WO1999066033A1 - Expression ciblee sur la surface de la proteine d'enveloppe du virus c de l'hepatite modifiee - Google Patents

Expression ciblee sur la surface de la proteine d'enveloppe du virus c de l'hepatite modifiee Download PDF

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WO1999066033A1
WO1999066033A1 PCT/US1999/012665 US9912665W WO9966033A1 WO 1999066033 A1 WO1999066033 A1 WO 1999066033A1 US 9912665 W US9912665 W US 9912665W WO 9966033 A1 WO9966033 A1 WO 9966033A1
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protein
hcv
antibodies
truncated
envelope
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PCT/US1999/012665
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English (en)
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Xavier Forns
Suzanne U. Emerson
Jens Bukh
Robert H. Purcell
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The Government Of The United States Of America, Represented By The Secretary, Department Of Healtha Nd Human Services
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Priority to AU43350/99A priority Critical patent/AU4335099A/en
Publication of WO1999066033A1 publication Critical patent/WO1999066033A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • C07K16/109—Hepatitis C virus; Hepatitis G virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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 is in the field of hepatitis C virus (HCV) vaccines and diagnostic assays.
  • the invention relates to chimeric genes comprising an endoplasmic reticulum signal sequence and a coding sequence which encodes a truncated HCV envelope protein fused at its carboxy terminus to a plasma membrane anchor sequence which serves to direct the truncated HCV envelope protein to the host cell surface. More specifically, the invention relates to expression vectors which comprise these chimeric genes, and DNA based vaccines which employ the expression vectors as immunogens to produce protective antibodies to HCV in a mammal.
  • the invention also relates to the use of host cells expressing truncated envelope protein on their cell surface in diagnostic assays to screen sera for the presence of antibodies to HCV envelope proteins, as antigens in the screening of phage display combinatorial libraries, and as reagents to develop tissue culture systems suitable for testing anti- HCV envelope antibodies for neutralizing activity.
  • HCV is a positive-sense single-stranded RNA virus that belongs to the Flaviviridae family (Houghton M: Hepatitis C viruses. In: Fields BN, Knipe DM, Howley PM, eds. Virology. Volume 1. 3rd ed. Philadelphia: Lippincott-Raven Publishers, 1996; 1035-1058).
  • the 9.6 kb genome contains a single long open reading frame encoding a polyprotein that is cleaved into at least 10 structural and nonstructural proteins.
  • the structural proteins consist of the capsid protein and two envelope glycoproteins (El and E2).
  • the envelope proteins exhibit the greatest genetic heterogeneity (Bukh J.
  • HCV Hepatitis C virus
  • HCV Hepatitis C: global prevalence. Weekly Epidemiological Record 1997; 72:341-348.
  • Individuals with chronic HCV infection are at increased risk for the development of liver cirrhosis and hepatocellular carcinoma (Houghton M: Hepatitis C viruses. In: Fields BN, Knipe DM, Howley PM, eds. Virology. Volume 1. 3rd ed. Philadelphia: Lippincott-Raven Publishers, 1996; 1035-1058).
  • HCV World Health Organization. Hepatitis C: global prevalence. Weekly Epidemiological Record 1997; 72:341-348.
  • Houghton M Hepatitis C viruses. In: Fields BN, Knipe DM, Howley PM, eds. Virology. Volume 1. 3rd ed. Philadelphia: Lippincott-Raven Publishers, 1996; 1035-1058.
  • Despite the near elimination of HCV transmission via blood transfusion it is believed that approximately 25,000 new cases of HCV infection occur every year in the U
  • HCV Hepatitis C: global prevalence. Weekly Epidemiological Record 1997; 72:341-348) and the majority of these lead to chronic infection. Moreover, natural infection with HCV seems not to elicit protective immunity: studies in thalassemic children, as well as studies in experimentally infected chimpanzees, have shown that reinfection with HCV can occur (Farci P, et al, Science (1992); 258:135-140. Lai ME, et al, Lancet (1994); 343: 388-390). Therefore, the development of an effective HCV vaccine is highly desirable.
  • DNA vaccination can induce humoral and cellular immune responses, and therefore, its potential might include both prevention of infection and immunotherapy of chronic infections.
  • One of the advantages of DNA vaccination is its versatility for studying the immunogenicity of multiple constructs containing different coding sequences, while avoiding the necessity of protein expression and purification.
  • the expression in the host of the foreign proteins encoded by DNA sequences facilitates a folding and presentation of the antigens that might better approach that occurring in the natural infection (28).
  • HCV-1 homologous strain
  • HCV-H77 closely related heterologous strain
  • HCV vaccines have focused on the envelope protein(s) since the success of any HCV vaccine will likely depend on the production of antibodies with adequate neutralizing activity.
  • E2 protein contains important neutralization epitopes (Farci P, et al, Proc Natl Acad Sci USA (1996); 93:15394-15399; Shimizu YK, et al, Virologyil996);223:409-412; Choo Q-L, et al, Proc Natl Acad Sci USA (1994); 91: 1294-1298; Houghton M, et al, Minerva Medica: (1997): 656-659; Weiner AJ, et al, Proc Natl Acad Sci U S A (1992);89:3468-3472; Zibert A, et al, Hepatology (1997);25:1245-1249).
  • the E2 protein was demonstrated to be targeted to the cell surface if the carboxy-terminal 29 amino acids of the E2 protein are replaced by a membrane anchor (Cocquerel L, et al, J Virol (1998); 72: 2183-2191).
  • the surface-expressed form of the HCV E2 protein was not tested as an immunogen.
  • the protein conformation and antigen presentation of the E2 protein are dependent on its sequence composition and possibly on glycosylation patterns (and changing the secretory pathway of E2 by targeting the protein to the cell surface might change its glycosylation pattern), it is unclear whether a surface-expressed E2 protein will function effectively as an immunogen.
  • the present invention relates to a chimeric gene which comprises i) an endoplasmic reticulum signal sequence; and ii) a coding sequence which encodes a truncated hepatitis C virus (HCV) envelope protein fused at its carboxy-terminus to a plasma membrane anchor sequence.
  • HCV hepatitis C virus
  • the nucleic acid sequences contained in the chimeric gene may be DNA, cDNA, RNA or any synthetic variant thereof capable of directing host cell synthesis of a polypeptide.
  • the truncated HCV envelope protein encoded by the coding sequence may be an envelope 1 (El) or envelope 2 (E2) protein or the El and E2 proteins coexpressed in tandem.
  • the invention further relates to expression vectors comprising the chimeric genes of the invention and to pharmaceutical compositions and DNA-based vaccines which comprise the expression vector.
  • the invention also relates to methods of preventing or treating HCV in a mammal comprising administering the expression vectors of the invention to a mammal in an amount effective to stimulate the production of protective antibodies.
  • the invention also provides a kit for the treatment or prevention of
  • kits comprising an expression vector of the invention useful as an immunogen in generating protective antibodies to HCV.
  • the invention further relates to the use of the expression vectors of the invention as immunogens to generate antibodies to the truncated envelope protein(s), preferably neutralizing antibodies.
  • the invention therefore relates to the use of such antibodies in passive immonoprophylaxis and to pharmaceutical compositions which comprise these antibodies.
  • the invention also relates to transformation of host cells with expression vectors comprising the chimeric genes of the invention to produce host cells which express truncated HCV envelope protein on their cell surface.
  • the invention further relates to the use of host cells expressing truncated HCV envelope protein on their cell surface as reagents in diagnostic assays to detect antibodies to HCV in biological samples such as sera or as a capture antigen in a panning procedure to obtain genes expressing monoclonal antibodies to HCV from combinatorial phage display libraries.
  • the invention therefore relates to monoclonal antibodies obtained from the screening of phage display libraries with cells expressing truncated HCV envelope protein on their cell surface, to pharmaceutical compositions which comprise these monoclonal antibodies, and to the use of these antibodies in passive immunoprophylaxis.
  • the invention also relates to the infection of host cells which express truncated HCV envelope protein on their cell surface with enveloped viruses that acquire their envelope protein(s) by budding through the plasma membrane.
  • the enveloped virus used to infect the host cell will then incorporate the surface-expressed HCV envelope protein into its envelope when budding from the cell and the resulting pseudoviruses may be used for screening antibodies to HCV envelope protein(s) for neutralizing activity.
  • Figure 1 illustrates the construction of pE2 and pE2surf.
  • pE2 the signal sequence within El and the entire sequence of E2 and p7 (aa 364-809) were cloned into the expression vector pcDNA 3.1. (Invitrogen).
  • pE2surf DNA encoding a truncated form of E2 lacking 31 carboxy-terminal amino acids as well as p7 (aa 384-715) was cloned into pDisplay.
  • This fragment was cloned in frame between a signal peptide sequence (to direct the protein to the secretory pathway) and the platelet-derived growth factor receptor (PDGFR) transmembrane domain sequence (for expression of the protein on the cell surface) included in the vector.
  • CMV human cytomegalovirus immediate-early promoter/enhancer.
  • T7 T7 promoter/priming site.
  • pA polyadenylation signal.
  • Figure 2 shows Western blotting analysis of expressed HCV E2 glycoprotein.
  • pE2 plasmids pE2, pE2surf, or pDisplay
  • cells were lysed and the lysates were submitted to SDS-PAGE.
  • H79 anti-HCV positive plasma
  • cells transfected with pE2 (lane 1) and pE2surf (lane 2) showed a specific protein of the expected size (approximately 68 kD), which did not appear in cells transfected with pDisplay (lane 3).
  • Figure 3 shows detection of expressed HCV E2 glycoprotein via indirect immunofluorescence.
  • staining was performed on fixed and permeabilized cells, cells transfected with pE2 and pE2surf both showed intracytoplasmic expression of HCV E2 glycoprotein.
  • staining was performed on live cells, only cells transfected with pE2surf showed expression of E2 glycoprotein on the cell surface.
  • Figure 4 shows anti-E2 response in BALB-C mice after DNA vaccination with pDisplay, pE2 or pE2surf.
  • Intramuscular injection groups A, B and C.
  • Gene gun groups D, E and F.
  • Anti-E2 optical density values of the 5 mice included in each group are depicted at each time point.
  • Figure 5 shows anti-E2 response in rhesus macaques after DNA vaccination with pDisplay, pE2 or pE2surf.
  • Anti-E2 optical density values of the 5 macaques are depicted at each time point. Empty bars: immunization with pE2; filled bars: immunization with pE2surf; hatched bars: immunization with pDisplay.
  • Figure 6 shows antibodies against synthetic peptides of the HCV E2 glycoprotein in mice that developed anti-E2 by ELISA after vaccination.
  • Overlapping peptides (16 mer) comprising amino acid positions 384 to 809 were tested in an ELISA with prebleed sera (empty bars) and postvaccination sera (filled bars). Mice are identified by a capital letter, which indicates the vaccination group, and a number from 1 to 5, which coincides with the location of the animals at each time point in Figure 4.
  • plasma samples from patient H H77 and H79
  • the position of HVRl and the major reactive epitope described in the text are indicated.
  • the HCV envelope proteins El and E2 are typically retained in the endoplasmic reticulum.
  • the present invention relates to the modification of the El and/or E2 proteins to permit their expression, either alone or in tandem, on the plasma membrane surface of the cell.
  • the present invention relates to chimeric genes compnsing:
  • endoplasmic reticulum (ER) signal sequence is meant a nucleic acid sequence which encodes a continuous stretch of amino acids, typically about 15 to about 25 residues in length, which are known in the art to be generally located at the amino terminus of proteins and are capable of targeting proteins to the endoplasmic reticulum.
  • ER signal sequences are known to those of skill in the art (see, for example, van Heijne, G. J. Mol. Biol.. (1985) 184:99-105) and those of skill in the art would understand that even though their amino acid sequences may vary, such ER signal sequences are functionally interchangeable.
  • ER signal sequences which may be used in the chimeric genes of the invention include, but are not limited to, the 20-carboxy-terminal amino acids of the full-length HCV El protein (amino acids 364-383 of the HCV polyprotem), which serves as the natural signal sequence of the E2 protein or the murine Ig kappa-chain V-J2-C signal peptide sequence contained in the pDisplay vector.
  • the coding sequence contained in the chimeric gene of the invention encodes a truncated HCV envelope protein fused at its carboxy-terminus to a plasma membrane anchor sequence.
  • the anchor sequence causes the truncated envelope protein to become embedded in the plasma membrane of a host cell such that the envelope protein is expressed on the surface of the host cell and is exposed to the extracellular environment.
  • carboxy-terminus of the envelope protein is preferably immediately adjacent to the plasma membrane anchor sequence, the carboxy-terminus of the envelope protein and the anchor sequence may be separated by intervening amino acid sequence.
  • the truncated HCV envelope protein encoded by the coding sequence is a truncated El or E2 protein where it is understood that the full-length El protein consists of amino acids 192-383 of the HCV polyprotem and the full-length E2 protein consists of amino acids 384-746 of the HCV polyprotem.
  • Sequence encoding the full-length HCV El and E2 proteins of HCV isolates can be obtained from computer data bases such as GenBank or EMBL and the truncations described below can be readily introduced into these sequences by methods known to those of skill in the art.
  • the El and E2 sequences used in the chimeric genes of the invention can be obtained from the sequences of infectious clones of HCV genotypes la and lb [see Kolykhalov, A.A. et al (1997) Science 277, 570-574; (1997) and Figures 4 and 14 of copending U.S. Serial No. 09/014,416, hereby incorporated by reference].
  • the amino acid sequences of the truncated envelope proteins of this invention encompass the native amino acid sequences of known HCV isolates as well as conservative substitutions of the native sequences which result in an amino acid sequence that is immunogenically equivalent to the native amino acid sequence.
  • Examples of conservative substitutions of native El or E2 amino acid sequences include the substitution of one polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • Constant substitutions also encompasses one or more residues chemically derivatized by reaction of a functional side group.
  • Examples of such derivatized molecules include but are not limited to, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloracetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-imbenzylhistidine.
  • chemical derivatives those proteins which contain one or more naturally-occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5 -hydroxy lysine may be substituted for lysine; 3- methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.
  • the truncated HCV envelope protein is a truncated E2 protein
  • the approximately 30 carboxy-terminal amino acids of E2 have been identified as an ER retention sequence and its removal and replacement with a plasma membrane anchor sequence is believed to be critical for expression of the truncated E2 protein on the cell surface.
  • the truncated E2 protein contains a truncation of at least the 20 carboxy-terminal amino acids of the full-length E2 protein, more preferably, a truncation of at least the 25 carboxy-terminal amino acids, and most preferably, a truncation of at least about the 30 carboxy-terminal amino acids.
  • the pE2surf protein disclosed in the Examples contains the entire E2 protein minus the 31 carboxy-terminal amino acids.
  • the truncated E2 protein may be further truncated (in addition to the carboxy-terminal truncation described above) at its carboxy-and/or amino terminus so long as the resultant truncated E2 protein is capable of being expressed on the cell surface in a form recognized by anti-E2 antibodies.
  • the E2 protein is further truncated at its carboxy-terminus since the amino terminus (amino acids from 384 to 412 of the HCV polyprotein) of the E2 protein is recognized as a hypervariable region which is believed to contain important epitopes for virus neutralization.
  • the truncated E2 protein is preferably at least about 290 amino acids in length, more preferably at least about 320 amino acids in length and most preferably at least about 330 amino acids in length.
  • the truncated envelope protein is a truncated El protein
  • the truncated El protein contain a truncation of at least the 20 carboxy- terminal amino acids of the full-length El protein, more preferably, a truncation of at least the 30 carboxy-terminal amino acids and most preferably, a truncation of at least about the 35 carboxy-terminal amino acids.
  • the truncated El protein contains a deletion of the 36 carboxy-terminal amino acids of the HCV El protein.
  • the truncated El protein may be further truncated at its carboxy and/or amino terminus so long as the truncated El protein is capable of being expressed on the cell surface in a form recognized by anti- El antibodies.
  • the El protein is at least about 69 amino acids in length, more preferably at least about 123 amino acids in length and most preferably at least about 155 amino acids in length.
  • Assays for determining whether a truncated El or E2 protein is expressed on the cell surface in a form recognized by anti-El or anti-E2 antibodies respectively are known to those of skill in the art and include immunofluorescence or flow cytometry using anti-envelope antibodies.
  • the truncated envelope protein encoded by the coding sequence consists of El and E2 proteins expressed in tandem.
  • the plasma membrane anchor sequence will be fused to the carboxy-terminus of a truncated E2 protein from which at least 20, more preferably, at least 25, and most preferably at least about 30 carboxy-terminal amino acids have been removed.
  • the El protein to be expressed in tandem with the truncated E2 protein may be either a full-length El protein or a truncated El protein containing carboxy-and/or amino terminal truncations.
  • the preferred amino to carboxy order is El protein, E2 protein, plasma membrane anchor sequence sequence.
  • the order of the El and E2 sequences in the chimeric gene may be reversed such that the amino to carboxy order is E2, and, then truncated El fused at its carboxy-terminus to a plasma membrane anchor sequence.
  • each of the El and E2 proteins to be expressed in tandem may each be fused to a plasma membrane anchor sequence at their respective carboxy-terminii.
  • the coding sequence encodes the signal sequence in core, the El and E2 proteins in tandem, the coding sequence encodes the entire El sequence and the sequence of the E2 protein up to amino acid 715 of the HCV polyprotein.
  • E1-E2 protein expression of E1-E2 protein on the surface of a cell in a form recognized by anti-envelope antibodies can be readily assessed by methods known to those of ordinary skill in the art such as immunofluorescence or flow cytometry.
  • plasma membrane anchor sequence as used in the chimeric gene of the invention is meant a nucleic acid sequence which encodes an amino acid sequence that allows for retention of at least part of the protein in the plasma membrane of a cell.
  • a plasma membrane anchor sequence encodes a sequence of hydrophobic amino acids of sufficient length to span the lipid bilayer of the plasma membrane.
  • hydrophobic sequences are known in the art as transmembrane domains and are typically found at the carboxy-terminus of many proteins found on the surface of cells or virions. These transmembrane domains are typically at least 20 to 30 amino acids in length and are followed by charged cytoplasmic domains of varying lengths. It is therefore understood that the plasma membrane anchor sequence encoded by the coding sequence of the invention may contain in addition to a transmembrane domain of a virion or a protein found on the surface of a cell, a cytoplasmic domain.
  • the encoded plasma membrane anchor sequence is at least twenty amino acids in length, more preferably, from about 20 to about 100 amino acids in length, and most preferably, from about 30 to about 70 amino acids in length.
  • plasma membrane anchor sequences include, but are not limited to, hydrophobic transmembrane domains of receptors such as those for insulin and for a number of growth factors including platelet-derived growth factor (PDGF) and epidermal growth factor (EFG), as well as the transmembrane domains of viral proteins that are anchored in the lipid envelope of the intact virion such as the transmembrane domains of the vesicular stomatitis and rabies virus G proteins.
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • Preferred plasma membrane anchor sequences for inclusion in the chimeric genes of the invention are sequences which encode the 50 amino acid transmembrane domain of the PDGF receptor as contained in the pDisplay vector described in the Examples the carboxy-terminal 64 and 37 amino acids respectively of the CD4 and decay accelerating factor (DAF) proteins (these sequences constitute the transmembrane and cytoplasmic domains of the CD4 and DAF proteins and the 49 carboxy-terminal amino acids of the VSV G protein (also constituting the transmembrane and cytoplasmic domains of the VSV G protein).
  • DAF decay accelerating factor
  • transmembrane domains suitable for use as plasma membrane anchor sequences in the chimeric genes of the invention are known or could be readily identified by carrying out carboxy-terminal deletions of known plasma membrane or viral envelope proteins (see, for example, Men et al (J. Virol. (1991) 65; 1400-1407).
  • the present invention therefore relates to insertion of the chimeric gene of the invention into a suitable expression vector that functions in eukaryotic cells, preferably in mammalian cells.
  • suitable it is meant that the vector is capable of carrying and expressing a chimeric gene of the invention.
  • the expression vector therefore contains at least one promoter and any other sequences necessary or preferred for appropriate transcription and translation of the chimeric gene.
  • Preferred expression vectors include, but are not limited to, plasmid vectors.
  • the invention relates to the use of expression vectors containing the chimeric genes of the invention as immunogens to produce protective antibodies to HCV.
  • Direct transfer of the chimeric gene of the invention to a mammal, preferably a primate, more preferably a human, may be accomplished by injection by needle or by use of other DNA delivery devices such as the gene gun.
  • Possible routes of administration of the expression vector include, but are not limited to, intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal and intranasal.
  • chimeric genes comprising envelope proteins of isolates from multiple genotypes of HCV may be administered together to provide protection against challenge with multiple genotypes of HCV.
  • a polycistronic vector in which multiple different chimeric genes may be expressed from a single vector is created by placing expression of each gene under control of an internal ribosomal entry site (IRES) (Molla, a. et al. Nature. 356:255-257 (1992); Gong, S.K. et al. J. of Virol. 263:1651-1660 (1989)).
  • IRS internal ribosomal entry site
  • the expression vectors containing the chimeric genes of the invention may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition.
  • Suitable amounts of material to administer for prophylactic and therapeutic purposes will vary depending on the route selected and the immunogen (truncated El, truncated E2, etc.) administered.
  • the vaccines of the present invention may be administered once or periodically until a suitable titer of anti-HCV antibodies appear in the blood.
  • a suitable amount of expression vector to be used for prophylactic purposes might be expected to fall in the range of from about 1 ⁇ g to about 5 mg, more preferably from about 100 ⁇ g to about 5 mg, and most preferably from about 1 mg to about 2 mg. Such administration will, of course, occur prior to any sign of HCV infection. Further, one of skill in the art will readily understand that the amount of vector to be used will depend on the size and species of animal the vector is to be administered to.
  • a vaccine of the present invention may be employed in sterile liquid forms such as solutions or suspensions.
  • Any inert carrier is preferably used, such as saline or phosphate-buffered saline, or any such carrier in which the expression vector of the present invention can be suitably suspended.
  • the vaccines may be in the form of single dose preparations or in multi-dose flasks which can be utilized for mass- vaccination programs of both animals and humans.
  • specific adjuvants such as CpG motifs (Krieg, A.K. et al.(1995) Nature 374:546 and Krieg et al. (1996)) L Lab. Clin. Med.. 128:128) may prove useful with DNA-based vaccines.
  • the DNA-based vaccines will normally exist as physically discrete units suitable as a unitary dosage for animals, especially mammals, and most especially humans, wherein each unit will contain a predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent.
  • the dose of said vaccine or inoculum according to the present invention is administered at least once.
  • a second or booster dose may be administered at some time after the initial dose.
  • the need for, and timing of, such booster dose will, of course, be determined within the sound judgment of the administrator of such vaccine or inoculum and according to sound principles well known in the art. For example, such booster dose could reasonably be expected to be advantageous at some time between about 2 weeks to about 6 months following the initial vaccination.
  • the chimeric genes of the present invention can also be administered for purposes of therapy, where a mammal, especially a primate, and most especially a human, is already infected, as shown by well-known diagnostic measures.
  • expression vectors containing the chimeric genes of the present invention are used for such therapeutic purposes, much of the same criteria will apply as when they are used as a vaccine, except that inoculation will occur post-infection.
  • the therapeutic agent comprises a pharmaceutical composition containing a sufficient amount of the expression vector so as to elicit a therapeutically effective response in the organism to be treated.
  • the amount of pharmaceutical composition to be administered will, as for vaccines, vary depending on the immunogen contained therein and on the route of administration.
  • the therapeutic agent according to the present invention can thus be administered by, subcutaneous, intramuscular, intradermal or intranasal routes.
  • amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Of course, the actual amounts will vary depending on the route of administration as well as the sex, age, and clinical status of the subject which, in the case of human patients, is to be determined with the sound judgment of the clinician.
  • the therapeutic agent of the present invention can be employed in sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the expression vectors of the present invention can be suitably suspended.
  • the therapeutic agents may be in the form of single dose preparations or in the multi-dose flasks, which can be utilized for mass-treatment programs of both animals and humans.
  • the expression vectors of the present invention when used as therapeutic agents, they may be administered as a single dose or as a series of doses, depending on the situation as determined by the person conducting the treatment.
  • expression vectors containing the chimeric genes of the invention are useful for preparing antibodies to the truncated envelope protein(s) since the truncated envelope proteins expressed on the surface of the cell are believed to be more native than purified recombinantly expressed envelope proteins. These antibodies can then be used directly as antiviral agents or they may be used in immunoassays to detect the presence of the hepatitis C virus in patient sera..
  • a host animal can be immunized using expression vectors containing the chimeric genes of the invention. The host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the envelope protein(s) of the virus particles.
  • the gamma globulin fraction or the IgG antibodies can be obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art.
  • the antibodies are substantially free of many of the adverse side effects, which may be associated with other anti-viral agents such as drugs.
  • the antibody compositions can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the Fc portion of a foreign species antibody or using an antibody of the same species as the host animal, for example, the use of antibodies from human human hybridomas.
  • Humanized antibodies i.e., nonimmunogenic in a human
  • Such chimeric antibodies may contain the reactive or antigen-binding portion of an antibody from one species and the Fc portion of an antibody (nonimmunogenic) from a different species.
  • chimeric antibodies include, but are not limited to, non-human mammal-human chimeras, rodent-human chimeras, murine-human and rat-human chimeras (Robinson et al., International Patent Application 184,187; Taniguchi M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., PCT Application WO 86/01533; Cabilly et al, 1987 Proc. Natl. Acad. Sci. USA 84:3439; Nishimura et al., 1987 Cane. Res. 47:999; Wood et al, 1985 Nature 314:446; Shaw et al., 1988 J.
  • the antibodies or antigen binding fragments may also be produced by genetic engineering.
  • the technology for expression of both heavy and light chain genes in E. coli is the subject of the PCT patent applications; publication number WO 901443, WO901443, and WO 9014424 and in Huse et al., 1989 Science 246:1275- 1281.
  • the antibodies can also be used as a means of enhancing the immune response.
  • the antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody.
  • antibodies reactive with the HCV envelope proteins can be passively administered alone or in conjunction with another anti-viral agent to a host infected with an HCV to enhance the immune response and/or the effectiveness of an antiviral drug.
  • antibodies to the envelope protein(s) can be induced by administered anti-idiotype antibodies as immunogens.
  • a purified antibody preparation prepared as described above is used to induce anti-idiotype antibody in a host animal, the composition is administered to the host animal in a suitable diluent. Following administration, usually repeated administration, the host produces anti-idiotype antibody.
  • antibodies produced by the same species as the host animal can be used or the Fc region of the administered antibodies can be removed.
  • serum or plasma is removed to provide an antibody composition.
  • the composition can be purified as described above for anti- envelope antibodies, or by affinity chromatography using anti-envelope antibodies bound to the affinity matrix.
  • the anti-idiotype antibodies produced are similar in conformation to the authentic envelope amino acid sequence and may be used to prepare an HCV vaccine rather than using an expression vector encoding a truncated envelope protein of the invention.
  • the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutaneously or the like in an effective concentration in a physiologically suitable diluent with or without adjuvant.
  • One or more booster injections may be desirable.
  • the expression vectors containing the chimeric genes of the invention are also intended for use in producing antiserum designed for pre- or post-exposure prophylaxis.
  • a chimeric gene or a mixture of chimeric genes encoding truncated envelope proteins from HCV isolates belonging to different genotypes is administered by injection to human volunteers, according to known methods for producing human antisera.
  • Antibody response to the expressed envelope proteins is monitored during a several-week period following immunization by periodic serum sampling to detect the presence of anti-envelope serum antibodies using immunoassay methods known to one of skill in the art.
  • the antiserum from immunized individuals may be administered as a pre-exposure prophylactic measure for individuals who are at risk of contracting infection.
  • the antiserum is also useful in treating an individual post-exposure, analogous to the use of high titer antiserum against hepatitis B virus for post-exposure prophylaxis.
  • Monoclonal anti-envelope protein antibodies or anti-idiotype antibodies can be produced by methods well known to those of ordinary skill in the art using the expression vectors of the invention as immunogens.
  • monoclonal antibodies specific for HCV envelope protein(s) may be obtained by using host cells transfected with the expression vectors of the invention as panning agents to screen combinatorial phage antibody libraries.
  • the combinatorial libraries to be screened may be constructed from individuals infected with HCV or immunized with recombinantly expressed HCV proteins or with DNA based vaccines encoding such proteins.
  • the monoclonal antibodies obtained by panning combinatorial phage display libraries with cells which express truncated envelope protein on their cell surface may, as described above for the antibodies produced in response to immunization with an expression vector of the invention, be of particular utility therapeutically, prophylactically and as diagnostic reagents.
  • the present invention further relates to the production of host cells expressing envelope protein(s) on their cell surface by culturing a host cell transformed or transfected with the chimeric gene of the invention under conditions such that the truncated envelope protein encoded by the chimeric gene is produced and expressed on the surface of the cell.
  • Suitable host cells from which the chimeric gene of the invention may be expressed include primary cell cultures and tissue culture cells such as Huh7, CHO, Vero and COS-7 cells.
  • the host cells expressing the truncated envelope protein(s) on their cell surface may be used diagnostically as a reagent to screen biological samples such as serum for the presence of anti-envelope antibodies using methods known to one of ordinary skill in the art, including, but not limited to, immunofluoresence and flow cytometry.
  • immunoassays such as Western blots and ELIS
  • truncated envelope protein purified from such lysates in immunoassays such as Western blots and ELIS As.
  • lysis and purification of the host cell envelope proteins would not be as advantageous as using the intact host cell as an antigen since the surface-expressed envelope proteins of the present invention are better antigens and immunogens than intracellularly expressed envelope proteins and are believed to be more native than purified recombinantly expressed envelope proteins.
  • the present invention therefore relates to kits comprising the host cells of the invention.
  • the present invention further relates to the use of host cells which express truncated HCV envelope proteins on their cell surface to develop a tissue culture system for generating pseudovirions useful for identifying antibodies to HCV which exhibit neutralizing activity.
  • the lack of a reliable cell culture system for HCV represents a major obstacle for the study of virus neutralization since the present methods for demonstrating neutralizing activity of anti-HCV antibodies are either indirect (i.e. in vitro binding assays) or prohibitively time-consuming and expensive in that they require incubating antibodies or antiserum with HCV and then injecting the HCV into sero-negative primates such as chimpanzees.
  • the host cells of the invention provide a solution to the lack of a reliable cell culture system for HCV since one may infect the host cells of the invention with an enveloped virus which acquires its envelope by budding through the plasma membrane.
  • Such an enveloped virus when used to infect host cells which express a truncated HCV envelope protein on their surface, would incorporate the truncated HCV envelope protein into its envelope when budding from the cell and this resulting pseudovirus could then be used to test the neutralization activity of antibodies to HCV.
  • the neutralizing activity of antibodies or antiserum may be readily determined by incubating pseudovirions with the antibody or antiserum and measuring the reduction in plaque number in susceptible cells as compared to the plaque number in susceptible cells exposed to pseudovirions not treated with antibody or antiserum (or perhaps treated with pre-immune serum).
  • "Susceptible cells” are cells that the HCV envelope proteins can attach to and the pseudovirion can replicate in. Such pseudovirions could therefore be incorporated in a kit or used as a diagnostic reagent.
  • Preferred envelope viruses for use in infecting the host cells of the invention are viruses which grow well in cell culture (and in particular, in many types of cells) and form easily detectable plaques.
  • the virus utilized to infect the host cell and thereby incorporate the expressed truncated HCV envelope protein into its envelope is a virus which typically contains at least one envelope protein in its virion.
  • the host cell would preferably be infected with a virus that typically contains only a single envelope protein in its virion such as the vesicular stomatitis virus (VSV).
  • VSV vesicular stomatitis virus
  • the host cell has been transfected with a chimeric gene encoding both El and E2 proteins such that two HCV envelope proteins are expressed on the surface of the host cell
  • the host cell is preferably infected with viruses which have two envelope proteins such as members of the alphaviruses family including Sinbis virus.
  • HCV proteins encoded by these plasmids had sequences identical to those of an infectious cPNA clone of strain H77 (genotype la ) (Yanagi M, et al, Proc Natl Acad Sci USA (1997); 94: 8738- 8743).
  • One construct (pE2) contained the signal sequence located at the carboxy terminus of El and the entire E2 and p7 genes of HCV (aa 364-809) in the pcONA3.1 expression vector (Invitrogen, Carlsbad, CA) (Fig. 1).
  • the other construct contained a truncated form of E2 (aa 384-715) lacking the carboxy-terminal hydrophobic domain.
  • the truncated E2 sequence was cloned, in frame, into the pPisplay expression vector (Invitrogen) between a leader sequence which targeted the HCV protein to the secretory pathway that determines posttranslational modifications, and the transmembrane domain of the platelet-derived growth factor receptor (PPGFR), that anchored the HCV protein to the plasma membrane (Fig. 1).
  • the pE2 surf construct replaced the 31 C-terminal amino acids of the E2 protein with the transmembrane domain of PPGFR.
  • the inserts of the two HCV constructs were amplified by PCR from plasmids containing the full-length sequence of strain H77 ) (Yanagi M, et al, Proc Natl Acad Sci USA (1997); 94: 8738-8743) using the following primers.
  • Amplifications were performed from 2 ⁇ l of plasmid ONA (0.1 ng/ ⁇ l) in a reaction mixture of 5 ⁇ l 10X KlenTaq PCR buffer (Clontech, Palo Alto, CA), 1.25 ⁇ l 10 mM dNTPs (Pharmacia, Uppsala, Sweden), 1 ⁇ l 10 ⁇ M sense primer, 1 ⁇ l 10 ⁇ M antisense primer, 1 ⁇ l 5 OX Advantage KlenTaq Polymerase mix (Clontech), and water to a final volume of 50 ⁇ l.
  • the PCR was performed in a Robocycler thermal cycler (Stratagene, La Jo 11a, CA) for 25 cycles with denaturation at 99 °C for 35 sec, annealing at 67 °C for 30 sec and elongation at 68°C for 2 min.
  • the E2-p7 region of HCV was amplified using a sense primer (SEQ. IP. No: 4) containing an Nhel restriction site and an antisense primer (SEQ. IP. NO: 1) containing a HmdIII restriction site and two termination codons immediately following the carboxy terminus of p7 (aa 809) (Table 1).
  • the PCR products were digested with Mel and H dlLI (New England Biolabs, Beverly, MA) and cloned into the digested expression vector pcONA3.1, by using T4 ligase (Promega, Madison, WI).
  • the truncated form of E2 was amplified using a sense primer containing (SEQ. IP. NO: 2) a BgUl restriction site and an antisense primer (SEQ. IP. NO: 3) containing a Pstl restriction site as shown above.
  • SEQ. IP. NO: 2 a sense primer containing (SEQ. IP. NO: 2) a BgUl restriction site and an antisense primer (SEQ. IP. NO: 3) containing a Pstl restriction site as shown above.
  • the PCR products digested with Bgl ⁇ l and Pstl were cloned into the digested expression vector pOisplay.
  • E. coli P ⁇ 5 alfa library-competent cells (Gibco/BRL, Gaithersburg, MP) were transformed and plated on LB agar containing ampicillin (100 ⁇ g/ml) (Sigma, St Louis, MO). Clones containing the correct insert were grown at 37 °C in the presence of ampicillin.
  • ONA was prepared from 100 ml of bacterial cultures with the modified alkaline lysis method with Endofree Plasmid Purification Kit (Qiagen, Hilden, Germany) to remove endotoxins. Sequence analysis (both strands of plasmid PNA) confirmed that the HCV constructs encoded the authentic HCV protein.
  • HCV E2 protein in mammalian cells COS-7 and Huh7 cells were cultured in the presence of Oulbecco's modified Eagle's medium (GIBCO/BRL) containing 10% bovine serum (Bio Whittaker, Walkersville, MO) and penicillin-streptomycin (Sigma). Cells were transfected at 60-80% confluency with pE2, pE2surf or with pPisplay (without HCV sequences) using Superfect Reagent (Qiagen), according to the manufacturer's instructions. After about 48 hours cells were tested for the expression of HCV glycoprotein E2 by Western blot analysis and indirect immunofluorescence. Western blotting Transfected cells grown in 60 mm dishes were lysed (lysis buffer: 1%
  • NP40 1 mM EPTA, 50 mM Tris HC1, pH 7.5
  • SPS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • membranes were incubated with a goat anti-human immunoglobulin conjugated to horseradish peroxidase (dilution 1:5000) (Pierce, Rockford, IL) for 1 hour at room temperature. After washing, the membranes were incubated with substrate (Supersignal Chemiluminescent Substrate, Pierce) and exposed to X-ray films.
  • substrate Supersignal Chemiluminescent Substrate, Pierce
  • E2 glycoprotein After transfection with pE2, pE2surf or pPisplay, cells were fixed and permeabilized with cold acetone for 5 min. Thereafter, cells were incubated for 40 min at room temperature with a 1:100 dilution of either plasma H79 or plasma from an anti-HCV negative blood donor. After washing, cells were incubated with a fluorescein-isothiocyanate (FITC)-conjugated goat anti-human IgG (1 :100 dilution) (Pierce) for 30 min at room temperature. After washing, slides were mounted and examined for immunofluorescence. Immunofluorescence staining for cell surface-expressed E2 was performed as described above, except that cells were neither fixed nor permeabilized (live cells).
  • FITC fluorescein-isothiocyanate
  • mice Six groups, of 5 BALB-C mice each (6 weeks old), were used in this study. All animals received humane care in compliance with the National Institutes of Health's guidelines. Groups A, B and C were immunized by intramuscular injection of plasmid PNA and groups P, E and F were immunized intraepidermally by gene gun. Group A and P mice were negative controls and were immunized with p Pisplay, groups B and E were immunized with pE2, and groups C and F mice were immunized with pE2surf. All mice were immunized at weeks 0, 3 and 6. Mice were bled at weeks 0, 3, 6, 8, 11 and 14.
  • mice from the intramuscular group were injected with 25 ⁇ l of sterile saline containing 25 ⁇ g of plasmid PNA.
  • PNA bullets were generated. Briefly, 200 ⁇ g of plasmid PNA were vortexed gently with 100 mg of 1.6 micron gold particles (Bio-Rad) in a solution of calcium chloride and spermidine. PNA was allowed to precipitate onto the particles at room temperature for 15 min. The particles were washed three times in cold 100% ethanol to remove unbound PNA and resuspended in ethanol at a particle concentration of 7 mg/ml.
  • the PNA gold particles were deposited onto the inside wall of Tefzel tubing (2.3 mm, I.P., McMaster-Carr, Inc.) by rotation and allowed to dry. The tubing was cut into 1.25 mm lengths to make the bullets and stored desiccated at - 20 °C.
  • Mice were injected with PNA-coated particles from four bullets in well- separated sites on the shaved abdomen, using an Accell gene delivery device at a pressure of 400 psi helium. Each mouse received approximately 8 ⁇ g of PNA.
  • Rhesus macaques were injected intramuscularly into the hiccps femoris muscle with 1 ml of endotoxin-free PBS containing 1 mg of PNA.
  • the EIA uses polystyrene beads, coated with recombinant HCV E2 antigen (genotype la) that was expressed in Chinese hamster ovary cells.
  • Sera to be tested were diluted 1:41 in specimen diluent (50 mM Tris HC1, 10% fetal calf serum, 0.2% Triton X, 0.1 % sodium azide, pH 7.5). After washing, samples were incubated with horseradish peroxidase-labeled anti-mouse antibodies or anti-monkey antibodies. Following washing, chromagen was added and optical densities were read at 492 nni in a Quantum II spectrophotometer (Abbott Laboratories).
  • the positive cut-off was established at 3 times the optical density value of samples from negative control mice or monkeys.
  • a 1:41 dilution of sera in specimen diluent was progressively diluted in control mouse sera and tested as described above.
  • Sera obtained from the mice following vaccination (week 14) and macaques (week 10) were tested for reactivity against the E2 glycoprotein by immunofluorescence, in Huh7 cells transfected with pE2, pE2surf or pPisplay, as described above.
  • the sera were tested at a 1/50 dilution and a FITC -conjugated goat anti-mouse IgG (Pierce) at a 1/100 dilution was used as a secondary antibody.
  • mice pre- and post-vaccination sera (week 14) and macaques (week 10), as well as plasmas H77 and H79 obtained from patient H, were tested for reactivity against the 52 peptides at a dilution of 1/350 for mice and at 1/50 for macaques (1/150 for H77 and H79) by standard ELISA (Lofstrand Laboratories, Rockville, MP). All tests were performed in duplicate. The positive cut-off value was established at 3 times the optical density value of the negative control sample (pre- vaccination sera for mice and macaques, sample H77 for patient H).
  • HCV expression vectors Two HCV expression vectors were constructed: pE2, which contained sequences of the entire E2 and p7 genes of HCV preceded by the signal sequence within El (aa 364-809) and pE2surf, which contained a truncated form of E2 (aa 384- 715) that excluded the last 31 amino acids of E2 and p7 (Fig 1).
  • pE2surf the carboxy-terminal hydrophobic domain of E2 was replaced by the transmembrane domain of the PPGFR (Fig. 1).
  • the HCV proteins encoded by these plasmids have amino acid sequences identical to those of an infectious cPNA clone of strain H77 (genotype la ) (Yanagi M, et al, Proc Natl Acad Sci USA (1997); 94: 8738-8743).
  • the ability of pE2 and pE2surf to express E2 glycoprotein in mammalian cells was tested following transfection of the plasmids into Huh7 and COS-7 cells. About two days after transfection, cells were lysed and the lysates were submitted to SOS-PAGE. Because of the extra protein sequences added to the truncated E2surf, both E2 and E2surf have a similar size.
  • HCV E2 glycoprotein When cells were treated with acetone, HCV E2 glycoprotein was detected in the cytosol of cells transfected with pE2, as well as in cells transfected with pE2surf, but not in cells that had been transfected with the control plasmid pPisplay (Fig 3). When live cells were stained (no fixation/permeabilization steps), the E2 glycoprotein was detected on the surface of cells transfected with pE2surf, but not in cells transfected with pE2 or with the control plasmid (Fig 3). Therefore, truncated E2 was expressed on the cell surface and recognized by human antibodies produced during a natural infection.
  • mice were injected intramuscularly or intraepidermally with pE2, pE2surf or pPisplay at weeks 0, 3 and 6 and tested for antibodies against E2 by ELISA at weeks 0, 3, 6, 8, 11 and by immunofluorescence at week 14.
  • pE2surf surface-expressed E2
  • Fig 4C None of the animals that were inoculated intramuscularly with the intracellular form of E2 (pE2) or the control plasmid (pPisplay) developed anti-E2 antibodies (Fig. 4A and B).
  • mice that were injected via gene gun a more uniform response was observed in terms of antibody production.
  • ELISA Fig. 4P, E, and F
  • immunofluorescence data not shown
  • all 5 animals vaccinated with pE2 and all 5 animals vaccinated with pE2surf developed anti-E2 antibodies whereas none of the control mice developed anti-E2.
  • all 10 mice developed anti-E2 within the gene gun group, compared with only 2 of 10 mice within the intramuscular group (p ⁇ 0.01).
  • There was an excellent correlation between results obtained by ELISA and those obtained by immunofluorescence for detection of anti-E2 data not shown).
  • mice positive for anti-E2 by ELISA recognized both the E2 protein expressed intracellularly and on the cell surface.
  • mouse E5 from group E which tested borderline negative for anti-E2 by ELISA, was positive by immunofluorescence.
  • the remaining 18 mice negative for anti-E2 by ELISA were also negative by immunofluorescence.
  • mice vaccinated via gene gun In mice vaccinated via gene gun, the surface-expressed E2 glycoprotein elicited an earlier and stronger immune response compared to the intracellular form of E2.
  • Three of 5 animals (designated F1-F5) were positive for anti-E2 after the first immunization with pE2surf (week 3) and all of them had anti-E2 after the second immunization.
  • animals vaccinated with the intracellular form of E2 did not develop antibodies before the second immunization and only 2 animals were positive for anti-E2 after the second immunization.
  • the relative anti-E2 titers after completing the immunization schedule are shown in Table 1.
  • Table 1 Relative ELISA anti-E2 titers in mice vaccinated via gene gun* a .
  • mice inoculated with pE2surf had higher titers of anti-E2 at all time points compared to the animals inoculated with pE2.
  • a recombinant E2 glycoprotein directed to the cell surface was shown to be more immunogenic than an intracellular form following DNA immunization in mice.
  • the animals immunized with pE2surf developed an earlier and stronger humoral immune response than those immunized with pE2.
  • these differences did not reach statistical significance, probably because only a few animals were included in each group. Similar results were obtained in rhesus macaques, which were immunized only by intramuscular injection.
  • peptide 520 was recognized as a major linear epitope in 4 of the 10 anti- E2 positive mice vaccinated via gene gun (mouse E2 vaccinated with pE2 and mice F3, F4 and F5 vaccinated with pE2surf).
  • Sera from two additional anti-E2 positive mice (El and F2) recognized several other linear epitopes.
  • major linear epitopes could not be identified (data from mouse E5, which was positive only by immunofluorescence and negative for anti-E2 by ELISA are not shown).
  • Sera from the vaccinated mice that were negative for anti-E2 did not react specifically with any of the peptides (data not shown).
  • Antibodies to the linear epitopes representing the HVRl region of HCV were not detected. (Fig. 6).
  • mice vaccinated with the surface-expressed E2 (via gene gun) recognized the same major linear epitope as recognized by plasma H79 from patient H and from a significant proportion of HCV infected patients (Mink et al (1994) Virology 200: 246-255). Since this linear epitope was immunogenic in our HCV E2 surface-expressed protein, one might speculate that antigenic presentation, hence conformation of E2 on the cell surface, resembled that of the E2 protein contained in an HCV virion.
  • a major linear epitope was recognized by se m of one macaque (Tl 19) vaccinated with pE2surf; this epitope was represented by peptide 640 (aa 640- 655). Sera from the remaining monkeys did not react with any of the peptides (data not shown).
  • HCV hepatitis C virus
  • chimpanzees with a positive anti-E2 immune response are challenged with a virulent HCV monoclonal pool.

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Abstract

La présente invention décrit comment les protéines d'enveloppes du virus de l'hépatite C tronquées peuvent être ciblées sur la membrane plasmatique et comment les formes tronquées de la protéine d'enveloppe exprimée à la surface de la cellule sont plus immunogènes que la protéine d'enveloppe exprimée au niveau intracellulaire. En outre, les cellules hôtes exprimant une forme tronquée de la protéine d'enveloppe sur leur surface cellulaire présentent une grande utilité comme antigènes dans les dosages diagnostiques pour détecter la présence d'anticorps anti-VHC, comme agent pour la détection de bibliothèques de combinaisons pour identifier les anticorps monoclonaux spécifiques à la protéine ou aux protéines d'enveloppes du virus de l'hépatite C et comme système de culture tissulaire pour générer des pseudovirions permettant d'identifier les anticorps qui présentent une activité de neutralisation.
PCT/US1999/012665 1998-06-18 1999-06-04 Expression ciblee sur la surface de la proteine d'enveloppe du virus c de l'hepatite modifiee WO1999066033A1 (fr)

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EP1783218A1 (fr) * 2004-06-25 2007-05-09 Advanced Life Science Institute, Inc. Arn du vhc ayant une nouvelle sequence
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Publication number Priority date Publication date Assignee Title
US6992032B2 (en) 1999-09-29 2006-01-31 Phillips Petroleum Company Organometal catalyst compositions
WO2002010417A2 (fr) * 2000-08-02 2002-02-07 Xencor Procedes et compositions pour la construction et l'utilisation de virus a enveloppe comme particules de presentation
WO2002010417A3 (fr) * 2000-08-02 2002-10-17 Xencor Inc Procedes et compositions pour la construction et l'utilisation de virus a enveloppe comme particules de presentation
EP1783218A1 (fr) * 2004-06-25 2007-05-09 Advanced Life Science Institute, Inc. Arn du vhc ayant une nouvelle sequence
EP1783218A4 (fr) * 2004-06-25 2009-02-18 Advanced Life Science Inst Inc Arn du vhc ayant une nouvelle sequence
US20130323282A1 (en) * 2010-11-26 2013-12-05 Heidi Drummer Compositions and methods
US9884109B2 (en) * 2010-11-26 2018-02-06 The Macfarlane Burnet Institute For Medical Research And Public Health Ltd Compositions and methods

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