AU708174B2 - Purified hepatitis C virus envelope proteins for diagnostic and therapeutic use - Google Patents

Description

WO 96/04385 PCT/EP95/03031 '1 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 oligomeric El and/or E2 and/or E1/E2 envelope proteins in assays for monitoring disease, and/or diagnosis of disease, and/or treatment of disease.

The invention also relates to epitopes of the El and/or E2 envelope proteins and monoclonal antibodies thereto, as well their use in diagnosis, prophylaxis or treatment.

Backqround of the invention The E2 protein purified from cell lysates according to the methods described in the present invention reacts with approximately 95% of patient sera. This reactivity is similar to the reactivity obtained with E2 secreted from CHO cells (Spaete et al., 1992).

However, the intracellularly expressed form of E2 may more closely resemble the native viral envelope protein because it contains high mannose carbohydrate motifs, whereas the E2 protein secreted from CHO cells is further modified with galactose and sialic acid sugar moieties. When the aminoterminal half of E2 is expressed in the baculovirus system, only about 13 to 21% of sera from several patient groups can be detected (Inoue et al., 1992). After expression of E2 from E. coli, the reactivity of HCV sera was even lower and ranged from 14 (Yokosuka et al., 1992) to 17% (Mita et al., 1992).

About 75% of HCV sera (and 95% of chronic patients) are anti-El positive using the purified, vaccinia-expressed recombinant El protein of the present invention, in sharp contrast with the results of Kohara et al. (1992) and Hsu et al. (1993). Kohara

I

-2et 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 baculovirusexpressed 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. Therefore, the vaccinia virus system may be used for selecting the best *expression constructs and for limited upscaling, and large-scale expression and S: 15 purification of single or specific oligomeric envelope proteins containing high-mannose carbohydrates may be achieved when expressed from several yeast strains. In the case of hepatitis B for example, manufacturing of HBsAg from mammalian cells was much more costly compared with yeast-derived hepatitis B vaccines.

SUMMARY OF THE INVENTION The present invention provides new purification methods for recombinantly expressed El and/or E2 and/or E1/E2 proteins such that said recombinant proteins are directly useable for diagnostic and vaccine purposes as single or specific oligomeric recombinant proteins free from contaminants instead of aggregates.

-2a- The present invention also provides 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.

According to a first aspect the present invention provides a method for purifying recombinant HCV single or specific oligomeric envelope proteins selected from the group consisting of El and/or E2 and/or E1/E2, wherein a disulphide bond cleavage or reduction step is carried out with a disulphide bond cleavage agent on the recombinantly expressed protein.

According to a second aspect the present invention provides a method according to 0 the first aspect, further comprising at least the following steps: lysing recombinant El and/or E2 and/or E1/E2 expressing host cells, possibly in the presence of an SH blocking agent such as N-ethylmaleimide

(NEM),

recovering said HCV envelope proteins by affinity purification such as by means of lectin-chromotography, such as lentil-lectin chromatography, or by means of 15 immunoaffinity using anti-El and/or anti-E2 specific monoclonal antibodies, reduction or cleavage of the disulphide bonds with a disulphide bond cleaving agent, such as DTT, preferably also in the presence of an SH blocking agent, such as NEM or Biotin-NEM, and, recovering the reduced El and/or E2 and/or E1/E2 envelope proteins by gelfiltration and possibly also by a subsequent Ni2+-IMAC chromatography and desalting step.

According to a third aspect the present invention provides an isolated HCV envelope protein obtained by a method according to the first aspect.

2b According to a fourth aspect the present invention provides a method according to the first or second aspect, further comprising at least the following steps: growing a host cell transformed with a recombinant vector comprising a nucleotide sequence allowing the expression of a HCV single or specific oligomeric El and/or E2 and/or E1/E2 protein in a suitable culture medium, causing expression of said vector under suitable conditions, and, lysing said transformed host cells, preferably in the presence of an SH group blocking agent, such as N-ethylmaleimide

(NEM),

recovering said HCV envelope protein by affinity purification by means of for instance lectin-chromatography or immunoaffinity chromatography using anti-El and/or anti-E2 specific monoclonal antibodies, with said lectin being preferably lentillectin, followed by, incubation of the eluate of the previous step with a disulphide bond cleavage agent, such as DTT, preferably also in the presence of an SH group blocking agent, such as 15 NEM or Biotin-NEM, and, isolating the HCV single or specific oligomeric El and/or E2 and/or E1/E2 protein by means of gelfiltration and possibly also by means of an additional Ni 2

-IMAC

chromatography and desalting step.

a According to a fifth aspect the present invention provides an isolated HCV envelope protein according to the third aspect, for use as a medicament.

According to a sixth aspect the present invention provides an isolated HCV envelope protein according to the third aspect, for use as a vaccine for immunising a mammal, preferably humans, against HCV, comprising administrating an effective amount of said composition possibly accompanied by pharmaceutically acceptable adjuvants, to produce an immune response.

According to a seventh 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 the third aspect, to produce an immune response.

According to an eighth aspect the present invention provides a vaccine composition for immunising a mammal, preferably humans, against HCV, comprising an effective amount of an isolated HCV envelope protein according to the third aspect possibly accompanied by pharmaceutically acceptable adjuvants.

According to a ninth aspect the present invention provides an isolated HCV envelope protein according to the third aspect, for in vitro detection of HCV antibodies present in a biological sample.

According to a tenth aspect the present invention provides a method for in vitro ,o 15 diagnosis of HCV antibodies present in a biological sample, comprising at least the o following steps: contacting said biological sample with an isolated HCV envelope protein according to the third aspect, preferably in an immobilised form under appropriate conditions a. which allow the formation of an immune complex, *a C 20 b) removing unbound components, a.c) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies being conjugated to a detectable label under appropriate conditions, -3ad) detecting the presence of said immune complexes visually or mechanically.

According to an eleventh 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 the third aspect, preferably in an immobilised form on a solid substrate, 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 a twelfth aspect the present invention provides the use of an isolated HCV envelope protein according to the third aspect, comprising HCV El protein, more particularly HCV single El proteins, for in vitro monitoring HCV disease or prognosing the response to treatment, particularly with interferon, of patients suffering from HCV infection comprising: o.

15 incubating a biological sample from a patient with HCV infection with an El protein a or a suitable part thereof under conditions allowing the formation of an immunological complex, Sremoving unbound components, calculating the anti-E 1 titers present in said sample at the start of and during the %o 20 course of treatment, 99 9 monitoring the natural course of HCV disease, or prognosing the response to 999999 treatment of said patient on the basis of the amount of anti-E I titers found in said sample at the start of treatment and/or during the course of treatment.

-3b- According to a thirteenth aspect the present invention provides a kit for monitoring HCV disease or prognosing the response to treatment, particularly with interferon, of patients suffering from HCV infection comprising: at least one isolated HCV envelope protein, more particularly an El protein according to the third aspect, 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, possibly also an automated scanning and interpretation device for inferring a decrease of anti-E 1 titers during the progression of treatment.

According to a fourteenth aspect the present invention provides a serotyping assay for detecting one or more serological types of HCV present in a biological sample, more particularly for detecting antibodies of the different types of HCV to be detected 15 combined in one assay format, comprising at least the following steps:

S.

a) contacting the biological sample to be analysed for the presence ofHCV antibodies of one or more serological types, with at least one isolated HCV El and/or E2 and/or l /E2 protein according to the third aspect, preferentially in an immobilised form :o under appropriate conditions which allow the formation of an immune complex, b) removing unbound components, *O e O c) incubating the immune complexes formed with heterologous antibodies, with said a heterologous antibodies being conjugated to a detectable label under appropriate conditions, 3cd) 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.

According to a fifteenth aspect the present invention provides a kit for serotyping one or more serological types of HCV present in a biological sample, more particularly for detecting the antibodies to these serological types of HCV comprising: at least one isolated HCV El and/or E2 and/or El/E2 protein according to the third aspect, 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, possibly also an automated scanning and interpretation device for detecting the presence of one or more serological types present from the observed binding pattern.

15 According to a sixteenth aspect the present invention provides an isolated HCV envelope protein according to the third aspect, to raise upon immunisation an El and/or E2 specific monoclonal antibody.

According to a seventeenth aspect the present invention provides an isolated HCV :envelope protein according to the third aspect, for the preparation of an immunoassay kit.

S According to an eighteenth aspect the present invention provides the use of an isolated HCV envelope protein according to the third aspect, for detecting HCV antibodies present in a biological sample.

-3d- 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".

Other embodiments and advantages of the present invention will be clear from the following description.

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

The term 'hepatitis C virus single envelope proteins' refers to a polypeptide or an analogue thereof mimotopes) comprising an amino acid sequence (and/or amino acid analogues) defining at least one HCV epitope of either the El or the E2 region.

a

**I

*o WO 96/04385 PCT/EP95/03031 4 These single envelope proteins in the broad sense of the word may be both monomeric or homo-oligomeric forms of recombinantly expressed envelope proteins. Typically, the sequences defining the epitope correspond to the amino acid sequence of either the El or the E2 region of HCV (either identically or via substitution of analogues of the native S 5 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 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 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 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 NS1, is believed to span amino acids 384-809 or 384-746 (Grakoui et al., 1993) of the HCV polyprotein and to also be an envelope protein. Upon expression in a vaccinia system (glycosylated), it is believed to have an apparent gel molecular weight of about 72 kDa.

It is understood that these protein endpoints are approximations 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 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 WO 96/04385 PCT/EP95/03031 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 advantageous to adsorb the polyclonal serum first with the denatured antigen and see if it retains antibodies to the antigen of interest.

The HCV antigens of the present invention can be made by any recombinant method that provides the epitope of intrest. 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 (Kniskern et al., 1994) or glycosylation mutants derived by means of vanadate resistence selection (Ballou et al., 1991)) may be ideally suited for production of secreted high-mannose-type sugars; whereas proteins secreted from mammalian cells may contain modifications including galactose or sialic acids which may be undesirable for certain diagnostic or vaccine applications. However, it may also be possible and sufficient for certain applications, as it is known for proteins, to express the antigen in other recombinant hosts (such as E. coli) and renature the protein after recovery.

The term 'fusion polypeptide' intends a polypeptide in which the HCV antigen(s) are part of a single continuous chain of amino acids, which chain does not occur in nature. The HCV antigens may be connected directly to each other by peptide bonds or be separated by intervening amino acid sequences. The fusion polypeptides may also contain amino acid sequences exogenous to HCV.

WO 96/04385 PCT/EP95/03031 6 The term 'solid phase' intends a solid body to which the individual HCV antigens or the fusion polypeptide comprised of HCV antigens are bound covalently or by noncovalent means such as hydrophobic adsorption.

The term 'biological sample' intends a fluid or tissue of a mammalian individual an anthropoid, a human) that commonly contains antibodies produced by the individual, more particularly antibodies against HCV. The fluid or tissue may also contain HCV antigen. Such components are known in the art and include, without limitation, blood, plasma, serum, urine, spinal fluid, lymph fluid, secretions of the respiratory, intestinal or genitourinary tracts, tears, saliva, milk, white blood cells and myelomas.

Body components include biological liquids. The term 'biological liquid' refers to a fluid obtained from an organism. Some biological fluids are used as a source of other products, such as clotting factors 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 immunologically cross-reactive with natural El, and includes El proteins of genotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other newly identified HCV type or subtype.

'E2' as used herein refers to a protein or polypeptide expressed within the first 900 amino acids of an HCV polyprotein, sometimes referred to as the NS1 protein. In its natural form it is a 72 kDa glycoprotein that is found in strong association with membranes. In most natural HCV strains, the E2 protein is encoded in the viral polyprotein following the 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 WO 96/04385 PCT/EP95/03031 7 position 809. The term 'E2' as used herein also includes analogs and truncated forms that are immunologically cross-reactive with natural E2. For example, insertions of multiple codons between codon 383 and 384, as well as deletions of amino acids 384- 387 have been reported by Kato et al. (1992).

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

The term 'specific oligomeric' E1 and/or E2 and/or E1/E2 envelope proteins refers to all possible oligomeric forms of recombinantly expressed 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' E1 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 E1 and/or E2 and/or E1/E2 recombinantly expressed proteins. These single or specific oligomeric envelope proteins according to the present invention can be further defined by the following formula 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= 1 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. E1/E1 dimers, E1/E1/E1 trimers or E1/E1/E1/E1 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 homooligomers of El or E2 are all 'homo-oligomers' within the scope of this definition. The oligomers may contain one, two, or several different monomers of El or E2 obtained from different types or subtypes of hepatitis C virus including for example those described in an international application published under WO 94/25601 and European application No. 94870166.9 both by the present applicants. Such mixed oligomers are still homo-oligomers within the scope of this invention, and may allow more universal diagnosis, prophylaxis or treatment of HCV.

The term 'purified' as applied to proteins herein refers to a composition wherein the desired protein comprises at least 35% of the total protein component in the composition. The desired protein preferably comprises at least 40%, more preferably at least about 50%, more preferably at least about 60%, still more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least WO 96/04385 PCT/EP95/03031 8 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 pure, preferably, 90%, more preferably 95%, more preferably 97%, more preferably 98%, more preferably 99%, even more preferably 99.5%, and most preferably the contaminating proteins should be undetectable by conventional methods like SDS-PAGE and silver staining.

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

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 eukaryotes are yeasts, particularly species within Saccharomvces, Schizosaccharomyces, Kluveromyces Pichia Pichia astoris), Hansenula (e.g.

Hansenula o orpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zyqosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K.

Jactis are the most commonly used yeast hosts, and are convenient fungal hosts.

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

The term 'higher eukaryote' refers to host cells derived from higher animals, such as mammals, reptiles, insects, and the like. Presently preferred higher eukaryote host cells are derived from Chinese hamster CHO), monkey COS and Vero cells), baby hamster kidney (BHK), pig kidney (PK15), rabbit kidney 13 cells (RK1 the human osteosarcoma cell line 143 B, the human cell line HeLa and human hepatoma cell lines like Hep G2, and insect cell lines Spodoptera fruqiperda). The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like.

WO 96/04385 PCT/EP95/03031 9 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 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 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 WO 96/04385 PCT/EP95/03031 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.

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

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

A 'coding sequence' is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries 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.

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 a, 1 b, 1 c, 1 d, 1 e, 1 f, 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, 5b, 6a, 6b, 6c, 7a, 7b, 7c, 8a, 8b, 9a, 9b, 10a, or any other newly defined HCV (sub)type. It is to be understood that the amino acids constituting the epitope need not be part of a linear sequence, but may be interspersed by any number of amino acids, thus forming a conformational epitope.

WO 96/04385 PCT/EP95/03031 11 The term 'immunogenic' refers to the ability of a substance to cause a humoral and/or cellular response, whether alone or when linked to a carrier, in the presence or absence of an adjuvant. 'Neutralization' refers to an immune response that blocks the infectivity, either partially or fully, of an infectious agent. A 'vaccine' is an immunogenic composition capable of eliciting protection against HCV, whether partial or complete.

A vaccine may also be useful for treatment of an individual, in which case it is called a therapeutic vaccine.

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

The term 'effective amount' refers to an amount of epitope-bearing polypeptide sufficient to induce an immunogenic response in the individual to which it is administered, or to otherwise detectably immunoreact in its intended system immunoassay). Preferably, the effective amount is sufficient to effect treatment, as defined above. The exact amount necessary will vary according to the application. For vaccine applications or for the generation of polyclonal antiserum antibodies, for example, the effective amount may vary depending on the species, age, and general condition of the individual, the severity of the condition being treated, the particular polypeptide selected and its mode of administration, etc. It is also believed that effective amounts will be found within a relatively large, non-critical range. An appropriate effective amount can be readily determined using only routine experimentation.

Preferred ranges of El and/or E2 and/or E1/E2 single or specific oligomeric envelope proteins for prophylaxis of HCV disease are 0.01 to 100 /g/dose, preferably 0.1 to pg/dose. Several doses may be needed per individual in order to achieve a sufficient immune response and subsequent protection against HCV disease.

Detailed description of the invention More particularly, the present invention contemplates a method for isolating or purifying recombinant HCV single or specific oligomeric envelope protein selected from the group consisting of E1 and/or E2 and/or E1/E2, characterized in that upon lysing the transformed host cells to isolate the recombinantly expressed protein a disulphide bond cleavage or reduction step is carried out with a 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 WO 96/04385 PCTIEP95/03031 12 disulphide bond linked with contaminants.

The proteins according to the present invention are recombinantly expressed in lower or higher eukaryotic cells or in prokaryotes. The recombinant proteins of the present invention are preferably glycosylated and may contain high-mannose-type, hybrid, or complex glycosylations. Preferentially said proteins are expressed from mammalian cell lines as discussed in detail in the Examples section, or in yeast such as in mutant yeast strains also as 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 retained in the ER or Golgi Apparatus of mammalian cells or are retained in or secreted from yeast cells, preferably secreted from yeast mutant strains such as the mnn9 mutant (Kniskern et al., 1994), or from mutants that have been selected by means of vanadate resistence (Ballou et al., 1991).

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

vaccinia) proteins or of other HCV envelope proteins (single or oligomeric), and form aspecific intermolecular bridges. This results in the formation of 'aggregates' of HCV envelope proteins together with contaminating proteins. It was also shown in WO 92/08734 that 'aggregates' were obtained after purification, but it was not described which protein interactions were involved. 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 non-covalent protein-protein interactions. The present invention thus provides a means for selectively cleaving the disulphide bonds under specific conditions and for separating the cleaved proteins from contaminating proteins which greatly interfere with diagnostic, prophylactic and therapeutic applications. The free thiol groups WO 96/04385 PCT/EP95/03031 13 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 are modified into cysteic acid (Moore et al., 1963).

Sulfitolysis (R-S-S-R 2 R-SO 3 for example by means of sulphite (SO2 3) 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, cysteine, glutathione Red, e-mercapto-ethylamine, or thioglycollic acid, of which DTT and 9-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).

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 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 mM.

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) WO 96/04385 PCT/EP95/03031 14 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.

A particularly preferred method for obtaining disulphide bond cleavage employs a combination of a classical disulphide bond cleavage agent as detailed above and a detergent (also as detailed above). As contemplated in the Examples section, the particular combination of a low concentration of DTT (1.5 to 7.5 mM) and about 3.5 of Empigen-BB is proven to be a particularly preferred combination of reducing agent and detergent for the purification of recombinantly expressed 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 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, 1 988) 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) 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) WO 96/04385 PCT/EP95/03031 N-ethylmaleimide (NEM; Benesch et al., 1956) N-(4-dimethylamino-3,5-dinitrophenyl) maleimide or Tuppy's maleimide which provides a color to the protein P-chloromercuribenzoate (Grassetti et al., 1969) 4-vinylpyridine (Friedman and Krull, 1969) can be liberated after reaction by acid hydrolysis acrylonitrile, can be liberated after reaction by acid hydrolysis (Weil and Seibles, 1961) NEM-biotin obtained from Sigma B1267) 2 2 '-dithiopyridine (Grassetti and Murray, 1967) 4 ,4'-dithiopyridine (Grassetti and Murray, 1967) 6 6 '-dithiodinicontinic acid (DTDNA; Brown and Cunnigham, 1970) 2, 2 '-dithiobis-(5'-nitropyridine) (DTNP; 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 of thiol groups of the same protein or enzyme molecule. One may conclude that this variation in reactivity of the thiol groups is due to the steric environment of these groups, such as the shape of the molecule and the surrounding groups of atoms and their charges, as well as to the size, shape and charge of the reagent molecule or ion. Frequently the presence of adequate concentrations of denaturants such as sodium dodecylsulfate, urea or guanidine hydrochoride will cause sufficient unfolding of the protein molecule to permit equal access to all of the reagents for thiol groups. By varying the concentration of denaturant, the degree of unfolding can be controlled and in this way thiol groups with different degrees of reactivity may be revealed. Although up to date most of the work reported has been done with p-chloromercuribenzoate, N-ethylmaleimide and 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 intermolecular aggregates upon recombinant expression and purification of El and E2 (envelope) proteins are also within the scope of the present invention.

A preferred SH group blocking reagent according to the present invention is N- WO 96/04385 PCT/EP95/03031 16 ethylmaleimide (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 after any other process for cleaving disulphide bridges. Said SH group blocking reagent may also be modified with any group capable of providing a detectable label and/or any group aiding in the immobilization of said recombinant protein to a solid substrate, e.g. biotinylated

NEM.

Methods for cleaving cysteine bridges and blocking free cysteines have also been described in Darbre (1987), 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 E1 and/or E2 and/or E1/E2 expressing host cells, preferably in the presence of an SH group blocking agent, such as N-ethylmaleimide

(NEM),

and possibly a suitable detergent, preferably Empigen-BB, recovering said HCV envelope protein by affinity purification for instance by means lectin-chromatography, such as lentil-lectin chromatography, or immunoaffinity chromatography using anti-El and/or anti-E2 specific monoclonal antibodies, followed by, reduction or cleavage of disulphide bonds with a disulphide bond cleaving agent, such as DTT, preferably also in the presence of an SH group blocking agent, such as NEM or Biotin-NEM, and, recovering the reduced HCV El and/or E2 and/or E1/E2 envelope proteins for instance by gelfiltration (size exclusion chromatography or molecular sieving) and possibly also by an additional Ni2+-IMAC chromatography and desalting step.

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

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

(AUA).

Preferably said method is usable to purify single or specific oligomeric

HCV

envelope protein produced intracellularly as detailed above.

For secreted El or E2 or E1/E2 oligomers, lectins binding complex sugars such WO 96/04385 PCT/EP95/03031 17 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 consisting of El and/or E2 and/or El/E2, characterized as being isolated or purified by a method as defined above.

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

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

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

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

The segment of the HCV cDNA encoding the desired El and/or E2 sequence inserted into the vector sequence may be attached to a signal sequence. Said signal sequence may be that from a non-HCV source, e.g. the IgG or tissue plasminogen activator (tpa) leader sequence for expression in mammalian cells, or the a-mating factor sequence for expression into yeast cells, but particularly preferred constructs according to the present invention contain signal sequences appearing in the HCV genome before the respective start points of the El and E2 proteins. The segment of the HCV cDNA encoding the desired El and/or E2 sequence inserted into the vector may also include deletions e.g. of the hydrophobic domain(s) as illustrated in the examples section, or of WO 96/04385 PCT/EP95/03031 18 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 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 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 forms to which a 5'-terminal ATG codon and a 3'terminal stop codon has been added, or forms which have a factor Xa cleavage site and/or 3 to 10, preferably 6 Histidine codons have been added.

A variety of vectors may be used to obtain recombinant expression of HCV single or specific oligomeric envelope proteins of the present invention. Lower eukaryotes such as yeasts and glycosylation mutant strains are typically transformed with plasmids, or are transformed with a recombinant virus. The vectors may replicate within the host WO 96/04385 PCT/EP95/03031 19 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 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 Autoqrapha californica 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 cells are known in the art.

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

The present invention also relates to recombinant El and/or E2 and/or E1/E2 proteins characterized in that at least one of their glycosylation sites has been removed and are consequently termed glycosylation mutants. As explained in the Examples section, different glycosylation mutants may be desired to diagnose (screening, confirmation, prognosis, etc.) and prevent HCV disease according to the patient in question. An E2 protein glycosylation mutant lacking the GLY4 has for instance been found to improve the reactivity of certain sera in diagnosis. These glycosylation mutants

I

WO 96/04385 PCT/EP95/03031 are preferably purified according to the method disclosed in the present invention. Also contemplated within the present invention are recombinant vectors carrying the nucleic acid insert encoding such a El and/or E2 and/or E1!E2 glycosylation mutant as well as host cells tranformed with such a recombinant vector.

The present invention also relates to recombinant vectors including a polynucleotide which also forms part of the present invention. The present invention relates more particularly to the recombinant nucleic acids as represented in SEQ ID NO 3, 5, 7, 9, 11, 13, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47 and 49, or parts thereof.

The present invention also contemplates host cells transformed with a recombinant vector as defined above, wherein said vector comprises a nucleotide sequence encoding HCV El and/or E2 and/or E1/E2 protein as defined above in addition to a regulatory sequence operably linked to said HCV El and/or E2 and/or El/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 hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, PK15, RK1 3 and a number of other cell lines.

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

A preferred method for isolating or purifying HCV envelope proteins as defined above is further characterized as comprising at least the following steps: growing a host cell as defined above transformed with a recombinant vector according to the present invention or with a known recombinant vector expressing El and/or E2 and/or El/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 WO 96/04385 PCT/EP95/03031 21 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-E 1 and/or anti-E2 specific monoclonal antibodies, with said lectin being preferably lentillectin or GNA, followed by, incubation of the eluate of the previous step with a disulphide bond cleavage means, such as DTT, preferably followed by incubation with an SH group blocking agent, such as NEM or Biotin-NEM, and, isolating the HCV single or specific oligomeric El and/or E2 and/or El/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 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.

Ni2+-IMAC chromatography followed by a desalting step is preferably used for contructs bearing a (His), as described by Janknecht et al., 1991, and Hochuli et al., 1988.

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

The present invention further relates to a composition comprising at least one of the following El peptides as listed in Table 3: E1-31 (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), WO 96/04385 PCT/EP95/03031 22 E1-35A (SEQ ID NO 59) spanning amino acids 208 to 227 of the El V2 region (epitope B), 1 bE1 (SEQ ID NO 53) spanning amino acids 192 to 228 of El regions (V1, C1, and V2 regions (containing epitope E1-51 (SEQ ID NO 66) spanning amino acids 301 to 320 of the El region, El-53 (SEQ ID NO 67) spanning amino acids 313 to 332 of the El C4 region (epitope A), 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), Env 69 or E2-69 (SEQ ID NO 73) spanning amino acid positions 409 to 428 of the E2 region (epitope A), Env 23 or E2-23 (SEQ ID NO 86) spanning positions 583 to 602 of the E2 region (epitope E), Env 25 or E2-25 (SEQ ID NO 87) spanning positions 595 to 614 of the E2 region (epitope E), Env 27 or E2-27 (SEQ ID NO 88) spanning positions 607 to 626 of the E2 region (epitope E), Env 17B or E2-17B (SEQ ID NO 83) spanning positions 547 to 566 of the E2 region (epitope D), Env 13B or E2-13B (SEQ ID NO 82) spanning positions 523 to 542 of the E2 region (epitope C; recognized by monoclonal antibody 16A6E7, see 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 WO 96/04385 PCT/EP95/03031 23 reactive with any of the polypeptides or peptides as defined above, and with said antibody being preferably a monoclonal antibody.

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

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

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

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

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

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

Antibodies directed to peptides or single or specific oligomeric envelope proteins derived from a certain genotype may be used as a medicament, more particularly for incorporation into an immunoassay for the detection of HCV genotypes (for detecting I i WO 96/04385 PCT/EP95/03031 24 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 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: contacting said biological sample with any of the El and/or E2 specific monoclonal antibodies as defined above, preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex, (ii) removing unbound components, (iii) incubating the immune complexes formed with heterologous antibodies, which specifically bind to the antibodies present in the sample to be analyzed, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immune complexes visually or mechanically by means of densitometry, fluorimetry, colorimetry).

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

The present invention also relates to a composition comprising El and/or E2 WO 96/04385 PCT/EP95/03031 and/or E1 /E2 recombinant HCV proteins purified according to the method of the present invention or a composition comprising at least one peptides as specified above for use as a medicament.

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

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

Also, the present invention relates to a vaccine composition for immunizing a mammal, preferably humans, against HCV, comprising HCV single or specific oligomeric proteins or peptides derived from the 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 1 protein is type-specific. Cocktails including type 1 E2 protein and El 1 proteins derived from several genotypes may be particularly advantageous. Cocktails containing a molar excess of El versus E2 or E2 versus El may also be particularly useful.

Immunogenic compositions may be administered to animals to induce production of antibodies, either to provide a source of antibodies or to induce protective immunity in the animal.

Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.

Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, WO 96/04385 PCT/EP95/03031 26 amino acid copolymers; and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: aluminim hydroxide (alum), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) as found in U.S. Patent No. 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 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 (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).

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 (t

~I

WO 96/04385 PCTEP95/03031 27 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 pg/dose, more particularly from 0.1 to 100 ug/dose.

The single or specific oligomeric envelope proteins may also serve as vaccine 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 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 V 1 region (encompassing amino acid positions 191 to 202), the V2 region (encompassing amino acid positions 213 to 223), the V3 region (encompassing amino acid positions 230 to 242), the V4 region (encompassing amino acid positions 230 to 242), the V5 region (encompassing amino acid positions 294 to 303) and the V6 region (encompassing amino acid positions 329 to 336). Another useful location for insertion of haptens is the hydrophobic region (encompassing approximately amino acid positions 264 to 293). It is shown in the present invention that this region can be deleted without affecting the reactivity of the deleted El protein with antisera. Therefore, haptens may be inserted at the site of the deletion.

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

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

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

The present invention also relates to a method for in vitro diagnosis of HCV antibodies present in a biological sample, comprising at least the following steps WO 96/04385 PCTIEP95/03031 contacting said biological sample with a composition comprising any of the envelope peptide or proteins as defined above, preferably in an immobilized form under appropriate conditions which allow the formation of an immune complex, wherein said peptide or protein can be a biotinylated peptide or protein which is covalently bound to a solid substrate by means of streptavidin or avidin complexes, (ii) removing unbound components, (iii) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies having conjugated to a detectable label under appropriate conditions, (iv) detecting the presence of said immune complexes visually or mechanically 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 at least one peptide or protein composition as defined above, possibly in combination with other polypeptides or peptides from HCV or other types of HCV, with said peptides or proteins being preferentially immobilized on a solid substrate, more preferably on different microwells of the same ELISA plate, and even more preferentially on one and the same membrane strip, a buffer or components necessary for producing the buffer enabling binding reaction between these polypeptides or peptides and the antibodies against HCV present in the biological sample, means for detecting the immune complexes formed in the preceding 0 binding reaction, possibly also including an automated scanning and interpretation device for inferring the HCV genotypes present in the sample from the observed binding pattern.

The immunoassay methods according to the present invention utilize single or WO 96/04385 PCT/EP95/03031 29 specific oligomeric antigens from the El and/or E2 domains that maintain linear (in case of peptides) and conformational epitopes (single or specific oligomeric proteins) recognized by antibodies in the sera from individuals infected with HCV. It is within the scope of the invention to use for instance single or specific oligomeric antigens, dimeric antigens, as well as combinations of single or specific oligomeric antigens. The HCV El and E2 antigens of the present invention may be employed in virtually any assay format that employs a known antigen to detect antibodies. Of course, a format that denatures the HCV conformational epitope should be avoided or adapted. A common feature of all of these assays is that the antigen is contacted with the body component suspected of containing HCV antibodies under conditions that permit the antigen to bind to any such antibody present in the component. Such conditions will typically be physiologic temperature, pH and ionic strenght using an excess of antigen. The incubation of the antigen with the specimen is followed by detection of immune complexes comprised of the antigen.

Design of the immunoassays is subject to a great deal of variation, and many formats are known in the art. Protocols may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the immune complex are also known; examples of which are assays which utilize biotin and avidin or streptavidin, and enzyme-labeled and mediated immunoassays, such as 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 sheets or microtiter wells), polystyrene latex in beads or microtiter plates, polyvinylidine fluoride (known as ImmunolonTM), diazotized paper, nylon membranes, activated beads, and Protein A beads. For example, Dynatech ImmunolonTM 1 or Immunlon"T 2 microtiter plates or 0.25 inch polystyrene beads (Precision Plastic Ball) can be used in the heterogeneous format. The solid support containing the antigenic polypeptides is typically washed after separating it from the test sample, and prior to detection of bound antibodies. Both standard and competitive formats are know in the art.

WO 96/04385 PCT/EP95/03031 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 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 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 El /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 WO 96/04385 PCT/EP95/03031 31 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 reaction between HCV antibodies, if any, and the HCV antigen. Detecting whether anti-HCV antibody HCV antigen complexes are formed as a result of the reacting. Blood contributed to the blood supply is from donors that do not exhibit antibodies to the native HCV antigens, El or E2.

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 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, microparticles, microwells of a reaction tray, test tubes and magnetic beads. The signal generating compound can include an enzyme, a luminescent compound, a chromogen, a radioactive element and a chemiluminescent compound. Examples of enzymes include alkaline phosphatase, horseradish peroxidase and beta-galactosidase. Examples of enhancer compounds include biotin, anti-biotin and avidin. Examples of enhancer J r WO 96/04385 PCT/EP95/03031 32 compounds binding members include biotin, anti-biotin and avidin. In order to block the effects of rheumatoid factor-like substances, the test sample is subjected to conditions sufficient to block the effect of rheumatoid factor-like substances. These conditions comprise contacting the test sample with a quantity of anti-human IgG to form a mixture, and incubating the mixture for a time and under conditions sufficient to form a reaction mixture product substantially free of rheumatoid factor-like substance.

The present invention further contemplates the use of E1 proteins, or parts thereof, more particularly HCV single or specific oligomeric 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 E1 protein or a suitable part thereof under conditions allowing the formation of an immunological complex, removing unbound components, calculating the anti-El titers present in said sample (for example at the start of and/or during the course of (interferon) therapy), monitoring the natural course of HCV disease, or prognosing the response to treatment of said patient on the basis of the amount anti-El titers found in said sample at the start of treatment and/or during the course of treatment.

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 E1 peptides as listed in Table 3 were found to be useful for in vitro monitoring HCV disease or prognosing the response to interferon treatment of patients suffering from HCV infection: E1-31 (SEQ ID NO 56) spanning amino acids 181 to 200 of the Core/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 WO 96/04385 PCT/EP95/03031 33 (epitope

B),

1ibEl (SEQ ID NO 53) spanning amino acids 192 to 228 of El regions (V1, C1, and V2 regions (containing epitope 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),

(SEQ ID NO 68) spanning 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 HCV infection comprising: at least one El protein or El peptide, more particularly an El protein or El peptide as defined above, a buffer or components necessary for producing the buffer enabling the binding reaction between these proteins or peptides and the anti-El antibodies present in a biological sample, means for detecting the immune complexes formed in the preceding binding reaction, possibly also an automated scanning and interpretation device for inferring a decrease of anti-El1 titers during the progression of treatment.

It is to be understood that also E2 protein and peptides according to the present invention can be used to a certain degree to monitor/prognose HCV treatment as indicated above for the 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 WO 96/04385 PCT/EP95/03031 34 antibodies of one or more serological types, with at least one of the El and/or E2 and/or E1/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 b.uffer enabling the binding reaction between these proteins or peptides and the anti-El antibodies present in a biological sample, means for detecting the immune complexes formed in the preceding binding reaction, possibly also an automated scanning and interpretation device for detecting the presence of one or more serological types present from the observed binding pattern.

The present invention also relates to the use of a peptide or protein composition as defined above, for immobilization on a solid substrate and incorporation into a reversed phase hybridization assay, preferably for immobilization as parallel lines onto a solid support such as a membrane strip, for determining the presence or the genotype of HCV according to a method as defined above. Combination with other type-specific WO 96/04385 PCT/EP95/03031 antigens from other HCV polyprotein regions also lies within the scope of the present invention.

Ar WO 96/04385 PCT/EP95/03031 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 of plasmid pgs ATA 18 Restriction map of plasmid pMS 66 Restriction map of plasmid pv HCV-11A Anti-E1 levels in non-responders to IFN treatment Anti-E1 levels in responders to IFN treatment Anti-E1 levels in patients with complete response to IFN treatment Anti-E1 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-E1 reactivity competed with peptides Competition of reactivity of anti-E1 monoclonal antibodies with peptides Anti-E1 (epitope 1) levels in non-responders to IFN treatment Anti-E1 (epitope 1) levels in responders to IFN treatment Anti-E1 (epitope 2) levels in non-responders to IFN treatment Anti-E1 (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 amino acid sequences of the respective El or E2 proteins as shown in the sequence listing.

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 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.

30 Figure 22: Figure 23: WO 96/04385 PCT/EP95/03031 Figure 24: Figure 25: Figure 26: Figure 27: ELISA results obtained from fractions obtained after gelfiltration chromatography of 4 different El purifications of cell lysates infected with vvHCV39 (type 1b), vvHCV40 (type 1b), vvHCV62 (type 3a), and vvHCV63 (type Profiles obtained from purifications of El proteins of type 1b type 3a and type 5a (from RK13 cells infected with vvHCV39, vvHCV62, and vvHCV63, respectively; purified on lentil lectin and reduced as in example 5.2 5.3) and a standard The peaks indicated with and represent pure El protein peaks (see Figure 24, El reactivity mainly in fractions 26 to Silver staining of an SDS-PAGE as described in example 4 of a raw lysate of El vvHCV40 (type 1 b) (lane pool 1 of the gelfiltration of 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).

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

Siver staining of an SDS-PAGE gel of the gelfiltration fractions of vvHCV- 39 (Els, type 1b) 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 fraction 26 construct 62, lane 11: fraction 27 construct 62, lane 12: fraction 28 construct 62, lane 13: fraction 29 construct 62, lane 14: Figure 28:

M

fr4 WO 96/04385 PCT/EP95/03031 Figure 29: Figure 30: Figure 31A Figure 31B: Figure 32: Figure 33: Figure 34: fraction 30 construct 62, lane 15: fraction 31 construct 62.

Western Blot analysis with anti-E1 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).

OD

2 8 0 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).

OD

2 8 0 profile (continuous line) of the lentil-lectin gelfiltration chromatography E2 protein pool from RK1 3 cells infected with vvHCV44 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).

OD

2 8 0 profile (continuous line) of the lentil-lectin gelfiltration chromatography E2 protein pool from RK1 3 cells infected with vvHCV44 in which the E2 pool was reduced and blocked according to Example 5.3 (reduced conditions). The dotted line represents the E2 reactivity as detected by ELISA (as in example 6).

Ni2+-IMAC chromatography and ELISA reactivity of the E2 protein as expressed from vvHCV44 after gelfiltration under reducing conditions as shown in Figure 31B.

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

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.

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.

S 30 Figure 35A: WO 96/04385 Figure 35B: PCT/EP95/03031 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.

Figure 36: and NR grou Figure 37: Figure 38: Figure 39: Figure 40: Figure 41: Figure 42A: Figure 42B: Average El antibody (E1Ab) and E2 antibody (E2Ab) levels in the LTR ips.

Averages El antibody (E1Ab) levels for non-responders (NR) and long term responders (LTR) for type 1b and type 3a.

Relative map positions of the anti-E2 monoclonal antibodies.

Partial deglycosylation of HCV El envelope protein. The lysate of vvHCV1OA-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).

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

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

In vitro mutagenesis of HCV El glycoprotein (part First step of PCR amplification.

In vitro mutagensis of HCV El glycoprotein (part Overlap extension and nested PCR.

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

Analysis of El glycoprotein mutants by Western blot expressed in HeLa (left) and RK13 (right) cells. Lane 1: wild type VV (vaccinia virus), Lane 2: original El protein (vvHCV-OA), Lane 3: El mutant Gly-1 (vvHCV-81), Lane 4: El mutant Gly-2 (vvHCV-82), Lane 5: El mutant Gly-3 (vvHCV- 83), Lane 6: El mutant Gly-4 (vvHCV-84), Lane 7: El mutant Lane 8: El mutant Gly-6 (vvHCV-86).

Figure 43: Figure 44A: WO 96/04385 PCT/EP95/03031 Figure 44B: Analysis of El glycosylation mutant vaccinia viruses by PCR amplification/restriction. Lane 1: El (vvHCV-10A), BspE I, Lane 2: E1.GLY-1 (vvHCV-81), BspE/, 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: E1 (vvHCV-1OA), Stu I, Lane 11: E1.GLY-4 (vvHCV-84), Stul, Lane 13: E1 (vvHCV-10A), Sma Lane 14: (vvHCV-85), Snma, Lane 16: El (vvHCV-10A), Stu/, Lane 17: E1.GLY-6 (vvHCV-86), Stu I, Lane 3 6 9 12 15 Low Molecular Weight Marker, pBluescript SK+, Msp 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 280C, medium samples were taken. The equivalent of 2 0 0 pl of culture 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 280C, medium samples were taken.

The equivalent of 350 pl 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 El protein as despected in Example 1.

Table 2 Summary of Anti-E1 tests Table 3 Synthetic peptides for competition studies Table 4: Changes of envelope antibody levels over time.

Table 5: Difference between LTR and NR Table 6: Competition experiments between murine E2 monoclonal antibodies Table 7: Primers for construction of El glycosylation mutants Table 8: Analysis of El glycosylation mutants by ELISA WO 96/04385 PCT/EP95/03031 41 Example 1: Cloning and expression of the hepatitis C virs E1 protein 1. Construction of vaccinia virus recombination vectors The pgptATA 18 vaccinia recombination plasmid is a modified version of pATA1 8 (Stunnenberg et al, 1988) with an additional insertion containing the E. coli xanthine guanine phosphoribosyl transferase gene under the control of the vaccinia virus 13 intermediate promoter (Figure 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 Hindlll-cut pATA18 vector. This created an extra Pac I restriction site (Figure The original Hindll site was not restored.

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 of pgptATA18 (Figure 3).

Oligonucleotide linker with SEQ ID NO 2/95: CCGGG GAGGCCTGCACGTGATCGAGGGCAGACACCATCACCAC CACTAATAGTTAATTAA CTGCA3 3' C CTCCGGACGTGCACTAGCTCCCGTCTGTGGTAGTGGTGGTAGTGATTATCAATTAATT

G

XmaI PstI WO 96/04385 PCT/EP95/03031 42 Example 2. Construction of HCV recombinant plasmids 2.1. Constructs encoding different forms of the E1 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 reombination vectors: HCCI9A (SEQ ID NO HCCI10A (SEQ ID NO HCCI11A (SEQ ID NO HCCI12A (SEQ ID NO HCCI13A (SEQ ID NO 11), and HCCI17A (SEQ ID NO 13) as depicted in Figure 21. cDNA fragments containing the Elcoding regions were cleaved by EcoRI and Hindlll restriction from the respective pSP72 plasmids and inserted into the EcoRI/Hindlll-cut pgptATA-18 vaccinia recombination vector (described in example downstream of the 11K vaccinia virus late promoter.

The respective plasmids were designated pvHCV-9A, pvHCV-1 OA, pvHCV-1 1 A, pvHCV- 12A, pvHCV-13A and pvHCV-17A, of which pvHCV-11A is shown in Figure 4.

2.2. Hydrophobic region El deletion mutants Clone HCCI37, 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 HCCI10A with primer sets HCPr52 (SEQ ID NO 16)/HCPr107 (SEQ ID NO 19) and HCPr108 (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 pSP72HCCI37 containing clone HCCI37 (SEQ ID 15) was selected.

A recombinant vaccinia plasmid containing the full-length El cDNA lacking hydrophobic domain I was constructed by inserting the HCV sequence surrounding the deletion (fragment cleaved by Xma I and BamH I from the vector pSP72-HCCI37) into the Xma I-Bam H I sites of the vaccinia plasmid pvHCV-1 A. The resulting plasmid was named WO 96/04385 PCT/EP95/03031 43 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 Ell-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 E1 carboxyterminus (theoretically extending to around amino acids 337-340) was not completely included in construct pvHCV-38, a 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-1 8 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 HCCI62 (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 HCCI63 (SEQ ID NO 31) was derived from a type 5a-infected child with post-transfusion hepatitis (serum BE95, clone PC-4-1, SEQ ID NO 45 in WO 94/25601).

2.4. E2 constructs The HCV E2 PCR fragment 22 was obtained from serum BE11 (genotype 1 b) by means of primers HCPrl09 (SEQ ID NO 33) and HCPr72 (SEQ ID NO 34) using techniques of RNA preparation, reverse-transcription and PCR, as described in Stuyver al., ,O993, anu mte fragment was cloned into the Sma I-cut pSP72 vector. Clone HCCI22A (SEQ ID NO 35) was cut with Ncol/AlwNI or by BamHI/AlwNI and the sticky ends of the fragments were blunted (Ncol and BamHI sites with Klenow DNA Polymerase I (Boehringer), and AlwNI with T4 DNA polymerase (Boehringer)). The WO 96/04385 PCT/EP95/03031 44 BamHI/AlwNI cDNA fragment was then inserted into the vaccinia pgsATA-18 vector that had been linearized by EcoR I and Hind III cleavage and of which the cohesive ends had been filled with Klenow DNA Polymerase (Boehringer). The resulting plasmid was named pvHCV-41 and encoded the E2 region from amino acids Met347 to Gln673, including 37 amino acids (from Met347 to Gly383) of the El protein that can serve as signal sequence. The same HCV cDNA was inserted into the EcoR I and Bbr PI-cut vector pMS66, that had subsequently been blunt ended with Klenow DNA Polymerase.

The resulting plasmid was named pvHCV-42 and also encoded amino acids 347 to 683.

The Ncol/AlwNI fragment was inserted in a similar way into the same sites of pgsATA- 18 (pvHCV-43) or pMS-66 vaccinia vectors (pvHCV-44). pvHCV-43 and pvHCV-44 encoded amino acids 364 to 673 of the HCV polyprotein, of which amino acids 364 to 383 were derived from the natural carboxyterminal region of the El protein encoding the signal sequence for E2, and amino acids 384 to 673 of the mature E2 protein.

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) per cell). Two hours later, the vaccinia recombination plasmid was transfected into the infected cells in the form of a calcium phosphate coprecipitate containing 500 ng of the plasmid DNA to allow homologous recombination (Graham van der Eb, 1973; Mackett et al., 1985). Recombinant viruses expressing the Escherichia coli xanthine-guanine phosphoribosyl transferase (gpt) protein were selected on rabbit kidney RK13 cells incubated in selection medium (EMEM containing 25 pg/ml mycophenolic acid (MPA), 250pg/ml xanthine, and 1 5 /g/ml hypoxanthine; Falkner and Moss, 1988; Janknecht et al, 1991). Single recombinant viruses were purified on fresh L- WO 96/04385 PCT/EP95/03031 monolayers of RK1 3 cells under a 0.9% agarose overlay in selection medium. Thymidine kinase deficient 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 pl) 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-10A 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 p/ of the virus solution was added per 106 cells such that the m.o.i. was 3, and incubated for 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.

Example 4: Analysis of recombinant proteins by means of western blottin The infected cells were washed two times with PBS, directly lysed with lysis buffer (50 mM Tris.HCI pH 7.5, 150 mM NaCI, 1% Triton X-1 00, 5 mM MgCI 1 /g/ml aprotinin (Sigma, Bornem, Belgium)) or detached from the flasks by incubation in 50 mM Tris.HCL pH 7.5/ 10 mM EDTA/ 150 mM NaCI for 5 min, and collected by centrifugation (5 min at 1000g). The cell pellet was then resuspended in 200 pl lysis buffer (50 mM Tris.HCL pH 8.0, 2 mM EDTA, 150 mM NaCI, 5 mM MgCI 2 aprotinin, 1% Triton X-100) per 106 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 n WO 96/04385 PCT/EP95/03031 46 (SDS-PAGE). The proteins were then electro-transferred from the gel to a nitrocellulose sheet (Amersham) using a Hoefer HSI transfer unit cooled to 4°C for 2 hr at 100 V constant voltage, in transfer buffer (25 mM Tris.HCI pH 8.0, 192 mM glycine, 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 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/O.1 Tween 20, the nitrocellulose filters were incubated with alkaline phosphatase substrate solution diluted in Blotto/O.1 Tween 20. After washing with 0.1 Tween 20 in PBS, the filters were incubated with alkaline phosphatase substrate solution (100 mM Tris.HCI pH 9.5, 100 mM NaCI, 5 mM MgCI,, 0,38 pg/ml nitroblue tetrazolium, 0.165 p/g/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.HCI pH 7.5, 150 mM NaCI, 1% Triton X-100, 5 mM MgCI 2 1 pg/ml aprotinin (Sigma, Bornem, Belgium) containing 2 mM biotinylated

N-

ethylmaleimide (biotin-NEM) (Sigma) was added per 10" cells at 4°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 JA-14 rotor at 14,000 rpm (30100 g at for 1 hour at 4'C.

A

WO 96/04385 PCT/EP95/03031 47 5.2. Lectin Chromatography The cleared cell lysate was loaded at a rate of 1 ml/min on a 0.8 by 10 cm Lentillectin Sepharose 4B column (Pharmacia) that had been equilibrated with 5 column volumes of lysis buffer at a rate of 1ml/min. The lentil-lectin column was washed with to 10 column volumes of buffer 1 (0.1M potassium phosphate pH 7.3, 500 mM KCI, 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 MgCI 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 vvHCV39 (type 1 vvHCV40 (type 1 vvHCV62 (type 3a), and vvHCV63 (type 5a). Figure 23 shows the profiles obtained from the values shown in Figure 22. These results show that the-lectin affinity column can be employed for envelope proteins of the different types of HCV.

5.3. Concentration and partial reduction The El- or E2-positive fractions were pooled and concentrated on a Centricon 30 kDa (Amicon) by centrifugation for 3 hours at 5,000 rpm in a Beckman JA-20 rotor at 4°C. In some experiments the El- or E2-positive fractions were pooled and concentrated by nitrogen evaporation. An equivalent of 3.108 cells was concentrated to approximately 200 pl. For partial reduction, 30% Empigen-BB (Calbiochem, San Diego, CA, USA) was added to this 200 pl 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 37 NEM (1M in dimethylsulphoxide) was subsequently added to a final concentration of 50 mM and left to react for another 30 min at 37°C to block the free sulphydryl groups.

L WO 96/04385 PCT/EP95/03031 48 5.4. Gel filtration chromatography 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/ p sample loop of the Smart System (Pharmacia) and PBS/3% Empigen-BB buffer was added for gelfiltration. Fractions of 250 pl were collected from Vo to 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), vvHCV40 (type 1b), vvHCV62 (type 3a), and vvHCV63 (type 5a). Figure shows the profiles obtained from purifications of El proteins of types b, 3a, and (from RK13 cells infected with vvHCV39, vvHCV62, 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 peaks 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 E1 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 streptavidin-alkaline phosphatase as shown in Figure 27. It can be readily observed that, amongst others, the 29 kDa and 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 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 which shows a faster mobility on SDS/PAGE because of the presence of only 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- WO 96/04385 PCT/EP95/03031 49 aggregated and free of contaminants. The subtype lb El protein purified from 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 of Hijikata 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 2 8 0 profile (continuous line) of the lentil lectin chromatography. The dotted line represents the E2 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 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 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 pg of purified E2 protein and a 30 mM imidazole wash. The pure E2 protein could be easily recovered by a 200 mM imidazole elution step. 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 2 of RK13 cells infected with vvHCV11A (or for the production of El or vvHCV41, vvHCV42, vvHCV43, or vvHCV44 for production of E2 protein, the procedures described in examples 5.1 to 5.5 allow the w WO 96/04385 PCT/EP95/03031 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 30-40%, 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-E1 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/pl or 100 /1 or 200 pl) per well of a 5 /g/ml solution of Streptavidin (Boehringer Mannheim) in PBS for 16 hours at 4°C or for 1 hour at 37 C. Alternatively, the wells were coated with 1 volume of 5 pg/ml of Galanthus nivalis agglutinin (GNA) in 50 mM sodium carbonate buffer pH 9.6 for 16 hours at 4 C or for 1 hour at 37 In the case of coating with GNA, the plates were washed 2 times with 400 pl 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 °C or for 16 hours at 4 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 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°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 diluted to a concentration of 20 ng/ml in Sample Diluent of the Innotest HCV Ab Ill kit and 1 volume of the solution was left to react with the El or E2 protein for 1 hour at 37°C. The microwells were washed times with 400 pl of Washing Solution of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium). The bound antibodies were detected by incubating each well for 1 hour at 37°C with a goat anti-human or anti-mouse IgG, peroxidase-conjugated secondary antibody (DAKO, Glostrup, Denmark) diluted 1/80,000 in 1 volume of Conjugate Diluent of the Innotest HCV Ab III kit (Innogenetics, Zwijndrecht, Belgium), WO 96/04385 PCT/EP95/03031 51 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°C after washing of the plates 3 times with 400 pl 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-El 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 anti-envelope antibodies may prove more useful in a clinical setting. To investigate the possibility of the use of anti-E1 antibody titer and anti-E2 antibody titer as prognostic markers for outcome of hepatitis C disease, a series of IFN-a treated patients with long-term 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 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 1b) was compared with 9 patients showing non-complete responses to treatment (NR, follow up 1 to 4 years, 6 type 1b and 3 type 3a). Type lb (vvHCV-39, see example and 3a El (vvHCV-62, see example proteins 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 1 b hepatitis C virus were tested for reactivity with purified type 1 b El protein, while samples of a type 3a infection were tested for reactivity of anti-type 3a E1 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-E1 signal-to-noise ratios of these patients followed during the course of interferon WO 96/04385 PCT/EP95/03031 52 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 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-El antibody titers had decreased 6.9 times in LTR but only 1.5 times in NR. At the end of follow up, the anti-El titers had declined by a factor of 22.5 in the patients with sustained response and even slightly increased in NR. Therefore, based on these data, decrease of anti-El antibody levels during monitoring of IFN-a therapy correlates with long-term, sustained response to treatment. The anti-El assay may be very useful for prognosis of long-term response to IFN treatment, or to treatment of the hepatitis C disease in general.

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 seroconversion for anti-HBsAg antibodies. Also in many other virus infections, the virus is eliminated when anti-envelope antibodies are raised. However, in the experiments of the present invention, anti-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, this non-obvious finding may be very important and useful for clinical diagnosis of HCV infections. As shown in Figures 9, 10, 11, and 12, anti-E2 levels behaved very differently in the same patients studied and no obvious decline in titers was observed as for anti-El 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-El antibodies at the start of treatment, or monitoring the patient during the course of infection and measuring the anti-El titer, may become a useful marker for clinical diagnosis of hepatitis C.

Furthermore, the use of more defined regions of the El or E2 proteins may become desirable, as shown in example 7.3.

WO 96/04385 PCT/EP95/03031 53 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 1 b and 3a-infected LTR and further supplemented the cohort with a matched series of NR, such that both groups included 14 patients each. Some partial responders (PR) and responders with relapse (RR) were also analyzed.

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-a 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 (Table Within the type 1b-infected group, LTR also had higher El antibody levels than NR at the initiation of treatment [P 0.05]; the limited number of type 3ainfected NR did not allow statistical analysis.

Of antibody levels monitored in LTR during the 1.5-year follow up period, only El antibodies cleared rapidly compared with levels measured at initiation of treatment [P 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 3infected 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 ,oidU ALT levels. The minute quantities of viral RNA, still present in the RR group during treatment, seemed to be capable of anti-E B cell stimulation. Anti-El monitoring may therefore not only be able to discriminate LTR from NR, but also from RR.

WO 96/04385 PCT/EP95/03031 54 7.3. Monitoring of antibodies of defined reqions 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 Agtl 1 libraries predominantly yielded linear epitopes dispersed throughout the core and non-structural regions, and analysis of the envelope regions had to await cloning and expression of the E1/E2 region in mammalian cells. This approach sharply contrasts with many other viral infections of which epitopes to the envelope regions had already been mapped long before the deciphering of the genomic structure.

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

As no HCV vaccines or tests allowing clinical diagnosis and prognosis of hepatitis

C

disease are available today, the characterization of viral envelope regions exposed to immune surveillance may significantly contribute to new directions in HCV diagnosis and prophylaxis.

Several 20-mer peptides (Table 3) that overlapped each other by 8 amino acids, were synthesized according to a previously described method (EP-A-O 489 968) based on the HC-J1 sequence (Okamoto et al., 1990). None of these, except peptide (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 pg/ml of El peptide with the 1/20 diluted human serum in sample diluent.

Figure 13 shows the results of reactivity of human sera to the recombinant El (expressed from vvHCV-40) protein, in the presence of single or of a mixture of El peptides. 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 the same peptides, when tested on the recombinant El protein. Some of the murine monoclonal antibodies obtained from Baibi/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 WO 96/04385 PCT/EP95/03031 detected. This finding was surprising, since the env53 and env31 peptides had not shown any reactivity when coated directly to the solid phase.

Therefore peptides were synthesized using technology described by applicant previously (in WO 93/18054). The following peptides were synthesized: peptide NH2-SNSSEAADMIMHTPGCV-GKbiotin (SEQ ID NO 51) spanning amino acids 208 to 227 of the HCV polyprotein in the El region peptide biotin-env53 ('epitope A') biotin-GG-ITGHRMAWDMMMNWSPTTAL-COOH (SEQ ID NO 52) spanning amino acids to 313 of 332 of the HCV polyprotein in the E1 region peptide 1bE1 ('epitope B') H2N-YEVRNVSGIYHVTNDCSNSSIVYEAADMIMHTPGCGK -biotin(SEQID NO 53) spanning amino acids 192 to 228 of the HCV polyprotein in the El region and compared with the reactivities of peptides Ela-BB (biotin-GG- TPTVATRDGKLPATQLRRHIDLL, SEQ ID NO 54) and Elb-BB (biotin-GG- TPTLAARDASVPTTTIRRHVDLL, SEQ ID NO 55) which are derived from the same region of sequences of genotype 1 a and 1 b 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 (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 remiission of disease, while the anti-lbE1 antibodies (epitope B) were present almost exclusively in long term responders at the start of IFN treatment. Therefore, anti-1 bE1 (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 WO 96/04385 PCT/EP95/03031 56 advantageously used for the detection of cross-reactive antibodies (antibodies that cross-react between major genotypes) and antibodies to the env53 region may be very useful for universal El antigen detection in serum or liver tissue. Monoclonal antibodies that recognized the env53 region were reacted with a random epitope library. In 4 clones that reacted upon immunoscreening with the monoclonal antibody 5E1 A1 0, the sequence -GWD- was present. Because of its analogy with the universal HCV sequence present in all HCV variants in the env53 region, the sequence AWD is thought to contain the essential sequence of the env53 cross-reactive murine epitope. The env31 clearly also contains a variable region which may contain an epitope in the amino terminal sequence -YQVRNSTGL- (SEQ ID NO 93) and may be useful for diagnosis.

Env31 or E1-31 as shown in Table 3, is a part of the peptide 1bE1. Peptides El-33 and El-51 also reacted to some extent with the murine antibodies, and peptide E1-55 (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-E1 antibodies, especially in patients with a long-term response to treatment. Therefore, it is clear that the decrease in anti-envelope antibodies could not be measured as efficiently with an assay employing a recombinant E1/E2 protein as with a single anti-E1 or anti-E2 protein.

The anti-E2 response would clearly blur the anti-E1 response in an assay measuring both kinds of antibodies at the same time. Therefore, the ability to test anti-envelope antibodies to the single El and E2 proteins, was shown to be useful.

7.4. Mappinq 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, 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 4' WO 96/04385 PCT/EP95/03031 57 antibodies in the supernatant of hybridomas 15C8C1, 12D11F1, 9G3E6, 8G10D1H9, 10D3C4, 4H6B2, 17F2C2, 5H6A7, 15B7A2 recognizing conformational epitopes were purified by means of protein A affinity chromatography and 1 mg/ml of the resulting IgG's were biotinylated in borate buffer in the presence of biotin. Biotinylated antibodies were separated from free biotin by means of gelfiltration chromatography. Pooled biotinylated antibody fractions were diluted 100 to 10,000 times. E2 protein bound to the solid phase was detected by the biotinylated IgG in the presence of 100 times the amount of non-biotinylated competing antibody and subsequently detected by alkaline phosphatase labeled streptavidin.

Percentages of competition are given in Table 6. Based on these results, 4 conformational anti-E2 epitope regions (epitopes F, 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).

Example 8: El glycosylation mutants 8.1. Introduction The El protein encoded by vvHCV1 OA, 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 vvHCV1OA-infected or vv. .v I A-,Iec rted I 3 cells with decreasing concentrations of glycosidases (PNGase F or Endoglycosidase H, (Boehringer Mannhein Biochemica) according to the .manufacturer's instructions), such that the proteins in the lysate (including El) are partially deglycosylated (Fig. 39 and 40, respectively).

E

WO 96/04385 PCT/EP95/03031 58 Mutants devoid of some of their glycosylation sites could allow the selection of envelope proteins with improved immunological reactivity. For HIV for example, gpl proteins lacking certain selected sugar-addition motifs, have been found to be particularly useful for diagnostic or vaccine purpose. The addition of a new oligosaccharide side chain in the hemagglutinin protein of an escape mutant of the A/Hong Kong/3/68 (H3N2) influenza virus prevents reactivity with a neutralizing monoclonal antibody (Skehel et al, 1984). When novel glycosylation sites were introduced into the influenza hemaglutinin protein by site-specific mutagenesis, dramatic antigenic changes were observed, suggesting that the carbohydrates serve as a modulator of antigenicity (Gallagher et al., 1988). In another analysis, the 8 carbohydrate-addition motifs of the surface protein gp70 of the Friend Murine Leukemia Virus were deleted. Although seven of the mutations did not affect virus infectivity, mutation of the fourth glycosylation signal with respect to the amino terminus resulted in a non-infectious phenotype (Kayman et al., 1991). Furthermore, it is known in the art that addition of N-linked carbohydrate chains is important for stabilization of folding intermediates and thus for efficient folding, prevention of malfolding and degradation in the endoplasmic reticulum, oligomerization, biological activity, and transport of glycoproteins (see reviews by Rose 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 1b El protein are required for proper folding and reactivity, since some are absent in certain (sub)types. The fourth carbohydrate motif (on Asn251), present in types 1 b, 6a, 7, 8, and 9, is absent in all other types know today. This sugar-addition motif may be mutated to yield a type lb 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 V1 region (on Asn), while it is present on all other isolates (Stuyver et al., 1994) However, even among the completely conserved sugar-addition motifs, the presence of the carbohydrate may not be required for folding, but may have a role in evasion of immune surveillance. Therefore, identification of the carbohydrate addition motifs which are not required for proper folding (and reactivity) is not obvious, and each mutant has to be analyzed and tested for reactivity. Mutagenesis of a glycosylation motif (NXS or NXT sequences) can be achieved by either mutating the codons for N, S, or T, in such a way that these codons encode amino acids different from N in the case of N, and/or amino L I WO 96/04385 PCT/EP95/03031 59 acids different from S or T in the case of S and in the case of T. Alternatively, the X position may be mutated into P, since it is known that NPS or NPT are not frequently modified with carbohydrates. After establishing which carbohydrate-addition motifs are required for folding and/or reactivity and which are not, combinations of such mutations may be made.

8.2. Mutaqenesis of the E1 protein All mutations were performed on the E1 sequence of clone HCCI10A (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 11 K 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 Gin 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).

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-1OA) 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 the sequence of N-linked glycosylation sites.

For site-directed mutagenesis, the 'mispriming' or 'overlap extension' (Horton, 19.) vvas usu. TIe 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 Table 7) and with the respective 3' antisense GLY# primers. The second fragment Q t WO 96/04385 PCT/EP95/03031 (product OVR#) was amplified with the 3' antisense TK, primer and the respective 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 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 alvcosvlation mutants Starting from the 6 plasmids containing the mutant El sequences as described in example 8.2, recombinant vaccinia viruses were generated by recombination with wt vaccinia virus as described in example 2.5. Briefly, 175 cm 2 -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 IS I* WO 96/04385 PCT/EP95/03031 61 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 indicate that removal of the 1st (GLY1), 3rd (GLY3), and 6th (GLY6) glycosylation motifs reduces reactivity of some sera, while removal of the 2nd and 5th site does not. Removal of GLY4 seems to improve the reactivity of certain sera. These data indicate that different patients react differently to the glycosylation mutants of the present invention. Thus, such mutant El proteins may be useful for the diagnosis (screening, confirmation, prognosis, etc.) and prevention of HCV disease.

Example 9: Expression of HCV E2 protein in lvcosvlation-deficient easts The E2 sequence corresponding to clone HCCL41 was provided with the amating 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 highmannose type glycosylations upon expression of such a construct in S. cerevisiae strains (Figure 45). This resulted in a too high level of heterogeneity and in shielding of reactivity, which is not desirable for either vaccine or diagnostic purposes. To overcome his 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 WO 96/04385 PCT/EP95/03031 62 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 present invention which guarantee the elimination of contaminating proteins and which preserve the conformation, and thus the reactivity of the HCV envelope proteins. The amounts of purified HCV envelope protein needed for diagnostic screening assays are in the range of grams per year. For vaccine purposes, even higher amounts of envelope protein would be needed. Therefore, the vaccinia virus system may be used for selecting the best expression constructs and for limited upscaling, and large-scale expression and purification of single or specific oligomeric envelope proteins containing high-mannose carbohydrates may be achieved when expressed from several yeast strains. In the case of hepatitis B for example, manufacturing of HBsAg from mammalian cells was much more costly compared with yeast-derived hepatitis

B

vaccines.

The purification method 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 but also less related virusses such as Hepatitis B Virus (mainly for the purification of HBsAg).

I Te envelupe 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.

WO 96/04385 63PC/9/301 PCT/EP95/03031 Table 1: Recombinant vaccinial plasmids and viruses Plasmid name Name cDNA subclone Length (nt/aa) Vector used construction for insertion pvHCV-13A Els EcoR I Hind 111 472/1 57 pgptATA-18 pvHCV-1 2A Els EcoR I Hind III 1 472/158 pg ptATA- 18 pvHCV-9A Ell EcoR I Hi nd 111 631/211 pgptATA-1 8 pvHCV- 11A Els EcoR I Hind 111 625/207 pgptATA-18 pvHCV-17A Els EcoR I Hind 111 625/208 pg ptATA- 18 pvHCV-lOA El EcoR I Hind 111 783/262 pg ptATA- 18 pvHCV-1 8A COREs -Acc I (KI) EcoR I (KI1) 403/130 pg ptATA- 18 PvHCV-34 CORE Acc I (KI) Fsp 1 595/197 pgptATA-18 pvHCV-33 CORE-El Acc I (KI) 1150/380 pg ptATA- 18 CORE-Elb.his EcoR I BamH I (KI1) 1032/352 pMS-66 pvHCV-36 CORE-El n.his EcoR I Nco I (KI) 1106/376 pMS-66 pvHCV-37 ElIA Xma I BamH 1 711/239 pvHCV-1OA pvHCV-38 ElAs EcoR I BstE 11 553/183 pvHCV-11 lA pvHCV-39 ElAb EcoR I BamH 1 960/313 pgsATA-18 E1Ab.his EcoR I BamH I (KI) 960/323 pMS-66 pvHCV-41 E2bs BamH I (KI)-AIwN I (T4) 1005/331 pgsATA-18 pvHCV-42 E2bs.his BamH I (KI)-AIwN I (T4) 1005/341 pMS-66 -pvHCV-43 E2ns Nco I (KI) AlwN I (T4) 932/314 pgsATA- 18 -pvHCV-44 E2ns.his Nco I (KI) AlwN I (14) 1 932/32 1 pMS-66 -pvHCV-62 El1s (type 3a) EcoR I Hind 111 625/207 pgsATA-18 -pvHCV-63 Els (type 5) EcoR I Hind 111 625/207 pgsATA-18 -pvHCV-64 E2 BamH I Hind 111 1410/463 pgsATA-18 E1-E2 BamH I Hind 111 2072/691 pvHCV-66 CORE-E1l-E2 BamH I Hind 111 2427/809 pvHCV-33 nt: nucleotide aa: aminoacid Ki: Kienow DNA Pol filling Position: aminoacid position in the HCV polyprotein sequence 14: T4 DNA Pol filling SUBTITUTE SHEET (RULE 26) WO 96/04385 PCTJEP95/03031 54 Table 1 cogntinued: Recombinant vacciniap lasmids and viruses Plasmid HCV cDNA subclone Vector Name Name IConstruction J Length used for I (ntlaa) insertion j pvHCV-81 El *-GLY 1 EcoRi BamH 1 783/262 pvHCV-1OA pvHCV-82 El *-GLY 2 EcoRi BamH 1 783/262 pvHCV-lOA pvHCV.-83 El *-GLY 3 EcoRi BamH 1 783/262 pvHCV-lOA pvHCV-84 El *-GLY 4 EcoRI BamH 1 783/262 pvHCV-1OA El *-GLY 5 EcoRi BamH 1 783/262 pvHCV-lOA pvHCV-86 El *.-GLY 6 EcoRi BamH 1 783/262 pvHCV-lOA nt: nucleotide aa: aminoacid KI: Klenow DNA Pol filling Position: ammnoacid position in the HCV polyprotein sequence T4: T4 DNA Pal filling SUBSTITUTE SHEET (RULE 26)

_M

WO 96/04385 PCT/EP95/03031 Table 2 Summary of anti-El tests SIN SD (mean anti-E1 titer) Start of treatment End of treatment Follow-up LTR 6.94+2.29 4.48 2.69 (1:568) 2.99 2.69 (1:175) (1:3946) NR 5.77 3.77 5.29 3.99 6.08 3.73 (1:1607) (1:1060) (1:1978) LTR Long-term, sustained response for more than 1 year NR No response, response with relapse, or partial response WO 96/04385 PCTJEP95/0303 1 Table 3 Synthetic peptides for competition studies

PROTEIN

PEPTIDE AMINO ACID SEQUENCE El-31 El-33 El-37 El-39 El1-41 El -43 El -45 El -49 El-51 El-53 El-57 El-59 El-63

LLSCLTVPASAYQVRNSTGL

QVRNSTGLYHVTNDCPNSSI

NDCPNSSIVYEAHDAILHTP

SNSS iVYEAADM IM HTPG CV HDA ILHTPG CVPC VREGN VS CVREGNVS

RCWVAMTPTVAT

AMTPTVATRDG

KLPATQLRR

LPATQLRRHIDLLVGSATLC

LVGSATLCSALYVGDLCGSV

QLFTFSPR RH WTTQGCNCS

I

TQGCNCSIYPGHITGHRMAW

ITGHRMAWDMMMNWSPTAAL

NWSPTAALVMAQLLRIPQAI

LLRIPQAILDMIAGAHWGVL

AGAHWGVLAGIAYFSMVGNM

VVLLLFAGVDAETIVSGG

QA

POSITION

181-200 193-2 12 205-224 208-227 217-236 229-248 241-260 3-272 265-284 289-308 301-320 313-332 325-344 3 37-35 6 349-368 373-392 SEQ ID NO 56 57 58 59 61 62 63 64 66 67 68 69 71 WO 96/04385 PCT/EP95/0303 1 E2-67 E2-69 E2-$3B E2-$1 B E2-1 B E2-3B E2-7B E2-9B E2-1 1lB E2-1 3B E2-1 7B E2-1 9B E2-21 E2-23 E2-25 E2-27 E2-29 E2-31 E2-33 E2-35

SGLVSLFTPGAKQNIQLINT

QNIQLINTNGSWHINSTALN

LNCNESLNTG WWLAGLIYQH

K

AG LIYQH KFN SSG CPE RLAS

GCPERLASCRPLTDFDQGWG

TDFDQGWGPISYANGSGPDQ

ANGSGPDQRPYCWHYPPKPC

WHYPPKPCGIVPAKSVCGPV

AKSVCGPVYCFTPSPVVVGT

PSPVVVGTTDRSGAPTYSWG

GAPTYSWGENDTDVFVLNNT

GNWFGCTWMNSTGFTKVCGA

GFTKVCGAPPVCIGGAGNNT

IGGAGNNTLHCPTDCFRKHP

TDCFRKHPDATYSRCGSGPW

SRCGSGPWITPRCLVDYPYR

CLVDYPYRLWHYPCTINYTI

PCTINYTIFKIRMYVGGVEH

MYVGGVEHRLEAACNWTPGE

ACNWTPGERCDLEDRDRSEL

EDRDRSELSPLLLTTTQWQV

397-416 409-428 427-446 439-458 451-470 463-482 475-494 487-506 499-5 18 511-530 52 3-542 547-5 66 9-5 78 57 1-590 83-602 595-6 14 607-626 619-638 63 1-650 643-662 655-674 72 73 74 76 7 7 78 79 81 82 83 84 86 87 88 89 91 92

,A

h i I W

L-

Table 4. Change of Envelope Antibody levels over time (complete study, 28 patients) Wilcoxon Signed Rank test (P values) ElAb NR ElAb NR ElAb NR ElAb LTR ElAb LTR ElAb LTR type l b type 3a All type l b type 3a E2Ab NR El Ab LTR End of therapy* 0.1167 0.2604 0.285 0.005-8----0.043-, -0.0499** 0.0186*' 0.0640 6 months follow up* 0.86 0.72 13 0.5930 0.Q004 7 0.043** 0.063 0.04326 0.0464'- 12 months follow up* 0.7989 0.3105 1 0.0051" 0.0679 0.0277-' 0.0869 0.0058** Data were compared wivale banda ntainohrp P values 0.05 0 Table 5. Difference between LTR and NR (complete study) Mann-Withney ElAb SIN El1Ab titers ElAb SIN ElAb SIN E2Ab SiN U test (P values) All All type l b type 3a All initiation of therapy 0.0257' 0.05 0.68 0.1078 End of therapy 0.1742 0.1295 6 months follow up, 1 0.6099 0.42 5 0.3081 12 months follow up 0.67 0.23 0.4386 0.6629 P values <c 0.05

M

Ml 4

M

Table 6. Competition experiments between murine E2 monoclonal antibodies Decrease of anti-E2 reactivity of biotinylated anti-E2 mabs competitor 17H1QF4D1O 2F1OH1O 16A6E7 10D3C4 4H6B2 17C2F2 9G3E6 12D11F1 15C8G1 8G1OD1H9 17H1OF4D1O 2F1OH1O E 16A6E7 IN 10OD3C4 1 4HI612 I 17G2F2 2 9G3E6 N 12D 11 F1 N 10 1 92 82 75 68 26 18 11 6 4

ND

43

ND

ND

082 0 4

ND

15C8C1 ND 8G1OD1H9 2 competitor controls 15137A2 0 51-6A7 0 23C12H9 ND ND, not done 0 9 1 2 0 12 IND 2 12 10 9 0 8 0 0 ND 4 ND

.I

I I Table 7. Primers SEQ ID NO. 96 GPT 5'-GTTTAACCACTGCATGATG-3' SEQ ID NO. 97 TKR 5'-GTCCCATCGAGTGCGGCTAC-3' SEQ ID NO. 98 GLYl 5'-CGTGACATGGTACATTCCGGACACTTGGCGCACTTCATAAGCGGA- 3 SEQ ID NO. 99 GLY2 5'-TG CCTCATACACAATG GAG CTCTG GGACGAGTCGTTCGTGAC- 3 SEQ ID NO. 100O GLY3 5'-TACCCAGCAGCGGGAGCTCTGTTGCTCCCGAACGCAGGGCAC- 3 SEQ ID NO. 101 GLY4 5'-TGTCGTGGTGGGGACGGAGGCCTGCCTAGCTGCGAGCGTGGG- 3 SEQ ID NO. 102 GLY5 5'-CGTTATGTGGCCCGGGTAGATTGAGCACTGGCAGTCCTGCACCGTCTC- 3 SEQ ID NO. 1 C13 GLY6 5'-CAGGGCCGTTGTAGGCCTCCACTGCATCATCATATCCCAAGC- 3 SEQ ID NO. 104 OVR1 5 '-CCGGAATGTACCATGTCACGAACGAC-3' SEQ IQ NO. 105 OVR2 5'-GCTCCATTGTGTATGAGGCAGCGG-3' SEQ ID NO. 106 OVR3 5'-GAGCTCCCGCTGCTGGGTAGCGC3' SEQ ID NO. 107 OVR4 5'-CCTCCGTCCCCACCACGACAATACG-3' SEQ ID NO. 108 OVR5 5'-CTACCCGGGCCACATAACGGGTCACCG-3' SEQ ID NO. 109 OVR6 5'-GGAGGCCTACAACGGCCCTGGTGG-3' SEQ ID NO. 110 GPT-2 5'-TTCTATCGATTAAATAGAATTC -3' SEQ ID NO. 111 TKR-2 5'-GCCATACGCTGACAGCCGATCCC-3' nucleotides underlined represent additional restriction site nucleotides in bold represent mutations with respect to the original HCCI 1OA sequence Taje8Anj)~p~j_~ EltMM ans1

SERUM

SN GLYI SN GLY2* SN GLY3 SN GLY4 SN GLY5 SN GLY6 SN El SN GLYl ,SN GLY2 ~SN GLY3 ~SN GLY4 q..SN GLY5 C-:SN GLY6 r

T

-SN El mGLYI/El r~GLY2/E I 'GLY3IEl GLY4/El GLY6IEl GLYlIEl GLY2/E 1 GLY3IE 1 GLY4IEl 1 GLY6/E I 1.802462 2.400795 1.642718 2.578154 2.482051 2.031487 2.828205 13 5.685561 7.556682 7.930538 8.176816 8.883408 8.005561 8. 825112 2.120971 1.76818 1.715477 3.824 038 1.7921761 1.495737 2.227036 14 3.233604 2.567613 2.763055 6.561122 2.940334 2.499952 3. 183771 3 1.40387 1 2.325495 2.261646 3.874605 2.409344 2.131613 2.512792 15 3.763498 3.621928 3.016099 5.707668 3.125561 2.621704 3.067265 4 1.205597 2.639308 2.354748 1.499387 2.627358 2.527925 2.790881 5 2.120191 2.459019 1.591818 3.15 1.7 15311 2 .494 833 3. 131579 6 2.866913 5.043993 4.833742 4.7 1302 4.964765 4.784027 4.869128 7 1.950345 2.146302 1.96692 4. 198751 2.13912 2.02069 2.287753 19 1.93476 2. 127712 1.980185 3.813321 2.442804 1.506716 2.7712 18 8 1.866 183 1.595477 1.482099 3.959542 1.576336 1.496489 1.954 198 20 2.4717 1 2.92 1288 2.557384 3.002535 3. 126761 2.665433 3.678068 9 1.730193 1.688973 1.602222 3.710507 1. 708937 1.704976 1.805556 21 4.378633 4.680101 4.268633 4.293038 4.64557 2.781063 5.35443 10 2.468162 2.482212 2.191558 5.17084 1 3.02 1807 2.677757 2.6 16822 22 1. 188748 1.15078 1 0.97767 2.393011 1.153656 1.280743 1. 167286 11 1.220654 1.467582 1.4642 16 4.250784 1.562092 1.529608 1.557 19 23 2. 158889 1.66 1914 1.336775 3.68213 1.8 17901 1.475062 2.083333 12 1.629403 2.070524 1.72 1164 3.955153 2.07278 1.744221 2.593886 24 1.706992 1.632785 1.20376 2.48 1585 1.6382 11 1.7 16423 1.78252 16 17 18 1.985105 2.317721 6.675179 3.055649 2.933792 7.65433 2.945628 2.515305 5.775357 5.684498 5.604813 6.4125 3.338912 2.654224 5.424107 2.572385 2.363301 5.194107 3.280335 2.980354 7.191964 Sum

S/N

59.88534 69.65243 62.09872 102.6978 69.2651 1 61. 32 181 76.54068 Average

SIN

2.495223 2.,902 185 2.58744 7 4.279076 2,886046 2.555075 3. 189195

SERUM

1 0.637316 0.848876 0.580834 0.911587 0.877607 0.718296 13 0.644248 0.85627 0.898633 0.92654 1.006606 0.907134 2 0.952:374 0.793961 0.770296 1.717097 0.8054147 0.671626 14 1.015(652 0.8064169 0.8671356 2.060802 0.923538 0.785217 3 0.55869 0.925463 0.900053 1.541952 0.958831 0.848305 15 1.226988 1. 180833 0.983319 1.860833 1.019006 0.854737 4 0.431977 0.94569 0.84373 0.537245 0.941408 0.90578 16 0.605153 0.931505 0.897966 1.732902 1.017857 0.784184 5 0.677036 0.785233 0.508312 1.005882 0.547746 0.796669 17 0.777666 0.984377 0.843962 1.880587 0.890574 0.79296 6 0.588794 1.0359 13 0.992733 0.967939 1.019642 0.982522 18 0.928144 1.064289 0.803029 0.89162 0.75419 0.72221 7 0.852516 0.93817 0.859761 1.8353 17 0.935031 0.883264 19 0.698162 0.76779 0.714554 1.376045 0.88 1491 0.543702 8 0. 95496 1 0.816436 0.7584 18 2.026172 0.806641 0.765781 20 0.672013 0.794245 0.695306 0.816335 0.850109 0.724683 9 0.958261 0.935431 0.887385 2.05505 0.946488 0.944294 21 0.817759 0.87406 1 0.797215 0.801773 0.867612 0.519395 10 0.94319 0.94856 0.837488 1.976 1.154762 1.023286 22 1.018386 0.98586 0.837558 2.050064 0.988323 1.097 197 11 0.783882 0.942455 0.940294 2.72978 1.003 148 0.982288 23 1.036267 0.797719 0.64 1652 1.767422 0.872593 0.70803 12 0.628 171 0.798232 0.663547 1.524798 0.799102 0.672435 24 0.957628 0.915998 0.675314 1.392 178 0.919042 0.962919 Sum E1/GLY# 19.36524 21.67384 19.19921 36.38592 21.78679 19. 5969 1 Average E1/GLY# 0.806885 0.903077 0.799967 1.5 1608 0.907783 0.8 16538 WO 96/04385 PCT/EP95/03031 73

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WO 96/04385 PCT/E-P95/0303 1 74 Grassetti, D. Murray, J. (1969) Analyt. Chim. Acta. 46, 139.

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77 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 78

TAACTGCA

68 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 GGT TGC TCT TTC TCT ATC TTC CTC TTG GCT TTA 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 ATT CCA GCT TCC GCT TAT GAG GTG CGC AAC GTG TCC GGG ATG 96 Leu Thr Ile Pro Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Met 25 TAC CAT GTC ACG AAC GAC TGC TCC AAC TCA AGC ATT 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 Glu 55 AAC AAC TCT TCC CGC TGC TGG GTA GCG CTC ACC CCC ACG CTC GCA GCT 240 Asn Asn Ser Ser Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala 70 75 AGG AAC GCC AGC GTC CCC ACC 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 GCG GCT GCT CTC TGT TCC GCT ATG TAC GTG GGG GAT CTC 336 Leu Val Gly Ala Ala Ala Leu Cys Ser Ala Met Tyr Val Gly Asp Leu 100 105 110 TGC GGA TCT GTC TTC CTC GTC TCC CAG CTG TTC ACC ATC TCG CCT CGC 384 Cys Gly Ser Val Phe Leu Val Ser Gin Leu Phe Thr Ile Ser Pro Arg 115 120 125 CGG CAT GAG ACG GTG CAG GAC TGC AAT TGC TCA ATC TAT CCC GGC CAC 432 nm His Glu Thr val Gin Asp Cys Asn Cys Ser Ile Tyr Pro Gly His 130 135 140 ATA ACA GGT CAC CGT ATG GCT TGG GAT ATG ATG ATG AAC TGG TCG CCT 480 Ile Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro 145 150 155 160 ACA ACG GCC CTG GTG GTA TCG CAG CTG CTC CGG ATC CCA CAA GCT GTC 528 79 Thr Thr Ala Leu Val Val Ser Gin Leu Leu Arg Ile 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 TTT 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 Ile Phe Leu Leu Ala Leu Leu Ser Cys 1 5 10 576 624 Leu Tyr Ala Asn Arg Leu Cys Arg Ile 145 Thr Val Tyr Thr His Asp Asn Asn Val Gly His 130 Thr Thr Asp Tyr Ile Pro Val Thr Met Ile Ser Ser Ala Ser Gly Ala 100 Ser Val 115 Glu Thr Gly His Ala Leu Met Val 180 Ser Met 195 Ala Ser Asn Asp Met His Arg Cys 70 Val Pro Ala Ala Phe Leu Val Gin Arg Met 150 Val Val 165 Ala Gly Ala Cys Thr 55 Trp Thr Leu Val Asp 135 Ala Ser Ala Tyr Ser 40 Pro Val Thr Cys Ser 120 Cys Trp Gin His Trp 200 Glu 25 Asn Gly Ala Thr Ser 105 Gin Asn Asp Leu Trp 185 Val Ser Cys Leu Ile 90 Ala Leu Cys Met Leu 170 Gly Arg Ser Val Thr Arg Met Phe Ser Met 155 Arg Val Asn Val Ile Val Pro Cys Pro Thr Arg His Tyr Val Thr Ile 125 Ile Tyr 140 Met Asn Ile Pro Leu Ala Ser Tyr Val Leu Val Gly 110 Ser Pro Trp Gin Gly 190 Gly Glu Arg Ala Asp Asp Pro Gly Ser Ala 175 Leu Val Gly Asn Ala Lys Val Leu Ile Val Met Leu 205 Leu Phe Ala Leu 210 80 INFORMATION FOR SEQ ID NO: 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: mat peptide LOCATION: 1..789 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATG TTG GGT AAG GTC ATC GAT ACC CTT Met Leu Gly Lys Val Ile Asp Thr Leu 1

GTG

Val

GCC

Ala

ACA

Thr

CTG

Leu

TCC

Ser

TAT

Tyr

GTT

Val

CTC

Leu

GTC

Val 145

ATT

Ile

CAT

His

TTG

Leu

CTG

Leu

TAC

Tyr

GCG

Ala 100

AAC

Asn

AGG

Arg Leu 5

CCG

Pro

GGC

Gly

CCC

Pro

ACC

Thr

CAT

His

GAC

Asp

AAC

Asn

AAC

Asn Val

CTC

Leu

GTC

Val

GGT

Gly

GTT

Val 70

GTC

Val

ATG

Met

TCT

Ser

GCC

Ala Gly 150

GGC

Gly

GTT

Val

TCT

Ser

GCT

Ala

AAC

Asn

ATG

Met

CGC

Arg 120

GTC

Val Ala Ala

ACA

Thr 10

CCC

Pro

GAG

Glu

TCT

Ser

GCT

Ala

TGC

Cys 90

ACC

Thr

TGG

Trp

ACC

Thr

TTC

Phe Cys Gly Phe Ala

CTA

Leu

GAC

Asp

ATC

Ile

TAT

Tyr 75

TCC

Ser

CCC

Pro

GTA

Val

ACG

Thr

TGT

Cys 155 GGG -GGC Gly Gly GGC GTG Gly Val TTC CTC Phe Leu GAA GTG Glu Val AAC TCA Asn Ser GGG TGC Gly Cys GCG CTC Ala Leu 125 ACA ATA Thr Ile 140 TCC GCT Ser Ala TGC GGC TTC GCC GAC CTC Asp

GCC

Ala

TAT

Tyr

GCT

Ala

AAC

Asn

ATT

Ile

CCC

Pro

CCC

Pro

CGC

Arg

TAC

Tyr Leu

AGG

Arg

GCA

Ala

TTG

Leu

GTG

Val

GTG

Val

TGC

Cys

ACG

Thr

CAC

His

GTG

Val 160 96 144 192 240 288 336 384 432 GGG GAC CTC TGC GGA TCT GTC Gly Asp Leu Cys Gly Ser Val 165 TTC CTC GTC TCC CAG CTG TTC Phe Leu Val Ser Gln Leu Phe ACC ATC Thr Ile 175 81 TCG CCT CGC CGG CAT GAG ACG GTG CAG GAC TGC AAT TGC Ser Pro Arg Arg His Glu Thr Val Gin Asp Cys Asn Cys 180 185 CCC GGC CAC ATA ACG GGT CAC CGT ATG GCT TGG GAT ATG Pro Gly His Ile Thr Gly His Arg Met Ala Trp Asp Met 195 200 205 TGG TCG CCT ACA ACG GCC CTG GTG GTA TCG CAG CTG CTC Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gin Leu Leu 210 215 220 CAA GCT GTC GTG GAC ATG GTG GCG GGG GCC CAT TGG GGA Gin Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly 225 230 235 GGT CTC GCC TAC TAT TCC ATG GTG GGG AAC TGG GCT AAG Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys 245 250 GTG ATG CTA CTC TTT GCT CCC TAATAG 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: TCA ATC Ser Ile 190 ATG ATG Met Met CGG ATC Arg Ile GTC CTG Val Leu GTT TTG Val Leu 255

TAT

Tyr

AAC

Asn

CCA

Pro

GCG

Ala 240

ATT

Ile 576 624 672 720 768 Met Leu Gly Lys Val Ile Asp Thr Leu Val Ala Thr Leu Ser Tyr Val Leu Val 145 Gly Leu Gly Ser Gly Glu Arg Ala 130 Asp Tyr Ile Ala His Asn Leu Cys Leu Met Tyr Ala Ala 100 Glu Asn 115 Ala Arg Leu Leu Leu Val Gly Val 70 Val Met Ser Ala Gly 150 Gly Val 40 Ser Ala Asn Met Arg 120 Val Ala Thr Pro Glu Ser Ala Cys Thr Trp Thr Phe Cys Gly Phe Ala Leu Asp Ile Tyr 75 Ser Pro Val Thr Cys 155 Gly Gly Phe Glu Asn Gly Ala Thr 140 Ser Gly Ala Val Asn Leu Leu Val Arg Ser Ser Cys Val 110 Leu Thr 125 Ile Arg Ala Met Asp Leu Ala Arg Tyr Ala Ala Leu Asn Val Ile Val Pro Cys Pro Thr Arg His Tyr Val 160 Gly Asp Leu Cys Ser Val Phe Leu Val Ser Gin Leu Phe Thr Ile 175 82 Ser Pro Arg Arg His Glu Thr Val Gin Asp Cys Asn Cys Ser Ile Tyr 180 185 190 Pro Gly His Iie 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 (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..630 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..627 (xi) 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 Lu Ser Cs 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 83 TAT GAG GCA GCG GAC ATG ATC ATG CAC ACC CCC Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro 100 105 GTT CGG GAG AAC AAC TCT TCC CGC TGC TGG GTA Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val 115 120 CTC GCA GCT AGG AAC GCC AGC GTC CCC ACT ACG Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr 130 135 GTC GAT TTG CTC GTT GGG GCG GCT GCT TTC TGT Val Asp Leu Leu Val Gly Ala Ala Ala Phe Cys 145 150 155 GGG GAT CTC TGC GGA TCT GTC TTC CTC GTC TCC Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser 165 170 TCG CCT CGC CGG CAT GAG ACG GTG CAG GAC TGC Ser Pro Arg Arg His Glu Thr Val Gin Asp Cys 180 185 CCC GGC CAC ATA ACA GGT CAC CGT ATG GCT TGG Pro Gly His Ile Thr Gly His Arg Met Ala Trp 195 200 TGG TAATAG Trp 210 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 209 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 1 5 10 Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 25 Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 40 Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 55 Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr 70 75 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser 90 Tyr Glu Ala Ala Asp Met Ile Met His Thr Pro 100 105 Val Arg Glu Asn Asn Ser Ser Arg Cys Trp Val 115 120 Leu Ala Ala Arg Asn Ala Ser Val Pro Thr Thr GGG TGC I Gly Cys GCG CTC Ala Leu 125 ACA ATA Thr Ile 140 TCC GCT Ser Ala CAG CTG Gin Leu AAT TGC Asn Cys GAT ATG Asp Met 205

GTG

Val 110

ACC

Thr

CGA

Arg

ATG

Met

TTC

Phe

TCA

Ser 190

ATG

Met CCC TGC Pro Cys CCC ACG Pro Thr CGC CAC Arg His TAC GTG Tyr Val 160 ACC ATC Thr Ile 175 ATC TAT Ile Tyr ATG AAC Met Asn 336 384 432 480 528 576 624 Gly Gly Gly Phe 31u Asn Gly Ala rhr Phe Ala Gly Ala Val Asn Leu Leu Val Arg Ser Ser Cys Val 110 Leu Thr 125 Ile Arg Asp Leu Ala Arg Tyr Ala Ala Leu Asn Val Ile Val Pro Cys Pro Thr Arg His 84 130 135 140 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 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 483 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..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 Glu 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 85 100

TGC

Cys

CGG

Arg

GTA

Val 145 (2) GGA TCT GTT TTC CTT GTT TCC CAG CTG TTC A Gly Ser Val Phe Leu Val Ser Gin Leu Phe T 115 120 CAT CAA ACA GTA CAG GAC TGC AAC TGC TCA A His Gin Thr Val Gin Asp Cys Asn Cys Ser I 130 135 1 TCA GGT CAC CGC ATG GCT TGG GAT ATG ATG A Ser Gly His Arg Met Ala Trp Asp Met Met M 150 155 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 159 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110 LCC TTC TCA CCT CGC 'hr Phe Ser Pro Arg 125 ,TC TAT CCC GGC CAT le Tyr Pro Gly His TG AAC TGG TCC TAATAG et Asn Trp Ser 160 384 432 483 Met Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser 1 Leu Tyr Ala Gly Arg Leu Cys Arg Val 145 Ala Asn Met Arg Val Ala Phe Val Arg 10 'al Arg Asn Val Ser Gly ;er Ser Ile Val Tyr Glu ys Val Pro Cys Val Arg eu Thr Pro Thr Leu Ala le Arg Arg His Val Asp 90 .la Met Tyr Val Gly Asp 110 eu Phe Thr Phe Ser Pro 125 ys Ser Ile Tyr Pro Gly 140 et Met Met Asn Trp Ser 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

_E

86 ;iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..477 (ix) FEATURE: NAME/KEY: mat_pe LOCATION: 1..474 ptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATG TCC GGT TGC Met Ser Gly Cys TCT TTC TCT ATC TTC CTC Ser Phe Ser Ile Phe 1

CTG

Leu

TAC

Tyr

GCG

Ala

GGC

Gly

AGG

Arg

CTC

Leu

TGC

Cys

CGG

Arg

GTA

Val 145

ACC

Thr

CAT

His

GAC

Asp

AAC

Asn

AAC

Asn

GTT

Val

GGA

Gly

CAT

His 130

TCA

Ser

ATA

Ile

GTC

Val

ATG

Met

TCC

Ser

GCC

Ala

GGG

Gly

TCT

Ser 115

CAA

Gin

GGT

Gly 5 CCA GCT Pro Ala ACG AAC Thr Asn ATC ATG Ile Met TCC CGT Ser Arg AGC GTC Ser Val GCT GCT Ala Ala 100 GTT TTC Val Phe ACA GTA Thr Val CAC CGC His Arg TCC GCT TAT GAA Ser Ala GAC TGC Asp Cys CAC ACC His Thr 55 TGC TGG Cys Trp 70 CCC ACA Pro Thr GCT TTC Ala Phe CTT GTT Leu Val CAG GAC Gin Asp 135 ATG GCT Met Ala 150 Tyr

TCC

Ser 40

CCC

Pro

GTG

Val

ACG

Thr

TGT

Cys

TCC

Ser 120

TGC

Cys Glu 25

AAC

Asn

GGG

Gly

GCG

Ala

ACA

Thr

TCC

Ser 105

CAG

Gin

AAC

Asn Leu

GTG

Val

TCA

Ser

TGC

Cys

CTC

Leu

ATA

Ile 90

GCT

Ala

CTG

Leu

TGC

Cys TTG GCC Leu Ala

CGC

Arg

AGC

Ser

GTG

Val

ACT

Thr 75

CGA

Arg

ATG

Met

TTC

Phe

TCA

Ser

AAC

Asn

ATA

Ile

CCC

Pro

CCC

Pro

CGC

Arg

TAC

Tyr

ACC

Thr

ATC

Ile 140

GTG

Val

GTG

Val

TGC

Cys

ACG

Thr

CAC

His

GTG

Val

TTC

Phe 125

TAT

Tyr

TCC

Ser

TAT

Tyr

GTT

Val

CTC

Leu

GTC

Val

GGG

Gly 110

TCA

Ser

CCC

Pro

GGG

Gly

GAG

Glu

CGG

Arg

GCG

Ala

GAT

Asp

GAT

Asp

CCT

Pro

GGC

Gly CTG CTG TCC TGT Leu Leu Ser Cys

GTG

Val

GCA

Ala

GAG

Glu

GCC

Ala

TTG

Leu

CTC

Leu

CGC

Arg

CAT

His 96 144 192 240 288 336 384 432 480 TGG GAT ATG Trp Asp Met ATG ATG AAC TGG TAATAG Met Met Asn Trp 155 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 Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys 1 5 10 Leu Tyr Ala Gly Arg Leu Cys Arg Val 145 Thr Ile His Val Asp Met Asn Ser Asn Ala Val Gly Gly Ser 115 His Gin 130 Ser Gly Pro Ala Thr Asn Ile Met Ser Arg Ser Val Ala Ala 100 Val Phe Thr Val His Arg Ser Asp His Cys 70 Pro Ala Leu Gin Met 150 Ala Tyr Cys Ser 40 Thr Pro 55 Trp Val Thr Thr Phe Cys Val Ser 120 Asp Cys 135 Ala Trp 87 Glu Val Arg 25 Asn Ser Ser Gly Cys Val Ala Leu Thr 75 Thr Ile Arg 90 Ser Ala Met 105 Gin Leu Phe Asn Cys Ser Asp Met Met 155 Asn Val Ser Gly Val Ile Val Tyr Glu Ala Pro Cys Val Arg Glu Pro Thr Leu Ala Ala Arg His Val Asp Leu Tyr Val Gly Asp Leu 110 Thr Phe Ser Pro Arg 125 Ile Tyr Pro Gly His 140 Met Asn Trp (2) 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 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..633 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..630 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CTG GGT AAG GCC ATC GAT ACC CTT ACG TGC GGC TTC GCC GAC CTC Leu Gly Lys Ala Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu 5 10 GGG TAC ATT CCG CTC GTC GGC GCC CCC CTA GGG GGC GCT GCC AGG Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg 25 CTG GCG CAT GGC GTC CGG GTT CTG GAA GAC GGC GTG AAC TAT GCA Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala 40 GGG AAT TTG CCT GGT TGC TCT TTC TCT ATC TTC CTC TTG GCT TTA Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu 55

ATG

Met 1

GTG

Val

GCC

Ala

ACA

Thr 88 CTG TCC TGT CTA ACC ATT CCA GCT TCC GCT TAC 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 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 GCG GCT AGG AAC GCC AGC ATC CCC ACT ACA ACA ATA CGA CGC CAC 432 Leu Ala Ala Arg Asn Ala Ser Ile Pro Thr Thr Thr Ile Arg Arg His 130 135 140 GTC GAT TTG CTC GTT GGG GCG GCT GCT TTC 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 CTG TTC 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 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 TAC TAATAG 640 Trp Tyr 640 210 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 210 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: Met Leu Gly Lys Ala Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu 1 5 10 Val 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 Val 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 Arg Asn Val 70 75 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val I I 89 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 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 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: 90 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 (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES (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 91 (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: mat peptide LOCATION: 1..717 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:

ATG

Met 1

GTG

Val

GCC

Ala

ACA

Thr

CTG

Leu

TCC

Ser

TAT

Tyr

GTT

Val

CTC

Leu TTG GGT AAG GTC ATC GAT ACC CTT ACA TGC GGC Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly

GGG

Gly

CTG

Leu

GGG

Gly

TCC

Ser

GGG

Gly

GAG

Glu

CGG

Arg

GCA

Ala 130

TAC

Tyr

GCG

Ala

AAT

Asn

TGT

Cys

ATG

Met

GCA

Ala

GAG

Glu 115

GCT

Ala

ATT

Ile

CAT

His

TTG

Leu

CTG

Leu

TAC

Tyr

GCG

Ala 100

AAC

Asn

AGG

Arg

CTC

Leu

GTC

Val

GGT

Gly

GTT

Val 70

GTC

Val

ATG

Met

TCT

Ser

GCC

Ala

GTC

Val

CGG

Arg

TGC

Cys 55

CCA

Pro

ACG

Thr

ATC

Ile

TCC

Ser

AGC

Ser 135

GCC

Ala 25

CTG

Leu

TTC

Phe

TCC

Ser

GAC

Asp

CAC

His 105

TGC

Cys

CCC

Pro

CTA

Leu

GAC

Asp

ATC

Ile

TAT

Tyr 75

TCC

Ser

CCC

Pro

GTA

Val

ACG

Thr

GGG

Gly

GGC

Gly

TTC

Phe

GAA

Glu

AAC

Asn

GGG

Gly

GCG

Ala

ACA

Thr 140 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 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 Cys Val Pro Cys 110 CTC ACC CCC ACG Leu Thr Pro Thr 125 ATA CGA CGC CAC Ile Arg Arg His 48 96 144 192 240 288 336 384 432 92 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 TCG CCT ACA ACG GCC CTG GTG 576 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 CCA CAA GCT GTC GTG GAC ATG GTG GCG 624 Val Ser Gin Leu Leu Arg Ile Pro Gin Ala Val Val Asp Met Val Ala 195 200 205 GGG GCC CAT TGG GGA GTC CTG GCG GGT CTC GCC TAC TAT TCC ATG GTG 672 Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val 210 215 220 GGG AAC TGG GCT AAG GTT TTG ATT GTG ATG CTA CTC TTT GCT CCC TAATAG 723 Gly Asn Trp Ala Lys Val Leu Ile Val Met Leu Leu Phe Ala Pro 225 230 235 240 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 Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu 1 5 10 Val 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 Val 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 Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val 70 75 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val 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 Val Pro Thr Thr Thr Ile Arg Arg His 130 135 140 Val Asp Ser Gin Leu Phe Thr Ile Ser Pro Arg Arg His Glu Thr Val 145 150 155 160 Gin Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Ile Thr Gly His Arg 165 170 175 I I 93 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 TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..558 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..555 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: ATG TTG 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 Leu 1 5 10 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 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 TTG 192 Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu 55 CTG TCC TGT CTG ACC GTT 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 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 94 115 120 125 CTC GCA GCT AGG AAC GCC AGC GTC CCC ACC ACG ACA ATA CGA CGC CAC 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 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 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 Met Ala Trp Asp Met Met Met Asn Trp 180 185 INFORMATION FOR SEQ ID NO: 24: SEQUENCE CHARACTERISTICS: LENGTH: 185 amino acids TYPE: amino acid TOPOLOGY: linear (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 Val Ala Thr Leu Ser Tyr Val Leu Val 145 Gin Met Gly Leu Gly Ser Gly Glu Arg Ala 130 Asp Asp Ala Tyr Ala Asn Cys Met Ala Glu 115 Ala Ser Cys Trp Ala Leu Phe Ser Asp His 105 Cys Pro Ser Pro Trp 185 Gly Gly Gly Val Phe Leu Glu Val Asn Ser Gly Cys Ala Leu 125 Thr Ile 140 Arg His Ile Thr Ala Asn Leu Arg Ser Val 110 Thr Arg Glu Gly INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 606 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..603 (ix) FEATURE: NAME/KEY: mat peptide LOCATION: 1..600 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATG TTG GGT AAG GTC ATC Met Leu Gly Lys Val Ile 1 5 GTG GGG TAC ATT CCG CTC Val Gly Tyr Ile Pro Leu GCC CTG GCG CAT GGC GTC Ala Leu Ala His Gly Val ACA GGG AAT TTG CCC GGT Thr Gly Asn Leu Pro Gly CTG TCC TGT CTG ACC GTT Leu Ser Cys Leu Thr Val 70 TCC GGG ATG TAC CAT GTC Ser Gly Met Tyr His Val TAT GAG GCA GCG GAC ATG Tyr Glu Ala Ala Asp Met 100 GTT CGG GAG AAC AAC TCT Val Arg Glu Asn Asn Ser 115 CTC GCA GCT AGG AAC GCC Leu Ala Ala Arg Asn Ala 130 GTC GAT TCC CAG CTG TTC Val Asp Ser Gin Leu Phe 145 150 CAG GAC TGC AAT TGC TCA Gin Asp Cys Asn Cys Ser 165 ATG GCT TGG GAT ATG ATG GAT ACC CTT ACA TGC GGC TTC GCC GAC Asp Thr Leu Thr Cys Gly Phe Ala 10

CCC

Pro

GAG

Glu

TCT

Ser

GCT

Ala

TGC

Cys 90

ACC

Thr

TGG

Trp

ACC

Thr

CCT

Pro

GGC

Gly 170

TCG

CTA

Leu

GAC

Asp

ATC

Ile

TAT

Tyr 75

TCC

Ser

CCC

Pro

GTA

Val

ACG

Thr

CGC

Arg 155

CAC

His

CCT

Asp

GCC

Ala

TAT

Tyr

GCT

Ala

AAC

Asn

ATT

Ile

CCC

Pro

CCC

Pro

CGC

Arg

ACG

Thr

CAC

His 175

CTG

CTC

Leu

AGG

Arg

GCA

Ala

TTG

Leu

GTG

Val

GTG

Val

TGC

Cys

ACG

Thr

CAC

His

GTG

Val 160

CGT

Arg

GTG

48 96 144 192 240 288 336 384 432 480 528 576 96 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 Val Ala Thr Leu Ser Tyr Val Leu Val 145 Gly Leu Gly Ser Gly Glu Arg Ala 130 Asp Tyr Ala Asn Cys Met Ala Glu 115 Ala Ser Ile His Leu Leu Tyr Ala 100 Asn Arg Gin Pro Gly Pro Thr His Asp Asn Asn Leu Cys 165 Leu Val Gly Val Arg Val 40 Gly Cys Ser 55 Val Pro Ala 70 Val Thr Asn Met Ile Met Ser Ser Arg 120 Ala Ser Val 135 Phe Thr Ile 150 Ser Ile Tyr Ala Pro 25 Leu Glu Phe Ser Ser Ala Asp Cys 90 His Thr 105 Cys Trp Pro Thr Ser Pro Pro Gly 170 Leu Asp Ile Tyr 75 Ser Pro Val Thr Arg 155 His Gly Gly Phe Glu Asn Gly Ala Thr 140 Arg Ile Gly Ala Val Asn Leu Leu Val Arg Ser Ser Cys Val 110 Leu Thr 125 Ile Arg His Glu Thr Gly Asp Leu Ala Arg Tyr Ala Ala Leu Asn Val Ile Val Pro Cys Pro Thr Arg His Thr Val 160 His Arg 175 Gin Asp Cys Asn Asp Met 180 Leu Leu Met Arg Asn Trp 185 Leu Ser Pro Thr Thr Ala Leu Val 190 INFORMATION FOR SEQ ID NO: 27: SEQUENCE CHARACTERISTICS: LENGTH: 636 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 97 (iii) (iii) HYPOTHETICAL: NO ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..633 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1..630 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: TTG OGT AAG GTC ATC GAT ACC CTT ACA TGC G Leu Gly Lys Val Ile Asp Thr Leu Thr Cvs G

ATG

Met GC TTC GCC GAC CTC ly Phe Ala Asp Leu 1

GTG

Val

GCC

Ala

ACA

Thr

CTG

Leu

TCC

Ser

TAT

Tyr

GTT

Val

CTC

Leu

GTC

Val 145

CAG

Gin

ATG

Met

GTA

Va1

CAT

GGG

Gly

CTG

Leu

GGG

Gly

TCC

Ser

GGG

Gly

GAG

Glu

CGG

Arg

OCA

Ala 130

GAT

Asp

GAC

Asp

OCT

Ala

TCG

Ser

CAC

TAC ATT Tyr Ile GCG CAT Ala His AAT TTG Asn Leu TGT CTG Cys Leu ATG TAC Met Tyr OCA GCG Ala Ala 100 GAG AAC Glu Asn 115 OCT AGO Ala Arg TCC CAG Ser Gin TGC AAT Cys Asn TGG GAT Trp Asp 160 CAG CTO Gin Leu 195

TAATAG

5

CCG

Pro

GGC

Oly

CCC

Pro

ACC

Thr

CAT

His

GAC

Asp

AAC

Asn

AAC

Asn

CTG

Leu

TGC

Cys 165

ATO

Met

CTC

Leu

CTC

Leu

GTC

Val

GGT

Gly

OTT

Val 70

GTC

Val

ATO

Met

TCT

Ser 0CC Ala

TTC

Phe 150

TCA

Ser

ATO.

Met

CGG

Arg

GTC

Val

CGG

Arg

TGC

Cys 55

CCA

Pro

ACO

Thr

ATC

Ile

TCC

Ser

AGC

Ser 135

ACC

Thr

ATC

Ile

ATO

Met

ATC

Ile

GGC

Gly

OTT

Val 40

TCT

Ser

OCT

Ala

AAC

Asn

ATO

Met

CGC

Arg 120

OTC

Vai

ATC

Ile

TAT

Tyr

AAC

Asn

GTG

Va1 200 0CC Ala 25

CTG

Leu

TTC

Phe

TCC

Ser

GAC

Asp

CAC

His 105

TGC

Cys

CCC

Pro

TCG

Ser

CCC

Pro

TGG

Trp 18b

ATC

Ile 10

CCC

Pro

GAO

Glu

TCT

Ser

OCT

Ala

TGC

Cys 90

ACC

Thr

TGG

Trp

ACC

Thr

CCT

Pro

GGC

Gly 170

TCO

Ser

GAO

Glu

CTA

Leu

GAC

Asp

ATC

Ile

TAT

Tyr 75

TCC

Ser

CCC

Pro

OTA

Val

ACO

Thr

CGC

Arg 155

CAC

His

CCT

Pro

GGC

Oly

GGG

Gly

GGC

Gly

TTC

Phe

OAA

Glu

AAC

Asn

GGG

Gly

GCG

Ala

ACA

Thr 140

CGG

Arg

ATA

Ile

ACA

Thr

AGA

Arg

GGC

Oly

GTG

Va1

CTC

Leu

GTG

Val

TCA

Ser

TGC

Cys

CTC

Leu 125

ATA

Ile

CAT

His

ACO

Thr

ACG

Thr

CAC

His 205

OCT

Ala

AAC

Asn

TTG

Leu

CGC

Arg

AGC

Ser

GTG

Val 110

ACC

Thr

CGA

Arg

GAG

Glu

GGT

Gly 0CC Ala 190

CAT

His 0CC Ala

TAT

Tyr

OCT

Ala

AAC

Asn

ATT

Ile

CCC

Pro

CCC

Pro

CGC

Arg

ACO

Thr

CAC

His 175

CTG

Leu

CAC

His

AGO

Arg

OCA

Ala

TTO

Leu

GTG

Val

GTG

Val

TGC

Cys

ACG

Thr

CAC

His

GTG

Val 160

CGT

Arg

GTG

Val

CAC

His 96 144 192 240 288 336 384 432 480 528 576 624 98 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 Gly Phe Ala val Ala Thr Leu Ser Tyr Val Leu Val 145 Gin Met Val His Gly Leu Gly Ser Gly Glu Arg Ala 130 Asp Asp Ala Ser His 210 Leu Val Gly Val 70 Val Met Ser Ala Phe 150 Ser Met Arg Gly Val Ser Ala Asn Met Arg 120 Val Ile Tyr Asn Val 200 Leu Asp Ile Tyr 75 Ser Pro Val Thr Arg 155 His Pro Gly Gly Gly Gly Val Phe Leu Glu Val Asn Ser Gly Cys Ala Leu 125 Thr Ile 140 Arg His Ile Thr Thr Thr Arg 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 His 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 99 (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .627 (ix) FEATURE: NAME/KEY: mat-peptide (B3) LOCATION: 1. .624 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: ATG GGT AAG GTC ATC GAT Met Gly Lys Val Ile Asp

ACC

Thr 1

GGG

Gly

CTT

Leu

GGG

Gly

TCT

Ser

GGC

Gly

GAG

Giu

CAG

Gin

OCA

Ala

GAC

Asp 145

GAC.

Asp

CCT

Pro

TAC

Tyr

GCG

Ala

AAT

Asn 50

TGC

Cys

CTC

Leu

GCC

Ala

GAC

Asp

GTC

Val1 130

CTA

Leu 1ATG M4et

CGT

Arg

ATC

Ile

CAT

His 35

TTG

Leu

TTA

Leu

TAT

Tyr

GAT

Asp

GGC

Gly 115

AAG

Lys

TTA

Leu

TGT

Cys

CGC

Arg

CCG

Pro 20

GGC

Giy

CCC

Pro

ATT

Ile

GTC

Val

GAC

Asp 100

AAT

Asn

TAC

Tyr

GTG

Val1

GGG

Gly

CAT

His 180 5

CTC

Leu

GTG

Val1

GGT

Gly

CAT

His

CTT

Leu

GTT

Val1

ACA

Thr

GTC

Val

GGC

Gly

GCT

Ala 165

CAA

Gin GTC GGC Val Gly AGG GCC Arg Ala TGC TCC Cys Ser 55 CCA OCA Pro Ala 70 ACC AAC Thr Asn ATT CTG Ile Leu TCC ACG Ser Thr GGA GCA Gly Ala 135 GCG GCC Ala Ala 150 GTC TTC Val Phe ACG GTC Thr Val CTT ACG TGC GGA TTC Leu Thr Cys Gly Phe 10 GCT CCC GTA GGA GGC Ala Pro Val Gly Gly 25 CTT GAA GAC GGG ATA Leu Giu Asp Gly Ile 40 TTT TCT ATT TTC CTT Phe Ser Ile Phe Leu GCT AGT CTA GAG TGG Ala Ser Leu Giu Trp 75 GAC TGT TCC AAT AGC Asp Cys Ser Asn Ser 90 CAC ACA CCC GGC TGC His Thr Pro Gly Cys 105 TGC TGG ACC CCA GTG Cys Trp, Thr Pro Val 12 0 WCC ACC GCT TCG ATA I'hr Thr Ala Ser Ile 140 kCG ATG TGC TCT GCG Chr Met Cys Ser Ala 155 'TC GTG GGA CAA GCC eu Val Gly Gin Ala 170 ,AG ACC TGT AAC TGC fln Thr Cys Asn Cys 185

GCC

Ala

GTC

Val1

AAT

Asn

CTC

Leu

CGG

Arg

AGT

Ser

ATA

Ile

ACA

Thr 125

CGC

Arg

CTC

Leu

TTC

Phe

TCG

Ser GAT CTC ATG Asp Leu Met

GCA

Ala

TTC

Phe

GCT

Al a

AAT

Asn

ATT

Ile

CCT

Pro 110

CCT

Pro

AGT

Ser

TAC

Tyr

ACG

Thr

CTG

Leu 190

AGA

Arg

GCA

Ala

CTG

Leu

ACG

Thr

GTG

Val

TGT

Cys

ACA

Thr

CAT

His

GTG

Val

TTC

Phe 175

TAC

Tyr

GCC

Ala

ACA

Thr

TTC

Phe

TCT

Ser

TAC

Tyr

GTC

Val1

GTG

Val1

GTG

Val1

GGT

Gly 160

AGA

Arg

CCA

Pro 96 144 192 240 288 336 384 432 480 528 576 GGC CAT C~TT Gly His Leu 195

TAATAG

TCA nn rAT cna Ser Gly His Arg ATG OCT TaG nAT AT ATO TACG Met Ala Trp Asp Met Met Met Asn Trp 200 205 100 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 Gly Phe Ala Asp Leu Met Gly Leu Gly Ser Gly Glu Gln Ala Asp 145 Asp Pro Gly Tyr Ala Asn Cys Leu Ala Asp Val 130 Leu Met Arg His Ile His Leu Leu Tyr Asp Gly 115 Lys Leu Cys Arg Leu 195 Pro Gly Pro Ile Val Asp 100 Asn Tyr Val Gly His 180 Ser Leu Val Val Arg Gly Cys His Pro 70 Leu Thr Val Ile Thr Ser Val Gly Gly Ala 150 Ala Val 165 Gin Thr Gly Ala Ser 55 Ala Asn Leu Thr Ala 135 Ala Phe Val SAla Leu 40 Phe Ala Asp His Cys 120 Thr Thr Leu Gin Met 200 Pro 25 Glu Ser Ser Cys Thr 105 Trp Thr Met Val Thr 185 Val Asp Ile Leu Ser 90 Pro Thr Ala Cys Gly 170 Cys Gly Gly Phe Glu 75 Asn Gly Pro Ser Ser 155 Gin Asn Gly Val Ile Asn Leu Leu Trp Arg Ser Ser Cys Ile Val Thr 125 Ile Arg 140 Ala Leu Ala Phe Cys Ser Ala Phe Ala Asn Ile Pro 110 Pro Ser Tyr Thr Leu 190 Arg Ala Leu Thr Val Cys Thr His Val Phe 175 Tyr Ala Thr Phe Ser Tyr Val Val Val Gly 160 Arg Pro Gly His Arg Ala Trp Asp Met Met 205 Met Asn Trp 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 101 (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 CTT GCT CTT CTC 192 Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Ile Leu Ala Leu Leu 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 GGG 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 Glu Ala Asp Asn Leu Ile Leu His Ala Pro Gly Cys Val Pro Cys Val 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 Ile Thr Pro Thr Leu 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 GCG TTA TAC GTA GGA 480 Asp Tyr Leu Ala Gly Gly Ala Ala Leu Cys Ser Ala Leu Tyr Val Gly 145 150 155 160 GAC GCG TGT GGG GCA CTA TTC TTG GTA GGC CAA ATG TTC ACC TAT AGG 528 Asp Ala Cys Gly Ala Leu Phe Leu Val Gly Gin Met Phe Thr Tyr Arg 165 170 175 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 Ile Tyr Ser 180 185 190 GGC CAT GTT ACC GGC CAC CGG ATG GCA TGG GAT ATG ATG ATG AAC TGG 624 Gly His Val Thr Gly His Arg Met Ala Trp Asp Met Met Met Asn Trp 195 200 205 TAATAG 630 INFORMATION FOR SEQ ID NO: 32: ww 102 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 ic Gly Leu Gly Ser Gly Glu Met Ser Asp 145 Asp Tyr Ala Asn Cys Ile Ala Thr Ala 130 Tyr Ala Ile His Leu Leu Tyr Asp Gly 115 Pro Leu Cys Pro Gly Pro Thr His Asn 100 Asn Ser Ala Gly Leu Val Gly Val Arg Val Gly Cys Ser 55 Val Pro Ala 70 Val Thr Asn Leu Ile Leu Val Ser Arg Leu Gly Ala 135 Gly Gly Ala 150 Ala Leu Phe 165.

Ala Thr Val Gly Leu 40 Phe Ser Asp His Cys 120 Val Ala Leu Gin SPro 25 Glu Ser Ala Cys Ala 105 Trp Thr Leu Val Asn 185 Ile Gly Gly Val Ala Arg Ala Asp Ile Val Pro 90 Pro Val Ala Cys Gly 170 Cys Gly Val Asn Tyr Ala Thr Phe Pro 75 Asn Gly Gin Pro Ser 155 Gin Asn Ile Leu Tyr Arg Ser Ser Cys Val Ile Thr 125 Leu Arg 140 Ala Leu Met Phe Cys Ser Ala Asn Ile Pro 110 Pro Arg Tyr Thr Ile Leu Ala Val Cys Thr Ala Val Tyr 175 Tyr Leu Ser Tyr Val Leu Val Gly 160 Arg Ser Pro Arg Gin His 180 190 Gly His Val Thr Gly His Arg Met 195 200 Ala Trp Asp Met Met Asn Trp INFORMATION FOR SEQ ID NO: 33: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: 103 TGGGATATGA TGATGAACTG

GTC

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

TGG

Trp 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GAT ATG ATG ATG AAC TGG TCG CCT ACA ACG GCC Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala 5 10 CAG CTG CTC CGG ATC CCA CAA GCT GTC GTG GAC ATG Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met 25 CAT TGG GGA GTC CTG GCG GGC CTC GCC TAC TAT TCC His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser 40 TGG GCT AAG GTT TTG GTT GTG ATG CTA CTC TTT GCC Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala 55 CTG GTG GTA TCG Leu Val Val Ser GTG GCG GGG GCC Val Ala Gly Ala ATG GTG GGG AAC Met Val Gly Asn GGC GTC GAC GGG Gly Val Asp Gly 144 192 240 CAT ACC CGC GTG TCA GGA GGG GCA GCA GCC TCC GAT ACC AGG GGC CTT 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 104 Ala Gin Lys Val Ser Leu Phe Ser Pro Gly Ser Ile Gin Leu Val Asn 90

ACC

Thr

TCC

Ser

AAC

Asn

AAG

Lys 145

TCG

Ser

ATT

Ile

AGC

Ser

AAC

Asn

CCG

Pro 225

TTC

Phe

AAC

Asn

GCC

Ala

ATG

Met

TTC

Phe 305

TTC

Phe

GAC

Asp

TGG

Trp

AAC

Asn

CTC

Leu

TCG

Ser 130

TTC

Phe

GAC

Asp

GTA

Val1

CCT

Pro

TGG

Trp 210

CCG

Pro

ACC

Thr

AAC

Asn

ACC

Thr

GTT

Vai 290

ACC

Thr

GAA

Giu

AGG

Arg

CAG

Gin GGC AGT Gly Ser 100 CAA ACA Gin Thr 115 TCT GGA Ser Gly GCT CAG Ala Gin CAG AGG Gin Arg CCC GCG Pro Ala 180 GTT GTG Val Val 195 GGG GCG Gly Ala CGA GGC Arg Gly AAG ACG Ljys Thr ACC TTG Thr Leu 260 TAC GCC Tyr Ala 275 CAT TAC His Tyr ATC TTC Ile Phe GCC GCA Ala Ala GAT AGA Asp Arg 340 ATA CTG Ile Leu

TGG

Trp

GGG

G ly

TGC

Cys

GGG

Gly

CCC

Pro 165

TCT

Ser

GTG

Val1

AAC

Asn

AAC

Asn

TGT

Cys 245

ACC

Thr

AGA

Arg

CCA

Pro

AAG

Lys

TGC

Cys 325

TCA

Ser

CCC

Pro

CAC

His

TTC

Phe

CCA

Pro

TGG

Trp 150

TAC

Tyr

CAG

G in

GG

Gly

GAC

Asp

TGG

Trp 230

GGG

Gly

TGC

Cys

TGC

Cys

TAT.

Tyr

GTT

Val 310

AAT

Asn

GAG

Giu

TGT

Cys

ATC

Ile

'TT

Phe

GAG

Giu 135

GGT

Giy

TGC

Cys

GTG

Val

ACG

Thr

TCG

Ser 215 TrC Phe

GGC

Giy

CCC

Pro

GGT

Giy

AGG

Arg 295

AGG

Arg

TGG

Trp

CTT

Leu

TCC

Ser

AAC

Asn

GCC

Ala 120

CGC

Arg

CCC

Pro

TGG

Trp

TGC

Cys

ACC

Thr 200

GAT

Asp

GGC

Gly

CCC

Pro

ACT

Thr

TCT

Ser 280

CTC

Leu

ATG

Met

ACT

Thr

AGC

Ser

TTC

Phe

AGG

Arg 105

GCA

Al a

TTG

Leu

CTC

Leu

CAC

His

GGT

Gly 185

GAT

Asp

GTG

Val1

TGT.

Cys

CCG

Pro

GAC

Asp 265

GGG

Gly

TGG

Trp

TAC

Tyr

CGA

Arg

CCG

Pro 345

ACC.

Thr

ACT

Thr

CTA

Leu

GCC

Ala

ACT

Thr

TAC

Tyr 170

CCA

Pro

CGG

Arg

CTG

Leu

ACA

Thr

TGC

Cys 250

TGT

Cys

CCC

Pro

CAC

His

GTG

Val1

GGA

Gly 330

CTG

Leu

ACC

Thr GCC CTG Ala Leu TTC TAC Phe Tyr AGC TGT Ser Cys 140 TAC ACT Tyr Thr 155 GCG CCT Ala Pro GTG TAT Vai Tyr TTT GGT Phe Gly ATT CTC Ile Leu 220 TGG ATG Trp, Met 235 AAC ATC Asn Ile TTT CGG Phe Arg TGG CTG Trp Leu TAC CCC Tyr Pro 300 GGG GGC Gly Gly 315 GAG CGT Giu Arg CTG CTG Leu Leu CTG CCG Leu Pro AAC TGC Asn Cys 110 AAA CAC Lys His 125 CGC TCC Arg Ser GAG CCT Glu Pro CGA CCG Arg Pro TGC TTC Cys Phe 190 GTC CCC Val Pro 205 AAC AAC Asn Asn AAT GGC Asn Gly GGG GGG Gly Gly AAG CAC Lys His 270 ACA CCT Thr Pro 285 TGC ACT Cys Thr GTG GAG Val Giu TGT GAC Cys Asp TCT ACA Ser Thr 350 GCC CTA Ala Leu

AAC

Asn

AAA

Lys

ATC

Ile

AAC

As n

TGT

Cys 175

ACC

Thr

ACG

Thr

ACG

Thr

ACT

Thr

GCC

Al a 255

CCC

Pro

AGG

Arg

GTC

Val1

CAC

His

TTG

Leu 335

ACA

Thr

TCC

Ser

GAC

Asp

TTC

Phe

GAC

Asp

AGC-

Ser 160

GGT

Gly

CCG

Pro

TAT

Tyr

CGG

Arg

GGG

Gly 240

GGC

Giy

GAG

Glu

TGT

Cys

AAC

Asn

AGG

Arg 320

GAG

Giu

GAG

Giu

ACC

Thr 336 384 432 480 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 105 355 360 GGC CTG ATC CAC CTC CAT CAG AAC ATC GTG GAC GT Gly Leu Ile His Leu His Gin Asn Ile Val Asp Va 370 375 38( GGT GTA GGG TCG GCG GTT GTC TCC CTT GTC ATC AAJ Gly Val Gly Ser Ala Val Val Ser Leu Val Ile Ly 385 390 395 CTG TTG CTC TTC CTT CTC CTG GCA GAC GCG CGC ATC Leu Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Ile 405 410 TGG ATG ATG CTG CTG ATA GCT CAA GCT GAG GCC GCC Trp Met Met Leu Leu Ile Ala Gin Ala Glu Ala Ala 420 425 GTG GTC CTC AAT GCG GCG GCC GTG GCC GGG GCG CAT Val Val Leu Asn Ala Ala Ala Val Ala Gly Ala His 435 440 TTC CTT GTG TTC TTC TGT GCT GCC TGG TAC ATC AAG Phe Leu Val Phe Phe Cys Ala Ala Trp Tyr Ile Lys 450 455 460 CCT GGT GCG GCA TAC GCC TTC TAT GGC GTG TGG CCG Pro Gly Ala Ala Tyr Ala Phe Tyr Gly Val Trp Pro 465 470 475 CTG CTG GCC TTA CCA CCA CGA GCT TAT GCC TAGTAA Leu Leu Ala Leu Pro Pro Arg Ala Tyr Ala 485 490 INFORMATION FOR SEQ ID NO: 36: SEQUENCE CHARACTERISTICS: LENGTH: 490 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: Trp Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala 1 5 10 Gin Leu Leu Arg Ile Pro Gin Ala Val Val Asp Met 25 His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser 40 Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala 55 His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp 70 75 Val Ser Leu Phe Ser Pro Gly Ser Ala Gin Lys Ile 90 Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu 100 105 Ser Leu Gin Thr Gly Phe Phe Ala Ala Leu Phe Tyr 115 120 365 3 CAA 1 Gin 0 A TGG s Trp C TGC e Cys

TTA

Leu

GGC

Gly 445

GGC

Gly

CTG

Leu

TAC

Tyr

GAG

Glu

GCC

Ala

GAG

Glu 430

ACT

Thr

AGG

Arg

CTC

Leu CTG TAC Leu Tyr TAT GTC Tyr Val 400 TGC TTA Cys Leu 415 AAC CTG Asn Leu CTT TCC Leu Ser CTG GTC Leu Val CTG CTT Leu Leu 480 1152 1200 1248 1296 1344 1392 1440 1476 Val Ser Gly Ala Gly Asn Asp Gly Gly Leu Val Asn Asn Asp Lys Phe 106 Axg Leu Ala Ser Asn Lys 145 Ser Ile Ser Asn Pro 225 Phe Asn Ala Met Phe 305 Phe Asp Trp Gly Gly 385 Leu Trp Val Phe Pro 465 Ser 130 Phe Asp Val Pro Trp 210 Pro Thr Asn Thr Val 290 Thr Glu Ser Ala Gin Pro Val 195 Gly Arg Lys Thr Tyr 275 His Ile Ala Gly Gin Arg Ala 180 Val Ala.

Gly.

Thr Leu 260 Ala Tyr Phe Ala Arg 340 Leu His Ser Phe Leu 420 Asn Phe Ala Cys Gly Pro 165 Ser Va1 Asn Asn Cys 245 Thr Arg Pro Lys Cys 325 Ser Pro Leu Ala Leu 405 Leu Ala Phe Tyr Pro Trp 150 Tyr Gin Gly Asp Trp 230 Gly Cys Cys Tyr Val 310 Asn Glu Cys His Val 390 Leu Ile Ala Cys Ala 470 lu 135 ly Cys Va1 rhr Ser 215 Phe ly Pro Gly Arg 295 Arg Trp Leu Ser Gin 375 Val Leu Ala Ala Ala 455 Phe Cys Arg Ser Ile Asp 140 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 Val Ala 410 Glu Gly Tyr Va1 Tyr 155 Ala Val Phe Ile Trp 235 Asn Phe Trp Tyr Gly 315 Glu Leu Leu Asp Ile 395 Arg Ala Ala Ile Trp 475 Thr Pro Tyr Gly Leu 220 Met Ile Arg Leu Pro 300 Gly Arg Leu Pro Va1 380 Lys Ile Ala His Lys 460 Glu Arg Cys Val 205 Asn Asn Gly Lys Thr 285 Cys Va1 Cys Ser Ala 365 Gin Trp Cys Leu Gly 445 Gly Pro Pro Phe 190 Pro Asn Gly Gly His 270 Pro Thr Glu Asp Thr 350 Leu Tyr Glu Ala Glu 430 Thr Arg Asn Cys 175 Thr Thr Thr Thr Ala 255 Pro Arg Va1 His Leu 335 Thr Ser Leu Tyr Cys 415 Asn Leu Leu Ser 160 Gly Pro Tyr Arg Gly 240 Gly Glu Cys Asn Arg 320 Glu Glu Thr Tyr Val 400 Leu Leu Ser Val Leu 480 Arg Asp Gln Ile 355 Leu Ile 370 Val Gly Leu Leu Met Met Val Leu 435 Leu Val 450 Gly Ala Pro Leu Leu Leu Leu Leu Ala Leu Pro 485 Pro Arg Aia Tyr Ala 490 107 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 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: G ATC CCA CAA GCT GTC GTG GAC ATG GTG GCG GGG GCC CAT TGG GGA Ile Pro Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly 1 5 10 GTC CTG Val

GTT

Val

GTG

Val

TTT

Phe

AGT

Ser

ACA

Thr

GGA

Gly

CAG

Gln

AGG

Arg

GCG

Ala 160 Leu

TTG

Leu

TCA

Ser

AGC

Ser

TGG

Trp

GGG

Gly

TGC

Cys

GGG

Gly

CCC

Pro 145

TCT

Ser GCG GGC Ala Gly GTT GTG Val Val GGA GGG Gly Gly CCC GGG Pro Gly CAC ATC His Ile TTC TTT Phe Phe CCA GAG Pro Glu 115 TGG GGT Trp Gly 130 TAC TGC' Tyr Cys CAG GTG Gln Val CTC GCC TAC TAT TCC ATG GTG GGG AAC TGG Leu

ATG

Met

GCA

Ala

TCG

Ser

AAC

Asn

GCC

Ala 100

CGC

Arg

CCC

Pro TGr Trp Ala

CTA

Leu

GCA

Ala

GCT

Ala

AGG

Arg 85

GCA

Ala

TTG

Leu

CTC

Leu

HCA

His Tyr

CTC

Leu

GCC

Ala

CAG

Gln 70

ACT

Thr

CTA

Leu

GCC

Ala

ACT

Thr

TAC

Tyr 150 Tyr Ser TTT GCC Phe Ala 40 TCC GAT Ser Asp 55 AAA ATC Lys Ile GCC CTG Ala Leu TTC TAC Phe Tyr AGC TGT Ser Cys 120 TAC ACT Tyr Thr 135 Ala Pro Met 25

GGC

Gly

ACC

Thr

CAG

Gln

AAC

Asn

AAA

Lys 105

CGC

Arg

GAG

Glu Arg Val

GTC

Val

AGG

Arg

CTC

Leu

TGC

Cys 90

CAC

His

TCC

Ser

CCT

Pro Pro

TTC

Phe 170 Gly

GAC

Asp

GGC

Gly

GTA

Val

AAC

Asn

AAA

Lys

ATC

Ile

AAC

Asn Cys 155 Asn

GGG

Gly

CTT

Leu

AAC

Asn

GAC

Asp

TTC

Phe

GAC

Asp

AGC

Ser 140 jGl Gly Trp

CAT

His

GTG

Val

ACC

Thr

TCC

Ser

AAC

Asn

AAG

Lys 125

TCG

Ser

ATT

Ile GCT AAG Ala Lys ACC CGC Thr Arg TCC CTC Ser Leu AAC GGC Asn Gly CTC CAA Leu Gin TCG TCT Ser Ser 110 TTC GCT Phe Ala GAC CAG Asp Gin GTA CCC Val Pro 94 142 190 238 286 334 382 430 478 TGC GGT Cys Gly 165 CCA GTG TAT TGC Pro Val Tyr Cys ACC CCG AGC CCT Thr Pro Ser Pro

I

108 GTG GTG GGG ACG ACC Val Val Gly Thr Thr GAT CGG TTT GGT GTC Asp Arg Phe Gly Val

GCG

Ala

GGC

Gly

ACG

Thr

TTG

Leu 240

GCC

Ala

TAC

Tyr

TTC

Phe

GCA

Ala

AGA

Arg 320

AAC

Asn

AAC

Asn

TGT

Cys 225

ACC

Thr

AGA

Arg

CCA

Pro

AAG

Lys

TGC

Cys 305

TCA

GAC

Asp

TGG

Trp 210

GGG

Gly

TGC

Cys

TGC

Cys

TAT

Tyr

GTT

Val 290

AAT

Asn

GAG

TCG

Ser 195

TTC

Phe

GGC

Gly

CCC

Pro

GGT

Gly

AGG

Arg 275

AGG

Arg

TGG

Trp

CTT

180

GAT

Asp

GGC

Gly

CCC

Pro

ACT

Thr

TCT

Ser 260

CTC

Leu

ATG

Met

ACT

Thr

GTG

Val

TGT

Cys

CCG

Pro

GAC

Asp 245

GGG

Gly

TGG

Trp

TAC

Tyr

CGA

Arg

CTG

Leu

ACA

Thr

TGC

Cys 230

TGT

Cys

CCC

Pro

CAC

His

GTG

Val

GGA

Gly 310 ATT CTC Ile Leu 200 TGG ATG Trp Met 215 AAC ATC Asn Ile TTT CGG Phe Arg TGG CTG Trp Leu TAC CCC Tyr Pro 280 GGG GGC Gly Gly 295 GAG CGT Glu Arg 185

AAC

Asn

AAT

Asn

GGG

Gly

AAG

Lys

ACA

Thr 265

TGC

Cys

GTG

Val

TGT

Cys

CCC

Pro

AAC

Asn

GGC

Gly

GGG

Gly

CAC

His 250

CCT

Pro

ACT

Thr

GAG

Glu

GAC

Asp ACG TAT AAC TGG GGG Thr Tyr Asn

ACG

Thr

ACT

Thr

GCC

Ala 235

CCC

Pro

AGG

Arg

GTC

Val

CAC

His

TTG

Leu 315

CGG

Arg

GGG

Gly 220

GGC

Gly

GAG

Glu

TGT

Cys

AAC

Asn

AGG

Arg 300

GAG

Glu

CCG

Pro 205

TTC

Phe

AAC

Asn

GCC

Ala

ATG

Met

TTC

Phe 285

TTC

Phe

GAC

Asp Trp Gly 190 CCG CGA Pro Arg ACC AAG Thr Lys AAC ACC Asn Thr ACC TAC Thr Tyr 255 GTT CAT Val His 270 ACC ATC Thr Ile GAA GCC Glu Ala AGG GAT Arg Asp 622 670 718 766 814 862 910 958 1006 1021 AGC CCG CTG CTG CTG TCT Ser Glu Leu Ser Pro Leu Leu Leu Ser 325 ACA ACA GAG TGG Thr Thr Glu Trp 330 CAG AGT Gin Ser 335 GGC AGA GCT TAATTA Gly Arg Ala INFORMATION FOR SEQ ID NO: 38: 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 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 Ph Ala Gly val Astp Gly HJ l Iri. y Vdl 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 109 Trp Giy Cys Gly Pro 145 Ser Val1 Asn Asn Cys 225 Thr Arg Pro Lys Cys 305 Ser Arg His Phe Pro Trp 130 Tyr Gin Gly Asp Trp 210 Gly Cys Cys Tyr Val1 290 Asn Giu Ala Ile Phe Glu 115 Gly Cys Val Thr Ser 195 Phe Gly Pro Gly Mrg 275 Arg Trp Leu Asn Ala 100 Arg Pro Trp Cys Thr 180 Asp Giy Pro Thr Ser 260 Leu Met Thr Ser Arg Ala Leu Leu His Giy 165 Asp Val1 Cys Pro Asp 245 Gly Trp Tyr Arg Pro 325 Thr Ala Leu Asn Leu Phe Ala Ser Thr Tyr 135 Tyr Ala 150 Pro Val Mrg Phe Leu Ile Thr Trp 215 Cys Asn 230 Cys Phe Pro Trp His Tyr Val Gly 295 Gly Glu 310 Leu Leu Tyr Cys 120 Thr Pro Tyr Giy Leu 200 Met Ile Mrg Leu Pro 280 Gly Mrg Leu Lys 105 Arg Glu Arg Cys Val 185 Asn Asn Gly Lys Thr 265 Cys Val1 Cys Ser Cys 90 His Ser Pro Pro Phe 170 Pro Asn Giy Gly His 250 Pro Thr Giu Asp Thr 330 Asn Lys Ile Asn Cys 155 Thr Thr Thr Thr Ala 235 Pro Arg Val His Leu 315 Thr Asp Phe Asp Ser 140 Gly Pro Tyr Arg Gly 220 Gly Giu Cys Asn Arg 300 Giu Giu Ser Asn Lys 125 Ser Ile Ser Asn Pro 205 Phe Asn Ala Met Phe 285 Phe Asp Trp Leu Ser 110 Phe Asp Val1 Pro Trp 190 Pro Thr Asn Thr Val1 270 Thr Giu Gln Ser Aila Gin Pro Val1 175 Gly Mrg Lys Thr Tyr 255 His Ile Ala Thr Gly Gin Arg Ala 160 Val Al a Gly Thr Leu 240 Al a Tyr Phe Aila Mrg 320 Gly Arg Asp Gin Ser 335 INFORMATION FOR SEQ ID NO: 39: SEQUENCE CHARACTERISTICS: LENGTH: 1034 base pairs TYPE: nucieic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: 110 NAME/KEY: CDS 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 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 GGT AAG Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys 25

GTT

Val

GTG

Val1

TTT

Phe

AGT

Ser

ACA

Thr

GGA

Gly

CAG

Gln

AGG

Arg

GCG

Ala 160

GTG

Val

GCG

Ala

GGC

Gly

ACG

Thr TTG GTT GTG ATG Leu

TCA

Ser

AGC

Ser

TGG

Trp

GGG

Gly

TGC

Gys

GGG

Gly

CCC

Pro 145

TCT

Ser

GTG

Val1

AAC

Asn kAC ksn

TGT

-ys 225 Val

GGA

Gly

CCC

Pro

CAC

His

TTC

Phe

CCA

Pro

TGG

Trp 130

TAG

Tyr

GAG

Gin

GGG

Gly

GAG

Asp

TGG

Trp 210

GGG

GlyC Val1

GGG

Gly

GGG

Gly

ATC

Ile

TT

Phe

GAG

Glu 115

GGT

Gly

TGC

Cys

GTG

Val1 kCG rhr rCG 3er 195

ETC

?he

'GC

fly Met

GCA

Ala

TG

Ser

AAC

Asn

GCC

Ala 100

CGG

Arg

CCC

Pro

TGG

Trp

TGC

Gys

ACC

Thr 180

GAT

Asp

GGC

Gly

CCC

Pro GTA CTC TTT GCC GGC GTC GAG GGG CAT ACC GG Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg 40

GGA

Ala

GCT

Ala

AGG

Arg 85

GGA

Ala

TTG

Leu

GTC

Leu

CAC

His GGT4 Gly 165

GAT

Asp

GTG

Val

TGT

:ys

CCG

Pro TGC GAT Ser Asp 55 AAA ATG ACC AGG GGC CTT GTG TCC CTC Arg Gly Leu Val Ser CTC GTA AAC ACC Gin Lys Ile Gin

ACT

Thr

GTA

Leu

GCC

Ala

ACT

Thr

TAG

Tyr 150

CCA

Pro

CGG

krg

CTG

eu

),CA

Chr

PGC

'ys ~30

GGG

Ala

TTC

Phe

AGC

Ser

TAG

Tyr 135

GG

Ala

GTG

Val

TTT

Phe

PSTT

Ile r~c rrp 215

GTG

Leu

TAG

Tyr

TGT

Cys 120

ACT

Thr

GGT

Pro

TAT

Tyr

GGT

Gly

GTC

Leu 200 ATr.

Met

AAG

Asn

AAA

Lys 105

G

Arg

GAG

Glu

GGA

Arg

TG

Cys

GTG

Val1 185

AAG

Asn lAsn Leu

TGG

Cys 90

GAG

His

TC

Ser

GCT

Pro

CCG

Pro

TTG

Phe 170

CCC

Pro

AAG

Asn GOC-r Gly Val

AAC

Asn

AAA

Lys

ATG

Ile

AAG

Asn

TGT

Gys 155

ACC

Thr

AG

Thr

ACG

Thr Thr

GCC

Ala 235 Asn

GAG

Asp

TTC

Phe

GAG

Asp

AGG

Ser 140

GGT

Gly

GCG

Pro

TAT

Tyr

GG

Arg Gly 220 Thr

TGG

Ser

AAG

Asn

AAG

Lys 125

TG

Ser

ATT

Ile

AGG

Ser

A.AC

Asn

CCG

Pro 205 Phe

AAC

As n

CTC

Leu

TG

Ser 110

TTG

Phe

GAG

Asp

GTA

Val

GCT

Pro

TGG

Trp 190

CG

Pro Thr Leu

GGC

Gly

CAA

Gin

TGT

Ser

GGT

Ala

GAG

Gin

CCC

Pro

GTT

Val 175

GGG

Gly

GA

Arg Lys 142 190 238 286 334 382 430 478 526 574 622 6 7 0 718 AAC ATC GGG GGG Asn Ilie Gly Gly GGG AAG AAC AGC Gly Asn Asn Thr ~1_111 111 TTG ACC TGC CCC ACT GAC TGT TTT CGG AAG CAC CC Leu Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pr 240 245 250 GCC AGA TGC GGT TCT GGG CCC TGG CTG ACA CCT AGC Ala Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arc 260 265 TAC CCA TAT AGG CTC TGG CAC TAC CCC TGC ACT GTC Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys Thr Val 275 280 TTC AAG GTT AGG ATG TAC GTG GGG GGC GTG GAG CAC Phe Lys Val Arg Met Tyr Val Gly Gly Val Glu His 290 295 GCA TGC AAT TGG ACT CGA GGA GAG CGT TGT GAC TTG Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu 305 310 315 AGA TCA GAG CTT AGC CCG CTG CTG CTG TCT ACA ACA Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr 320 325 330 CAG ACA CCA TCA CCA CCA TCA CTA AT AG 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: 1le Pro Gin Ala Val Val Asp Met Val Ala Gly Ala 1 5 10 Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn 25 Leu Val Val Met Leu Leu Phe Ala Gly Val Asp Gly 40 ier Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu 55 ;er Pro Gly Ser Ala Gin Lys Ile Gin Leu Val Asn 70 75 'rp His Ile Asn Arg Thr Ala Leu Asn Cys Asn Asp 90 hly Phe Phe Ala Ala Leu Phe Tyr Lys His Lys Phe 100 105 -ys Pro Glu Arg Leu Ala Ser Cys Arg Ser Ile Asp 115 120 ;ly Trp Gly Pro Leu Thr Tyr Thr Glu Pro Asn Ser 130 135 140 ro Tyr Cys Trp His Tyr Ala Pro Arg Pro Cys Gly 45 150 155 C GAG o Glu

STGT

SCys

AAC

SAsn

SAGG

Arg 300

GAG

Glu

GGT

Gly His I Trp I His T Val S Thr A Ser L Asn S 1 Lys P 125 Ser A Ile V GCC ACC Ala Thr ATG GTT Met Val 270 TTC ACC Phe Thr 285 TTC GAA Phe Glu GAC AGG Asp Arg GAT CGA Asp Arg

TAC

Tyr 255

CAT

His

ATC

Ile

GCC

Ala

GAT

Asp

GGG

Gly 335 766 814 862 910 958 1006 1034

I

I

G

C

G

P

1 :rp Gly Val la Lys Val 'hr Arg Val er Leu Phe sn Gly Ser eu Gin Thr er Ser Gly he Ala Gin sp Gin Arg al Pro Ala 160 112 Ser Gin Val Cys Gly Pro Val Tyr Cys Phe Thr Pro Ser Pro Val Val 165 170 175 Val Gly Thr Thr Asp Arg Phe Gly Val Pro Thr Tyr Asn Trp Gly Ala 180 185 190 Asn Asp Ser Asp Val Leu Ile Leu Asn Asn Thr Arg Pro Pro Arg 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 225 230 235 240 Thr Cys Pro Thr Asp Cys Phe Arg Lys His Pro Glu Ala Thr Tyr Ala 245 250 255 Arg Cys Gly Ser Gly Pro Trp Leu Thr Pro Arg 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 Arg Met Tyr Val Gly Gly Val Glu His Arg Phe Glu Ala Ala 290 295 300 Cys Asn Trp Thr Arg Gly Glu Arg Cys Asp Leu Glu Asp Arg Asp Arg 305 310 315 320 Ser Glu Leu Ser Pro Leu Leu Leu Ser Thr Thr Gly Asp Arg Gly Gin 325 330 335 Thr Pro Ser Pro Pro Ser Leu 340 INFORMATION FOR SEQ ID NO: 41: 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: mat_peptide LOCATION: 1..939 (xi) SEQUENCE DESCRIPTION: SEO ID NO: 41: ATG GTG GGG AAC TGG GCT AAG GTT TTG GTT GTG ATG CTA CTC TTT GCC 48 Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala 1 5 10 GGC GTC GAC GGG CAT ACC CGC GTG TCA GGA GGG GCA GCA GCC TCC GAT 96 Gly Val Asp Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp 113

AC(

Thi

CAG

Gir

AAC

Asn

AAA

Lys

CGC

Arg

GAG

Glu

CGA

Arg

TGC

Cys 145

GTC

Val1 *AGG GGC *Arg Gly CTC GTA Leu Val *TGC AAC Cys Asn CAC AAA Hius Lys TCC ATC Ser Ile CCT AAC Pro Asn 11is CCG TGT Pro Cys 130 TTC ACC Phe Thr CCC ACG Pro Thr

CT

Le

AAI

As2

GA(

Asi Trc Phe

GAC

Asp 100

AGC

Ser

GGT

Gly

'CG

ro

'AT

.'yr TGTG TO u Val Se: CACC AA( ni Thr Asr TCC CTC Ser Let 7C AAC TCG Asn Ser AAG TTC Lys Phe TCG GAC Ser Asp ATT GTA Ile Val AGC CCT Ser Pro 150 AAC TGG CTC TTT rLeu Phe 40 GGC AGT SGly Ser 55 CAA ACA Gin Thr TCT GGA Ser Gly GCT CAG Ala Gin CAG AGG Gin Arg 120 CCC GCG Pro Ala 135 GTT GTG Vai Val GGG GCG

AG(

Sei

TGC

Trp

GGG

Gly

TGC

Cys

GGG

Gly 105

CCC

Pro rCT Ser

,TG

al1 ccC Prc

CAC

His

TTC

Phe

CCA

Pro 90

TGG

Trp

TAC

Tyr

CAG

Gin

GGG

Gly

GAC

Asp 170 GGG TCG )Gly Ser ATC AAC Ile Asn TTT GCC Phe Ala 75 GAG CGC Glu Arg GGT CCC Gly Pro TGC TGG Cys Trp GTG TGC Val Cys 140 ACG ACC Thr Thr 155 TCG GAT C Ser Asp I

GC'

Al~

AGC

Arc

GCA

Ala

TTG

Leu

CTC

Leu

CAC

[-is 125

GGT

G

1 y

'AT

ksp

TG

tal T CAG AAA a Gin Lys ACT GCC Thr Ala CTA TTC Leu Phe GCC AGC Ala Ser ACT TAC Thr Tyr 110 TAC GCG Tyr Ala CCA GTG Pro Val CGG TTTC Arg PheC CTG ATT C Leu Ile L

ATC

Ile

CTG

Leu

TAC

Tyr

TGT

Cys

ACT

Thr

CCT

Pro

TAT

Tyr 3GT fly

TC

jCu 144 192 240 288 336 384 432 480 528 Asn Trp Gly Ala Asn 165 AAC AAC Asn Asn AAT GGC Asn Gly GGG GGG Gly Gly 210 AAG CAC Lys His 225 ACA CCT Thr Pro TGC ACT Cys Thr '3G GAG ~P Val Glu

ACG

Thr

ACT

Thr 195

GCC

Ala

CCC

Pro

AGG

Arg

GTC

Val1

CAC

His 275

CGG

Mrg 180

GGG

Gly

GGC

Gly

GAG

Glu

TGT

Cys

AAC

Asn 260

AGG

Arg

CCG

Pro

TTC

Phe

AAC

Asn

GCC

Ala

ATG

Met 245

TTC

Phe Yc' Phe

CCG

Pro

ACC

Thr

AAC

Asn

ACC

Thr 230

GTT

Val1

ACC

Thr LiAA Glu CGA GGC AAC TGG TTC GGC TGT Mrg Gly Asn Trp Phe Gly Cys 185 ACA TGG ATG Thr Trp Met 190 ACG TGT Thr Cys 200 TTG ACC Leu Thr GCC AGA Ala Arg TAC CCA Tyr Pro TTC AAG Phe Lys 265 GCA TGC Ala Cys 280

GGG

Gly

TGC

Cys

TGC

Cys

TAT

Tyr 250

GTT

Val1

AAT

Asn

GGC

Gly

CCC

Pro

GGT

Gly 235

AGG

Mrg

AGG

Arg

TGG

Trp

CCC

Pro

ACT

Thr 220

TCT

Ser

CTC

Leu

ATG

Met

ACT

Thr

AGC

Ser 300 CCG TGC AAC Pro Cys Asn 205 GAC TGT TTT Asp Cys Phe GGG CCC TGG Gly Pro Trp TGG CAC TAC Trp His Tyr 255 TAG GTG GGG Tyr Val Gly 270 CGA GGA GAG Arg Gly Glu 285 CCG CTG CTG Pro Leu Leu

ATC

ile

CGG

Mrg

CTG

iLeu 240

CCC

?'ro

GGC

Gly

CGT

.Arg

CTG

:,eu 576 624 672 720 768 816 864 912 TGT GAC TTG GAG GAC AGG GAT AGA TCA GAG CTT Cys Asp Leu Glu Asp Mrg Asp Arg Ser Glu Leu 290 295 114 TCT ACA ACA GAG TGG CAG AGC TTA ATT AAT TAG 945 Ser Thr Thr Glu 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 Val Leu Val Val Met Leu Leu Phe Ala 1 5 10 Gly Val Asp Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp 25 Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gin Lys Ile 40 Gin Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu 55 Asn Cys Asn Asp Ser Leu Gin Thr Gly Phe Phe Ala Ala Leu Phe Tyr 70 75 Lys His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys 90 Arg Ser Ile Asp Lys Phe Ala Gin Gly Trp Gly Pro Leu Thr Tyr Thr 100 105 110 Glu Pro Asn Ser Ser Asp Gin Arg Pro Tyr Cys Trp His Tyr Ala Pro 115 120 125 Arg Pro Cys Gly Ile Val Pro Ala Ser Gin Val Cys Gly Pro Val Tyr 130 135 140 Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly 145 150 155 160 Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile Leu 165 170 175 Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp Met 180 185 190 Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile 195 200 205 Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe Arg 210 215 220 Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp Leu 225 230 235 240 Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arq Leu TrD His Tvr Pro 245 250 255 Cys Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly 260 265 270 Val Glu His Arg Phe Glu Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg 275 280 285 115 Cys Asp Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu 290 295 300 Ser Thr Thr Glu 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 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..958 (ix) FEATURE: NAME/KEY: matpeptide LOCATION: 1..955 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: ATG GTG GGG AAC TGG GCT AAG GTT TTG GTT GTG ATG CTA CTC TTT GCC 48 Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala 1 5 10 GGC GTC GAC GGG CAT ACC CGC GTG TCA GGA GGG GCA GCA GCC TCC GAT 96 Gly Val Asp Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp 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 CTG 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 70 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 TTC 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 Glu 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 130 135 140 TGC TTC ACC CCG AGC CCT GTT GTG GTG GGG ACG ACC GAT CGG TTT GGT 480 116 Cys Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe 145 150 155 GTC CCC ACG TAT AAC TGG GGG GCG AAC GAC TCG GAT GTG CTG ATT C Val Pro Thr Tyr Asn Trp Gly Ala Asn Asp Ser Asp Val Leu Ile L 165 170 175 AAC AAC ACG CGG CCG CCG CGA GGC AAC TGG TTC GGC TGT ACA TGG A Asn Asn Thr Arg Pro Pro Arg Gly Asn Trp Phe Gly Cys Thr Trp M 180 185 190 AAT GGC ACT GGG TTC ACC AAG ACG TGT GGG GGC CCC CCG TGC AAC A Asn Gly Thr Gly Phe Thr Lys Thr Cys Gly GIy Pro Pro Cys Asn I 195 200 205 GGG GGG GCC GGC AAC AAC ACC TTG ACC TGC CCC ACT GAC TGT TTT C Gly Gly Ala Gly Asn Asn Thr Leu Thr Cys Pro Thr Asp Cys Phe A 210 215 220 AAG CAC CCC GAG GCC ACC TAC GCC AGA TGC GGT TCT GGG CCC TGG C: Lys His Pro Glu Ala Thr Tyr Ala Arg Cys Gly Ser Gly Pro Trp LE 225 230 235 24 ACA CCT AGG TGT ATG GTT CAT TAC CCA TAT AGG CTC TGG CAC TAC CC Thr Pro Arg Cys Met Val His Tyr Pro Tyr Arg Leu Trp His Tyr Pr 245 250 255 TGC ACT GTC AAC TTC ACC ATC TTC AAG GTT AGG ATG TAC GTG GGG GG 275 280 285 TGT GAC TTG GAG GAC AGG GAT AGA TCA GAG CTT AGC CCG CTG CTG CT

TAG

INFORMATION FOR SEQ ID NO: 44: SEQUENCE

CHARACTERISTICS:

LENGTH: 319 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: Met Val Gly Asn Trp Ala Lys Val Leu Val Val Met Leu Leu Phe Ala 1 5 10 Gly Val Asp Gly His Thr Arg Val Ser Gly Gly Ala Ala Ala Ser Asp 25 Thr Arg Gly Leu Val Ser Leu Phe Ser Pro Gly Ser Ala Gin Lys ile 40 Gin Leu Val Asn Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ala Leu 55 3ly 160

:TC

jeu

,TG

let

TC

le 3G rg rG

C

!C

y

T

g u -ic 528 576 624 672 720 768 816 864 912 958 961 117 Gly Phe Phe Ala Ala Leu Phe Asn Cys Asn Asp Ser Leu Gin Thr Lys His Lys Phe Asn Ser Ser Gly Cys Pro Glu Arg Leu Ala Ser Cys Arg Ser Giu Pro Arg Pro 130 Cys Phe 145 Val Pro Asn Asn Asn Gly Gly Gly 210 Lys His 225 Thr Pro Cys Thr Val Glu Cys Asp 290 Ile Asn 115 Cys Thr Thr Thr Thr 195 Ala Pro Arg Val His 275 Leu Asp 100 Ser Gly Pro Tyr Arg 180 Gly Gly Giu Cys Asn 260 Arg Glu Lys Phe Ala Ser Ile Ser Asn 165 Pro Phe Asn Al a Met 245 Phe Phe A.sp Asp Val1 Pro 150 Trp Pro Thr Asn Thr 230 Val1 Thr Glu Arg Gin Pro 135 *Val Gly Arg Lys Thr 215 Tyr His Ile Ala Asp 295 Gly Gin Arg 120 Al a Val Ala Gly Thr 200 Leu Ala Tyr Phe Ala 280 Arg Gln Gly 105 Pro Ser Val1 Asn Asn 185 Cys Thr Arg Pro Lys 265 Cys Ser Thr Trp Tyr Gin Gly Asp 170 Trp Gly Cys Cys Tyr 250 Val1 Asn Glu Pro *Gly Cys Val Thr 155 Ser Phe G ly Pro Gly 235 Arg Arg Trp, Leu Ser 315 Pro Trp Cys 140 Thr Asp Gly Pro Thr 220 Ser ELeu M1et rhr Ser 300 Pro Leu His 125 Gly Asp Val Cys Pro 205 Asp Gly Trp Tyr Arg 285 Pro Pro Thr 110 Tyr Pro Arg Leu Thr 190 Cys Cys Pro His Val 270 Gly Leu Ser Tyr Ala Val1 Phe Ile 175 Trp Asn Phe Trp Tyr 255 Gly Glu Leu Leu Thr Pro Tyr Gly 160 Leu Met Ile Arg Leu 240 Pro Gly Arg Ser Thr Thr Gly Asp Arg 305 310 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: matpeptide 118 LOCATION: 1. .1389 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ATG GTG GCG GGG GCC CAT TGG GGA GTC CTG GCG GGC CTC GCC TAC TAT Met Vai Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr 1 5 10 TCC ATG GTG GGG AAC TGG Ser Met Val Gly Asn Trp GCT AAG GTT TTG GTT GTG ATG CTA CTC 'TTT Ala~ uys Val Leu Val Val M 25TC GG GG

GCC

Al a

GAT

Asp

ATC

Ile

CTG

Leu

TAC

Tyr

TGT

Cys

ACT

Thr

CCT

Pro 145

TAT

Tyr

GGT

Gly

CTC

Leu

ATG

Met

ATC

225

GGC

Gly

ACC

Thr

CAG

Gin

AAC

Asn

AAA

Lys

CGC

Arg

GAG

Giu 130

CGA

Arg

TGC

Cys1

GTC

Val kiAC2 %sn2 k.AT ),sn 3GG C ,Jly C GTC GAC Val Asp AGG GGC Arg Gly GGG CAT ACC CGC GTC Gly His Thr Arg Val 40 CTT GTG TCC CTC TTI Leu Vai Ser Leu Phe Ser Giy Gly A AGC CCC GGG T

CTC

Leu

TGC

Cys

CAC

His

TCC

Ser 115

CCT

Pro

CCG

Pro

TTC

Phe

~CC

Pro

%JAC

%sn 195

,GC

;iy

GG

GTA

Val

AAC

Asn

AAA

Lys 100

ATC

Ile

AAC

Asn

TGT

Cys

ACC

Thr

ACG

Thr 180

ACG

Thr

ACT

Thr

GCC

Ala

AAC

Asn

GAC

Asp

TTC

Phe

GAC

Asp

AGC

Ser

GGT

Gly

CCG

Pro 165

TAT

T'yr

CGG

krg 3GG Gly 3GC 3ly.

ACC AAC GGC AGT Thr Asn Gly Ser 70 TCC CTC CAA ACA Ser

AAC

Asn

AAG

Lys

TCG

Ser

ATT

Ile 150

AGC

Ser

AAC

Asn

CCG

Pro

TTC

Phe

AAC

Asn 230 Leu

TCG

Ser

TTC

Phe

GAC

Asp 135

GTA

Val1

CCT

Pro

TGG

Trp

CCG

Pro

ACC

Thr 215

AAC

Asn Gin

TCT

Ser

GCT

Al a 120

CAG

Gin

CCC

Pro

GTT

Val

GGG

Gly.

CGA

Arg 200

AAG

Lys

ACC

Thr Thr

GGA

Gly 105

CAG

Gin

AGG

Arg

GCG

Ala

GTG

Val1

GCG

Al a 185

GGC

Gly

ACG

rhr

TTG

Leu Ser

TGG

Trp

GGG

Gly 90

TGC

Cys

GGG

Gly

CCC

Pro

TCT

Ser

GTG

Val1 170

AAC

Asn

AAC

Asn

TGT

Cys

ACC

Thr Pro

CAC

His 75

TTC

Phe

CCA

Pro

TGG

Trp

TAC

Tyr

CAG

Gin 155

GGG

Gly

GAC

Asp

TGG

Trp

GGG

Gly

TGC

Cys 235 Gly

ATC

Ile

TTT

Phe

GAG

Glu

GT

Gly

TGC

Cys 140

GTG

Val1

ACG

Thr

TCG

Ser

TTC

Phe

GGC

Gly 220

CCC

Pro

S

A

A

GI

A:

C(

CC

12

TC

Ty.

TG

AC

Th

G.A

As G1 20 Pr rh let Leu Leu Phe ;CA GCA GCC TCC .la Ala Ala Ser CG GCT CAG AAA er Ala Gin Lys AC AGG ACT GCC sn Arg Thr Ala CC GCA CTA TTC la Ala Leu Phe 3C TTG GCC AGC Leu Ala Ser 110 'C CTC ACT TAC :o Leu Thr Tyr G CAC TAC GCG p His Tyr Ala IC GGT CCA GTG 's Gly Pro Val 160 'C OAT CGG TTT Lr Asp Arg Phe 175 TGTG CTG ATT p Val Leu Ile 190 C TGT ACA TGG y Cys Thr Trp C CCG TOC AAC o Pro Cys Asn T GAC TOT -rTT r Asp Cys Phe 240 96 144 192 240 288 336 384 432 480 528 576 624 672 720 CGG AAG CAC Arg Lys His CCC GAG GCC ACC TAC AGA TGC GGT TCT GGG CCC Pro Giu 245 Ala Thr Tyr Arg 250 Cys Gly Ser Gly Pro 255

CTG

Leu

CCC

Pro

GGC

Gly

CGT

Arg 305

CTG

Leu

CCG

Pro

GTG

Val

AAA~

Lys

ATC

Ile 385 GCC 'I Ala I CAT G His G AAG G Lys G

ACA

Thr

TGC

Cys

GTG

Val1 290

TGT

Cys

TCT

Ser

GCC

Ala

CAA

TGG

rrp 370 rGC rTA ~eu GC2 ;ly

GC

~ly1 CCT AGG TOT Pro Arg Cys 260 ACT GTC AAC Thr Val Asn 275 GAG CAC AG Giu His Arg GAC TTG GAG Asp Leu Glu ACA ACA GAG Thr Thr Giu 325 CTA TCC ACC Leu Ser Thr 340

ATG

Met

TTC

Phe

TTC

Phe

GAC

Asp 310

TG

Trp

GC

Gly GTT CAT Vai His ACC ATC Thr Ile 280 GAA GCC Giu Ala 295 AGG OAT Arg Asp CAG ATA Gin Ile CTO ATC Leu Ile 119 TAC CCA TAT AGO Tyr Pro Tyr Arg 265 TTC AAG OTT AGO Phe Lys Val Arg GCA TOC AAT TOO Aia Cys Asn Trp 300 AGA TCA GAG CTT- Arg Ser Oiu Leu 315 CTO CCC TOT TC Leu Pro Cys Ser 330 CAC CTC CAT CAG His Leu His Gin 345

CTC

Leu

ATO

Met 285

ACT

Thr

AGC

Ser

TTC

Phe

AAC

Asn TOG CAC TA Trp His Ty 270 TAC OTO 00 Tyr Vai Gil COA OGA GA Arg Giy Oh CCO CTG CT( Pro Leu LeL 320 ACC ACC CTG Thr Thr Leu 335 ATC OTO GAC Ile Vai Asp 350

C

r

G

y 8i6 864 912 960 1008 1056 1104 1152 1200 i2 48 1296 1344 rAC T'yr 355 33AG 3lu 3CC la

;AG

;iu

~CT

'hr ,00 .rg 35

CTG

Leu

TAT

Tyr

TOC

Cys

AAC

Asn

CTT

Leu 420

CTO

Leu

TAC

Tyr

OTC

Val1

TTA

Leu

CTO

Leu 405

TCC

Ser

OTC

Val1

GT

Gly

CTO

Leu

TOO

Trp 390

OTO

Val1

TTC

Phe

CCT

Pro

OTA

Val

TTO

Leu 375

ATO

Met

OTC

Vai

CTT

Leu

GOT

Gly 000 Giy 360

CTC

Leu

ATO

Met

CTC

Leu

OTO

Vai

OCO

Aia 440 TCO OCO Ser Ala TTC CTT Phe Leu CTG CTO Leu Leu AAT GCO Asn Ala 410 TTC TTC Phe Phe 425 OCA TAC Ala Tyr

OTT

Vali

CTC

Leu

ATA

Ile 395

OCO

Ala

TOT

Cys

GCC

Aila OTC TCC Val Ser 365 CTO OCA Leu Aia 380 OCT CAA Aia Gin 0CC OTO Ala Vai OCT 0CC Ala Aia TTC TAT Phe Tyr 445

CTT

Leu

OAC

Asp

OCT

Ala 0CC Aila

TOG

Trp 430

GOC

Gly

GTC

Vali

OCO

Ala

GAO

Olu 000 Oly 415

TAC

Tyr

OTO

Val

ATC

Ile

COC

Arg 0CC Ala 400

OCO

Al a

ATC

Ile

TOO

Trp 4 CCO CTO CTC CTO CTT CTO CTG 0CC TTA CCA CCA CGA OCT TAT 0CC TAGTAA Pro Leu Leu Leu Leu Leu Leu Ala Leu Pro Pro Arg Aia Tyr Ala 450 455 460 INFORMATION FOR SEQ ID NO: 46: SEQUENCE CHARACTERISTICS: LENGTH: 463 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION- qfn Tn Mrn. Met Val Ala Gly Ala His Trp Gly Val Leu Ala Oly Leu Ala Tyr Tyr 1 5 10 Ser Met Val Oly Asn Trp, Ala Lys Val Leu Val Val Met Leu Leu ?he 25 1395 Ala Gly Val Asp Gly His Thr Arg Val 40 120 Ser Gly Gly Ala Ala Ala Ser Asp Thr Arg Gly Leu Val Ser Leu Phe Ser Pr 55 Asn Ile Leu Tyr Cys Thr Pro 145 Tyr Gly Leu Met Ile 225 Arg Leu Pro Gly Arg 305 Leu Pro I Vai C Lys '1 Gin Leu Val Asn Thr Gly Ser Trr Asn Lys Arg Glu 130 Arg Cys Val1 Asn Asn 210 Gly Lys rhr Cys la 1 290 ;er ;ln rp Cys His Ser 115 Pro Pro Phe Pro Asn 195 Gly Gly His Pro Thr 275 Glu Asp Thr Leu Tyr 355 Glu Asn ILys 100 Ile Asn Cys Thr Thr 180 Thr Thr Ala Pro Arg 260 Val His Leu Thr Ser 340 Leu Tyr Asp Phe Asp Ser Gly Pro 165 Tyr Arg Giy Gly Glu 245 Cys Asn Arg Giu Glu 325 ['hr ['yr la 1 70 Ser Asn Lys Ser Ile 150 Ser Asn Pro Phe Asn 230 Ala Met Phe Phe Asp 310 Trp C Gly I Gly Leu L Leu Gin Thr Set Phe Asp 135 Val1 Pro Trp Pro Thr 215 Asn rhr la 1 ['hr 3lu ~95 krg ln ~eu Tal ~eu 175 *Ser Ala 120 Gin Pro Val Gly Arg 200 Lys Thr Tyr His Ile 280 Ala.

Asp.

Ile Ile Gly 360 Leu Giy 105 Gin Arg Ala Val1 Al a 185 Gly Thr Leu Aia Tyr 265 Phe Al a Arg Lieu -His 345 Ser Phe Gly 90 'Cys Gly Pro Ser Val 170 Asn Asn Cys Thr Arg 250 Pro Lys Cys Ser Pro 330 Leu Ala Leu I Hi~ PhE Prc Trp Tyr Gin 155 Gly Asp Trp Gly Cys 235 Cys Tyr Val1 ksn G1u 315 :ys -[is fal ~eu oGly s Ile SPhe Glu Gly Cys 140 *Val *Thr Ser Phe Gly 220 Pro Gly Arg I Arg Trp 1* 300 Leu S Ser P Gin A Val S 3 Leu A 380 Se: Asi Al~ Arc Pro 125 Trp Cys Thr Asp Gly 205 Pro ['hr 3er ~eu ~et 'hr ;er 'he ~sn er l1a r Al~ 'i Arc a Ala Leu 110 Leu His Gly Asp Val1 190 Cys Pro Asp Gly Trp 270 Tyr Arg Pro Thr Ile 350 Leu Asp aGin Lys Thr Ala Leu Phe IAla Thr Tyr Tyr Ala Pro Val 160 Arg Phe 175 Leu Ile Thr Trp Cys Asn Cys Phe 240 Pro Trp 255 His Tyr Val Gly Gly Giu Leu Leu 320 Thr Leu 335 Val Asp Val Ile Ala Arg Cys Ala Cys Leu Trp 390 Met Met Leu Leu AlGiAaGi Ala Gln Ala Glu 121 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 CTT 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 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 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 TTG 192 Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu 55 CTG TCC TGT CTG ACC GTT 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 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 122 115 CTC OCA GCT Leu Ala Ala 130 GTC GAT ?TG Val Asp Leu 145 GGG GAC CTC Gly Asp Leu TCG CCT CGC Ser Pro Arg CCC GGC CAC Pro Gly His 195 TGG TCG CCT Trp Ser Pro 210 CAA GCT GTC Gin Ala Val 225 GGC CTC GCC Gly Leu Ala GTG ATG CTA C Val Met Leu L

AGG

Arg

CTC

Leu

TGC

Cys

CGG

Arg 180

ATA

Ile

A~CA

rhr 3TGC lal I

[AC

[yr2 2

AA

As:

GT.

Va

GG)

Glj 165

CAT

His

ACG

Thr kCG rhr 3AC ~sp

.'AT

yr :45 120 C GCC AGC GTC CCC n Ala Ser Val Pro 135 rGGG GCG GCT GCT L Gly Ala Ala Ala 150 STCT GTC ITC CTC 'Ser Val Phe Leu GAG ACG GTG CAG Giu Thr Val Gin 185 GGT CAC CGT ATG Gly His Arg Met 200 GCC CTG GTG GTA Ala Leu Val Val 215 ATG GTG GCG GGG Met Val Ala Gly 230 TCC ATG GTG GGG Ser Met Val Gly GCC GGC GTC GAC G Ala Gly Val Asp G 265

ACC

Thr

TTC

Phe

GTC

Val1 170

GAC

Asp

GCT

Ala

TCG

Ser 3CCC kla 1 AC2 ~sn 2 ~50

~GGC

Ily H

AC

Th TGr Cy~ 15!

TC(

Sez

TGC

Cys

TGG

Trp

CAG

31n

'AT

is ~35

'GG

rp

'AT

i1s 12 G ACA AT r Thr Ii 140 T TCC GC' s Ser AL.

SCAG CT( Gin Lei AAT TG( Asn CyE GAT ATC Asp Met 205 CTG CTC Leu Leu 220 TGG GGA Trp Gly GCT AAG Ala Lys ACC CGC Thr Arg A CGA CGC CAC e Arg Arg His T' ATG TAC GTG a Met Tyr Val 160 3 TTC ACC ATC 1 Phe Thr Ile 175 TCA ATC TAT Ser Ile Tyr 190 IATG ATG AAC .Met Met Asn CGG ATC CCA Arg Ile Pro GTC CTG GCG Vai Leu Ala 240 GTT TTG GTT Val Leu Val 255 GTG TCA GGA Val Ser Gly 432 480 528 576 624 672 720 768 816 .eu Phe 260

GGG

Gly

GGG

Giy

ATC

Ile 305

TTT

Phe

GAG

Glu

GGT

Gly

TGC

Cys

GTG

Val 385

GCA

Ala

TCG

Ser 290

AAC

Asn

GCC

Ala

CGC

Arg

CC

Pro

TGG

rrp 370

GCA

Ala 275

GCT

Ala

AGG

Arg

GCA

Ala

TTG

Leu

CTC

Leu 355

CAC

His

GCC

Ala

CAG

Gin

ACT

Thr

CTA

Leu

GCC

Al a 340

ACT

Thr

TAC

TCC

Ser

AAA

Lys

GCC

Al a

TTC

Phe 325

AGC

Ser

TAC

Tyr (rrr.

GAT

Asp

ATC

Ile

CTG

Leu 310

TAC

Tyr

TGT

Cys

ACT

Thr ACC AGG Thr Arg 280 CAG CTC Gin Leu 295 AAC TGC Asn Cys AAA CAC Lys His CGC TCC Arg Ser GAG CCT Glu Pro 360 Arg Pro 375

GGC

Gly

GTA

Val

AAC

Asn

AAA

Lys

ATC

Ile 345

AAC

Asn Cys

CTT

Leu

AAC

Asn

GAC

Asp

TTC

Phe 330

GAC

Asp

AGC

Ser Gly

GTG

Val1

ACC

Thr

TCC

Ser 315

AAC

Asn

AAG

Lys

TCG

Ser Ile

AGC

Ser 395

TCC

Ser

AAC

Asn 300

CTC

Leu

TCG

Ser

TTC

Phe

GAC

Asp Val1 380

CTC

Leu 285

GGC

Giy

CAA

Gin

TCT

Ser

GCT

Ala

CAG

Gin 365 Pro

TTT

Phe

AGT

Ser

ACA

Thr

GGA

Gly

CAG

Gin 350

AGG

Arg Ala3

AGC

Ser

TGG

Trp

GGG

Gly

TGC

Cys 335

GGG

Gly

CCC

Pro

TCT

Ser

CCC

Pro

CAC

His

ITC

Phe 320

CCA

Pro

TGG

Trp

TAC

Tyr

CAG

Gin

GGG

Gly 400 864 912 960 1008 1056 1104 1152 1200 T'yr Ala Pro rGC GGT CCA GTG 'ys Gly Pro Val TGC TTC ACC CCG Cys Phe Thr Pro CCT GTT GTG GTG Pro Val Val Val

AC

Th

TC(

Se~

TT(

PhE

GGC

Gij

CCC

Pro 465

GGT

Gly

AGG

Arg

AGG

Arg

TGG

Trp, G ACC GAT CGG r Thr Asp Arg G GAT GTG CTG r Asp Val Leu 420 GGC TGT ACA Gly Cys Thr 435 CCC CCG TGC Pro Pro Cys 450 ACT GAC TGT Thr Asp Cys TCT GGG CCC Ser Gly Pro CTC TGG CAC Leu Trp His 500 ATG TAC GTG Met Tyr Val C 515 ACT CGA GGA G Thr Arg Gly G 530 Tr.

Ph~ 401

AT)

Ile

TGG

Trp

AAC

Asn

TTT

Phe

TGG

rrp 485 rAC ['yr

GG

fly

;AG

[GGT GTC SGly Val CTC AAC Leu Asn ATG AAT Met Asn ATC GGG Ile Gly 455 CGG AAG Arg Lys 470 CTG ACA Leu Thr CCC TGC Pro Cys GGC GTGC Giy Val C CGT TGTC Arg Cys A 535 CCC ACG Pro Thr AAC ACG Asn Thr 425 GGC ACT Gly Thr 440 GGG GCC Gly Ala CAC CCC His Pro CCT AGG Pro Arg %.CT GTC r'hr Val 505 ,AG CAC2 ;lu His 1 20 AC TTG G sp Leu G 123 TAT AAC Tyr Asn 410 CGG CCG Arg Pro GGG TTC Gly Phe GGC AAC Gly Asn GAG GCC Glu Ala 475 rGT ATG Cys Met 490 kAC TTC .7 ks5n Phe I3 GG TTC G ~rg Phe G AG GAC ~lu Asp A 5

TG(

Trj

CC(

Prc

ACC

Thr

AAC

Asn 460

A.CC

rhr 3TT Ia 1

~CC

h r

AA

i u

GG

rg G GGG GCG SGly Ala CGA GGC Arg Gly 430 AAG ACG Lys Thr 445 ACC TTG Thr Leu TAC GCC Tyr Ala CAT TAC His Tyr ATC TTC Ile Phe 510 GCC GCA Ala Ala C 525 GAT AGA I Asp Arg S

AA(

Asi 41~

AAC

Asr

TGT

Cys

ACC

Thr

AGA

Arg

CCA

Pro 495 kAG Lys

E'GC

~ys

'CA

er

CGAC

-1 Asp

TGG

ITrp

GG

Gly

TGC

Cys

TGC

Cys 480

TAT

Tyr

GTT

Val

AAT

Asn

GAG

Giu 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 1824 1872 1920 1968 2016 CTT AGC Leu Ser 545 TCC TTC Ser Phe CAG AAC Gin Asn GTC TCC Val Ser CTG GCA Leu Ala 610 GCT CAA Ala Gin 625 GCC GTG Ala Vai GCT GCC Ala Ala

CCG

Pro

ACC

Thr

ATC

Ile

CTT

Leu 595

GAC

Asp

GCT

Ala

GCC

Ala

TGG

CTG CTG Leu Leu ACC CTG Thr Leu 565 GTG GAC Val Asp 580 GTC ATC Val Ile GCG CGC Ala Arg GAG GCC Glu Ala GGG GCG Gly Ala TAC ATC CTG TCT ACA Leu 550

CCG

Pro

GTG

Val

AAA

Lys

ATC

Ile

GCC

Ala 630

CAT

His Ser

GCC

Ala

CAA

Gin

TGG

Trp

TGC

Cys 615

TTA

Leu

GGC

Gly Thr

CTA

Leu

TAC

Tyr

GAG

Glu 600

GCC

Ala

GAG

Giu

ACT

ACA GAG TGG Thr Giu Trp 555 TCC ACC GGC Ser Thr Gly 570 CTG TAC GGT Leu Tyr Gly 585 TAT GTC CTG Tyr Val Leu TGC TTA TGG Cys Leu Trp AAC CTG GTG Asn Leu Val 635 CTT TCC TTC Leu Ser Phe CAG ATA CTG CCC TGT Gin Ile Leu Pro Cys CTG ATC Leu Ile GTA GGG Val Gly TTG CTC Leu Leu 605 ATG ATG Met Met 620 GTC CTC Val Leu CTT GTG Leu Val GGT GCG Gly Ala CAC CTC CAT His Leu His 575 TCG GCG GTT Ser Ala Val 590 TTC CTT CTC Phe Leu Leu CTG CTG ATA Leu Leu Ile AAT GCG GCG Asn Ala Ala 640 TTC TTC TGT Phe Phe Cvs 655 GCA TAC GCC Ala Tyr Ala 650 Trp Tyr 660 Ile AAG GGC AGG Lys Gly Arg CTG GTC Leu Val 665

CCT

Pro TTC TAT GGC GTG TGG CCG CTG CTC CTG CTT CTG CTG GCC TTA CCA CCA 2064 124 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 2082 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: Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu 1 5 10 Val 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 Val Asn Tyr Ala 40 Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu 55 60 Leu Ser Cys Leu Thr Val Pro Ala Ser Ala Tyr Glu Val Arg Asn Val 70 75 Ser Gly Met Tyr His Val Thr Asn Asp Cys Ser Asn Ser Ser Ile Val 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 Val 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 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 Ser Pro Thr Thr Ala Leu Val Val Ser Gin Leu Leu Arg Ile Pro 210 215 220 Gln Ala Val Val Asp Met Val Ala Gly Ala His Trp Gly Val Leu Ala 22 230 235 240 Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala Lys Val Leu Val 245 250 255 Val Met Leu Leu Phe Ala Gly Val Asp Gly His Thr Arg Val Ser Gly 260 265 270 Gly Ala Ala Ala Ser Asp 275 125 Thr Arg Gly Leu 280 Val Ser Leu Phe Ser Pro 285 Gly Ser 290 lie Asn 305 Phe Ala Glu Arg Gly Pro Cys Trp 370 Val Cys 385 Thr Thr Ser Asp Phe Gly Gly Pro 450 Pro Thr 465 Gly Ser Arg Leu Arg Met Trp Thr 530 Leu Ser I 545 Ser Phe Gin Asn I Val Ser I Leu Ala 610 Ala Gin 625 Ala Gin Lys Ile Gin Leu Val Asn Thr Ar Al Leu Leu 355 His Gly Asp Val Cys 435 Pro Asp Gly Trp Tyr 515 k.rg ?ro rhr le .eu ~sp la Thi Leu Ala 340 Thr Tyr Pro Arg Leu 420 Thr Cys Cys Pro His 500 Val Gly Leu Thr Va1 580 Va1 Ala Glu 295 Ala Leu Asn 310 Phe Tyr Lys 325 Ser Cys Arg Tyr Thr Glu Ala Pro Arg 375 Val Tyr Cys 390 Phe Gly Val 405 Ile Leu Asn Trp Met Asn Asn Ile Gly 455 Phe Arg Lys 470 Trp Leu Thr 485 Tyr Pro Cys Gly Gly Val C Glu Arg Cys 1 535 Leu Leu Ser 1 550 Leu Pro Ala L 565 Asp Val Gin I Ile Lys Trp G 6 Arg Ile Cys A 615 Ala Ala Leu G 630 Asn Gly Ser Trp His 300 Leu Gin Thr Gly Phe Cys Asn As His Ser Pro 360 Pro Phe Pro Asn Gly 440 Gly His ?ro rhr lu j20 ~sp 'hr leu 'yr ;lu .00 la Ilu Lys lie 345 Asn Cys Thr Thr Thr 425 Thr Ala Pro Arg Val 505 His Leu Thr Ser Leu 585 Tyr Cys i Asn Ph 33' As Sei Gl Pro Tyr 410 Arg Gly Gly Glu Cys 490 Asn Arg Glu 31u rhr 570 Tyr al Leu Leu p Ser 315 e Asn 0 Lys Ser Ile Ser 395 Asn Pro Phe Asn.

Ala 475 Met Phe Phe Asp Trp 555 Gly I Gly I Leu I Trp r' Val 635 Sei Phe Asp Val 380 Pro Trp Pro Thr Asn 460 Thr Val rhr Glu krg 540 ,ln eu lal 4eu let Tal Ser Ala Gin 365 Pro Val Gly Arg Lys 445 Thr Tyr His Ile Ala 525 Asp Ile Ile Gly Leu I 605 Met Leu 1 Gil Glr Arc Ala Val Ala Gly 430 Thr Leu Ala Tyr Phe 510 Ala Arg Leu His Ser 590 Phe eu %sn r Cys 335 1 Gly Pro Ser Va1 Asn 415 Asn Cys Thr Arg Pro 495 Lys Cys Ser Pro Leu 575 Ala Leu Leu Ala 320 Pro Trp Tyr Gin Gly 400 Asp Trp Gly Cys Cys 480 Tyr Va1 Asn Glu Cys 560 His Val Leu Ile Ala 640 126 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

ATG

Met 1

CGC

Arg

GGA

Gly

ACT

Thr

ATC

Ile

TAC

Tyr

CTC

Leu (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..2430 (ix) FEATURE: NAME/KEY: mat_peptide LOCATION: 1..2427 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: AGC ACG AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT Ser Thr Asn Pro Lys Pro Gin Arg Lys Thr Lys Arg 5 10 CGC CCA CAG GAC GTC AAG TTC CCG GGC GGT GGT CAG Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gin 25 GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly 40 AGG AAG ACT TCC GAG CGG TCG CAA CCT CGT GGG AGG Arg Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly Arg 55 CCC AAG GCT CGC CGA CCC GAG GGT AGG GCC TGG GCT Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala 70 75 CCT TGG CCC CTC TAT GGC AAT GAG GGC ATG GGG TGG Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly Trp 90 CTG TCA rrCC CC TCT C nCCT A.o TGG...

Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro 100 105 AAC ACC AAC Asn Thr Asn

ATC

Ile

GTG

Val

CGA

Arg

CAG

Gin

GCA

Ala

ACA

Thr 110

GTT

Val

CGC

Arg

CAA

Gin

CCC

Pro

GGA

Gly

GAC

Asp

GGT

Gly

GCG

Ala

CCT

Pro

GGG

Gly

TGG

Trp

CCC

Pro 96 144 192 240 288 336 CGG CGT AGG Arg Arg Arg 115 TCG CGT AAT TTG Ser Arg Asn Leu AAG GTC ATC GAT Lys Val Ile Asp

ACC

Thr 125 CTT ACA TGC Leu Thr Cys TTC GCC GAC Phe Ala Asp 127 CTC GTG GGG TAC ATT CCG CTC GTC Leu Val Gly Tyr Ile Pro Leu Val GGC GCC CCC CTA Gly Ala Pro Leu 130 GGG GGC Gly Gly 145 GGC GTG Gly Val TTC CTC 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 Gin 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 385 135 GCT GCC Ala Ala AAC TAT Asn Tyr TTG GCT Leu Ala 180 CGC AAC Arg Asn 195 AGC ATT Ser Ile GTG CCC Val Pro ACC CCC Thr Pro CGA CGC Arg Arg 260 ATG TAC Met Tyr 275 TTC ACC Phe Thr TCA ATC Ser Ile ATG ATG Met Met CGG ANTC Arg Ile 340 GTC CTG Val Leu 355 GTT TTG Vai Leu GTG TCA Vai Ser

AGG

Arg

GCA

Ala 165

TTG

Leu

GTG

Val1

GTG

Val1

TGC

Cys

ACG

Thr 245

CAC

His

GTG

Vai

ATC

Ile

TAT

Tyr

AAC

Asn 325

CCA

Pro

GCG

Ala

GTT

Val1

GGA

Gly GCC CTG GCG Ala Leu Ala 150 ACA GGG AAT Thr Gly Asn CTG TCC TGT Leu Ser Cys TCC GGG ATG Ser Gly Met 200 TAT GAG GCA Tyr Giu Ala 215 GTT CGG GAG Val Arg Glu 230 CTC GCA GCT Leu Ala Ala GTC GAT TTG Val Asp Leu GGG GAC CTC Gly Asp Leu 280 TCG CCT CGC Ser Pro Arg 295 CCC GGC CAC Pro Gly His 310 TGG TCG CCT Trp Ser Pro CAA GCT GTC Gin Ala Val GGC CTC GCC Gly Leu Ala 360 GTG ATG CTA Vai Met Leu 375 GGG GCA GCA Gly Ala Ala 390 CAT GGC His Gly TTG CCC Leu Pro 170 CTG ACC Leu Thr 185 TAC CAT Tyr His GCG GAC Ala Asp AAC AAC Asn Asn AGG AAC Arg Asn 250 CTC GTT Leu Val 265 TGC GGA Cys Gly CGG CAT Arg His ATA ACG Ile Thr ACA ACG Thr Thr 330 GTG GAC Val Asp 345 TAC TAT Tyr Tyr CTC TTT Leu Phe GCC TCC Ala Ser 140 GTC CGG Val Arg 155 GGT TGC Gly Cys GTT CCA Val Pro GTC ACG Val Thr ATG ATC Met Ile 220 TCT TCC Ser Ser 235 GCC AGC Ala Ser GGG GCG Gly Ala TCT GTC Ser Val GAG ACG Giu Thr 300 GGT CAC Gly His 315 GCC CTG Ala Leu ATG GTG Met Val TCC ATG Ser Met GCC GGC Ala Gly 380 GAT ACC Asp Thr 395

GTT

Val1

TCT

Ser

GCT

Al a

AAC

Asn 205

ATG

Met

CGC

Arg

GTC

Val1

GCT

Ala

TTC

Phe 285

GTG

Val

CGT

Arg

GTG

Val1

GCG

Ala

GTG

Val1 365

GTC

Val1

AGG

Arg

CTG

Leu

TTC

Phe

TCC

Ser 190

GAC

Asp

CAC

His

TGC

Cys

CCC

Pro

GCT

Ala 270

CTC

Leu

CAG

Gin

ATG

Met

GTA

Val

GGG

Gly 350

GGG

Gly

GAC

Asp

GGC

Gly GAG GAC Giu Asp 160 TCT ATC Ser Ile 175 GCT TAT Ala Tyr TGC TCC Cys Ser ACC CCC Thr Pro TGG GTA Trp Val 240 ACC ACG Thr Thr 255 TTC TGT Phe Cys GTC TCC Val Ser GAC TGC Asp Cys GCT TGG Ala Trp 320 TCG CAG Ser Gin 335 GCC CAT Ala His AAC TGG Asn Trp GGG CAT Gly His CTT GTG Leu Val 400 480 528 576 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 TCC CTC TITT AGC CCC GGG TCG GCT CAG AAA ATC CAG CTC GTA AAC ACC 128 Ser Leu Phe Ser Pro Cly Ser Ala Gin 405

AAC

Asn

CTC

Leu

TCG

Ser

TTC

Phe 465

GAC

Asp

GTA

Val

CCT

Pro

TGG

Trp

CCG

Pro 545

ACC

Thr

AAC

Asn

ACC

Thr

GTT

Val1

ACC

Thr 625

GAA

Glu

AGG

Arg

CAG

Gin GGC AGT Gly Ser CAA ACA Gin Thr 435 TCT GGA Ser Gly 450 GCT CAG Ala Gin CAG AGG Gin Arg CCC GCG Pro Ala GTT GTG Val Val 515 GGG GG Gly Ala 530 CGA GGC Arg Gly AAG ACG Lys Thr ACC TTG Thr Leu TAG GCC Tyr Ala 595 CAT TAC His Tyr 610 ATC TTC Ile Phe GCC GCA Ala Ala GAT AGA Asp Arg ATA CTG Ile Leu

TGG

Trp 420

GGG

Gly

TGC

Gys

GGG

Gly

CCC

Pro

TCT

Ser 500

GTG

Val1

AAC

Asn

AAC

Asn

TGT

Cys

ACC

Thr 580

AGA

Arg

CCA

Pro

AAG

L~ys

TGC

Cys

TCA

Ser 660

CCC

Pro

CAC

His

TTC

Phe

CCA

Pro

TG

Trp,

TAC

Tyr 485

GAG

Gln

GGG

Gly

GAC

Asp

TGG

Trp

GGG

Gly 565

TGC

Gys rGC :Cys

TAT

Tyr

GTT

Val

AAT

Asn 645

GAG

Giu

TGT

Cys

ATC

Ile

TTT

Phe

GAG

Glu

GGT

Giy 470

TGC

Cys

GTG

Val

ACG

Thr

TG

Ser TrC Phe 550

GCC

Gly

CCC

Pro

GT

Gly

AGG

Arg

AG

Arg 630

TGG

Trp

GTT

Leu

TCC

Ser AAC AGG Asn Arg GGC GCA Ala Ala 440 CGC TTG Arg Leu 455 CCC CTC Pro Leu TGG CAC Trp His TGG GGT Cys Gly ACC GAT Thr Asp 520 GAT GTG Asp Val 535 GGC TGT Gly Cys CCC GCG Pro Pro ACT GAG Thr Asp TGT CCC Ser Gly 600 CTC TGG Leu Trp 615 ATG TAG Met Tyr ACT GGA Thr Arg AGG CCC Ser Pro TTC ACC Phe Thr

ACT

Thr 425

CTA

Leu

CC

Ala

ACT

Thr

TAG

Tyr

GCA

Pro 505

CGG

Arg

GTG

Leu

ACA

Thr

TGC

Cys.

TGT

Gys 585

CCC

Pro

GAG

His

GTG

Val

GGA

Cly

CTG

Leu 665

ACC

Thr Lys 410

GCC

Ala

TTC

Phe

AGC

Ser

TAG

Tyr

C

Ala 490

GTC

Val

TTT

Phe

ATT

Ile

TGO

Trp

AAG

Asn 570

TTT

Phe

TGG

Trp

TAG

Tyr

GGG

Gly

GAG

Glu 650

GTG

Leu

CG

Leu

GTG

Leu

TAG

Tyr

TGT

Cys

ACT

Thr 475

GCT

Pro

TAT

Tyr

GT

Gly

CTC

Leu

ATG

Met 555

ATC

Ile

CCC

Arg

CTG

Leu

CCC

Pro

GCC

Gly 635

CGT

Arg

CTG

Leu

CCC

Pro

AAC

Asn

AAA

Lys

CC

Arg 460

GAG

Giu

GGA

Arg

TGG

Cys

GTC

Val1

AAC

Asn 540

AAT

Asn

GGG

Cly

AAC

Lys

ACA

Thr

TC

Cys 620

GTG

Val1

TGT

Gys

TCT

Ser

CC

Al a Ile Gin Leu Val Asn Thr

TGC

Gys

CAC

His 445

TCC

Ser

GCT

Pro

CCC

Pro

TTC

Phe

CCC

Pro 525

AAC

Asn

GCC

Cly

CCC

Gly

CAC

His

GGT

Pro 605

ACT

Thr

GAG

Giu

GAG

Asp

ACA

Thr

CTA

Leu

AAG

Asn 430

AAA

Lys

ATG

Ile

AAC

Asn

TGT

Cys

ACC

Thr 510

ACG

Thr

ACG

Thr

ACT

Thr

CC

Al a

CCC

Pro 590

AGC

Arg

GTC

Val

CAG

His

TTG

Leu

ACA

Thr 670

TCC

Ser 415

GAG

Asp

TTC

Phe

GAG

Asp

AGC

Ser

GGT

Gly 495

CCG

Pro

TAT

Tyr

CGG

Arg

GGG

Gly

GCC

Gly 575

GAG

Giu

TOT

Gys

AAC

Asn

AGG

Arg

GAG

Glu 655

GAG

Giu

ACC

Thr

TCC

Ser

AAC

Asn

AAG

Lys

TCG

Ser 480

ATT

Ile

AGC

Ser

AAG

Asn

CCG

Pro

TTC

Phe 560

AAC

Asn

CC

Ala

ATG

Met

TTC

Phe

TTC

Phe 640

GAG

Asp

TGG

Trp

GC

Gly 1296 1344 1392 1440 1488 1536 *i1584 1632 1680 1728 1776 1824 1872 1920 1968 2016 2064 129

CTG

Leu

GTA

Val 705

TTG

Leu

ATG

Met

GTC

Val

CTT

Leu

GGT

Gly 785

CTG

Leu

ATC

Ile 690

GGG

Gly

CTC

Leu

ATG

Met

CTC

Leu

GTG

Val 770

GCG

Ala

GCC

Ala 675 SCAC CTC CAT CAG His Leu His Gin TCG GCG GTT GTC Ser Ala Val Val 710 TTC CTT CTC CTG Phe Leu Leu Leu 725 CTG CTG ATA GCT Leu Leu Ile Ala 740 AAT GCG GCG GCC Asn Ala Ala Ala 755 TTC TTC TGT GCT Phe Phe Cys Ala GCA TAC GCC TTC Ala Tyr Ala Phe 790 TTA CCA CCA CGA Leu Pro Pro Arg 805 680 685 SAAC ATC GTG GAC GTG CAA TAC CTG Asn Ile Val Asp Val Gin Tyr Leu 695 700 TCC CTT GTC ATC AAA TGG GAG TAT Ser Leu Val Ile Lys Trp Glu Tyr 715 GCA GAC GCG CGC ATC TGC GCC TGC Ala Asp Ala Arg Ile Cys Ala Cys 730 CAA GCT GAG GCC GCC TTA GAG AAC Gin Ala Glu Ala Ala Leu Glu Asn 745 750 GTG GCC GGG GCG CAT GGC ACT CTT Val Ala Gly Ala His Gly ThrLeu 760 765 GCC TGG TAC ATC AAG GGC AGG CTG Ala Trp Tyr Ile Lys Gly Arg Leu 775 780 TAT GGC GTG TGG CCG CTG CTC CTG Tyr Gly Val Trp Pro Leu Leu Leu 795 GCT TAT GCC TAGTAA Ala Tyr Ala 810 TAC GGT Tyr Gly GTC CTG Val Leu 720 TTA TGG Leu Trp 735 CTG GTG Leu Val TCC TTC Ser Phe GTC CCT Val Pro CTT CTG Leu Leu 800 2112 2160 2208 2256 2304 2352 2400 2433

C

T

I

T

L

A

INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 809 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: let Ser Thr Asn Pro Lys Pro Gin Arg Lys Thr Lys 1 5 10 rg Arg Pro Gin Asp Val Lys Phe Pro Gly Gly Gly 25 ;ly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu 40 'hr Arg Lys Thr Ser Glu Arg Ser Gin Pro Arg Gly 55 le Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp 70 75 yr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Met Gly 90 eu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly 100 105 rg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp 115 120 Arg Gin Gly Arg Ala Trp Pro Thr 125 Thr Val Arg Gin Pro Gly Asp Thr Asn Gly Ala Pro Gly Trp Pro Cys Gly Phe 130 Gly Gly 145 Gly Val Phe Leu Glu Val Asn Ser 210 Gly Cys 225 Al a Al a Asn Leu Arg 195 Ser Val Asp Leu Ala Arg Tyr Ala 165 Ala Leu 180 Asn Val Ile Val Pro Cys Val1 Ala 150 Thr Leu Ser Tyr Val 230 Gly Tyr Ile 135 Leu Ala His Gly Asn Leu Ser Cys Leu 185 Gly Met Tyr 200 Giu Ala Ala 215 Arg Giu Asn 1 Pro Giy Pro 170 Thr His Asp Asn 30 pLeu Val Giy 140 Val Arg Val 155 Giy Cys Ser Val Pro Ala Val Thr Asn 205 Met Ile Met 220 Ser Ser Arg 235 Ala Leu Phe Ser 190 Asp His Cys Pro Giu Ser 175 Ala Cys Thr Trp Leu Asp 160 Ile Tyr Ser Pro Val1 240 Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser Val Pro 245 250 Thr Thr 255 Thr Ser Gin Asn 305 Asp Leu Trp, Ala Thr 385 Ser Asn Leu Ser Phe 465 Asp Ile Ala Leu 290 Cys Met Leu Gly Lys 370 Arg Leu Gly Gln Ser 450 kl a 31n Arg Met 275 Phe Ser Met Arg Val1 355 Val Val Phe Ser Thr 435 Gly Gin Arg Arc 260 Tyr Thr Ile Met Ile 340 Leu Leu Ser Ser Trp 420 Giy Cys Giy Pro His Val Asp Lei Val Ile Tyr Asn 325 Pro Ala Val Gly Pro 405 His Phe Pro Trp Tyr 485 Gly Ser Pro 310 Trp Gin Gly Val Gly 390 Gly Ile Phe Glu Gly 470 Cys Asp Pro 295 Gly Ser Ala Leu Met 375 Ala Ser ksn k.la krg 155 Pro Vrp Leu 280 Mrg His Pro Val1 Ala 360 Leu Ala.

Ala Arg Ala 440 Leu Leu His Let 265 Cys Ile Thr Val 345 Tyr Leu Al a Gin rhr 425 Leu kla r'hr ['yr Val Gly His Thr Thr 330 Asp Tyr Phe Ser Lys 410 Ala Phe Ser Tyr Ala 490 Gly Ser Giu Gly 315 Ala Met Ser Ala Asp 395 Ile Leu T'yr Thr 475 Pro Ala Ala Ala Phe Val Phe 285 Thr Val 300 His Arg Leu Val Val Ala Met Val 365 Gly Val 380 Thr Arg Gin Leu Asn Cys Lys His 445 Arg Ser 460 Glu Pro Mrg Pro 270 Leu Gir Met Val Gly 350 Gly Asp Gly Val1 As n 430 Lys Ile Asn 1Val Asp Ala Ser 335 Ala Asn Gly Leu Asn 415 Asp Phe Asp Ser Gly 495 Cys Ser Cys Trp 320 Gin His Trp His Val1 400 Thr Ser Asn L~ys Ser 480 Ile Val Pro Ala Ser Gin Val 500 Pro Val Val Vai Gly Thr 515 131 Pro Val T} 505 Arg Phe Gi Pro Ser Asn 525 Trp Gly 530 Pro Arg 545 Thr Lys Asn Thr Thr Tyr Val His 610 Ala Asn Asp Ser Gly Thr Leu Ala 595 Tyr Asn Cys Thr 580 Mrg Pro Thr 625 Giu Arg Gin Leu Val1 705 Leu Met Val1 Leu Gly 785 Leu Ile Phe Lys Val Ala Asp Ile Ile 690 Gly Leu Met Leu Val 770 Ala Al a Ala Mrg Leu 675 His Ser Phe Leu Asn 755 Phe Al a Leu Cys Ser 660 Pro Leu Ala Leu Leu 740 Ala Phe Tyr Pro Asn 645 Giu Cys His Val1 Leu 725 Ile Ala Cys Ala Pro 805 Phe 550 rGly Pro Gly Mrg Arg 630 Trp Leu Ser Gin Val 710 Leu Ala Ala Ala Phe 790 Arg Pro Pro Cys Vai Leu Il Cys Thr Trr Thr Ser Leu 615 Met Thr Ser Phe Asn 695 Ser kla 'ln lal ~75 ['yr lia *Asp *Gly 600 Trp Tyr Arg Pro Thr 680 Ile Leu Asp Ala Ala 760 Trp Gly N Tyr I Cys 585 Pro His Val1 Gly Leu 665 Thr Val1 Val Ala G1u 745 31y C'yr Tal l a Asr 570C Phe Trp Tyr Gly Glu 650 Leu Leu Asp Ile Axg 730 Al a Ala Ile Trp Leu Asn 540 Met Asn 555 Sle Gly Arg Lys Leu Thr Pro Cys 620 Gly Val 635 Mrg Cys Leu Ser Pro Ala Val Gin 700 Lys Trp, 715 Ile Cys I Ala Leu C His Gly I Asr Glj G i His Pro 605 Thr Glu Asp Thr Leu 685 I'yr 31u l a flu 1 Thr Thr Ala Pro 590 Arg Val1 His Leu Thr 670 Ser Leu Tyr Cys Asn 750 Arc Gil G i) 575 Glu Cys Asn Arg Glu 655 Glu Thr Tyr L~eu 735 4 eu gPro SPhe 560 'Asn Ala Met Phe Phe 640 Asp Trp Gly Gly Leu 720 Trp, Val1 Phe Lys Pro 795 765 Gly Arg 780 Leu Leu Leu Val Leu Leu INFORMATION FOR SEQ ID NO: 51: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 132 (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 (ii) MOLECULE TYPE: peptide (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 1..22 (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: LENGTH: 37 amino acids 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: 133 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 Gln Leu Arg Arg His Ile Asp Leu Leu 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: 134 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 Ile 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 Ile 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 135 (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 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: 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 SeT 1 5 10 Ala Thr Leu Cys INFORMATION FOR SEQ ID NO: 64: 136 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 Asn Cys Ser Ile INFORMATION FOR SEQ ID NO: 66: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: Thr Gln 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 137 (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 Gin Leu Leu Arg Ile 1 5 10 Pro Gin Ala Ile INFORMATION FOR SEQ ID NO: 69: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: Leu Leu Arg Ile Pro Gin 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 10 Val Gly Asn Met 138 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 Ile 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 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 Gln Leu Ile Asn Thr Asn Gly Gin Trp His Ile 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: peptide 139 (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 Ile 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: Ala Gly Leu Ile Tyr Gin His Lys Phe Asn Ser Ser Gly Cys Pro Glu 1 5 10 Arg Leu Ala Ser INFORMATION FOR SEQ ID NO: 76: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single 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 Phe 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 DESCRIPTTON: 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 140 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 Gln Arg Pro Tyr Cys Trp His Tyr Pro 1 5 10 Pro Lys Pro Cys INFORMATION FOR SEQ ID NO: 79: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: Trp His Tyr Pro Pro Lys Pro Cys Gly Ile Val Pro Ala Lys Ser Val 1 5 10 Cys Gly Pro 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: 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: peptide 141 (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: 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 Ile Gly Gly Ala 1 5 10 Gly Asn Asn Thr 142 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 10 Tyr Pro Tyr Arg INFORMATION FOR SEQ ID NO: 88: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 143 (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 1 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 DESCRITION.: 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 144 INFORMATION FOR SEQ ID NO: 92: SEQUENCE CHARACTERISTICS: LENGTH: 20 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) Glu SEQUENCE DESCRIPTION: SEQ ID NO: 92: Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Thr Thr Thr 10 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 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 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 I ll I Illll 145 (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:

II

146 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 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 INFOPRMATIO' FOR SEQ ID NO: 102: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 147 (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

(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: 148 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 149 (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 on

I

Claims (14)

1. Method for purifying recombinant HCV single or specific oligomeric envelope proteins selected from the group consisting of El and/or E2 and/or E1/E2, wherein a disulphide bond cleavage or reduction step is carried out with a disulphide bond cleavage agent on the recombinantly expressed protein.

2. Method according to claim 1, wherein said disulphide bond cleavage or reduction step is carried out under partial cleavage or reducing conditions.

3. Method according to claim 1 or claim 2, wherein said disulphide bond cleavage agent is dithiothreitol (DTT), preferably in a concentration range of 0.1 to 50 mM, preferably 0.1 to 20 mM, more preferably 0.5 to 10 mM.

4. Method according to claim 1, wherein said disulphide bond cleavage agent is combined with a detergent. Method according to claim 4, wherein said detergent is Empigen-BB, preferably at 15 a concentration of 1 to 10%, more preferably at a concentration of

6. Method according to claim 1 or claim 2, wherein said disulphide bond cleaving agent comprises a combination of a classical disulphide bond cleavage agent, such as DTT, and a detergent, such as Empigen-BB. S'0 Method according to any one of claims 1 to 6, further comprising the step of 20 blocking disulphide bond reformation with an SH group blocking agent.

8. Method according to claim 7, wherein said SH group blocking agent is N- ethylmaleimide (NEM) or a derivative thereof.

9. Method according to claim 7, wherein said step of blocking the disulphide bond reformation is brought about by low pH conditions. 151 Method according to any one of claims 1 to 9, further comprising at least the following steps: lysing recombinant El and/or E2 and/or E1/E2 expressing host cells, possibly in the presence of an SH blocking agent such as N-ethylmaleimide (NEM), recovering said HCV envelope proteins by affinity purification such as by means of lectin-chromotography, such as lentil-lectin chromatography, or by means of immunoaffinity using anti-El and/or anti-E2 specific monoclonal antibodies, *reduction or cleavage of the disulphide bonds with a disulphide bond cleaving agent, such as DTT, preferably also in the presence of an SH blocking agent, such as NEM 10 or Biotin-NEM, and, recovering the reduced El and/or E2 and/or E1/E2 envelope proteins by gelfiltration and possibly also by a subsequent Ni+-IMAC chromatography and desalting step.

11. An isolated HCV envelope protein obtained by a method according to any one of claims 1 to 4*

12. An isolated HCV envelope protein according to claim 11, wherein said recombinant HCV envelope proteins are expressed from recombinant mammalian cells such as by using a vaccinia virus based system.

13. An isolated HCV envelope protein according to claim 11, wherein said recombinant HCV envelope proteins are expressed from recombinant yeast cells.

14. Method according to any one of claims 1 to 10, further comprising at least the fnlollnwin step:o+ m^-po. -152- growing a host cell transformed with a recombinant vector comprising a nucleotide sequence allowing the expression of a HCV single or specific oligomeric El and/or E2 and/or E1/E2 protein in a suitable culture medium, causing expression of said vector under suitable conditions, and, lysing said transformed host cells, preferably in the presence of an SH group blocking agent, such as N-ethylmaleimide (NEM), recovering said HCV envelope protein by affinity purification by means of for instance lectin-chromatography or immunoaffinity chromatography using anti-E1 1 and/or anti-E2 specific monoclonal antibodies, with said lectin being preferably lentil- 10 lectin, followed by, incubation of the eluate of the previous step with a disulphide bond cleavage agent, such as DTT, preferably also in the presence of an SH group blocking agent, such as NEM or Biotin-NEM, and, isolating the HCV single or specific oligomeric El and/or E2 and/or El/E2 protein by 15 means of gelfiltration and possibly also by means of an additional Ni2-IMAC chromatography and desalting step. An isolated HCV envelope protein according to any one of claims 11 to 13, for use as a medicament.

16. An isolated HCV envelope protein according to any one of claims 11 to 13, for use as a vaccine for immunising a mammal, preferably humans, against HCV, comprising adm iniktrin o an p-ff+t o 1 I adm----....itrating an effecte aou f sau id cmposition possibly accompanied by pharmaceutically acceptable adjuvants, to produce an immune response.

153- 17. 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 claims 11 to 13, to produce an immune response. 18. Method according to claim 17, wherein said mammal is human. 19. Vaccine composition for immunising a mammal, preferably humans, against HCV, comprising an effective amount of an isolated HCV envelope protein according to any one of claims 11 to 13, possibly accompanied by pharmaceutically acceptable adjuvants. 20. An isolated HCV envelope protein according to any one of claims 11 to 13, for in vitro detection of HCV antibodies present in a biological sample. 10 21. Method for in vitro diagnosis of HCV antibodies present in a biological sample, comprising at least the following steps: a) contacting said biological sample with an isolated HCV envelope protein according to any one of claims 11 to 13, preferably in an immobilised form under appropriate conditions which allow the formation of an immune complex, 15 b) removing unbound components, c) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies being conjugated to a detectable label under appropriate conditions, detecting the presence of said immune complexes visually or mechanically. 22. Kit for determining the presence of HCV antibodies present in a biological sample, CAmnrcinn.T at least one isolated HCV envelope protein according to any one of claims 11 to 13, preferably in an immobilised form on a solid substrate, -154- 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. 23. Use of an isolated HCV envelope protein according to any one of claims 11 to 13, comprising HCV El protein, more particularly HCV single El proteins, for in vitro monitoring HCV disease or prognosing the response to treatment, particularly with interferon, of patients suffering from HCV infection comprising: incubating a biological sample from a patient with HCV infection with an El protein 10 or a suitable part thereof under conditions allowing the formation of an immunological complex, removing unbound components, *0 calculating the anti-E l titers present in said sample at the start of and during the course of treatment, S 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. 24. Kit for monitoring HCV disease or prognosing the response to treatment, particularly with interferon, of patients suffering from HCV infection comprising: at least one isolated HCV envelope protein, more particularly an El protein according v UIIj l, ui k, lalllms 1 I t 13,

155- 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, possibly also an automated scanning and interpretation device for inferring a decrease of anti-El titers during the progression of treatment. 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 *w 10 steps: a) contacting the biological sample to be analysed for the presence of HCV antibodies of one or more serological types, with at least one isolated HCV El and/or E2 and/or E1/E2 protein according to any one of claims 11 to 13, preferentially in an immobilised form under appropriate conditions which allow the formation of an immune complex, b) removing unbound components, c) incubating the immune complexes formed with heterologous antibodies, with said heterologous antibodies being conjugated to a detectable label under appropriate conditions, detecting the presence of said immune complexes visually or mechanically by means nfrhPncjtnmi-trIr l Inrit., means of densito .lrIIy coluimtcuy) and inferring the presence of one or more HCV serological types present from the observed binding pattern. -156- 26. 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 isolated HCV El and/or E2 and/or El/E2 protein according to any one of claims 11 to 13, 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, *i means for detecting the immune complexes formed in the preceding binding reaction, 10 possibly also an automated scanning and interpretation device for detecting the presence of one or more serological types present from the observed binding pattern. S::t 27. An isolated HCV envelope protein according to any one of claims 11 to 13 to raise upon immunisation an E 1 and/or E2 specific monoclonal antibody. 28. An isolated HCV envelope protein according to any one of claims 11 to 13, for the preparation of an immunoassay kit. 29. Use of an isolated HCV envelope protein according to any one of claims 11 to 13, for detecting HCV antibodies present in a biological sample. Use of an isolated HCV envelope protein according to any one of claims 11 to 13 for the manufacture of a medicament for immunising a mammal against HCV. 31. Use according to claim 30, wherein said mammal is human. 32. Method according to any one of claims 1 to 10 and substantially as herein described with reference to any one of the examples. -157- 33. An isolated HCV envelope protein according to any one of claims 11 to 13 and substantially as herein described with reference to any one of the examples and/or any one of the figures. 34. Method according to claim 17 or claim 18 and substantially as herein described with reference to any one of the examples. A vaccine composition according to claim 19 and substantially as herein described with reference to any one of the examples. 36. Method according to claim 21 and substantially as herein described with reference o• o• to any one of the examples. S10 37. Kit according to claim 22 and substantially as herein described with reference to any one of the examples. 38. Use according to claim 23 and substantially as herein described with reference to S: any one of the examples. 39. Kit according to claim 24 and substantially as herein described with reference to any one of the examples. Serotyping assay according to claim 25 and substantially as herein described with reference to any one of the examples. 41. Kit according to claim 26 and substantially as herein described with reference to any one of the examples. DATED this 2nd day of June 1999 INNOGENETICS N.V. Attorney: IVAN A. RAJKOVIC Fellow Institute of Patent Attorneys of Australia of BALDWIN SHELSTON WATERS