AU757962B2 - Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use - Google Patents

Description

-1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

.o, Name of Applicant/s: Actual Inventor/s: Address for Service: Invention Title: Innogenetics N.V.

Geert Maertens and Fons Bosman and Guy De Martynoff and Marie- Ange Buyse BALDWIN SHELSTON WATERS MARGARET STREET SYDNEY NSW 2000 'PURIFIED HEPATITIS C VIRUS ENVELOPE PROTEINS FOR DIAGNOSTIC AND THERAPEUTIC USE' Details of Original Application No. 33824/95 dated 31 JUL 1995 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 25884AUP01 la- PURIFIED HEPATITIS C VIRUS ENVELOPE PROTEINS FOR DIAGNOSTIC AND THERAPEUTIC USE Field of the invention The present invention relates to the general fields of recombinant protein expression, purification of recombinant proteins, synthetic peptides, diagnosis of HCV infection, prophylactic treatment against HCV infection and to the prognosis/monitoring of the clinical efficiency of treatment of an individual with chronic hepatitis, or the prognosis/monitoring of natural disease.

More particularly, the present invention relates to purification methods for hepatitis C virus envelope proteins, the use in diagnosis, prophylaxis or therapy of HCV envelope proteins purified according to the methods described in the present invention, the use of single or specific oligomneric El and/or E2 and/or El/E2 envelope proteins in assays for monitoring disease, and/or diagnosis of disease, and/or treatment of disease. The invention also relates to epitopes of the El and/or E2 envelope proteins and monoclonal antibodies **:thereto, as well their use in diagnosis, prophylaxis or treatment.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Background of the invention The E2 protein purified from cell lysates according to the methods described in the present invention reacts with approximately 95% of patient sera. This reactivity is similar to the reactivity obtained with E2 secreted from CHO cells (Spaete et al., 1992). However, the intracellularly expressed form of E2 may more closely resemble the native viral envelope protein because it contains high mannose carbohydrate motifs, whereas the E2 protein secreted from CHO cells is flurther modified with galactose and sialic acid sugar moieties. When the aminoterminal half of E2 is expressed in the baculovirus system, only about 13 to 21% of sera from several patient groups can be detected (Jnoue et al., 1992).

After expression of E2 from E. coli, the reactivity of HCV sera was even lower and ranged P~AL~from 14 (Yokosuka et al., 1992) to 17% (Mita et al., 1992).

-2- About 75% ofHCV sera (and 95% of chronic patients) are anti-El positive using the purified, vaccinia-expressed recombinant El protein of the present invention, in sharp contrast with the results ofKohara et al. (1992) and Hsu et al. (1993). Kohara et al. used a vaccinia-virus expressed El protein and detected anti-El antibodies in 7 to 23% of patients, while Hsu et al. only detected 14/50 sera using baculovirus-expressed El.

These results show that not only a good expression system but also a good purification protocol are required to reach a high reactivity of the envelope proteins with human patient sera. This can be obtained using the proper expression system and/or purification protocols of the present invention which guarantee the conservation of the natural folding of the protein and the purification protocols of the present invention which guarantee the elimination of contaminating proteins and which preserve the conformation, and thus the reactivity of the HCV envelope proteins. The amounts of purified HCV envelope protein needed for diagnostic screening assays are in the range of grams per year.

For vaccine purposes, even higher amounts of envelope protein would be needed.

15 Therefore, the vaccinia virus system may be used for selecting the best expression S•constructs and for limited upscaling, and large-scale expression and purification of single or specific oligomeric envelope proteins containing high-mannose carbohydrates may be achieved when expressed from several yeast strains. In the case of hepatitis B for example, manufacturing of HBsAg from mammalian cells was much more costly compared with 20 yeast-derived hepatitis B vaccines.

eee* •Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

The present invention relates to a new purification method for recombinantly expressed El and/or E2 and/or El/E2 proteins such that said recombinant proteins are directly usable for diagnostic and vaccine purposes as single or specific oligomeric recombinant proteins free from contaminants instead of aggregates.

-3- The present invention also relates to compositions comprising purified (single or specific oligomeric) recombinant El and/or E2 and/or E1/E2 glycoproteins comprising conformational epitopes from the El and/or E2 domains of HCV.

The present invention further relates to novel recombinant vector constructs for recombinantly expressing El and/or E2 and/or E1/E2 proteins, as well as host cells transformed with said vector constructs.

The present invention also relates to a method for producing and purifying recombinant HCV El and/or E2 and/or E1/E2 proteins.

The present invention also relates to diagnostic and immunogenic uses of the recombinant HCV El and/or E2 and/or E1/E2 proteins of the present invention, as well as kits for diagnostic use, vaccines or therapeutics comprising any of the recombinant HCV El and/or E2 and/or E1/E2 proteins of the present invention.

The present invention further relates to a new use of El, E2, and/or E1/E2 proteins, or suitable parts thereof, for monitoring/prognosing the response to treatment of patients 15 with interferon) suffering from HCV infection.

The present invention relates to the use of the recombinant El, E2, and/or E1/E2 proteins of the present invention in HCV screening and confirmatory antibody tests.

o* The present invention relates to El and/or E2 peptides which can be used for diagnosis of HCV infection and for raising antibodies. Such peptides may also be used to 20 isolate human monoclonal antibodies.

The present invention also relates to monoclonal antibodies, more particularly human monoclonal antibodies or mouse monoclonal antibodies which are humanized, which react specifically with El and/or E2 epitopes, either comprised in peptides or conformational epitopes comprised in recombinant proteins.

The present invention relates to possible uses of anti-El or anti-E2 monoclonal antibodies for HCV antigen detection or for therapy of chronic HCV infection.

The present invention further relates to kits for monitoring/prognosing the response to treatment with interferon) of patients suffering from HCV infection or monitoring/prognosing the outcome of the disease.

Summary of the Invention According to a first aspect, the present invention provides an isolated HCV single or specific oligomeric envelope protein selected from the group consisting of El and/or E2 and/or El/E2, having a purity degree of at least 80%, characterised by the absence of aggregates.

According to a second aspect, the present invention provides an isolated HCV 10 single or specific oligomeric envelope protein selected from the group consisting of El and/or E2 and/or El/E2, having a purity degree of at least 90%, characterised by the absence of aggregates.

According to a third aspect, the present invention provides an isolated HCV single or specific oligomeric envelope protein selected from the group consisting of El and/or E2 and/or El/E2, having a purity degree of at least 95%, characterised by the absence of aggregates.

According to a fourth aspect, the present invention provides a method for immunising a mammal against HCV, comprising the steps of administering to said *mammal an effective amount of an isolated HCV envelope protein according to any one of the first to third aspects, to produce an immune response.

oo•According to a fifth aspect, the present invention provides a vaccine composition o for immunizing a mammal against HCV, comprising an effective amount of an isolated HCV envelope protein according to any one of the first to third aspects, optionally accompanied by pharmaceutically acceptable adjuvants.

According to a sixth aspect, the present invention provides a method for in vitro diagnosis of HCV antibodies present in a biological sample, comprising at least the following steps: contacting said biological sample with an isolated HCV envelope protein according to any one of the first to third aspects, under appropriate conditions which allow the formation of an immune complex, 7(ii) removing unbound components, -4a- (iii) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies being conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immune complexes visually or mechanically.

According to a seventh aspect, the present invention provides a kit for determining the presence of HCV antibodies present in a biological sample, comprising: at least one isolated HCV envelope protein according to any one of the first to third aspects, a buffer or components necessary for producing the buffer enabling binding reaction between these proteins and antibodies against HCV present in said biological sample, a means for detecting the immune complexes formed in the preceding binding reaction.

According to an eighth aspect, the present invention provides use of an isolated HCV envelope protein according to any one of the first to third aspects, comprising HCV El protein, for in vitro monitoring HCV disease or prognosing the response to treatment of patients suffering from HCV infection comprising: incubating a biological sample from a patient with HCV infection with an El 20 protein or a suitable part thereof under conditions allowing the formation of an immunological complex, removing unbound components, calculating the anti-El titers present in said sample at the start of and during the *a*SSS course of treatment, monitoring the natural course of HCV disease, or prognosing the response to treatment of said patient on the basis of the amount of anti-El titers found in said sample at the start of treatment and/or during the course of treatment.

4b According to a ninth aspect, the present invention provides a kit for monitoring HCV disease or prognosing the response to treatment of patients suffering from HCV infection comprising: at least one isolated HCV envelope protein according to any one of the first to third aspects, a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins and the anti-E antibodies present in a biological sample, means for detecting the immune complexes formed in the preceding binding reaction, and optionally also an automated scanning and interpretation device for inferring a decrease of anti-E titers during the progression of treatment.

According to a tenth aspect, the present invention provides a serotyping assay for detecting one or more serological types ofHCV present in a biological sample, comprising at least the following steps contacting the biological sample to be analyzed for the presence of HCV Sg.. antibodies of one or more serological types, with at least one isolated HCV El and/or E2 and/or El/E2 protein according to any one of the first to third aspects, under appropriate conditions which allow the formation of an immune complex, (ii) removing unbound components, (iii) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies being conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immune complexes visually or mechanically by means of densitometry, fluorimetry, colorimetry) and inferring the presence of one or more HCV serological types present from the observed binding pattern.

4c According to an eleventh aspect, the present invention provides a kit for serotyping one or more serological types of HCV present in a biological sample, comprising: at least one isolated HCV El and/or E2 and/or El/E2 protein according to any one of the first to third aspects, a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins and the anti-E antibodies present in a biological sample, means for detecting the immune complexes formed in the preceding binding reaction, and optionally also an automated scanning and interpretation device for detecting the presence of one or more serological types present from the observed binding pattern.

According to a twelfth aspect, the present invention provides use of an isolated HCV envelope protein according to any one of the first to third aspects, for detecting HCV antibodies present in a biological sample.

According to a thirteenth aspect,the present invention provides use of an isolated HCV envelope protein according to any one of the first to third aspects, for the manufacture of a medicament for immunising a mammal against HCV.

20 Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of"including, but not limited to".

Definitions The following definitions serve to illustrate the different terms and expressions used in the present invention.

The term 'hepatitis C virus single envelope protein' refers to a polypeptide or an analogue thereof(e.g. mimotopes) comprising an amino acid sequence (and/or amino acid Sanalogues) defining at least one HCV epitope of either the El or the E2 region. These 4d single envelope proteins in the broad sense of the word may be both monomeric or homooligomeric forms ofrecombinantly expressed envelope proteins. Typically, the sequences defining the epitope correspond to the amino acid sequence of either the El or the E2 region of HCV (either identically or via substitution of analogues of the native amino acid residue that do not destroy the epitope). In general, the epitope-defining sequence will be 3 or more amino acids in length, more typically, 5 or more amino acids in length, more typically 8 or more amino acids in length, and even more typically 10 or more amino acids in length. With respect to conformational epitopes, the length of the epitope-defining sequence can be subject to wide variations, since it is believed that these epitopes are formed by the three-dimensional shape of the antigen folding). Thus, the amino acids defining the epitope can be relatively few in number, but widely dispersed along the length of the molecule being brought into the correct epitope conformation via folding. The portions of the antigen between the residues defining the epitope may not be critical to the conformational structure of the epitope. For example, deletion or substitution of these o, 15 intervening sequences may not affect the conformational epitope provided sequences critical to epitope conformation are maintained cysteines involved in disulfide bonding, glycosylation sites, etc.). A conformational epitope may also be formed by 2 or more essential regions of subunits of a homooligomer or heterooligomer.

The HCV antigens of the present invention comprise conformational epitopes from 20 the El and/or E2 (envelope) domains of HCV. The El domain, which is believed to correspond to the viral envelope protein, is currently estimated to span amino acids 192- 383 of the HCV polyprotein (Hijikata et al., 1991). Upon expression in a mammalian system (glycosylated), it is believed to have an approximate molecular weight of 35 kDa as determined via SDS-PAGE. The E2 protein, previously called NS 1, is believed to span amino acids 384-809 or 384-746 (Grakoui et al., 1993) of the HCV polyprotein and to also be an envelope protein. Upon expression in a vaccinia system (glycosylated), it is believed to have an apparent gel molecular weight of about 72 kDa. It is understood that these protein endpoints are approximations the carboxy terminal end of E2 could lie somewhere in the 730-820 amino acid region, e.g. ending at amino acid 730, 735, 740, 742, 744, 745, preferably 746, 747, 748, 750, 760, 770, 780, 790, 800, 809, 810, 820). The 10 E2 protein may also be expressed together with the El, P7 (aa 747-809), NS2 (aa 810- 1026), NS4A (aa 1658-1711) or NS4B (aa 1712-1972). Expression together with these other HCV proteins may be important for obtaining the correct protein folding.

It is also understood that the isolates used in the examples section of the present invention were not intended to limit the scope of the invention and that any HCV isolate from type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or any other new genotype of HCV is a suitable source of El and/or E2 sequence for the practice of the present invention.

The El and E2 antigens used in the present invention may be full-length viral proteins, substantially full-length versions thereof, or functional fragments thereof (e.g.

fragments which are not missing sequence essential to the formation or retention of an epitope). Furthermore, the HCV antigens of the present invention can also include other sequences that do not block or prevent the formation of the conformational epitope of interest. The presence or absence of a conformational epitope can be readily determined though screening the antigen of interest with an antibody (polyclonal serum or monoclonal to the conformational epitope) and comparing its reactivity to that of a denatured version of the antigen which retains only linear epitopes (if any). In such screening using polyclonal antibodies, it may be adyantageous to adsorb the polyclonal serum first with the denatured antigen and see if it retains antibodies to the antigen of interest.

The HCV antigens of the present invention can be made by any recombinant method that provides the epitope of intrest. For example, recombinant intracellular expression in mammalian or insect cells is a preferred method to provide glycosylated El and/or E2 antigens in 'native' conformation as is the case for the natural HCV antigens.

Yeast cells and mutant yeast strains mnn 9 mutant (Kniskem et al., 1994) or glycosylation mutants derived by means ofvanadate resistence selection (Ballou et al., 1991)) may be ideally suited for production of secreted high-mannose-type sugars; whereas proteins secreted from mammalian cells may contain modifications including galactose or sialic acids which may be undesirable for certain diagnostic or vaccine applications.

However, it may also be possible and sufficient for certain applications, as it is known for proteins, to express the antigen in other recombinant hosts (such as E. coli) and renature the protein after recovery.

The term 'fusion polypeptide' intends a polypeptide in which the HCV antigen(s) 10 are part of a single continuous chain of amino acids, which chain does not occur in nature.

i "The HCV antigens may be connected directly to each other by peptide bonds or be separated by intervening amino acid sequences. The fusion polypeptides may also contain amino acid sequences exogenous to HCV.

The term 'solid phase' intends a solid body to which the individual HCV antigens or 15 the fusion polypeptide comprised of HCV antigens are bound covalently or by noncovalent means such as hydrophobic adsorption.

The term 'biological sample' intends a fluid or tissue of a mammalian individual an anthropoid, a human) that commonly contains antibodies produced by the individual, more particularly antibodies against HCV. The fluid or tissue may also contain 20 HCV antigen. Such components are known in the art and include, without limitation, blood, plasma, serum, urine, spinal fluid, lymph fluid, secretions of the respiratory, intestinal or genitourinary tracts, tears, saliva, milk, white blood cells and myelomas. Body components include biological liquids. The term 'biological liquid' refers to a fluid obtained from an organism. Some biological fluids are used as a source of other products, such as clotting factors Factor VIII;C), serum albumin, growth hormone and the like.

In such cases, it is important that the source of biological fluid be free of contamination by virus such as HCV.

The term 'immunologically reactive' means that the antigen in question will react specifically with anti-HCV antibodies present in a body component from an HCV infected individual.

The term 'immune complex' intends the combination formed when an antibody binds to an epitope on an antigen.

'El' as used herein refers to a protein or polypeptide expressed within the first 400 amino acids of an HCV polyprotein, sometimes referred to as the E, ENV or S protein. In its natural form it is a 35 kDa glycoprotein which is found in strong association with membranes. In most natural HCV strains, the El protein is encoded in the viral polyprotein following the C (core) protein. The El protein extends from approximately amino acid (aa) 192 to about aa 383 of the full-length polyprotein.

The term 'El' as used herein also includes analogs and truncated forms that are 10 immunologically cross-reactive with natural E and includes El proteins of genotypes 1, i 3, 4, 5, 6, 7, 8, 9, 10, or any other newly identified HCV type or subtype.

'E2' as used herein refers to a protein or polypeptide expressed within the first 900 amino acids of an HCV polyprotein, sometimes referred to as the NS1 protein. In its natural form it is a 72 kDa glycoprotein that is found in strong association with S 15 membranes. In most natural HCV strains, the E2 protein is encoded in the viral polyprotein following the El protein. The E2 protein extends from approximately amino acid position 384 to amino acid position 746, another form of E2 extends to amino acid position 809.

The term 'E2' as used herein also includes analogs and truncated forms that are immunologically cross-reactive with natural E2. For example, insertions of multiple 20 codons between codon 383 and 384, as well as deletions of amino acids 384-387 have been reported by Kato et al. (1992).

'E1/E2' as used herein refers to an oligomeric form of envelope proteins containing at least one El component and at least one E2 component.

The term 'specific oligomeric' El and/or E2 and/or E1/E2 envelope proteins refers to all possible oligomeric forms ofrecombinantly expressed El and/or E2 envelope proteins which are not aggregates. El and/or E2 specific oligomeric envelope proteins are also referred to as homo-oligomeric El or E2 envelope proteins (see below).

The term 'single or specific oligomeric' El and/or E2 and/or E1/E2 envelope proteins refers to single monomeric El or E2 proteins (single in the strict sense of the word) as well as specific oligomeric El and/or E2 and/or E1/E2 recombinantly expressed proteins. These single or specific oligomeric envelope proteins according to the present invention can be further defined by the following formula (El)x(E2)y wherein x can be a number between 0 and 100, and y can be a number between o and 100, provided that x and y are not both 0. With x=l and y=0 said envelope proteins include monomeric El.

The term 'homo-oligomer' as used herein refers to a complex of El and/or E2 containing more than one El or E2 monomer, e.g. El/El dimers, El/El/El trimers or El/El/El/El tetramers and E2/E2 dimers, E2/E2/E2 trimers or E2/E2/E2/E2 tetramers, El pentamers and hexamers, E2 pentamers and hexamers or any higher-order homo-oligomers of El or E2 are all 'homo-oligomers' within the scope of this definition. The oligomers may contain one, two, or several different monomers of El or E2 obtained from different types 10 or subtypes of hepatitis C virus including for example those described in an international •i application published under WO 94/25601 and European application No. 94870166.9 both by the present applicants. Such mixed oligomers are still homo-oligomers within the scope of this invention, and may allow more universal diagnosis, prophylaxis or treatment of

HCV.

15 The term 'purified' as applied to proteins herein refers to a composition wherein the desired protein comprises at least 35% of the total protein component in the composition.

The desired protein preferably comprises at least 40%, more preferably at least about more preferably at least about 60%, still more preferably at least about 70%, even more e preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% of the total protein component. The composition may contain other compounds such as carbohydrates, salts, lipids, solvents, and the like, withouth affecting the determination of the percentage purity as used herein. An 'isolated' HCV protein intends an HCV protein composition that is at least 35% pure.

The term 'essentially purified proteins' refers to proteins purified such that they can be used for in vitro diagnostic methods and as a therapeutic compound. These proteins are substantially free from cellular proteins, vector-derived proteins or other HCV viral components. Usually these proteins are purified to homogeneity (at least 80% pure, preferably, 90%, more preferably 95%, more preferably 97%, more preferably 98%, more preferably 99%, even more preferably 99.5%, and most preferably the contaminating proteins should be undetectable by conventional methods like SDS-PAGE and silver staining.

-9- The term 'recombinantly expressed' used within the context of the present invention refers to the fact that the proteins of the present invention are produced by recombinant expression methods be it in prokaryotes, or lower or higher eukaryotes as discussed in detail below.

The term 'lower eukaryote' refers to host cells such as yeast, fungi and the like.

Lower eukaryotes are generally (but not necessarily) unicellular. Preferred lower euklaryotes are yeasts, particularly species within Saccharomyces, Schizosaccharomyces, Kluveromyces, Pichia Pichia pastoris), Hansenula Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomvces, Zvyosaccharomyces and the like.

10 Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts, and are convenient fungal hosts.

The term 'prokaryotes' refers to hosts such as E.coli, Lactobacillus, Lactococcus, Salmonella, Streptococcus, Bacillus subtilis or Streptomvces. Also these hosts are contemplated within the present invention.

15 The term 'higher eukaryote' refers to host cells derived from higher animals, such as mammals, reptiles, insects, and the like. Presently preferred higher eukaryote host cells are derived from Chinese hamster CHO), monkey COS and Vero cells), baby hamster kidney (BHK), pig kidney (PK15), rabbit kidney 13 cells (RK13), the human osteosarcoma cell line 143 B, the human cell line HeLa and human hepatoma cell lines like S* 20 Hep G2, and insect cell lines Spodoptera frugiperda). The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively the host cells may also be transgenic animals.

The term 'polypeptide' refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude postexpression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogues of an amino acid (including, for example, unnatural amino acids, PNA, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

The term 'recombinant polynucleotide or nucleic acid' intends a polynucleotide or nucleic acid of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of a polynucleotide with which it is associated in nature, is linked to a polynucleotide other than that to which it is linked in nature, or does not occur in nature.

The term 'recombinant host cells', 'host cells', 'cells', 'cell lines', 'cell cultures', and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be or have been, used as recipients for a •recombinant vector or other transfer polynucleotide, and include the progeny of the 10 original cell which has been transfected. It is understood that the progeny of a single •parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

The term 'replicon' is any genetic element, a plasmid, a chromosome, a virus, a cosmid, etc., that behaves as an autonomous unit of polynucleotide replication within a cell; capable of replication under its own control.

The term 'vector' is a replicon further comprising sequences providing replication and/or expression of a desired open reading frame.

"The term 'control sequence' refers to polynucleotide sequences which are necessary 20. to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and terminators; in eukaryotes, generally, such control sequences include promoters, terminators and, in some instances, enhancers. The term 'control sequences' is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences which govern secretion.

The term 'promoter' is a nucleotide sequence which is comprised of consensus sequences which allow the binding of RNA polymerase to the DNA template in a manner such that mRNA production initiates at the normal transcription initiation site for the adjacent structural gene.

-11- The expression 'operably linked' refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence 'operably linked' to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

An 'open reading frame' (ORF) is a region of a polynucleotide sequence which encodes a polypeptide and does not contain stop codons; this region may represent a portion of a coding sequence or a total coding sequence.

A 'coding sequence' is a polynucleotide sequence which is transcribed into mRNA 10 and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include but is not limited to mRNA, DNA (including cDNA), and recombinant polynucleotide sequences.

15 As used herein, 'epitope' or 'antigenic determinant' means an amino acid sequence that is immunoreactive. Generally an epitope consists of at least 3 to 4 amino acids, and more usually, consists of at least 5 or 6 amino acids, sometimes the epitope consists of about 7 to 8, or even about 10 amino acids. As used herein, an epitope of a designated polypeptide denotes epitopes with the same amino acid sequence as the epitope in the designated polypeptide, and immunologic equivalents thereof. Such equivalents also include strain, subtype (=genotype), or type(group)-specific variants, e.g. of the currently known sequences or strains belonging to genotypes la, lb, Ic, Id, le, If, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 3a, 3b, 3c, 3d, 3e, 3f, 3g, 4a, 4b, 4c, 4d, 4e, 4f, 4g, 4h, 4i, 4j, 4k, 41, 5a, 6a, 6b, 6c, 7a, 7b, 7c, 8a, 8b, 9a, 9b, 10a, or any other newly defined HCV (sub)type. It is to be understood that the amino acids constituting the epitope need not be part of a linear sequence, but may be interspersed by any number of amino acids, thus forming a conformational epitope.

The term 'immunogenic' refers to the ability of a substance to cause a humoral and/or cellular response, whether alone or when linked to a carrier, in the presence or absence of an adjuvant. 'Neutralization' refers to an immune response that blocks the infectivity, either partially or fully, of an infectious agent. A 'vaccine' is an immunogenic -12composition capable of eliciting protection against HCV, whether partial or complete. A vaccine may also be useful for treatment of an individual, in which case it is called a therapeutic vaccine.

The term 'therapeutic' refers to a composition capable of treating HCV infection.

The term 'effective amount' refers to an amount of epitope-bearing polypeptide sufficient to induce an immunogenic response in the individual to which it is administered, or to otherwise detectably immunoreact in its intended system immunoassay).

Preferably, the effective amount is sufficient to effect treatment, as defined above. The exact amount necessary will vary according to the application. For vaccine applications or 10 for the generation of polyclonal antiserum antibodies, for example, the effective amount •may vary depending on the species, age, and general condition of the individual, the severity of the condition being treated, the particular polypeptide selected and its mode of administration, etc. It is also believed that effective amounts will be found within a relatively large, non-critical range. An appropriate effective amount can be readily 15 determined using only routine experimentation. Preferred ranges of El and/or E2 and/or E1/E2 single or specific oligomeric envelope proteins for prophylaxis of HCV disease are 0.01 to 100 tLg/dose, preferably 0.1 to 50 g/dose. Several doses may be needed per individual in order to achieve a sufficient immune response and subsequent protection against HCV disease.

Detailed description of the invention More particularly, the present invention contemplates a method for isolating or purifying recombinant HCV single or specific oligomeric envelope protein selected from the group consisting of El and/or E2 and/or E1/E2, characterized in that upon lysing the transformed host cells to isolate the recombinantly expressed protein a disulphide bond cleavage or reduction step is carried out with a disculphide bond cleaving agent.

The essence of these 'single or specific oligomeric' envelope proteins of the invention is that they are free from contaminating proteins and that they are not disulphide bond linked with contaminants.

-13- The proteins according to the present invention are recombinantly expressed in lower or higher eukaryotic cells or in prokaryotes. The recombinant proteins of the present invention are preferably glycosylated and may contain high-mannose-type, hybrid, or complex glycosylations. Preferentially said proteins are expressed from mammalian cell lines as discussed in detail in the Examples section, or in yeast such as in mutant yeast strains also as detailed in the Examples section.

The proteins according to the present invention may be secreted or expressed within components of the cell, such as the ER or the Golgi Apparatus. Preferably, however, the proteins of the present invention bear high-mannose-type glycosylations and are 10 retained in the ER or Golgi Apparatus of mammalian cells or are retained in or secreted from yeast cells, preferably secreted from yeast mutant strains such as the mnn9 mutant (Kniskem et al., 1994), or from mutants that have been selected by means of vanadate resistence (Ballou et al., 1991).

Upon expression of HCV envelope proteins, the present inventors could show that 15 some of the free thiol groups of cysteines not involved in intra- or inter-molecular disulphide bridges, react with cysteines of host or expression-system-derived (e.g.

vaccinia) proteins or of other HCV envelope proteins (single or oligomeric), and form aspecific intermolecular bridges. This results in the formation of'aggregates' of HCV S* envelope proteins together with contaminating proteins. It was also shown in WO 92/08734 that 'aggregates' were obtained after purification, but it was not described which protein interactions were involved. In patent application WO 92/08734, recombinant E1/E2 protein expressed with the vaccinia virus system were partially purified as aggregates and only found to be 70% pure, rendering the purified aggregates not useful for diagnostic, prophylactic or therapeutic purposes.

Therefore, a major aim of the present invention resides in the separation of single or specific-oligomeric HCV envelope proteins from contaminating proteins, and to use the purified proteins 95% pure) for diagnostic, prophylactic and therapeutic purposes. To those purposes, the present inventors have been able to provide evidence that aggregated protein complexes ('aggregates') are formed on the basis of disulphide bridges and noncovalent protein-protein interactions. The present invention thus provides a means for selectively cleaving the disulphide bonds under specific conditions and for separating the 14cleaved proteins from contaminating proteins which greatly interfere with diagnostic, prophylactic and therapeutic applications. The free thiol groups may be blocked (reversibly or irreversibly) in order to prevent the reformation of disulphide bridges, or may be left to oxidize and oligomerize with other envelope proteins (see definition homo-oligomer). It is to be understood that such protein oligomers are essentially different from the 'aggregates' described in WO 92/08734 and WO 94/01778, since the level of contaminating proteins is undetectable.

Said disuphide bond cleavage may also be achieved by: performic acid oxidation by means of cysteic acid in which case the cysteine residues 10 are modified into cysteic acid (Moore et al., 1963).

Sulfitolysis (R-S-S-R 2 R-SO3) for example by means of sulphite (SO 2 together with a proper oxidant such as Cu 2 in which case the cysteine is modified into S-sulphocysteine (Bailey and Cole, 1959).

Reduction by means of mercaptans, such as dithiotreitol (DDT), 1-mercapto-ethanol, 15 cysteine, glutathione Red, e-mercapto-ethylamine, or thioglycollic acid, of which DTT and B-mercapto-ethanol are commonly used (Cleland, 1964), is the preferred method of this invention because the method can be performed in a water environment and because the cysteine remains unmodified.

Reduction by means of a phosphine Bu 3 P) (Ruegg and Rudinger, 1977).

20 All these compounds are thus to be regarded as agents or means for cleaving disulphide bonds according to the present invention.

Said disulphide bond cleavage (or reducing) step of the present invention is preferably a partial disulphide bond cleavage (reducing) step (carried out under partial cleavage or reducing conditions).

A preferred disulphide bond cleavage or reducing agent according to the present invention is dithiothreitol (DTT). Partial reduction is obtained by using a low concentration of said reducing agent, i.e. for DTT for example in the concentration range of about 0.1 to about 50 mM, preferably about 0.1 to about 20 mM, preferably about 0.5 to about 10 mM, preferably more than 1 mM, more than 2 mM or more than 5 mM, more preferably about 1.5 mM, about 2.0 mM, about 2.5 mM, about 5 mM or about 7.5 mM.

15 Said disulphide bond cleavage step may also be carried out in the presence of a suitable detergent (as an example of a means for cleaving disulphide bonds or in combination with a cleaving agent) able to dissociate the expressed proteins, such as DecylPEG, EMPIGEN-BB, NP-40, sodium cholate, Triton X-100.

Said reduction or cleavage step (preferably a partial reduction or cleavage step) is carried out preferably in in the presence of (with) a detergent. A preferred detergent according to the present invention is Empigen-BB. The amount of detergent used is preferably in the range of 1 to 10 preferably more than more preferably about of a detergent such as Empigen-BB.

10 A particularly preferred method for obtaining disulphide bond cleavage employs a combination of a classical disulphide bond cleavage agent as detailed above and a detergent (also as detailed above). As contemplated in the Examples section, the particular combination of a low concentration of DTT (1.5 to 7.5 mM) and about 3.5 of Empigen- BB is proven to be a particularly preferred combination of reducing agent and detergent for S 15 the purification of recombinantly expressed El and E2 proteins. Upon gelfiltration chromatography, said partial reduction is shown to result in the production of possibly dimeric El protein and separation of this El protein from contaminating proteins that cause false reactivity upon use in immunoassays.

It is, however, to be understood that also any other combination of any reducing S* 20 agent known in the art with any detergent or other means known in the art to make the cysteines better accessible is also within the scope of the present invention, insofar as said combination reaches the same goal of disulphide bridge cleavage as the preferred combination examplified in the present invention.

Apart from reducing the disulphide bonds, a disulphide bond cleaving means according to the present invention may also include any disulphide bridge exchanging agents (competitive agent being either organic or proteinaeous, see for instance Creighton, 1988) known in the art which allows the following type of reaction to occur: R1 S- S R2 R3 SH R1 S- S R3 +R2 SH R1, R2: compounds of protein aggregates R3 SH: competitive agent (organic, proteinaeous) 16- The term 'disulphide bridge exchanging agent' is to be interpretated as including disulphide bond reforming as well as disulphide bond blocking agents.

The present invention also relates to methods for purifying or isolating HCV single or specific oligomeric envelelope proteins as set out above further including the use of any SH group blocking or binding reagent known in the art such as chosen from the following list: Glutathion 5,5'-dithiobis-(2-nitrobenzoic acid) or bis-(3-carboxy-4-nitrophenyl)-disulphide (DTNB or Ellman's reagent) (Elmann, 1959) 10 N-ethylmaleimide (NEM; Benesch et al., 1956) N-(4-dimethylamino-3,5-dinitrophenyl) maleimide or Tuppy's maleimide which Sprovides a color to the protein P-chloromercuribenzoate (Grassetti et al., 1969) 4-vinylpyridine (Friedman and Krull, 1969) can be liberated after reaction by acid 15 hydrolysis acrylonitrile, can be liberated after reaction by acid hydrolysis (Weil and Seibles, 1961) NEM-biotin obtained from Sigma B1267) 2,2'-dithiopyridine (Grassetti and Murray, 1967) 20 4,4'-dithiopyridine (Grassetti and Murray, 1967) 6,6'-dithiodinicontinic acid (DTDNA; Brown and Cunnigham, 1970) 2,2'-dithiobis-(5'-nitropyridine) (DTNP; US patent 3597160) or other dithiobis (heterocyclic derivative) compounds (Grassetti and Murray, 1969) A survey of the publications cited shows that often different reagents for sulphydryl groups will react with varying numbers ofthiol groups of the same protein or enzyme molecule. One may conclude that this variation in reactivity of the thiol groups is due to the steric environment of these groups, such as the shape of the molecule and the surrounding groups of atoms and their charges, as well as to the size, shape and charge of the reagent molecule or ion. Frequently the presence of adequate concentrations of denaturants such as sodium dodecylsulfate, urea or guanidine hydrochoride will cause sufficient unfolding of the protein molecule to permit equal access to all of the reagents for -17thiol groups. By varying the concentration of denaturant, the degree of unfolding can be controlled and in this way thiol groups with different degrees of reactivity may be revealed.

Although up to date most of the work reported has been done with pchloromercuribenzoate, N-ethylmaleimide and DTNB, it is likely that the other more recently developed reagents may prove equally useful. Because of their varying structures, it seems likely, in fact, that they may respond differently to changes in the steric environment of the thiol groups.

Alternatively, conditions such as low pH (preferably lower than pH 6) for preventing free SH groups from oxidizing and thus preventing the formation of large 10 intermolecular aggregates upon recombinant expression and purification of El and E2 S(envelope) proteins are also within the scope of the present invention.

«*O

A preferred SH group blocking reagent according to the present invention is Nethylmaleimide (NEM). Said SH group blocking reagent may-be administrated during lysis of the recombinant host cells and after the above-mentioned partial reduction process or S 15 after any other process for cleaving disulphide bridges. Said SH group blocking reagent may also be modified with any group capable of providing a detectable label and/or any

S**

group aiding in the immobilization of said recombinant protein to a solid substrate, e.g.

biotinylated NEM.

Methods for cleaving cysteine bridges and blocking free cysteines have also been 20 described in Darbre (1987), Means and Feeney (1971), and by Wong (1993).

A method to purify single or specific oligomeric recombinant El and/or E2 and/or E1/E2 proteins according to the present invention as defined above is further characterized as comprising the following steps: lysing recombinant El and/or E2 and/or E1/E2 expressing host cells, preferably in the presence of an SH group blocking agent, such as N-ethylmaleimide (NEM), and possibly a suitable detergent, preferably Empigen-BB, recovering said HCV envelope protein by affinity purification for instance by means lectin-chromatography, such as lentil-lectin chromatography, or immunoaffinity chromatography using anti-El and/or anti-E2 specific monoclonal antibodies, followed by, -18reduction or cleavage of disulphide bonds with a disulphide bond cleaving agent, such as DTT, preferably also in the presence of an SH group blocking agent, such as NEM or Biotin-NEM, and, recovering the reduced HCV El and/or E2 and/or E1/E2 envelope proteins for instance by gelfiltration (size exclusion chromatography or molecular sieving) and possibly also by an additional Ni2+-IMAC chromatography and desalting step.

It is to be understood that the above-mentioned recovery steps may also be carried out using any other suitable technique known by the person skilled in the art.

Preferred lectin-chromatography systems include Galanthus nivalis agglutinin 10 (GNA) chromatography, or Lens culinaris agglutinin (LCA) (lentil) lectin chromatography as illustrated in the Examples section. Other useful lectins include those recognizing high-mannose type sugars, such as Narcissus pseudonarcissus agglutinin (NPA), Pisum sativum agglutinin (PSA), or Allium ursinum agglutinin (AUA).

Preferably said method is usable to purify single or specific oligomeric HCV 15 envelope protein produced intracellularly as detailed above.

For secreted El or E2 or E1/E2 oligomers, lectins binding complex sugars such as Ricinus communis agglutinin I (RCA are preferred lectins.

The present invention more particularly contemplates essentially purified recombinant HCV single or specific oligomeric envelope proteins, selected from the group *0 20 consisting of E and/or E2 and/or E1/E2, characterized as being isolated or purified by a method as defined above.

The present invention more particularly relates to the purification or isolation of recombinant envelope proteins which are expressed from recombinant mammalian cells such as vaccinia.

The present invention also relates to the purification or isolation of recombinant envelope proteins which are expressed from recombinant yeast cells.

The present invention equally relates to the purification or isolation of recombinant envelope proteins which are expressed from recombinant bacterial (prokaryotic) cells.

The present invention also contemplates a recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic promoter sequence -19followed by a nucleotide sequence allowing the expression of the single or specific oligomeric El and/or E2 and/or El/E2 of the invention.

Particularly, the present invention contemplates a recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic promoter sequence followed by a nucleotide sequence allowing the expression of the single El or El of the invention.

Particularly, the present invention contemplates a recombinant vector comprising a vector sequence, an appropriate prokaryotic, eukaryotic or viral or synthetic promoter 1 sequence followed by a nucleotide sequence allowing the expression of the single El or E2 10 of the invention.

Di The segment of the HCV cDNA encoding the desired El and/or E2 sequence inserted into the vector sequence may be attached to a signal sequence. Said signal sequence may be that from a non-HCV source, e.g. the IgG or tissue plasminogen activator So(tpa) leader sequence for expression in mammalian cells, or the c-mating factor sequence 15 for expression into yeast cells, but particularly preferred constructs according to the present invention contain signal sequences appearing in the HCV genome before the respective start points of the El and E2 proteins. The segment of the HCV cDNA encoding the .ooooi desired El and/or E2 sequence inserted into the vector may also include deletions e.g. of •the hydrophobic domain(s) as illustrated in the examples section, or of the E2 hypervariable region I.

More particularly, the recombinant vectors according to the present invention encompass a nucleic acid having an HCV cDNA segment encoding the polyprotein starting in the region between amino acid positions 1 and 192 and ending in the region between positions 250 and 400 of the HCV polyprotein, more preferably ending in the region between positions 250 and 341, even more preferably ending in the region between positions 290 and 341 for expression of the HCV single El protein. Most preferably, the present recombinant vector encompasses a recombinant nucleic acid having a HCV cDNA seqment encoding part of the HCV polyprotein starting in the region between positions 117 and 192, and ending at any position in the region between positions 263 and 326, for expression of HCV single El protein. Also within the scope of the present invention are forms that have the first hydrophobic domain deleted (positions 264 to 293 plus or minus 8 amino acids), or forms to which a 5'-terminal ATG codon and a 3'-terminal stop codon has been added, or forms which have a factor Xa cleavage site and/or 3 to 10, preferably 6 Histidine codons have been added.

More particularly, the recombinant vectors according to the present invention encompass a nucleic acid having an HCV cDNA segment encoding the polyprotein starting in the region between amino acid positions 290 and 406 and ending in the region between positions 600 and 820 of the HCV polyprotein, more preferably starting in the region between positions 322 and 406, even more preferably starting in the region between positions 347 and 406, even still more preferably starting in the region between positions S: 10 364 and 406 for expression of the HCV single E2 protein. Most preferably, the present recombinant vector encompasses a recombinant nucleic acid having a HCV cDNA seqment encoding the polyprotein starting in the region between positions 290 and 406, and ending at any position of positions 623, 650, 661, 673, 710, 715, 720, 746 or 809, for expression of HCV single E2 protein. Also within the scope of the present invention are 15 forms to which a 5'-terminal ATG codon and a 3'-terminal stop codon has been added, or forms which have a factor Xa cleavage site and/or 3 to 10, preferably 6 Histidine codons have been added.

A variety of vectors may be used to obtain recombinant expression of HCV single or specific oligomeric envelope proteins of the present invention. Lower eukaryotes such 20 as yeasts and glycosylation mutant strains are typically transformed with plasmids, or are transformed with a recombinant virus. The vectors may replicate within the host independently, or may integrate into the host cell genome.

Higher eukaryotes may be transformed with vectors, or may be infected with a recombinant virus, for example a recombinant vaccinia virus. Techniques and vectors for the insertion of foreign DNA into vaccinia virus are well known in the art, and utilize, for example homologous recombination. A wide variety of viral promoter sequences, possibly terminator sequences and poly(A)-addition sequences, possibly enhancer sequences and possibly amplification sequences, all required for the mammalian expression, are available in the art. Vaccinia is particularly preferred since vaccinia halts the expression of host cell proteins. Vaccinia is also very much preferred since it allows the expression of El and E2 proteins of HCV in cells or individuals which are immunized with the live recombinant -21 vaccinia virus. For vaccination of humans the avipox and Ankara Modified Virus (AMV) are particularly useful vectors.

Also known are insect expression transfer vectors derived from baculovirus Autographa califorica nuclear polyhedrosis virus (AcNPV), which is a helperindependent viral expression vector. Expression vectors derived from this system usually use the strong viral polyhedrin gene promoter to drive the expression of heterologous genes. Different vectors as well as methods for the introduction of heterologous DNA into the desired site of baculovirus are available to the man skilled in the art for baculovirus expression. Also different signals for posttranslational modification recognized by insect 10 cells are known in the art.

Also included within the scope of the present invention is a method for producing purified recombinant single or specific oligomeric HCV El or E2 or E1/E2 proteins, wherein the cysteine residues involved in aggregates formation are replaced at the level of :the nucleic acid sequence by other residues such that aggregate formation is prevented.

15 The recombinant proteins expressed by recombinant vectors caarying such a mutated El and/or E2 protein encoding nucleic acid are also within the scope of the present invention.

The present invention also relates to recombinant El and/or E2 and/or E1/E2 proteins characterized in that at least one of their glycosylation sites has been removed and are consequently termed glycosylation mutants. As explained in the Examples section, 20 different glycosylation mutants may be desired to diagnose (screening, confirmation, prognosis, etc.) and prevent HCV disease according to the patient in question. An E2 protein glycosylation mutant lacking the GLY4 has for instance been found to improve the reactivity of certain sera in diagnosis. These glycosylation mutants are preferably purified according to the method disclosed in the present invention. Also contemplated within the present invention are recombinant vectors carrying the nucleic acid insert encoding such a El and/or E2 and/or E1/E2 glycosylation mutant as well as host cells tranformed with such a recombinant vector.

The present invention also relates to recombinant vectors including a polynucleotide which also forms part of the present invention. The present invention relates more particularly to the recombinant nucleic acids as represented in SEQ ID NO 3, 7, 9, 11, 13, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 and 49, or parts thereof.

-22- The present invention also contemplates host cells transformed with a recombinant vector as defined above, wherein said vector comprises a nucleotide sequence encoding HCV El and/or E2 and/or El/E2 protein as defined above in addition to a regulatory sequence operably linked to said HCV El and/or E2 and/or E1/E2 sequence and capable of regulating the expression of said HCV El and/or E2 and/or E1/E2 protein.

Eukaryotic hosts include lower and higher eukaryotic hosts as described in the definitions section. Lower eukaryotic hosts include yeast cells well known in the art.

Higher eukaryotic hosts mainly include mammalian cell lines known in the art and include many immortalized cell lines available from the ATCC, inluding HeLa cells, Chinese 10 hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, PK15, RK13 and a number of other cell lines.

The present invention relates particularly to a recombinant El and/or E2 and/or E1/E2 protein expressed by a host cell as defined above containing a recombinany vector S- as defined above. These recombinant proteins are particularly purified according to the 15 method of the present invention.

A preferred method for isolating or purifying HCV envelope proteins as defined above is further characterized as comprising at least the following steps: growing a host cell as defined above transformed with a recombinant vector according to the present invention or with a known recombinant vector expressing 20 El and/or E2 and/or E1/E2 HCV envelope proteins in a suitable culture medium, causing expression of said vector sequence as defined above under suitable conditions, and, lysing said transformed host cells, preferably in the presence of a SH group blocking agent, such as N-ethylmaleimide (NEM), and possibly a suitable detergent, preferably Empigen-BB, recovering said HCV envelope protein by affinity purification such as by means of lectin-chromatography or immunoaffinity chromatography using anti-El and/or anti-E2 specific monoclonal antibodies, with said lectin being preferably lentillectin or GNA, followed by, -23incubation of the eluate of the previous step with a disulphide bond cleavage means, such as DTT, preferably followed by incubation with an SH group blocking agent, such as NEM or Biotin-NEM, and, isolating the HCV single or specific oligomeric El and/or E2 and/or E1/E2 proteins such as by means of gelfiltration and possibly also by a subsequent Ni2+-IMAC chromatography followed by a desalting step.

As a result of the above-mentioned proces, El and/or E2 and/or E1/E2 proteins may be produced in a form which elute differently from the large aggregates containing vector-derived components and/or cell components in the void volume of the gelfiltration 10 column or the IMAC collumn as illustrated in the Examples section. The disulphide bridge cleavage step advantageously also eliminates the false reactivity due to the presence of host and/or expression-system-derived proteins. The presence of NEM and a suitable detergent during lysis of the cells may already partly or even completely prevent the aggregation between the HCV envelope proteins and contaminants.

15 Ni 2 +-IMAC chromatography followed by a desalting step is preferably used for contructs bearing a (His) 6 as described by Janknecht et al., 1991, and Hochuli et al., 1988.

The present invention also relates to a method for producing monoclonal antibodies in small animals such as mice or rats, as well as a method for screening and isolating human B-cells that recognize anti-HCV antibodies, using the HCV single or specific 20 oligomeric envelope proteins of the present invention.

The present invention further relates to a composition comprising at least one of the following El peptides as listed in Table 3: El-31 (SEQ ID NO 56) spanning amino acids 181 to 200 of the Core/El V1 region, E1-33 (SEQ ID NO 57) spanning amino acids 193 to 212 of the El region, E1-35 (SEQ ID NO 58) spanning amino acids 205 to 224 of the El V2 region (epitope

B),

E1-35A (SEQ ID NO 59) spanning amino acids 208 to 227 of the El V2 region (epitope

B),

lbEl (SEQ ID NO 53) spanning amino acids 192 to 228 of El regions (VI, C1, and V2 regions (containing epitope -24- E1-51 (SEQ ID NO 66) spanning amino acids 301 to 320 of the El region, E1-53 (SEQ ID NO 67) spanning amino acids 313 to 332 of the El C4 region (epitope

A),

E1-55 (SEQ ID NO 68) spanning amino acids 325 to 344 of the El region.

The present invention also relates to a composition comprising at least one of the following E2 peptides as listed in Table 3: Env 67 or E2-67 (SEQ ID NO 72) spanning amino acid positions 397 to 416 of the E2 region (epitope A, recognized by monoclonal antibody 2F10H10, see Figure 19), t10 Env 69 or E2-69 (SEQ ID NO 73) spanning amino acid positions 409 to 428 of the E2 region (epitope A), *f.

S" Env 23 or E2-23 (SEQ ID NO 86) spanning positions 583 to 602 of the E2 region (epitope

E),

Env 25 or E2-25 (SEQ ID NO 87) spanning positions 595 to 614 of the E2 region 15 (epitope E), Env 27 or E2-27 (SEQ ID NO 88) spanning positions 607 to 626 of the E2 region (epitope E), Env 17B or E2-17B (SEQ ID NO 83) spanning positions 547 to 566 of the E2 region (epitope D), 20 Env 13B or E2-13B (SEQ ID NO 82) spanning positions 523 to 542 of the E2 region (epitope C; recognized by monoclonal antibody 16A6E7, see Figure 19).

The present invention also relates to a composition comprising at least one of the following E2 conformational epitopes: epitope F recognized by monoclonal antibodies 15C8C1, 12D11F1 and 8G10D1H9, epitope G recognized by monoclonal antibody 9G3E6, epitope H (or C) recognized by monoclonal antibody 10D3C4 and 4H6B2, or, epitope I recognized by monoclonal antibody 17F2C2.

The present invention also relates to an El or E2 specific antibody raised upon immunization with a peptide or protein composition, with said antibody being specifically reactive with any of the polypeptides or peptides as defined above, and with said antibody being preferably a monoclonal antibody.

The present invention also relates to an El or E2 specific antibody screened from a variable chain library in plasmids or phages or from a population of human B-cells by means of a process known in the art, with said antibody being reactive with any of the polypeptides or peptides as defined above, and with said antibody being preferably a monoclonal antibody.

The El or E2 specific monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly from a mouse or rat, immunized against the HCV polypeptides or peptides according to the invention, as defined above on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the polypeptides which has been initially 9* used for the immunization of the animals.

S. 15 The antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.

The monoclonal antibodies according to this preferred embodiment of the invention may be humanized versions of mouse monoclonal antibodies made by means of recombinant DNA technology, departing from parts of mouse and/or human genomic 20 DNA sequences coding for H and L chains from cDNA or genomic clones coding for H and L chains.

Alternatively the monoclonal antibodies according to this preferred embodiment of the invention may be human monoclonal antibodies. These antibodies according to the present embodiment of the invention can also be derived from human peripheral blood lymphocytes of patients infected with HCV, or vaccinated against HCV. Such human monoclonal antibodies are prepared, for instance, by means of human peripheral blood lymphocytes (PBL) repopulation of severe combined immune deficiency (SCID) mice (for recent review, see Duchosal et al., 1992).

The invention also relates to the use of the proteins or peptides of the invention, for the selection of recombinant antibodies by the process of repertoire cloning (Persson et al., 1991).

-26- Antibodies directed to peptides or single or specific oligomeric envelope proteins derived from a certain genotype may be used as a medicament, more particularly for incorporation into an immunoassay for the detection of HCV genotypes (for detecting the presence of HCV El or E2 antigen), for prognosing/monitoring of HCV disease, or as therapeutic agents.

Alternatively, the present invention also relates to the use of any of the abovespecified El or E2 specific monoclonal antibodies for the preparation of an immunoassay kit for detecting the presence of El or E2 antigen in a biological sample, for the preparation of a kit for prognosing/monitoring of HCV disease or for the preparation of a HCV 10 medicament.

The present invention also relates to the a method for in vitro diagnosis or detection of HCV antigen present in a biological sample, comprising at least the following steps: 0 contacting said biological sample with any of the El and/or E2 specific monoclonal antibodies as defined above, preferably in an immobilized form 15 under appropriate conditions which allow the formation of an immune complex, (ii) removing unbound components, (iii) incubating the immune complexes formed with heterologous antibodies, which specifically bind to the antibodies present in the sample to be 20 analyzed, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immune complexes visually or mechanically by means of densitometry, fluorimetry, colorimetry).

The present invention also relates to a kit for in vitro diagnosis of HCV antigen present in a biological sample, comprising: at least one monoclonal antibody as defined above, with said antibody being preferentially immobilized on a solid substrate, a buffer or components necessary for producing the buffer enabling binding reaction between these antibodies and the HCV antigens present in the biological sample, -27a means for detecting the immune complexes formed in the preceding binding reaction, possibly also including an automated scanning and interpretation device for inferring the HCV antigens present in the sample from the observed binding pattern.

The present invention also relates to a composition comprising El and/or E2 and/or El/E2 recombinant HCV proteins purified according to the method of the present invention or a composition comprising at least one peptides as specified above for use as a medicament.

O The present invention more particularly relates to a composition comprising at least S•one of the above-specified envelope peptides or a recombinant envelope protein composition as defined above, for use as a vaccine for immunizing a mammal, preferably humans, against HCV, comprising administering a sufficient amount of the composition possibly accompanied by pharmaceutically acceptable adjuvant(s), to produce an immune 15 response.

More particularly, the present invention relates to the use of any of the compositions as described here above for the preparation of a vaccine as described above.

Also, the present invention relates to a vaccine composition for immunizing a mammal, preferably humans, against HCV, comprising HCV single or specific oligomeric "20 proteins or peptides derived from the El and/or the E2 region as described above.

Immunogenic compositions can be prepared according to methods known in the art. The present compositions comprise an immunogenic amount of a recombinant El and/or E2 and/or El/E2 single or specific oligomeric proteins as defined above or El or E2 peptides as defined above, usually combined with a pharmaceutically acceptable carrier, preferably further comprising an adjuvant.

The single or specific oligomeric envelope proteins of the present invention, either El and/or E2 and/or El/E2, are expected to provide a particularly useful vaccine antigen, since the formation of antibodies to either El or E2 may be more desirable than to the other envelope protein, and since the E2 protein is cross-reactive between HCV types and the El protein is type-specific. Cocktails including type 1 E2 protein and El proteins derived from several genotypes may be particularly advantageous. Cocktails containing a molar excess -28of El versus E2 or E2 versus El may also be particularly useful. Immunogenic compositions may be administered to animals to induce production of antibodies, either to provide a source of antibodies or to induce protective immunity in the animal.

Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers; and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

10 Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to aluminim hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S. Patent No. 4,606,918, N-acetyl-normuramyl-L-alanyl-Disoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE) and RIBI, which S 15 contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween emulsion. Any of the 3 components MPL, TDM or CWS may also be used alone or combined 2 by 2. Additionally, adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA) or SAF-1 (Syntex) may be used. Further, Complete Freund's Adjuvant 20 (CFA) and Incomplete Freund's Adjuvant (IFA) may be used for non-human applications and research purposes.

The immunogenic compositions typically will contain pharmaceutically acceptable vehicles, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, preservatives, and the like, may be included in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect. The El and E2 proteins may also be incorporated into Immune Stimulating Complexes together with saponins, for example Quil A (ISCOMS).

-29- Immunogenic compositions used as vaccines comprise a'sufficient amount' or 'an immunologically effective amount' of the envelope proteins of the present invention, as well as any other of the above mentioned components, as needed. 'Immunologically effective amount', means that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment, as defined above. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, the strain of infecting HCV, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Usually, the amount will vary from 0.01 to 1000 tg/dose, more particularly from 0.1 to 100 jig/dose.

The single or specific oligomeric envelope proteins may also serve as vaccine 15 carriers to present homologous T cell epitopes or B cell epitopes from the core, NS2, NS3, NS4 or NS5 regions) or heterologous (non-HCV) haptens, in the same manner as Hepatitis B surface antigen (see European Patent Application 174,444). In this use, envelope proteins provide an immunogenic carrier capable of stimulating an immune response to haptens or antigens conjugated to the aggregate. The antigen may be 20 conjugated either by conventional chemical methods, or may be cloned into the gene encoding El and/or E2 at a location corresponding to a hydrophilic region of the protein.

Such hydrophylic regions include the V1 region (encompassing amino acid positions 191 to 202), the V2 region (encompassing amino acid positions 213 to 223), the V3 region (encompassing amino acid positions 230 to 242), the V4 region (encompassing amino acid positions 230 to 242), the V5 region (encompassing amino acid positions 294 to 303) and the V6 region (encompassing amino acid positions 329 to 336). Another useful location for insertion of haptens is the hydrophobic region (encompassing approximately amino acid positions 264 to 293). It is shown in the present invention that this region can be deleted without affecting the reactivity of the deleted El protein with antisera. Therefore, haptens may be inserted at the site of the deletion.

The immunogenic compositions are conventionally administered parenterally, typically by injection, for example, subcutaneously or intramuscularly. Additional formulations suitable for other methods of administration include oral formulations and suppositories. Dosage treatment may be a single dose schedule or a multiple dose schedule.

The vaccine may be administered in conjunction with other immunoregulatory agents.

The present invention also relates to a composition comprising peptides or polypeptides as described above, for in vitro detection of HCV antibodies present in a biological sample.

The present invention also relates to the use of a composition as described above 10 for the preparation of an immunoassay kit for detecting HCV antibodies present in a biological sample.

The present invention also relates to a method for in vitro diagnosis of HCV antibodies present in a biological sample, comprising at least the following steps S: contacting said biological sample with a composition comprising any of the 15 envelope peptide or proteins as defined above, preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex, wherein said peptide or protein can be a biotinylated peptide or protein which is covalently bound to a solid substrate by means of streptavidin or avidin complexes, 20 (ii) removing unbound components, (iii) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immune complexes visually or mechanically by means of densitometry, fluorimetry, colorimetry).

Alternatively, the present invention also relates to competition immunoassay formats in which recombinantly produced purified single or specific oligomeric protein El and/or E2 and/or E1/E2 proteins as disclosed above are used in combination with El and/or E2 peptides in order to compete for HCV antibodies present in a biological sample.

The present invention also relates to a kit for determining the presence of HCV antibodies, in a biological sample, comprising -31 at least one peptide or protein composition as defined above, possibly in combination with other polypeptides or peptides from HCV or other types of HCV, with said peptides or proteins being preferentially immobilized on a solid substrate, more preferably on different microwells of the same ELISA plate, and even more preferentially on one and the same membrane strip, a buffer or components necessary for producing the buffer enabling binding reaction between these polypeptides or peptides and the antibodies against .HCV present in the biological sample, SI 10 means for detecting the immune complexes formed in the preceding binding reaction, possibly also including an automated scanning and interpretation device for inferring the HCV genotypes present in the sample from the observed binding pattern.

o 15 The immunoassay methods according to the present invention utilize single or specific oligomeric antigens from the El and/or E2 domains that maintain linear (in case of peptides) and conformational epitopes (single or specific oligomeric proteins) recognized by antibodies in the sera from individuals infected with HCV. It is within the scope of the invention to use for instance single or specific oligomeric antigens, dimeric antigens, as .20 well as combinations of single or specific oligomeric antigens. The HCV El and E2 antigens of the present invention may be employed in virtually any assay format that employs a known antigen to detect antibodies. Of course, a format that denatures the HCV conformational epitope should be avoided or adapted. A common feature of all of these assays is that the antigen is contacted with the body component suspected of containing HCV antibodies under conditions that permit the antigen to bind to any such antibody present in the component. Such conditions will typically be physiologic temperature, pH and ionic strenght using an excess of antigen. The incubation of the antigen with the specimen is followed by detection of immune complexes comprised of the antigen.

Design of the immunoassays is subject to a great deal of variation, and many formats are known in the art. Protocols may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the -32labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin or streptavidin, and enzymelabeled and mediated immunoassays, such as ELISA assays.

The immunoassay may be, without limitation, in a heterogeneous or in a homogeneous format, and of a standard or competitive type. In a heterogeneous format, the polypeptide is typically bound to a solid matrix or support to facilitate separation of the sample from the polypeptide after incubation. Examples of solid supports that can be used are nitrocellulose in membrane or microtiter well form), polyvinyl chloride in 10 sheets or microtiter wells), polystyrene latex in beads or microtiter plates, polyvinylidine fluoride (known as ImmunolonT), diazotized paper, nylon membranes, 0activated beads, and Protein A beads. For example, Dynatech Immunolon 1 or o Immunlon T M 2 microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball) can be used in the heterogeneous format. The solid support containing the antigenic 15 polypeptides is typically washed after separating it from the test sample, and prior to detection of bound antibodies. Both standard and competitive formats are know in the art.

In a homogeneous format, the test sample is incubated with the combination of antigens in solution. For example, it may be under conditions that will precipitate any antigen-antibody complexes which are formed. Both standard and competitive formats for o 20 these assays are known in the art.

In a standard format, the amount of HCV antibodies in the antibody-antigen complexes is directly monitored. This may be accomplished by determining whether labeled anti-xenogeneic anti-human) antibodies which recognize an epitope on anti- HCV antibodies will bind due to complex formation. In a competitive format, the amount of HCV antibodies in the sample is deduced by monitoring the competitive effect on the binding of a known amount of labeled antibody (or other competing ligand) in the complex.

Complexes formed comprising anti-HCV antibody (or in the case of competitive assays, the amount of competing antibody) are detected by any of a number of known techniques, depending on the format. For example, unlabeled HCV antibodies in the -33 complex may be detected using a conjugate of anti-xenogeneic Ig complexed with a label an enzyme label).

In an immunoprecipitation or agglutination assay format the reaction between the HCV antigens and the antibody forms a network that precipitates from the solution or suspension and forms a visible layer or film of precipitate. If no anti-HCV antibody is present in the test specimen, no visible precipitate is formed.

There currently exist three specific types of particle agglutination (PA) assays.

These assays are used for the detection of antibodies to various antigens when coated to a support. One type of this assay is the hemagglutination assay using red blood cells (RBCs) that are sensitized by passively adsorbing antigen (or antibody) to the RBC. The addition of specific antigen antibodies present in the body component, if any, causes the RBCs coated with the purified antigen to agglutinate.

To eliminate potential non-specific reactions in the hemagglutination assay, two artificial carriers may be used instead of RBC in the PA. The most common of these are latex particles. However, gelatin particles may also be used. The assays utilizing either of these carriers are based on passive agglutination of the particles coated with purified antigens.

The HCV single or specififc oligomeric El and/or E2 and/or E1/E2 antigens of the present invention comprised of conformational epitopes will typically be packaged in the form of a kit for use in these immunoassays. The kit will normally contain in separate containers the native HCV antigen, control antibody formulations (positive and/or negative), labeled antibody when the assay format requires the same and signal generating reagents enzyme substrate) if the label does not generate a signal directly. The native HCV antigen may be already bound to a solid matrix or separate with reagents for binding it to the matrix. Instructions written, tape, CD-ROM, etc.) for carrying out the assay usually will be included in the kit.

Immunoassays that utilize the native HCV antigen are useful in screening blood for the preparation of a supply from which potentially infective HCV is lacking. The method for the preparation of the blood supply comprises the following steps. Reacting a body component, preferably blood or a blood component, from the individual donating blood with HCV El and/or E2 proteins of the present invention to allow an immunological -34reaction between HCV antibodies, if any, and the HCV antigen. Detecting whether anti- HCV antibody HCV antigen complexes are formed as a result of the reacting. Blood contributed to the blood supply is from donors that do not exhibit antibodies to the native HCV antigens, El or E2.

In cases of a positive reactivity to the HCV antigen, it is preferable to repeat the immunoassay to lessen the possibility of false positives. For example, in the large scale screening of blood for the production of blood products blood transfusion, plasma, Factor VIII, immunoglobulin, etc.) 'screening' tests are typically formatted to increase sensitivity (to insure no contaminated blood passes) at the expense of specificity; i.e. the 10 false-positive rate is increased. Thus, it is typical to only defer for further testing those donors who are 'repeatedly reactive'; i.e. positive in two or more runs of the immunoassay on the donated sample. However, for confirmation of HCV-positivity, the 'confirmation' tests are typically formatted to increase specificity (to insure that no false-positive samples are confirmed) at the expense of sensitivity. Therefore the purification method described in the present invention for El and E2 will be very advantageous for including single or specific oligomeric envelope proteins into HCV diagnostic assays.

The solid phase selected can include polymeric or glass beads, nitrocellulose, S microparticles, microwells of a reaction tray, test tubes and magnetic beads. The signal S* generating compound can include an enzyme, a luminescent compound, a chromogen, a 20 radioactive element and a chemiluminescent compound. Examples of enzymes include :alkaline phosphatase, horseradish peroxidase and beta-galactosidase. Examples of enhancer compounds include biotin, anti-biotin and avidin. Examples of enhancer compounds binding members include biotin, anti-biotin and avidin. In order to block the effects of rheumatoid factor-like substances, the test sample is subjected to conditions sufficient to block the effect of rheumatoid factor-like substances. These conditions comprise contacting the test sample with a quantity of anti-human IgG to form a mixture, and incubating the mixture for a time and under conditions sufficient to form a reaction mixture product substantially free of rheumatoid factor-like substance.

The present invention further contemplates the use of El proteins, or parts thereof, more particularly HCV single or specific oligomeric El proteins as defined above, for in vitro monitoring HCV disease or prognosing the response to treatment (for instance with Interferon) of patients suffering from HCV infection comprising: incubating a biological sample from a patient with hepatitis C infection with an El protein or a suitable part thereof under conditions allowing the formation of an immunological complex, removing unbound components, calculating the anti-El titers present in said sample (for example at the start of and/or during the course of (interferon) therapy), monitoring the natural course of HCV disease, or prognosing the response to treatment of said patient on the basis of the amount anti-E 1 titers found in said sample at the start of treatment and/or during the course of treatment.

Patients who show a decrease of 2, 3, 4, 5, 7, 10, 15, or preferably more than times of the initial anti-El titers could be concluded to be long-term, sustained responders to HCV therapy, more particularly to interferon therapy. It is illustrated in the Examples section, that an anti-El assay may be very useful for prognosing long-term response to IFN treatment, or to treatment of Hepatitis C virus disease in general.

More particularly the following El peptides as listed in Table 3 were found to be useful for in vitro monitoring HCV disease or prognosing the response to interferon treatment of patients suffering from HCV infection: El-31 (SEQ ID NO 56) spanning amino acids 181 to 200 of the Core/El VI region, E1-33 (SEQ ID NO 57) spanning amino acids 193 to 212 of the El region, E1-35 (SEQ ID NO 58) spanning amino acids 205 to 224 of the El V2 region (epitope B), E1-35A (SEQ ID NO 59) spanning amino acids 208 to 227 of the El V2 region (epitope

B),

lbEl (SEQ ID NO 53) spanning amino acids 192 to 228 of El regions (VI, Cl, and V2 regions (containing epitope El-51 (SEQ ID NO 66) spanning amino acids 301 to 320 of the El region, -36- E1-53 (SEQ ID NO 67) spanning amino acids 313 to 332 of the El C4 region (epitope A), E1-55 (SEQ ID NO 68) spamnning amino acids 325 to 344 of the El region.

It is to be understood that smaller fragments of the above-mentioned peptides also fall within the scope of the present invention. Said smaller fragments can be easily prepared by chemical synthesis and can be tested for their ability to be used in an assay as detailed above and in the Examples section.

The present invention also relates to a kit for monitoring HCV disease or prognosing the response to treatment (for instance to interferon) of patients suffering from 10 HCV infection comprising: at least one El protein or El peptide, more particularly an El protein or El peptide as defined above, a buffer or components necessary for producing the buffer enabling the :binding reaction between these proteins or peptides and the anti-E antibodies present in a biological sample, means for detecting the immune complexes formed in the preceding binding reaction, possibly also an automated scanning and interpretation device for inferring a decrease of anti-El titers during the progression of treatment.

20 It is to be understood that also E2 protein and peptides according to the present S• invention can be used to a certain degree to monitor/prognose HCV treatment as indicated above for the El proteins or peptides because also the anti-E2 levels decrease in comparison to antibodies to the other HCV antigens. It is to be understood, however, that it might be possible to determine certain epitopes in the E2 region which would also be suited for use in an test for monitoring/prognosing HCV disease.

The present invention also relates to a serotyping assay for detecting one or more serological types of HCV present in a biological sample, more particularly for detecting antibodies of the different types of HCV to be detected combined in one assay format, comprising at least the following steps contacting the biological sample to be analyzed for the presence of HCV antibodies of one or more serological types, with at least one of the El -37and/or E2 and/or El/E2 protein compositions or at least one of the El or E2 peptide compositions as defined above, preferantially in an immobilized form under appropriate conditions which allow the formation of an immune complex, (ii) removing unbound components, (iii) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies being conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immune complexes visually or mechanically by means of densitometry, fluorimetry, colorimetry) and inferring the presence of one or more HCV serological types present from the observed binding pattern.

It is to be understood that the compositions of proteins or peptides used in this method are recombinantly expressed type-specific envelope proteins or type-specific peptides.

The present invention further relates to a kit for serotyping one or more serological types of HCV present in a biological sample, more particularly for detecting the antibodies to these serological types of HCV comprising: at least one El and/or E2 and/or El/E2 protein or El or E2 peptide, as defined above, a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins or peptides and the anti-El 1 antibodies present in a biological sample, means for detecting the immune complexes formed in the preceding binding reaction, possibly also an automated scanning and interpretation device for detecting the presence of one or more serological types present from the observed binding pattern.

The present invention also relates to the use of a peptide or protein composition as defined above, for immobilization on a solid substrate and incorporation into a reversed phase hybridization assay, preferably for immobilization as parallel lines onto a solid -38support such as a membrane strip, for determining the presence or the genotype of HCV according to a method as defined above. Combination with other type-specific antigens from other HCV polyprotein regions also lies within the scope of the present invention.

Figure and Table legends Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11: Figure 12: Figure 13 Figure 14 Figure 15 Figure 16 Figure 17: Figure 18 Figure 19 Figure 20: Figure 21: Restriction map of plasmid pgpt ATA 18 Restriction map ofplasmid pgs ATA 18 Restriction map ofplasmid pMS 66 Restriction map of plasmid pv HCV-11A Anti-El levels in non-responders to IFN treatment Anti-El levels in responders to IFN treatment Anti-El levels in patients with complete response to IFN treatment Anti-El levels in incomplete responders to IFN treatment Anti-E2 levels in non-responders to IFN treatment Anti-E2 levels in responders to IFN treatment Anti-E2 levels in incomplete responders to IFN treatment Anti-E2 levels in complete responders to IFN treatment Human anti-El reactivity competed with peptides Competition of reactivity of anti-El monoclonal antibodies with peptides Anti-E1 (epitope 1) levels in non-responders to IFN treatment Anti-El (epitope 1) levels in responders to IFN treatment Anti-E1 (epitope 2) levels in non-responders to IFN treatment Anti-El (epitope 2) levels in responders to IFN treatment Competition of reactivity of anti-E2 monoclonal antibodies with peptides Human anti-E2 reactivity competed with peptides Nucleic acid sequences of the present invention. The nucleic acid sequences encoding an El or E2 protein according to the present invention may be translated (SEQ ID NO 3 to 13, 21-31, 35 and 41-49 are translated in a reading frame starting from residue number 1, SEQ ID NO 37-39 are translated in a reading frame starting from residue number into the -39amino acid sequences of the respective El or E2 proteins as shown in the sequence listing.

Figure 22: ELISA results obtained from lentil lectin chromatography eluate fractions of 4 different El purifications of cell lysates infected with vvHCV39 (type lb), vvHCV40 (type lb), vvHCV62 (type 3a), and vvHCV63 (type Figure 23: Elution profiles obtained from the lentil lectin chromatography of the 4 different El constructs on the basis of the values as shown in Figure 22.

Figure 24: ELISA results obtained from fractions obtained after gelfiltration chromatography of 4 different El purifications of cell lysates infected with vvHCV39 (type lb), vvHCV40 (type lb), vvHCV62 (type 3a), and *o.

vvHCV63 (type Figure 25: Profiles obtained from purifications of El proteins of type lb type 3a and type 5a (from RK13 cells infected with vvHCV39, wHCV62, and vvHCV63, respectively; purified on lentil lectin and reduced as in example 5.2 5.3) and a standard The peaks indicated with and represent pure El protein peaks (see Figure 24, El reactivity mainly in fractions 26 to Figure 26: Silver staining of an SDS-PAGE as described in example 4 of a raw lysate of El vvHCV40 (type lb) (lane pool 1 of the gelfiltration representing fractions 10 to 17 as shown in Figure 25 (lane pool 2 of the gelfiltration of vvHCV40 representing fractions 18 to 25 as shown in Figure 25 (lane and El pool (fractions 26 to 30) (lane 4).

Figure 27: Streptavidine-alkaline phosphatase blot of the fractions of the gelfiltration of El constructs 39 (type lb) and 62 (type 3a). The proteins were labelled with NEM-biotin. Lane 1: start gelfiltration construct 39, lane 2: fraction 26 construct 39, lane 3: fraction 27 construct 39, lane 4: fraction 28 construct 39, lane 5: fraction 29 construct 39, lane 6: fraction 30 construct 39, lane 7 fraction 31 construct 39, lane 8: molecular weight marker, lane 9: start gelfiltration construct 62, lane 10: fraction 26 construct 62, lane 11: fraction 27 construct 62, lane 12: fraction 28 construct 62, lane 13: fraction 29 construct 62, lane 14: fraction 30 construct 62, lane 15: fraction 31 construct 62.

Figure 28: Siver staining of an SDS-PAGE gel of the gelfiltration fractions of vvHCV- 39 (Els, type Ib) and vvHCV-62 (Els, type 3a) run under identical conditions as Figure 26. Lane 1: start gelfiltration construct 39, lane 2: fraction 26 construct 39, lane 3: fraction 27 construct 39, lane 4: fraction 28 construct 39, lane 5: fraction 29 construct 39, lane 6: fraction 30 construct 39, lane 7 fraction 31 construct 39, lane 8: molecular weight marker, lane 9: start gelfiltration construct 62, lane 10: fraction 26 construct 62, lane 11: fraction 27 construct 62, lane 12: fraction 28 construct 62, lane 13: fraction 29 construct 62, lane 14: fraction 30 construct 62, lane 15: fraction 31 S* construct 62.

Figure 29: Western Blot analysis with anti-El mouse monoclonal antibody 5E1A10 giving a complete overview of the purification procedure. Lane 1: crude lysate, Lane 2: flow through of lentil chromagtography, Lane 3: wash with Empigen BB after lentil chromatography, Lane 4: Eluate of lentil chromatography, Lane 5: Flow through during concentration of the lentil eluate, Lane 6: Pool of El after Size Exclusion Chromatography (gelfiltration).

Figure 30: OD 2 80 profile (continuous line) of the lentil lectin chromatography of E2 protein from RK13 cells infected with vvHCV44. The dotted line represents the E2 reactivity as detected by ELISA (as in example 6).

Figure 31A: OD 280 profile (continuous line) of the lentil-lectin gelfiltration chromatography E2 protein pool from RK13 cells infected with wHCV44 in which the E2 pool is applied immediately on the gelfiltration column (non-reduced conditions). The dotted line represents the E2 reactivity as detected by ELISA (as in example 6).

Figure 31 B: OD 28 0 profile (continuous line) of the lentil-lectin gelfiltration chromatography E2 protein pool from RK13 cells infected with vvHCV44 in which the E2 pool was reduced and blocked according to Example 5.3 -41- (reduced conditions). The dotted line represents the E2 reactivity as detected by ELISA (as in example 6).

2+ Figure 32: Ni -IMAC chromatography and ELISA reactivity of the E2 protein as expressed from wHCV44 after gelfiltration under reducing conditions as shown in Figure 31B.

Figure 33: Silver staining of an SDS-PAGE of 0.5 tg of purified E2 protein recovered by a 200 mM imidazole elution step (lane 2) and a 30mM imidazole wash (lane 1) of the Ni2+-IMAC chromatography as shown in Figure 32.

Figure 34: OD profiles of a desalting step of the purified E2 protein recovered by 200 mM immidazole as shown in Figure 33, intended to remove imidazole.

Figure 35A: Antibody levels to the different HCV antigens (Core 1, Core 2, E2HCVR, NS3) for NR and LTR followed during treatment and over a period of 6 to 12 months after treatment determined by means of the LIAscan method.

The average values are indicated by the curves with the open squares.

Figure 35B: Antibody levels to the different HCV antigens (NS4, NS5, El and E2) for NR and LTR followed during treatment and over a period of 6 to 12 months after treatment determined by means of the LIAscan method. The avergae vallues are indicated by the curve with the open squares.

20 Figure 36: Average El antibody (E1Ab) and E2 antibody (E2Ab) levels in the LTR and NR groups.

Figure 37: Averages El antibody (E1Ab) levels for non-responders (NR) and long term responders (LTR) for type lb and type 3a.

Figure 38: Relative map positions of the anti-E2 monoclonal antibodies.

Figure 39: Partial deglycosylation of HCV El envelope protein. The lysate of vvHCV 10A-infected RK13 cells were incubated with different concentrations of glycosidases according to the manufacturer's instructions.

Right panel: Glycopeptidase F (PNGase Left panel: Endoglycosidase H (Endo H).

Figure 40: Partial deglycosylation of HCV E2 envelope proteins. The lysate of vvHCV64-infected (E2) and vvHCV41-infected (E2s)RK13 cells were 9* -42incubated with different concentrations of Glycopeptidase F (PNGase F) according to the manufacturer's instructions.

Figure 41: In vitro mutagenesis of HCV El glycoproteins. Map of the mutated sequences and the creation of new restriction sites.

Figure 42A: In vitro mutagenesis of HCV El glycoprotein (part First step of PCR amplification.

Figure 42B: In vitro mutagensis of HCV El glycoprotein (part Overlap extension and nested PCR.

Figure 43: In vitro mutagesesis of HCV El glycoproteins. Map of the PCR mutated fragments (GLY-# and OVR-#) synthesized during the first step of amplification.

Figure 44A: Analysis of El glycoprotein mutants by Western blot expressed in HeLa

S

(left) and RK13 (right) cells. Lane 1: wild type VV (vaccinia virus), Lane 2: original El protein (vvHCV-10A), Lane 3: El mutant Gly-1 (wHCV-81), Lane 4: El mutant Gly-2 (wHCV-82), Lane 5: El mutant Gly-3 (wHCV- 83), Lane 6: El mutant Gly-4 (vvHCV-84), Lane 7: El mutant (vvHCV-85), Lane 8: El mutant Gly-6 (vvHCV-86).

Figure 44B: Analysis of El glycosylation mutant vaccinia viruses by PCR amplification/restriction. Lane 1: El (vvHCV-1OA), BspE I, Lane 2: E1.GLY-1 (vvHCV-81), BspE I, Lane 4: El (vvHCV-10A), Sac I, Lane E1.GLY-2 (vvHCV-82), Sac I, Lane 7: El (vvHCV-10A), Sac I, Lane 8: E1.GLY-3 (vvHCV-83), Sac I, Lane 10: El (vvHCV-10A), Stu I, Lane 11: E1.GLY-4 (vvHCV-84), Stu I, Lane 13: El (vvHCV-O1A), Sma I, Lane 14: (wHCV-85), Sma I, Lane 16: El (wHCV-1OA), Stu I, Lane 17: E1.GLY-6 (vvHCV-86), Stu I, Lane 3 6 9 12 15 Low Molecular Weight Marker, pBluescript SK+, Msp I.

Figure 45: SDS polyacrylamide gel electrophoresis of recombinant E2 expressed in S.

cerevisiae. Innoculates were grown in leucine selective medium for 72 hrs.

and diluted 1/15 in complete medium. After 10 days of culture at 28 0

C,

medium samples were taken. The equivalent of 200 p1 of culture 43 supernatant concentrated by speedvac was loaded on the gel. Two independent transformants were analysed.

Figure 46: SDS polyacrylamide gel electrophoresis of recombinant E2 expressed in a glycosylation deficient S. cerevisiae mutant. Innoculae were grown in leucine selective medium for 72 hrs. and diluted 1/15 in complete medium.

After 10 days of culture at 28°C, medium samples were taken. The equivalent of 350 Ll of culture supernatant, concentrated by ion exchange chromatography, was loaded on the gel.

Table 1 Features of the respective clones and primers used for amplification for constructing the different forms of the E1 protein as despected in Example 1.

Table 2 Summary of Anti-El tests V649 oTable 3 Synthetic peptides for competition studies Table 4: Changes of envelope antibody levels over time.

Table 5: Difference between LTR and NR Table 6: Competition experiments between murine E2 monoclonal antibodies Table 7: Primers for construction of El glycosylation mutants 0 Table 8: Analysis of El glycosylation mutants by ELISA Example 1: Cloning and expression of the hepatitis C virus El protein *e 1. Construction of vaccinia virus recombination vectors The pgptATA18 vaccinia recombination plasmid is a modified version of pATA 18 (Stunnenberg et al, 1988) with an additional insertion containing the E. coli xanthine guanine phosphoribosyl transferase gene under the control of the vaccinia virus I3 intermediate promoter (Figure The plasmid pgsATA18 was constructed by inserting an oligonucleotide linker with SEQ ID NO 1/94, containing stop codons in the three reading frames, into the Pst I and HindIII-cut pATA18 vector. This created an extra Pac I restriction site (Figure The original HindIII site was not restored.

-44- Oligonucleotide linker with SEQ ID NO 1/94: 5' G GCATGC AAGCTT AATTAATT 3' 3' ACGTC CGTACG TTCGAA TTAATTAA TCGA PstI SphI HindIII Pac I (HindIII) In order to facilitate rapid and efficient purification by means of Ni 2 chelation of engineered histidine stretches fused to the recombinant proteins, the vaccinia recombination vector pMS66 was designed to express secreted proteins with an additional carboxy-terminal histidine tag. An oligonucleotide linker with SEQ ID NO 2/95, containing unique sites for 3 restriction enzymes generating blunt ends (Sma I, Stu I and Pml I/Bbr PI) was synthesized in such a way that the carboxy-terminal end of any cDNA could be inserted in frame with a sequence encoding the protease factor Xa cleavage site followed by a nucleotide sequence encoding 6 histidines and 2 stop codons (a new Pac I •restriction site was also created downstream the 3'end). This oligonucleotide with SEQ ID NO 2/95 was introduced between the Xma I and Pst I sites ofpgptATA18 (Figure 3).

Oligonucleotide linker with SEQ ID NO 2/95: CCGGG GAGGCCTGCACGTGATCGAGGGCAGACACCATCACCACCATCACTAATAGTTAATTAA CTGCA 3' 3' C CTCCGGACGTGCACTAGCTCCCGTCTGTGGTAGTGGTGGTAGTGATTATCAATTAATT G XmaI PstI Example 2. Construction of HCV recombinant plasmids 2.1. Constructs encoding different forms of the El protein Polymerase Chain Reaction (PCR) products were derived from the serum samples by RNA preparation and subsequent reverse-transcription and PCR as described previously (Stuyver et al., 1993b). Table 1 shows the features of the respective clones and the primers used for amplification. The PCR fragments were cloned into the Sma I-cut pSP72 (Promega) plasmids. The following clones were selected for insertion into vaccinia 10 reombination vectors: HCC19A (SEQ ID NO HCCll0A (SEQ ID NO HCCll 1A •(SEQ ID NO HCC112A (SEQ ID NO HCCl13A (SEQ ID NO 11), and HCCll7A (SEQ ID NO 13) as depicted in Figure 21. cDNA fragments containing the El-coding regions were cleaved by EcoRI and HindIII restriction from the respective pSP72 plasmids and inserted into the EcoRI/HindIII-cut pgptATA-18 vaccinia recombination vector 15 (described in example downstream of the 11K vaccinia virus late promoter. The respective plasmids were designated pvHCV-9A, pvHCV-1OA, pvHCV-11 A, pvHCV- 12A, pvHCV-13A and pvHCV-17A, of which pvHCV-11A is shown in Figure 4.

S.2.2. Hydrophobic region El deletion mutants Clone HCC137, containing a deletion of codons Asp264 to Val287 (nucleotides 790 to 861, region encoding hydrophobic domain I) was generated as follows: 2 PCR fragments were generated from clone HCCll0A with primer sets HCPr52 (SEQ ID NO 16)/HCPrl07 (SEQ ID NO 19) and HCPrl08 (SEQ ID NO 20)/HCPR54 (SEQ ID NO 18).

These primers are shown in Figure 21. The two PCR fragments were purified from agarose gel after electrophoresis and 1 ng of each fragment was used together as template for PCR by means of primers HCPr52 (SEQ ID NO 16) and HCPr54 (SEQ ID NO 18). The resulting fragment was cloned into the Sma I-cut pSP72 vector and clones containing the deletion were readily identified because of the deletion of 24 codons (72 base pairs).

Plasmid pSP72HCC137 containing clone HCC137 (SEQ ID 15) was selected. A recombinant vaccinia plasmid containing the full-length El cDNA lacking hydrophobic -46domain I was constructed by inserting the HCV sequence surrounding the deletion (fragment cleaved by Xma I and BamH I from the vector pSP72-HCC137) into the Xma I- Bam H I sites of the vaccinia plasmid pvHCV-1OA. The resulting plasmid was named pvHCV-37. After confirmatory sequencing, the amino-terminal region containing the internal deletion was isolated from this vector pvHCV-37 (cleavage by EcoR I and BstE II) and reinserted into the Eco RI and Bst EII-cut pvHCV-11A plasmid. This construct was expected to express an El protein with both hydrophobic domains deleted and was named pvHCV-38. The El-coding region of clone HCC138 is represented by SEQ ID NO 23.

As the hydrophilic region at the El carboxyterminus (theoretically extending to 10 around amino acids 337-340) was not completely included in construct pvHCV-38, a S•larger El region lacking hydrophobic domain I was isolated from the pvHCV-37 plasmid by EcoR I/Bam HI cleavage and cloned into an EcoRI/BamHI-cut pgsATA-18 vector. The resulting plasmid was named pvHCV-39 and contained clone HCC139 (SEQ ID NO The same fragment was cleaved from the pvHCV-37 vector by BamH I (of which the sticky ends were filled with Klenow DNA Polymerase I (Boehringer)) and subsequently by EcoR I cohesive end). This sequence was inserted into the EcoRI and Bbr PI-cut vector pMS-66. This resulted in clone HCC140 (SEQ ID NO 27) in plasmid containing a 6 histidine tail at its carboxy-terminal end.

2.3. El of other genotypes Clone HCC162 (SEQ ID NO 29) was derived from a type 3a-infected patient with chronic hepatitis C (serum BR36, clone BR36-9-13, SEQ ID NO 19 in WO 94/25601, and see also Stuyver et al. 1993a) and HCC163 (SEQ ID NO 31) was derived from a type infected child with post-transfusion hepatitis (serum BE95, clone PC-4-1, SEQ ID NO in WO 94/25601).

2.4. E2 constructs The HCV E2 PCR fragment 22 was obtained from serum BE11 (genotype Ib) by means of primers HCPrl09 (SEQ ID NO 33) and HCPr72 (SEQ ID NO 34) using -47techniques of RNA preparation, reverse-transcription and PCR, as described in Stuyver et al., 1993b, and the fragment was cloned into the Sma I-cut pSP72 vector. Clone HCC122A (SEQ ID NO 35) was cut with NcoI/AlwNI or by BamHI/AlwNI and the sticky ends of the fragments were blunted (NcoI and BamHI sites with Klenow DNA Polymerase I (Boehringer), and AlwNI with T4 DNA polymerase (Boehringer)). The BamHI/AlwNI cDNA fragment was then inserted into the vaccinia pgsATA-18 vector that had been linearized by EcoR I and Hind III cleavage and of which the cohesive ends had been filled with Klenow DNA Polymerase (Boehringer). The resulting plasmid was named pvHCV- 41 and encoded the E2 region from amino acids Met347 to Gln673, including 37 amino 10 acids (from Met347 to Gly383) of the El protein that can serve as signal sequence. The isame HCV cDNA was inserted into the EcoR I and Bbr PI-cut vector pMS66, that had subsequently been blunt ended with Klenow DNA Polymerase. The resulting plasmid was named pvHCV-42 and also encoded amino acids 347 to 683. The NcoI/AlwNI fragment was inserted in a similar way into the same sites ofpgsATA-18 (pvHCV-43) or pMS-66 vaccinia vectors (pvHCV-44). pvHCV-43 and pvHCV-44 encoded amino acids 364 to 673 of the HCV polyprotein, of which amino acids 364 to 383 were derived from the natural carboxyterminal region of the El protein encoding the signal sequence for E2, and amino acids 384 to 673 of the mature E2 protein.

20 2.5. Generation of recombinant HCV-vaccinia viruses Rabbit kidney RK13 cells (ATCC CCL 37), human osteosarcoma 143B thymidine kinase deficient (TK) (ATCC CRL 8303), HeLa (ATCC CCL and Hep G2 (ATCC HB 8065) cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, Md, USA). The cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10 foetal calf serum, and with Earle's salts (EMEM) for RK13 and 143 B and with glucose (4 g/l) for Hep G2. The vaccinia virus WR strain (Western Reserve, ATTC VR119) was routinely propagated in either 143B or RK13 cells, as described previously (Panicali Paoletti, 1982; Piccini et al., 1987; Mackett et al., 1982, 1984, and 1986). A confluent monolayer of 143B cells was infected with wild type vaccinia virus at a multiplicity of infection of 0.1 0.1 plaque forming unit (PFU) -48per cell). Two hours later, the vaccinia recombination plasmid was transfected into the infected cells in the form of a calcium phosphate coprecipitate containing 500 ng of the plasmid DNA to allow homologous recombination (Graham van der Eb, 1973; Mackett et al., 1985). Recombinant viruses expressing the Escherichia coli xanthine-guanine phosphoribosyl transferase (gpt) protein were selected on rabbit kidney RK13 cells incubated in selection medium (EMEM containing 25 ptg/ml mycophenolic acid (MPA), 250 ig/ml xanthine, and 15 p.g/ml hypoxanthine; Falkner and Moss, 1988; Janknecht et al, 1991). Single recombinant viruses were purified on fresh monolayers of RK13 cells under °a 0.9% agarose overlay in selection medium. Thymidine kinase deficient (TK") 10 recombinant viruses were selected and then plaque purified on fresh monolayers of human 143B cells in the presence of 25 [g/ml 5-bromo-2'-deoxyuridine. Stocks of purified recombinant HCV-vaccinia viruses were prepared by infecting either human 143 B or rabbit RK13 cells at an m.o.i. of 0.05 (Mackett et al, 1988). The insertion of the HCV cDNA fragment in the recombinant vaccinia viruses was confirmed on an aliquot (50 Ll) of the cell lysate after the MPA selection by means of PCR with the primers used to clone the respective HCV fragments (see Table The recombinant vaccinia-HCV viruses were named according to the vaccinia recombination plasmid number, e.g. the recombinant vaccinia virus vvHCV-O1A was derived from recombining the wild type WR strain with the pvHCV-1OA plasmid.

Example 3: infection of cells with recombinant vaccinia viruses A confluent monolayer of RK13 cells was infected at a m.o.i. of 3 with the recombinant HCV-vaccinia viruses as described in example 2 For infection, the cell monolayer was washed twice with phosphate-buffered saline pH 7.4 (PBS) and the recombinant vaccinia virus stock was diluted in MEM medium. Two hundred 1l of the virus solution was added per 10 cells such that the m.o.i. was 3, and incubated for 45 min at 24°C. The virus solution was aspirated and 2 ml of complete growth medium (see example 2) was added per 106 cells. The cells were incubated for 24 hr at 37 0 C during which expression of the HCV proteins took place.

-49- Example 4: Analysis of recombinant proteins by means of western blotting The infected cells were washed two times with PBS, directly lysed with lysis buffer mM Tris.HCI pH 7.5, 150 mM NaC1, 1% Triton X-100, 5 mM MgCl 2 1 ug/ml aprotinin (Sigma, Bomem, Belgium)) or detached from the flasks by incubation in 50 mM Tris.HCL pH 7.5/ 10 mM EDTA/ 150 mM NaCl for 5 min, and collected by centrifugation (5 min at 1000g). The cell pellet was then resuspended in 200 p1 lysis buffer (50 mM 10 Tris.HCL pH 8.0, 2 mM EDTA, 150 mM NaC1, 5 mM MgCl2, aprotinin, 1% Triton X- 100) per 10 6 cells. The cell lysates were cleared for 5 min at 14,000 rpm in an Eppendorf centrifuge to remove the insoluble debris. Proteins of 20 pl lysate were separated by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then electro-transferred from the gel to a nitrocellulose sheet (Amersham) using a 15 Hoefer HSI transfer unit cooled to 4 0 C for 2 hr at 100 V constant voltage, in transfer buffer mM Tris.HCI pH 8.0, 192 mM glycine, 20% methanol). Nitrocellulose filters were blocked with Blotto (5 fat-free instant milk powder in PBS; Johnson et al., 1981) and incubated with primary antibodies diluted in Blotto/0.1 Tween 20. Usually, a human negative control serum or serum of a patient infected with HCV were 200 times 20 diluted and preincubated for 1 hour at room temperature with 200 times diluted wild type vaccinia virus-infected cell lysate in order to decrease the non-specific binding. After washing with Blotto/0.1% Tween 20, the nitrocellulose filters were incubated with alkaline phosphatase substrate solution diluted in Blotto/0.1 Tween 20. After washing with 0.1% Tween 20 in PBS, the filters were incubated with alkaline phosphatase substrate solution (100 mM Tris.HCl pH 9.5, 100 mM NaC1, 5 mM MgCl2, 0,38 gg/ml nitroblue tetrazolium, 0.165 tg/ml 5-bromo-4-chloro-3-indolylphosphate). All steps, except the electrotransfer, were performed at room temperature.

Example 5: Purification of recombinant El or E2 protein 5.1. Lysis Infected RK13 cells (carrying El or E2 constructs) were washed 2 times with phosphate-buffered saline (PBS) and detached from the culture recipients by incubation in PBS containing 10 mM EDTA. The detached cells were washed twice with PBS and 1 ml of lysis buffer (50 mM Tris.HCl pH 7.5, 150 mM NaC1, 1% Triton X-100, 5 mM MgCl 2 1 :g/ml aprotinin (Sigma, Bomem, Belgium) containing 2 mM biotinylated N- 10 ethylmaleimide (biotin-NEM) (Sigma) was added per 10 5 cells at 4 0 C. This lysate was homogenized with a type B douncer and left at room temperature for 0.5 hours. Another volumes of lysis buffer containing 10 mM N-ethylmaleimide (NEM, Aldrich, Bornem, Belgium) was added to the primary lysate and the mixture was left at room temperature for 15 min. Insoluble cell debris was cleared from the solution by centrifugation in a Beckman 15 JA-14 rotor at 14,000 rpm (30100 g at rmx) for 1 hour at 4 0

C.

5.2. Lectin Chromatography The cleared cell lysate was loaded at a rate of Iml/min on a 0.8 by 10 cm Lentil- 20 lectin Sepharose 4B column (Pharmacia) that had been equilibrated with 5 column volumes of lysis buffer at a rate of Iml/min. The lentil-lectin column was washed with 5 to column volumes of buffer 1 (0.1M potassium phosphate pH 7.3, 500 mM KC1, glycerol, 1 mM 6-NH 2 -hexanoic acid, 1 mM MgCI 2 and 1% DecylPEG (KWANT, Bedum, The Netherlands). In some experiments, the column was subsequently washed with 10 column volumes of buffer 1 containing 0.5% Empigen-BB (Calbiochem, San Diego, CA, USA) instead of 1% DecylPEG. The bound material was eluted by applying elution buffer (10 mM potassium phosphate pH 7.3, 5% glycerol, 1 mM hexanoic acid, 1mM MgC12, 0.5% Empigen-BB, and 0.5 M a-methyl-mannopyranoside). The eluted material was fractionated and fractions were screened for the presence of El or E2 protein by means of ELISA as described in example 6. Figure 22 shows ELISA results obtained from lentil lectin eluate fractions of 4 different El purifications of cell lysates infected with -51 wHCV39 (type Ib), vvHCV40 (type Ib), vvHCV62 (type 3a), and vvHCV63 (type Figure 23 shows the profiles obtained from the values shown in Figure 22. These results show that the lectin affinity column can be employed for envelope proteins of the different types of HCV.

5.3. Concentration and partial reduction The El- or E2-positive fractions were pooled and concentrated on a Centricon kDa (Amicon) by centrifugation for 3 hours at 5,000 rpm in a Beckman JA-20 rotor at 4 0

C.

S. 10 In some experiments the El or E2-positive fractions were pooled and concentrated by ft 8 •nitrogen evaporation. An equivalent of 3.10 cells was concentrated to approximately 200 pl. For partial reduction, 30% Empigen-BB (Calbiochem, San Diego, CA, USA) was added to this 200 p1 to a final concentration of 3.5 and 1M DTT in H 2 0 was subsequently added to a final concentration of 1.5 to 7.5 mM and incubated for 30 min at 15 37 NEM (1M in dimethylsulphoxide) was subsequently added to a final concentration of 50 mM and left to react for another 30 min at 37C to block the free sulphydryl groups.

5.4. Gel filtration chromatography 20 A Superdex-200 HR 10/20 column (Pharmacia) was equilibrated with 3 column volumes PBS/3% Empigen-BB. The reduced mixture was injected in a 500 p1 sample loop of the Smart System (Pharmacia) and PBS/3% Empigen-BB buffer was added for gelfiltration. Fractions of 250 p l were collected from Vo to V t The fractions were screened for the presence of El or E2 protein as described in example 6.

Figure 24 shows ELISA results obtained from fractions obtained after gelfiltration chromatography of 4 different El purifications of cell lysates infected with vvHCV39 (type lb), wHCV40 (type lb), vvHCV62 (type 3a), and wHCV63 (type 5a). Figure shows the profiles obtained from purifications of El proteins of types lb, 3a, and 5a (from RK13 cells infected with vvHCV39, vHCV62, and vvHCV63, respectively; purified on lentil lectin and reduced as in the previous examples). The peaks indicated with and represent pure El protein peaks (El reactivity mainly in fractions 26 to 30). These -52peaks show very similar molecular weights of approximately 70 kDa, corresponding to dimeric El protein. Other peaks in the three profiles represent vaccinia virus and/or cellular proteins which could be separated from El only because of the reduction step as outlined in example 5.3. and because of the subsequent gelfiltration step in the presence of the proper detergent. As shown in Figure 26 pool 1 (representing fractions 10 to 17) and pool 2 (representing fractions 18 to 25) contain contaminating proteins not present in the El pool (fractions 26 to 30). The El peak fractions were ran on SDS/PAGE and blotted as described in example 4. Proteins labelled with NEM-biotin were detected by streptavidinalkaline phosphatase as shown in Figure 27. It can be readily observed that, amongst 10 others, the 29 kDa and 45kDa contaminating proteins present before the gelfiltration chromatography (lane 1) are only present at very low levels in the fractions 26 to 30. The band at approximately 65kDa represents the El dimeric form that could not be entirely disrupted into the monomeric El form. Similar results were obtained for the type 3a El protein (lanes 10 to 15), which shows a faster mobility on SDS/PAGE because of the 5: 15 presence of only 5 carbohydrates instead of 6. Figure 28 shows a silver stain of an SDS/PAGE gel run in identical conditions as in Figure 26. A complete overview of the purification procedure is given in Figure 29.

The presence of purified El protein was further confirmed by means of western blotting as described in example 4. The dimeric El protein appeared to be non-aggregated 20 and free of contaminants. The subtype Ib El protein purified cells according to the above scheme was aminoterminally sequenced on an 477 Perkins- Elmer sequencer and appeared to contain a tyrosine as first residue. This confirmed that the El protein had been cleaved by the signal peptidase at the correct position (between A191 and Y192) from its signal sequence. This confirms the finding ofHijikata et al. (1991) that the aminoterminus of the mature El protein starts at amino acid position 192.

Purification of the E2 protein The E2 protein (amino acids 384 to 673) was purified from RK13 cells infected with vvHCV44 as indicated in Examples 5.1 to 5.4. Figure 30 shows the OD 280 profile (continuous line) of the lentil lectin chromatography. The dotted line represents the E2 -53 reactivity as detected by ELISA (see example Figure 31 shows the same profiles obtained from gelfiltration chromatography of the lentil-lectin E2 pool (see Figure 30), part of which was reduced and blocked according to the methods as set out in example and part of which was immediately applied to the column. Both parts of the E2 pool were run on separate gelfiltration columns. It could be demonstrated that E2 forms covalently-linked aggregates with contaminating proteins if no reduction has been performed. After reduction and blocking, the majority of contaminating proteins segregated into the Vo fraction. Other contaminating proteins copurified with the E2 protein, were not covalently linked to the E2 protein any more because these contaminants could be removed in a 10 subsequent step. Figure 32 shows an additional Ni2+-IMAC purification step carried out for the E2 protein purification. This affinity purification step employs the 6 histidine residues added to the E2 protein as expressed from vvHCV44. Contaminating proteins either run through the column or can be removed by a 30 mM imidazole wash. Figure 33 shows a silver-stained SDS/PAGE of 0.5 pg of purified E2 protein and a 30 mM imidazole wash.

15 The pure E2 protein could be easily recovered by a 200 mM imidazole elution step. Figure 34 shows an additional desalting step intended to remove imidazole and to be able to switch to the desired buffer, e.g. PBS, carbonate buffer, saline.

Starting from about 50,000 cm of RK13 cells infected with wHCV 11A (or vvHCV40) for the production of El or vvHCV41, vvHCV42, vvHCV43, or vvHCV44 for *i 20 production ofE2 protein, the procedures described in examples 5.1 to 5.5 allow the purification of approximately 1.3 mg of El protein and 0.6 mg of E2 protein.

It should also be remarked that secreted E2 protein (constituting approximately 60-70% being in the intracellular form) is chracterized by aggregate formation (contrary to expectations). The same problem is thus posed to purify secreted E2. The secreted E2 can be purified as disclosed above.

Example 6: ELISA for the detection of anti-El or anti-E2 antibodies or for the detection of El or E2 proteins Maxisorb microwell plates (Nunc, Roskilde, Denmark) were coated with 1 volume 50 ptl or 100 tl or 200 tl) per well of a 5 p.g/ml solution of Streptavidin (Boehringer -54- Mannheim) in PBS for 16 hours at 4 0 C or for 1 hour at 37 0 C. Alternatively, the wells were coated with 1 volume of 5 plg/ml of Galanthus nivalis agglutinin (GNA) in 50 mM sodium carbonate buffer pH 9.6 for 16 hours at 4 0 C or for 1 hour at 37 0 C. In the case of coating with GNA, the plates were washed 2 times with 400 [l of Washing Solution of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium). Unbound coating surfaces were blocked with 1.5 to 2 volumes of blocking solution casein and 0.1% NaN 3 in PBS) for 1 hour at 37 0 C or for 16 hours at 4 0 C. Blocking solution was aspirated. Purified El or E2 was diluted to 100-1000 ng/ml (concentration measured at A 280 nm) or column fractions to be screened for El or E2 (see example or El or E2 in non-purified cell 10 lysates (example were diluted 20 times in blocking solution, and 1 volume of the El or E2 solution was added to each well and incubated for 1 hour at 37 0 C on the Streptavidin- or GNA-coated plates. The microwells were washed 3 times with 1 volume of Washing Solution of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium).

Serum samples were diluted 20 times or monoclonal anti-El or anti-E2 antibodies were 15 diluted to a concentration of 20 ng/ml in Sample Diluent of the Innotest HCV Ab III kit and 1 volume of the solution was left to react with the El or E2 protein for 1 hour at 37 0

C.

The microwells were washed 5 times with 400 il of Washing Solution of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium). The bound antibodies were detected by incubating each well for 1 hour at 37 0 C with a goat anti-human or anti-mouse IgG, 20 peroxidase-conjugated secondary antibody (DAKO, Glostrup, Denmark) diluted 1/80,000 in 1 volume of Conjugate Diluent of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium), and color development was obtained by addition of substrate of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium) diluted 100 times in 1 volume of Substrate Solution of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium) for 30 min at 24 0 C after washing of the plates 3 times with 400 tl of Washing Solution of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium).

Example 7: Follow up of patient groups with different clinical profiles 7.1. Monitoring of anti-E and anti-E2 antibodies The current hepatitis C virus (HCV) diagnostic assays have been developed for screening and confirmation of the presence of HCV antibodies. Such assays do not seem to provide information useful for monitoring of treatment or for prognosis of the outcome of disease. However, as is the case for hepatitis B, detection and quantification of antienvelope antibodies may prove more useful in a clinical setting. To investigate the 10 possibility of the use of anti-E 1 antibody titer and anti-E2 antibody titer as prognostic markers for outcome of hepatitis C disease, a series of IFN-ca treated patients with longterm sustained response (defined as patients with normal transaminase levels and negative HCV-RNA test (PCR in the 5' non-coding region) in the blood for a period of at least 1 year after treatment) was compared with patients showing no response or showing 15. biochemical response with relapse at the end of treatment.

A group of 8 IFN-a treated patients with long-term sustained response (LTR, follow up 1 to 3.5 years, 3 type 3a and 5 type lb) was compared with 9 patients showing non-complete responses to treatment (NR, follow up 1 to 4 years, 6 type lb and 3 type 3a).

Type lb (vvHCV-39, see example and 3a El (vvHCV-62, see example proteins 20 were expressed by the vaccinia virus system (see examples 3 and 4) and purified to homogeneity (example The samples derived from patients infected with a type Ib hepatitis C virus were tested for reactivity with purified type lb El protein, while samples of a type 3a infection were tested for reactivity of anti-type 3a El antibodies in an ELISA as desribed in example 6. The genotypes of hepatitis C viruses infecting the different patients were determined by means of the Inno-LiPA genotyping assay (Innogenetics, Zwijndrecht, Belgium). Figure 5 shows the anti-El signal-to-noise ratios of these patients followed during the course of interferon treatment and during the follow-up period after treatment. LTR cases consistently showed rapidly declining anti-El levels (with complete negativation in 3 cases), while anti-El levels of NR cases remained approximately constant. Some of the obtained anti-El data are shown in Table 2 as average S/N ratios -56- SD (mean anti-El titer). The anti-El titer could be deduced from the signal to noise ratio as show in Figures 5, 6, 7, and 8.

Already at the end of treatment, marked differences could be observed between the 2 groups. Anti-E1 antibody titers had decreased 6.9 times in LTR but only 1.5 times in NR.

At the end of follow up, the anti-El1 titers had declined by a factor of 22.5 in the patients with sustained response and even slightly increased in NR. Therefore, based on these data, decrease of anti-E 1 antibody levels during monitoring of IFN-a therapy correlates with long-term, sustained response to treatment. The anti-El assay may be very useful for 1 prognosis of long-term response to IFN treatment, or to treatment of the hepatitis C disease in general.

This finding was not expected. On the contrary, the inventors had expected the anti- El antibody levels to increase during the course of IFN treatment in patients with long term response. As is the case for hepatitis B, the virus is cleared as a consequence of the •V seroconversion for anti-HBsAg antibodies. Also in many other virus infections, the virus is 15 eliminated when anti-envelope antibodies are raised. However, in the experiments of the present invention, anti-El antibodies clearly decreased in patients with a long-term response to treatment, while the antibody-level remained approximately at the same level in non-responding patients. Although the outcome of these experiments was not expected, S this non-obvious finding may be very important and useful for clinical diagnosis of HCV 20 infections. As shown in Figures 9, 10, 1 l,.and 12, anti-E2 levels behaved very differently in the same patients studied and no obvious decline in titers was observed as for anti-E 1 antibodies. Figure 35 gives a complete overview of the pilot study.

As can be deduced from Table 2, the anti-El titers were on average at least 2 times higher at the start of treatment in long term responders compared with incomplete responders to treatment. Therefore, measuring the titer of anti-E1 antibodies at the start of treatment, or monitoring the patient during the course of infection and measuring the anti- E l titer, may become a useful marker for clinical diagnosis of hepatitis C. Furthermore, the use of more defined regions of the El or E2 proteins may become desirable, as shown in example 7.3.

-57- 7.2. Analysis of El and E2 antibodies in a larger patient cohort The pilot study lead the inventors to conclude that, in case infection was completely cleared, antibodies to the HCV envelope proteins changed more rapidly than antibodies to the more conventionally studied HCV antigens, with El antibodies changing most vigorously. We therefore included more type lb and 3 a-infected LTR and further supplemented the cohort with a matched series of NR, such that both groups included 14 patients each. Some partial responders (PR) and responders with relapse (RR) were also analyzed.

10 Figure 36 depicts average El antibody (E1Ab) and E2 antibody (E2Ab) levels in •the LTR and NR groups and Tables 4 and 5 show the statistical analyses. In this larger *cohort, higher El antibody levels before IFN-i therapy were associated with LTR (P 0.03). Since much higher El antibody levels were observed in type 3a-infected patients "compared with type 1 b-infected patients (Figure 37), the genotype was taken into account S1: is (Table Within the type lb-infected group, LTR also had higher El antibody levels than NR at the initiation of treatment [P 0.05]; the limited number of type 3a-infected NR did not allow statistical analysis.

Of antibody levels monitored in LTR during the 1.5-year follow up period, only El 1 antibodies cleared rapidly compared with levels measured at initiation of treatment [P 20 0.0058, end of therapy; P 0.0047 and P 0.0051 at 6 and 12 months after therapy, respectively]. This clearance remained significant within type 1- or type 3-infected LTR (average P values 0.05). These data confirmed the initial finding that E1Ab levels decrease rapidly in the early phase of resolvement. This feature seems to be independent of viral genotype. In NR, PR, or RR, no changes in any of the antibodies measured were observed throughout the follow up period. In patients who responded favourably to treatment with normalization of ALT levels and HCV-RNA negative during treatment, there was a marked difference between sustained responders (LTR) and responders with a relapse In contrast to LTR, RR did not show any decreasing El antibody levels, indicating the presence of occult HCV infection that could neither be demonstrated by PCR or other classical techniques for detection of HCV-RNA, nor by raised ALT levels.

The minute quantities of viral RNA, still present in the RR group during treatment, seemed -58to be capable of anti-El B cell stimulation. Anti-E1 monitoring may therefore not only be able to discriminate LTR from NR, but also from RR.

7.3. Monitoring of antibodies of defined regions of the El protein Although the molecular biological approach of identifying HCV antigens resulted in unprecedented breakthrough in the development of viral diagnostics, the method of immune screening of Xgtl 1 libraries predominantly yielded linear epitopes dispersed throughout the core and non-structural regions, and analysis of the envelope regions had to 10 await cloning and expression of the E1/E2 region in mammalian cells. This approach sharply contrasts with many other viral infections of which epitopes to the envelope Sregions had already been mapped long before the deciphering of the genomic structure.

Such epitopes and corresponding antibodies often had neutralizing activity useful for vaccine development and/or allowed the development of diagnostic assays with'clinical or I: s15 prognostic significance antibodies to hepatitis B surface antigen).

As no HCV vaccines or tests allowing clinical diagnosis and prognosis of hepatitis C disease are available today, the characterization of viral envelope regions exposed to immune surveillance may significantly contribute to new directions in HCV diagnosis and prophylaxis.

20 Several 20-mer peptides (Table 3) that overlapped each other by 8 amino acids, were synthesized according to a previously described method (EP-A-0 489 968) based on the HC-J1 sequence (Okamoto et al., 1990). None of these, except peptide env35 (also referred to as E1-35), was able to detect antibodies in sera of approximately 200 HCV cases. Only 2 sera reacted slightly with the env35 peptide. However, by means of the anti- El ELISA as described in example 6, it was possible to discover additional epitopes as follows: The anti-El ELISA as described in example 6 was modified by mixing 50 p.g/ml of El peptide with the 1/20 diluted human serum in sample diluent. Figure 13 shows the results of reactivity of human sera to the recombinant El (expressed from protein, in the presence of single or of a mixture of El peptides. While only 2% of the sera could be detected by means of El peptides coated on strips in a Line Immunoassay format, over half of the sera contained anti-El antibodies which could be competed by means of -59the same peptides, when tested on the recombinant El protein. Some of the murine monoclonal antibodies obtained from Balb/C mice after injection with purified El protein were subsequently competed for reactivity to El with the single peptides (Figure 14).

Clearly, the region of env53 contained the predominant epitope, as the addition of env53 could substantially compete reactivity of several sera with El, and antibodies to the env31 region were also detected. This finding was surprising, since the env53 and env31 peptides had not shown any reactivity when coated directly to the solid phase.

Therefore peptides were synthesized using technology described by applicant previously (in WO 93/18054). The following peptides were synthesized: 10 peptide

NH

2 -SNSSEAADMIMHTPGCV-GKbiotin (SEQ ID NO 51) spanning amino acids 208 to 227 of the HCV polyprotein in the El region peptide biotin-env53 ('epitope A') biotin-GG-ITGHRMAWDMMMNWSPTTAL-COOH (SEQ ID NO 52) 15 spanning amino acids to 313 of 332 of the HCV polyprotein in the El region peptide IbEl ('epitope B')

H

2 N-YEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGCGK -biotin (SEQ ID NO 53) 20 spanning amino acids 192 to 228 of the HCV polyprotein in the El region and compared with the reactivities of peptides Ela-BB (biotin-GG- TPTVATRDGKLPATQLRRHIDLL, SEQ ID NO 54) and Elb-BB (biotin-GG- TPTLAARDASVPTTTIRRHVDLL, SEQ ID NO 55) which are derived from the same region of sequences of genotype la and lb respectively and which have been described at the IXth international virology meeting in Glasgow, 1993 ('epitope Reactivity of a panel of HCV sera was tested on epitopes A, B and C and epitope B was also compared with env35A (of 47 HCV-positive sera, 8 were positive on epitope B and none reacted with Reactivity towards epitopes A, B, and C was tested directly to the biotinylated peptides (50 pg/ml) bound to streptavidin-coated plates as described in example 6. Clearly, epitopes A and B were most reactive while epitopes C and env35A-biotin were much less reactive. The same series of patients that had been monitored for their reactivity towards the complete El protein (example was tested for reactivity towards epitopes A, B, and C. Little reactivity was seen to epitope C, while as shown in Figures 15, 16, 17, and 18, epitopes A and B reacted with the majority of sera. However, antibodies to the most reactive epitope (epitope A) did not seem to predict remission of disease, while the anti- IbEl antibodies (epitope B) were present almost exclusively in long term responders at the start of IFN treatment. Therefore, anti-lbEl (epitope B) antibodies and anti-env53 (epitope A) antibodies could be shown to be useful markers for prognosis of hepatitis C disease.

The env53 epitope may be advantageously used for the detection of cross-reactive antibodies (antibodies that cross-react between major genotypes) and antibodies to the 10 env53 region may be very useful for universal El antigen detection in serum or liver tissue.

Monoclonal antibodies that recognized the env53 region were reacted with a random epitope library. In 4 clones that reacted upon immunoscreening with the monoclonal antibody 5E1A10, the sequence -GWD- was present. Because of its analogy with the universal HCV sequence present in all HCV variants in the env53 region, the sequence 15 AWD is thought to contain the essential sequence of the env53 cross-reactive murine epitope. The env31 clearly also contains a variable region which may contain an epitope in the amino terminal sequence -YQVRNSTGL- (SEQ ID NO 93) and may be useful for diagnosis. Env31 or E1-31 as shown in Table 3, is a part of the peptide lbEl. Peptides El- )33 and El-51 also reacted to some extent with the murine antibodies, and peptide E1-55 20 (containing the variable region 6 spanning amino acid positions 329-336) also reacted with some of the patient sera.

Anti-E2 antibodies clearly followed a different pattern than the anti-El antibodies, especially in patients with a long-term response to treatment. Therefore, it is clear that the decrease in anti-envelope antibodies could not be measured as efficiently with an assay employing a recombinant E1/E2 protein as with a single anti-El or anti-E2 protein. The anti-E2 response would clearly blur the anti-El response in an assay measuring both kinds of antibodies at the same time. Therefore, the ability to test anti-envelope antibodies to the single El and E2 proteins, was shown to be useful.

-61 7.4. Mapping of anti-E2 antibodies Of the 24 anti-E2 Mabs only three could be competed for reactivity to recombinant E2 by peptides, two of which reacted with the HVRI region (peptides E2-67 and E2-69, designated as epitope A) and one which recognized an epitope competed by peptide E2- 13B (epitope The majority of murine antibodies recognized conformational anti-E2 epitopes (Figure 19). A human response to HVRI (epitope and to a lesser extent HVRII (epitope B) and a third linear epitope region (competed by peptides E2-23, E2-25 or E2-27, designated epitope E) and a fourth linear epitope region (competed by peptide E2-17B, 10 epitope D) could also frequently be observed, but the majority of sera reacted with •conformational epitopes (Figure 20). These conformational epitopes could be grouped according to their relative positions as follows: the IgG antibodies in the supernatant of hybridomas 15C8C1, 12D11F1, 9G3E6, 8G10D1H9, 10D3C4, 4H6B2, 17F2C2, 5H6A7, 15B7A2 recognizing conformational epitopes were purified by means of protein A affinity 15 chromatography and 1 mg/ml of the resulting IgG's were biotinylated in borate buffer in the presence of biotin. Biotinylated antibodies were separated from free biotin by means of gelfiltration chromatography. Pooled biotinylated antibody fractions were diluted 100 to 10,000 times. E2 protein bound to the solid phase was detected by the biotinylated IgG in the presence of 100 times the amount of non-biotinylated competing antibody and 20 subsequently detected by alkaline phosphatase labeled streptavidin.

Percentages of competition are given in Table 6. Based on these results, 4 conformational anti-E2 epitope regions (epitopes F, G, H and I) could be delineated (Figure 38). Alternatively, these Mabs may recognize mutant linear epitopes not represented by the peptides used in this study. Mabs 4H6B2 and 10D3C4 competed reactivity of 16A6E7, but unlike 16A6E7, they did not recognize peptide E2-13B. These Mabs may recognize variants of the same linear epitope (epitope C) or recognize a conformational epitope which is sterically hindered or changes conformation after binding of 16A6E7 to the E2-13B region (epitope H).

-62- Example 8: El glycosylation mutants 8.1. Introduction The El protein encoded by vvHCV1OA, and the E2 protein encoded by vvHCV41 to 44 expressed from mammalian cells contain 6 and 11 carbohydrate moieties, respectively. This could be shown by incubating the lysate of vvHCV10A-infected or vvHCV44-infected RK13 cells with decreasing concentrations of glycosidases (PNGase F or Endoglycosidase H, (Boehringer Mannhein Biochemica) according to the 10 manufacturer's instructions), such that the proteins in the lysate (including E1) are partially deglycosylated (Fig. 39 and 40, respectively).

Mutants devoid of some of their glycosylation sites could allow the selection of envelope proteins with improved immunological reactivity. For HIV for example, *0 proteins lacking certain selected sugar-addition motifs, have been found to be particularly 15 useful for diagnostic or vaccine purpose. The addition of a new oligosaccharide side chain in the hemagglutinin protein of an escape mutant of the A/Hong Kong/3/68 (H3N2) influenza virus prevents reactivity with a neutralizing monoclonal antibody (Skehel et al, 1984). When novel glycosylation sites were introduced into the influenza hemaglutinin protein by site-specific mutagenesis, dramatic antigenic changes were observed, suggesting 20 that the carbohydrates serve as a modulator of antigenicity (Gallagher et al., 1988). In another analysis, the 8 carbohydrate-addition motifs of the surface protein gp70 of the Friend Murine Leukemia Virus were deleted. Although seven of the mutations did not affect virus infectivity, mutation of the fourth glycosylation signal with respect to the amino terminus resulted in a non-infectious phenotype (Kayman et al., 1991). Furthermore, it is known in the art that addition of N-linked carbohydrate chains is important for stabilization of folding intermediates and thus for efficient folding, prevention of malfolding and degradation in the endoplasmic reticulum, oligomerization, biological activity, and transport of glycoproteins (see reviews by Rose et al., 1988; Doms et al., 1993; Helenius, 1994).

After alignment of the different envelope protein sequences of HCV genotypes, it may be inferred that not all 6 glycosylation sites on the HCV subtype lb El protein are -63 required for proper folding and reactivity, since some are absent in certain (sub)types. The fourth carbohydrate motif (on Asn251), present in types Ib, 6a, 7, 8, and 9, is absent in all other types know today. This sugar-addition motif may be mutated to yield a type Ib El protein with improved reactivity. Also the type 2b sequences show an extra glycosylation site in the V5 region (on Asn299). The isolate S83, belonging to genotype 2c, even lacks the first carbohydrate motif in the Vi region (on Asn), while it is present on all other isolates (Stuyver et al., 1994) However, even among the completely conserved sugaraddition motifs, the presence of the carbohydrate may not be required for folding, but may have a role in evasion of immune surveillance. Therefore, identification of the 10 carbohydrate addition motifs which are not required for proper folding (and reactivity) is *not obvious, and each mutant has to be analyzed and tested for reactivity. Mutagenesis of a glycosylation motif (NXS or NXT sequences) can be achieved by either mutating the codons for N, S, or T, in such a way that these codons encode amino acids different from N in the case ofN, and/or amino acids different from S or T in the case of S and in the case of 15 T. Alternatively, the X position may be mutated into P, since it is known that NPS or NPT are not frequently modified with carbohydrates. After establishing which carbohydrateaddition motifs are required for folding and/or reactivity and which are not, combinations of such mutations may be made.

20 8.2. Mutagenesis of the El protein All mutations were performed on the El sequence of clone HCCl1OA (SEQ ID NO. The first round of PCR was performed using sense primer 'GPT' (see Table 7) targetting the GPT sequence located upstream of the vaccinia 11K late promoter, and an antisense primer (designated GLY#, with representing the number of the glycosylation site, see Fig. 41) containing the desired base change to obtain the mutagenesis. The six GLY# primers (each specific for a given glycosylation site) were designed such that: Modification of the codon encoding for the N-glycosylated Asn (AAC or AAT) to a Gln codon (CAA or CAG). Glutamine was chosen because it is very similar to asparagine (both amino acids are neutral and contain non-polar residues, glutamine has a longer side chain (one more -CH 2 group).

-64- The introduction of silent mutations in one or several of the codons downstream of the glycosylation site, in order to create a new unique or rare a second Smal site for restriction enzyme site. Without modifying the amino acid sequence, this mutation will provide a way to distinguish the mutated sequences from the original El sequence (pvHCV- OA) or from each other (Figure 41) This additional restriction site may also be useful for the construction of new hybrid (double, triple, etc.) glycosylation mutants.

18 nucleotides extend 5' of the first mismatched nucleotide and 12 to 16 nucleotides extend to the 3' end. Table 7 depicts the sequences of the six GLY# primers overlapping 10 the sequence of N-linked glycosylation sites.

For site-directed mutagenesis, the 'mispriming' or 'overlap extension' (Horton, 1993) was used. The concept is illustrated in Figures 42 and 43. First, two separate fragments were amplified from the target gene for each mutated site. The PCR product obtained from the 5' end (product GLY#) was amplified with the 5' sense GPT primer (see 15 Table 7) and with the respective 3' antisense GLY# primers. The second fragment (product OVR#) was amplified with the 3' antisense TKR primer and the respective 5' sense primers (OVR# primers, see Table 7, Figure 43).

The OVR# primers target part of the GLY# primer sequence. Therefore, the two groups of PCR products share an overlap region of identical sequence. When these 20 intermediate products are mixed (GLY-1 with OVR-1, GLY-2 with OVR-2, etc.), melted at high temperature, and reannealed, the top sense strand of product GLY# can anneal to the antisense strand of product OVR# (and vice versa) in such a way that the two strands act as primers for one another (see Fig. Extension of the annealed overlap by Taq polymerase during two PCR cycles created the full-length mutant molecule E1Gly#, which carries the mutation destroying the glycosylation site number Sufficient quantities of the E1GLY# products for cloning were generated in a third PCR by means of a common set of two internal nested primers. These two new primers are respectively overlapping the 3' end of the vaccinia 11K promoter (sense GPT-2 primer) and the 5' end of the vaccinia thymidine kinase locus (antisense TKR-2 primer, see Table All PCR conditions were performed as described in Stuyver et al. (1993).

Each of these PCR products was cloned by EcoRI/BamHI cleavage into the EcoRI/BamHI-cut vaccinia vector containing the original El sequence (pvHCV-1OA).

The selected clones were analyzed for length of insert by EcoRI/BamH I cleavage and for the presence of each new restriction site. The sequences overlapping the mutated sites were confirmed by double-stranded sequencing.

8.3. Analysis of El glycosylation mutants S* Starting from the 6 plasmids containing the mutant El sequences as described in 10 example 8.2, recombinant vaccinia viruses were generated by recombination with wt vaccinia virus as described in example 2.5. Briefly, 175 cm flasks of subconfluent RK13 cells were infected with the 6 recombinant vaccinia viruses carrying the mutant El sequences, as well as with the vvHCV-1OA (carrying the non-mutated El sequence) and wt vaccinia viruses. Cells were lysed after 24 hours of infection and analyzed on western blot as described in example 4 (see Figure 44A). All mutants showed a faster mobility (corresponding to a smaller molecular weight of approximately 2 to 3 kDa) on SDS-PAGE than the original El protein; confirming that one carbohydrate moiety was not added.

Recombinant viruses were also analyzed by PCR and restriction enzyme analysis to confirm the identity of the different mutants. Figure 44B shows that all mutants (as shown in Figure 41) contained the expected additional restriction sites. Another part of the cell lysate was used to test the reactivity of the different mutant by ELISA. The lysates were diluted 20 times and added to microwell plates coated with the lectin GNA as described in example 6. Captured (mutant) El glycoproteins were left to react with 20-times diluted sera of 24 HCV-infected patients as described in example 6. Signal to noise values (OD of GLY#/OD of wt) for the six mutants and El are shown in Table 8. The table also shows the ratios between S/N values of GLY# and El proteins. It should be understood that the approach to use cell lysates of the different mutants for comparison of reactivity with patient sera may result in observations that are the consequence of different expression levels rather then reactivity levels. Such difficulties can be overcome by purification of the different mutants as described in example 5, and by testing identical quantities of all the different El proteins. However, the results shown in table 5 already -66indicate that removal of the 1st (GLY1), 3rd (GLY3), and 6th (GLY6) glycosylation motifs reduces reactivity of some sera, while removal of the 2nd and 5th site does not. Removal of GLY4 seems to improve the reactivity of certain sera. These data indicate that different patients react differently to the glycosylation mutants of the present invention. Thus, such mutant El proteins may be useful for the diagnosis (screening, confirmation, prognosis, etc.) and prevention ofHCV disease.

Example 9: Expression of HCV E2 protein in glvcosylation-deficient yeasts 10 The E2 sequence corresponding to clone HCCL41 was provided with the a-mating factor pre/pro signal sequence, inserted in a yeast expression vector and S. cerevisiae cells transformed with this construct secreted E2 protein into the growth medium. It was observed that most glycosylation sites were modified with high-mannose type glycosylations upon expression of such a construct in S. cerevisiae strains (Figure 45). This S 15 resulted in a too high level of heterogeneity and in shielding of reactivity, which is not desirable for either vaccine or diagnostic purposes. To overcome this problem, S cerevisiae mutants with modified glycosylation pathways were generated by means of selection of vanadate-resistant clones. Such clones were analyzed for modified glycosylation pathways by analysis of the molecular weight and heterogeneity of the glycoprotein invertase. This allowed us to identify different glycosylation deficient S.

cerevisiae mutants. The E2 protein was subsequently expressed in some of the selected mutants and left to react with a monoclonal antibody as described in example 7, on western blot as described in example 4 (Figure 46).

Example 10. General utility The present results show that not only a good expression system but also a good purification protocol are required to reach a high reactivity of the HCV envelope proteins with human patient sera. This can be obtained using the proper HCV envelope protein expression system and/or purification protocols of the present invention which guarantee the conservation of the natural folding of the protein and the purification protocols of the -67present invention which guarantee the elimination of contaminating proteins and which preserve the conformation, and thus the reactivity of the HCV envelope proteins. The amounts of purified HCV envelope protein needed for diagnostic screening assays are in the range of grams per year. For vaccine purposes, even higher amounts of envelope protein would be needed. Therefore, the vaccinia virus system may be used for selecting the best expression constructs and for limited upscaling, and large-scale expression and purification of single or specific oligomeric envelope proteins containing high-mannose carbohydrates may be achieved when expressed from several yeast strains. In the case of hepatitis B for example, manufacturing of HBsAg from mammalian cells was much more 10 costly compared with yeast-derived hepatitis B vaccines.

The purification method dislcosed in the present invention may also be used for 'viral envelope proteins' in general. Examples are those derived from Flaviviruses, the newly discovered GB-A, GB-B and GB-C Hepatitis viruses, Pestiviruses (such as Bovine viral Diarrhoea Virus (BVDV), Hog Cholera Virus (HCV), Border Disease Virus (BDV)), 15 but also less related virusses such as Hepatitis B Virus (mainly for the purification of HBsAg).

The envelope protein purification method of the present invention may be used for intra- as well as extracellularly expressed proteins in lower or higher eukaryotic cells or in prokaryotes as set out in the detailed description section.

68 Table 1: Recombinant-vaccinia plasmids and viruses Plasmid name Name cDNA. subclone Length (ntlaa) Vector used construction for insertion pvHCV-13)A Els EcoR I- Hind 111 472/157 pgptATA- 18 pvHCV-12A Els EcoR I Hind 111 472/158 pgptATA- 18 pvHCV.-9A ElI EcoR I Hind 111 631/211 pgptATA- 18 pvHCV- 11 A Els EcoR I Hind 111 625/207 pgptATA- 18 pvHCV-17A Els EcoR I Hind 111 625/208 pgptATA- 18 pvHCV-1OA ElI EcoR I Hind 111 783/262 pgptATA-18 pvHC V-I 8A COREs Acc I (KI) EcoR I (Ki) 403/130 pgptATA- 18 pvHCV-34 CORE Acc I (KI) Fsp I 595/197 pgptATA- 18 pvHCV-33 CORE-El Acc I (KL) 1150/380 pgptATA- 18 pvHCV-35 CORE- EcoR I BamH I (Ki) 1032/352 pMS-66 Elb.his pvHCV-36 CORE- EcoR I Nco I (KI) 1106/376 pMS-66 Eln.his pvHCV-37 ElA Xma I- BamHI1 711/239 pvHCV-1OA pvHCV-38 ElAs EcoR I- BstEI11 553/183 pvHCV- I1IA pvHCV-39 BlAb EcoR I BaniH I 960/3 13 pgsATA- 18 pvHCV-40 ElAb.his EcoR I BamH I (KI) 960/323 pMS-66 pvHCV-41 E2bs BamH I (K1)-AlwN I (T4) 1005/33 1 pgsATA- 18 pvHCV-42 E2bs.his BamH I (KI)-A~wN I (T4) 1005/341 pMS-66 pvHCV-43 E2ns Nco I (KI) AiwN I (T4) 932/314 pgsATA-1 8 pvHCV-44 E2ns.his Nco I (KI) AlwN I (T4) 932/32 1 pMS-66 pvHCV-62 Els (type 3a) EcoR I Hind 111 625/207 pgsATA- 18 pvHCV-63 El1s (type 5) EcoR I Hind 111 625/207 pgsATA- 18 pvHCV-64 E2 BamH I Hind 111 1410/463 pgsATA- 18 EI-E2 BamH I Hind 111 2072/69 1 pvHCV-lOA pvHCV-66 CORE-E1-E2 BamH I Hind 111 2427/809 pvHCV-33 nt: nucleotide aa: aminoacid Ki: Kienow DNA Pol filling T4: T4 DNA Pol filling Position: aminoacid position in the HCV polyprotein. sequence 69 Table 1 continued: Recombinant vaccinia plasmids and viruses Plasmnid HCV cDNA subclone Vector Name Name Construction Length used for (ntlaa) insertion pvHCV-81 El*-GLY 1 EcoRi BamH 1 783/262 pvHCV-lIOA pvHCV-82 E1*-GLY 2 EcoRI Ban-H 1 783/262 pvHCV-1OA pvHCV-83 E 1 *GLY 3 EcoRl BamHI1 783/262 pvHCV-lOA pvHCV-84 El*-GLY 4 EcoRi BamH 1 783/262 pvHCV-1OA pvHCV-85 E1*-GLY 5 EcoRi BamH 1 783/262 pvHCV-lOA pvHCV-86 El *-GLY 6 EcoRi BamH lI 783/262 pvHCV- IOA S nt: nucleotide aa: aminoacid 1(1: Klenow DNA Pol filling T4: T4 DNA Pol filling Position: am-inoacid position in the HCV polyprotein sequence

S

70 Table 2 :Summary of anti-El tests SIN SD (mean anti-El titer) es..

es 0

OS

~0@gW* 0

S@S*

00 0 0 6 Start of treatment End of treatment Follow-up LTR 6.94 2.29 (1:3946) 4.48 2.69 (1:568) 2.99 2.69 (1:175)* NR 5.77 3.77 (1:1607) 5.29 3.99 (1:1060) 6.08 3.73 (1:1978) LTR :Long-term, sustained response for more than 1 year NR :No response, response with relapse, or partial response 00 0* 0 OS 0

SO

0 @0 71 Table 3 Synthetic peptides for competition studies PROTEIN PEPTIDE AMINO ACID SEQUENCE POSITION SEQ ID NO El EI-31 LLSCLTVPASAYQVRNSTGL 181-200 56 .El1-33 QVRINSTGLYHVTNDCPNS SI 193-212 57 NDCPNSSIVYEAHiDAJLHTP 205-224 58 SNS SIVYEAADMJMHTPGCV 208-227 59 El-37 HDAILHTPGCVPCVREGNVS 217-236 *E1-39 CVREGNVSRCWVAMTPTVAT 229-248 61 *EI-41 AMTPTVATRDGKLPATQLRR 241-260 62 ElI-43 LPATQLR.RIIDLLVGSATLC 253-272 63 LVGSATLCSALYVGDLCGSV 265-284 64 **E1-49 QLFTFSPRRIIWTTQGCNCSI 289-308 *El-51 TQGCNCSIYPGHITGHRMAW 301-320 66 *:EI-53 ITGHRMAWDMMMNWSPTAAL 313-332 67 0 0EI-55 NWSPTAALVMAQLLRIPQAI 325-344 68 EI-57 LLRIPQAILDMIAGA}IWGVL 337-356 69 EI-59 AGAHWGVLAGIAYFSMVGNM 349-368 *EI-63 VVLLLFAGVDAETIVSGGQA 373-392 71 72 E2 E2-67 SGLVSLFTPGAKQNIQLINT 397-416 72 E2-69 QNIQLINTNGSWIIINSTALN 409-428 73 E2-$3)B LNCNESLNTGWWLAGLIYQHK 427-446 74 E2-$ IB AGLIYQHKFNSSGCPERLAS 439-458 E2-1B GCPERLASCRPLTDFDQGWG 45 1-470 76 E2-3B TDFDQGWGPISYANGSGPDQ 463-482 77 ANGSGPDQRPYCWHYPPKPC 475-494 78 :.E2-7B WHYPPKPCGJVPAKSVCGPV 487-506 79 E2-9B AKSVCGPVYCFTPSPVVVGT 499-518 E2-1 IlB PSPVVVGTTDRSGAPTYSWG 511-530 81 E2- 13B GAPTYSWGENDTDVFVLNNT 523-542 82 E2-17B GNWFGCTWMNSTGFTKVCGA 547-566 83 *E2-19B GFTKVCGAPPVCIGGAGNNT 55 9-578 84 E2-2 1 IGGAGNNTHPDFK{ 571-590 E2-23 TDCFRKHPDATYSRCGSGPW 583-602 86 E2-25 SRCGSGPWITPRCLVDYPYR 595-614 87 E2-27 CLVDYPYRLWHYPCTJNYTJ 607-626 88 E-29 PCTINYTIFKIRMYVGGVEH 61963 89 E2-31 MYVGGVEHRLEAACNWTPGE 63 1-650 E2-33 ACNWTPGERCDLEDRDRSEL 643-662 91 E2-35 EDRDRSELSPLLLTTTQWQV 655-674 92 .6 6 6 6 *66 *66 6 6.

6 6 6 Table 4. Change of Envelope Antibody levels over time (complete study, 28 patients) Wilcoxon Sig-ned Rank test (P values) ElAbNR All EIAb NR type l b EIAbNR type 3 a BlAb LTR EIAb LTR All type l b EIAb LTR type 3a E2Ab NR All EIAb LTR All End of therapy* 0.1167 0.2604 0.285 0.0058** 0.043** 0.0499** 0.0 186*' 0.0640 6 months follow up* 0.86 0.7213 0.5930 0.0047* 0.043* 0.063 0.04326 0.0464* 12 months follow up* 0.7989 0.3105 1 -0.005 1 0.0679 0.0277* 0.0869 0.0058** Data were compared with values obtained at initiation of therapy 6P values 0.05 Table 5. Difference between LTR and NR (complete study) Mann-Withney U test (P values) BlAb S/N All ElIAb titers ElAb S/N All type lb BlAb S/N type 3a E2Ab S/N All Initiation of therapy 0.0257* 0.05* 0.68 0.1078 End of therapy 0.1742 0.1295 6 months follow up 1 0.6099 0.425 0.3081 12 months follow up 0.67 0. 23 0.43 86 0.6629 *P values 0.05 Table 6. Competition experiments between murine E2 monoclonal antibodies Decrease of anti-E2 reactivity of biotinylated anti-E2 mabs competitor 17H10F4D10 2F101-10 16A6E7 10D3C4 41-6132 17C2F2 9G3E6 12D11F1 15C8C1 8G1OD1H9 17H10F4D10 62 10 ND 11 ND 5 6 30 ND 2F1OH1O 90 1 ND 30 ND 0 4 12 ND 16A6E7 ND ND ND ND ND ND ND ND ND 10D3C4 11 50 92 -94 26 28 43 53 4H6B2 ND ND 82 ND ND ND ND ND ND 17C2F2 2 ND 75 ND 56 -11 10 0 0 9G3E6 ND ND 68 ND 11 ND 60 76 ND 12D11F1 ND ND 26 ND 13 ND ND 88 ND 15C8C1 ND ND 18 ND 10 ND ND ND ND 8G1OD1H9 2 2 11 ND 15 ND 67 082 81 competitor controls 15137A2 0 0 9 15 10 9 0 0 0 51-6A7 0 2 0 12 8 0 0 4 0 0 23C12H9 ND ND 2 12 ND 4 ND ND ND 2 ND, not done Ta l 7. Primers... SE ID NO 96 GPT *GT* *CTG.A.. SE ID NO 97 TK .C- SEQ ID NO. 961 GPT4 5'-GTTACTG CGAG3 TCTGTCGGGGG SEQ ID NO. 972 TKR 5'-GTCATCGTGCGGTAC-3'GATGCGCTGACTCC3 SEQ ID NO. 983 GLY6 5'-CGGCTGTAATCCGGCACTCT CGCATATAAGCG-3 SEQ ID NO. 994 GVl2 SEQ ID NO. 100 GLY3 5'-GTCCATGAGCGGGAGCCGGCTCGACCGGCC3 SEQ ID NO. 101 GLY4 5'-GTCTG GTGGGGAGGCCGCCTGTGGGGTG SEQ ID NO. 102 GLY5 SEQ ID NO. 03 GLY6 5'-CAGGCCGTTGGCC TACTCACATATTCCAGC SEQ ID NO. 104 OVR1 5'-CGGAAGTACCAGCAACG ACCGGG-3 SEQ ID NO. 110 OVR2 5'-GCTATGTTATGAGC -3 SEQ ID NO. Ill TKR-2 5'-GCCATACGCTCACAGCCGATCCC-3' nucleotides underlined represent additional restriction site nucleotides in bold represent mutations with respect to the original HCCI11OA sequence 0* 0 T-SERUM nalX5s-fElg QyMSA

SERUM

SN GLY1 SN GLY2 SN GLY3 SN GLY4 SN GLY5 SN GLY6 SN E1 SN GLY1 SN GLY2 SN GLY3 SN GLY4 SN GLY5 SN GLY6 SN E1 ,GLYl/El GLY2/E1 GLY3/E 1 GLY4/Ei GLY5/E1 GLY6/E1 GLYI1/E1 GLY2/E1 GI.Y3/E1 GLY4LE1 GLY5/E1 I GLYG/FI 1 1 1.802462 2.400795 1.642718 2.570154 2.482051 2.031487 2.828205 13 5.685561 7.556682 7.930538 8.176816 8.883408 8.005561 8.825112

SERUM

1 0.637316 0.848876 0.580B34 0.911587 0.877607 0.718296 13 0.644248 0.85627 0.098633 0.92654 1.006606 0.907134 2 2.120971 1.76810 1.715477 3.824038 1.793761 1.495737 2.227036 14 3.233604 2.567613 2.763055 6.561122 2.940334 2.499952 3.183771 2 0.952374 0.793961 0.770296 1.717097 0.805447 0.671626 14 1.015652 0.806469 0.OG7056 2.060802 0.9235308 0.7085217 3 1.403871 2.325495 2.261646 3.874605 2.409344 2.131613 2.512792 15 3.763490 3.621920 3.016099 5.707668 3.125561 2.621704 3.067265 3 0.55869 0.925463 0.900053 1.541952 0.958831 0.848305 15 1.226988 1.180033 0.983319 1,060033 1.019006 0.054737 4 1.205597.

2.639300 2.354748 1.499387 2.627358 2.527925 2.790881 16 1.905105 3.055649 2.945628 5.684490 3.338912 2.572385 3.200335 4 0.431977 0.94569 0.84373 0.537245 0.941408 0.90578 16 0.605153 0.931505 0.097966 1.732902 1.017857 0.704104 5 6 2.120191 2.866913 2.459019 5.043993 1.591810 4.033742 3.15 4.71302 1.715311 4.964765 2.494833 4.784027 3.131579 4.869128 17 18 2.317721 6.675179 2.933792 7.65433 2.515305 5.775357 5.604813 6.4125 2.654224 5.424107 2.363301 5.194107 2.980354 7.191964 5 6 0.677036 0.508794 0.785233 1.035913 0.508312 0.992733 1.005882 0.967939 0.547746 1.019642 0.796669 0.982522 17. 10 0.777666 0.920144 0.984377 1.064289 0.843962 0.803029 1.800587 0.09162 0.890574 0.75419 0.79296 0.72221 7 1.950345 2.146302 1.96692 4.198751 2.13912 2.02069 2.287753 19 1.93476 2.127712 1.900185 3.813321 2.442804 1.506716 2.771218 7 0.852516 0.9381.7 0.859761 1.835317 0.935031 0.803264 19 0.690162 0.76779 0.714554 1.376045 0.881491 0.543702 8 1.866183 1.595477 1.402099 3.959542 1.576336 1.496409 1.954198 20 2.47171 2.921280 2.557384 3.002535 3.126761 2.665433 3.678060 8 0.954961 0.816436 0.758418 2.026172 0.006641 0.765781 20 0.672013 0.794245 0.695306 0.816335 0.850109 0.724683 9 1.730193 1.608973 1.602222 3.710507 1.708937 1.704976 1.805556 21 4.378633 4.600101 4.268633 4.293038 4.64557 2.781063 5.35443 9 0.958261 0.935431 0.087385 2.05505 0.946488 0.944294 21 0.017759 0.874061 0.797215 0.001773 0.867612 0.519395 10 2.4680162 2.4082212 2.191558 5.170841 3.021807 2.677757 2.616822 22 1.1088748 1.150701 0.97767 2.393011 1.153656 1.280743 1.167206 10 0.94319 0.94056 0.837480 1.976 1.154762 1.023286 22 1.010306 0.98586 0.037550 2.050064 0.908323 1.097197 11 1.220654 1.467502 1.464216 4.250784 1.562092 1.529608 1.55719 23 2.150809 1.661914 1.336775 3.68213 1.017901 1.475062 2.003333 11 0.783002 0.942455 0.940294 2.72978 1.003148 0.982280 23 1.036267 0.797719 0.641652 1.767422 0.872593 0.70003 12 1.629403 2.070524 1.721164 3.955153 2.07278 1.744221 2.593886 24 1.706992 1.632785 1.20376 2.481585 1.638211 1.716423 1.78252 12 0,620171 0.790232 0.663547 1.524798 0.799102 0.672435 24 0.957628 0.915998 0.675314 1.392178 0.919042 0.962919 Sum

S/N

59.00534 69.65243 62.09872 102.6978 69.26511 61.32181 76.54068 Sumi E1I/GLY# 19.36524 21.67304 19.19921 36.38592 21.78679 19.59691 Avera o

SIN

2.495223 2.902105 2.507447 4.279076 2.006046 2.555075 3.109195 Average E I/GLYII// 0.006005 0.903077 0.799967 1.51600 0.907703 0.016530 -78-

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82_ SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: Innogenetics N.V.

STREET: Industriepark Zwijnaarde 7 Bus 4 CITY: Gent COUNTRY: Belgium POSTAL CODE (ZIP): 9052 TELEPHONE: 00-32-09.241.07.11 TELEFAX: 00-32-09.241.07.99 (ii) TITLE OF INVENTION: Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use.

(iii) NUMBER OF SEQUENCES: 111 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/EP/95/03031 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GGCATGCAAG CTTAATTAAT T 21 INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 68 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: CCGGGGAGGC CTGCACGTGA TCGAGGGCAG ACACCATCAC CACCATCACT AATAGTTAAT 6C 83

TAACTGCA

INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 642 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA* (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .639 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1. .636 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATG CCC GOT TGC TCT TrC TCT ATC TTC Met Pro Gly Cys Ser Phe Ser Ile Phe CTC TTG OCT TTIA CTO TCC TOT Leu Leu Ala Leu Leu Ser Cys

AAC

Asn

AGO

Arg

CTC

Leu

TOC

Cys

COO

Arg

ATA

Ile 145 ACC A T Thr Ile CAT OTC His Val 35 GAC ATO Asp Met AAC TCT Asn Ser AAC 0CC Asn Ala OTT 000 Val Gly OGA TCT Oly Ser 115 CAT GAG His Glu 130 ACA GOT Thr Oly

OCT

Ala

AAC

As n

ATG

Met

CGC

Arg

GTC

Val1

OCT

Al a

TTC

Phe

OTG

Val1

COT

Arg

TCC

Ser

GAC

Asp

CAC

His

TOC

Cys

CCC

Pro

OCT

Al a

CTC

Leu

CAG

G In

ATG

Me t GAG OTO Glu Val AAC TCA Asn Ser 000 TGC Gly Cys OCO CTC Ala Leu ACA ATA Thr Ile TCC OCT Ser Ala 105 CAG CTG Gln Leu AAT TOC Asn Cys OAT ATO Asp Met

GTO

Val1

OTO

Val1

TOC

Cys

ACO

Thr

CAC

His

OTG

Val1

ATC

Ile 125

TAT

Tyr

AAC

As n 240 150 155 ACA ACO 0CC CTG OTG OTA TCG CAG CTO CTC COG ATC CCA CAA OCT CTC 84 Thr Thr Ala Leu Val Val Ser Gin Leu Leu Arg le Pro Gin Ala Val 165 170 175 GTG GAC ATG GTG GCG GGG GCC CAT TGG GGA GTC CTG GCG GGC CTC GCC Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala 180 185 190 TAC TAT TCC ATG GTG GGG AAC TGG GCT AAG GTT TTG ATT GTG ATG CTA Tyr Tyr Ser Met Val Gly Asn Trp, Ala Lys Val Leu Ile Val Met Leu 195 200 205 CTC TT'r GCT CTC TAATAG Leu Phe Ala Leu 210 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 212 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Pro Gly Cys Ser Phe Ser le Phe Leu Leu Ala Leu Leu Ser CYs 1 5 10 Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Met 25 Tyr His Val Thr Asn Asp Cys Ser Asn Se: Se: Ile-Val Tyr Glu Ala 40 Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu 55 Asn Asn Ser Se: Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala 70 75 Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp "eu 90 Leu Val Gly Ala Ala Ala Leu Cys Ser Ala Met Tyr Val Gly Asp Leu 100 105 110 Cys Gly Se: Val Phe Leu Val Ser Gin Leu Phe Thr Ile Ser Pro Ar g 115 120 125 Arg His Glu Thr Val Gin Asp Cys Asn Cys 5cr Ile Tyr Pro Giv: ?i s 130 135 140 Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asri Tro Ser :ro 145 150 15516 Thr Thr Ala Leu Val Val Ser Gin Leu Leu Arg Ile Pro Gin Ala ValI 165 170 175 Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala In 190lriq Val Gly Asn Ala Lys Val Leu Ile Val Met: eu 205 195 Leu Phe Ala Leu 85 INFORMATION FOR SEQ ID NO: Wi SEQUENCE CHARACTERISTICS: LENGTH: 795 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .792 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1. .789 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: a.

a a.

a *aa.

a. a. a a.

ATG 'ITG GGT AAG Met Leu Gly Lys 1 GTG GGG TAC ATT Val Gly Tyr Ile 20 GCC CTG GCG CAT Ala Leu Ala His ACA GGG AAT TTG Thr Gly Asn Leu 50 CTG TCC TGT CTG Leu Ser Cys Leu 65 TCC GGG ATG TAC Ser Gly Met Tyr TAT GAG GCA GCG Tyr GlU Ala Ala 100 GTT CGG GAG AAC Val Arg Glu Asn 115 CTC GCA GCT AGG Leu Ala Ala Arg 130 GTC GAT TTG CTC Val Asp Leu Leu 145 GTC ATC GAT ACC CTr ACA TGC GGC Val Ile Asp Thr Leu Thr Cys Gly 10 CCC CTA Pro Leu GAG GAC Glu Asp TCT ATC Ser Ile GCT TAT Ala Tyr 75 TGC TCC Cys Ser ACC CCC Thr Pro TGG GTA Trp Val

GGG

Gly

GGC

C ly

TTC

Phe

GAA

Glu

AAC

Asn

GGG

C ly

GCG

Ala

ACA

Thr 140

TCC

Ser TTC CCC GAC CTC Phe Ala Asp Leu GGC GCT GCC AGG Gly Ala Ala Arg GTG AAC TAT GCA Val Asn Tyr Ala CTC TTG GCT TTG Leu Leu Ala Leu GTG CGC AAC GTG Val Arg Asn Val TCA AGC ATT GTG Ser Ser Ile Val TGC GTG CCC TGC Cvs Val Pro Cys 110 CTC ACC CCC ACG Leu Thr Pro Thr 125 ATA CGA CGC CAC Ile Arg Arg His GCT ATG TAC GTG Ala Met Tyr Val 160 336 GGG GAC CTC TGC GGA TCT GTC rrC CTC Gly Asp Leu Cys Gly Ser Val Phe Leu 165 TCC CAG CTG TTC Ser Gin Leu Phe ACC ATC 528 Thr Ile 175 86 TCG CCT CGC CGG CAT GAG ACG GTG CAG GAC TGC AAT TGC TCA ATC TAT 5 7 6 Ser Pro Arg Arg His Giu Thr Val Gin Asp Cys As n Cys Ser Ile Tyr 180 185 190 CCC GGC CAC ATA ACG GGT CAC CGT ATG GCT TGG GAT ATG ATG ATG AAC 624 Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn 195 200 205 TGG TCG CCT ACA ACG GCC CTG GTG GTA TCG CAG CTG CTC CGG ATC CCA 672 Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gin Leu Leu Arg Ile Pro 210 215 220 CAA GCT GTC GTG GAC ATG GTG GCG GGG GCC CAT TGG GGA GTC CTG GCG 720 Gin Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu Aia 225 230 235 240 GGT CTC GCC TAC TAT TCC ATG GTG GGG AAC TGG GCT AAG GTT TTG A7I' 768 Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val Leu Ile *245 250 255 GTG ATG CTA CTC TTT OCT CCC TAATAG 795 Val Met Leu Leu Phe Ala Pro 260 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 263 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu 1 5 10 Val Oly Tyr Ile Pro Leu Val Oly Ala Pro Leu Gly Gly Ala Ala Arg 25 *Ala Leu Ala His Gly Val Ar-g Val Leu Giu Asp Oly Val Asn Tyr Ala 40 4 Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu 55 Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Giu Val Ara Asn Val 70 75 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val 90 Tyr Olu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro -ys 100 105 liC Vai Arg Giu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr 115 120 125 Leu Ala Ala Arg Asn Ala Se: Val Pro Thr Thr Thr Ile Ara Arg iS 130 135 140 Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met: Tyr Val 145 150 15510 Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gin Leu Phe Thr Ile 165 170 175 87 Ser Pro Arg Arg His Glu Thr Val Gin Asp Cys Asn Cys Ser Ile Tyr 180 185 190 Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn 195 200 205 Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gin Leu Leu Arg Ile Pro 210 215 220 Gin Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu Ala 225 230 235 240 Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val Leu Ile 245 250 255 Val Met Leu Leu Phe Ala Pro 260 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 633 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..630 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..627 i SEQUENCE DESCRIPTION: SEQ ID NO: 7: ATG TTG GGT AAG GTC ATC GAT ACC CTT ACG TGC GGC TTC GCC GAC CTC 48 Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu 1 5 10 ATG GGG TAC ATT CCG CTC GTC GGC GCC CCC CTA GGG GGT GCT GCC AGA 96 Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg 25 GCC CTG GCG CAT GGC GTC CGG GTT CTG GAA GAC GGC GTG AAC TAT GCA 144 Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala 40 ACA GGG AAT TTG CCT GGT TGC TCT TTC TCT ATC TTC CTC TTG GCT TTA 192 Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser ile Phe Leu Leu Ala Leu 55 CTG TCC TGT CTG ACC ATT CCA GCT TCC GCT TAT GAG GTG CGC AAC GTG 240 Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val 70 75 TCC GGG ATG TAC CAT GTC ACG AAC GAC TGC TCC AAC TCA AGC ATT GTG 288 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val 90 88 TAT GAG GCA GCG GAC ATG ATC ATG CAC ACC CCC GGG TGC GTG CCC TGC 336 Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys 100 105 110 GTT CGG GAG AAC AAC TCT TCC CGC TGC TGG GTA GCG CTC ACC CCC ACG 384 Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr 115 120 125 CTC GCA GCT AGG AAC GCC AGC GTC CCC ACT ACG ACA ATA CGA CGC CAC 432 Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His 130 135 140 GTC GAT TTG CTC GTT GGG, GCG GCT GCT 'rrC TGT TCC GCT ATG TAC GTG 480 Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val 145 150 155 160 GGG GAT CTC TGC GGA TCT GTC TTC CTC GTC TCC CAG C'TO 77C ACC ATC 528 Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gin Leu Phe Thr Ile *165 170 175 .TCG CCT CGC CGG CAT GAG ACG GTG CAG GAC TGC AAT TGC TCA ATC TAT 576 Ser Pro Arg Arg His Glu Thr Val Gin Asp Cys Asn Cys Ser Ile Tyr .180 185 190 *CCC GGC CAC ATA ACA GGT CAC CGT ATG GCT TGG GAT ATG ATG ATG AAC 624 ***Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn ***195 200 205 TGG TAATAG 633 OV, Trp 210 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 209 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: MtLeu Gly Lys Val Ile Asp Thr Leu Thr Cys Giy Phe Ala Asp Leu 1 5 10 iS Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg 25 Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Vai Asn Tyr Ala 40 Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu 55 Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arq Asn Val 70 75 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val 90 Tyr Giu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys 100 105 110 Vai Arg Giu Asn Asn Ser Ser Ar g Cys Trp Val Ala Leu Thr Pro Thr 115 120 125 Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His 89 130 135 140 Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val 145 150 155 160 Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gin Leu Phe Thr Ile 165 170 175 Ser Pro Arg Arg His Glu Thr Val Gin Asp Cys Asn Cys Ser Ile Tyr 180 185 190 Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn 195 200 205 Trp I INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 483 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S: (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..480 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..477 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATG CCC GGT TGC TCT TTC TCT ATC TTC CTC TTG GCC CTG CTG TCC TGT 48 Met Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys 1 5 10 CTG ACC ATA CCA GCT TCC GCT TAT GAA GTG CGC AAC GTG TCC GGG GTG 96 Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Val 25 TAC CAT GTC ACG AAC GAC TGC TCC AAC TCA AGC ATA GTG TAT GAG GCA 144 Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala 40 GCG GAC ATG ATC ATG CAC ACC CCC GGG TGC GTG CCC TGC GTT CGG GAG 192 Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg C-lu 55 GGC AAC TCC TCC CGT TGC TGG GTG GCG CTC ACT CCC ACG CTC GCG GCC 240 Gly Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala 70 75 AGG AAC GCC AGC GTC CCC ACA ACG ACA ATA CGA CGC CAC GTC GAT TTG 288 Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu 90 CTC GTT GGG GCT GCT GCT TTC TGT TCC GCT ATG TAC GTG GGG GAT CTC 336 Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val Gly Asp Leu 90 100 105 110 TGC GGA TCT GTT TTC CTT GTT TCC CAG CTG TTC ACC TTC TCA CCT CGC 384 Cys Gly Ser Val Phe Leu Val Ser Gin Leu Phe Thr Phe Ser Pro Arg 115 120 125 CGG CAT CAA ACA GTA CAG GAC TGC AAC TGC TCA ATC TAT CCC GGC CAT 432 Arg His Gin Thr Val Gin Asp Cys Asn Cys Ser Ile Tyr Pro Gly His 130 135 140 GTA TCA GGT CAC CGC ATG GCT TGG GAT ATG ATG ATG AAC TGG TCC TAATAG 483 Val Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser 145 150 155 160 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: I LENGTH: 159 amino acids TYPE: amino acid TOPOLOGY: linear 4 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: *1 i Met Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys 1 5 10 Leu Thr Ile Pro Ala Ser Al.a Tyr Glu Val Arg Asn Val Ser Gly Val S* 20 25 4** Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala 40 Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu 55 Gly Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala 70 75 *Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu 85 90 Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val Gly Asp Leu 100 105 110 Cys Gly Ser Val Phe Leu Val Ser Gin Leu Phe Thr Phe Ser Pro Arg 115 120 125 Arg His Gin Thr Val Gin Asp Cys Asn Cys Ser Ile Tyr Pro Gly His 130 135 140 Val Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser 145 150 155 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 480 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO 91 (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .477 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1. .474 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATG TCC GGT TGC TCT 'ITC TCT ATC I-rC CTC Met Ser Gly Cys Ser Phe Ser Ile Phe Leu AAC GCC Asn Ala GTT GG Val Giy GGA TCT Gly Ser 115 CAT CAA His Gin 130 TCA GGT Ser Giy

GCT

Ala

AAC

Asn

ATG

Met

CGT

Arg

GTC

Val1 85

GCT

Ala

TTC

Phe

GTA

Val1

CGC

Arg

OCT

Ala

TGC

Cys

ACC

Thr 55

TGG

Trp

ACA

Thr

TTC

Phe

GTT

Val1

GAC

Asp 135

GCT

Al a TTG GCC CTG CTG Lau Aia Leu Leu CGC .AAC GTG TCC Arg Asn Val Ser AGC ATA GTG TAT Ser Ile Val Tyr GTG CCC TGC GTT Val Pro Cys Val ACT CCC ACG CTC Thr Pro Thr Leu 75 CGA CGC CAC GTC Arg Arg His Val ATG TAC GTG GGG Met Tyr Val Gly 110 TTC ACC TTC TCA Phe Thr Phe Ser 125 TCA ATC TAT CCC Ser Ile ryr Pro 140 ATG ATG AAC TGG Met met Asn Trp 155 TCC TOT Ser Cys GGG GTG Gly Val GAG GCA Glu Ala CGG GAG Arg Glu GCG GCC Ala Ala GAT TT G Aso Leu GAT CTC Asp Leu CCT CGC Pro Arg GGC CAT Glv Hi_4s

TAATAG

48 96 144 192 240 288 336 384 432 480 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 158 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Met Ser Oly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys 1 5 10 92 Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Val 25 Tyr His Val Thr Asn Asp Cys Ser Asn- Ser Ser Ile Val Tyr Glu Ala 40 Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu 55 Gly Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala 70 75 Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu 90 Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val Gly Asp Leu 100 1.05 110 Cys Gly Ser Val Phe Leu Val Ser Gin Leu Phe Thr Phe 5cr Pro Arg 115 120 125 *Arg His Gin Thr Val Gin Asp Cys Asn Cys Ser Ile Tyr Pro Gly His 130 135 140 Val Ser Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp *145 150 155 INFORMATION FOR SEQ, ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 636 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO *o (ix) FEATURE: NAME/KEY: CDS o: LOCATION: 1. .633 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1. .630 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: ATG CTG GGT AAG GCC ATC GAT ACC CTT ACG TGC GGC TTC GCC GAC CTC 48 Met Leu Gly Lys Ala Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu 1 5 10 1 GTG GGG TAC ATT CCG CTC GTC GGC GCC CCC CTA GGG GGC GCT GCC AGG 96 Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg 25 GCC CTG GCG CAT GGC GTC CGG GTT CTG GAA GAC GGC GTG AAC TAT GCA 144 Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Al a 40 ACA GGG AAT TTG CCT GGT TGC TCT T~TC TCT ATC TTC CTC TTG GCT 7TA 1-92 Thr Gly Asri Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala '_-2u 55 93 CTG TCC Leu Ser TGT CTA ACC ATT Cys Leu Thr Ile 0..

-:0.0 ATG TAC Met Tyr GCA GCG Ala Ala 100 GAG AAC Glu Asn 115 GCT AGO Ala Arg TrG CTC Leu Leu CTC TGC Leu Cys CGC CGG Arg Arg 180 CAC ATA His Ile 195

TAATAG

CCA OCT TCC OCT Pro Ala Ser Ala ACG AAC GAC TGC Thr Asn Asp Cys ATC ATG CAC ACC Ile Met His Thr 105 TCC CGC TGC TGG Ser Arg Cys Trp 120 AGC ATC CCC ACT Ser Ile Pro Thr 135 GCG GCT GCT TTC Ala Ala Ala Phe GTC TTC CTC GTC Val Phe Leu Val 170 ACG GTG CAG GAC Thr Val Gin Asp 185

GTG

Val

TCA

Ser

TGC

Cys

CTC

Leu 125

ATA

Ile

GCT

Al a

CTG

Leu

TGC

Cys

ATG

Met 205 ACG GGT CAC CGT Thr Gly His Arg ATG GCT TGG GAT Met Ala Trp Asp ATO ATG AAC Met Met Asn INFORMATION FOR SEQ ID NO: 14: met Val1 Ala Thr Leu Se r SEQUENCE CHARACTERISTICS: LENGTH: 210 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Leu Oly Lys Ala Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Lau 10 Gly Tyr Ile Pro Leu Val Oly Ala Pro Leu Oly Gly Ala Ala Arg 25 Leu Ala His Gly Val Arg Val Leu Glu Asp Oly Val Asn Tyr Ala 40 Oly Asn Leu Pro Oly Cys Ser Phe Ser Ilie Phe Lau Leu Ala Lau 55 Ser Cys Leu Thr Ilie Pro Ala Ser Ala Tyr Glu Val Arg Asn Val 70 75 Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val 94 90 Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys 100 105 110 Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr 115 120 125 Leu Ala Ala Arg Asn Ala Ser Ile Pro Thr Thr Thr Ile Arg Arg His 130 135 140 Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys Ser Ala Met Tyr Val 145 150 155 160 Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser Gln Leu Phe Thr Ile 165 170 175 Ser Pro Arg Arg His Glu Thr Val Gln Asp Cys Asn Cys Ser Ile Tyr 180 185 190 SPro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn 195 200 205 Trp Tyr 210 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATGCCCGGTT GCTCTTTCTC TATCTT 26 INFORMATION FOR SEQ ID NO: 16: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: ATGTTGGGTA AGGTCATCGA TACCCT 26 INFORMATION FOR SEQ ID NO: 17: 95 SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CTATTAGGAC CAGTTCATCA TCATATCCCA INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA o (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES e (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CTATTACCAG TTCATCATCA TATCCCA 27 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: ATACGACGCC ACGTCGATTC CCAGCTGTTC ACCATC 36 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO 96 (iii) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GATGGTGAAC AGCTGGGAAT CGACGTGGCG TCGTAT INFORMATION FOR SEQ ID NO: 21: SEQUENCE CHARACTERISTICS: LENGTH: 723 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .720 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1. .717 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: a. a.

a a a.

a a a

ATG

Met 1

GTG

Val1

GCC

Ala

ACA

Thr

CTG

Leu

TCC

Se r

TAT

Tyr

GTT

Val1 TTG GGT AAG GTC ATC Leu Gly Lys Val Ile GAT ACC CTT ACA TGC GGC TTC GCC GAC CTC Asp Thr Leu Thr Cys Gly Phe Ala

T

Ile

CAT

His

TTG

Leu

CTG

Leu

TAC

Tyr

GCG

Ala 100

AAC

As n 5 CCG CTC Pro Leu GGC GTC Gly Val CCC GGT Pro Gly ACC GT Thr Val 70 CAT GTC His Val GAC ATG Asp Met AAC TCT Asn Ser

GTC

Val

CGG

Arg

TGC

Cys

CCA

Pro

ACG

Thr

ATC

Ile

TCC

Ser

CTA

Leu

GAC

Asp

ATC

lle

TAT

Tyr

TCC

Ser

CCC

Pro

GTA

Val1 Asp Leu GCC AGG Ala Arg TAT GCA Tyr Ala GCT 'ITG Ala Lau AAC C-TG Asn Val ATT GTG ile Val CCC TGC Pro Cys CCC AC-G Pro -hr 288 CTC GCA GCT AGG AAC GCC AGC GTC CCC ACC ACG ACA ATA CGA CGC CAC Thr Thr Ile Arg Arg Leu Ala 130 Ala Arg Asn Ala Ser 135 Val Pro Thr 97 GTC GAT TCC CAG CTG i-rC ACC ATC TCG CCT CGC CGG CAT Val Asp Ser Gin Leu Phe Thr Ile Ser Pro Arg Arg His 145 150 155 CAG GAC TGC AAT TGC TCA ATC TAT CCC GGC CAC ATA ACG Gin Asp Cys Asn Cys Ser Ile Tyr Pro Giy His Ile Thr 165 170 ATG GCT TGG GAT ATG ATG ATG AAC TGG TCG CCT ACA ACG Met Aia Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr 180 185 GTA TCG CAG CTG CTC CGG ATC CCA CAA GCT GTC GTG GAC Vai Ser Gin Leu Leu Arg Ile Pro Gin Aia Val Val A-sp 195 200 205 GGG GCC CAT TGG GGA GTC CTG GCG GGT CTC GCC TAC TAT Giy Aia His Trp Gly Vai Leu Aia Giy Leu Aia Tyr Tyr 210 215 220 GGG AAC TGG GCT AAG GTr TrG, ATT GTG ATG CTA CTC TTT Gly Asn Trp Ala Lys Val Leu Ile Vai Met Leu Leu*Phe 225 230 235 INFORMATION FOR SEQ ID NO: 22: SEQUENCE CHARACTERISTICS: LENGTH: 239 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: Met Leu Giy Lys Val Ile Asp Thr Leu Thr Cys Gly Phe 1 5 10 Vai Gly Tyr Ilie Pro Leu Vai Giy Ala Pro Leu Gly Gly 25 Ala Leu Aia His Gly Val Arg Val Leu Glu Asp Gly Val 35 40 Thr Gly Asn Leu Pro Giy Cys Ser Phe Ser Ile Phe Leu 55 Leu Ser Cys Leu Thr Val Pro Aia Ser Ala Tyr Giu Val 70 75 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser 90 Tyr Glu Ala Ala Asp Met le Met His Thr Pro Gly Cys 100 105 Val Arg Giu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu 115 120 125 Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile 130 135 140 Val Asp Ser Gin Leu Phe Thr Ile Ser Pro Arg Arg His 145 150 155 Gin Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr 165 170 Giu

GGT

Gly

GCC

Ala 190

ATG

Met

TCC

Ser

GCT.

Ala Al a Al a As n Leu Arg Se r VaI 110 Thr Arg Glu Thr

CAC

His 175

CTG

Leu

GTG

Val

ATG

Met

CCC

Pro Asp Ala Tyr Ala Asn Ile Pro Pro Arg Thr Val1 160

CGT

Arg

GTG

Val1

GCG

Ala

GTG

Val1

TAATAG

240 GAG ACG GTG 480 528 576 624 672 723 Leu Arg Al a Leu Val1 Val1 Cys Thr His Val1 160 Arg Gly His 175 -1 98 Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val 180 185 190 Val Ser Gin Leu Leu Arg Ile Pro Gin Ala Val Val Asp Met Val Ala 195 200 205 Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val 210 215 220 Gly Asn Trp Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Pro 225 230 235 INFORMATION FOR SEQ ID NO: 23: SEQUENCE CHARACTERISTICS: LENGTH: 561 base pairs TYPE: nucleic acid STRANDEDNESS: single V TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 00 (iii) HYPOTHETICAL: NO .0.0 (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDSoptd LOCATION: 1. .558 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: ATG TrG GGT AAG GTC ATC GAT ACC CTT ACA TGC GGC TTC GCC GAC CTC 48 Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Lau 1 5 10 GTG GGG TAC AITT CCG CTC GTC GGC GCC CCC CTA GGG GGC GCT DCC AGG 96 *.Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Ar g 2025 30 GCC CTG GCG CAT GGC GTC CGG GTT CTG GAG GAC GGC GTG AhC TAT GCA 144 Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala 40 ACA GGG AAT 'VTD CCC GGT TGC TCT TTC TCT ATC i-rC CTC TTG GCT 77 G19 Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala eu 55 CTG TCC TGT CTG ACC GTT CCA GCT TCC GCT TAT DAA GTG CGC AAC GTG 240 Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Ary Asn Val 70 75 TCC OGG ATG TAC CAT GTC ACG AAC GAC TDC TCC AAC TCA ADC ATT GTG 28 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser ile Val 90 TAT GAD GCA GCG GAC ATD ATC ATD CAC ACC CCC GGG TGC DTG CCC DGC 336 Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys 100 105 110 DTT CGG DAD AAC AAC TCT TCC CDC TDC TDD GTA DCD CTC ACC CCC CG 334 Val Arg Dlu Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro 7hr 99 115 120 125 CTC GCA GCT AGG AAC GCC AGC GTC CCC ACC ACG ACA ATA CGA CGC CAC 432 Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr Thr Ile Arg Arg His 130 135 140 GTC GAT TCC CAG CTG TTC ACC ATC TCG CCT CGC CGG CAT GAG ACG GTG 480 Val Asp Ser Gin Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val 145 150 155 160 CAG GAC TGC AAT TGC TCA ATC TAT CCC GGC CAC ATA ACG GGT CAC CGT 528 Gin Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg 165 170 175 ATG GCT TGG GAT ATG ATG ATG AAC TGG TAATAG 561 Met Ala Trp Asp Met Met Met Asn Trp 180 185 V INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 185 amino acids TYPE: amino acid TOPOLOGY: linear 0(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu *1 5 10 *Val Gly T'yr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala A.rg 25 Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala *35 40 Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ilie Phe Leu Leu Ala Leu 55 Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val 70 75 *.:Ser Gly Met Tryr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val *85 90 Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys 100 105 110 Val Arg Glu Asn Asn 5cr Ser Arg Cys Trp Val Ala Leu Thr Pro Thr 115 120 125 Leu Ala Ala Az-g Asn Ala Ser Val Pro Thr Thr Thr Ile Ary Ara is 130 135 140 Val Asp Ser Gin Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val 145 150 155 Gin Asp Cys Asn Cys 5cr Ile Tyr Pro Gly His Ile Thr Gly His Arg 165 170 175 Met Ala Trp Asp Met Met Met Asn Trp 180 185 INFORM4ATION FOR SEQ ID NO: 100 SEQUENCE CHARACTERISTICS: LENGTH: 606 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cONA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..603 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1..600 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: a.

ATG TTG GGT Met Leu Gly AAG GTC ATC GAT ACC CTT Lys Val Ile Asp Thr Leu ACA TGC GGC TTC Thr Cys Gly Phe GCC GAC CTC Ala Asp Leu is

ACA

Thr

CTG

Leu

TCC

Ser

TAT

Tyt

GTT

Val

CTC

Leu

GTC

Val 145

CAG

Gin

ATT

Ile

CAT

His

TTG

Leu

CTG

Leu

TAC

Tyr

GCG

Ala 100

AAC'

Asn

AGG

Arg

CAG

Gin 5

CCG

Pro

GGC

Gly

CCC

Pro

ACC

Thr

CAT

His

GAC

Asp

AAC

Asn

AAC

Asn

CTG

Leu

TGC

Cys 165

CTC

Leu

GTC

Va1

GGT

Gly

GTT

Va1 70

GTC

Va1

ATG

Met

TCT

Ser

GCC

Ala

TTC

Phe 150

TCA

Ser

GTC

Val

CGG

Arg

TGC

Cys

CCA

Pro

ACG

Thr

ATC

Ile

TCC

Ser

AGC

Ser 135

ACC

Thr

ATC

Ile

GGC

Cly

GTT

Val 40

TCT

Ser

GCT

Ala

AAC

Asn

ATG

Met

CGC

Arg 120

CTC

Val

ATC

Ile

TAT

Tyr

GCC

Ala 25

CTG

Leu

TTC

Phe

TCC

Ser

CAC

Asp

CAC

His 105

TGC

Cys

CCC

Pro

TCG

Ser

CCC

Pro 10

CCC

Pro

GAG

Glu

TCT

Ser

GCT

Ala

TGC

Cys 90

ACC

Thr

TGG

Trp

ACC

Thr

CCT

Pro

GGC

Gly 170

GGG

Gly

GGC

Gly

TTC

Phe

CAA

Glu

AAC

Asn

GGG

Cly

GCG

Ala

ACA

Thr 140

CGG

Arg

ATA

Ile

GGC

Gly

GTG

Val

CTC

Leu

GTG

Va1

TCA

Ser

TGC

Cys

CTC

Leu 125

ATA

Ile

CAT

His

ACG

Thr

GCT

Ala

AAC

Asn

TTG

Leu

CGC

Arg

AGC

Ser

GTG

Va1 110

ACC

Thr

CGA

Arg

GAG

Glu

GGT

Gly

TAT

Tyr

GCT

Ala

AAC

Asn

ATT

Ile

CCC

Pro

CCC

Pro

CGC

Arg

ACG

Thr

CAC

His 175

GCA

Ala

TTG

Leu

GTG

Val

GTG

Va1

TGC

Cys

ACG

Thr

CAC

His

GTG

Va1 160

CGT

Ar g CCC AGG Ala Arg 48 96 144 192 240 288 336 384 432 480 528 GAC TGC AAT Asp Cys Asn ATO GCT TGG GAT ATG ATG ATG AAC TOG TCG CCT ACA ACG GCC CTG GTG 101 met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val 180 185 190 GTA TCG CAG CTG CTC CGG ATC CTC TAATAG Val Ser Gin Leu Leu Arg Ile Leu 195 200 INFORMATION FOR SEQ ID NO: 26: SEQUENCE CHARACTERISTICS: LENGTH: 200 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala 0 60 Ile His Leu Leu Tyr Ala 100 Asn Arg Gin Asn Asp 180 Leu Pro Gly Pro Thr His Asp As n As n Leu Cys 165 Met Leu Val1 Arg Cys Pro Thr Ile Ser Ser 135 Thr Ile Met Ile Gly Ala Val Leu Ser Phe Ala Ser Asn Asp met His 105 Arg Cys 120 Val Pro Ile Ser Tyr Pro Asn Trp 185 Leu 200 Gly Val1 Leu Val1 Ser Cys Leu 125 Ile His Thr Asp Leu Ala Arg Tyr Ala Ala Leu Asn Val Ile Val Pro Cys Pro Thr Arg His Thr Val 160 His Arg Ser Pro Thr Thr Ala Leu Val 190 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 636 base pairs TYPE: nucleic acid CC) STRLANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA 102 (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .633 (ix) FEATURE: NAME/KEY: mat peptide LOCATION: 1. .630 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: ATG 11TG GGT AAG Met Leu Gly Lys 1 GTG GGG Val Gly GCC CTG Ala Leu ACA GGG Thr Gly 50

GTC

Val1 5

CCG

Pro

GGC

Gly ATC GAT ACC CTT ACA TGC GGC Ile Asp Thr Leu Thr Cys Gly TTC GCC GAC CTC Phe Ala Asp Leu GGC GCT GCC AGG Gly Ala Ala Arg GTG AAC TAT GCA Val Asn Tyr Ala CTC TTG GCT ITG Leu Leu Ala Leu GTG CGC AAC GTG Val Arg Asn Val TCA AGC ATT CTG Ser Ser Ile Val TTG CCC GGT Leu Pro Gly

CTG

Leu 65

TCC

Ser

TAT

Tyr

GTT

Val

CTC

Le'u

GTC

Val1 145

CAG

Glri

ATG

Met

GTA

Val

CAT

TCC TGT CTG Ser Cys Leu GGG ATG TAC Gly Met Tyr GAG GCA GCG Glu Ala Ala 100 CGG GAG AAC Arg Glu Asn 115 GCA GCT AGG Ala Ale Arg 130 GAT TCC CAG ASP Ser Gln GAC TGC AAT Asp Cys Asn GCT TGG GAT Ala Trp Asp 180 TCG CAG CTG Ser Gln Leu 195 CAC TAATAG TGC TCT TTC TCT ATC Cys Ser Phe Ser Ile CCA GCT TCC GCT TAT Pro Ala Ser Ala Tyr 75 ACG AAC GAC TGC TCC Thr Asn Asp Cys Ser ATC ATG CAC ACC CCC Ile Met His Thr Pro 105 TCC CGC TGC TGG GTA Ser Arg Cys Trp Val 120 AGC GTC CCC ACC ACG Ser Val Pro Thr Thr 135 ACC ATC TCG CCT CGC Thr Ile Ser Pro Arg 155 ATC TAT CCC GGC CAC Ile Tyr Pro Gly His 110 ATG AAC TGG TCG CCT Met Asn Trp Ser Pro 185 GGG TGC Gly Cys GCG CTC Ala Leu 125 ACA ATA Thr Ile 140 CGG CAT Arg His ATA ACG Ile Thr ACA ACG Thr Thr AGA CAC Arg His 205 432 CTC CGG ATC Leu Arg Ile ATC GAG GGC Ile Glu Gly CAT CAC CAC His His 'His 103 His His 210 INFORMATION FOR SEQ ID NO: 28: SEQUENCE CHARACTERISTICS: LENGTH: 210 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys G 1 5 10 Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu G 20 Ala Leu Ala His Gly Val Arg Val Leu Glu Asp G 40 Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile P] Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr G: 65 70 75 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser A: 85 90 Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro G.

100 105 Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val A: 115 120 Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr TI 130 135 1 Val Asp Ser Gin Leu Phe Thr Ile Ser Pro Arg A: 145 150 155 Gin Asp Cys Asn Cys Ser Ile Tyr Pro Gly His I 165 170 Met Ala Trp Asp Met Met Met Asn Trp Ser Pro TI 180 185 Val Ser Gin Leu Leu Arg lie Val Ile Glu Gly A: 195 200 His His 210 INFORMATION FOR SEQ ID NO: 29: SEQUENCE CHARACTERISTICS: LENGTH: 630 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO ly Phe Ala Gly Val Leu Val Ser Cys Leu 125 Ile His Thr Thr His 205 Asp Leu Ala Arg Tyr Ala Ala Leu Asn Val Ile Val Pro Cys Pro Thr Arg His Thr Val 160 His Arg 175 Leu Val His :is 104 (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .627 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1. .624 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: ATG GGT AAG GTC ATC GAT ACC CTT ACG TGC GGA TTC GCC GAT CTC ATG met Gly Lys Val Ile Asp Thr Leu Thr

S

*5S* *5

S

*5

S

S.

1

GGG

Gly

CTT

Leu

GGG

Gly

TCT

Ser

GGC

G ly

GAG

Glu

CAG

Gin

GCA

Ala

GAC

Asp 145

GAC

Asp

CCT

Pro

CCG

Pro

GGC

Gly

CCC

Pro

ATT

Ile

OTC

Val1

GAC

Asp 100

AAT

As n

TAC

Tyr

GTG

Val1

GGG

Gly

CAT

His 180 5

CTC

Leu

GTG

Val1

GGT

Gly

CAT

His

CIW

Leu 85

GTT

Val1

ACA

Thr

GTC

Val1

GGC

Gly

GCT

Ala 165

CAA

Gin

GGC

Gly

GCC

Ala

TCC

Ser

GCA

Ala

AAC

Asn

CTG

Leu

ACG

Thr

GCA

Ala 135

GCC

Ala

TTC

Phe

OTC

-Val1 Cys 10

GTA

Val

GAC

Asp

ATT

Ile

CTA

Leu 7CC Ser 90

CCC

Pro

ACC

Thr

OCT

Al a

TGC

Cys

OA

Oly 170

TOT

Cys

GTC

Val1

AAT

Asn

CTC

Leu

COG

Arg

AGT

Ser

ATA

Ile

ACA

Thr 125

CGC

Arg

CTC

Leu

TTC

Phe

TCO

Se r

ATG

Met 205 Gly Phe Ala Asp Leu Met AGA 0CC Arg Ala GCA ACA Ala Thr CTO 'ITC Leu Phe ACO TCT Thr Ser 070 TAC Val Tyr TGT OTC Cys Val ACA 070 Thr Val CAT OTG His Val GTG GOT Val Gly TTC AGA Phe Ar g 175 TAC CCA Tyr Pro 48 96 144 192 240 288 336 384 432 480 528 576 624 GGC CAT CTT Oly His Leu 195

TAATAG

TCA GGA CAT CGA Ser Oly His Arg GCT TGGOAT ATG Ala Trp Asp Met ATG AAC TOG Met Asn 7ro 105 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 208 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Gly Lys Val Ile Asp Thr Leu Thr Cys Gly PC 1 5 10 Gly Tyr Ile Pro Leu Val Gly Ala Pro Val Gly GI Leu Ala His Gly Val Arg Ala Leu Giu Asp Gly I.

35 40 Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe L, 50 Ser Cys Leu Ile His Pro Ala Ala Ser Leu Glu T: 6570 Gly Leu Tyr Val Leu Thr Asn Asp Cys Ser Asn S 90 Giu Ala Asp Asp Val Ile Leu His Thr Pro Gly C, 100 105 Gin Asp Gly Asn Thr Ser Thr Cys Trp Thr Pro V 115 120 Ala Val Lys Tyr Val Gly Ala Thr Thr Ala Ser I 130 135 1 Asp Leu Leu Val Gly Ala Ala Thr Met Cys Ser A 145 150 155 Asp Met Cys Gly Ala Vai Phe Leu Val Gly Gin A 165 170 Pro Arg Arg His Gin Thr Val Gin Thr Cys Asn C 180 185 Gly His Leu Ser Gly His Ar; Met Ala Trp Asp M 195 200 Val1 As n Leu Ar; Ser Ile Thr 125 Ar; Leu Phe Ser met 205 Arg Ala Leu Thr Val1 Cys Thr His Vai Phe 175 Tyr As n Ala Thr Phe Ser TVr Val Val Val1 G ly Arg Pro Tro he Ala Asp Leu Met is INFORMATION FOR SEQ ID NO: 31: SEQUENCE CHARACTERISTICS: LENGTH: 630 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO 106- (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .627 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1. .624 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: ATG GGT AAG GTC ATC GAT ACC CTA ACG TGC GGA TTC GCC GAT CTC ATG 48 Met Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met 1 5 10 GGG TAT ATC CCG CTC GTA GGC GGC CCC ATT GGG GGC GTC GCA AGG GCT 96 Gly Tyr Ile Pro Leu Val Gly Gly Pro Ile Gly Gly Val Ala Arg Ala 25 *CTC GCA CAC GGT GTG AGG GTC CTT GAG GAC GGG GTA AAC TAT GCA ACA 144 ~Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr 40 *GGG AAT TTA CCC GGT TGC TCT TTC TCT ATC TTT ATT CIT GCT CTT CTC 192 ***Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Ile Leu Ala Leu Leu 50 55 TCG TGT CTG ACC GTT CCG GCC TCT GCA GTT CCC TAC CGA AAT GCC TCT 240 Ser Cys Leu Thr Val Pro Ala Ser Ala Val Pro Tyr Arg Asn Ala Ser 70 75 0OGGG ATT TAT CAT GTT ACC AAT GAT TGC CCA AAC TCT TCC ATA GTC TAT 288 *Gly Ile Tyr His Val Thr Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr 90 GAG GCA GAT AAC CTG ATC CTA CAC GCA CCT GGT TGC GTG CCT TGT GTC 336 0.0. Glu Ala Asp Asn Leu Ile Leu His Ala Pro Gly Cys Val Pro Cys Val ft...*100 105 110 ATG ACA GGT AAT GTG AGT AGA TGC TGG GTC CAA ATT ACC CCT ACA CTG 384 Met Thr Gly Asn Val Ser Arg Cys Trp Val Gin Ilie Thr Pro Thr L-eu ***115 120 125 **TCA GCC CCG AGC CTC GGA GCA GTC ACG GCT CCT CTT CGG AGA GCC GTT 432 Ser Ala Pro Ser Leu Gly Ala Val Thr Ala Pro Leu Arg Arg Ala Val 130 135 140 GAC TAC CTA GCG GGA GGG GCT GCC CTC TGC TCC GC1G TTA TAC GTA GGA 480 Asp Tyr Leu Ala Gly Gly Ala Ala Leu Cys Ser Ala Leu Tyr Val Gly 14-5 150 155 160 GAC GCG TGT GGG GCA CTA ITC 'TTO GTA GGC CAA ATG TTC ACC TAT AGG 528 Asp Ala Cys Gly Ala Leu Phe Leu Val Gly Gin Met Phe Thr Tyfr Arg 165 170 1-75 CCT CGC CAG CAC GCT ACG GTG CAG AAC TGC AAC TGT TCC ATT TAC AGT 576 Pro Arg Gin His Ala Thr Val Gin Asn Cys Asn Cys Ser le T yr Sc 180 185 *190 GGC CAT GTT ACC GGC CAC CGG ATG GCA TGG GAT ATG ATG ATG AAC 7-GG o62 4 Gly His Val Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn 7 rp 195 200 205 TAATAG 630 INFORMATION FOR SEQ ID NO: 32: 107 SEQUENCE CHARACTERISTICS: LENGTH: 208 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: Met Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met 1 5 10 Gly Tyr Ile Pro Leu Val Gly Gly Pro Ile Gly Gly Val Ala Arg Ala 25 Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr 40 ,se. Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Ile Leu Ala Leu Leu 50 55 Ser Cys Leu Thr Val Pro Ala Ser Ala Val Pro Tyr Arg Asn Ala Ser 65 70 75 Gly Ile Tyr His Val Thr Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr 85 90 Glu Ala Asp Asn Leu Ile Leu His Ala Pro Gly Cys Val Pro Cys Val 100 105 110 Met Thr Gly Asn Val Ser Arg Cys Trp Val Gin Ile Thr Pro Thr Leu er* 115 120 125 Ser Ala Pro Ser Leu Gly Ala Val Thr Ala Pro Leu Arg Arg Ala Val 130 135 140 *Asp Tyr Leu Ala Gly Gly Ala Ala Leu Cys Ser Ala Leu Tyr Val Gly 145 150 155 160

C

Asp Ala Cys Gly Ala Leu Phe Leu Val Gly Gin Met Phe Thr Tyr Arg 165 170 175 SPro Arg Gin His Ala Thr Val Gin Asn Cys Asn Cys Ser Ile Tyr Ser 180 185 190 Gly His Val Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp 195 200 205 INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: 108 TGGGATATGA TGATGAACTG GTC 23 INFORMATION FOR SEQ ID NO: 34: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: CTATTATGGT GGTAAGCCAC AGAGCAGGAG INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1476 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..1473 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..1470 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TGG GAT ATG ATG ATG AAC TGG TCG CCT ACA ACG GCC CTG GTG GTA TCG 48 Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ser 1 5 10 CAG CTG CTC CGG ATC CCA CAA GCT GTC GTG GAC ATG GTG GCG GGG GCC 96 Gin Leu Leu Arg Ile Pro Gin Ala Val Val Asp Met Val Ala Gly Ala 25 CAT TGG GGA GTC CTG GCG GGC CTC GCC TAC TAT TCC ATG GTG GGG AAC 144 His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn 40 TGG GCT AAG GTT TTG GTT GTG ATG CTA CTC TTT GCC GGC GTC GAC GGG 192 Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly 55 CAT ACC CGC GTG TCA GGA GGG GCA GCA GCC TCC GAT ACC AGG GGC CTT 240 His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu 70 75 GTG TCC CTC TTT AGC CCC GGG TCG GCT CAG AAA ATC CAG CTC GTA AAC 288 109 Val Ser Leu Phe Ser Pro Gly Ser Ala Gin Lys Ile Gin Leu Val Asn 90 ACC AAC GGC AGT TGG CAC ATC AAC AGG ACT GCC CTG AAC TGC AAC GAC 336 Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp 100 105 110 TCC CTC CAA ACA GGG TTC TrT GCC GCA CTA TTC TAC AAA CAC AAA TTC 384 Ser Leu Gin Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe 115 120 125 AAC TCG TCT GGA TGC CCA GAG CGC TTG GCC AGC TGT CGC TCC ATC GAC 432 Asn Ser Ser Gly Cys Pro Giu Arg Leu Ala Ser Cys Arg Ser Ile Asp 130 135 140 AAG TTC GCT CAG GGG TGG GGT CCC CTC ACT TAC ACT GAG CCT AAC AGC 480 Lys Phe Ala Gin Gly Trp Gly Pro Leu Thr Tyr Thr Giu Pro Asn Ser 145 150 155 160 TCG GAC CAG AGG CCC TAC TGC TGG CAC TAC GCG CCT CGA CCG TGT GGT 528 Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly 165 170 175 ATT GTA CCC GCG TCT CAG GTG TGC GGT CCA GTG TAT TGC TTC ACC CCG 576 Ile Val Pro Ala Ser Gin Val Cys Gly Pro Val Tyr Cys Phe Thr Pro 180 185 190 AGC CCT GTT GTG GTG GGG ACG ACC GAT CGG TTT GGT GTC CCC ACG TAT 624 Ser Pro Val Val val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr 195 200 205 AAC TGG GGG GCG AAC GAC TCG GAT GTG CTG ATT CTC AAC AAC ACG CGG 672 Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg 210 215 220 CCG CCG CGA GGC AAC TGG TTC GGC TGT ACA TGG ATG AAT GGC ACT GGG 720 0. Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly 225 230 235 240 TTC ACC AAG ACG TGT GGG GGC CCC CCG TGC AAC ATC GGG GGG GCC GGC 768 Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly 245 250 255 AAC AAC ACC TTG ACC TGC CCC ACT GAC TGT TTT CGG AAG CAC CCC GAG 816 Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu 260 265 270 GCC ACC TAC GCC AGA TGC GGT TCT GGG CCC TGG CTG ACA CCT AGG TGT 864 Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys 275 280 285 ATG GTT CAT TAC CCA TAT AGG CTC TGG CAC TAC CCC TGC ACT GTC AAC 912 Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn 290 295 300 TTC ACC ATC TC AAG GT- AGG ATG TAC GTG GGG GGC GTG GAG CAC AGG 960 Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val Giu His Ar g 305 310 315 320 TTC GAA GCC GCA TGC AAT TGG ACT CGA GGA GAG CGT TGT GAC TTG GAG 1008 Phe Giu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arq Cys Asp Leu Glu 325 330 335 GAC AGG GAT AGA TCA GAG CTI AGC CCG CTG CTG CTG TCT ACA ACA GAG 1056 Asp Arg Asp Arg Ser Giu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu 340 345 350 TGG CAG ATA CTG CCC TGT TCC TTC ACC ACC CTG CCG GCC CTA TCC ACC 1104 Trp Gin Ile Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala Leu Ser Thr 110 355 360 365 GGC CTG ATC C-AC CTC CAT CAG A-AC ATC GTG GAC GTG CAA TAC CTG TAC 1152 Gly Leu Ile His Leu His Gin Asn Ile Val Asp Val Gin Tyr Leu Tyr 370 375 380 GGT GTA GGG TCG GCG G'rr GTC TCC C IT GTC ATC AAA TGG GAG TAT GTC 1200 Gly Val Gly Ser Ala Val Val Ser Leu Val Ile Lys Trp Giu Tyr Val 385 .390 395 400 CTG 'rrG CTC T1'C CTT CTC CTG GCA GAC GCG CGC ATC TGC GCC TGC TTA 1248 Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Ile Cys Alia Cys Leu 405 410 415 TGG ATG ATG CTG CTG ATA GCT CA-A GCT GAG GCC GCC TTA GAG .'AAC CTG 1296 Trp Met Met Leu Leu Ile Ala Gin Ala Giu Ala Ala Leu Glu Asn Leu 420 425 430 *..GTG GTC CTC AAT GCG GCG GCC GTG GCC GGG GCG CAT GGC ACT CTT TCC 1344 00 Val Val Leu Asn Ala Ala Ala Val Ala Gly Ala His Gly .Thr Leu Ser .:435 440 445 0.0:TTC CTI- GTG ITC TTC TGT OCT GCC TGG TAC ATC AAG GGC AGG CTG GTC 1392 Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val .00 450 455 460 .000 0.CCT GGT GCG GCA TAC GCC TrC TAT GGC GTG TGG CCG CTG CTC CTG CTT 1440 Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu 465 470 475 480 CTG CTG GCC TTA CCA CCA CGA GCT TAT GCC TAGTAA 1476 *Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala 485 490 o ooos o(2) INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 490 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ser 1 5 10 Gin Leu Leu Arg Ile Pro Gin Ala Val Val Asp Met Val Ala Gly Ala 25 His Tro Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn 40 Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly 55 His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu 70 75 Val Ser Leu Phe Ser Pro Gly Ser Ala Gin Lys Ile Gin Leu Val Asn 90 Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asfl Cys Asn Asp 100 105 110 Ser Leu Gin Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys ?he 115 120 125 111 Ser Gly Cys Pro Glu Arg Leu Ala Ser 135 Asn Ser 130 Cys Arg Ser Ile Asp 140 Lys 145 Ser Ile Ser Asn Pro 225 Phe Asn Ala Met Phe 305 Phe Aso Trp Gly Gly 3985 Leu Trp Val Phe Pro 465 Phe Asp Val Pro Trp 210 Pro Thr Asn Thr Val 290 Thr Glu Arg Gin Leu 370 Val Leu Met Val Leu 450 Gly Ala I Gin Pro Val 195 Gly Arg Lys Thr Tyr 275 His Ile Ala Asp Ile 355 Ile Gly Leu Met Leu 435 Vai Ala Gln Arg Ala 180 Val Ala Gly Thr Leu 260 Ala Tyr Phe Ala Arg 340 Leu His Ser Phe Leu 420 Asn Phe Ala Gly Pro 165 Ser Va1 Asn Asn Cys 245 Thr Arg Pro Lys ys 325 Ser Pro Leu Ala Leu 405 Leu Ala Phe Tyr Trp 150 Tyr Gin Gly Asp Trp 230 Gly Cys Cys Tyr Va1 310 Asn Glu Cys His Val 390 Leu Ile Ala Cys Ala 470 Gly Cys Val Thr Ser 215 Phe Gly Pro Gly Arg 295 Arg Trp Leu Ser Gin 375 Va1 Leu Ala Ala Ala 455 Phe Pro Trp Cys Thr 200 Asp Gly Pro Thr Ser 280 Leu Met Thr Ser Phe 360 Asn Ser Ala Gin Val 440 Ala Tyr Leu His Gly 185 Asp Val Cys Pro Asp 265 Gly Trp Tyr Arg Pro 345 Thr Ile Leu Asp Ala 425 Ala Trp Gly Thr Tyr 170 Pro Arg Leu Thr Cys 250 Cys Pro His Val Gly 330 Leu Thr Val Va1 Ala 410 Glu Gly Tyr Val Tyr 155 Ala Val Phe Ile Trp I 235 Asn Phe Trp Tyr Gly 315 Glu Leu Leu Asp Ile 395 Arg Ala Ala Ile Trp 475 rhr Pro ryr Gly Leu 220 M4et Ile Arg Leu Pro 300 Gly Arg Leu Pro Val 380 Lys Ile Ala His Lys 460 Pro Glu Pro Arg Pro Cys. Phe 190 Val Pro 205 Asn Asn Asn Gly Gly Gly Lys His 270 Thr Pro 285 Cys Thr Val Glu Cys Asp Ser Thr 350 Ala Leu 365 Gin Tyr Trp Glu Cvs Ala Leu Glu 430 Gly Thr 445 Gly Arr Leu Leu Asn Cys 175 Thr Thr Thr Thr Ala 255 Pro Arg Va1 His Leu 335 Thr Ser Leu Tyr Cys 415 Asn Leu Leu Leu Ser 160 Gly Pro Tyr Arg Gly 240 Gly Glu Cys Asn 320 Glu Glu Thr Tyr Val 400 Zeu Leu Ser Val 480 Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala 485 490 112 INFORMATION FOR SEQ ID NO: 37: SEQUENCE CHARACTERISTICS: LENGTH: 1021 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 2..1018 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 2..1015 SEQUENCE DESCRIPTION: SEQ ID NO: 37: !G ATC CCA CAA GCT GTC GTG GAC ATG GTG GCG GGG GCC CAT TGG GGA 46 Ile Pro Gln Ala Val'Val Asp Met Val Ala Gly Ala His Trp Gly 1 5 10 GTC CTG GCG GGC CTC GCC TAC TAT TCC ATG GTG GGG AAC TGG GCT AAG 94 Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys 20 25 GTT TTG GTT GTG ATG CTA CTC TTT GCC GGC GTC GAC GGG CAT ACC CGC 142 Val Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg 40 GTG TCA GGA GGG GCA GCA GCC TCC GAT ACC AGG GGC CTT GTG TCC CTC 190 Val Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu Val Ser Leu 55 TTT AGC CCC GGG TCG GCT CAG AAA ATC CAG CTC GTA AAC ACC AAC GGC 238 Phe Ser Pro Gly Ser Ala Gln Lys Ile Gln Leu Val Asn Thr Asn Gly 65 70 75 AGT TGG CAC ATC AAC AGG ACT GCC CTG AAC TGC AAC GAC TCC CTC CAA 286 Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Gln 85 90 ACA GGG TTC TTT GCC GCA CTA TTC TAC AAA CAC AAA TTC AAC TCG TCT 334 Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn Ser Ser 100 105 110 GGA TGC CCA GAG CGC TTG GCC AGC TGT CGC TCC ATC GAC AAG TTC OCT 382 Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys Phe Ala 115 120 125 CAG GGG TGG GGT CCC CTC ACT TAC ACT GAG CCT AAC AGC TCG GAC CAG 430 Gin Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser Asp Gin 130 135 140 AGG CCC TAC TGC TGG CAC TAC GCG CCT CGA CCG TGT GGT ATT GTA CCC 478 Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro 145 150 155 GCG TCT CAG GTG TGC GGT CCA GTG TAT TGC TTC ACC CCG AGC CCT GTT 526 Ala Ser Gin Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val 160 165 170 175 113 GTG GTG GGG ACG ACC GAT CGG TTT GGT GTC CCC ACG TAT AAC a a a a a. a Val Val GCG AAC Ala Asn GGC AAC Gly Asn ACG TGT Thr Cys 225 TTG ACC Leu Thr 240 GCC AGA Ala Arg TAC CCA Tyr Pro TTC AAG Phe Lys GCA TGC Ala Cys 305 AGA TCA Arg Ser 320 GGC AGA Gly Arg Thr Thr Asp 180 TCG GAT GTG Ser Asp Val 195 'r'c GGC TGT Phe Gly Cys GGC CCC CCG Gly Pro Pro CCC ACT GAC Pro Thr Asp 245 GGT TCT GGG Gly Ser Gly 260 AGG CTC TGG Arg Leu Trp 275 AGG ATG TAC Arg Met Tyr TGG ACT CGA Trp Thr Arg CTT AGC CCG Leu Ser Pro 325

TAATTA

CTG CTG CTG TCT Leu Leu Leu Ser ACA GAG TGG CAG Thr Glu Tro Gin 1006 1021 INFORMATION FOR SEQ ID NO: 38: Wi SEQUENCE CHARACTERISTICS: LENGTH: 338 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: Ile Pro Gin Ala Val Val Asp Mez Val Ala Gly Ala His Trp Gly Val.

1 5 10 Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val 25 Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg Val 40 Ser Gly Gly Ala Ala Ala Ser Asp Thr kArg Gly Leu Val Ser Leu ?he 55 Ser Pro Gly Ser Ala Gin Lys Ile Gin Leu Val Asn Thr Asrn Gly ser 114 70 75 Trp His Ile Asn Ar; Thr Ala Leu Asn Cys Asn Asp Ser Leu Gin Thr 90 Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn Ser Ser Gly 100 105 110 Cys Pro Giu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys Phe Ala Gin 115 120 125 Gly Trp Gly Pro Leu Thr Tyr Thr Giu Pro Asn Ser Ser Asp Gin Ar; 130 .135 140 Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile Val Pro Ala 145 150 155 160 Ser Gin Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val *165 170 175 Val Giy Thr Thr Asp Ar; Phe Gly Val Pro Thr Tyr Asn Trp Gly Ala *180 185 190 :.Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Ar; Pro Pro Ar; Gly 195 200 205 *Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe Thr Lys Thr 210 215 220 Cys Gly Gly Pro Pro Cys Asn Ile Gly Gly Ala Gly Asn Asn Thr Leu ~*225 230 23S 240 Thr Cys Pro Thr Asp Cys Phe Ar; Lys His Pro Glu Ala Thr Tyr Ala 245 250 255 Ar; Cys Gly Ser Gly Pro Trp Leu Thr Pro Ar; Cys Met Val His Tyr 260 265 270 Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile Phe 275 280 285 :Lys Val Ar; Met Tyr Val Gly Gly Val Glu His Ar; Phe Glu Ala Ala :290 295 300 Cys Asn Trp Thr Ar; Gly Glu Ar; Cys Asp Leu Giu Asp Ar; Asp Ar; 305 310 315 320 Ser Giu Leu Ser Pro Leu Leu Leu Ser Thr Thr Glu Trp Gin Ser Gly 325 330 335 Arg Ala INFORMATION FOR SEQ 1D NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 1034 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: 115 NAME/KEY: COS LOCATION: 2. .1032 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 2. .1029 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: G ATC CCA CAA GCT GTC GTG GAC ATG GTG GCG GGG GCC CAT TGG GGA Ile Pro Gin Ala Val Vai Asp Met Val Ala Gly Ala His Trp Gly 1 5 10 is GTC CTG GCG GGC CTC GCC TAC TAT TCC ATG Val Leu GTT ITG Val Leu GTG TCA Val Ser T'IT AGC Phe Ser AGT TGG Ser Trp ACA GGG Thr Gly GGA TGC Gly Cys CAG GGG Gin Giy AGG CCC Arg Pro 145 GCG TCT Ala Ser 160 GTG GTG Val Val GCG AAC Ala~ Asn GGC AAC Gly Asn Ala

GTT

Val1

GGA

G ly 50

CCC

Pro

CAC

His

TTC

Phe

CCA

Pro

TGG

Trp 130

TAC

Tyr

CAG

Gin

GGG

G ly

GAC

Asp

TGG

Trp 210 Gly Leu GTG ATG Val Met 35 GGG GCA Gly Ala GGG TCG Gly Ser ATC AAC Ile Asn =r GCC Phe Ala 100 GAG CGC Glu Arg 115 GGT CCC Gly Pro TGC TGG Cys Trp GTG TGC Val Cys ACG ACC Thr Thr 180 TCG GAT Ser Asp 195 TTrC GGC Phe Giy Ala Tyr CTA CTC Leu Leu GCA CC Ala Ala GCT CAG Ala Gin 70 AGG ACT Arg Thr 85 GCA CTA Ala Leu TTG GCC Leu Ala CTC ACT Leu Thr CAC TAC His Tyr 150 GGT CCA Gly Pro 165 GAT CGG Asp Arg GTG CTG Val Leu TGT ACA Cys Thr Tyr T1T Phe

TCC

Ser 55

AAA

Lys

GCC

Ala

TTC

Phe

AGC

Ser

TAC

Tyr 135

GCG

Ala

GTG

Val1

TTT

Phe

ATT

Ile

TGG

Trp 215 Ser Met 25 GCC GCC Ala Gly 40 GAT ACC Asp Thr ATC CAG Ile Gin CTG AAC Leu Asn TAC AAA Tyr Lys 105 TGT CGC Cys Arg 120 ACT GAG Thr Glu CCT CGA Pro Arg TAT TGC Tyr Cys GGT GTC Gly Val 185 CTC AAC Leu Asn 200 ATG AAT Met Asn GTG GGG Val Gly GTC GAC Val Asp AGG GCC Arg Gly CTC GTA Leu Val TGC AAC Cys Asn 90 CAC AAA H is Lys TCC ATC Ser Ile CCT AAC Pro Asn CCG TGT Pro Cys 155 TTC ACC Phe Thr 170 CCC ACG Pro Thr AAC ACG Asn Thr GGC ACT Gly Thr AAC TGG GCT A.AG Asn Trp Ala Lys GGG CAT ACC CGC Gly His Thr Arg CTT GTG TCC CTC Leu Val Ser Leu AAC ACC AAC GGC Asn Thr Asn Gly GAC TCC CTC CAA Asp Ser Leu Gin TTC AAC TCG TCT Phe Asn Ser Ser GAC AAG TTC GCT Asp Lys Phe Ala 125 AGC TCG GAC CAG 5cr Ser Asp Gin 140 GGT ATT GTA CCC Gly Ile Val Pro CCG AGC CCT GTT Pro Ser Pro Val TAT AAC TGG GGG Tyr Asn Trp Gly 190 CGG CCG CCG CGA Arg Pro Pro Ar-g 205 GGG TTC ACC AAG Gly Phe Thr Lys 220 670 ACG TGT GGG GGC CCC CCG TGC AAC ATC GGG GGG GCC GGC Gly Ala Gly AAC AAC ACC Asn Asn Thr Thr Cys 225 Gly Gly Pro Pro Asn Ile Gly 116 TTG ACC TGC CCC ACT GAC TGT TTT CGG AAG CAC CCC GAG GCC ACC TAC 766 Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr 240 245 250 255 GCC AGA TGC GGT TCT GGG CCC TGG CTG ACA CCT AGG TGT ATG GTT CAT 814 Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met Val His 260 265 270 TAC CCA TAT AGG CTC TGG CAC TAC CCC TGC ACT GTC AAC TTC ACC ATC 862 Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val Asn Phe Thr Ile 275 280 285 TTC AAG GTT AGG ATG TAC GTG GGG GGC GTG GAG CAC AGG TTC GAA GCC 910 Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His Arg Phe Glu Ala 290 295 300 GCA TGC AAT TGG ACT CGA GGA GAG CGT TGT GAC TTG GAG GAC AGG GAT 958 Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp 305 310 315 AGA TCA GAG CTT AGC CCG CTG CTG CTG TCT ACA ACA GGT GAT CGA GGG 1006 Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gly Asp Arg Gly 320 325 330 335 *.CAG ACA CCA TCA CCA CCA TCA CTA AT AG 1034 Gin Thr Pro Ser Pro Pro Ser Leu 340 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 343 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ile Pro Gin Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val 1 5 10 Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val 25 Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg Val 40 Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu Val Ser Leu Phe 55 Ser Pro Gly Ser Ala Gin Lys Ile Gin Leu Val Asn Thr Asn Gly Ser 70 75 Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser Leu Gin Thr 90 Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn Ser Ser Gly 100 105 110 Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys Phe Ala Gln 115 120 125 Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser Asp Gin Arg 130 135 140 Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile'Val Pro Ala 145 150 155 160 117 Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val Ser Gin Val Cys Val1 Asn Asn Cys 225 thr Arg Pro Lys Cys 305 Ser Thr Gly Leu 200 Met Ile Arg Leu Pro 280 Gly Arg Trp Gly 190.

Pro Arg Thr Lys Asn Thr Thr Tyr 255 Val His 270 Thr Ile Glu Ala Arg Asp Leu Ser Thr Thr Gly Asp Arg Gly Gin 330 335 S. 5 C S INFORMATION FOR SEQ ID NO: 41: Wi SEQUENCE CHARACTERISTICS: LENGTH: 945 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .942 (ix) FEATURE: NAME/KEY:- matpeptide LOCATION: 1. .939 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: ATG GTG GOG AAC TOG OCT AAG GTT TTG GTT OTO ATG CTA CTC TTT GCC Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala 1 5 10 GOC OTC GAC 000 CAT ACC COC OTO TCA OGA 000 OCA OCA 0CC TCC OAT Oly Val Asp Oly His Thr Arg Val Ser Oly Oly Ala Ala Ala Set Asp -118 25 ACC AGG GGC CTT GTG TCC CTC TTT AGC CCC GGG TCG GCT CAG AAA ATC 144 Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gin Lys Ile 40 CAG CTC GTA AAC ACC AAC GGC AGT TGG CAC ATC AAC AGG ACT GCC CTIG 192 Gin Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu 55 AAC TGC AAC GAC TCC CTC CAA ACA GGG TTC TTT GCC GCA CTA TTC TAC 240 Asn Cys Asn Asp Ser Leu Gin Thr Gly Phe Phe Ala Ala Leu Phe Tyr 6570 75 AAA CAC AAA TTC AAC TCG TCT GGA TGC CCA GAG CGC TTG GCC AGC TGT 288 Lys His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys 90 *CGC TCC ATC GAC AAG TrC GCT CAG GGG TGG GGT CCC CTC ACT TAC ACT 336 *.:Arg Ser Ile Asp Lys Phe Ala Gin Gly Trp Gly Pro Leu Thr Tyr Thr .100 105 110 *GAG CCT .AAC AGC TCG GAC CAG AGG CCC TAC TGC TGG CAC TAC GCG CCT 384 ***Giu Pro Asn Ser Ser Asp Gin Arg Pro Tyr Cys Trp His Tyr Ala Pro *115 120 125 CGA CCG TGT GGT ATT GTA CCC GCG TCT CAG GTG TGC GGT CCA GTG TAT 432 Arg Pro Cys Gly Ile Val Pro Ala Ser Gin Val Cys Gly Pro Val Tyr 0 V 130 135 .140 TGC TTC ACC CCG AGC CCT GT'r OTG GTG GGO ACG ACC GAT C GG TTT GGT 480 Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly 145 150 155 GTC CCC ACG TAT AAC TGG GOG GCG AAC GAC TCG GAT OTG CTG ATT CTC 528 Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu le Leu 165 170 175 AAC AAC ACG CGG CCG CCG CGA GGC AAC TGG TTC GGC TGT ACA TOG A TG 576 Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met 0* 180 185 190 *.*AAT GGC ACT GGG TTC ACC AAG ACG TGT GGG GGC CCC CCG TGC AAC ATC 624 Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn :ile 195 200 205 GGG GGG GCC GGC AAC AAC ACC TTG ACC TGC CCC ACT GAC TGT TTT CGG 672 Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg 210 215 220 AAG CAC CCC GAG GCC ACC TAC GCC AGA TGC GGT TCT GGG CCC TGG '-TG 720 Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Tro Lau 225 230 235 2410 ACA CCT AGG TGT ATG GTT CAT TAC CCA TAT AGO CTC TGG CAC TAC ZCC 768 Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr =ro 245 250 255 TGC ACT GTC AAC 1-rC ACC ATC T-LC AAG GI T AGG ATG TAC GTG GGG G GC 816 Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly 260 265 270 GTG GAG CAC AGG TTC GAA GCC GCA TGC AAT TGG ACT CGA GGA GAG Z CG:T 864 Val Giu His Ary Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Giu -r g 275 280 285 TOT GAC TT GAG GAC AGO GAT AGA TCA GAG CTT AGC CCC CTG CTG ZCTG 912 Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu -2U 290 295 300 119 TCT ACA ACA GAG TGG CAG AGC TTA ATT AAT TAG Ser Thr Thr Giu Trp Gin Ser Leu Ile Asn 305 310 INFORMATION FOR SEQ ID NO: 42: SEQUENCE CHARACTERISTICS: LENGTH: 314 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: Met Val Gly Asn Trp Ala Lys Vai Leu Val Val Met Leu Leu Phe Ala 1 5 10 Gly Thr Gin Asn Lys Arg Glu Arg Cys 145 Val Asn Asn Gly Lys 225 Thr Cys His Val Thr Ser Asn Lys Ser Ile Ser Asn 165 Pro Phe Asn Ala Met 245 Phe Thr Ser Asn Leu 70 Ser Phe Asp Va1 Pro 150 Trp Pro Thr Asn Thr 230 Val Thr Arg Leu Gly Gin Ser Ala Gin Pro 135 Val Gly Arg Lys Thr 215 Tyr His Ile Ser Ser Trp Gly Cys Gly 105 Pro Ser Val Asn Asn 185 Cys Thr Arg Pro Lys 265 Gly Gly Ile Phe 75 Glu Gly Cys Va1 Thr 155 Ser Phe Gly.

Pro Gly 235 Arg Arg Ala Ala Arg Ala Leu Leu His 125 Gly Asp Val Cys Pro 205 Asp Gly Tro Tyr Arg 285 Ser Lys Ala Phe Ser Tyr Ala Va1 Phe Ile 175 Trp Asn Phe Tro Tyr 255 Gly Asp Ile Leu Tyr CTs Thr Pro Tyr Gly 160 Leu Met Ile ArgU :,eu 240 ±-ro Gly Val Glu His Arg Phe Giu Ala Ala Cys Asn Trp Thr Gl,, Glu A4rg -120 Cys Asp Leu Giu Asp Arg Asp Arg Ser Giu Leu Ser Pro Leu Leu Leu 290 295 300 Ser Thr Thr Giu Trp Gin Ser Leu Ile Asn 305 310 INFORMATION FOR SEQ ID NO: 43: SEQUENCE CHARACTERISTICS: LENGTH: 961 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

S

S S

*SS*

S S (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .958 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1. .955 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: ATO OTO 000 AAC TGG GCT AAG OTT TTG GTT GTO A Met Val Gly Asn Trp Ala Lys Val Leu Val Val M 1 5 10 TG CTA CTC TTT et Leu Leu Phe

GCC

Al a GOC OTC Oly Val ACC AGG Thr Arg CAG CTC Gin Leu AAC TOC Asn Cys AAA CAC Lys His CGC TCC Arg Ser GAG CCT Olu Pro COA CCG Arg Pro 130 GAC 000 Asp Oly GOC CTT Oly Leu OTA AAC Val Asn AAC GAC Asn Asp AAA TTIC Lys Phe ATC GAC Ile Asp 100 AAC AOC Asn Ser 115 TOT GOT Cys Gly

CAT

His

GTG

Val

ACC

Thr

TCC

Ser

AAC

As n

AAG

Lys

TCG

Ser

ATT

Ilie

ACC

Thr

TCC

Ser

AAC

As n

CTC

Leu 70

TCG

Ser Phe

GAC

Asp

OTA

COC

Arg

CTC

Leu

GOC

O ly 55

CAA

Gin

TCT

Ser

OCT

Ala

CAG

Gin

CCC

GTG

Val1

TTT

Phe 40

AGT

Ser

ACA

Thr

GGA

Gly

CAG

Gin

AGO

Arg 120

GCG

TCA OGA Ser Oly 25 AOC CCC Ser Pro TOG CAC Trp His 000 TTC Gly Phe TOC CCA Cys Pro 90 000 TG Oly Trp 105 CCC TAC Pro Tyr TCT CAG Ser Gin G00

GG

Gly

ATC

Ile

TTT

Phe 75

GAG

Giu

GT

Gly

TOGC

Cys

GTO

Val1

OCA

Aia

TCO

Ser

AAC

Asn 0CC Aila

CGC

Arg

CCC

Pro

TG

Trp

TGC

Cys 140 OCA 0CC Ala Ala OCT CAG Ala Gin AGO ACT Arg Thr OCA CTA Ala Leu TTG 0CC Leu Ala CTC ACT Leu Thr 11*0 CAC TAC His Ty r 125 GOT CCA Oly Pro

TCC

Ser

AAA

Lys 0CC Ala

TTC

Phe

AGC

Ser

TAC

Tyr

OCO

Al a

OTO

Val1

GAT

Asp

ATC

ile

CTG

Leu

TAC

Tyr

TOT

Cys

ACT

Thr

CCT

Pro

TAT

Tyr 48 96 192 240 288 336 384 432 480 Val Pro Ala 135 TGC TTC ACC CCO AGC CCT OTT OTO OTOG 000 ACO ACC OAT CGG TTT SGT 121 Cys 145

GTC

Val1

AAC

Asn

AAT

Asn

GGG

Gly

:AAG

.9 Lys 225

ACA

Thr

TGC

Cys 5. GTG Val

*TGT

Cys

TCT

Ser S. 305 .9 TAG Pro

TAT

Tyr

CGG

Arg 180

GGG

G ly

GGC

Gly

GAG

Glu

TGT

Cys

AAC

Asn 260

AGG

Arg

GAG

Glu

GGT

Gly Ser

AAC

As n 165

CCG

Pro

TTC

Phe

AAC

Asn

GCC

Ala

ATG

Met 245

TTC

Phe

TTC

Phe

GAC

Asp

GAT

Asp Pro 150

TG

Trp

CCG

Pro

ACC

Thr

AAC

As n

ACC

Thr 230

GTT

Val

ACC

Thr

GAA

Glu

AGO

Arg

CGA

Arg 310 Val Val Val Gly Thr Thr Asp Arg Phe

GG

Gly

CGA

Arg

AAG

Lys

ACC

Thr 215

TAC

Tyr

CAT

His

ATC

Ile

GCC

Ala

GAT

Asp 295 000 Gly Gly 160

CTC

Leu

ATG

Met

ATC

Ile

CGG

Arg

CTG

Leu 240

CCC

Pro

GGC

Gly

CGT

Arg

CTG

Leu CCA TCA Pro Ser 315 CCA CCA TCA CTA A Pro Pro Ser Leu INFORMATION FOR SEQ ID NO: 44: Met 1 G ly Thr Gin Wi SEQUENCE CHARACTERISTICS: LENGTH: 319 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: Vai Giy Asn Trp Ala Lys Vai Leu Val Val Met Le'i Leu Phe Ala 510 Val Asp Gly His Thr Arg Val Ser Gly Giy Ala Ala Ala Ser Asp 25 Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gin Lys Ile 40 Leu Vai Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu 55 122 Asn Cys Asn Asp Ser Leu Gin Thr Gly Phe 70 0 C *0 *000 *0 S S 000 S *500

S

0* 0 *00*

S

*5*0

S

4 0 .5 S 0* S 0*

S.

Lys Arg Glu Arg Cys 145 Val1 Asn As n Gly Lys 225 Thr Cys Val1 Cys Ser Hi s Ser Pro Pro 130 Phe Pro Asn Gly Gly 210 His Pro Thr G 1u Asp 290 Thr Lys Ile Asn 115 Cys Thr Thr Thr Thr 195 Ala Pro Arg Val1 His 275 Leu Thr Phe Asp 100 Ser Gly Pro Tyr.

Arg 180 Gly Gly Giu Cys Asn 260 Arg Glu Gly.

ksn 85 -,ys Ser Ile Ser Asn 165 Pro Phe Asn Ala Met 245 Phe Phe Asp Asp Ser Phe Asp Val Pro 150 Trp Pro Thr As n Thr 230 Val1 Thr Glu Arg Arg 310 Ser Al a Gin Pro 135 Val1 Gly Arg Lys Thr 215 Tyr His Ile Al a Asp 295 Gly Gin Arg 120 Ala Val Al a Gly Thr 200 Leu Al a Tyr Phe Ala 280 Arg cys Gly 105 Pro Ser Val1 Asn Asn 185 Cys Thr Arg Pro Lys 265 Cys Ser Pro 90 Trp Tyr Gin Gly Asp 170 Trp Gly Cys Cys Tyr 250 Val As n G lu Pro Phe Glu Giy Cys Val1 Thr 155 Ser Phe Gly Pro Gly 235 Arg Arg *Trp *Leu Ser 315 Arg Pro Trp Cys 140 Thr Asp G ly Pro Thr 220 Ser Leu Met Thr Ser 300 Pro Leu Ala Leu Thr 110 His Tyr 125 Gly Pro Asp Arg Val Leu Cys Thr 190 Pro Cys 205 ASP Cys Gly Pro Trp His Tyr Val 270 Arg Gly 285 Pro Leu Pro Ser Ser T'yr Ala Val Phe Ile 175 Trp Asn Phe Trp.

Tyr 255 Gly Glu Leu Leu -ys rhr Pro Tyr Gly 160 Leu Met Ile Ax g Leu 240 Pro Gly Arg Leu Ala Ala Leu Phe Gly Gln Thr INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1395 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .1392 (ix) FEATURE: NAME/KEY: mat peptide -123 LOCATION: 1. .1389 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATG GTG GCG GGG GCC Met Val Ala Gly Ala CAT TGG GGA GTC CTG His Trp Gly Val Leu GCG GGC CTC Ala Gly Leu GCC TAC TAT Ala Tyr Tyr *0 ~q

S

S

S S

S.

@55550

S

OS

550e 0

OS

S

550 S .555 60 0@ S *5O* 50 0@ 0 005S 0 0 505050 5 00

S

*5 0 00 @0 1

TCC

Ser 0CC Al a

GAT

Asp

ATC

Ile 65

CTG

Leu

TAC

Tyr

TOT

Cys

ACT

Thr

CCT

Pro 145

TAT

Tyr

GOT

Gly

CTC

Leu

ATG

Met

ATC

Ile 225

CGG

Arg

GG

Gly 20

GAC

Asp

GGC

Gly

GTA

Val

AAC

Asn

AAA

Lys 100

ATC

Ile

AAC

Asn

TGT

Cys

ACC

Thr

ACG

Thr 180

ACG

Thr

ACT

Thr 0CC Ala

CCC

Pro 5

AAC

Asn

GG

Gly

CTT

Leu

AAC

Asn

GAC

Asp

TTC

Phe

GAC

Asp

AGC

S er

GGT

Gly

CCG

Pro 165

TAT

Tyr

CG

Arg 000 Oly

GOC

Oly

GAG

Olu 245

TOO

Trp

CAT

His

OTG

Val

ACC

Thr 70

TCC

Ser

AAC

As n

AAG

Lys

TCG

Ser

ATT

Ile 150

AOC

Ser

AAC

As n

CCO

Pro

TTC

Phe

AAC

Asn 230 0CC Al a

OCT

Al a

ACC

Thr

TCC

Ser 55

AAC

As n

CTC

Leu

TCO

Ser

'FTC

Phe

GAC

Asp 135

OTA

Val

CCT

Pro

TOO

Trp

CCG

Pro

ACC

Thr 215

AAC

Asn

ACC

Thr

AAG

Lys

COC

Arg 40

CTC

Leu

GGC

Gly

CAA

Gin

TCT

Ser

OCT

Al a 120

CAG

Gin

CCC

Pro OTr Val1 000 O ly

COA

Arg 200

AAG

Lys

ACC

Thr

TAC

Tyr 10 Leu

TCA

Ser

AOC

Ser

TOO

Trp 000 Gly 90

TOC

Cys 000 Gly

CCC

Pro

TCT

Ser

OTO

Val1 170

AAC

Asn

AAC

Asn

TOT

Cys

ACC

Thr

AGA

Arg 250

GTO

Val1 000 O ly 000 Gly

ATC

Ile

TTT

Phe

GAG

Oiu

GOT

Oly

TOC

Cys 140

GTG

Val1

ACO

Thr

TCO

Ser

TTC

Phe

GOC

Oly 220

CCC

Pro

GT

Gly

ATO

Met

OCA

Al a

TCO

Ser

AAC

As n 0CC Ala

COC

Arg

CCC

Pro 125

TGO

Trp

TOC

Cys

ACC

Thr

OAT

Asp

GOC

Oly 205

CCC

Pro

ACT

Thr

TCT

Ser

CTC

Leu 0CC Ala

CAG

Gln

ACT

Thr

CTA

Leu 0CC Ala

ACT

Thr

TAC

Tyr

CCA

Pro

COO

Arg 17,5

CTG

Leu

ACA

Thr

TOC

Cys

TOT

Cys

CCC

Pro 255 720 124

CTG

Leu

CCC

Pro

GGC

Gly

CGT

Arg 305

CTG

Leu

**.*CCG

j Pro

GTG

Val1

.AAA

Lys

ATC

385 Ala

CAT

His

:AAG

Lys

CCG

Pro TAC CCA TAT Tyr Pro Tyr 265 TTC AAG GT Phe Lys Val GCA TOC AAT Ala Cys Asn AGA TCA GAG Arg Ser Glu 315 CTG CCC TGT Leu Pro Cys 330 CAC CTC CAT His Leu His 345 TCG GCG GTT Ser Ala Val TTC C IT CTC Phe Leu Leu CTG CTG ATA Leu Leu Ile 395 AAT GCG GCG Asn Ala Ala 410 TTC TTC TGT Phe Phe Cys 425 GCA. TAC GCC Ala Tyr Ala

AGG

Arg

AGG

Arg

TGG

Trp 300

CTT

Leu

TCC

Ser

CAG

Glri

GTC

Val1

CTG

Leu 380

GCT

Al a

GCC

Ala

GCT

Al a

TTC

Phe 816 864 912 960 1008 1056 110 4 1152 1200 1248 1296 -395

TTA

Leu CCA CGA GCT TAT GCC TAGTAA Pro Arg 460 Ala Tyr Ala INFORMATION FOR SEQ ID NO: 46: SEQUENCE CHARACTERISTICS: LENGTH: 463 amino acids TYPE: amino acid TOPOLOGY: linear (iiJ) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: Met Val Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr iY-r 1 5 10 1 Ser Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu ?he 25 3C 125 Val Ser Gly Ala Asp Ile Leu Tyr Cys Thr Pro 145 Tyr G ly Leu Met Ile 225 Arg Leu Pro Gly Arg 305 Leu Pro Val1 Lys Gly Thr Gin Asn Lys Arg G iu 130 Arg Cys Val Asn Asn 210 G ly Lys Thr Cys Val1 290 Cys Ser Al a Gin Trp 370 Val Arg Leu Cys His Ser 115 Pro Pro Phe Pro Asn 195 Gly Gly His Pro Thr 275 Glu Asp Thr Leu Tyr 355 G iu Asp Gly Vai Asn Lys 100 Ile Asn Cys Thr Thr 180 Thr Thr Ala Pro Arg 260 Val1 His Leu Thr Ser 340 Leu Tyr

G

1 y Leu As n Asp Phe Asp S-er Gly Pro 165 Tyr Arg Gly Gly Giu 245 Cys As n Arg Glu Glu 325 Thr Tyr Val1 His Val Thr 70 Ser Asn Lys Ser Ile 150 Ser As n Pro Phe Asn 230 Al a Met Phe Phe Asp Trp Gly G ly Leu Thr Ser 55 Asn Leu Ser Phe Asp 135 Val1 Pro Trp Pro Thr 215 Asri Thr Val1 Thr Giu 295 Arg Gin Leu Val1 Leu 375 Arg 40 Leu Gly Gln Ser Al a 120 Gin Pro Val1 Gly Arg 200 Lys Thr Tyr His Ile 280 Ala Asp Ile Ile G ly 360 Leu Phe Ser Thr Gly 105 Gin Arg Ala Val Ala 185 Gly Thr Leu Ala Tyr- 265 Phe Ala Arg Leu His 345 Ser Phe Ser Trp Gly 90 Cys Gly Pro Ser Val1 170 As n Asn Cys Thr Arg 250 Pro Lys Cys Ser Pro 330 Leu Ala Leu Pro His 75 Phe Pro Tro Tyr Gin 155 Gly Asp Trp, Gly Cys 235 Cys Tyr Val1 Asn Glu 315 Cys His Val1 Leu Gly Giy Ile Phe Giu Gly Cys 140 Val1 Thr Ser Phe G ly 220 Pro Gly Arg Arg Trp 300 Leu Ser Gin Val Leu 380 Ala Ser Asn Aila Arg Pro 125 Trp Cys Thr Aso G iy 205 Pro Thr Ser Leu Met 285 Thr Ser Phe As n Ser 365 Ala Al a Ala Arg Ala Leu 110 Leu His Gly Asp Val1 190 Cys Pro Asp Giy Trp 270 Tyr Arg Pro Thr Ile 350 Leu Asp Ala Gin Thr Leu Ala Thr Tyr Pro Arg 175 Leu Thr Cys Cys Pro 255 His Val1 G ly Leu Thr 335 Val1 Val1 Ala Ser Lys Ala Phe Ser Tyr Ala Val1 160 Phe Ile Trp, As n Phe 240 Tro Tyr Gly Glu Leu 320.

Leu Asp Ile Arg Ile Cys Ala Cys Leu Trp 390 Met Met Leu Leu Ile Ala Gin Ala Giu Aia 395 400 126 Ala Leu Glu Asn Leu Val Val Leu Asn Ala Ala Ala Val Ala Gly Ala 405 410 415 His Gly Thr Leu Ser Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile 420 425 430 Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp 435 440 445 Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala 450 455 460 INFORMATION FOR SEQ ID NO: 47: SEQUENCE CHARACTERISTICS: LENGTH: 2082 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .2079 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1. .2076 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: *AAT TTG GGT AAG GTC ATC GAT ACC C'1r ACA TGC GGC TTC GCC GAC CTC 48 Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu 1 5 10 *GTG GOG TAC ATr CCG CTC GTC GGC GCC CCC CTA GGG GGC GCT GCC AGG 96 **:Val Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Ar g 25 GCC CTG GCG CAT GGC GTC CGG GTIT CTG GAG GAC GGC GTG AAC TAT GCA 144 Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala 40 ACA GGG AAT TTG CCC GGT TGC TCT TTC TCT ATC TTC CTC TTG GCT 7.G 192 Thr Gly Asn Leu Pro Gly Cys Ser ?he Ser Ile Phe Leu Leu Ala Leu 55 CTG TCC TGT CTG ACC G TT CCA GCT TCC GCT TAT GAA GTG CGC AAC GTG 240 Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val 70 75 TCC GGG ATG TAC CAT GTC ACG AAC GAC TGC TCC AAC TCA AGC ATT GTG 288 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val 90 TAT GAG GCA GCG GAC ATG ATC ATG CAC ACC CCC GGG TGC GTG CCC T.Gc 336 Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys Val Pro Cys 100 105 110 GTT CGG GAG AAC AAC TCT TCC CGC *TGC TGG GTA GCG CTC ACC CCC ACG 384 Val Arg Glu Asri Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro 7hr 127 125 B. B B B

AAC

As n

GTT

Val1

GGA

Gly 165

CAT

His

ACG

Thr

ACG

Thr

GAC

Asp

TAT

Tyr 245 Phe

TCC

Ser

AAA

Lys

GCC

Al a TrC Phe 325

AGC

Ser

TAC

Tyr

GCG

Al a

GCC

Ala

GGG

Giy 150

TCT

Ser

GAG

Glu

GGT

Gly

GCC

Ala

ATG

Met 230

TCC

Ser

GCC

Ala

GAT

Asp

ATC

Ile

CTG

Leu 310

TAC

Tyr

TGT

Cys

ACT

Thr

CCT

Pro

AGC

Ser 135

GCG

Al a

GTC

Val1

ACG

Thr

CAC

His

CTG

Leu 215

GTG

Val1

ATG

Met

GGC

Giy

ACC

Thr

CAG

Gin 295

AAC

Asn

AAA

Lys

CGC

Arg

GAG

Giu

CGA

Arg 375 ACA ATA Thr Ile 140 TCC GCT Ser Ala CAG CTG Gin Leu AAT TGC Asn Cys GAT ATG Asp Met 205 CTG CTC Leu Leu 220 TGG GGA Trp Gly GCT AAG Ala Lys ACC CGC Thr Ar g TCC CTC Ser Leu 285 AAC GGC Asn Gly 300 CTC CAA Leu Gin TCG TCT Ser Ser TTC GCT Phe Ala GAC CAG Asp Gin 365 GTA CCC Val Pro 380 CGA CC Arg Arg ATG TAC Met TPyr TTC ACC Phe Thr 175 TCA ATC Ser Ile 190 ATG ATG met met CGG ATC Arg Ile GTC CTG Val Leu GTT TTG Val Leu 255 GTG TCA Val Ser 270 TTT AGC Phe Ser AGT TGG Ser Trp ACA CCC Thr Cly GCA TGC Cly Cys 335 CAG CCC Gin Cly 350 AGG CCC Arg Pro C TCT Ala Ser

CAC

His

GTG

Val1 160

ATC

Ile

TAT

Tyr

AAC

Asn

CCA

Pro

GCG

Al a 240

GTT

Val1

GGA

Gly

CCC

Pro

CAC

H is

=-TC

Phe 320

CCA

Pro

TCGG

Tr o

TAC

Tvr

CAG

Gin 432 480 5 28 576 624 672 720 768 816 86 4 912 960 1008 1056 12.04 1152 1200 CTC TCC GGT CCA GTG TAT TGC TTC ACC CCC ACC CCT Val Cys Gly Pro Val Cys Phe Thr Pro Ser Pro OTT GTG GTG GG Val Val Val Clv 40-0 128 a a.

*4 *aaa..

a. a a ACG ACC GAT CGG Thr Thr Asp Arg TCG GAT GTG CTG Ser Asp Val Leu 420 TTC GGC TGT ACA Phe Gly Cys Thr 435 GGC CCC CCG TGC Gly Pro Pro Cys 450 CCC ACT GAC TGT Pro Thr Asp Cys 465 GGT T :T GGG CCC Gly Ser Gly Pro AGG CTC TGG CAC Arg Leu Trp His 500 AGG ATG TAC GTG Arg Met Tyr Val 515 TGG ACT CGA GGA Trp Thr Arg Gly 530 Cr1' AGC CCG CTG Leu Ser Pro Leu 545 TCC TTC ACC ACC Ser Phe Thr Thr CAG AAC ATC GTG Gin Asn Ile Vai 580 GTQ TCC CTT GTC Val Ser Leu Val 595 CTG GCA GAC GCG Leu Ala Asp Ala 610 GCT CAA GCT GAG Ala Gin Ala Giu 625 GCC GTG GCC GGG Ala Vai Ala Gly GCT GCC TGG TAC Ala Ala Trp Tyr TTr GGT GTC CCC ACG Phe Gly Val Pro Thr 405 ArT' CTC AAC AAC ACG Ile Leu Asn Asn Thr 425 TOG ATG AAT GGC ACT Trp Met Asn Gly Thr 440 AAC ATC GGG GGG GCC Asn Ile Gly Giy Ala 455 TTT CGG AAG CAC CCC Phe Arg Lys His Pro 470 TGG CTG ACA CCT AGG Trp Leu Thr Pro Arg 4 85 TAC CCC TGC ACT GTC Tyr Pro Cys Thr Vai 505 GGG GGC GTG GAG CAC Gly Gly Val Giu His 520 GAG CGT TGT GAC TTG Giu Arg Cys Asp Leu 535 CTG CTG TCT ACA ACA Leu Leu Ser Thr Thr 550 CTG CCG GCC CTA TCC Leu Pro Ala Leu Ser 565 GAC GTG CAA TAC CTG Asp Val Gin Tyr Leu 585 ATC AAA TGG GAG TAT Ile Lys Trp Glu Tyr 600 CGC ATC TGC GCC TGC Arg Ile Cys Ala Cys 615 GCC GCC TTA GAG AAC Ala Ala Leu Glu Asn 630 GCG CAT GGC ACT CTT Ala His Gly Thr Leu 645 ATC AAG GGC AGG CTG Ile Lys Gly Arg Leu 665 Tyr 410

CGG

Arg

GGG

G ly

GGC

Gly

GAG

Glu

TGT

Cys 490

AAC

As n

AGG

Arg

GAG

Giu

GAG

Glu

ACC

Thr 570

TAC

Tyr

GTC

Val1

TTA

Leu

CTG

Leu

TCC

Ser 650

GTC

Va I Asri Trp Gly Ala Asn Asp 415 CCG CCG CGA GGC AAC TGG Pro Pro Arg Gly Asn Trp 430 TTC ACC AAG ACG TGT GGG Phe Thr Lys Thr Cys Gly 445 AAC AAC ACC TTG ACC TGC Asn Asn Thr Leu Thr Cys 460 GCC ACC TAC GCC AGA TGC Ala Thr Tyr Ala Arg Cys 475 480 ATG GTT CAT TAC CCA TAT Met Val His Tyr Pro Tyr 495 TTC ACC ATC TTC AAG GTT Phe Thr Ile Phe Lys Val 510 TTC GAA GCC GCA TGC AAT Phe Giu Ala Ala Cys Asn 525 GAC AGG GAT AGA TCA GAG Asp Arg Asp Ara Ser Giu 540 TGG CAG ATA CTG CCC TGT Trp Gin Ile Leu Pro Cys 555 560 GGC CTG ATC CAC CTC CAT Giy Leu Ile His Leu His 575 GGT GTA GGG TCG GCG GTT Gly Val Gly Ser Ala Val 590 CTG TTG CTC TTC CTT CTC Leu Leu Leu Phe Leu Leu 605 TGG ATG ATOG CTG CTG ATA Trp Met Me: Leu Leu 1 620 GTG GTC CTC AAT GCG GCG Val Val Leu Asn Ala Ala 635 640 'rrC CTT GTG TTC TTrc TGT Phe Leu Val Phe Phe Cys 655 CCT GGT GCG GCA TAC GCC Pro Gly Ala Ala Tyr Ala 670 TAT AAC TGG GGG GCG AAC GAC 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 19-58 2016 2064 TTC TAT GGC GTG TGG CCG CTG CTC CTG CTT CTG CTG GCC TTA CCA CCA -129 Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro 675 680 685 CGA GCT TAT GCC TAGTAA Arg Ala Tyr Ala 690 INFORMATION FOR SEQ ID NO: 48: SEQUENCE CHARACTERISTICS: LENGTH: 692 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: 2082

S

S.

Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala I le His Leu Leu Tyr Ala 100 As n Arg Leu Cys Arg 180 Ile Thr Val1 Tyr Pro Gly Pro Thr His Asp Asn As n Val1 Gly 165 His Thr Thr Asp Tyr 245 Pro Glu Ser Ala Cys 90 Thr Trp Thr Phe Val1 170 Asp Ala Ser Ala As n 250 Leu Asp Ile Tyr Ser Pro Val1 Thr Cys 155 Ser Cys Trrp Gin His 235 Trp Gly Val1 Leu Val1 Ser Cys Leu 125 Ile Al a Leu Cys Me- 205 Leu GlIy Lvs Asp Leu Ala Arg Tlyr Ala Ala Leu Asn Val Ile Val Pro Cys Pro Th r Arg His Ty r Val Thr 11ie 175 Ile Tv r Met Asn le Pro Leu Ala 240 Leu Val 255 Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg Val. Ser zl~y 265 270 130 Gly Ala Ala Ala Ser Asp 275 S. a

S

Gly Ile 305 Phe Glu Gly ys Val 385 Thr Ser Phe Gly Pro 465 Gly Arg Arg Trp Leu 545 Ser Gin Val Leu er ksn kla krg Pro Trp 370 Cys rhr Asp Gly Pro 450 Thr Ser Leu Met Thr 530 Ser Phe Asn Ser Ale 610 Ala C Arg Ala Leu Leu 355 His Gly Asp.

Val Cys 435 Pro Aso Gly Trp Tyr 515 Arg Pro Thr Ile Leu 595 Asp 31n rhr Leu kla 340 rhr Tyr Pro Arg Leu 420 Thr Cys Cys Pro His 500 Val Gly Leu Thr Val 580 Val Ala Lys Ala Phe 325 Ser Tyr Ala Va1 Phe 405 Ile Trp Asn Phe Trp 485 Tyr Gly Glu Leu Leu 565 Asp Ile Arg Ile Leu 310 Tyr Cys Thr Pro Tyr 390 Gly Leu Met Ile Arg 470 Leu Pro Gly Arg Leu 550 Pro Va1 Lys Ile Thr Arg 280 Gin Leu 295 Asn Cys Lys His Arg Ser Glu Pro 360 Arg Pro 375 Cys Phe Val Pro Asn Asn Asn Gly 440 Gly Gly 455 Lys His Thr Pro Cys Thr Val Glu 520 Cys Asp 535 Ser Thr Ala Leu Gin Tyr Trp Glu 600 Cys Ala 615 Va1 Asn Lys Ile 345 Asn Cys Thr Thr Thr 425 Thr Ala Pro Arg Val 505 His Leu Thr Ser Leu 585 Tyr Cys Asn Asp Phe 330 Asp Ser Gly Pro Tyr 410 Arg Gly Gly Glu Cys 490 Asn Arg Glu Glu Thr 570 Tyr Val Leu Gly Leu Vai Ser Leu Phe Ser Pro Thr Ser 315 Asn Lys Ser Ile Ser 395 Asn Pro Phe Asn Ala 475 Met Phe Phe Asp Trp 555 Gly Gly Leu Tr- Asn 300 Leu Ser Phe Asp Val 380 Pro Trp Pro Thr Asn 460 Thr Val Thr Glu Arg 540 Gin Leu Va1 Leu Met 620 285 Gly Gin Ser Ala Gin 365 Pro Va1 Gly Arg Lys 445 Thr Tyr His Ile Ala 525 Asp Ile Ile Gly Leu 605 Met Ser Thr Gly Gin 350 Arg Ala Va1 Ala Gly 430 Thr Leu Ala Tyr Phe 510 Ala Arg Leu His Ser 590 Phe Leu Trp Gly Cys 335 Gly Pro Ser Val Asn 415 Asn Cys Thr Arg Pro 495 Lys Cys Ser Pro Leu 575 Ala Leu Leu His Phe 320 Pro Trp Tyr Gin Gly 400 Asp Trp Gly ys Cys 480 Tyr Val Asn Glu Cys 560 His Val Leu Ile Gln Ala Glu Ala Leu Glu Asn Leu Val Leu Asn Ala 131 Ala Val Ala Gly Ala His Gly Thr Leu Ser Phe Leu Val Phe Phe Cys 645 650 655 Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro Gly Ala Ala Tyr Ala 660 665 670 Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro 675 680 685 Arg Ala Tyr Ala 690 INFORMATION FOR SEQ ID NO: 49: SEQUENCE CHARACTERISTICS: LENGTH: 2433 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: 0 NAME/KEY: CDS LOCATION: 1..2430 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1..2427 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: ATG AGC ACG AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC ACC AAC 48 Met Ser Thr Asn Pro Lys Pro Gin Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 CGC CGC CCA CAG GAC GTC AAG TTC CCG GGC GGT GGT CAG ATC GTT GOT 96 Arg Arg Pro Gin Asp Val Lys Phe Pro Gly Gly Gly Gin Ile Val Gly 20 25 GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG CGC GCG 144 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 40 ACT AGG AAG ACT TCC GAG CGG TCG CAA CCT CGT GGG AGG CGA CAA CCT 192 Thr Arg Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly Arg Arg Gin Pro 55 ATC CCC AAG GCT CGC CGA CCC GAG GGT AGG GCC TGG GCT CAG CCC GGG 240 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gin Pro Gly 70 75 TAC CCT TOOGG CCC CTC TAT GGC AAT GAG GGC ATG GGG TOOGG GCA GGA TGG 288 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp Ala Gly Trp 90 CTC CTG TCA CCC CGC GGC TCT CGG CCT AOT TGG GGC CCT ACA GAC CCC 336 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC GAT ACC CTT ACA TOC 384 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cvys 115 120 125 132 GGC TTC Gly Phe 130 GCC GAC CTC GTG GGG TAC ATr Ala Asp Leu Val Gly Tyr Ile 135 CCG CTC GTC GGC GCC CCC CTA Pro Leu Val Gly Ala Pro Leu 140 432 a.

a.

a a a. a a a GGG GGC C Gly Gly 1 145 GGC GTG Gly Val 'rC C'rc Phe Leu GAA GTG Glu Val AAC TCA Asn Ser 210 GGG TGC Gly Cys 225 GCG CTC Ala Leu ACA ATA Thr Ile TCC GCT Ser Ala CAG CTG Gln Leu 290 AAT TGC Asn Cys 305 GAT ATG Asp Met CTG CTC Leu Leu TGG GGA Trp Gly GCT AAG Ala Lys 370 ACC CGC Thr Arg

;CT

kAC ksn .eu

CGC

krg 1 95 kGC Ser

GTG

Val1

ACC

Thr

CGA

Arg

ATG

Met 275 Phe

TCA

S er

ATG

Met

CGG

Arg

GTC

Val1 355

GTI

Val1

GTC

ValI

GCC

Ala

TATC

Tyr

GCT

Ala 180

AAC

Asn Ile

CCC

Pro

CCC

Pro

CGC

Arg 260

TAC

Tyr

ACC

Thr

ATC

Ile

ATG

Met

ATC

Ile 340

CTG

Leu TT7G Leu

TCA

*Ser k GG !Lrg

;CA

kla 165 rT G Leu

GTG

Val1

GTG

Val

I'GC

cys

ACG

Thr 245

C.AC

His

GTG

Val

ATC

Ile

TAT

Tyr

AAC

Asn 325

CCA

Pro

GCG

Ala

GTT

Vai

GGA

Gly

GCC(

Ala I IS0

ACAC

Thr

CTG

Leu

TCC

Ser

TAT

Tyr GTr Val 230

CTC

Leu

GTC

Val

GGG

Gly

TCG

Ser

CCC

Pro 310

TGG

Trp

CAA

Gin

GGC

Gly

*GTG

Val1

GGG

*Gly 390

.TG

~eu

;GG

;ly 3er

~GG

.Ay

GAG

Glu 215

CGG

Arg

GCA

Ala

GAT

Asp

GAC

Asp

CCT

Pro 295

GGC

Gly

TCG

Ser

GCT

Al a

CTC

Leu

ATG

Met 375

GCA

Ala GCG CAT Ala His AAT TTG Asn Leu TGT CTG Cys Leu 185 ATG TAC Met Tyr 200 GCA GCG Ala Ala GAG AAC.

Glu Asn GCT AGG Ala Arg I-rG CTC Leu Leu 265 CTC TGC Leu Cys 280 CGC CGG Ar g Ar g CAC ATA His Ile CCT ACA Pro Thr GTC GTG Val Val 345 GCC TAC Ala Tyr 360 CTA CTC Leu Leu GCA GCC Ala Ala

;GC

Ily

CCC

Pro 170

ACC

rhr

CAT

H~is

GAC

Asp

AAC

Asn

AAC

Asn 250

GTT

Val1

GGA

G ly

CAT

His

ACG

Thr

ACG

Thr 330

GAC

Asp

TAT

Tyr

TTT

Phe

GTC

Val1 155

GGT

G ly

GTT

Val

GTC

Val1

ATG

Met

TCT

Ser 235

GCC

Al a

GGG

G ly

TCT

Ser

GAG

Glu

GGT

Gly 315

GCC

Ala

ATG

Met

TCC

Ser

GCC

Al a CGG C Arg N TGC I) Cys

CCAC

Pro

ACG

Thr

ATC

Ile 220

TCC

Ser

AGC

Ser

GCG

Ala.

GTC

Val1

ACG

Thr 300

CAC

His

CTG

Leu

GTG

Val1

ATG

Met

GGC

Gly 380 ,TT CTG GAG GAC Pal Leu Glu Asp 160 ~CT TTC TCT ATC ;er Phe Ser Ile 175 3CT TCC GCT TAT kla Ser Ala Tyr 190 kIAC GAC TGC TCC %sn Asp Cys Ser 205 k.TG CAC ACC CCC 41et His Thr Pro CGC TGC TGG GTA Arg Cys Trp Val 240 GTC CCC ACC ACG Val Pro Thr Thr 255 GCT GCT TTC TGT Ala Ala Phe Cys 270 TTC CTC GTC TCC Phe Leu Val Ser 285 GTG CAG GAC TGC Val Gln Asp Cys CGT ATG GCT TGG Arg Met Ala Trp 320 GTG GTA TCG CAG Val Val Ser Gin 335 GCG GGG GCC CAT Ala Gly Ala His 350 GTG GGG AAC TGG Val Gly Asn Trp 365 GTC GAC GGG CAT Val Asp Gly !-is AGG GGC CTT GTG Arg Gly Leu Val 480 528 576 624 672- 720 768 816 864 912 960 1008 1056 1104 1152 1200 1243 TCC GAT ACC Ser Asp Thr 395 TCC CTC ri-r AGC CCC GGG TCG GCT CAG AAA ATC CAG C-1C GTA AAC ACC 133- Ser Leu Phe Ser Pro Gly Ser Ala Gin LysIle Gin Leu Val ASrI Thr 405 410 415 AAC GGC AGT TGG CAC ATC AAC AGG ACT GCC CTG AAC TGC AAC GAC TCC 1296 Asri Gly Ser Trp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp Ser 420 425 430 CTC CAA ACA GGG TTC TTT GCC GCA CTA 'rrC TAC AAA CAC AAA TTC AAC 1344 Leu Gin Thr Gly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe Asn 435 440 445 TCG TCT GGA TGC CCA GAG CGC TrG GCC AGC TGT CGC TCC ATC GAC AAG 1392 Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp Lys 450 455 460 TTC GCT CAG GGG TGG 0 -T CCC CTC ACT TAC ACT GAG CCT AAC AGC TCG 1440 Phe Ala Gin Gly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser Ser 465 470 475 480 GAC CAG AGG CCC TAC TGC TGG CAC TAC GCG CCT CGA CCG TGT GGT AIT 1488 Asp Gin Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly Ile *485 490 495 GTA CCC GCG TCT CAG GIG TGC GGT CCA GTG TAT TGC TTC ACC CCG AGC 1536 Val Pro Ala Ser Gin Val Cys Giy Pro Val Tyr Cys Phe Thr Pro Ser 500 505 510 CCT GTT GTG GTG GGG ACG ACC GAT CGG Trr GGT GTC CCC ACG TAT AAC .1584 Pro Val Val Val Gly Thr Thr Asp Arg Phe Giy Val Pro Thr Tyr Asn 515 520 525 *TGG GGG GCG AAC GAC TCG GAT GIG CTG AIT CTC AAC AAC ACG CGG CCG 1632 *Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro 530 535 540 CCG CGA GGC AAC TGG TIC GGC TGT ACA TGG ATG AAT GGC ACT GGG TTC 1680 Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Gly Thr Gly Phe 545 550 555 560 *ACC AAG ACG TGT GGG GGC CCC CCG TGC AAC ATC GGG GGG GCC GGC AAC 1728 Thr Lys Ihr Cys Giy Gly Pro Pro Cys Asn Ile Giy Giy Ala Gly Asn *::565 570 575 *.AAC ACC TTG ACC TGC C CC ACT GAC TGT TT CGG AAG CAC CCC GAG GCC 1776 .Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys Hi-Js Pro Giu Ala 580 585 590 ACC TAC GCC AGA TGC GGT TCT GGG C CC TGG CIG ACA CCT AGG TGT ATG 1824 Ih.r Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg Cys Met 595 600 605 GTT CAT TAC CCA TAT AGG CTC TGG CAC TAC CCC TGC ACT GTC AAC 1C 1872 Val His Tyr Pro Tyr Ar-g Leu Trp His Tyr Pro Cys Th r Val Asn Pne 610 615 620 ACC AIC TTC AAG GTT AGG ATG TAC GTG GGG GGC GIG GAG CAC AGG 117C 1920 Ihr'Ile Phe Lys Val Arg Met Tyr Val Gly Giy Val Glu His Arg ?he 625 630 635 640 GAA GCC GCA TGC AAT TGG ACT CGA GGA GAG CGT TGT GAC TTG GAG GAC 1968 Giu Ala Ala Cys Asn Trp Thr Arg Gly Giu Arg Cys Asp Leu Giu Asp 645 650 655 AGG GAT AGA TCA GAG CIT AGC CCG CTG CTG CTG TCT ACA ACA GAG TGG 2016 Arg Asp Arg Ser Giu Leu Ser Pro Leu Leu Leu Ser Ihr Thr Giu Trp 660 665 670 CAG ATA CTG CCC TGT ICC ITC ACC ACC CTG CCG GCC CTA TCC ACC GGC 2064 Gin Ile Leu Pro Cys Ser Phe Thr Ihr Leu Pro Ala Leu Ser Thr Gly 134 675 680 685 CTG ATC CAC CTC CAT CAG AAC ATC GTG GAC GTG CAA TAC CTG TAC GGT 2112 Leu Ile His Leu His Gin Asn Ile Val Asp Val Gin Tyr Leu Tyr Gly 690 695 700 GTA GGG TCG GCG G'rr GTC TCC CTT GTC ATC AAA TGG GAG TAT GTC CTG 2160 Val Gly Ser Ala Val Val Ser Leu Val Ile Lys Trp, Giu Tyr Val Leu 705 710 715 720 TTG CTC TTC CTT CTC CTG GCA GAC GCG CGC ATC TGC GCC TGC TTA TGG 2208 Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Ile Cys Ala Cys Leu Trp 725 730 735 ATG ATG CTG CTG ATA GCT CAA GCT GAG GCC GCC TTA GAG AAC CTG GTG 2256 Met Met Leu Leu Ile Ala Gin Ala Glu Ala Ala Leu Glu Asn Leu Val 740 745 750 GTC CTC AAT GCG GCG GCC GTG GCC GGG GCG CAT GGC ACT CTT TCC TTC 2304 Val Leu Asn Ala Ala Ala Val Ala Gly Ala His Gly Thr Leu Ser Phe .:755 760 765 GTG T!'C ITC TGT GCT GCC TGG TAC ATC AAG GGC AGG CTG GTC.CCT 2352 *.Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys Gly Arg Leu Val Pro GT770 775 780 *Gly GCG GCA TAC GCC TTC TAT GGC GTG TGG CCG CTG CTC CTG CTT CTG 2400 GyAla Ala Tyr Ala Phe Tyr Gly Val Trp Pro Leu Leu Leu Leu Leu 785 790 795 B00 CTG GCC ITrA CCA CCA CGA GCT TAT GCC TAGTAA 2433 Leu Ala Leu Pro Pro Arg Ala Tyr Ala 805 810 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 809 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ Ib NO: Met Ser Thr Asn Pro Lys Pro Gin Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 Arg Ar-g Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gin Ile Val Gly 25 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 40 Thr Arg Lys Thr Ser Giu Arg Ser Gin Pro Ara Gly Arg A-rg Gin Pro 55 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gin Pro G2.y 70 75 TrPro Trp Pro Leu Tyr Gly Asn Giu Gly Met Gly Trp Ala Gly Trp 90 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro 100 105 110 Ar4 Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 135 a a a a

S

a.

*0 a. a a a Phe Ala 130 Gly Ala Val Asn Leu Leu Val Arg 195 Ser Ser 210 -Cys Val Leu Thr Ile Arg Ala Met 275 Leu Phe 290 Cys Ser Met Met Leu Arg Gly Val 355 Lys Val 370 Arg Val Leu Phe Gly Ser Gin Thr 435 Ser Gly 450 Ala Gin Leu Val Gly 135 Arg Ala Leu 150 Ala Thr Gly 165 Leu Leu Ser Val Ser Gly Val Tyr Glu 215 Cys Val Arg 230 Thr Leu Ala 245 His Val Asp Val Gly Asp Ile Ser Pro 295 Tyr Pro Gly 310 Asn Trp Ser 325 Pro Gin Ala Ala Gly Leu Val Val Met 375 Gly Gly Ala 390 Pro Gly Ser 405 His Ile Asn Phe Phe Ala Pro Glu Arg 455 Trp Gly Pro 470 Al a Asn Cys Met 200 Ala Glu Ala Leu Leu 280 Arg His Pro Val1 Ala 360 Leu Ala Al a Arg Al a 440 Leu Leu Val1 155 Gly Val1 Val Met Ser 235 Ala Gly Ser Glu Gly 315 Ala Met Ser Ala Asp 395 Ile Leu Tyr Cys Thr 475 Val1 Ser Ala Asn 205 Met Arg Val1 Al a Phe 285 Val1 Ara Val1 Al a Val1 365 Val.

Arg Leu Cys His 445 Ser Pro Tyr Ile Pro Leu Val Gly Ala Pro Leu Asp Gin Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys 485 490 Gly Ile 495 136 Val Pro Ala Ser Gin Val Cys Giy Pro Val Ty r Cys Phe 9# 9 .9 9* 9 9 .9.

9.

.9 9 9 9* Pro Trp Pro 545 Thr Asn Thr Val Thr 625 Glu Arg Gin Leu Val1 705 Leu Met Val, Leu Gly 785 Leu 500 Val1 Asn Asfl Cys Thr 580 Arg Pro Lys Cys Ser 660 Pro Leu Ala Leu Leu 740 Ala Phe TIyr Pro Gly Asp Tro Gly 565 Cys Cys Tyr Val Asn 645 Giu Cys His Val1 Leu 725 Ile Ala Cys Al a Pro 805 Thr Ser Phe 550 Gly Pro Gly Arg Arg 630 Trp Leu Ser Gin Val1 710 Leu Ala Al a Al a Phe 790 Arg Thr Asp 535 Gly Pro Thr Ser Leu 615 Met Thr Ser Phe Asn 695 Ser Ala Gin Val1 Ala 775 Tyr Al a Asp 520 Val1 Cys Pro Asp Gly 600 Trp Tyr Arg Pro Thr 680 Ile Leu Asp Al a Ala 760 Trp Gly Tyr 505 Arg Leu Thr Cys Cys 585 Pro His Val Gly Leu 665 Thr Val1 Val1 Ala Glu 745 Gly Tyr Vali Al a G ly Leu Met 555 I le Arg Leu Pro Gly 635 Arg Leu Pro Val1 Lvs 715 Ile Al a His Lys Pro 795 Val1 As n 540 Asn Gly Lys Thr Cys 620 Val1 Cys Ser Al a Gin 700 Trp Cys Leu Gly Gly 780 Leu Pro 525 As n Gly Gly His Pro 605 Thr G iu Asp Thr Leu 685 Tyr G iu Al a Giu Thr 765 Arg Leu Thr 510 Thr Thr Thr Ala Pro 590 Arg Val1 His Leu Thr 670 Ser Leu Tyr Cys As n 750 Leu Leu Leu Pro Ser Tyr Asn Arg Pro Gly Phe 560 Gly Asn 575 Glu Ala Cys Met Asn Phe Arg Phe 640 Giu Asp 655 Glu Trp Thr Gly Tyr Gly Val Leu 720 Leu Tro 735 Leu Val Ser ?he Vai Pro Leu Leu 800 INFORMATION FOR SEQ ID NO: 51: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 137 (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 1..17 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: Ser Asn Ser Ser Glu Ala Ala Asp Met Ile Met His Thr Pro Gly Cys 1 5 10 Val INFORMATION FOR SEQ ID NO: 52: SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear S(ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 1..22 o* (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: Gly Gly Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp 1 5 10 Ser Pro Thr Thr Ala Leu INFORMATION FOR SEQ ID NO: 53: SEQUENCE CHARACTERISTICS: o LENGTH: 37 amino acids o* TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 1..37 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: Tyr Glu Val Arg Asn Val Ser Gly Ile Tyr His Val Thr Asn Asp Cys 1 5 10 Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met His Thr 25 Pro Gly Cys Gly Lys INFORMATION FOR SEQ ID NO: 54: 138 SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 1..25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: Gly Gly Thr Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Ala Thr 1 5 10 Gin Leu Arg Arg His Ile Asp Leu Leu o 20 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 25 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 1..25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Gly Gly Thr Pro Thr Leu Ala Ala Arg Asp Ala Ser Val Pro Thr Thr 1 5 10 Thr Ile Arg Arg His Val Asp Leu Leu INFORMATION FOR SEQ ID NO: 56: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: Leu Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Gin Val Arg Asn 1 5 10 Ser Thr Gly Leu INFORMATION FOR SEQ ID NO: 57: 139 SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: Gin Val Arg Asn Ser Thr Gly Leu Tyr His Val Thr Asn Asp Cys Pro 1 5 10 Asn Ser Ser Ile INFORMATION FOR SEQ ID NO: 58: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: Asn Asp Cys Pro Asn Ser Ser Ile Val Tyr Glu Ala His Asp Ala Iie 1 5 10 Leu His Thr Pro INFORMATION FOR SEQ ID NO: 59: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: Ser Asn Ser Ser Iie Val Tyr Glu Ala Ala Asp Met Ile Met His Thr 1 5 10 Pro Gly Cys Val INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 140 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: His Asp Ala Ile Leu His Thr Pro Gly Val Pro Cys Val Arg Glu Gly 1 5 10 Asn Val Ser INFORMATION FOR SEQ ID NO: 61: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: ease Cys Val Arg Glu Gly Asn Val Ser Arg Cys Trp Val Ala Met Thr Pro 1 5 10 Thr Val Ala Thr INFORMATION FOR SEQ ID NO: 62: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: Ala Met Thr Pro Thr Val Ala Thr Arg Asp Gly Lys Leu Pro Ala Thr 1 5 10 Gin Leu Arg Arg INFORMATION FOR SEQ ID NO: 63: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: Leu Pro Ala Thr Gin Leu Arg Arg His Ile Asp Leu Leu Val Gly Ser 1 5 10 Ala Thr Leu Cys INFORMATION FOR SEQ ID NO: 64: 141 SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: Leu Val Gly Ser Ala Thr Leu Cys Ser Ala Leu Tyr Val Gly Asp Leu 1 5 10 Cys Gly Ser Val INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Gln Leu Phe Thr Phe Ser Pro Arg Arg His Trp Thr Thr Gln Gly Cys 1 5 10 S• Asn Cys Ser Ile INFORMATION FOR SEQ ID NO: 66: SEQUENCE CHARACTERISTICS: S* LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: Thr Gin Gly Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His 1 5 10 Arg Met Ala Trp INFORMATION FOR SEQ ID NO: 67: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 142 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro 1 5 10 Thr Ala Ala Leu INFORMATION FOR SEQ ID NO: 68: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: Asn Trp Ser Pro Thr Ala Ala Leu Val Met Ala Gln Leu Leu Arg Ile 1 5 10 Pro Gln Ala Ile INFORMATION FOR SEQ ID NO: 69: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid S(C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: Leu Leu Arg Ile Pro Gln Ala Ile Leu Asp Met Ile Ala Gly Ala His 1 5 10 Trp Gly Val Leu INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ala Gly Ala His Trp Gly Val Leu Ala Gly Ile Ala Tyr Phe Ser Me: 1 5 1 0 Val Gly Asn Met 143 INFORMATION FOR SEQ ID NO: 71: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: Val Val Leu Leu Leu Phe Ala Gly Val Asp Ala Glu Thr lie Val Ser 1 5 10 Gly Gly Gin Ala INFORMATION FOR SEQ ID NO: 72: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: Ser Gly Leu Val Ser Leu Phe Thr Pro Gly Ala Lys Gin Asn Ile Gin 1 5 10 1 Leu Ile Asn Thr INFORMATION FOR SEQ ID NO: 73: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: Gin Asn Ile Gin Leu Ile Asn Thr Asn Gly Gin Trp His le Asn Ser 1 5 10 Thr Ala Leu Asn INFORMATION FOR SEQ ID NO: 74: SEQUENCE

CHARACTERISTICS:

LENGTH: 21 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: pepcide 144 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: Leu Asn Cys Asn Glu Ser Leu Asn Thr Gly Trp Trp Leu Ala Gly Leu 1 5 10 Iie Tyr Gin His Lys INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: SAla Gly Leu Ile Tyr Gin His Lys Phe Asn Ser Ser Gly Cys Pro Glu 1 5 10 Arg Leu Ala Ser *2 INFORMATION FOR SEQ ID NO: 76: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single S(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Pro Leu Thr Asp ?he Asp 1 5 10 Gin Gly Trp Gly INFORMATION FOR SEQ ID NO: 77: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: Thr Asp Phe Asp Gin Gly Trp Gly Pro Ile Ser Tyr Ala Asn Gly Ser 1 5 10 Gly Pro Asp Gin 145 INFORMATION FOR SEQ ID NO: 78: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: Ala Asn Gly Ser Gly Pro Asp Gin Arg Pro Tyr Cys Trp His Tyr Pro S' 1 5 10 Pro Lys Pro Cys **0 S(2) INFORMATION FOR SEQ ID NO: 79: 0 9000 t SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: Trp His Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Lys Ser Val 5 10 Cys Gly Pro Val 9 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ala Lys Ser Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val 1 5 10 Val Val Gly Thr INFORMATION FOR SEQ ID NO: 81: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: pepride 146 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Ser Gly Ala Pro Thr 1 5 10 Tyr Ser Trp Gly INFORMATION FOR SEQ ID NO: 82: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: o Gly Ala Pro Thr Tyr Ser Trp Gly Glu Asn Asp Thr Asp Val Phe Val 1 5 10 Leu Asn Asn Thr INFORMATION FOR SEQ ID NO: 83: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: Gly Asn Trp Phe Gly Cys Thr Trp Met Asn Ser Thr Gly Phe Thr Lys 1 5 10 Val Cys Gly Ala INFORMATION FOR SEQ ID NO: 84: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: Gly Phe Thr Lys Val Cys Gly Ala Pro Pro Val Cys lie Gly Gly Ala 10 Gly Asn Asn Thr 147 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ile Gly Gly Ala Gly Asn Asn Thr Leu His Cys Pro Thr Asp Cys Arg 1 5 10 Lys His Pro INFORMATION FOR SEQ ID NO: 86: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: Thr Asp Cys Phe Arg Lys His Pro Asp Ala Thr Tyr Ser Arg Cys Gly 1 5 10 Ser Gly Pro Trp INFORMATION FOR SEQ ID NO: 87: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE .DESCRIPTION: SEQ ID NO: 87: Ser Arg Cys Gly Ser Gly Pro Trp Ile Thr Pro Arg Cys Leu Val Asp 1 5 1 0 1 5 Tyr Pro Tyr Arg INFORMATION FOR SEQ ID NO: 88: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear tii) MOLECULE TYPE: peptide 148 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: Cys Leu Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Ile 1 5 10 Asn Tyr Thr Ile INFORMATION FOR SEQ ID NO: 89: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: Pro Cys Thr Ile Asn Tyr Thr Ile Phe Lys Ile Arg Met Tyr Val Gly 1 5 10 Gly Val Glu His INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Tyr Val Gly Gly Val Glu His Arg Leu Glu Ala Ala Cys Asn Trp S1 5 10 Thr Pro Gly Glu INFORMATION FOR SEQ ID NO: 91: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: Ala Cys Asn Trp Thr Pro Gly Glu Arg Cys .Asp Leu Glu Asp Arg Asp 1 5 10 Arg Ser Glu Leu 149 INFORMATION FOR SEQ ID NO: 92: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92: Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Thr Thr Thr 1 5 Gin Trp Gin Val INFORMATION FOR SEQ ID NO: 93: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: Tyr Gln Val Arg Asn Ser Thr Gly Leu 1 INFORMATION FOR SEQ ID NO: 94: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid S" STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94: ACGTCCGTAC GTTCGAATTA ATTAATCGA 29 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO 150 (iii) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CCTCCGGACG TGCACTAGCT CCCGTCTGTG GTAGTGGTGG TAGTGATTAT CAATTAATTG INFORMATION FOR SEQ ID NO: 96: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96: GTTTAACCAC TGCATGATG 19 INFORMATION FOR SEQ ID NO: 97: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: GTCCCATCGA GTGCGGCTAC INFORMATION FOR SEQ ID NO: 98: SEQUENCE CHARACTERISTICS: LENGTH: 45 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98: 151 CGTGACATGG TACATTCCGG ACACTTGGCG CACTTCATAA GCGGA INFORMATION FOR SEQ ID NO: 99: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99: TGCCTCATAC ACAATGGAGC TCTGGGACGA GTCGTTCGTG AC 42 INFORMATION FOR SEQ ID NO: 100: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs S TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: TACCCAGCAG CGGGAGCTCT GTTGCTCCCG AACGCAGGGC AC 42 INFORMATION FOR SEQ ID NO: 101: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101: TGTCGTGGTG GGGACGGAGG CCTGCCTAGC TGCGAGCGTG GG 42 INFORMATION FOR SEQ ID NO: 102: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 152 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102: CGTTATGTGG CCCGGGTAGA TTGAGCACTG GCAGTCCTGC ACCGTCTC 48 INFORMATION FOR SEQ ID NO: 103: SEQUENCE CHARACTERISTICS: LENGTH: 42 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO S. (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103: CAGGGCCGTT CTAGGCCTCC ACTGCATCAT CATATCCCAA GC 42 INFORMATION FOR SEQ ID NO: 104: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104: CCGGAATGTA CCATGTCACG AACGAC 26 INFORMATION FOR SEQ ID NO: 105: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: 153 GCTCCATTGT GTATGAGGCA GCGG 24 INFORMATION FOR SEQ ID NO: 106: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106: GAGCTCCCGC TGCTGGGTAG CGC 23 INFORMATION FOR SEQ ID NO: 107: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107: CCTCCGTCCC CACCACGACA ATACG INFORMATION FOR SEQ ID NO: 108: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: CTACCCGGGC CACATAACGG GTCACCG 27 INFORMATION FOR SEQ ID NO: 109: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 154 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109: GGAGGCCTAC AACGGCCCTG GTGG 24 INFORMATION FOR SEQ ID NO: 110: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110: •TTCTATCGAT TAAATAGAAT TC 22 INFORMATION FOR SEQ ID NO: 111: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111: GCCATACGCT CACAGCCGAT CCC 23

Claims (12)

1. An isolated HCV single or specific oligomeric envelope protein selected from the group consisting of El and/or E2 and/or El/E2, having a purity degree of at least characterised by the absence of aggregates.

2. An isolated HCV single or specific oligomeric envelope protein selected from the group consisting of El and/or E2 and/or E1/E2, having a purity degree of at least characterised by the absence of aggregates.

3. An isolated HCV single or specific oligomeric envelope protein selected from the group consisting of El and/or E2 and/or El/E2, having a purity degree of at least characterised by the absence of aggregates. An isolated HCV envelope protein according to any one of claims 1 to 3, wherein said isolated HCV envelope proteins are expressed from recombinant mammalian cells such as by using a vaccinia virus based system. An isolated HCV envelope protein according to any one of claims 1 to 3, wherein 15 said isolated HCV envelope proteins are expressed from recombinant yeast cells.

6. An isolated HCV envelope protein according to any one of claims 1 to 5, for use as a medicament. An isolated HCV envelope protein according to any one of claims 1 to 5, for use as a vaccine for immunizing a mammal against HCV, comprising administering an effective amount of said composition, optionally accompanied by pharmaceutically acceptable adjuvants, to produce an immune response

8. An isolated HCV envelope protein according to claim 7 wherein said mammal is human.

9. A method for immunising a mammal against HCV, comprising the steps of administering to said mammal an effective amount of an isolated HCV envelope protein 9 -according to any one of claims 1 to 5, to produce an immune response. -156- A method according to claim 9, wherein said mammal is human.

11. A vaccine composition for immunizing a mammal against HCV, comprising an effective amount of an isolated HCV envelope protein according to any one of claims 1 to 5, optionally accompanied by pharmaceutically acceptable adjuvants.

12. A vaccine composition according to claim 11 wherein said mammal is human.

13. An isolated HCV envelope protein according to any one of claims 1 to 5, for in vitro detection of HCV antibodies present in a biological sample. S. 14. A method for in vitro diagnosis of HCV antibodies present in a biological sample, comprising at least the following steps: .0 10 contacting said biological sample with an isolated HCV envelope protein S•°according to any one of claims 1 to 5, under appropriate conditions which allow the formation of an immune complex, (ii) removing unbound components, oooo° (iii) incubating the immune complexes formed with heterologous antibodies, 15 with said heterologous antibodies being conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immune complexes visually or mechanically. A method according to claim 14 wherein said isolated HCV envelope protein is in an immobilised form.

16. A kit for determining the presence of HCV antibodies present in a biological sample, comprising:

157- at least one isolated HCV envelope protein according to any one of claims 1 to a buffer or components necessary for producing the buffer enabling binding reaction between these proteins and antibodies against HCV present in said biological sample, a means for detecting the immune complexes formed in the preceding binding reaction. 17. A kit according to claim 16 wherein said at least one isolated HCV envelope o protein is in an immobilised form on a solid substrate. 18. Use of an isolated HCV envelope protein according to any one of claims 1 to 10 comprising HCV El protein, for in vitro monitoring HCV disease or prognosing the response to treatment of patients suffering from HCV infection comprising: S- incubating a biological sample from a patient with HCV infection with an E 1 protein or a suitable part thereof under conditions allowing the formation of an .ooot° S• immunological complex, .15 removing unbound components, ocalculating the anti-El titers present in said sample at the start of and during the course of treatment, monitoring the natural course of HCV disease, or prognosing the response to treatment of said patient on the basis of the amount of anti-El titers found in said sample at the start of treatment and/or during the course of treatment. 19. Use according to claim 18 wherein the HCV El protein is a HCV single El protein. -158 Use according to any one of claims 18 or 19 wherein said treatment is treatment with interferon. 21. A kit for monitoring HCV disease or prognosing the response to treatment of patients suffering from HCV infection comprising: at least one isolated HCV envelope protein according to any one of claims 1 to a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins and the anti-El antibodies present in a biological S. 0oeo sample, o6 means for detecting the immune complexes formed in the preceding binding 10 reaction, and a optionally also an automated scanning and interpretation device for inferring a *0 decrease of anti-E 1 titers during the progression of treatment. 9694 022. A kit according to claim 21 wherein said at least one isolated HCV envelope 60000: S protein is an El protein. 15 23. A kit according to claim 21 or claim 22, wherein said treatment is treatment with 0. 0 interferon. 24. A serotyping assay for detecting one or more serological types of HCV present in a biological sample, comprising at least the following steps contacting the biological sample to be analyzed for the presence of HCV antibodies of one or more serological types, with at least one isolated HCV El and/or E2 and/or El/E2 protein according to any one of claims 1 to 5, under appropriate conditions which allow the formation of an immune complex,

159- (ii) removing unbound components, (iii) incubating the immune complexes formed with heterologous antibodies. with said heterologous antibodies being conjugated to a detectable label under appropriate conditions. (iv) detecting the presence of said immune complexes visually or mechanically by means of densitometry, fluorimetry, colorimetry) i: and inferring the presence of one or more HCV serological types present from the observed binding pattern. A serotyping assay according to claim 24 for detecting antibodies of the different types of HCV combined in one assay format. 26. A serotyping assay according to claim 24 or claim 25 wherein said at least one isolated HCV El and/or E2 and/or E1/E2 protein is in an immobilised form. 27. A kit for serotyping one or more serological types of HCV present in a biological sample, comprising: 15 at least one isolated HCV El and/or E2 and/or E1/E2 protein according to any one of claims 1-to a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins and the anti-El antibodies present in a biological sample, means for detecting the immune complexes formed in the preceding binding reaction, and -160- optionally also an automated scanning and interpretation device for detecting the presence of one or more serological types present from the observed binding pattern. 28. A kit according to claim 27 for detecting antibodies to said serological types of HCV. 29. An isolated HCV envelope protein according to any one of claims 1 to 5. to raise upon immunization an E 1 and/or E2 specific monoclonal antibody. An isolated HCV envelope protein according to any one of claims 1 to 5, for the preparation of an immunoassay kit. 31. Use of an isolated HCV envelope protein according to any one of claims 1 to C tbfor detecting HCV antibodies present in a biological sample. 32. Use of an isolated HCV envelope protein according to any one of claims 1 to for the manufacture of a medicament for immunising a mammal against HCV. 33. Use according to claim 32 wherein said mammal is human. 15 34. An isolated HCV single or specific oligomeric envelope protein, substantially as herein described with reference to one or more of the examples but excluding comparative examples. A method for immunising a mammal against HCV, substantially as herein described with reference to one or more of the examples but excluding comparative examples. 36. A vaccine composition for immunising a mammal against HCV, substantially as herein described with reference to one or more of the examples but excluding comparative examples. 161 37. A method for in vitro diagnosis of HCV antibodies present in a biological sample, substantially as herein described with reference to one or more of the examples but excluding comparative examples. 38. A kit for determining the presence of HCV antibodies present in a biological sample, substantially as herein described with reference to one or more of the examples but excluding comparative examples. S39. Use of an isolated HCV single or specific oligomeric envelope protein, substantially as herein described with reference to one or more of the examples but excluding comparative examples. 40. A kit for monitoring HCV disease or prognosing the response to treatment of patients suffering from HCV infection, substantially as herein described with reference to one or more of the examples but excluding comparative examples. S41. A serotyping assay for detecting one or more serological types of HCV present in a •biological sample, substantially as herein described with reference to one or more of the examples but excluding comparative examples. 42. A kit for serotyping one or more serological types of HCV present in a biological sample, substantially as herein described with reference to one or more of the examples but excluding comparative examples. DATED this 29th Day of October, 1999 INNOGENETICS N.V. Attorney: IVAN A. RAJKOVIC Fellow Institute of Patent and Trade Mark Attorneys of Australia of BALDWMn SI 4 FLSTON WATERS