructs for the prevention and/or treatment of respiratory tract infections
US8945567B2 (en) 2009-06-05 2015-02-03 Ablynx N.V. Monovalent, bivalent and trivalent anti human respiratory syncytial virus (HRSV) nanobody constructs for the prevention and/or treatment of respiratory tract infections
US11028151B2 (en) 2009-06-05 2021-06-08 Ablynx N.V. Monovalent, bivalent and trivalent anti human respiratory syncytial virus (HRSV) nanobody constructs for the prevention and/or treatment of respiratory tract infections
US9644022B2 (en) 2009-11-30 2017-05-09 Ablynx N.V. Amino acid sequences directed against human respiratory syncytial virus (HRSV) and polypeptides comprising the same for the prevention and/or treatment of respiratory tract infections
US9579377B2 (en) 2011-06-14 2017-02-28 Globeimmune, Inc. Yeast-based compositions and methods for the treatment or prevention of hepatitis delta virus infection
US9987352B2 (en) 2011-06-14 2018-06-05 Globeimmune, Inc. Yeast-based compositions and methods for the treatment or prevention of hepatitis delta virus infection
CN103732250A (zh) * 2011-06-14 2014-04-16 全球免疫股份有限公司 基于酵母的治疗或预防丁型肝炎病毒感染的组合物和方法
RU2570553C2 (ru) * 2013-11-20 2015-12-10 Федеральное бюджетное учреждение "Всероссийский научно-исследовательский институт лесоводства и механизации лесного хозяйства (ФБУ ВНИИЛМ) Способ очистки вирусных полиэдров
US10493141B2 (en) 2014-09-17 2019-12-03 The University Of Iowa Research Foundation Viral RNA segments as immunomodulatory agents and vaccine components
US20190284230A1 (en) * 2016-09-29 2019-09-19 Macfarlane Burnet Institute For Medical Research And Public Health Limited Assembled glycoproteins
CN110049993A (zh) * 2016-09-29 2019-07-23 麦克法兰布奈特医疗研究与公共健康研究所有限公司 组装的糖蛋白
IL265524B (en) * 2016-09-29 2022-10-01 Macfarlane Burnet Institute For Medical Res And Public Health Limited complex glycoproteins
WO2018058177A1 (fr) * 2016-09-29 2018-04-05 Macfarlane Burnet Institute For Medical Research And Public Health Limited Glycoprotéines assemblées
IL265524B2 (en) * 2016-09-29 2023-02-01 Macfarlane Burnet Institute For Medical Res And Public Health Limited complex glycoproteins
US12162906B2 (en) 2016-09-29 2024-12-10 Macfarlane Burnet Institute For Medical Research And Public Health Limited Method for preparing multimeric forms of the hepatitis c virus (HCV) envelope glycoprotein 2 (HCV E2)
WO2020117760A1 (fr) * 2018-12-03 2020-06-11 Duke University Procédé de purification de nanoparticules et de protéines recombinantes

Also Published As

Publication number Publication date
AU2002361160A1 (en) 2003-06-30
CA2468690A1 (fr) 2003-06-26
KR20040076869A (ko) 2004-09-03
EP1461080A2 (fr) 2004-09-29
WO2003051912A3 (fr) 2004-03-04
RU2319505C2 (ru) 2008-03-20
MXPA04005891A (es) 2004-09-13
JP2005516939A (ja) 2005-06-09
WO2003051912A9 (fr) 2004-07-15
NZ533396A (en) 2005-04-29
AU2002361160B2 (en) 2008-07-03
RU2004116914A (ru) 2005-04-20
IL162236A0 (en) 2005-11-20
CN1622828A (zh) 2005-06-01
BR0215081A (pt) 2004-10-19

Similar Documents

Publication Publication Date Title
US6890737B1 (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
AU2002361160B2 (en) Purified Hepatitis C virus envelope proteins for diagnostic and therapeutic use
US7101561B2 (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US20040126395A1 (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
AU2002238502B2 (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US7108855B2 (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
US20030095980A1 (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
AU757962B2 (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
AU708174B2 (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
ZA200405218B (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use.
NZ521299A (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use
ZA200207272B (en) Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use.
JP2004525885A (ja) 診断用および治療用の精製ｃ型肝炎ウイルスエンベロープタンパク質

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 2002361160

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2468690

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 162236

Country of ref document: IL

WWE Wipo information: entry into national phase

Ref document number: 533396

Country of ref document: NZ

Ref document number: 1600/DELNP/2004

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: PA/a/2004/005891

Country of ref document: MX

Ref document number: 2002796675

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2003552792

Country of ref document: JP

Ref document number: 1020047009720

Country of ref document: KR

COP Corrected version of pamphlet

Free format text: PAGE 52/69, DRAWINGS, ADDED

WWE Wipo information: entry into national phase

Ref document number: 2004116914

Country of ref document: RU

WWE Wipo information: entry into national phase

Ref document number: 20028282027

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 2002796675

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 533396

Country of ref document: NZ

WWG Wipo information: grant in national office

Ref document number: 533396

Country of ref document: NZ

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载