WO1999066033A1 - Surface targeted expression of a modified hepatitis c virus envelope protein - Google Patents

Abstract

The present invention discloses that truncated HCV envelope proteins can be targeted to the plasma membrane and that truncated forms of the envelope protein expressed on the cell surface were more immunogenic than intracellularly expressed envelope protein. Further, host cells expressing a truncated form of the envelope protein on their cell surface are disclosed as useful as antigens in diagnostic assays to detect the presence of anti-HCV antibodies, as a panning agent for screening combinatorial libraries to identify monoclonal antibodies specific for HCV envelope protein(s), and as a tissue culture system for generating pseudovirions useful for identifying antibodies which exhibit neutralizing activity.

Description

TITLE OF INVENTION

SURFACE TARGETED EXPRESSION OF A MODIFIED HEPATITIS C VIRUS

ENVELOPE PROTEIN

FIELD OF INVENTION

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.

BACKGROUND OF INVENTION

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. et al, Proc Natl Acad Sci USA (1993); 90:8234-8238; Okamoto H, et al, Virology (1992); 188: 331-341) and a region in the amino terminal end of E2 has been shown to bε extraordinarily heterogeneous (hypervariable region 1 [HVRl]) (Hijikata M, et al, Biochem Biophvs Res Commun (1991); 175: 220-228; Weiner AJ, et al, Virology 1991; 180: 842-848). In addition, a protein known as p7 is found as a fusion with E2 as well as a cleaved species, but it is not known whether p7 is a structural or nonstructural protein (Rice MR:, Virology, (1996); 931-960). Hepatitis C virus (HCV) is one of the main etiological agents of chronic liver disease worldwide (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). About 170 million people globally (4 million people in the U.S.) are chronically infected with HCV (World Health Organization. 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). 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.S. (World Health Organization. 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.

In recent years, vaccination with DNA has been shown to elicit protective immune responses against several pathogens (Fynan EF, et al, Proc Natl Acad Sci USA (1993); 90:11478-11482. Wang B. et al. Proc Natl Acad Sci USA (1993); 90:4156-4160. Sedegah M, et al. Proc Natl Acad Sci USA (1994): 91:9866- 9870. Xiang ZQ, et al, Virology (1994); 199:132-140. Michel ML, et al, Proc Natl Acad Sci USA (1995); 92:5307-5311. Sizemore DR, et al, Science (1995): 270:299- 302. Bourne N, J Infect Pis (1996); 173:800-807), including human immunodeficiency virus and flaviviruses (Boyer JD, et al, Nature Med (1997); 3:526- 532. Schmaljohn C, et al, J Virol (1997); 71 :9563-9569). 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. In addition, 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).

The analysis of the immune response against the structural proteins of HCV generated by injection of DNA constructs into mice has therefore become an important approach for vaccine development; (Major ME, et al, J Virol (1995); 69:5798-5805; Tokushige K, et al, Hepatology 1996; 24:14-20; Inchauspe G, et al, Vaccine (1997); 15:853-856; Lagging LM, et al, J Virol (1995); 69:5859-5863; Nakano I, et al, J Virol (1997); 71 :7101-7109;Saito T, et al, Gastroenterology (1997); 112:1321-1330). These studies have shown that DNA immunization of mice can generate antibodies against the structural proteins of HCV (Major ME, et al, J Virol (1995); 69:5798-5805; Tokushige K, et al, Hepatology 1996; 24:14-20; Inchauspe G, et al, Vaccine (1997); 15:853-856; Lagging LM, et al, J Virol (1995); 69:5859-5863; Nakano I, et al, J Virol (1997); 71:7101-7109;Saito T, et al, Gastroenterology (1997); 112:1321-1330; Tedeschi V, et al, Hepatology (1997); 25:459-462). However, while an experimental vaccine produced from expressed envelope glycoproteins has been shown to induce protection in chimpanzees against a low dose challenge with an homologous strain (HCV-1) (Choo Q-L, et al, Proc Natl Acad Sci USA (1994); 91 : 1294-1298), challenge with a closely related heterologous strain (HCV-H77) resulted in infection (Houghton M, et al, Minerva Medica; (1997): 656-659). Thus, the development of an effective vaccine against HCV remains a great challenge.

To date, the development of HCV vaccines has 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. In particular, a major focus has been the E2 protein because it is believed that E2 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).

It is well established that the E2 glycoprotein expressed alone or with El (E1-E2 heterodimer) is retained in the endoplasmic reticulum (Rice MR:, Virology, (1996); 931-960). However, secreted forms of the envelope E2 protein were expressed by removing the hydrophobic domain of the protein at the carboxy-terminus (Nakano I, et al, J Virol (1997); 71:7101-7109; Saito T, et al, Gastroenterology (1997); 112:1321-1330). More recently, 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). However, the surface-expressed form of the HCV E2 protein was not tested as an immunogen. Further, since 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.

SUMMARY OF INVENTION

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.

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

HCV, the kit 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. BRIEF DESCRIPTION OF FIGURES

Figure 1 illustrates the construction of pE2 and pE2surf. For 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). For 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. Two days after transfection of Huh7 cells with plasmids pE2, pE2surf, or pDisplay, cells were lysed and the lysates were submitted to SDS-PAGE. When stained with anti-HCV positive plasma (H79), 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. Two days after transfection of Huh7 cells with plasmids pE2, pE2surf or pDisplay, cells were examined by indirect immunofluorescence after staining with anti-HCV positive plasma H79. When staining was performed on fixed and permeabilized cells, cells transfected with pE2 and pE2surf both showed intracytoplasmic expression of HCV E2 glycoprotein. When 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. Arrows: time of vaccination. 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. As a reference, plasma samples from patient H (H77 and H79) were tested for linear epitopes. The position of HVRl and the major reactive epitope described in the text are indicated.

DESCRIPTION OF INVENTION

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.

In particular, the present invention relates to chimeric genes compnsing:

(i) an endoplasmic reticulum signal sequence; and

(ii) a coding sequence which encodes a truncated HCV envelope protein fused at its carboxy- terminus to a plasma membrane anchor sequence.

By 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. Such 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. Examples of 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. Of course, those skilled in the art would readily understand that while the 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.

In one embodiment, 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. Alternatively, 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]. Of course, it is understood that 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.

"Conservative 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. Also included as chemical derivatives are 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.

Where 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. Thus, 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.

For example, the pE2surf protein disclosed in the Examples contains the entire E2 protein minus the 31 carboxy-terminal amino acids.

Of course, it is understood that 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. Preferably, 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. However, since three immunogenic domains have been mapped within the E2 protein to amino acids 384 to 443, amino acids 504 to 554, and amino acids 609 to 674 of the HCV polyprotein (Nakano et al, J. of Virol. (1997) 71:7101-7109) it may be desirable to avoid deletions of the carboxy-terminus which extend in further than amino acid 674. Of course, amino-terminal truncations may also be made such as deleting the hypervariable region in an attempt to reveal more conserved epitopes.

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.

Where the truncated envelope protein is a truncated El protein, it is preferred that 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. In a preferred embodiment, the truncated El protein contains a deletion of the 36 carboxy-terminal amino acids of the HCV El protein.

Of course, as for the truncated E2 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. Preferably, 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. In an alternative embodiment, the truncated envelope protein encoded by the coding sequence consists of El and E2 proteins expressed in tandem.

Where the El and E2 proteins are to be expressed in tandem from the chimeric gene of the invention, 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.

In one embodiment, 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. In such an embodiment, the preferred amino to carboxy order is El protein, E2 protein, plasma membrane anchor sequence sequence. In an alternative embodiment, 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.

In yet another embodiment, 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.

In a preferred embodiment, where 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.

As with the truncated El or E2 proteins, 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.

By "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. At a minimum, a plasma membrane anchor sequence encodes a sequence of hydrophobic amino acids of sufficient length to span the lipid bilayer of the plasma membrane. Such 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.

Perferably, 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. Examples of 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.

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).

Of course, one of ordinary skill in the art would readily understand that other 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. By 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.

In yet another embodiment, 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.

Since the existence of different genotypes with a low degree of homology within the envelope proteins diminishes the hope of identifying conserved neutralization epitopes (Bukh J, et al, Sem Liver Pis (1995); 15:41-63), it is likely that a polyvalent vaccine will be needed to generate broadly reactive neutralizing antibodies. Thus, in a preferred embodiment, 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.

Accordingly, those of ordinary skill in the art would readily understand that multiple copies of different chimeric genes may be inserted into a single vector such that a host cell transformed or transfected with the vector will produce multiple envelope proteins. For example, 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)).

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. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. 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 μg to about 5 mg, more preferably from about 100 μg 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. Of course, 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. In order to increase the antibody level, 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. Subsequent doses may be administered as indicated. 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. When 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. Thus, when the expression vectors of the present invention are used as therapeutic agents in the treatment of 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. Of course, 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. One skilled in the art will certainly appreciate that the 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. Of course, when the expression vectors of the present invention are 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.

In addition to use as a vaccine, it is believed that 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.. To prepare antibodies, 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) may be produced, for example, by replacing an immunogenic portion of an antibody with a corresponding, but nonimmunogenic portion (i.e., chimeric antibodies). 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. Examples of 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. Natl. Cancer Inst. 80:15553, all incorporated herein by reference). General reviews of "humanized" chimeric antibodies are provided by Morrison S., 1985 Science 229:1202 and by Oi et al., 1986 BioTechniques 4:214. Suitable "humanized" antibodies can be alternatively produced by CDR or CEA substitution (Jones et al., 1986 Nature 321 :552; Verhoeyan et al., 1988 Science 239:1534; Biedleret al. 1988 J. Immunol. 141 :4053, all incorporated herein by reference).

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. Thus, 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.

Alternatively, antibodies to the envelope protein(s) can be induced by administered anti-idiotype antibodies as immunogens. Conveniently, 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. To eliminate an immunogenic response to the Fc region , antibodies produced by the same species as the host animal can be used or the Fc region of the administered antibodies can be removed. Following induction of anti- idiotype antibody in the host animal, 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.

When used as a means of inducing anti-HCV virus antibodies in an animal, 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. Here 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. For both in vivo use of antibodies to envelope proteins and anti- idiotype antibodies and diagnostic use, it may be preferable to use monoclonal antibodies. 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. Alternatively, 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.

Methods for producing combinatorial phage antibody libraries and for panning such libraries using intact cell surfaces as antigens are known to those of ordinary skill in the art. 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. Of course, one could use lysates of the host cells as antigens in immunoassays such as Western blots and ELIS As or alternatively, use truncated envelope protein purified from such lysates in immunoassays such as Western blots and ELIS As. However, one of skill in the art would understand that 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.

At present, 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. Of course, 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.

Where a single truncated HCV envelope protein is expressed on the surface of the host cell, 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).

Alternatively, where 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.

Any articles or patents referenced herein are hereby incorporated by reference. The following examples illustrate various aspects of the invention but are in no way intended to limit the scope thereof.

EXAMPLES

MATERIAL AND METHODS The original source of the HCV constructs used in this study was an acute phase plasma sample (H77) from patient H, who had posttranfusion hepatitis C (Feinstone SM, et al, J. Infect Pis (1981); 144: 588-598). For detection of E2 glycoprotein by immunoblot and immunofluorescence, we used plasma (H79) from patient H, obtained in the chronic phase two years after the onset of HCV infection. Plasmid construction, amplification and purification

Two expression vectors were prepared with HCV inserts downstream from the CMV immediate-early enhancer-promoter. The 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 (pE2surf) 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). Thus, 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.

For pE2,

SEQ. IP. NO: 4 (sense) 5' CGTCGCT GCATGGTGGGGAACTGGGCGAAGGTCCTGG 3' and SEQ. IP. NO: 1 (antisense)

5' ACGCGT GCΓTTTACTATGCGTATGCCCGCTGAGGCAACGCC 3'; and for pE2surf,

SEQ. IP. NO: 2 (sense)

5 ' ACGCGT G ΓCΓGAAACCCACGTCACCGGGGGAAATGCC

3'; and

SEQ. IP. NO: 3 (antisense)

5' ACGCGTCTGC GCTTAATGGCCCAGGACGCGATGCTTG 3' The restriction sites in the primers are shown in italics; start and stop codons are shown in bold.

Amplifications were performed from 2 μl of plasmid ONA (0.1 ng/μl) in a reaction mixture of 5 μl 10X KlenTaq PCR buffer (Clontech, Palo Alto, CA), 1.25 μl 10 mM dNTPs (Pharmacia, Uppsala, Sweden), 1 μl 10 μM sense primer, 1 μl 10 μM antisense primer, 1 μl 5 OX Advantage KlenTaq Polymerase mix (Clontech), and water to a final volume of 50 μl. 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. The PCR products digested with Bglϊl and Pstl (Promega) were cloned into the digested expression vector pOisplay.

E. coli PΗ5 alfa library-competent cells (Gibco/BRL, Gaithersburg, MP) were transformed and plated on LB agar containing ampicillin (100 μg/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. Expression of 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) for 20 min at 4 °C. After a short centrifugation (14,000 rpm for 5 min at 4°C), the cell lysates were submitted to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SPS-PAGE). Proteins were then transferred to nitrocellulose membranes (Novex, San Piego, CA) by using a Trans-Blot cell (Bio Rad, Hercules, CA). Membranes were incubated with plasma H79 (1 :750 dilution) overnight at 4 °C. Puplicate membranes were incubated with plasma (1:750 dilution) from an anti-HCV negative blood donor. Following standard washing procedures, 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.

Immunofluorescence

Cells grown in 4-well tissue culture chambers were screened for the expression of E2 glycoprotein after transfection with pE2, pE2surf or pPisplay. For detection of intracellularly expressed E2, 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).

PNA immunization in mice and rhesus macques

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.

Each hind leg muscle of mice from the intramuscular group was injected with 25 μl of sterile saline containing 25 μg of plasmid PNA. For the gene gun group, PNA bullets were generated. Briefly, 200 μg 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 8μg of PNA. Rhesus macaques were injected intramuscularly into the hiccps femoris muscle with 1 ml of endotoxin-free PBS containing 1 mg of PNA. Two animals (H403, H392) were immunized with pE2, two (L143, Tl 19) with pE2surf and the remaining animal (T120) with pPisplay. PNA immunization by the intramuscular route was repeated after 8 weeks; animals were bled weekly. Petection of specific HCV antibodies For detection of anti-E2 antibodies, an experimental EIA (Abbott Laboratories, North Chicago, IL) was used (Lesniewski R, et al, J Med Virol

(1995);45:415-422). 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. For titration of anti-E2, 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. Peptide synthesis and epitope mapping

A series of 52 peptides (16 mers; the last peptide being an 18 mer) beginning at aa 384 of HCV and encompassing the entire consensus sequence of the E2 and p7 proteins of H77 was synthesized (Pioneer Peptide Synthesis System Multiple Peptide Synthesis accessory; Perseptive Biosystems, Inc). Each peptide overlapped the adjacent peptide by 8 amino acids. All synthesized peptides were purified by HPLC.

Mouse 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).

Statistical analysis The Fisher's exact test was used to compare frequencies for categorical variables and the Mann- Whitney test to analyze differences for quantitative variables.

EXAMPLE 1

Expression of HCV E2 glycoprotein in mammalian cells

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). In 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. Immunostaining with chronic phase plasma (H79) from patient H showed a band of the predicted size (68kP) in lane 1 and lane 2 (transfected with pE2 or pE2surf, respectively), that was absent from lane 3 (transfected with the control plasmid pPisplay) (Fig. 2). Therefore, both E2 constructs expressed a protein that was immunoreactive with antibody to HCV. The cellular location of the expressed E2 glycoproteins was determined by indirect immunofluorescence microscopy. About two days after transfection, cells were stained with the H79 plasma. 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.

EXAMPLE 2

Specific antibody response to HCV E2 glycoprotein in mice and macaques.

BALB-C 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. Among the mice that were injected intramuscularly, 2 of the 5 animals inoculated with the surface-expressed E2 (pE2surf) developed anti-E2 antibodies (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). Among the mice that were injected via gene gun, a more uniform response was observed in terms of antibody production. In fact, as measured by ELISA (Fig. 4P, E, and F) or 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. In toto, 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). Sera from all 11 mice positive for anti-E2 by ELISA recognized both the E2 protein expressed intracellularly and on the cell surface. In addition, 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.

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. In contrast, animals vaccinated with the intracellular form of E2 (mice E1-E5) did not develop antibodies before the second immunization and only 2 animals were positive for anti-E2 after the second immunization. The ELISA optical density values were significantly higher in the group vaccinated with the surface-expressed E2 compared with the group vaccinated with the intracellular E2 at week 8 (p=0.03), and the differences reached near statistical significance at weeks 6 and 11 (p= 0.055 in both cases). 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.

Week postvaccination 8 11 14 mice immunized with pE2

El 656 2624 1312 E2 1312 2624 1312 E3 41 656 656 E4 164 656 656 E5 20.5 20.5 20.5 Mean 214 571 433 mice immunized with pE2 surf

FI 1312 2624 1312

F2 656 1312 656

F3 656 5248 2624

F4 2624 5248 2624 F5 656 1312 1312

Mean 1000 2630 1513

^a* Relative anti-E2 titers by end-point dilution. Mean values are expressed as geometric mean titers. The cut-off OO was 3 times higher than the OD of a pool of negative control mouse sera. b Mouse E5, negative by ELISA, was assigned an arbitrary value of one half of the screening dilution (20.5).

Again, mice inoculated with pE2surf had higher titers of anti-E2 at all time points compared to the animals inoculated with pE2. Thus, a recombinant E2 glycoprotein directed to the cell surface was shown to be more immunogenic than an intracellular form following DNA immunization in mice. In particular, the animals immunized with pE2surf developed an earlier and stronger humoral immune response than those immunized with pE2. However, 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. Neither of the two animals immunized with pE2 developed anti-E2, as detected by ELISA, but one animal was positive by immunofluorescence. Significantly, both animals immunized with pE2surf developed anti-E2, which was detected both by ELISA (Fig. 5) and by immunofluorescence. Anti-E2 were detected only after the second immunization.

Example 3

Mapping of linear epitopes in E2

To identify linear epitopes eliciting an immune response in the vaccinated BALB-C mice, serum was collected from mice prior to and following vaccination and compared for reactivity against 52 overlapping peptides (consensus amino acid sequence) of the E2-p7 region of strain H77 (Fig. 5). Human plasma samples H77 (negative for anti-HCV) and H79 (positive for anti-HCV) were used as a reference for epitope mapping.

One major linear epitope was recognized by chronic phase plasma (H79) of patient H; this epitope was represented by peptide 520 (aa 520-535).

Interestingly, 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. In the remaining 6 mice that developed anti-E2 antibodies, 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).

The majority of 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.

There was a clear discrepancy between results of assays detecting antibodies to linear versus conformational epitopes. Sera from some mice with high titers of anti-E2 did not recognize any of the overlapping peptides that represented the E2-p7 protein amino acid sequence, indicating that the majority (if not all) of the antibodies produced were directed against conformational epitopes.

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).

Example 4

Specific antibody response to HCV E2 glycoprotein in chimpanzees

The humoral immune response elicited by hepatitis C virus (HCV) E2 protein expressed in vivo after injection of plasmid DNA into two chimpanzees was analysed. A plasmid encoding a truncated form of the E2 protein targeted to the cell surface (pE2surf) was used as the immunogen. Ten milligrams of DNA was injected IM. The immunization schedule is zero, 4 and 8 weeks.

The results after the first two immunizations showed the one chimpanzee did not develop antibodies against E2, as detected by immunofluorescence. The second chimpanzee developed a positive anti-E2 response (titers approximately 1/1500 by immunofluorescence).

After the third immunization, chimpanzees with a positive anti-E2 immune response are challenged with a virulent HCV monoclonal pool.

In conclusion, the results presented in the examples suggest that presentation of HCV E2 on the cell surface may increase its immunogenicity while preserving its ability to react with antibodies generated during a natural infection.

Claims

1. A chimeric gene comprising:

(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.

2. The chimeric gene of claim 1, wherein the coding sequence encodes a truncated HCV envelope 2 protein.

3. The chimeric gene of claim 2, wherein the truncated HCV envelope 2 protein is at least about 332 amino acids in length.

4. The chimeric gene of claim 3, wherein the truncated HCV envelope 2 protein contains a truncation of about the 30 carboxy-terminal amino acids of the full-length envelope 2 protein.

5. The chimeric gene of claim 1, wherein the coding sequence encodes a truncated HCV envelope 1 protein.

6. The chimeric gene of claim 5, wherein the truncated envelope 1 protein is at least about 156 amino acids in length.

7. The chimeric gene of claim 6, wherein the truncated El protein contains a truncation at the carboxy-terminus of the El protein.

8. The chimeric gene of claim 7, wherein the truncation consists of about the 36 carboxy-terminal amino acids of the full-length El protein.

9. The chimeric gene of claim 1 , wherein the coding sequence encodes El and E2 proteins in tandem.

10. The chimeric gene of claim 9, wherein the E2 protein is truncated at its carboxy-terminus.

11. The chimeric gene of claim 10, wherein the truncation consists of about the 30 carboxy-terminal amino acids of the full-length E2 protein.

12. An expression vector comprising the chimeric gene of claim 1.

13. A host cell transformed or transfected with the expression vector of claim 12.

14. A method for expressing a truncated hepatitis C virus envelope protein on the surface of a cell comprising transforming a host cell with the expression vector of claim 12 under conditions which permit expression of the truncated envelope protein on the cell surface.

15. An assay for detecting antibodies to HCV in a biological sample, said assay comprising:

a) contacting the biological sample with the host cell of claim 13 under conditions which permit the formation of a complex between the antibodies and the envelope protein(s) expressed on the surface of the host cell; and

b) detecting the formation of said complex.

16. A method for identifying monoclonal antibodies having specific binding affinity for HCV envelope proteins, said method comprising:

a) exposing a combinatorial phage display library to the host cell of claim 13 under conditions which permit formation of a complex between the host cells and antibodies expressed by the phage display library; and b) recovering the phage from said complex.

17. The host cell of claim 13, wherein the host cell is further infected with an enveloped virus under conditions which permit the enveloped virus to incorporate the HCV envelope protein into its envelope and thereby form a pseudovirion.

18. A method for identifying antibodies which exhibit neutralizing activity to HCV, said method comprising:

a) exposing the pseudovirion produced by the host cell of claim 17 to the antibodies, and

b) measuring the number of viral plaques in susceptible host cells, a reduction in the number of plaques in the susceptible cells as compared to the number in susceptible cells incubated with pseudovirions not treated with antibody indicating that the antibodies have neutralizing activity.

19. A method for immunizing a mammal comprising administering the expression vector of claim 12 to the mammal in an amount effective to stimulate the production of protective antibodies to HCV.

20. Antibodies produced by the method of claim 19.

21. Antibodies identified by the method of claim 18.

22. A pharmaceutical composition comprising the antibodies of claims 20 or 21.

23. A pharmaceutical composition comprising the expression vector of claim 12.

24. A vaccine comprising the expression vector of claim 12.