AU750792B2 - 76 kDa, 32 kDa, and 50 kDa helicobacter polypeptides and corresponding polynucleotide molecules - Google Patents

Description

1 76 kDa, 32 kDa, and 50 kDa HELICOBACTER POLYPEPTIDES AND CORRESPONDING POLYNUCLEOTIDE MOLECULES The invention relates to Helicobacter polypeptides and corresponding polynucleotide molecules that can be used in methods to prevent or treat Helicobacter infection in mammals, such as humans.

Background of the Invention All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Helicobacter is a genus of spiral, gram-negative bacteria that colonize the gastrointestinal tracts of mammals. Several species colonize the 2 5 stomach, most notably H. pylori, H. heilmanii, H. felis, and H. mustelae.

Although H. pylori is the species most commonly associated with human infection, H. heilmanii and H. felis have also been isolated from humans, but at lower frequencies than H. pylori. Helicobacter infects over 50% of adult 30 populations in developed countries and nearly 100% in developing countries and some Pacific rim countries, making it one of the most prevalent infections worldwide.

Helicobacter is routinely recovered from gastric biopsies of humans 35 with histological evidence of gastritis and peptic ulceration. Indeed, H. pylori Ir (I la is now recognized as an important pathogen of humans, in that the chronic gastritis it causes is a risk factor for the development of peptic ulcer diseases and gastric carcinoma. It is thus highly desirable to develop safe and effective vaccines for preventing and treating Helicobacter infection.

A number of Helicobacter antigens have been characterized-or isolated. These include urease, which is composed of two structural subunits of approximately 30 and 67 kDa (Hu et al., Infect. Immun. 58:992, 1990; Dunn et al., J. Biol. Chem. 265:9464, 1990; Evans et al., Microbial Pathogenesis 10:15, e WO 98/43479 PCT/US98/06421 -2- 1991; Labigne et al., J. Bact., 173:1920, 1991); the 87 kDa vacuolar cytotoxin (VacA) (Cover et al., J. Biol. Chem. 267:10570, 1992; Phadnis et al., Infect.

Immun. 62:1557, 1994; WO 93/18150); a 128 kDa immunodominant antigen associated with the cytotoxin (CagA, also called TagA; WO 93/18150; U.S.

Patent No. 5,403,924); 13 and 58 kDa heat shock proteins HspA and HspB (Suerbaum et al., Mol. Microbiol. 14:959, 1994; WO 93/18150); a 54 kDa catalase (Hazell et al., J. Gen. Microbiol. 137:57, 1991); a 15 kDa histidine-rich protein (Hpn) (Gilbert et al., Infect. Immun. 63:2682, 1995); a 20 kDa membrane-associated lipoprotein (Kostrcynska et al., J. Bact. 176:5938, 1994); a 30 kDa outer membrane protein (BMlin et al., J. Clin. Microbiol. 33:381, 1995); a lactoferrin receptor (FR 2,724,936); and several porins, designated HopA, HopB, HopC, HopD, and HopE, which have molecular weights of 48-67 kDa (Exner et al., Infect. Immun. 63:1567, 1995; Doig et al., J. Bact.

177:5447, 1995). Some of these proteins have been proposed as potential vaccine antigens. In particular, urease is believed to be a vaccine candidate (WO 94/9823; WO 95/22987; WO 95/3824; Michetti et al., Gastroenterology 107:1002, 1994). Nevertheless, it is thought that several antigens may ultimately be necessary in a vaccine.

Summary of the Invention The invention provides polynucleotide molecules that encode a family of 76 kDa Helicobacter polypeptides, designated GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa polypeptide, designated GHPO 1360, and a 50 kDa polypeptide, designated GHPO 750, which can be used, in methods to prevent, treat, or diagnose Helicobacter infection. The polypeptides include those having the amino acid sequences -3shown in SEQ ID NOs:2-22 (even numbers), 66, and 68. Those skilled in the art will understand that the invention also includes polynucleotide molecules that encode mutants and derivatives of these polypeptides, which can result from the addition, deletion, or substitution of non-essential amino acids, as is described further below.

In addition to the polynucleotide molecules described above, the invention includes the corresponding polypeptides polypeptides encoded by the polynucleotide molecules of the invention, or fragments thereof), and monospecific antibodies that specifically bind to these polypeptides.

The present invention has many applications and includes expression cassettes, vectors, and cells transformed or transfected with the polynucleotides of the invention. Accordingly, the present invention provides methods for producing polypeptides of the invention in recombinant host systems and related expression cassettes, vectors, and transformed or transfected cells; (ii) live vaccine vectors, such as pox virus, Salmonella typhimurium, and Vibrio cholerae vectors, that contain polynucleotides of the invention (such vaccine vectors being useful in, methods for preventing or treating Helicobacter Sinfection) in combination with a diluent or carrier, and related pharmaceutical compositions and associated therapeutic and/or prophylactic methods; (iii) therapeutic and/or prophylactic methods involving administration of polynucleotide molecules, either in a naked form or formulated with a delivery vehicle, polypeptides or mixtures ofpolypeptides, or monospecific antibodies of the invention, and related pharmaceutical compositions; (iv) methods for -detecting the presence of Helicobacter in biological samples, which caninvolve the use ofpolynucleotide molecules, monospecific antibodies, or polypeptides of the invention; and methods for purifying polypeptides of the invention by antibody-based affinity chromatography.

3a For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

*o *o* ee WO 98/43479 PCT/US98/06421 -4- Brief Description of the Drawings Figure 1 is an alignment of the predicted amino acid sequences of GHPO 386 (SEQ ID NO:2), GHPO 789 (SEQ ID NO:4), and GHPO 1516 (SEQ ID NO:6), as well as a consensus sequence for the 76 kDa protein family.

Figure 2 is an alignment of the predicted amino acid sequences of GHPO 1197 (SEQ ID NO:8), GHPO 1180 (SEQ ID NO:10), GHPO 896 (SEQ ID NO:12), GHPO 711 (SEQ ID NO:14), GHPO 190 (SEQ ID NO:16), GHPO 185 (SEQ ID NO:18), GHPO 1417 (SEQ ID NO:20), and GHPO 1414 (SEQ ID NO:22), as well as a consensus sequence for the 76 kDa protein family.

Detailed Description Open reading frames (ORFs) encoding a family of new, full length, membrane-associated 76 kDa polypeptides, designated GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa polypeptide, designated GHPO 1360, and a 50 kDa polypeptide, designated GHPO 750, have been identified in the H. pylori genome. The amino acid sequences of the 76 kDa polypeptides are aligned in Figures 1 and 2. The 76 kDa, 32 kDa, and 50 kDa polypeptides can be used, for example, in vaccination methods for preventing or treating Helicobacter infection. For example, GHPO 750, GHPO 1360, GHPO 190, and GHPO 1516 have been shown to be protective antigens. By "protective antigen" is meant an antigen that is capable of reducing the infection level after challenge, relative to a positive control. Absolute protection from infection, although included in the invention, is not required.

The polypeptides of the invention (except GHPO 750, see below) are secreted polypeptides that can be produced in their mature forms as polypeptides that have been exported through class II or class III secretion WO 98/43479 PCT/US98/06421 pathways) or as precursors that include a signal peptide, which can be removed in the course of excretion/secretion by cleavage at the N-terminal end of the mature form. (The cleavage site is located at the C-terminal end of the signal peptide, adjacent to the mature form.) The cleavage site for the polypeptides of the invention and, thus, the first amino acid of the mature polypeptides, was putatively determined.

According to a first aspect of the invention, there are provided isolated polynucleotides that encode the precursor and mature forms of Helicobacter GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, GHPO 1414, GHPO 1360, and GHPO 750.

An isolated polynucleotide of the invention encodes: a polypeptide having an amino acid sequence that is homologous to a Helicobacter amino acid sequence of a polypeptide associated with the Helicobacter membrane, the Helicobacter amino acid sequence being selected from the group consisting of the amino acid sequences shown: -in SEQ ID NO:2, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 689 (GHPO 386); -in SEQ ID NO:4, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 713 (GHPO 789); -in SEQ ID NO:6, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 725 (GHPO 1516); WO 98/43479 PCT/US98/06421 -6- -in SEQ ID NO:8, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 691 (GHPO 1197); -in SEQ ID NO:10, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 652 (GHPO 1180); -in SEQ ID NO:12, beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1, and ending with an amino acid in position 673 (GHPO 896); -in SEQ ID NO:14, beginning with an amino acid in any one of positions -21 to 5, preferably in position -21 or position 1, and ending with an amino acid in position 619 (GHPO 711); -in SEQ ID NO:16, beginning with an amino acid in any one of positions -17 to 5, preferably in position -17 or position 1, and ending with an amino acid in position 635 (GHPO 190); -in SEQ ID NO: 18, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 626 (GHPO 185); -in SEQ ID NO:20, beginning with an amino acid in any one of positions -16 to 5, preferably in position -16 or position 1, and ending with an amino acid in position 467 (GHPO 1417); -in SEQ ID NO:22, beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1, and ending with an amino acid in position 673 (GHPO 1414); -in SEQ ID NO:66, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 279 (GHPO 1360); and WO 98/43479 PCT/US98/06421 -7- -in SEQ ID NO:68, beginning with an amino acid in position 1 and ending with an amino acid in position 399 (GHPO 750); or (ii) a derivative of the polypeptide.

The term "isolated polynucleotide" is defined as a polynucleotide that is removed from the environment in which it naturally occurs. For example, a naturally-occurring DNA molecule present in the genome of a living bacteria or as part of a gene bank is not isolated, but the same molecule, separated from the remaining part of the bacterial genome, as a result of, a cloning event (amplification), is "isolated." Typically, an isolated DNA molecule is free from DNA regions coding regions) with which it is immediately contiguous, at the 5' or 3' ends, in the naturally occurring genome.

Such isolated polynucleotides can be part of a vector or a composition and still be isolated, as such a vector or composition is not part of its natural environment.

A polynucleotide of the invention can consist of RNA or DNA cDNA, genomic DNA, or synthetic DNA), or modifications or combinations of RNA or DNA. The polynucleotide can be double-stranded or single-stranded and, if single-stranded, can be the coding (sense) strand or the non-coding (antisense) strand. The sequences that encode polypeptides of the invention, as shown in SEQ ID NOs:2-22 (even numbers), 66, and 68, can be the coding sequence as shown in SEQ ID NOs:1-21 (odd numbers), 65, and 67; a ribonucleotide sequence derived by transcription of or a different coding sequence that, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptides as the polynucleotide molecules having the sequences illustrated in any of SEQ ID NOs:1-21 (odd numbers), 65, and 67. The polypeptides of the invention can be ones that are naturally secreted or excreted by, H. felis, H. mustelae, H. heilmanii, or H. pylori.

WO 98/43479 PCT/US98/06421 -8- By "polypeptide" or "protein" is meant any chain of amino acids, regardless of length or post-translational modification glycosylation or phosphorylation). Both terms are used interchangeably in the present application.

(By "homologous amino acid sequence" is meant an amino acid sequence that differs from an amino acid sequence shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68, or an amino acid sequence encoded by the nucleotide sequence of any of SEQ ID NOs:1-21 (odd numbers), 65, and 67, by one or more non-conservative amino acid substitutions, deletions, or additions located at positions at which they do not destroy the specific antigenicity of the polypeptide. Preferably, such a sequence is at least more preferably at least 80%, and most preferably at least 90% identical to an amino acid sequence shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68.

Homologous amino acid sequences include sequences that are identical or substantially identical to an amino acid sequence as shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68. By "amino acid sequence that is substantially identical" is meant a sequence that is at least preferably at least 95%, more preferably at least 97%, and most preferably at least 99% identical to an amino acid sequence of reference and that differs from the sequence of reference, if at all, by a majority of conservative amino acid substitutions.

Conservative amino acid substitutions typically include substitutions among amino acids of the same class. These classes include, for example, amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; amino acids having basic side chains, such as lysine, arginine, and histidine; amino acids having acidic side chains, such as WO 98/43479 PCT/US98/06421 -9aspartic acid and glutamic acid; and amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.

Homology can be measured using sequence analysis software Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Similar amino acid sequences are aligned to obtain the maximum degree of homology identity). To this end, it may be necessary to artificially introduce gaps into the sequence. Once the optimal alignment has been set up, the degree of homology identity) is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions.

Homologous polynucleotide sequences are defined in a similar way.

Preferably, a homologous sequence is one that is at least 45%, more preferably at least 60%, and most preferably at least 85% identical to a coding sequence of any of SEQ ID NOs:1-21 (odd numbers), 65, and 67.

Polypeptides having a sequence homologous to one of the sequences shown in SEQ ID NOs:2-22 (even numbers), 66, and 68 include naturallyoccurring allelic variants, as well as mutants or any other non-naturally occurring variants that are analogous in terms of antigenicity, to a polypeptide having a sequence as shown in SEQ ID NOs:2-22 (even numbers), 66, and 68.

As is known in the art, an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not alter the biological function of the polypeptide. By "biological function" is meant a function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells. For example, the biological function of a WO 98/43479 PCT/US98/06421 porin is to allow the entry into cells of compounds present in the extracellular medium. The biological function is distinct from the antigenic function. A polypeptide can have more than one biological function.

Allelic variants are very common in nature. For example, a bacterial species, H. pylori, is usually represented by a variety of strains that differ from each other by minor allelic variations. Indeed, a polypeptide that fulfills the same biological function in different strains can have an amino acid sequence that is not identical in each of the strains. Such an allelic variation can be equally reflected at the polynucleotide level.

Support for the use of allelic variants of polypeptide antigens comes from, studies of the Helicobacter urease antigen. The amino acid sequence of Helicobacter urease varies widely from species to species, yet cross-species protection occurs, indicating that the urease molecule, when used as an immunogen, is highly tolerant of amino acid variations. Even among different strains of the single species H. pylori, there are amino acid sequence variations.

For example, although the amino acid sequences of the UreA and UreB subunits of H. pylori and H. felis ureases differ from one another by 26.5% and 11.8%, respectively (Ferrero et al., Molecular Microbiology 9(2):323-333, 1993), it has been shown that H. pylori urease protects mice from H. felis infection (Michetti et al., Gastroenterology 107:1002, 1994). In addition, it has been shown that the individual structural subunits of urease, UreA and UreB, which contain distinct amino acid sequences, are both protective antigens against Helicobacter infection (Michetti et al., supra): Similarly, Cuenca et al. (Gastroenterology 110:1770, 1996) showed that therapeutic immunization of H. mustelae-infected ferrets with H. pylori urease was effective at eradicating H. mustelae infection. Further, several urease WO 98/43479 PCT/US98/06421 -11variants have been reported to be effective vaccine antigens, including, e.g., recombinant UreA UreB apoenzyme expressed from pORV142 (UreA and UreB sequences derived from H. pylori strain CPM630; Lee et al., J. Infect.

Dis. 172:161, 1995); recombinant UreA UreB apoenzyme expressed from pORV214 (UreA and UreB sequences differ from H. pylori strain CPM630 by one and two amino acid changes, respectively; Lee et al., supra, 1995); a UreA-glutathione-S-transferase fusion protein (UreA sequence from H. pylori strain ATCC 43504; Thomas et al., Acta Gastro-Enterologica Belgica 56:54, 1993); UreA UreB holoenzyme purified from H. pylori strain NCTC 11637 (Marchetti et al., Science 267:1655, 1995); a UreA-MBP fusion protein (UreA from H. pylori strain 85P; Ferrero et al., Infection and Immunity 62:4981, 1994); a UreB-MBP fusion protein (UreB from H. pylori strain 85P; Ferrero et al., supra); a UreA-MBP fusion protein (UreA from H. felis strain ATCC 49179; Ferrero et al., supra); a UreB-MBP fusion protein (UreB from H. felis strain ATCC 49179; Ferrero et al., supra); and a 37 kDa fragment of UreB containing amino acids 220-569 (Dore-Davin et al., "A 37 kD fragment of UreB is sufficient to confer protection against Helicobacterfelis infection in mice"). Finally, Thomas et al. (supra) showed that oral immunization of mice with crude sonicates ofH. pylori protected mice from subsequent challenge with H. felis.

Polynucleotides, DNA molecules, encoding allelic variants can easily be obtained by polymerase chain reaction (PCR) amplification of genomic bacterial DNA extracted by conventional methods. This involves the use of synthetic oligonucleotide primers matching sequences that are upstream and downstream of the 5' and 3' ends of the coding region. Suitable primers can be designed based on the nucleotide sequence information provided in SEQ ID NOs:1-21 (odd numbers), 65, and 67. Typically, a primer consists of 10 to WO 98/43479 PCT/US98/06421 -12preferably 15 to 25 nucleotides. It can also be advantageous to select primers containing C and G nucleotides in proportions sufficient to ensure efficient hybridization, an amount of C and G nucleotides of at least preferably 50%, of the total nucleotide amount. Those skilled in the art can readily design primers that can be used to isolate the polynucleotides of the invention from different Helicobacter strains.

As an example, primers useful for cloning a polynucleotide molecule encoding a polypeptide having the amino acid sequence of unprocessed GHPO 386 (SEQ ID NO:2), including a signal peptide, are shown in SEQ ID NO:23 (matching at the 5' end) and in SEQ ID NO:25 (matching at the 3' end).

Primers useful for cloning a DNA molecule encoding a polypeptide having the amino acid sequence of mature GHPO 386 (amino acids 1-689 of SEQ ID NO:2), lacking a signal peptide, are shown in SEQ ID NO:24 (matching at the end) and in SEQ ID NO:25 (matching at the 3' end). Primers useful for cloning a DNA molecule encoding a polypeptide having the amino acid sequence of GHPO 1360 (SEQ ID NO:66), are shown in SEQ ID NO:78 (matching at the 5' end) and in SEQ ID NO:79 (matching at the 3' end). Use of these primers enables amplification of the entire gene encoding GHPO 1360.

Primers having sequences shown in SEQ ID NO:82 (matching at the 5' end of the coding sequence corresponding to the mature protein) and SEQ ID NO:79 (matching at the 3' end) can be used to amplify the portion of the gene encoding mature GHPO 1360. Experimental conditions for carrying out PCR can readily be determined by one skilled in the art and illustrations of carrying out PCR are provided in Examples 3 and 4.

Thus, the first aspect of the invention includes: WO 98/43479 PCT/US98/06421 -13isolated polynucleotide molecules DNA molecules) that can be amplified and/or cloned using the polymerase chain reaction from a Helicobacter, H. pylori, genome using either: a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:23, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID (unprocessed GHPO 386); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:26, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:28 (unprocessed GHPO 789); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:29, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:31 (unprocessed GHPO 1516); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:32, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:34 (unprocessed GHPO 1197); a 5' oligonucleotide primer having a sequence as shown in SEQ ID and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:37 (unprocessed GHPO 1180); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:38, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID (unprocessed GHPO 896); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:41, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:43 (unprocessed GHPO 711); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:44, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:46 (unprocessed GHPO 190); WO 98/43479 PCT/US98/06421 -14a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:47, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:49 (unprocessed GHPO 185); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:50, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:52 (unprocessed GHPO 1417); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:53, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID (unprocessed GHPO 1414); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:78, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:79 (unprocessed GHPO 1360); or a 5' oligonucleotide primer having a sequence as shown in SEQ ID and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:81 (GHPO 750); and (ii) isolated polynucleotide molecules DNA molecules) that can be amplified and/or cloned by the polymerase chain reaction from a Helicobacter, H. pylori, genome using either: a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:24, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID (mature GHPO 386); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:27, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:28 (mature GHPO 789); a 5' oligonucleotide primer having a sequence as shown in SEQ ID and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:31 (mature GHPO 1516); WO 98/43479 PCT/US98/06421 a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:33, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:34 (mature GHPO 1197); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:36, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:37 (mature GHPO 1180); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:39, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID (mature GHPO 896); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:42, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:43 (mature GHPO 711); a 5' oligonucleotide primer having a sequence as shown in SEQ ID and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:46 (mature GHPO 190); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:48, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:49 (mature GHPO 185); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:51, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:52 (mature GHPO 1417); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:54, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID (mature GHPO 1414); or a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:82, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:79 (mature GHPO 1360).

WO 98/43479 PCT/US98/06421 -16- The 5' ends of the primers described above can advantageously include a restriction endonuclease recognition site that contains, typically, 4 to 6 nucleotides. For example, the sequences 5'-GGATCC-3' (BamHI) or CTCGAG-3' (XhoI) can be used. Restriction sites can be selected by those skilled in the art so that the amplified DNA, when digested, if necessary, can be conveniently cloned into an appropriately digested vector, such as a plasmid vector. In addition, a 5' clamp GCC) can be included in the primers 5' to the restriction endonuclease recognition site.

Useful homologs that do not occur naturally can be designed using known methods for identifying regions of an antigen that are likely to be tolerant of amino acid sequence changes and/or deletions. For example, sequences of the antigen from different species can be compared to identify conserved sequences.

Polypeptide derivatives that are encoded by polynucleotides of the invention include, fragments, polypeptides having large internal deletions derived from full-length polypeptides, and fusion proteins. Polypeptide fragments of the invention can be derived from a polypeptide having a sequence homologous to the sequences of any of SEQ ID NOs:2-22 (even numbers), 66, and 68, to the extent that the fragments retain the substantial antigenicity of the parent polypeptide (specific antigenicity). Polypeptide derivatives can also be constructed by large internal deletions that remove a substantial part of the parent polypeptide, while retaining specific antigenicity.

Generally, polypeptide derivatives should be about at least 12 amino acids in length to maintain antigenicity. Advantageously, they can be at least 20 amino acids, preferably at least 50 amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length.

WO 98/43479 PCT/US98/06421 -17- Useful polypeptide derivatives, polypeptide fragments, can be designed using computer-assisted analysis of amino acid sequences in order to identify sites in protein antigens having potential as surface-exposed, antigenic regions (Hughes et al., Infect. Immun. 60(9):3497, 1992). For example, the Laser Gene Program from DNA Star can be used to obtain hydrophilicity, antigenic index, and intensity index plots for the polypeptides of the invention.

This program can also be used to obtain information about homologies of the polypeptides with known protein motifs. One skilled in the art can readily use the information provided in such plots to select peptide fragments for use as vaccine antigens. For example, fragments spanning regions of the plots in which the antigenic index is relatively high can be selected. One can also select fragments spanning regions in which both the antigenic index and the intensity plots are relatively high. Fragments containing conserved sequences, particularly hydrophilic conserved sequences, can also be selected.

Polypeptide fragments and polypeptides having large internal deletions can be used for revealing epitopes that are otherwise masked in the parent polypeptide and that may be of importance for inducing a protective T cell-dependent immune response. Deletions can also remove immunodominant regions of high variability among strains.

It is an accepted practice in the field of immunology to use fragments and variants of protein immunogens as vaccines, as all that is required to induce an immune response to a protein is a small 8 to 10 amino acids) immunogenic region of the protein. This has been done for a number of vaccines against pathogens other than Helicobacter. For example, short synthetic peptides corresponding to surface-exposed antigens of pathogens such as murine mammary tumor virus (peptide containing 11 amino acids; Dion et al., Virology 179:474-477, 1990), Semliki Forest virus (peptide containing 16 WO 98/43479 PCT/US98/06421 -18amino acids; Snijders et al., J. Gen. Virol. 72:557-565, 1991), and canine parvovirus (2 overlapping peptides, each containing 15 amino acids; Langeveld et al., Vaccine 12(15):1473-1480, 1994) have been shown to be effective vaccine antigens against their respective pathogens.

Polynucleotides encoding polypeptide fragments and polypeptides having large internal deletions can be constructed using standard methods (see, Ausubel et al., Current Protocols in Molecular Biology, John Wiley Sons Inc., 1994), for example, by PCR, including inverse PCR, by restriction enzyme treatment of the cloned DNA molecules, or by the method of Kunkel et al. (Proc. Natl. Acad. Sci. USA 82:448, 1985; biological material available at Stratagene).

A polypeptide derivative can also be produced as a fusion polypeptide that contains a polypeptide or a polypeptide derivative of the invention fused, at the or C-terminal end, to any other polypeptide (hereinafter referred to as a peptide tail). Such a product can be easily obtained by translation of a genetic fusion, a hybrid gene. Vectors for expressing fusion polypeptides are commercially available, and include the pMal-c2 or pMal-p2 systems of New England Biolabs, in which the peptide tail is a maltose binding protein, the glutathione-S-transferase system of Pharmacia, or the His-Tag system available from Novagen. These and other expression systems provide convenient means for further purification of polypeptides and derivatives of the invention.

Another particular example of fusion polypeptides included in invention includes a polypeptide or polypeptide derivative of the invention fused to a polypeptide having adjuvant activity, such as, subunit B of either cholera toxin or E. coli heat-labile toxin. Several possibilities can be WO 98/43479 PCT/US98/06421 -19used for producing such fusion proteins. First, the polypeptide of the invention can be fused to the N-terminal end or, preferably, to the C-terminal end of the polypeptide having adjuvant activity. Second, a polypeptide fragment of the invention can be fused within the amino acid sequence of the polypeptide having adjuvant activity.

Spacer sequences can also be included, if desired.

As stated above, the polynucleotides of the invention encode Helicobacter polypeptides in precursor or mature form. They can also encode hybrid precursors containing heterologous signal peptides, which can mature into polypeptides of the invention. By "heterologous signal peptide" is meant a signal peptide that is not found in the naturally-occurring precursor of a polypeptide of the invention.

A polynucleotide of the invention hybridizes, preferably under stringent conditions, to a polynucleotide having a sequence as shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67. Hybridization procedures are, described by Ausubel et al. (supra); Silhavy et al. (Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1984); and Davis et al. (A Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1980). Important parameters that can be considered for optimizing hybridization conditions are reflected in the following formula, which facilitates calculation of the melting temperature which is the temperature above which two complementary DNA strands separate from one another (Casey et al., Nucl. Acid Res. 4:1539, 1977): Tm 81.5 G+C) 1.6 log (positive ion concentration) 0.6 x formamide). Under appropriate stringency conditions, hybridization temperature (Th) is approximately 20 to 40°C, 20 to 250C, or, preferably, 30 to 40°C below the WO 98/43479 PCT/US98/06421 calculated Tm. Those skilled in the art will understand that optimal temperature and salt conditions can be readily determined empirically in preliminary experiments using conventional procedures. For example, stringent conditions can be achieved, both for pre-hybridizing and hybridizing incubations, within 4-16 hours at 42 C, in 6 x SSC containing formamide or (ii) within 4-16 hours at 65 0 C in an aqueous 6 x SSC solution (1 M NaCI, 0.1 M sodium citrate (pH For polynucleotides containing 30 to 600 nucleotides, the above formula is used and then is corrected by subtracting (600/polynucleotide size in base pairs). Stringency conditions are defined by a Th that is 5 to 10 C below Tm.

Hybridization conditions with oligonucleotides shorter than 20-30 bases do not precisely follow the rules set forth above. In such cases, the formula for calculating the Tm is as follows: Tm 4 x 2 For example, an 18 nucleotide fragment of 50% G+C would have an approximate Tm of 54°C.

A polynucleotide molecule of the invention, containing RNA, DNA, or modifications or combinations thereof, can have various applications. For example, a polynucleotide molecule can be used in a process for producing the encoded polypeptide in a recombinant host system, (ii) in the construction of vaccine vectors such as poxviruses, which are further used in methods and compositions for preventing and/or treating Helicobacter infection, (iii) as a vaccine agent, in a naked form or formulated with a delivery vehicle, and (iv) in the construction of attenuated Helicobacter strains that can over-express a polynucleotide of the invention or express it in a non-toxic, mutated form.

According to a second aspect of the invention, there is therefore provided an expression cassette containing a polynucleotide molecule of the invention placed under the control of elements a promoter) required for WO 98/43479 PCT/US98/06421 -21expression; (ii) an expression vector containing an expression cassette of the invention; (iii) a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention; as well as (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the invention, which involves culturing a procaryotic or eucaryotic cell transformed or transfected with an expression cassette and/or vector of the invention, under conditions that allow expression of the polynucleotide molecule of the invention and, recovering the encoded polypeptide or polypeptide derivative from the cell culture.

A recombinant expression system can be selected from procaryotic and eucaryotic hosts. Eucaryotic hosts include, for example, yeast cells Saccharomyces cerevisiae or Pichia pastoris), mammalian cells COS 1, NIH3T3, or JEG3 cells), arthropods cells Spodopterafrugiperda (SF9) cells), and plant cells. Preferably, a procaryotic host such as E. coli is used.

Bacterial and eucaryotic cells are available from a number of different sources that are known to those skilled in the art, the American Type Culture Collection (ATCC; Rockville, Maryland).

The choice of the expression cassette will depend on the host system selected, as well as the features desired for the expressed polypeptide. For example, it may be useful to produce a polypeptide of the invention in a particular lipidated form or any other form. Typically, an expression cassette includes a constitutive or inducible promoter that is functional in the selected host system; a ribosome binding site; a start codon (ATG); if necessary, a region encoding a signal peptide, a lipidation signal peptide; a polynucleotide molecule of the invention; a stop codon; and, optionally, a 3' terminal region (translation and/or transcription terminator). The signal peptide-encoding region is adjacent to the polynucleotide of the invention and WO 98/43479 PCT/US98/06421 -22is placed in the proper reading frame. The signal peptide-encoding region can be homologous or heterologous to the polynucleotide molecule encoding the mature polypeptide and it can be specific to the secretion apparatus of the host used for expression. The open reading frame constituted by the polynucleotide molecule of the invention, alone or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host system. Promoters and signal peptide-encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E. coli Patent No. 5,028,530; Cagnon et al., Protein Engineering 4(7):843, 1991); the promoter of the bacteriophage T7 RNA polymerase gene, which is functional in a number of E. coli strains expressing T7 polymerase Patent No. 4,952,496); the OspA lipidation signal peptide; and RlpB lipidation signal peptide (Takase et al., J. Bact. 169:5692, 1987).

The expression cassette is typically part of an expression vector, which is selected for its ability to replicate in the chosen expression system.

Expression vectors plasmids or viral vectors) can be chosen from, for example, those described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985, Supp. 1987) and can purchased from various commercial sources. Methods for transforming or transfecting host cells with expression vectors are well known in the art and will depend on the host system selected, as described in Ausubel et al. (supra).

Upon expression, a recombinant polypeptide of the invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane. The polypeptide WO 98/43479 PCT/US98/06421 -23can then be recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the cell culture. Typically, the recombinant polypeptide can be purified by antibody-based affinity purification or by any other method known to a person skilled in the art, such as by genetic fusion to a small affinity-binding domain. Antibody-based affinity purification methods are also available for purifying a polypeptide of the invention extracted from a Helicobacter strain. Antibodies useful for immunoaffinity purification of the polypeptides of the invention can be obtained using methods described below.

Polynucleotides of the invention can also be used in DNA vaccination methods, using either a viral or bacterial host as gene delivery vehicle (live vaccine vector) or administering the gene in a free form, e.g., inserted into a plasmid. Therapeutic or prophylactic efficacy of a polynucleotide of the invention can be evaluated as is described below.

Accordingly, in a third aspect of the invention, there is provided a vaccine vector such as a poxvirus, containing a polynucleotide molecule of the invention placed under the control of elements required for expression; (ii) a composition of matter containing a vaccine vector of the invention, together with a diluent or carrier; (iii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a vaccine vector of the invention; (iv) a method for inducing an immune response against Helicobacter in a mammal a human; alternatively, the method can be used in veterinary applications for treating or preventing Helicobacter infection of animals, e.g., cats or birds), which involves administering to the mammal an immunogenically effective amount of a vaccine vector of the invention to elicit an immune response, a protective or therapeutic immune response to Helicobacter; and a method for preventing and/or treating a Helicobacter WO 98/43479 PCT/US98/06421 -24- H. pylori, H. felis, H. mustelae, or H. heilmanii) infection, which involves administering a prophylactic or therapeutic amount of a vaccine vector of the invention to an individual in need. Additionally, the third aspect of the invention encompasses the use of a vaccine vector of the invention in the preparation of a medicament for preventing and/or treating Helicobacter infection.

A vaccine vector of the invention can express one or several polypeptides or derivatives of the invention, as well as at least one additional Helicobacter antigen such as a urease apoenzyme or a subunit, fragment, homolog, mutant, or derivative thereof. In addition, it can express a cytokine, such as interleukin-2 (IL-2) or interleukin-12 (IL-12), that enhances the immune response. Thus, a vaccine vector can include an additional polynucleotide molecules encoding, urease subunit A, B, or both, or a cytokine, placed under the control of elements required for expression in a mammalian cell.

Alternatively, a composition of the invention can include several vaccine vectors, each of which being capable of expressing a polypeptide or derivative of the invention. A composition can also contain a vaccine vector capable of expressing an additional Helicobacter antigen such as urease apoenzyme, a subunit, fragment, homolog, mutant, or derivative thereof, or a cytokine such as IL-2 or IL-12.

In vaccination methods for treating or preventing infection in a mammal, a vaccine vector of the invention can be administered by any conventional route in use in the vaccine field, for example, to a mucosal ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via a parenteral subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. Preferred routes depend WO 98/43479 PCT/US98/06421 upon the choice of the vaccine vector. The administration can be achieved in a single dose or repeated at intervals. The appropriate dosage depends on various parameters that are understood by those skilled in the art, such as the nature of the vaccine vector itself, the route of administration, and the condition of the.

mammal to be vaccinated the weight, age, and general health of the mammal).

Live vaccine vectors that can be used in the invention include viral vectors, such as adenoviruses and poxviruses, as well as bacterial vectors, e.g., Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bili de Calmette- Gu6rin (BCG), and Streptococcus. An example of an adenovirus vector, as well as a method for constructing an adenovirus vector capable of expressing a polynucleotide molecule of the invention, is described in U.S. Patent No.

4,920,209. Poxvirus vectors that can be used in the invention include, e.g., vaccinia and canary pox viruses, which are described in U.S. Patent No.

4,722,848 and U.S. Patent No. 5,364,773, respectively (also see, Tartaglia et al., Virology 188:217, 1992, for a description of a vaccinia virus vector, and Taylor et al, Vaccine 13:539, 1995, for a description of a canary poxvirus vector). Poxvirus vectors capable of expressing a polynucleotide of the invention can be obtained by homologous recombination, as described in Kieny et al. (Nature 312:163, 1984) so that the polynucleotide of the invention is inserted in the viral genome under appropriate conditions for expression in mammalian cells. Generally, the dose of viral vector vaccine, for therapeutic or prophylactic use, can be from about x 104 to about x 10", advantageously from about 1x10 7 to about lx10' 0 or, preferably, from about 1x10 7 to about lx10 9 plaque-forming units per kilogram. Preferably, viral vectors are administered parenterally, for example, in 3 doses that are 4 weeks apart.

Those skilled in the art will recognize that it is preferable to avoid adding a WO 98/43479 PCT/US98/06421 -26chemical adjuvant to a composition containing a viral vector of the invention and thereby minimizing the immune response to the viral vector itself.

Non-toxicogenic Vibrio cholerae mutant strains that can be used in live oral vaccines are described by Mekalanos et al. (Nature 306:551, 1983) and in U.S. Patent No. 4,882,278 (strain in which a substantial amount of the coding sequence of each of the two ctxA alleles has been deleted so that no functional cholerae toxin is produced); WO 92/11354 (strain in which the irgA locus is inactivated by mutation; this mutation can be combined in a single strain with ctxA mutations); and WO 94/1533 (deletion mutant lacking functional ctxA and attRSI DNA sequences). These strains can be genetically engineered to express heterologous antigens, as described in WO 94/19482.

An effective vaccine dose of a V cholerae strain capable of expressing a polypeptide or polypeptide derivative encoded by a polynucleotide molecule of the invention can contain, about Ixl10 to about 1x10 9 preferably about x 106 to about x 108 viable bacteria in an appropriate volume for the selected route of administration. Preferred routes of administration include all mucosal routes, but, most preferably, these vectors are administered intranasally or orally.

Attenuated Salmonella typhimurium strains, genetically engineered for recombinant expression of heterologous antigens, and their use as oral vaccines, are described by Nakayama et al. (Bio/Technology 6:693, 1988) and in WO 92/11361. Preferred routes of administration for these vectors include all mucosal routes. Most preferably, the vectors are administered intranasally or orally.

Others bacterial strains useful as vaccine vectors are described by High et al. (EMBO 11:1991, 1992) and Sizemore et al. (Science 270:299, 1995; Shigellaflexneri); Medaglini et al. (Proc. Natl. Acad. Sci. USA 92:6868, WO 98/43479 PCT/US98/06421 -27- 1995; (Streptococcus gordonii); Flynn (Cell. Mol. Biol. 40 (suppl. I):31, 1194), and in WO 88/6626, WO 90/0594, WO 91/13157, WO 92/1796, and WO 92/21376 (Bacille Calmette Guerin). In bacterial vectors, a polynucleotide of the invention can be inserted into the bacterial genome or it can remain in a free state, for example, carried on a plasmid.

An adjuvant can also be added to a composition containing a bacterial vector vaccine. A number of adjuvants that can be used are known to those skilled in the art. For example, preferred adjuvants can be selected from the list provided below.

According to a fourth aspect of the invention, there is also provided a composition of matter containing a polynucleotide of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polynucleotide of the invention; (iii) a method for inducing an immune response against Helicobacter, in a mammal, by administering to the mammal an immunogenically effective amount of a polynucleotide of the invention to elicit an immune response, a protective immune response to Helicobacter; and (iv) a method for preventing and/or treating a Helicobacter H. pylori, H.

felis, H. mustelae, or H. heilmanii) infection, by administering a prophylactic or therapeutic amount of a polynucleotide of the invention to an individual in need of such treatment. Additionally, the fourth aspect of the invention encompasses the use of a polynucleotide of the invention in the preparation of a medicament for preventing and/or treating Helicobacter infection. The fourth aspect of the invention preferably includes the use of a polynucleotide molecule placed under conditions for expression in a mammalian cell, in a plasmid that is unable to replicate in mammalian cells and to substantially integrate into a mammalian genome.

WO 98/43479 PCT/US98/06421 -28- Polynucleotides (for example, DNA or RNA molecules) of the invention can also be administered as such to a mammal as a vaccine. When a DNA molecule of the invention is used, it can be in the form of a plasmid that is unable to replicate in a mammalian cell and unable to integrate into the mammalian genome. Typically, a DNA molecule is placed under the control of a promoter suitable for expression in a mammalian cell. The promoter can function ubiquitously or tissue-specifically. Examples of non-tissue specific promoters include the early Cytomegalovirus (CMV) promoter Patent No. 4,168,062) and the Rous Sarcoma Virus promoter (Norton et al., Molec.

Cell Biol. 5:281, 1985). The desmin promoter (Li et al., Gene 78:243, 1989; Li et al., J. Biol. Chem. 266:6562, 1991; Li et al., J. Biol. Chem. 268:10403, 1993) is tissue-specific and drives expression in muscle cells. More generally, useful promoters and vectors are described, in WO 94/21797 and by Hartikka et al. (Human Gene Therapy 7:1205, 1996).

For DNA/RNA vaccination, the polynucleotide of the invention can encode a precursor or a mature form of a polypeptide of the invention. When it encodes a precursor form, the precursor sequence can be homologous or heterologous. In the latter case, a eucaryotic leader sequence can be used, such as the leader sequence of the tissue-type plasminogen factor (tPA).

A composition of the invention can contain one or several polynucleotides of the invention. It can also contain at least one additional polynucleotide eneeoding another Helicobacter antigen, such as urease subunit A, B, or both, or a fragment, derivative, mutant, or analog thereof. A polynucleotide encoding a cytokine, such as interleukin-2 (IL-2) or interleukin- 12 (IL-12), can also be added to the composition so that the immune response is enhanced. These additional polynucleotides are placed under appropriate control for expression. Advantageously, DNA molecules of the invention WO 98/43479 PCT/US98/06421 -29and/or additional DNA molecules to be included in the same composition are carried in the same plasmid.

Standard methods can be used in the preparation of therapeutic polynucleotides of the invention. For example, a polynucleotide can be used in a naked form, free of any delivery vehicles, such as anionic liposomes, cationic lipids, microparticles, gold microparticles, precipitating agents, e.g., calcium phosphate, or any other transfection-facilitating agent. In this case, the polynucleotide can be simply diluted in a physiologically acceptable solution, such as sterile saline or sterile buffered saline, with or without a carrier. When present, the carrier preferably is isotonic, hypotonic, or weakly hypertonic, and has a relatively low ionic strength, such as provided by a sucrose solution, e.g., a solution containing 20% sucrose.

Alternatively, a polynucleotide can be associated with agents that assist in cellular uptake. It can be, complemented with a chemical agent that modifies cellular permeability, such as bupivacaine (see, e.g., WO 94/16737), (ii) encapsulated into liposomes, or (iii) associated with cationic lipids or silica, gold, or tungsten microparticles.

Anionic and neutral liposomes are well-known in the art (see, e.g., Liposomes: A Practical Approach, RPC New Ed, IRL Press, 1990, for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides.

Cationic lipids can also be used for gene delivery. Such lipids include, for example, Lipofectin

T

which is also known as DOTMA 1- (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2bis(oleyloxy)-3-(trimethylammonio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine), and cholesterol derivatives. A description of these cationic lipids WO 98/43479 PCT/US98/06421 can be found in EP 187,702, WO 90/11092, U.S. Patent No. 5,283,185, WO 91/15501, WO 95/26356, and U.S. Patent No. 5,527,928. Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine; WO 90/11092). Other transfectionfacilitating compounds can be added to a formulation containing cationic liposomes. A number of them are described in, WO 93/18759, WO 93/19768, WO 94/25608, and WO 95/2397. They include, spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).

Gold or tungsten microparticles can also be used for gene delivery, as described in WO 91/359, WO 93/17706, and by Tang et al. (Nature 356:152, 1992). In this case, the microparticle-coated polynucleotides can be injected via intradermal or intraepidermal routes using a needleless injection device ("gene gun"), such as those described in U.S. Patent No. 4,945,050, U.S. Patent No. 5,015,580, and WO 94/24263.

The amount of DNA to be used in a vaccine recipient depends, e.g., on the strength of the promoter used in the DNA construct, the immunogenicity of the expressed gene product, the condition of the mammal intended for administration the weight, age, and general health of the mammal), the mode of administration, and the type of formulation. In general, a therapeutically or prophylactically effective dose from about 1 ug to about 1 mg, preferably, from about 10 ug to about 800 Lg, and, more preferably, from about 25 gg to about 250 gg, can be administered to human adults. The administration can be achieved in a single dose or repeated at intervals.

WO 98/43479 PCT/US98/06421 -31- The route of administration can be any conventional route used in the vaccine field. As general guidance, a polynucleotide of the invention can be administered via a mucosal surface, an ocular, intranasal, pulmonary, oral, intestinal, rectal, vaginal, or urinary tract surface, or via a parenteral route, e.g., by an intravenous, subcutaneous, intraperitoneal, intradermal, intraepidermal, or intramuscular route. The choice of administration route will depend on, e.g., the formulation that is selected. A polynucleotide formulated in association with bupivacaine is advantageously administered into muscle. When a neutral or anionic liposome or a cationic lipid, such as DOTMA, is used, the formulation can be advantageously injected via intravenous, intranasal (for example, by aerosolization), intramuscular, intradermal, and subcutaneous routes. A polynucleotide in a naked form can advantageously be administered via the intramuscular, intradermal, or subcutaneous routes. Although not absolutely required, such a composition can also contain an adjuvant. A systemic adjuvant that does not require concomitant administration in order to exhibit an adjuvant effect is preferable.

The sequence information provided in the present application enables the design of specific nucleotide probes and primers that can be used in diagnostic methods. Accordingly, in a fifth aspect of the invention, there is provided a nucleotide probe or primer having a sequence found in, or derived by degeneracy of the genetic code from, a sequence shown in any of SEQ ID NOs:1-21 (odd numbers), 65, and 67, or a complementary sequence thereof.

'The term "probe" as used in the present application refers to DNA (preferably single stranded) or RNA molecules (or modifications or combinations thereof) that hybridize under the stringent conditions, as defined above, to polynucleotide molecules having sequences homologous to those shown in any of SEQ ID NOs:l-21 (odd numbers), 65, and 67, or to a WO 98/43479 PCT/US98/06421 -32complementary or anti-sense sequence of any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67. Generally, probes are significantly shorter than the fulllength sequences shown in any of SEQ ID NOs: 1-21 (odd numbers), 65, and 67. For example, they can contain from about 5 to about 100, preferably from about 10 to about 80 nucleotides. In particular, probes have sequences that are at least 75%, preferably at least 85%, more preferably 95% homologous to a portion of a sequence as shown in any of SEQ ID NOs:1-21 (odd numbers), and 67, or a sequence complementary to such sequences.

Probes can contain modified bases, such as inosine, deoxycytidine, deoxyuridine, dimethylamino-5-deoxyuridine, or diamino-2, 6purine. Sugar or phosphate residues can also be modified or substituted. For example, a deoxyribose residue can be replaced by a polyamide (Nielsen et al., Science 254:1497, 1991) and phosphate residues can be replaced by ester groups such as diphosphate, alkyl, arylphosphonate, and phosphorothioate esters. In addition, the 2'-hydroxyl group on ribonucleotides can be modified by addition of, alkyl groups.

Probes of the invention can be used in diagnostic tests, or as capture or detection probes. Such capture probes can be immobilized on solid supports, directly or indirectly, by covalent means or by passive adsorption. A detection probe can be labeled by a detectable label, for example a label selected from radioactive isotopes; enzymes, such as peroxidase and alkaline phosphatase; enzymes that are able to hydrolyze a chromogenic, fluorogenic, or luminescent substrate; compounds that are chromogenic, fluorogenic, or luminescent; nucleotide base analogs; and biotin.

Probes of the invention can be used in any conventional hybridization method, such as in dot blot methods (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,-Cold WO 98/43479 PCT/US98/06421 -33- Spring Harbor, New York, 1982), Southern blot methods (Southern, J. Mol.

Biol. 98:503, 1975), northern blot methods (identical to Southern blot to the exception that RNA is used as a target), or a sandwich method (Dunn- et al., Cell 12:23, 1977). As is known in the art, the latter technique involves the use of a specific capture probe and a specific detection probe that have nucleotide sequences that are at least partially different from each other.

Primers used in the invention usually contain about 10 to nucleotides and are used to initiate enzymatic polymerization of DNA in an amplification process PCR), an elongation process, or a reverse transcription method. In a diagnostic method involving PCR, the primers can be labeled.

Thus, the invention also encompasses a reagent containing a probe of the invention for detecting and/or identifying the presence of Helicobacter in a biological material; (ii) a method for detecting and/or identifying the presence of Helicobacter in a biological material, in which a sample is recovered or derived from the biological material, DNA or RNA is extracted from the material and denatured, and the sample is exposed to a probe of the invention, for example, a capture probe, a detection probe, or both, under stringent hybridization conditions, so that hybridization is detected; and (iii) a method for detecting and/or identifying the presence of Helicobacter in a biological material, in which a sample is recovered or derived from the biological material, DNA is extracted therefrom, the extracted DNA is contacted with at least one, or, preferably two, primers of the invention, and amplified by the polymerase chain reaction, and an amplified DNA molecule is produced.

As mentioned above', polypeptides that can be produced by expression of the polynucleotides of the invention can be used as vaccirie WO 98/43479 PCT/US98/06421 -34antigens. Accordingly, a sixth aspect of the invention features a substantially purified polypeptide or polypeptide derivative having an amino acid sequence encoded by a polynucleotide of the invention.

A "substantially purified polypeptide" is defined as a polypeptide that is separated from the environment in which it naturally occurs and/or a polypeptide that is free of most of the other polypeptides that are present in the environment in which it was synthesized. The polypeptides of the invention can be purified from a natural source, such as a Helicobacter strain, or can be produced using recombinant methods.

Homologous polypeptides or polypeptide derivatives encoded by polynucleotides of the invention can be screened for specific antigenicity by testing cross-reactivity with an antiserum raised against a polypeptide having an amino acid sequence as shown in any of SEQ ID NOs:2-22 (even numbers), 66, and 68. Briefly, a monospecific hyperimmune antiserum can be raised against a purified reference polypeptide as such or as a fusion polypeptide, for example, an expression product of MBP, GST, or His-tag systems, or a synthetic peptide predicted to be antigenic. The homologous polypeptide or derivative that is screened for specific antigenicity can be produced as such or as a fusion polypeptide. In the latter case, and if the antiserum is also raised against a fusion polypeptide, two different fusion systems are employed.

Specific antigenicity can be determined using a number of methods, including Western blot (Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350, 1979), dot blot, and ELISA methods, as described below.

In a Western blot assay, the product to be screened, either as-apurified preparation or a total E. coli extract, is fractionated by SDS-PAGE, as described, for example, by Laemmli (Nature 227:680, 1970). After being transferred to a filter, such as a nitrocellulose membrane, the material is WO 98/43479 PCT/US98/06421 incubated with the monospecific hyperimmune antiserum, which is diluted in a range of dilutions from about 1:50 to about 1:5000, preferably from about 1:100 to about 1:500. Specific antigenicity is shown once a band corresponding to the product exhibits reactivity at any of the dilutions in the range.

In an ELISA assay, the product to be screened can be used as the coating antigen. A purified preparation is preferred, but a whole cell extract can also be used. Briefly, about 100 ll of a preparation of about 10 jig protein/ml is distributed into wells of a 96-well ELISA plate. The plate is incubated for about 2 hours at 37°C, then overnight at 4°C. The plate is washed with phosphate buffered saline (PBS) containing 0.05% Tween (PBS/Tween buffer) and the wells are saturated with 250 ul PBS containing 1% bovine serum albumin (BSA), to prevent non-specific antibody binding.

After 1 hour of incubation at 37°C, the plate is washed with PBS/Tween buffer.

The antiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA, and 100 tl dilutions are added to each well. The plate is incubated for minutes at 37oC, washed, and evaluated using standard methods. For example, a goat anti-rabbit peroxidase conjugate can be added to the wells when the specific antibodies used were raised in rabbits. Incubation is carried out for about 90 minutes at 37°C and the plate is washed. The reaction is developed with the appropriate substrate and the reaction is measured by colorimetry (absorbance measured spectrophotometrically). Under these experimental conditions, a positive reaction is shown once an O.D. value of is detected with a dilution of at least about 1:50, preferably of at least about 1:500.

In a dot blot assay, a purified product is preferred, although a whole cell extract can be used. Briefly, a solution of the product at a concentration of WO 98/43479 PCT/US98/06421 -36about 100 tg/ml is serially diluted two-fold with 50 mM Tris-HCI (pH One hundred pl of each dilution is applied to a filter, such as a 0.45 jIm nitrocellulose membrane, set in a 96-well dot blot apparatus (Biorad). The buffer is removed by applying vacuum to the system. Wells are washed by addition of 50 mM Tris-HCl (pH 7.5) and the membrane is air-dried. The membrane is saturated in blocking buffer (50 mM Tris-HCl (pH 0.15 M NaCI, 10 g/L skim milk) and incubated with an antiserum diluted from about 1:50 to about 1:5000, preferably about 1:500. The reaction is detected using standard methods. For example, a goat anti-rabbit peroxidase conjugate can be added to the wells when rabbit antibodies are used. Incubation is carried out for about 90 minutes at 37 0 C and the blot is washed. The reaction is developed with the appropriate substrate and stopped. The reaction is then measured visually by the appearance of a colored spot, by colorimetry. Under these experimental conditions, a positive reaction is associated with detection of a colored spot for reactions carried out with a dilution of at least about 1:50, preferably, of at least about 1:500. Therapeutic or prophylactic efficacy of a polypeptide or polypeptide derivative of the invention can be evaluated as is described below.

According to a seventh aspect of the invention, there is provided a composition of matter containing a polypeptide of the invention together with a diluent or carrier; (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a polypeptide of the invention; (iii) a method for inducing an immune response against Helicobacter in a mammal by administering to the mammal an immunogenically effective amount of a polypeptide of the invention to elicit an immune response, a protective immune response to Helicobacter; and (iv) a method for preventing and/or treating a Helicobacter H. pylori, H. felis, H. mustelae, or-H.

WO 98/43479 PCT/US98/06421 -37heilmanii) infection, by administering a prophylactic or therapeutic amount of a polypeptide of the invention to an individual in need of such treatment.

Additionally, this aspect of the invention includes the use of a polypeptide of the invention in the preparation of a medicament for preventing and/or treating Helicobacter infection.

The immunogenic compositions of the invention can be administered by any conventional route in use in the vaccine field, for example, to a mucosal ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface or via a parenteral subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. The choice of the administration route depends upon a number of parameters, such as the adjuvant used. For example, if a mucosal adjuvant is used, the intranasal or oral route will be preferred, and if a lipid formulation or an aluminum compound is used, a parenteral route will be preferred. In the latter case, the subcutaneous or intramuscular route is most preferred. The choice of administration route can also depend upon the nature of the vaccine agent. For example, a polypeptide of the invention fused to CTB or to LTB will be best administered to a mucosal surface.

A composition of the invention can contain one or several polypeptides or derivatives of the invention. It can also contain at least one additional Helicobacter antigen, such as the urease apoenzyme, or a subunit, fragment, homolog, mutant, or derivative thereof.

For use in a composition of the invention, a polypeptide or polypeptide derivative can be formulated into or with liposomes, such as neutral or anionic liposomes, microspheres, ISCOMS, or virus-like particles (VLPs), to facilitate delivery and/or enhance the immune response. These compounds are readily available to those skilled in the art; for example; see WO 98/43479 PCT/US98/06421 -38- Liposomes: A Practical Approach (supra). Adjuvants other than liposomes can also be used in the invention and are well known in the art (see, for example, the list provided below).

Administration can be achieved in a single dose or repeated as necessary at intervals that can be determined by one skilled in the art. For example, a priming dose can be followed by three booster doses at weekly or monthly intervals. An appropriate dose depends on various parameters, including the nature of the recipient whether the recipient is an adult or an infant), the particular vaccine antigen, the route and frequency of administration, the presence/absence or type of adjuvant, and the desired effect protection and/or treatment), and can be readily determined by one skilled in the art. In general, a vaccine antigen of the invention can be administered mucosally in an amount ranging from about 10 gg to about 500 mg, preferably from about 1 mg to about 200 mg. For a parenteral route of administration, the dose usually should not exceed about 1 mg, and is, preferably, about 100 pg.

When used as components of a vaccine, the polynucleotides and polypeptides of the invention can be used sequentially as part of a multi-step immunization process. For example, a mammal can be initially primed with a vaccine vector of the invention, such as a pox virus, via a parenteral route, and then boosted twice with a polypeptide encoded by the vaccine vector, e.g., via the mucosal route. In another example, liposomes associated with a polypeptide or polypeptide derivative of the invention can be used for priming, with boosting being carried out mucosally using a soluble polypeptide or polypeptide derivative of the invention, in combination with a mucosal adjuvant LT).

Polypeptides and polypeptide derivatives of the invention can also be used as diagnostic reagents for detecting the presence of anti-Helicobacter WO 98/43479 PCT/US98/06421 -39antibodies, in blood samples. Such polypeptides can be about 5 to about preferably, about 10 to about 50 amino acids in length and can be labeled or unlabeled, depending upon the diagnostic method. Diagnostic methods involving such a reagent are described below.

Upon expression of a polynucleotide molecule of the invention, a polypeptide or polypeptide derivative is produced and can be purified using known methods. For example, the polypeptide or polypeptide derivative can be produced as a fusion protein containing a fused tail that facilitates purification.

The fusion product can be used to immunize a small mammal, a mouse or a rabbit, in order to raise monospecific antibodies against the polypeptide or polypeptide derivative. The eighth aspect of the invention thus provides a monospecific antibody that binds to a polypeptide or polypeptide derivative of the invention.

By "monospecific antibody" is meant an antibody that is capable of reacting with a unique, naturally-occurring Helicobacter polypeptide. An antibody of the invention can be polyclonal or monoclonal. Monospecific antibodies can be recombinant, chimeric consisting of a variable region ofmurine origin and a human constant region), humanized a human immunoglobulin constant region and a variable region of animal, e.g., murine, origin), and/or single chain. Both polyclonal and monospecific antibodies can also be in the form of immunoglobulin fragments, F(ab)'2 or Fab fragments. The antibodies of the invention can be of any isotype, e.g., IgG or IgA, and polyclonal antibodies can be of a single isotype or can contain a mixture of isotypes.

The antibodies of the invention, which can be raised to a polypeptide or polypeptide derivative of the invention, can be produced and identified using standard immunological assays, Western blot assays, dot blot assays, or WO 98/43479 PCT/US98/06421 ELISA (see, Coligan et al., Current Protocols in Immunology, John Wiley Sons, Inc., New York, NY, 1994). The antibodies can be used in diagnostic methods to detect the presence of Helicobacter antigens in a sample, such as a biological sample. The antibodies can also be used in affinity chromatography methods for purifying a polypeptide or polypeptide derivative of the invention.

As is discussed further below, the antibodies can also be used in prophylactic and therapeutic passive immunization methods.

Accordingly, a ninth aspect of the invention provides a reagent for detecting the presence of Helicobacter in a biological sample that contains an antibody, polypeptide, or polypeptide derivative of the invention; and (ii) a diagnostic method for detecting the presence of Helicobacter in a biological sample, by contacting the biological sample with an antibody, a polypeptide, or a polypeptide derivative of the invention, so that an immune complex is formed, and detecting the complex as an indication of the presence of Helicobacter in the sample or the organism from which the sample was derived. The immune complex is formed between a component of the sample and the antibody, polypeptide, or polypeptide derivative, and that any unbound material can be removed prior to detecting the complex. A polypeptide reagent can be used for detecting the presence of anti-Helicobacter antibodies in a sample, a blood sample, while an antibody of the invention can be used for screening a sample, such as a gastric extract or biopsy sample, for the presence of Helicobacter polypeptides.

For use in diagnostic methods, the reagent the antibody, polypeptide, or polypeptide derivative of the invention) can be in a free state or can be immobilized on a solid support, such as, for example, on the interior surface of a tube or on the surface, or within pores, of a bead. Immobilization can be achieved using direct or indirect means. Direct means include passive WO 98/43479 PCT/US98/06421 -41adsorption non-covalent binding) or covalent binding between the support and the reagent. By "indirect means" is meant that an anti-reagent compound that interacts with the reagent is first attached to the solid support. For example, if a polypeptide reagent is used, an antibody that binds to it can serve as an anti-reagent, provided that it binds to an epitope that is not involved in recognition of antibodies in biological samples. Indirect means can also employ a ligand-receptor system, for example, a molecule, such as a vitamin, can be grafted onto the polypeptide reagent and the corresponding receptor can be immobilized on the solid phase. This concept is illustrated by the well known biotin-streptavidin system. Alternatively, indirect means can be used, by adding to the reagent a peptide tail, chemically or by genetic engineering, and immobilizing the grafted or fused product by passive adsorption or covalent linkage of the peptide tail.

According to a tenth aspect of the invention, there is provided a process for purifying, from a biological sample, a polypeptide or polypeptide derivative of the invention, which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific antibody of the invention.

For use in a purification process of the invention, the antibody can be polyclonal or monospecific, and preferably is of the IgG type. Purified IgGs can be prepared from an antiserum using standard methods (see, Coligan et al., supra). Conventional chromatography supports, as well as standard methods for grafting antibodies, are described, for example, by Harlow et al.

(Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988).

Briefly, a biological sample, such as an H. pylori extract, preferably in a buffer solution, is applied to a chromatography material, which is, WO 98/43479 PCT/US98/06421 -42preferably, equilibrated with the buffer used to dilute the biological sample, so that the polypeptide or polypeptide derivative of the invention the antigen) is allowed to adsorb onto the material. The chromatography material, such as a gel or a resin coupled to an antibody of the invention, can be in batch form or in a column. The unbound components are washed off and the antigen is eluted with an appropriate elution buffer, such as a glycine buffer, a buffer containing a chaotropic agent, guanidine HC1, or a buffer having high salt concentration 3 M MgCl 2 Eluted fractions are recovered and the presence of the antigen is detected, by measuring the absorbance at 280 nm.

An antibody of the invention can be screened for therapeutic efficacy as follows. According to an eleventh aspect of the invention, there is provided a composition of matter containing a monospecific antibody of the invention, together with a diluent or carrier; (ii) a pharmaceutical composition containing a therapeutically or prophylactically effective amount of a monospecific antibody of the invention; and (iii) a method for treating or preventing Helicobacter H. pylori, H. felis, H. mustelae, or H. heilmanii) infection, by administering a therapeutic or prophylactic amount of a monospecific antibody of the invention to an individual in need of such treatment. In addition, the eleventh aspect of the invention includes the use of a monospecific antibody of the invention in the preparation of a medicament for treating or preventing Helicobacter infection.

The monospecific antibody can be polyclonal or monoclonal, and is, preferably, predominantly of the IgA isotype. In passive immunizationmethods, the antibody is administered to a mucosal surface of a mammal, e.g., the gastric mucosa, orally or intragastrically, optionally, in the presence of a bicarbonate buffer. Alternatively, systemic administration, not requiring a WO 98/43479 PCT/US98/06421 -43bicarbonate buffer, can be carried out. A monospecific antibody of the invention can be administered as a single active agent or as a mixture with at least one additional monospecific antibody specific for a different Helicobacter polypeptide. The amount of antibody and the particular regimen used can be readily determined by one skilled in the art. For example, daily administration of about 100 to 1,000 mg of antibody over one week, or three doses per day of about 100 to 1,000 mg of antibody over two or three days, can be effective regimens for most purposes.

Therapeutic or prophylactic efficacy can be evaluated using standard methods in the art, by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, the H. felis mouse model and the procedures described by Lee et al. (Eur. J.

Gastroenterology Hepatology 7:303, 1995) or Lee et al. Infect. Dis.

172:161, 1995). Those skilled in the art will recognize that the H. felis strain of the model can be replaced with another Helicobacter strain. For example, the efficacy of polynucleotide molecules and polypeptides from H. pylori is, preferably, evaluated in a mouse model using an H. pylori strain. Protection can be determined by comparing the degree ofHelicobacter infection in the gastric tissue assessed by, for example, urease activity, bacterial counts, or gastritis, to that of a control group. Protection is shown when infection is reduced by comparison to the control group. Such an evaluation can be made for polynucleotides, vaccine vectors, polypeptides, and polypeptide derivatives, as well as for antibodies of the invention.

For example, various doses of an antibody of the invention can-be administered to the gastric mucosa of mice previously challenged with an H.

pylori strain, as described, by Lee et al. (supra). Then, after an appropriate period of time, the bacterial load of the mucosa can be estimated by WO 98/43479 PCT/US98/06421 -44assessing urease activity, as compared to a control. Reduced urease activity indicates that the antibody is therapeutically effective.

Adjuvants that can be used in any of the vaccine compositions described above are described as follows. Adjuvants for parenteral administration include, for example, aluminum compounds, such as aluminum hydroxide, aluminum phosphate, and aluminum hydroxy phosphate. The antigen can be precipitated with, or adsorbed onto, the aluminum compound using standard methods. Other adjuvants, such as RIBI (ImmunoChem, Hamilton, MT), can also be used in parenteral administration.

Adjuvants that can be used for mucosal administration include, for example, bacterial toxins, the cholera toxin the E. coli heat-labile toxin the Clostridium difficile toxin A, the pertussis toxin and combinations, subunits, toxoids, or mutants thereof. For example, a purified preparation of native cholera toxin subunit B (CTB) can be used. Fragments, homologs, derivatives, and fusions to any of these toxins can also be used, provided that they retain adjuvant activity. Preferably, a mutant having reduced toxicity is used. Suitable mutants are described, in WO 95/17211 (Arg-7-Lys CT mutant), WO 96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and Glu-129-Gly PT mutant). Additional LT mutants that can be used in the methods and compositions of the invention include, e.g., Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other adjuvants, such as the bacterial monophosphoryl lipid A (MPLA) of, E.

coli, Salmonella minnesota, Salmonella typhimurium, or Shigella flexneri; saponins, and polylactide glycolide (PLGA) microspheres, can also be used in mucosal administration. Adjuvants useful for both mucosal and parenteral administrations, such as polyphosphazene (WO 95/2415), can also be used.

WO 98/43479 PCT/US98/06421 Any pharmaceutical composition of the invention, containing a polynucleotide, polypeptide, polypeptide derivative, or antibody of the invention, can be manufactured using standard methods. It can be formulated with a pharmaceutically acceptable diluent or carrier, water or a saline solution, such as PBS, optionally, including a bicarbonate salt, such as sodium bicarbonate, 0.1 to 0.5 M. Bicarbonate can advantageously be added to compositions intended for oral or intragastric administration. In general, a diluent or carrier can be selected on the basis of the mode and route of administration, and standard pharmaceutical practice. Suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences, a standard reference text in this field and in the USP/NF.

The invention also includes methods in which gastroduodenal infections, such as Helicobacter infection, are treated by oral administration of a Helicobacter polypeptide of the invention and a mucosal adjuvant, in combination with an antibiotic, an antisecretory agent, a bismuth salt, an antacid, sucralfate, or a combination thereof. Examples of such compounds that can be administered with the vaccine antigen and an adjuvant are antibiotics, including, macrolides, tetracyclines, P-lactams, aminoglycosides, quinolones, penicillins, and derivatives thereof (specific examples of antibiotics that can be used in the invention include, e.g., amoxicillin, clarithromycin, tetracycline, metronidizole, erythromycin, cefuroxime, and erythromycin); antisecretory agents, including, H 2 receptor antagonists cimetidine, ranitidine, famotidine, nizatidine; and roxatidine), proton pump inhibitors omeprazole, lansoprazole, and pantoprazole), prostaglandin analogs misoprostil and enprostil), and anticholinergic agents pirenzepine, telenzepine, carbenoxolone, and WO 98/43479 PCT/US98/06421 -46proglumide); and bismuth salts, including colloidal bismuth subcitrate, tripotassium dicitrate bismuthate, bismuth subsalicylate, bicitropeptide, and pepto-bismol (see, Goodwin et al., Helicobacter pylori, Biology and Clinical Practice, CRC Press, Boca Raton, FL, pp 366-395, 1993; Physicians' Desk Reference, 49' t edn., Medical Economics Data Production Company, Montvale, New Jersey, 1995). In addition, compounds containing more than one of the above-listed components coupled together, ranitidine coupled to bismuth subcitrate, can be used. The invention also includes compositions for carrying out these methods, compositions containing a Helicobacter antigen (or antigens) of the invention, an adjuvant, and one or more of the above-listed compounds, in a pharmaceutically acceptable carrier or diluent.

Amounts of the above-listed compounds used in the methods and compositions of the invention can readily be determined by one skilled in the art. In addition, one skilled in the art can readily design treatment/immunization schedules. For example, the non-vaccine components can be administered on days 1-14, and the vaccine antigen adjuvant can be administered on days 7, 14, 21, and 28.

Methods and pharmaceutical compositions of the invention can be used to treat or to prevent Helicobacter infections and, accordingly, gastroduodenal diseases associated with these infections, including acute, chronic, and atrophic gastritis, and peptic ulcer diseases, gastric and duodenal ulcers.

A 76 kDa protein band containing GHPO 386, GHPO 789, and GHPO 1516 (hereinafter the "purified 76 kDa proteins"), GHPO 1360; and GHPO 750 were purified from Helicobacterpylori strain ATCC number 43579 (American Type Culture Collection, Rockville, Maryland) by immunoaffinity- WO 98/43479 PCT/US98/06421 -47based chromatography using the methods described below in Example 1, and were shown to be effective vaccine antigens as follows.

Groups of 10 mice each were orally immunized with 1, 5, or 25 tg of the purified 76 kDa proteins, purified GHPO 1360, or purified GHPO 750 in combination with 5 ug of the heat-labile enterotoxin (LT) of E. coli. Twenty five gg of recombinant urease, in combination with 5 gg LT, was used as a positive control, and 5 jig of LT in PBS was used as a negative control. The immunizations were carried out four times each, on days 0, 7, 14, and 21 of the experiment. On day 33, blood samples were collected from the mice and, on day 34, saliva samples were collected. On day 35, all of the mice were challenged by intragastric administration of 1 x 10' streptomycin-resistant, mouse-adapted H. pylori. On day 49, additional saliva samples were collected and, about two weeks after challenge, on days 52-53, the mice were sacrificed.

Stomachs were removed from the mice and were analyzed for Helicobacter infection by measuring urease activity in the intact stomach tissue and by a quantitative culture study (Table 1).

Briefly, these studies showed that the gastric urease activities in samples from mice immunized with all three amounts of the purified 76 kDa proteins 1, 5, and 25 ptg), in combination with LT, were generally lower than the gastric urease activities of samples from mice immunized with LT alone or mice that were not treated prior to challenge. Levels of gastric urease activity generally decreased with increasing amounts of the protein administered, with the gastric urease activity levels for the 25 jig doses generally approaching those of mice immunized with 25 ug of recombinant urease and LT.

The quantitative culture analyses showed that the levels of Helicobacter detected in the stomachs of mice immunized with the purified 76 WO 98/43479 PCT/US98/06421 -48kDa proteins, purified GHPO 1360, or purified GHPO 750, which generally decreased with increasing dosages, were less than the levels detected in the stomachs of control mice that were immunized with LT alone or untreated before Helicobacter challenge (Tables 1 and The percentages of mice protected by immunization with the purified 76 kDa proteins, purified GHPO 1360, or purified GHPO 750 met or approached the percentages of mice protected by treatment with urease (Tables 1 and These results show that the purified 76 kDa proteins, GHPO 1360, and GHPO 750 are effective vaccine antigens for use in preventing Helicobacter infection.

WO 98/43479 WO 9843479PCTIUS98/06421I -49- Table 1 Prophylactic Immunization with PMsv Antigens as Oral Dose Response Against H. pylori Challenge BALB/c mice Fisher's exact test CFU/ml (1/4 antrum) Wilcoxon rank sums mice infected infection status (based Mean SD test Antrum on quantitative A 55 CFU treatment group (based on quantitative ratios, treatment v. LT only control 0.148 O.D. group v. LT onlIy (group 11) Treatment cutoff) (group 11)) p-value p-value I jig 50 kDa LT 60% (6/10) 0.3034 30825 23210 0.1736 jig 50 kDa LT 40% (4/10) 0.0573 18910 16341 0.0588 jig 50 kDa LT 30% (3/10) 0.0198 22710)± 32397 0.0821 1 jig 32 kDa +LT 50% (5/10) 0.1409 44225± 87824 0.0756 pg 50kDa +LT 10% (1/10) 0.0011 11811 11579 0.0191 25jg5kDa +LT 0 0.0001 1608± 23917 0.01 14 jig rUre LT 0 0.0001 8208 8021 0.0179 LT 90% (9/10) 107340± 127949 (9/10) not determined 46173± 42325 0.2568 WO 98/43479 PCT/US98/06421 Table 2 Prophylactic Immunization with PMsv Antigens as Oral Dose Response Against H. pylori Challenge BALB/c mice Fisher's exact test CFU/ml (1/4 antrum) Wilcoxon rank sums mice infected infection status (based Mean SD test Antrum on quantitative A 5 so CFU treatment group (based on quantitative ratios, treatment v. LT only control A,s 5 0.148 O.D. group v. LT only (group 11) Treatment cutoff) (group 11)) p-value p-value 1 pg 76 kDa LT 56% 0.1409 39922 34708 0.2203 pg 76 kDa LT 80% 1 8802 7788 0.0864 pg 76 kDa LT 33% 0.0198 9712± 12183 0.0178 pg rUre LT 0 0.0001 8208 8021 0.0179 LT 90% (9/10) 107340 ±127949 (9/10) not determined 46173 42325 0.2568 The invention is further illustrated by the following examples.

Example 1 describes purification of GHPO 1516 (76 kDa), GHPO 1360 (32 kDa), and GHPO 750 (50 kDa) from Helicobacter cultures. Example 2 describes identification of genes, genes encoding 76 kDa proteins, such as GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 711, GHPO 190, GHPO 185, GHPO 1417, and GHPO 1414, a 32 kDa protein (GHPO 1360), and a 50 kDa protein (GHPO 750) in the Helicobacter genome, as well as identification of signal sequences, and primer design for amplification of genes lacking signal sequences. Example 3 describes cloning of DNA encoding GHPO 386, GHPO 789, GHPO 1516, GHPO 896, GHPO 1360, and GHPO 750 into a vector that provides a histidine tag, and production and purification of the resulting his-tagged fusion proteins. Example 4 describes methods for cloning DNA encoding the polypeptides of the invention so that they can be produced without His-tags, Example 5 describes methods WO 98/43479 PCT/US98/06421 -51for purifying recombinant polypeptides of the invention, and Example 6 describes use of the GHPO 1360 polypeptide as a serodiagnostic tool for H.

pylori infection EXAMPLE 1: Purification and partial sequence analysis of GHPO 1516 (76 kDa), GHPO 1360 (32 kDa), and GHPO 750 (50 kDa) protein from Helicobacter pylori 1.A. Culture and initial purification steps Frozen seeds from H. pylori strain ATCC 43579 are used to seed a cm 2 flask containing a biphasic medium (a solid phase made of Colombia gelose containing 6% fresh sheep blood and a liquid phase made of triptcase soja containing 20% fetal calf serum). After 24 hours of culturing under microaerophilic conditions, the liquid phase is used for seeding several 75 cm 2 flasks containing biphasic medium lacking sheep blood. After 24 hours of culture, the liquid phase is used to seed a 2 L biofermentor in triptcase soja liquid phase containing 10 g/L beta-cyclodextrine. At OD 1.5-1.8, this culture is diluted in a 10 L biofermentor containing the liquid medium. After 24 hours, the bacteria are spun in a centrifuge at 4,000 x g for 30 minutes at 4 0 C. A 10 L culture contains about 20 to 30 g (wet weight) bacteria.

The pellet obtained using the method described above is washed with 500 ml PBS (7.650 g NaC1, 0.724 g disodium phosphate, and 0.210 g monopotassium phosphate for one liter (pH for a one liter culture. The bacteria are then spun in a centrifuge again under the same conditions. The pellet (Cl) is suspended in 1% N-octyl-D-glucopyranoside (NOG; 30 ml/L; Sigma). The bacterial suspension is incubated for 1 hour at WO 98/43479 PCT/US98/06421 -52room temperature while stirring, spun in a centrifuge at 17,600 x g for minutes at 4 0 C, and the pellet (C2) is recovered.

The supernatant (S2) is dialyzed against PBS overnight at 4 0 C while stirring. The precipitate is recovered by centrifugation at 2,600 x g for 30 minutes at 4 0 C. The supernatant (S2d) is discarded and the pellet (Cs2d) is recovered and stored at -20 0

C.

The pellet (C2) is resuspended in 20 mM Tris-HC1 buffer (pH and 100 jpM Pefabloc (Buffer and is homogenized with an ultra-turrax (3821, Janke and Kungel). Lysozyme and EDTA are added at 0.1 mg/ml and 1 mM, respectively.

The homogenate is sonicated three times for 2 minutes each at 4 0

C,

and then is spun in an ultracentrifuge at 210,000 x g for 30 minutes at 4 0 C. The supernatant which contains the cytoplasmic and periplasmic proteins, is eliminated, while the pellet is recovered, washed with buffer A, and spun in an ultracentrifuge at 210,000 x g for 30 minutes at 4 0 C. The supernatant (S4) is eliminated and the pellet (C4) is stored at -20 0 C. This pellet (C4) contains membrane proteins.

The pellet (C4) is washed in 50 mM NaCO, (pH 9.5) and 100 gM Pefabloc (buffer The suspension is spun in an ultracentrifuge at 210,000 x g for 30 minutes at 4 0 C. The supernatant (S5) is eliminated, and the pellet (C5) is then washed and spun in an ultracentrifuge as is described above.

The supernatant (S6) is eliminated and the pellet (C6) is stored at -20 0

C.

WO 98/43479 PCT/US98/06421 -53- 1.B. Purification of the proteins of membrane fraction C4 by preparative

SDS-PAGE

SDS-PAGE is carried out according to the method of Laemmli (supra), using a biphasic gel consisting of a 5% polyacrylamide concentrating gel and a 10% polyacrylamide separating gel. The membrane fraction C4 is resuspended in buffer A, diluted in an equal volume of 2x sample buffer, and heated for 5 minutes at 95C. About 19 mg of protein is applied to the gel (16 x 12 cm; 5 mm thick). Pre-migration is carried out for 2 hours at 50 V, and is followed by migration overnight at 65 V. After Coomassie blue staining, five major bands are revealed that have apparent molecular weights of 87, 76, 54, 50, and 32 kDa. Bands at 50 and 32 kDa appear to be slightly contaminated with bands at 47 and 35 kDa, respectively.

A band corresponding to the purified 76 kDa proteins, 32 kDa protein (GHPO 1360), or 50 kDa protein (GHPO 750) is cut out from the gel and is pounded with an ultra-turrax in 10-20 ml extraction buffer (25 mM Tris- HCI (pH 8 M urea, 10% SDS, 100 gM phenyl methyl sulfonyl fluoride (PMSF), and 10 g.M Pefabloc (buffer Each homogenate is filtered through a Millipore AP20 filter under 7 bars at room temperature, washed with 5-10 ml buffer C, and then filtered again. Each filtrate is precipitated with three volumes of a 50/50 mixture of methanol and 75% isopropanol, and then is spun in a centrifuge at 240,000 x g for 16 hours at 10 0

C.

Each pellet is resuspended in 2 ml of 10 mM NaPO 4 (pH containing 1 M NaCI, 0.1% Sarkosyl, 100 p.M PMSF, and 6 M urea (buffer D).

The solubilized sample is dialyzed, in order, against 100 ml buffer D containing 4 M urea, 100 ml buffer D containing 2 M urea and 0.5% Sarkosyl, and twice against 100 ml buffer D that does not contain urea or Sarkosyl. The dialyses WO 98/43479 PCT/US98/06421 -54are carried out for 1 hour each while stirring at room temperature. The last dialysate is incubated for 30 minutes in an ice bath, and then is spun in a centrifuge at low speed for 10 minutes at 4°C. The supernatant is recovered, filtered through a Millipore filter (0.45 gm), and stored at -20 0

C.

1.C. Purification of the 76 kDa, 32 kDa, or 50 kDa protein by immunoaffinity-based chromatography 1.C.1. Antiserum preparation Specific polyclonal serum against the purified 76 kDa proteins, the 32 kDa protein (GHPO 1360), or the 50 kDa protein (GHPO 750), which are purified by preparative SDS-PAGE, is prepared by hyperimmunizing rabbits as follows. On day 0, a preparation containing 50 Rg of the protein mixed with complete Freund's adjuvant is administered subcutaneously to the rabbits at multiple sites. The rabbits are boosted at days 21 and 42 with 25 tg of the protein in incomplete Freund's adjuvant, and are sacrificed at day Complement is removed from the serum by heating for 30 minutes at 56 0

C.

The hyperimmune serum is then sterilized by filtration through a Millipore membrane (0.22 gm).

1.C.2. IgG purification The hyperimmune serum prepared as described above is applied to a Protein A Sepharose Fast Flow column (Pharmacia) that is equilibrated with 100 mM Tris-HCI (pH The column is washed with 10 column volumes of 100 mM Tris-HCl (pH and then with 10 column volumes of 10 mM Tris- HCl (pH IgGs are eluted in 0.1 M glycine buffer (pH and are collected as 5 ml fractions, to each of which 0.25 ml of Tris-HCl (pH 8.0) is added. The optical density of each fraction is measured at 280 nm, the IgGcontaining fractions are pooled together and, if necessary, frozen at -701C.

WO 98/43479 PCT/US98/06421 1.C.3. Preparation of the column An appropriate amount of CNBr-activated Sepharose 4B gel (Pharmacia; reference: 17-0430-01) is suspended in 1 mM NaCI buffer (1 g dry gel provides for 3.5 ml hydrated gel; 5 to 10 mg IgGs can be retained per ml of hydrated gel). The gel is then washed using a buchner by adding small quantities of 1 mM HC1. The total volume of 1 mM HCI that is used amounts to 200 ml/g of gel.

Purified IgGs are dialyzed for 4 hours at room temperature against volumes of 500 mM sodium phosphate buffer (pH The IgGs are then diluted to 3 mg/ml with the same buffer. IgGs are incubated with the gel overnight at 5+3 0 C while stirring. The gel is packed in a chromatography column and is washed with 2 column volumes of 500 mM phosphate buffer (pH The gel is then transferred to a tube and is incubated with 100 mM ethanolamine (pH and then it is washed with 2 column volumes of PBS.

The gel can be stored in PBS/merthiolate, 1/10,000.

1.C.4. Adsorption and elution The 76 kDa protein is adsorbed and eluted as follows. The membrane fraction Cs2d is suspended in 50 mM Tris-HCl (pH 2 mM EDTA, and then is filtered through a 0.45 pm membrane. The supernatant is applied to the column, which is equilibrated with 50 mM Tris-HCI (pH 2 mM EDTA, at a flow rate of about 10 ml/hour. The column is washed with column volumes of 50 mM Tris-HCl (pH 2 mM EDTA, and then with 2 to 6 volumes 10 mM phosphate buffer (pH 6.8).

The antigen is eluted with 100 mM glycine buffer (pH The eluate is collected in 3 ml fractions, to each of which is added 150 pl 1 M phosphate buffer (pH The optical density of each fraction is measured at WO 98/43479 PCT/US98/06421 -56- 280 nm, fractions containing the 76 kDa protein are pooled, and stored at -70 0

C.

Analysis by 10% SDS-PAGE reveals a single band at 76 kDa. Nterminal sequence was carried out on this purified 76 kDa preparation, and the sequence obtained is as follows: EDDGFYTSVGYQIGEAAQMV (SEQ ID NO:58).

The 32 kDa protein (GHPO 1360) or the 50 kDa protein (GHPO 750) is purified by immunoaffinity-based chromatography as follows. In order to separate the 32 or 50 kDa protein from the contaminating proteins (the 47 and 35 kDa proteins, respectively), membrane fraction C4 is solubilized in mM NaCO 3 (pH 9.5) for 30 minutes at room temperature under stirring and the preparation is centrifuged for 30 minutes at 200,000 x g at 4 0 C. The 47 and kDa proteins are insoluble in the NaCO 3 buffer and are eliminated in the pellet.

The supernatant is dialyzed against 50 mM Tris-HCL (pH 2 mM EDTA, and then is filtered through a 0.45 gtm membrane. The filtered supernatant is applied to the column, which is equilibrated with 50 mM Tris- HCL (pH 2 mM EDTA, at a flow rate of about 10 ml/hour. The column is washed with 20 column volumes of 50 mM Tris-HCL (pH 2 mM EDTA, and then with 2 to 6 volumes of 10 mM phosphate buffer (pH 6.8).

The antigen is eluted with 100 mM glycine buffer (pH The eluate is collected in 3 ml fractions, to each of which is added 150 4l 1 M phosphate buffer (pH The optical density of each fraction is measured at 280 nm, and fractions containing the 50 or 32 kDa protein are pooled and stored at -70 0

C.

Analysis of the purified protein by 10% SDS-PAGE reveals single bands at 50 and 32 kDa. N-terminal sequencing is carried out with the purified kDa protein preparation. The sequence found is as follows: MKEKFNRTKPHVNIGTIGHVDH (SEQ ID NO:73). Similarly, N-terminal WO 98/43479 PCT/US98/06421 -57and internal sequencing is carried out with the purified 32 kDa preparation.

The sequences found are as follows: AHNANNATHNTKK (SEQ ID NO:74) and KPAHNA (SEQ ID NO:75) (N-terminal), and IDKQPKAKK (SEQ ID NO:76) and FWAKKQAE (SEQ ID NO:77) (internal).

1.D. Purification of the 76 kDa protein from membrane fraction Cs2d and purification of the 32 kDa and 50 kDa proteins from membrane fraction C4 The 76 kDa protein can also be purified as follows. A 40 ml Q- Sepharose column (diameter: 2.5 cm; height: 8 cm) is prepared according to the manufacturer's instructions (Pharmacia). The column is washed and equilibrated with buffer B, containing 50 mM NaCO, (pH 100 gM Pefabloc, and 0.1% Zwittergent 3-14. The chromatography is monitored by measuring absorbance at 280 nm at the column exit.

One hundred and forty mg of protein from the membrane fraction Cs2d resuspended in buffer B are applied to the column. The column is washed with 0.1 M NaCI in buffer B, and then a 0.1-0.5 M NaCl gradient is applied to the column. The fraction eluted between 0.35 and 0.45 M NaCl is further purified on a 10 ml S-Sepharose column (diameter: 1.5 cm; height: cm; up to 10 mg protein/ml of gel), which is prepared according to the manufacturer's instructions (Pharmacia). The fraction obtained is dialyzed against 50 mM acetate (pH 5.0) containing 100 pM Pefabloc and 0.1% Zwittergent 3-14, and then is applied to the column, which is equilibrated with the acetate buffer.

The column is washed with the acetate buffer until the absorbance at 280 nm is stabilized (about 3 column volumes are required). Proteins are WO 98/43479 PCT/US98/06421 -58eluted with a 0-0.5 M NaCI gradient in acetate buffer. The fraction eluted at 0.15 M NaC1 is enriched with the 76 kDa protein.

The 32 kDa protein (GHPO 1360) can also be purified as follows.

Membrane fraction C4 is solubilized in 50 mM NaCObuffer (pH 9.5) at room temperature for 30 minutes under stirring. The suspension is then centrifuged at 200,000 x g for 30 minutes at 4 0 C. This allows the 32 and 35 kDa proteins to be separated, since the 35 kDa protein is insoluble in the NaCO 3 buffer. The supernatant is dialyzed against 50 mM NaPO, buffer (pH and then is applied to an SP-Sepharose column, which is equilibrated with the NaPO, buffer. The column is washed with the NaPO, buffer, and then an 0-0.5 M NaCI gradient is applied to the column. The fraction eluted between 0.26 and 0.31 M contains the 32 kDa protein.

The 50 kDa protein can also be purified as follows. Membrane fraction C4 is solubilized in 50 mM NaCO,buffer (pH 9.5) at room temperature for 30 minutes while stirring. The suspension is then centrifuged at 200,000 x g for 30 minutes at 4 0 C. This allows the 50 and 47 kDa proteins to be separated, since the 47 kDa protein is insoluble in the NaCO 3 buffer. The supernatant is dialyzed against 50 mM NaPO buffer (pH A 40 ml Q-Sepharose column (diameter: 2.5 cm; height: 8 cm) is prepared according to the manufacturer's instructions (Pharmacia), washed, and equilibrated with buffer B (pH 9.5) (50 mM NaCO,, 100 jtM Pefabloc, and 0.1% Zwittergent 3-14).

The chromatography is monitored by UV detection at 280 nm at the column exit. One hundred and forty mg of protein solubilized as is described above are applied to the column, which is then washed with buffer B until the absorbance at 280 nm is stabilized. The proteins are eluted with a 0.1- M NaCl gradient in buffer B (10 fold which is followed by washing in WO 98/43479 PCT/US98/06421 -59buffer B containing 0.5, and then 1, M NaCI (2 fold VT). The fractions are recovered, analyzed by SDS-PAGE, and pooled according to their electrophoretic profiles.

Fraction 9, which corresponds to the beginning of the washing at 1 M NaCI and contains acidic proteins, is further purified as follows. A 10 ml DEAE Sepharose column (diameter: 1.5 cm, height: 5 cm) is prepared according to the manufacturer's instructions (Pharmacia) (up to 10 mg protein/ml of gel). The column is washed and equilibrated with buffer B.

Chromatography is monitored as is described above.

Fraction 9 is dialyzed against buffer B and contains about 10 mg protein.

Fraction 9 is applied to the DEAE-Sepharose column. The column is washed with buffer B until the absorbance at 280 nm is stabilized. The proteins are eluted with a 0-0.5 M NaCl gradient in buffer B (10 fold followed by washing in buffer B, containing 1 M NaCl (2 fold Fractions are recovered and analyzed by SDS-PAGE. The 50 kDa protein is found in the fractions eluted at 0.3-0.4 M NaC1.

EXAMPLE 2: Identification of genes in the H. pylori genome, such as genes encoding the 76 kDa proteins, the 32 kDa protein (GHPO 1360), and the 50 kDa protein (GHPO 750) identification of signal sequences, and primer design for amplification of genes lacking signal sequences 2.A. Creating H. pylori genomic databases The H. pylori genome was provided as a text file containing a single contiguous string of nucleotides that had been determined to be 1.76 Megabases in length. The complete genome was split into 17 separate files using the program SPLIT (Creativity in Action), giving rise to 16 contigs, each WO 98/43479 PCT/US98/06421 containing 100,000 nucleotides, and a 1 7 th contig containing the remaining 76,000 nucleotides. A header was added to each of the 17 files using the format: >hpg0.txt (representing contig .hpgl.txt (representing contig etc.

The resulting 17 files, named hpg0 through hpgl6, were then copied together to form one file that represented the plus strand of the complete H. pylori genome.

The constructed database was given the designation A negative strand database of the H. pylori genome was created similarly by first creating a reverse complement of the positive strand using the program SeqPup (D.G.

Gilbert, Indiana University Biology Department) and then performing the same procedure as described above for the plus strand. This database was given the designation The regions predicted to encode open reading frames (ORFs) were defined for the complete H. pylori genome using the program GENEMARKTM (Borodovsky et al., Comp. Chem. 17:123, 1993). A database was created from a text file containing an annotated version of all ORFs predicted to be encoded by the H. pylori genome for both the plus and minus strands, and was given the designation Each ORF was assigned a number indicating its location on the genome and its position relative to other genes. No manipulation of the text file was required.

2.B. Searching the H. pylori databases The databases constructed as is described above were searched using the program FASTA (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988). FASTA was used for searching either a DNA sequence against either of the gene databases and/or or a peptide sequence against the ORF library TFASTX was used to search a peptide sequence against all possible reading frames of a DNA database and/or libraries). WO 98/43479 PCT/US98/06421 -61- Potential frameshifts also being resolved, FASTX was used for searching the translated reading frames of a DNA sequence against either a DNA database, or a peptide sequence against the protein database.

2.C. Isolation of DNA sequences from the H. pylori genome The FASTA searches against the constructed DNA databases identified exact nucleotide coordinates on one or more of the isolated contigs, and therefore the location of the target DNA. Once the exact location of the target sequence was known, the contig identified to carry the gene was exported into the software package MapDraw (DNAStar, Inc.) and the gene was isolated.

Gene sequences with flanking DNA was then excised and copied into the EditSeq. Software package (DNAStar, Inc.) for further analysis.

2.D. Identification of signal sequences The deduced protein encoded by a target gene sequence is analyzed using the PROTEAN software package (DNAStar, Inc.). This analysis predicts those areas of the protein that are hydrophobic by using the Kyte-Doolittle algorithm, and identifies any potential polar residues preceding the hydrophobic core region, which is typical for many signal sequences. For confirmation, the target protein is then searched against a PROSITE database (DNAStar, Inc.) consisting of motifs and signatures. Characteristic of many signal sequences and hydrophobic regions in general, is the identification of predicted prokaryotic lipid attachment sites. Where confirmation between the two approaches is apparent at the N-terminus of any protein, putative cleavage sites are sought. Specifically, this includes the presence of either an Alanine Serine or Glycine residue immediately after the core hydrophobic WO 98/43479 PCT/US98/06421 -62region. In the case oflipoproteins, a Cysteine residue would be identified as the +1 residue, post-cleavage.

2.E. Rational design of PCR primers based on the identification of signal sequences In order to clone gene sequences as N-terminus translational fusions for the generation of recombinant proteins with N-terminal Histidine tags, the gene sequence that specifies the signal sequence is omitted. The 5'-end of the genespecific portion of the N-terminal primer is designed to start at the first codon beyond the cleavage site. In the case oflipoproteins, the 5'-end of the Nterminal primer begins at the second codon, immediately after the modifiable residue at position +1 post-cleavage. The omission of the signal sequence from the recombinant allows for one-step purification, and potential problems associated with insertion of signal sequences in the membrane of the host strain carrying the hybrid construct are avoided.

EXAMPLE 3: Preparation of isolated DNA encoding GHPO 386, GHPO 789, GHPO 1516, GHPO 896, GHPO 1360, and GHPO 750, and production of these proteins as a histidine-tagged fusion proteins 3.A. Preparation of genomic DNA from Helicobacterpylori Helicobacter pylori strain ORV2001, stored in LB medium containing 50% glycerol at -70 0 C, is grown on Colombia agar containing 7% sheep blood for 48 hours under microaerophilic conditions (8-10% CO 2 5-7% O2, and 87% N 2 Cells are harvested, washed with PBS (pH and DNA is then extracted from the cells using the Rapid Prep Genomic DNA Isolation kit (Pharmacia Biotech).

WO 98/43479 WO 9843479PCT/US98/06421 -63- 3.B. PCR amplification DNA encoding GHPO 386, GHPO 789, GHPO 1516, GHPO 896, GHPO 1360, and GHPO odd numbers), 65, and 67 is amplified from genomic DNA, as can be prepared as is described above, by the Polymnerase Chain Reaction (PCR) using the following primers: GHPO 386: N-terminal primer: 5'-CTGAATTCGATTTCAAGGAGAAAACATGAAA-3' (SEQ ID NO: 59); and C-terminal primer: 5'-CCGCTCGAGTTAGTAAGCGAACACATAATT.3' (SEQ ID GHPO 789: N-terminal primer: 5'-CGCGGATCCGAATCCATTTAATCCAAAGG-3' (SEQ ID NO:6 1); and C-terminal primer: '-CCGCTCGAGTTAGTAAGCGAACACATAGTTCA-3' (SEQ ID NO:62).

GHPO 1516: N-terminal primer: 5'-CGCGGATCCGAATCCAATTTAATCCAAAAAGG-3' (SEQ ID NO:56); and C-terminal. piimer: 5'-CCGCTCGAGTTAAGTAAGCGAACACATATTCAA-3' (SEQ ID NO: 57).

14 GHPO 896: N-terminal primer: 5'-CGCGGATCCGAAGTTTCTTTGTATCAAAG-3' (SEQ ID NO:63); and C-terminal primer: WO 98/43479 PCT/US98/06421 -64- 5'-CCGCTCGAGTTAGTAAGCAAACACATAATTGTG-3' (SEQ ID NO:64).

GHPO 1360: N-terminal primer: 5'-CGCGGATCCGAATGAAAAAAAATATCTTAAAT-3' (SEQ ID NO:69); and C-terminal primer: 5'-CCGCTCGAGTTACTTGTTGATAACAATTTT-3' (SEQ ID GHPO 750: N-terminal primer: 5'-CGCGGATCCGAATGGCAAAAGAAAAGTTTAAC-3' (SEQ ID NO:71); and C-terminal primer: 5'-CCGCTCGAGTTATTCAATAATATTGCTCAC-3' (SEQ ID NO:72).

GHPO 711: N-terminal primer: 5'-GGGAATTCAAAAAAACGAAAAAAACG-3' (SEQ ID NO:83); and C-terminal primer: 5'-CCCCTCGAGTTAATAGGCAAACAC-3' (SEQ ID NO:84).

The N-terminal and C-terminal primers for each clone both include a clamp and a restriction enzyme recognition sequence for cloning purposes (BamHI (GGATCC) and Xhol (CTCGAG) recognition sequences).

Amplification of gene-specific DNA is carried out using a heat-stable DNA Polymerase Thermalase DNA Polymerase (Amresco)) according to the manufacturer's instructions. The reaction mixture, which is brought to a WO 98/43479 PCT/US98/06421 final volume of 100 il with distilled water, is as follows: dNTPs mix 200 gM ThermoPol buffer 10 Pl primers 300 nM each DNA template 50 ng DNA polymerase 2 units Appropriate amplification reaction conditions can readily be determined by one skilled in the art. In the present case, the following conditions were used. For GHPO 386 and GHPO 789, in a reaction containing Taq DNA polymerase (Appligene), a denaturing step was carried out at 95 C for seconds, followed by an annealing step at 50°C for one minute, and an extension step at 72°C for 2 minutes and 30 seconds. Twenty five cycles were carried out. For GHPO 896, in a reaction containing Taq DNA polymerase, a denaturing step was carried out at 97°C for 30 seconds, followed by an annealing step at 50°C for one minute, and an extension step at 72 C for 2 minutes and 30 seconds. Twenty five cycles were carried out. The same reaction conditions were used for GHPO 1516 as GHPO 896, except that Vent DNA polymerase was used for clone GHPO 1516, instead of Taq DNA polymerase, and the annealing temperature was 55°C. For GHPO 1360 and GHPO 750, Thermalase DNA polymerase was used. A denaturing step was carried out at 95 C for 30 seconds, followed by an annealing step at 55°C for one minute, and an extension step at 72 0 C for 2 minutes. Thirty cycles were carried out. For GHPO 711, Vent DNA polymerase was used. A denaturing step was carried out at 94°C for 30 seconds, followed by an annealing step at for 30 seconds, and an extension step at 72°C for 1 minute. Twenty five cycles were carried out.

WO 98/43479 PCT/US98/06421 -66- 3.C. Transformation and selection of transformants A single PCR product is thus amplified and is then digested at 37 0 C for 2 hours with BamHI and XhoI concurrently in a 20 u1 reaction volume. The digested product is ligated to similarly cleaved pET28a (Novagen) that is dephosphorylated prior to the ligation by treatment with Calf Intestinal Alkaline Phosphatase (CIP). The gene fusion constructed in this manner allows one-step affinity purification of the resulting fusion protein because of the presence of histidine residues at the N-terminus of the fusion protein, which are encoded by the vector.

The ligation reaction (20 gl) is carried out at 14°C overnight and then is used to transform 100 gl fresh E. coli XL 1-blue competent cells (Novagen).

The cells are incubated on ice for 2 hours, then heat-shocked at 42 C for seconds, and returned to ice for 90 seconds. The samples are then added to 1 ml LB broth in the absence of selection and grown at 37°C for 2 hours. The cells are then plated out on LB agar containing kanamycin (50 gg/ml) at a and neat dilution and incubated overnight at 37°C. The following day, colonies are picked onto secondary plates and incubated at 37 0 C overnight.

Five colonies are picked into 3 ml LB broth supplemented with kanamycin (100 gg/ml) and are grown overnight at 37°C. Plasmid DNA is extracted using the Quiagen mini-prep, method and is quantitated by agarose gel electrophoresis.

PCR is performed with the gene-specific primers under the conditions stated above and transformant DNA is confirmed to contain the desired insert.

If PCR-positive, one of the five plasmid DNA samples (500 ng) extracted from the E. coli XL 1-blue cells is used to transform competent BL21 (XDE3) E. coli competent cells (Novagen; as described previously). Transformants (10) are WO 98/43479 PCT/US98/06421 -67picked onto selective kanamycin (50 gg/ml) containing LB agar plates and stored as a research stock in LB containing 50% glycerol.

3.D. Purification of recombinant proteins One ml of frozen glycerol stock prepared as described in 3.C. is used to inoculate 50 ml of LB medium containing 25 gg/ml of kanamycin in a 250 ml Erlenmeyer flask. The flask is incubated at 37 0 C for 2 hours or until the absorbance at 600 nm (OD 600 reaches 0.4-1.0. The culture is stopped from growing by placing the flask at 4 0 C overnight. The following day, 10 ml of the overnight culture are used to inoculate 240 ml LB medium containing kanamycin (25 gg/ml), with the initial OD 600 about 0.02-0.04. Four flasks are inoculated for each ORF.

The cells are grown to an OD 600 of 1.0 (about 2 hours at 37°C), a 1 ml sample is harvested by centrifugation, and the sample is analyzed by SDS- PAGE to detect any leaky expression. The remaining culture is induced with 1 mM IPTG and the induced cultures are grown for an additional 2 hours at 37 0

C.

The final OD 600 is taken and the cells are harvested by centrifugation at 5,000 x g for 15 minutes at 4 0 C. The supernatant is discarded and the pellets are resuspended in 50 mM Tris-HCl (pH 2 mM EDTA. Two hundred and fifty ml of buffer are used for a 1 L culture and the cells are recovered by centrifugation at 12,000 x g for 20 minutes. The supernatant is discarded and the pellets are stored at -45 0

C.

3. E. Protein purification Pellets obtained from 3.D. are thawed and resuspended in 95 ml of mM Tris-HCI (pH Pefabloc and lysozyme are added to final WO 98/43479 PCT/US98/06421 -68concentrations of 100 ptM and 100 gg/ml, respectively. The mixture is homogenized with magnetic stirring at 5 0 C for 30 minutes. Benzonase (Merck) is added at a 1 U/ml final concentration, in the presence of 10 mM MgC1 2 to ensure total digestion of the DNA. The suspension is sonicated (Branson Sonifier 450) for 3 cycles of 2 minutes each at maximum output. The homogenate is spun in a centrifuge at 19,000 x g for 15 minutes and both the supernatant and the pellet are analyzed by SDS-PAGE to detect the cellular location of the target protein in the soluble or insoluble fractions, as is described further below.

3.E.1. Soluble fraction If the target protein is produced in a soluble form in the supernatant obtained in NaCl and imidazole are added to the supernatant to final concentrations of 50 mM Tris-HCl (pH 0.5 M NaCI, and 10 mM imidazole (buffer The mixture is filtered through a 0.45 jPm membrane and loaded onto an IMAC column (Pharmacia HiTrap chelating Sepharose; 1 ml) that has been charged with nickel ions according to the manufacturer's recommendations. After loading, the column is washed with 50 column volumes of buffer A and the recombinant target protein is eluted with 5 ml of buffer B (50 mM Tris-HC1 (pH 0.5 M NaCI, 500 mM imidazole).

The elution profile is monitored by measuring the absorbance of the fractions at 280 nm. Fractions corresponding to the protein peak are pooled, dialyzed against PBS containing 0.5 M arginine, filtered through a 0.22 pm membrane, and stored at -45 0

C.

3.E.2. Insoluble fraction If the target protein is expressed in the insoluble fraction (pellets obtained from purification is conducted under denaturing conditions.

NaCI, imidazole, and urea are added to the resuspended pellet to final WO 98/43479 PCT/US98/06421 -69concentrations of 50 mM Tris-HC1 (pH 0.5 M NaC1, 10 mM imidazole, and 6 M urea (buffer After complete solubilization, the mixture is filtered through a 0.45 gm membrane and loaded onto an IMAC column.

The purification procedures on the IMAC column are the same as described in 3.E. except that 6 M urea is included in all buffers used and column volumes of buffer C are used to wash the column after protein loading, instead of 50 column volumes.

The protein fractions eluted from the IMAC column with buffer D (buffer C containing 500 mM imidazole) are pooled. Arginine is added to the solution to final concentration of 0.5 M and the mixture is dialyzed against PBS containing 0.5 M arginine and various concentrations of urea (4 M, 3 M, 2 M, 1 M, and 0.5 M) to progressively decrease the concentration of urea. The final dialysate is filtered through a 0.22 jim membrane and stored at -45 0

C.

Alternatively, when the above purification process is not as efficient as it should be, two other processes may be used as follows. A first alternative involves the use of a mild denaturant, N-octyl glucoside (NOG). Briefly, a pellet obtained in 3.E. is homogenized in 5 mM imidazole, 500 mM sodium chloride, 20 mM Tris-HCl (pH 7.9) by microfluidization at a pressure of 15,000 psi and is clarified by centrifugation at 4,000-5,000 x g. The pellet is recovered, resuspended in 50 mM NaP04 (pH 7.5) containing 1-2% weight /volume NOG, and homogenized. The NOG-soluble impurities are removed by centrifugation. The pellet is extracted once more by repeating the preceding extraction step. The pellet is dissolved in 8 M urea, 50 mM Tris (pH The urea-solubilized protein is diluted with an equal volume of 2 M arginine, mM Tris (pH and is dialyzed against 1 M arginine for 24-48 hours to remove the urea. The final dialysate is filtered through a 0.22 gm membrane and stored at -45 0

C.

WO 98/43479 PCT/US98/06421 A second alternative involves the use of a strong denaturant, such as guanidine hydrochloride. Briefly, a pellet obtained in 3.E. is homogenized in mM imidazole, 500 mM sodium chloride, 20 mM Tris-HCI (pH 7.9) by microfluidization at a pressure of 15,000 psi and clarified by centrifugation at 4,000-5,000 x g. The pellet is recovered, resuspended in 6 M guanidine hydrochloride, and passed through an IMAC column charged with Ni The bound antigen is eluted with 8 M urea (pH Beta-mercaptoethanol is added to the eluted protein to a final concentration of 1 mM, then the eluted protein is passed through a Sephadex G-25 column equilibrated in 0.1 M acetic acid.

Protein eluted from the column is slowly added to 4 volumes of 50 mM phosphate buffer (pH The protein remains in solution.

3.F. Evaluation of the protective activity of the purified protein A protection test is described above that was carried out for testing the protective activity of the purified, native proteins. This test can also be used for testing the protective efficacy of recombinant proteins. Alternatively, the following test can be used.

Groups of 10 OF1 mice (IFFA Credo) are immunized rectally with gg of the purified recombinant protein, admixed with 1 jig of cholera toxin (Berna) in physiological buffer. Mice are immunized on days 0, 7, 14, and 21.

Fourteen days after the last immunization, the mice are challenged with H.

pylori strain ORV2Q01 grown in liquid media (the cells are grown on agar plates, as described in and, after harvest, the cells are resuspended in Brucella broth; the flasks are then incubated overnight at 37 0 Fourteen days after challenge, the mice are sacrificed and their stomachs are removed. The amount ofH. pylori is determined by measuring the urease activity in the stomach and by culture.

WO 98/43479 PCT/US98/06421 -71- 3.G. Production of monospecific polyclonal antibodies 3.G.1. Hyperimmune rabbit antiserum New Zealand rabbits are injected both subcutaneously and intramuscularly with 100 gg of a purified fusion polypeptide, as obtained in 3.E.1. or in the presence of Freund's complete adjuvant and in a total volume of approximately 2 ml. Twenty one and 42 days after the initial injection, booster doses, which are identical to priming doses, except that Freund's incomplete adjuvant is used, are administered in the same way.

Fifteen days after the last injection, animal serum is recovered, decomplemented, and filtered through a 0.45 gm membrane.

3.G.2. Mouse hyperimmune ascites fluid Ten mice are injected subcutaneously with 10-50 ig of a purified fusion polypeptide, as obtained in 3.E. 1. or in the presence of Freund's complete adjuvant and in a volume of approximately 200 gl. Seven and 14 days after the initial injection, booster doses, which are identical to the priming doses, except that Freund's incomplete adjuvant is used, are administered in the same way. Twenty one and 28 days after the initial infection, mice receive gg of the antigen alone intraperitoneally. On day 21, mice are also injected intraperitoneally with sarcoma 180/TG cells CM26684 (Lennette et al., Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections, Ed., Washington DC, American Public Health Association, 1979). Ascites fluid is collected 10-13 days after the last injection.

EXAMPLE 4: Methods for producing transcriptional fusions lacking Histags Methods for amplification and cloning ofDNA encoding the polypeptides of the invention as transcriptional fusions lacking His-tags are WO 98/43479 PCT/US98/06421 -72described as follows. Two PCR primers for each clone are designed based upon the sequences of the polynucleotides that encode them (SEQ ID NOs: 1-21 (odd numbers), 65, and 67). These primers can be used to amplify DNA encoding the polypeptides of the invention from any Helicobacterpylori strain, including, for example, ORV2001 and the H. pylori strain deposited with the American Type Culture Collection (ATCC, Rockville, Maryland) as ATCC number 43579, as well as from other Helicobacter species.

The N-terminal primers are designed to include the ribosome binding site of the target gene, the ATG start site, the signal sequence (if any), and the cleavage site. The N-terminal primers can include a 5 clamp and restriction endonuclease recognition site, such as that for BamHI (GGATCC), which facilitates subsequent cloning. Similarly, the C-terminal primers can include a restriction endonuclease recognition site, such as that for Xhol (CTCGAG), which can be used in subsequent cloning, and a TAA stop codon. Specific primers that can be used are listed above.

Amplification of a genes encoding the polypeptides of the invention can be carried out using Vent DNA polymerase (New England Biolabs) or Taq DNA polymerase (Appligene) under the conditions described above in Example 3. Alternatively, Thermalase DNA polymerase or Pwo DNA polymerase (Boehringer Mannheim) can be used, according to instructions provided by the manufacturers.

A single PCR product for each clone is amplified and can be cloned into BamHI-XhoI cleaved pET24, resulting in construction of transcriptional fusions that permit expression of the proteins without His-tags. The expressed products can be purified as denatured proteins that are refolded by dialysis into 1 M arginine.

WO 98/43479 PCT/US98/06421 -73- Cloning into pET 24 allows transcription of genes from the T7 promoter, which is supplied by the vector, but relies upon binding of the RNA-specific DNA polymerase to the intrinsic ribosome binding site of the genes, and thereby expression of the complete ORF. The amplification, digestion, and cloning protocols are as described above for constructing translational fusions.

EXAMPLE 5: Purification of the polypeptides of the invention by immunoaffinity Purification of specific IgGs An immune serum, as prepared as is described in section is applied to a protein A Sepharose Fast Flow column (Pharmacia) equilibrated in 100 mM Tris-HCl (pH The resin is washed by applying 10 column volumes of 100 mM Tris-HCl and 10 volumes of 10 mM Tris-HCl (pH 8.0) to the column. IgG antibodies are eluted with 0.1 M glycine buffer (pH 3.0) and are collected in 5 ml fractions to each of which is added 0.25 ml 1 M Tris-HCI (pH The optical density of the eluate is measured at 280 nm and the fractions containing the IgG antibodies are pooled, dialyzed against 50 mM Tris-HCl (pH and, if necessary, stored frozen at -70 C.

Preparation of the column An appropriate amount of CNBr-activated Sepharose 4B gel (1 g of dried gel provides for approximately 3.5 ml of hydrated gel; gel capacity is from 5 to 10 mg coupled IgG/ml of gel) manufactured by Pharmacia (17-0430- 01) is suspended in 1 mM HCI buffer and washed using a buchner by adding small quantities of 1 mM HCI buffer. The total volume of buffer is 200 ml per gram of gel.

WO 98/43479 PCT/US98/06421 -74- Purified IgG antibodies are dialyzed for 4 hours at 20+5 C against volumes of 500 mM sodium phosphate buffer (pH The antibodies are then diluted in 500 mM phosphate buffer (pH 7.5) to a final concentration of 3 mg/ml.

IgG antibodies are mixed with the gel overnight at 5±3 C. The gel is packed into a chromatography column and is washed with 2 column volumes of 500 mM phosphate buffer (pH and 1 column volume of 50 mM sodium phosphate buffer, containing 500 mM NaCI (pH The gel is then transferred to a tube, mixed with 100 mM ethanolamine (pH 7.5) for 4 hours at room temperature, and washed twice with 2 column volumes of PBS. The gel is then stored in 1/10,000 PBS/merthiolate. The amount of IgG antibodies coupled to the gel is determined by measuring the optical density (OD) at 280 nm of the IgG solution and the direct eluate, plus washings.

Adsorption and elution of the antigen An antigen solution in 50 mM Tris-HC1 (pH 2 mM EDTA, for example, the supernatant obtained in 3.E. 1. or the solubilized pellet obtained in after centrifugation and filtration through a 0.45 gm membrane, is applied to a column equilibrated with 50 mM Tris-HCI (pH 2 mM EDTA, at a flow rate of about 10 ml/hour. The column is then washed with 20 volumes of 50 mM Tris-HCl (pH 2 mM EDTA. Alternatively, adsorption can be achieved by mixing overnight at 5±3 C.

The adsorbed gel is washed with 2 to 6 volumes of 10 mM sodium phosphate buffer (pH 6.8) and the antigen is eluted with 100 mM glycine-buffer (pH The eluate is recovered in 3 ml fractions, to each of which is added 150 l1 of 1 M sodium phosphate buffer (pH Absorption is measured at WO 98/43479 PCT/US98/06421 280 nm for each fraction; those fractions containing the antigen are pooled and stored at 0

C.

EXAMPLE 6: The GHPO 1360 polypeptide is useful as a serodiagnostic tool for H. pylori infection The reactivity of patient sera against H. pylori proteins was analyzed by immunoblot technique. Briefly, total lysate ofH. pylori strain ORV2001 was subjected to SDS-PAGE electrophoresis (BioRad protean II system) on a 12.5% gel. Proteins were electrotransferred onto a nitrocellulose paper for immunoblot assay. After blocking, the nitrocellulose paper was incubated with patient sera (1:500 diluted in blocking buffer) for one hour at room temperature, washed, and further incubated with peroxidase-conjugated goat anti-human IgG. The positive bands were revealed by incubation with the appropriate substrates. The results showed that the H. pylori-positive ulcer patient sera react specifically with proteins having molecular weights between and 60 kDa and about 30 to 35 kDa. To identify the nature of these proteins, the reactivities of the patient sera were analyzed by immunoblot assay against purified proteins with similar molecular weights: urease (67 kDa and kDa), catalase (54 kDa), heat-shock protein B (60 kDa), and the GHPO 1360 polypeptide (32 kDa) expressed and purified as described in Example 5. All patient sera showed strong reactivity against the GHPO 1360 polypeptide, but the reactivities against other purified proteins were quite variable. These results show that the GHPO 1360 polypeptide is a useful antigen for use in diagnosis of H. pylori infection.

WO 98/43479 PCTIUS98/06421 -76- Other embodiments are within the following claims.

WO 98/43479 PCT/US98/06421 -77- SEQUENCE LISTING GENERAL INFORMATION APPLICANT: MERIEUX ORAVAX SOCIETE EN NOM COLLECTIF PASTEUR MERIEUX SERUMS ET VACCINS ET

AL.

(ii) TITLE OF THE INVENTION: 76 kDa, 30 kDa, and 50 kDa Helicobacter Polypeptides and Corresponding Polynucleotide Molecules (iii) NUMBER OF SEQUENCES: 84 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Clark Elbing LLP STREET: 176 Federal Street CITY: Boston STATE: MA COUNTRY: USA ZIP: 02110 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ for Windows Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US98/---- FILING DATE: 31-MAR-98

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/834,666 FILING DATE: 01-APR-1997 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/831,310 FILING DATE: 01-APR-1997 (viii) ATTORNEY/AGENT INFORMATION: NAME: Clark, Paul T.

REGISTRATION NUMBER: 30,162 REFERENCE/DOCKET NUMBER: 06132/037W01 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 617-428-0200 TELEFAX: 617-428-7045

TELEX:

INFORMATION FOR SEQ ID NO:1: SUBSTITUTE SHEET (RULE 26) WO 98/43479 -78- SEQUENCE CHARACTERISTICS: LENGTH: 2798 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: PCT/US98/06421 NAME/KEY: Coding Sequence LOCATION: 328...2451 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 328...385 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

TGGTCCTGGC

CGCGGAGTAG

TCCATTCTCC

AAAGCATCAA

GATTTTATGC

TAACCATTTT

ATTCCGAGGT

AGCAGTCCGG

GCAACCAATC

AATCAAAAAA

TATATTAACG

AATTTCAAGG

TCGAATCCTT GCACCCCAGC CATTTTTCCT TATTTTTTGG TAGCTCGTTG GGCTCATAAC CCAAAGGTCA GTGGTTCAAA CTTTAAACCA CACCACCACC AAACGAACCA AACGAAACAA ATGACAAAAT TTTTAAGAAA ATGACAAAAA AAAAAAAAAC AAATCTTGTG ATAAGATCTT ATTCTTTTAA AAGACTTATC AGAAAAC ATG AAA AAA ACC CTT TTA CTC TCT CTC Met Lys Lys Thr Leu Leu Leu Ser Leu 120 180 240 300 354

TCT

Ser CTC TCT CTC TCG Leu Ser Leu Ser

TTT

Phe TTG CTC CAC GCT GAA GAC GAC GGC TTT TAC Leu Leu His Ala Glu Asp Asp Gly Phe Tyr 402 ACA AGC GTG Thr Ser Val ACC AAA GGC Thr Lys Gly

GGC

Gly TAT CAA ATC GGT Tyr Gln Ile Gly

GAA

Glu 15 GCC GCT CAA ATG Ala Ala Gin Met GTG AAA AAC Val Lys Asn CTG AAC AAT Leu Asn Asn 450 ATT CAA GAG CTT TCA GAC AAT TAT GAA Ile Gin Glu Leu Ser Asp Asn Tyr Glu 30

AAG

Lys 498 CTT TTG Leu Leu AAT AAT TAC AGC Asn Asn Tyr Ser

ACC

Thr 45 CTA AAC ACC CTT Leu Asn Thr Leu

ATC

Ile AAA TTG TCC GCT Lys Leu Ser Ala

GAT

Asp CCG AGC GCG ATT Pro Ser Ala Ile

AAC

Asn 60 GAC GCA AGG GAT Asp Ala Arg Asp CTA GGC TCA AGC Leu Gly Ser Ser

TCT

Ser 546 594 642 AGG AAT TTG CTT Arg Asn Leu Leu

GAT

Asp GTC AAA ACC AAT Val Lys Thr Asn

TCC

Ser 80 CCC GCG TAT CAA Pro Ala Tyr Gin GCC GTG Ala Val CTT TTA GCA CTC AAT GCT GCA GTG GGG TTG TGG CAA GTT ACA AGC TAC SUBSTITUTE SHEET (RULE 26) WO 98/43479 Leu Leu Ala GCT TTT ACT Ala Phe Thr 105 PCTIUS98/06421 -79r Leu Trp Gin Val Leu Asn Ala Ala Val Thr Ser Tyr 100 GCT TGT GGT CCT Ala Cys Gly Pro

GGC

Gly 110 AGT AAC GAG AAT GCG AAT GGA GGG Ser Asn Glu Asn Ala Asn Gly Gly 115 738 ATC CAA Ile Gin 120 ACT TTT AAT AAT Thr Phe Asn Asn

GTG

Va1 125 CCA GGA CAA GAT ACG ACG ACC ATC ACT Pro Gly GIn As-p Thr Thr Thr Ile Thr 130 786

TGC

Cys 135 AAT TCG TAT TAT Asn Ser Tyr Tyr

GAG

Glu 140 CCA GGA CAT GGT Pro Gly His Gly

GGG

Gly 145 CCT ATA TCC ACT Pro Ile Ser Thr

GCA

Ala 150 AAT TAT GCG AAA Asn Tyr Ala Lys AAT CAA GCC TAT Asn Gin Ala Tyr

CAA

Gin 160 ATC ATC CAA AAG Ile Ile Gin Lys GCT TTG Ala Leu 165 ACA GCC AAT Thr Ala Asn ACT ACA AAA Thr Thr Lys 185

GGA

Gly 170 GCT AAT GGA GAT Ala Asn Gly Asp

GGG

Gly 175 GTC CCC GTT TTA Val Pro Val Leu AGC AAC ACC Ser Asn Thr 180 ACG GGG GGC Thr Gly Gly CTT GAT TTC ACT Leu Asp Phe Thr

ATC

Ile 190 AAT GGA GAC AAA Asn Gly Asp Lys

AGA

Arg 195 AAA CCA Lys Pro 200 AAT ACA CCT GAA Asn Thr Pro Glu TTC CCA TGG AGT Phe Pro Trp Ser

GAT

Asp 210 GGG AAA TAT ATT Gly Lys Tyr Ile

CAC

His 215 ACC CAA TGG ATT Thr Gin Trp Ile

AAC

Asn 220 ACA ATA GTA ACA Thr Ile Val Thr

CCA

Pro 225 ACA GAA ACA AAT Thr Glu Thr Asn 1026 1074 1122 AAC ACA GAA AAT Asn Thr Glu Asn

AAC

Asn 235 GCT CAA GAG CTT Ala Gin Glu Leu

TTA

Leu 240 AAA CAA GCG AGC Lys Gin Ala Ser ATC ATT Ile Ile 245 ATC ACT ACC Ile Thr Thr AGT TAT TGG Ser Tyr Trp 265

CTA

Leu 250 AAT GAG GCA TGC Asn Glu Ala Cys

CCA

Pro 255 AAC TTC CAA AAT Asn Phe Gin Asn GGT GGT AGA Gly Gly Arg 260 GGG ATG TTT Gly Met Phe 1170 1218 CAA GGG ATA AGC Gin Gly Ile Ser

GGC

Gly 270 AAT GGG ACA ATG Asn Gly Thr Met AAG AAT Lys Asn 280 GAA ATC AGC GCG Glu Ile Ser Ala CAA GGC ATG ATC Gin Gly Met Ile

GCT

Ala 290 AAC GCT CAA GAA Asn Ala Gin Glu 1266 1314

GCT

Ala 295 GTC GCG CAA AGC Val Ala Gin Ser

AAA

Lys 300 ATC GTT AGT GAA Ile Val Ser Glu

AAC

Asn 305 GCG CAA AAT CAA Ala Gin Asn Gin AAC TTG GAT ACT GGA AAA CCA TTC AAC CCT TAC ACG GAC GCC AGC TTT Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp Ala Ser Phe 1362 SUBSTITUTE SHEET (RULE 26) WO 98/43479 -80- PCT/US98/06421 315 GCG CAA AGC Ala Gin Ser CAA GCC GAA Gin Ala Glu 345

ATG

Met 330 CTC AAA AAC GCT Leu Lys Asn Ala

CAA

Gin 335 GCG CAA GCA GAG Ala Gin Ala Glu ATT TTA AAC Ile Leu Asn 340 ACA GCC TTT Thr Ala Phe CAA GTA GTA AAA Gin Val Val Lys

AAC

Asn 350 TTT GAA AAA ATC Phe Glu Lys Ile

CCT

Pro 355 1410 1458 1506 GTA TCA Val Ser 360 GAC TCT TTA GGG Asp Ser Leu Gly TGT TAT GAA GTG Cys Tyr Glu Val

CAA

Gin 370 GGG GGT GAG CGT Gly Gly Glu Arg

AGG

Arg 375 GGC ACC AAT CCA Gly Thr Asn Pro

GGT

Gly 380 CAG GTA ACT TCT Gin Val Thr Ser

AAC

Asn 385 ACT TGG GGA GCC Thr Trp Gly Ala

GGT

Gly 390 1554 1602 TGC GCG TAT GTG Cys Ala Tyr Val

AAA

Lys 395 CAA ACC ATA ACG Gin Thr Ile Thr

AAT

Asn 400 TTA GAC AAC AGC Leu Asp Asn Ser ATC GCT Ile Ala 405 CAC TTT GGC His Phe Gly GAC ACT CTA Asp Thr Leu 425

ACT

Thr 410 CAA GAG CAG CAG Gin Glu Gin Gin

ATA

Ile 415 CAG CAA GCC GAA Gin Gin Ala Glu AAC ATC GCT Asn Ile Ala 420 GGC AAC ACC Gly Asn Thr 1650 1698 GTG AAT TTC AAA Val Asn Phe Lys

TCT

Ser 430 AGA TAC AGC GAA Arg Tyr Ser Glu

TTA

Leu 435 TAT AAC Tyr Asn 440 AGC ATC ACC ACC Ser Ile Thr Thr

GCG

Ala 445 CTC TCC AAA GTC Leu Ser Lys Val AAC GCG CAA AGC Asn Ala Gin Ser

TTG

Leu 455 CAA AAC GTG GTG Gin Asn Val Val

AGC

Ser 460 AAA AAG AAT AAC Lys Lys Asn Asn

CCC

Pro 465 TAT AGC CCT CAA Tyr Ser Pro Gin

GGC

Gly 470 1746 1794 1842 ATA GAG ACC AAT Ile Glu Thr Asn

TAC

Tyr 475 TAC CTC AAT CAA Tyr Leu Asn Gin

AAT

Asn 480 TCT TAC AAC CAA ATC CAA Ser Tyr Asn Gin Ile Gin 485 ACC ATC AAC Thr Ile Asn GTC AAT TCT Val Asn Ser 505

CAA

Gin 490 GAA CTA GGG CGT Glu Leu Gly Arg

AAC

Asn 495 CCC TTT AGG AAA Pro Phe Arg Lys GTG GGC ATC Val Gly Ile 500 GGT ATT CAG Gly Ile Gin 1890 1938 CAA ACC AAC AAT Gin Thr Asn Asn

GGT

Gly 510 GCC ATG AAT GGG Ala Met Asn Gly GTG GGC Val Gly 520 TAT AAG CAA TTC TTT GGC CAA AAA AGA Tyr Lys Gin Phe Phe Gly Gin Lys Arg 525 TGG GGC GCT AGG Trp Gly Ala Arg 1986

TAT

Tyr 535 TAC GGC TTT TTT Tyr Gly Phe Phe

GAC

Asp 540 TAC AAC CAT GCG Tyr Asn His Ala

TTC

Phe 545 ATT AAA TCC AGC Ile Lys Ser Ser

TTC

Phe 550 2034 SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 -81- TTC AAC TCG GCT Phe Asn Ser Ala

TCT

Ser 555 GAT GTG TGG ACT Asp Val Trp Thr

TAT

Tyr 560 GGT TTT GGA GCG GAC GCT 2082 Gly Phe Gly Ala Asp Ala 565 CTT TAT AAC Leu Tyr Asn AAC AAG CTT Asn Lys Leu 585

TTC

Phe 570 ATC AAC GAT AAA Ile Asn Asp Lys

GCC

Ala 575 ACC AAT TTC TTA Thr Asn Phe Leu GGC AAA AAC Gly Lys Asn 580 GCG GGC ACT Ala Gly Thr 2130 2178 TCC GTG GGG CTT Ser Val Gly Leu

TTT

Phe 590 GGA GGG ATT GCG Gly Gly Ile Ala

TTA

Leu 595 TCA TGG Ser Trp 600 CTT AAT TCT GAG Leu Asn Ser Glu

TAT

Tyr 605 GTG AAT TTA GCC Val Asn Leu Ala GTG AAT AAC GTC Val Asn Asn Val

TAT

Tyr 615 AAC GCT AAA ATG Asn Ala Lys Met

AAT

Asn 620 GTG GCG AAT TTC Val Ala Asn Phe

CAA

Gin 625 TTC TTA TTC AAT Phe Leu Phe Asn 2226 2274 2322 GGA GTG AGG ATG Gly Val Arg Met

AAT

Asn 635 TTA GCC AGA TCC Leu Ala Arg Ser AAA AAA GGC AGC Lys Lys Gly Ser GAT CAT Asp His 645 GCG GCT CAG Ala Ala Gin ACG AAC TAT Thr Asn Tyr 665

CAT

His 650 GGG ATT GAA CTA Gly Ile Glu Leu

GGG

Gly 655 CTT AAA ATC CCC Leu Lys Ile Pro ACC ATC AAC Thr Ile Asn 660 AGA AGG CTT Arg Arg Leu 2370 2418 TAT TCT TTC ATG Tyr Ser Phe Met

GGG

Gly 670 GCT GAA CTC AAA Ala Glu Leu Lys TAT AGC Tyr Ser 680 GTG TAT TTG AAT Val Tyr Leu Asn

TAT

Tyr 685 GTG TTC GCT TAC Val Phe Ala Tyr TAAGCTTTTT GTGAAACTCC 2471

CTTTTTAAGG

TTTTTTAAGC

ACCCTTTCGT

AAACTCTTTT

CTTTTTTTAA

CGCTTTTTAA

GGTTTTTTTT

TTTTTTTTGA

TTTTAAACTC

TTTTAAACCC

ATTCTTTTTT

TCTTTAGGTT

TGAACTCTCT

ATTCTTTTTT

CCTTTTTTAA

TCTTTTTTTA

TGGGGGTTTG

TTATTTT

TTTTAAATTC

TTGAATTCTT

GGGATTTCTT

AGGGATTTCT

ATCTTTCTTT

TCTTTTTAAA

TGTTTTTAAG

TTTTTAAACT

TTTTAAAGCT

TTGCCAATCC

GAGATTTCTT

CTTTTTTTAA

CTTTTTTTTT

TTTTTGAAGT

CCACTACTTT

2531 2591 2651 2711 2771 2798 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 708 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...19 SUBSTITUTE SHEET (RULE 26) WO 98/43479 -82- OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: PCTIUS98/0642 I Met Lys Lys Thr Leu Leu Leu Ser Leu Ser Leu His Gly Giu Ser Asp Leu Asn Ala Arg Thr Asn Val Gly Gly Ser 110 Pro Gly Giy His Ala Tyr Asp Gly 175 Ile Asn 190 Phe Pro Ile Val Giu Leu Cys Pro 255 Gly Asn 270 Gin Gly Val Ser Phe Asn Ala Gin 335 Asn Phe 350 Cys Tyr Val Thr Ala Ala Asn Thr Asp Ser Leu Asn Gin Gly Gin 160 Val1 Giy Trp Thr Leu 240 Asn Gly Met Giu Pro 320 Al a Giu Giu Ser Giu Al a Tyr Leu Asn Pro Trp Giu Asp Giy 145 Ile Pro Asp Ser Pro 225 Lys Phe Thr Ile Asn 305 Tyr Gin Lys Val1 Asn Asp Gin Glu Ile Leu Ala Gin Asn Thr 130 Pro Ile Val Lys Asp 210 Thr Gin Gin Met Ala 290 Al a Thr Ala Ile Gin 370 Thr Asp Met Lys 35 Lys Gly Tyr Val Ala 115 Thr Ile Gin Leu Arg 195 Gly Giu Ala Asn Cys 275 Asn Gin Asp Giu Pro 355 Gly Trp Gly Phe Val Lys 20 Leu Asn Leu Ser Ser Ser Gin Ala 85 Thr Ser 100 Asn Giy Thr Ile Ser Thr Lys Ala 165 Ser Asn 180 Thr Gly Lys Tyr Thr Asn Ser Ile 245 Gly Gly 260 Gly Met Ala Gin Asn Gin Ala Ser 325 Ile Leu 340 Thr Ala Gly Glu Gly Ala Tyr Asn Asn Al a Ser 70 Val Tyr Gly Thr Al a 150 Leu Thr Gly Ile Ile 230 Ile Arg Phe Giu Asn 310 Phe As n Phe Arg Giy -10 Thr Thr Leu Asp 55 Arg Leu Ala Ile Cys 135 Asn Thr Thr Lys His 215 Asn Ile Ser Lys Ala 295 Asn Al a Gin Val1 Arg 375

CYS

Leu Ser Lys Leu 40 Pro Asn Leu Phe Gin 120 Asn Tyr Ala Thr Pro 200 Thr Thr Thr Tyr Asn 280 Val1 Leu Gln Ala Ser 360 Gly Ala Ser Vali Gly Asn Ser Leu Al a Thr 105 Thr Ser Ala Asn Lys 185 Asn Gin Giu Thr Trp 265 Giu Ala Asp Ser Giu 345 Asp Thr Tyr Leu Gly Ile Asn Ala Leu Leu Ala Phe Tyr Lys Gly 170 Leu Thr Trp Asn Leu 250 Gin Ile Gin Thr Met 330 Gin Ser Asn Val Ser Tyr Gin Tyr Ile Asp Asn Cys Asn Tyr Ile 155 Ala Asp Pro Ile As n 235 Asn Gly Ser Ser Gly 315 Leu Val1 Leu Pro Lys Phe Gin Giu Ser Asn Vali Al a Gly As n Giu 140 As n As n Phe Giu Asn 220 Al a Giu Ile Al a Lys 300 Lys Lys Val1 Gly Gly 380 Gin Leu Ile Leu Thr Asp Lys Al a Pro Val 125 Pro Gin Gly Thr Lys 205 Thr Gin Ala Ser Ile 285 Ile Pro Asn Lys Val 365 Gin Thr SUBSTITUTE SHEET (RULE 26) PCTIUS98/06421 WO 98/43479 -83- 390 Ile Gin Ser 430 Leu Lys Asn Arg Gly 510 Giy As n Trp Lys Phe 590 Vai Aia Arg Leu Giy 670 Val Thr Ile 41i5 Arg Ser As n Gin Asn 495 Aila Gin His Thr Al a 575 Giy Asn Asn Ser Giy 655 Aia Phe Asn Leu 400 Gin Gin Tyr Ser Lys Vai Asn Pro 465 Asn Ser 480 Pro Phe Met Asn Lys Arg Aia Phe 545 Tyr Giy 560 Thr Asn Giy Ile Leu Ala Phe Gin 625 Lys L~ys 640 Leu Lys Giu Leu Aia Tyr Asp Al a Giu Pro 450 Tyr Tyr Arg Gly Lys 530 Ilie Phe Phe Ala Thr 610 Phe Lys Ile Lys Asn Giu Leu 435 Asn Ser As n Lys Ile 515 Trp Lys Gly Leu Leu 595 Val Leu Gly Pro Tyr 675 Ser Asn 420 Gly Al a Pro Gin Val1 500 Gly Gly Ser Al a Gly 580 Al a Asn Phe Ser Thr 660 Arg Ile 405 Ile Asn Gin Gin Ile 485 Gly Ile Ala Ser Asp 565 Lys Gly Asn Asn Asp 645 Ile Arg Aia Aia Thr Ser Gly 470 Gin Ile Gin Arg Phe 550 Al a Asn Thr Val Met 630 His Asn Leu Hi s A~sp Tyr Leu 455 Ile Thr Val Val1 Tyr 535 Phe Leu Asn Ser Tyr 615 Gly Ala Thr Tyr Phe Thr Asn 440 Gin Giu Ile As n Gly 520 Tyr Asn Tyr Lys Trp 600 Asn Val Ala Asn Ser 680 Gly Leu 425 Ser Asn Thr Asn Ser 505 Tyr Gly Ser Asn Leu 585 Leu Al a Arg Gin Tyr 665 Vali Thr 410 'Val1 Ile Val1 Asn Gin 490 Gin Lys Phe Ala Phe 570 Ser Asn Lys Met His 650 Tyr Tyr Gin Asn Thr Val Tyr 475 Giu Thr Gin Phe Ser 555 Ile Val Ser Met Asn 635 Gly Ser Leu Giu Phe Thr Ser 460 Tyr Leu As n Phe Asp 540 Asp Asn Gly Glu Asn 620 Leu Ile Phe Asn Gin Lys Ala 445 Lys Leu Gly As n Phe 525 Tyr Val Asp Leu Tyr 605 Val Al a Giu Met Tyr 685 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 2699 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 199... .2397 OTHER INFORMATION: SUBSTITUTE SHEET (RULE 26) WO 98/43479 -84- NAME/KEY: Signal Sequence LOCATION: 199.. .259 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: PCT/US98/06421 TAAAATCCAA TTAAAAGCGT TCAAAGGTAA TCAAAATGAC AAAAAAAAAC GCTTTATGCT TGCGTTCTAA TGCAAATGCA TTCCAATGTA

CGCAAAAAAA

ATAATACCCC

TGAAATCCCT

CAAAAAATGA CGCAATTTTT AAATACATTC TAATAGCAAA AATACTAAAT CCAATTTAAT 120 180 231 CCAAAAAGGA GAAAAAAC ATG AAA Met Lys AAA CAC ATC Lys His Ile TCA TTA GCT TTA Ser Leu Ala Leu

GGC

Gly TCG CTT TTA GTT Ser Leu Leu Val

TCC

Ser ACT TTG AGC GCT GAA GAC GAC GGC TTT TAC ACA Thr Leu Ser Ala Giu Asp Asp Gly Phe Tyr Thr 279 AGC GTA GGC TAT CAG ATC GGT Ser Val Gly Tyr Gin Ile Gly GCC GCT CAA ATG Ala Ala Gin Met

GTA

Va1 ACA AAC ACC Thr Asn Thr AAA GGC Lys Gly ATC CAA CAG CTT Ile Gin Gin Leu

TCA

Ser 30 GAC AAT TAT GAA Asp Asn Tyr Glu

AAT

Asn TTG AAC AAC CTT Leu Asn Asn Leu

TTA

Leu ACG AGA TAC AGC Thr Arg Tyr Ser

ACC

Thr 45 CTA AAC ACC CTT ATC AAA TTG TCC GCT Leu Asn Thr Leu Ile Lys Leu Ser Ala 50

GAT

Asp CCG AGC GCA ATT Pro Ser Ala Ile

AAT

Asn GCG GTG CGG GAA Ala Vai Arg Glu

AAT

Asn 65 CTG GGC GCG AGC Leu Gly Ala Ser GCG AAG Ala Lys AAT TTG ATC Asn Leu Ile TTA GCG ATC Leu Ala Ile

GGC

Gly GAT AAA GCC AAC Asp Lys Ala Asn

TCC

Ser 80 CCC GCC TAT CAA Pro Ala Tyr Gin GCC GTG CTT Ala Val Leu GGC TAT GTG Gly Tyr Val 519 567 AAC GCG GCG GTA Asn Ala Ala Val TTT TGG AAT GTC Phe Trp Asn Val

GTG

Val 100 ACG CAA Thr Gin 105 TGT GGG GGT AAC Cys Gly Gly Asn

GCC

Ala 110 AAT GGT CAA GAA Asn Gly Gin Glu ACC TCT TCA ACC Thr Ser Ser Thr

ACC

Thr 120 ATC TTC AAC Ile Phe Asn AAC GAG Asn Glu 125 CAT AAG His Lys 140 CCA GGG TAT CGA Pro Gly Tyr Arg TCC ACT Ser Thr 130 TCC ATC ACT Ser Ile Thr

TGT

Cys 135 TCT TTG AAC GGG Ser Leu Asn Gly CCT GGA TAC TAT GGC CCT ATG AGC Pro Gly Tyr Tyr Gly Pro Met Ser 145 ATT GAG Ile Glu 150 711 759 AAT TTT AAA AAG CTT AAC GAA GCC TAT CAG ATC CTC CAA ACG GCT TTA Asn Phe Lys Lys Leu Asn Glu Ala Tyr Gin Ile Leu Gin Thr Ala Leu SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 -85- AAA AAC GGC Lys Asn Gly 170 TTA CCC GCG CTC Leu Pro Ala Leu

AAA

Lys 175 GAA AAC AAC GGG Glu Asn Asn Gly

AAG

Lys 180 GTC AGT GTA Val Ser Val ACC TAT Thr Tyr 185 ACC TAC ACA TGC Thr Tyr Thr Cys

TCA

Ser 190 GGG CAA GGG AAT Gly Gin Gly Asn AAC TGC TCG CCA Asn Cys Ser Pro

AGT

Ser 200 GTC AAC GGA ACC Val Asn Gly Thr

AAA

Lys 205 ACC ACA ACC CAA Thr Thr Thr Gin

ACC

Thr 210 ATA GAC GGC AAA Ile Asp Gly Lys

AGC

Ser 215 855 903 951 GTA ACC ACC ACG Val Thr Thr Thr AGT TCA AAA GTG Ser Ser Lys Val

GTT

Val 225 GGT AGC ATC GCT Gly Ser Ile Ala AGT GGC Ser Gly 230 AAC ACA TCA Asn Thr Ser GCT CAA GCG Ala Gin Ala 250

CAT

His 235 GTC ATC ACC AAC Val Ile Thr Asn

AAA

Lys 240 TTA GAC GGT GTG Leu Asp Gly Val CCT GAT AGC Pro Asp Ser 245 ACC ATC AAC Thr Ile Asn 999 1047 CTC TTA GCG CAA Leu Leu Ala Gin

GCG

Ala 255 AGC ACG CTC ATC Ser Thr Leu Ile

AAC

Asn 260 GAA GCA Glu Ala 265 TGC CCG TAT TTC Cys Pro Tyr Phe

CAT

His 270 GCT ACT AAT AGT Ala Thr Asn Ser GAG GCT AAC GCC Glu Ala Asn Ala

CCA

Pro 280 AAA TTC TCT ACT Lys Phe Ser Thr ACT GGG AAA ATA Thr Gly Lys Ile GGC GCT TTT TCA Gly Ala Phe Ser

GAA

Glu 295 1095 1143 1191 GAA ATC AGC GCG Glu Ile Ser Ala

ATC

Ile 300 CAA AAG ATG ATC Gin Lys Met Ile

ACG

Thr 305 GAC GCG CAA GAG Asp Ala Gin Glu CTA GTT Leu Val 310 AAT CAA ACG Asn Gin Thr AAT AAT AAT Asn Asn Asn 330

AGC

Ser 315 GTC ATT AAC AGC Val Ile Asn Ser

AAC

Asn 320 GAA CAA TCA ACT Glu Gin Ser Thr CCG GTA GGC Pro Val Gly 325 1239 GGC AAG CCT TTC Gly Lys Pro Phe CCT TTC ACG GAC GCA AGT TTT GCG Pro Phe Thr Asp Ala Ser Phe Ala 340 1287 CAA GGC Gin Gly 345 ATG CTC GCT AAC Met Leu Ala Asn

GCT

Ala 350 AGC GCG CAA GCT Ser Ala Gin Ala ATG CTC AAT TTA Met Leu Asn Leu GCC CAT CAG GTG GGG CAA Ala His Gin Val Gly Gin 360 365 GCC ATT AAC CCA Ala Ile Asn Pro AAT CTT AGC GAG Asn Leu Ser Glu

AAT

Asn 375 1335 1383 1431 TTT AAA AAT TTT Phe Lys Asn Phe

GTT

Val 380 ACA GGC TTT TTA Thr Gly Phe Leu

GCC

Ala 385 ACA TGC AAT AAC Thr Cys Asn Asn AAA TCA Lys Ser 390 SUBSTITUTE SHEET (RULE 26) WO 98/43479 ACA GCT GGC Thr Ala Gly ACT CAA ACT Thr Gin Thr 410 -86- PCTIUS98/06421 GGT GGC ACA CAA Gly Gly Thr Gin

GGT

Gly 400 TCA GCT CCA GGC Ser Ala Pro Gly ACA GTG ACC Thr Val Thr 405 ACC CTA ACG Thr Leu Thr 1479 1527 TTC GCT TCT GGT Phe Ala Ser Gly

TGC

Cys 415 GCG TAT GTG GAG Ala Tyr Val Glu

CAA

Gin 420 AAC TTA Asn Leu 425 GGC AAC AGC ATC Gly Asn Ser Ile

GCT

Ala 430 CAC TTT GGC ACT His Phe Gly Thr

CAA

Gin 435 GAG CAG CAG ATA Glu Gin Gin Ile

CAG

Gin 440 CAA GCC GAA AAC Gln Ala Glu Asn

ATC

Ile 445 GCT GAC ACT CTA Ala Asp Thr Leu

GTG

Val 450 AAT TTC AAA TCT Asn Phe Lys Ser

AGA

Arg 455 1575 1623 1671 TAC AGC Tyr Ser GAA TTA GGC Glu Leu Gly 460 AAC ACC TAT AAC Asn Thr Tyr Asn

AGC

Ser 465 ATC ACC ACC GCG Ile Thr Thr Ala CTC TCC Leu Ser 470 AAA GTC CCT Lys Val Pro AAC CCC TAT Asn Pro Tyr 490

AAC

Asn 475 GCG CAA AGC TTG Ala Gin Ser Leu

CAA

Gin 480 AAC GTG GTG AGC Asn Val Val Ser AAA AAG AAT Lys Lys Asn 485 CTC AAT CAA Leu Asn Gin 1719 1767 AGC CCT CAA GGC Ser Pro Gin Gly

ATA

Ile 495 GAG ACC AAT TAC Glu Thr Asn Tyr AAT TCT Asn Ser 505 TAC AAC CAA ATC Tyr Asn Gin Ile

CAA

Gin 510 ACC ATC AAC CAA Thr Ile Asn Gin

GAA

Glu 515 CTA GGG CGT AAC Leu Gly Arg Asn

CCC

Pro 520 TTT AGG AAA GTG Phe Arg Lys Val ATC GTC AAT TCT Ile Val Asn Ser

CAA

Gin 530 ACC AAC AAT GGT Thr Asn Asn Gly

GCC

Ala 535 1815 1863 1911 ATG AAT GGG ATC Met Asn Gly Ile

GGT

Gly 540 ATT CAG GTG GGC Ile Gin Val Gly

TAT

Tyr 545 AAG CAA TTC TTT Lys Gin Phe Phe GGC CAA Gly Gin 550 AAA AGA AAA Lys Arg Lys GCG TTC ATC Ala Phe Ile 570

TGG

Trp 555 GGC GCT AGG TAT Gly Ala Arg Tyr

TAC

Tyr 560 GGC TTT TTT GAT Gly Phe Phe Asp TAC AAC CAT Tyr Asn His 565 GTG TGG ACT Val Trp Thr 1959 2007 AAA TCC AGC TTT Lys Ser Ser Phe

TTC

Phe 575 AAC TCG GCT TCT Asn Ser Ala Ser TAT GGT Tyr Gly 585 TTT GGA GCG GAC Phe Gly Ala Asp

GCG

Ala 590 CTT TAT AAC TTC Leu Tyr Asn Phe

ATC

Ile 595 AAC GAT AAA GCC Asn Asp Lys Ala 2055 2103

ACC

Thr 600 AAT TTC TTA GGC Asn Phe Leu Gly

AAA

Lys 605 AAC AAC AAG CTT Asn Asn Lys Leu

TCT

Ser 610 TTG GGG CTT TTT Leu Gly Leu Phe GGG ATT GCG TTA GCG GGC ACT TCA TGG CTC AAT TCT GAG TAC GTG AAT 2151 SUBSTITUTE SHEET (RULE 26) WO 98/43479 -87- PCT/US98/06421 Gly Ile Ala Leu Ala 620 Gly Thr Ser Trp Leu 625 Asn Ser Glu Tyr Val Asn 630 TTA GCC ACC Leu Ala Thr TTC CAA TTC Phe Gin Phe 650

GTG

Val 635 AAT AAC GTC TAT Asn Asn Val Tyr

AAC

Asn 640 GCT AAA ATG AAT Ala Lys Met Asn GTG GCG AAT Val Ala Asn 645 GCC AGA TCC Ala Arg Ser 2199 2247 TTA TTC AAT ATG Leu Phe Asn Met GTG AGG ATG AAT Val Arg Met Asn

TTA

Leu 660 AAG AAA Lys Lys 665 AAA GGC AGC GAT Lys Gly Ser Asp

CAT

His 670 GCA GCT CAG CAT Ala Ala Gin His ATT GAG TTA GGG Ile Glu Leu Gly

CTT

Leu 680 AAA ATC CCC ACC Lys Ile Pro Thr AAC ACG AAC TAT Asn Thr Asn Tyr

TAT

Tyr 690 TCC TTT ATG GGG Ser Phe Met Gly

GCT

Ala 695 2295 2343 2391 GAA CTC AAA TAC Glu Leu Lys Tyr

AGA

Arg 700 AGG CTC TAT AGC Arg Leu Tyr Ser

GTG

Val 705 TAT TTG AAC TAT Tyr Leu Asn Tyr GTG TTC Val Phe 710 GCT TAC TAATGTTTGG CTCTTTGTGA AACTCCCTTT TTAAGGGGTT TTTTTTTGAA CT Ala Tyr 2449 CTCTTTTTAA ATTCTCTTTT TTTTTTTGAA TTCTTTGTTT TTTAAGGGAT TTCTTTTTTT TTTAAGGGGT TTCTTTTTAA

TTTGATCTTT

TAAAGAGATT TCTTTTTTTT TTAAGCTTTT TTTAAACCCT GAACTCCCTT TTTTGAACCC AGCTTTTTTG AAGTCTTTTT

AAGCTTTTTT

TTCGTTTTTA

TTTTTTTTAA

TTAAATTCTT

TTGAATTCTT

AACTCCCTTT

ACCCTCTTTT

TTTTTGGGGG

2509 2569 2629 2689 2699 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 733 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...20 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Lys Lys His Ile Leu Ser Leu Ala Leu Gly Ser Leu Leu Val Ser -15 -10 Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gin 1 SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCTIUS98/06421l -88- Ile Gly Glu Ala Ala Gin Met Val Thr Asn Thr Lys Gly Ile Gin Gin 20 Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser 35 Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn 50 55 Ala Val Arg Glu Asn Leu Gly Ala Ser Ala Lys Asn Leu Ile Gly Asp 70 Lys Ala Asn Ser Pro Ala Tyr Gin Ala Val Leu Leu Ala Ile Asn Ala 85 Ala Val Gly Phe Trp Asn Val Val Gly Tyr Val Thr Gin Cys Gly Gly 100 105 Asn Ala Asn Gly Gin Glu Ser Thr Ser Ser Thr Thr Ile Phe Asn Asn 110 115 120 Glu Pro Gly Tyr Arg Ser Thr Ser Ile Thr Cys Ser Leu Asn Gly His 125 130 135 140 Lys Pro Gly Tyr Tyr Gly Pro Met Ser Ile Glu Asn Phe Lys Lys Leu 145 150 155 Asn Glu Ala Tyr Gin Ile Leu Gin Thr Ala Leu Lys Asn Gly Leu Pro 160 165 170 Ala Leu Lys Glu Asn Asn Gly Lys Val Ser Val Thr Tyr Thr Tyr Thr 175 180 185 Cys Ser Gly Gin Gly Asn Asn Asn Cys Ser Pro Ser Val Asn Gly Thr 190 195 200 Lys Thr Thr Thr Gin Thr Ile Asp Gly Lys Ser Val Thr Thr Thr Ile 205 210 215 220 Ser Ser Lys Val Val Gly Ser Ile 'Ala Ser Gly Asn Thr Ser His Val 225 230 235 Ile Thr Asn Lys Leu Asp Gly Val Pro Asp Ser Ala Gin Ala Leu Leu 240 245 250 Ala Gin Ala Ser Thr Leu Ile Asn Thr Ile Asn Glu Ala Cys Pro Tyr 255 260 265 Phe His Ala Thr Asn Ser Ser Glu Ala Asn Ala Pro Lys Phe Ser Thr 270 275 280 Thr Thr Gly Lys Ile Cys Gly Ala Phe Ser Glu Glu Ile Ser Ala Ile 285 290 295 300 Gin Lys Met Ile Thr Asp Ala Gin Glu Leu Val Asn Gin Thr Ser Val 305 310 315 Ile Asn Ser Asn Glu Gin Ser Thr Pro Val Gly Asn Asn Asn Gly Lys 320 325 330 Pro Phe Asn Pro Phe Thr Asp Ala Ser Phe Ala Gin Gly Met Leu Ala 335 340 345 Asn Ala Ser Ala Gin Ala Lys Met Leu Asn Leu Ala His Gin Val Gly 350 355 360 Gin Ala Ile Asn Pro Glu Asn Leu Ser Glu Asn Phe Lys Asn Phe Val 365 370 375 380 Thr Gly Phe Leu Ala Thr Cys Asn Asn Lys Ser Thr Ala Gly Thr Gly 385 390 395 Gly Thr Gin Gly Ser Ala Pro Gly Thr Val Thr Thr Gin Thr Phe Ala 400 405 410 Ser Gly Cys Ala Tyr Val Glu Gin Thr Leu Thr Asn Leu Gly Asn Ser 415 420 425 Ile Ala His Phe Gly Thr Gin Glu Gin Gin Ile Gin Gin Ala Glu Asn 430 435 440 Ile Ala Asp Thr Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly 445 450 455 460 Asn Thr Tyr Asn Ser Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala JVI VVR SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/0642 1 Gin Gin Ile Gly 525 Ile Ala Ser Asp Lys 605 Gly Asn Asn Asp Ile 685 Arg Ser Gly Gin 510 Ile Gin Arg Phe Ala 590 Asn Thr Val Met His 670 Asn Leu Leu Ile 495 Thr Val Val Tyr Phe 575 Leu Asn Ser Tyr Gly 655 Ala Thr Tyr Gin 480 Glu Ile Asn Gly Tyr 560 Asn Tyr Lys Trp Asn 640 Val Ala Asn Ser 465 Asn Thr Asn Ser Tyr 545 Gly Ser Asn Leu Leu 625 Ala Arg Gin Tyr Val Val Asn Gin Gin 530 Lys Phe Ala Phe Ser 610 Asn Lys Met His Tyr 690 Tyr Val Tyr Glu 515 Thr Gin Phe Ser Ile 595 Leu Ser Met Asn Gly 675 Ser Leu Ser Tyr 500 Leu Asn Phe Asp Asp 580 Asn Gly Glu Asn Leu 660 Ile Phe Asn Lys 485 Leu Gly Asn Phe Tyr 565 Val Asp Leu Tyr Val 645 Ala Glu Met Tyr 470 Lys A Asn G Arg A Gly A 5 Gly G 550 Asn H Trp T Lys A Phe G 6 Val A 630 Ala A Arg S Leu G Gly A 6 Val P 710 sn iln .sn .la 35 In is hr la ly 15 sn sn er ly la 95 he Asn Pro Asn Ser 505 Pro Phe 520 Met Asn Lys Arg Ala Phe Tyr Gly 585 Thr Asn 600 Gly Ile Leu Ala Phe Gin Lys Lys 665 Leu Lys 680 Glu Leu Ala Tyr Tyr 490 Tyr Arg Gly Lys Ile 570 Phe Phe Ala Thr Phe 650 Lys Ile Lys 475 Ser Pro Asn Gin Lys Val Ile Gly 540 Trp Gly 555 Lys Ser Gly Ala Leu Gly Leu Ala 620 Val Asn 635 Leu Phe Gly Ser Pro Thr Tyr Arg 700 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2915 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 365...2597 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 365...425 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCTIUS98/06421 TTTTAGGCGA CAAAATCGCT TATGTTGGGG AGAAATAAAC CGCTCATAAG GGGCAAACGC GCAAAATCPA GCTCTTTAGT ATTTAATCTT ACACCACACA AAAAGCATCA AAATCAAAAA AAAAACGCTT TATGCTATAA TACCCCAAAT AATGCATTCC AATGTATGAA ATCCCTAATA AAAC ATG AAA AAA CAC ATC CTT TCA Met Lys Lys His Ile Leu Ser ATAAAGGCAA CCCGCACAAT TTCGCTCACA CCCAAAAAAG CGATTTTTAA AGAGGTTACG AAAAAATGCT AAAAGCCTTT TTATGGGCTA AATGACAAAA TTTTTAAGAA AATGACAAAA ACATTCTAAT AGCAPATGCG TTCTAATGCA CTAAATCCAA TTTAATCCAA AAAGGAGAAA TTA GCT TTA GGC TCG CTT TTA GTT Leu Ala Leu Gly Ser Leu Leu Val 120 180 240 300 360 409 457 TCC Ser ACT TTG AGC GCT Thr Leu Ser Ala

GAA

Glu 1 GAC GAC GGC TTT TAC ACA AGC GTA Asp Asp Gly Phe Tyr Thr Ser Val 5 GGC TAT Gly Tyr CAG ATC GGT Gin Ile Gly CAG CTT TCA Gin Leu Ser

GAA

Glu GCC GCT CAA ATG Ala Ala Gin Met

GTA

Val 20 ACA AAC ACC AAA GGC ATC CAA Thr Asn Thr Lys Gly Ile Gin GAC PAT TAT GAA Asp Asn Tyr Glu

PAT

Asn 35 TTG PAC AAC CTT Leu Asn Asn Leu

TTA

Leu ACG AGA TAC Thr Arg Tyr AGC ACC Ser Thr CTA AAC ACC CTT Leu Asn Thr Leu

ATC

Ile 50 AAA TTG TCC GCT Lys Leu Ser Ala

GAT

Asp CCG AGC GCA ATT Pro Ser Ala Ile AAT GCG GTG CGG GAA Asn Ala Val Arg Glu GAT AAA GCC AAC TCC Asp Lys Ala Asn Ser

PAT

Asn 65 CTG GGC GCG AGC Leu Gly Ala Ser

ACG

Thr 70 AAG PAT TTG ATC Lys Asn Leu Ile

GGC

Gly CCG GCG TAT CAA Pro Ala Tyr Gin

GCC

Ala GTG TTT TTA GCG Val Phe Leu Ala ATC AAC Ile Asn GCG GCG GTA Ala Aia Val AAC GGG AAC Asn Gly Asn 110

GGG

Gly TTG TGG PAT ACC Leu Trp Asn Thr GGC TAT GCG GTC Gly Tyr Ala Val ATG TGC GGG Met Cys Gly 105 TTT PAT GAC Phe Asn Asp GGC ACA GAG AGT GGG CCT GGC AGC GTG Gly Thr Giu Ser Gly Pro Gly Ser Val 115 CAA CCA Gin Pro 125 GGA CAG GAT TCC ACG CPA ATT ACT TGC Gly Gin Asp Ser Thr Gin Ile Thr Cys 130 CGC TTT GAA TCA Arg Phe Glu Ser

ACT

Thr 140 GGG CCT GGT AAA Gly Pro Gly Lys

AGC

Ser 145 ATG TCT ATT GAT Met Ser Ile Asp

GAA

Glu 150 TTC PAA AAA CTC Phe Lys Lys Leu

PAT

Asn 155 889 GAA GCC TAT CPA ATC ATC CAG CPA GCT Glu Ala Tyr Gin Ile Ile Gin Gin Ala 160

TTA

Leu 165 AAA PAT CPA AGT Lys Asn Gin Ser GGG TTT Gly Phe 170 CCT GAA TTA Pro Glu Leu

GGC

Gly 175 GGG PAC GGC ACA APA GTG ACT GTT PAT Cly Asn Giy Thr Lys Val Ser Val Asn 180 TAC PAT TAC Tyr Asn Tyr 185 985 SUBSTITUTE SHEET (RULE 26) WO 98/43479 GAA TGC AGA Giu Cys Arg 190 PCTIUS98/06421 CAA ACT GCT GAT Gin Thr Ala Asp

ATC

Ile 195 AAC GGC GGT GTG Asn Giy Gly Val

TAT

Tyr 200 CAG TTC TGC Gin Phe Cys 1033 AAG GCT Lys Aia 205 AAA AAT GGT AGT Lys Asn Gly Ser

AGT

Ser 210 AGC AGT AGT AAT Ser Ser Ser Asn

GGC

Gly 215 GGT AAT GGC AGT Giy Asn Gly Ser

AGC

Ser 220 ACG CAA~ ACA ACC Thr Gin Thr Thr

GCG

Ala 225 ACA ACC ACG CAA Thr Tflr Thr Gin GGC GTA ACG ATC Gly Vai Thr Ile 1081 1129 1177 ACT ACC TAT AAT Thr Thr Tyr Asn

AAT

As n 240 AAC AAA GCC ACC Asn Lys Ala Thr

GTC

Val1 245 AAA TTT GAC ATC Lys Phe Asp Ile ACC AAT Thr Asn 250 AAC GCT GAA Asn Ala Giu AAT ACG CAA Asn Thr Gin 270

CAG

Gin 255 CTG TTA AAT CAA Leu Leu Asn Gin

GCG

Ala 260 GCA AAC ATC ATG Ala Asn Ile Met CAA GTC CTT Gin Val Leu 265 AAC ACT CCA Asn Thr Pro 1225 1273 TGC CCT TTA GTG Cys Pro Leu Val

CGT

Arg 275 TCC ACG AAT AAC Ser Thr Asn Asn

GAA

Giu 280 GGG GGT Gly Gly 285 GGT CAA CCA TGG Gly Gin Pro Trp TTA AGC ACA TCC Leu Ser Thr Ser

GGG

Gly 295 AAT GCG TGC AGC Asn Ala Cys Ser 1321

ATC

Ile 300 TTC CAA CAA GAA Phe Gin Gin Glu

TTT

Phe 305 AGC CAG GTT ACT AGC ATG ATC AAA AAC Ser Gin Val Thr Ser Met Ile Lys Asn 310

GCC

Al a 315 1369 CAA GAA ATA ATC Gin Giu Ile Ile

GCG

Ala 320 CAA AGC AAA ATC Gin Ser Lys Ile

GTT

Val 325 AGT GAA AAC GCG Ser Glu Asn Ala CAA AAT Gin Asn 330 1417 CAA AAC AAC Gin Asn Asn AGO TTT GCG Ser Phe Ala 350 GAT ACT GGA AAA Asp Thr Gly Lys

CCA

Pro 340 TTC AAC CCT TAC Phe Asn Pro Tyr ACG GAC GCC Thr Asp Ala 345 OCA GAG ATG Ala Giu Met 1465 1513 CAA AGC ATG CTC Gin Ser Met Leu

AAA

Lys 355 AAC GCT CAA GCG Asn Ala Gin Ala TTC AAT Phe Asn 365 TTG AGC GAA CAA Leu Ser Gl Gin

GTG

Val 370 AAA AAG AAC TTG GAA GTC ATG AAA AAC Lys Lys Asn Leu Glu Val Met Lys Asn 1561 1609 AAC Asn 380 AAT AAT GTT AAC Asn Asn Val Asn

GAG

Giu 385 AAA TTA GCA GGA TTT GGG AAA GAA GAA Lys Leu Ala Gly Phe Gly Lys Glu Giu 390

GTA

V-

395 ATG ACC AAT TTT Met Thr Asn Phe

GTT

Val1 400 AGC GCC TTT TTG Ser Ala Phe Leu

GCA

Ala 405 AGC TGC AAA GAT GGT GGC Ser Cys Lys Asp Giy Gly 410 1657 ACA TTG CCT AAT OCA GGG GTT ACT TCT AAC ACT TGG GGG GCG GGT TGC 1705 SUBSTITUTE SHEET (RULE 26) WO 98/43479 Thr Leu Pro GCG TAT GTG Ala Tyr Val 430 PCT/US98/06421 Asn 415 Ala Gly Val Thr Ser 420 Asn Thr Trp Gly Ala Gly Cys 425 ATC GCT CAC Ile Ala His GGA GAG ACG ATA Gly Glu Thr Ile

AGC

Ser 435 GCC CTA ACC AAC Ala Leu Thr Asn

AGC

Ser 440 1753 TTT GGC Phe Gly 445 ACT CAA GAG CAG Thr Gin Glu Gin

CAG

Gin 450 ATA CAG CAA GCC Ile Gin Gin Ala

GAA

Glu 455 AAC ATC GCT GAC Asn Ile Ala Asp

ACT

Thr 460 CTA GTG AAT TTC Leu Val Asn Phe

AAA

Lys 465 TCT AGA TAC AGC Ser Arg Tyr Ser

GAA

Glu 470 TTA GGC AAC ACC Leu Gly Asn Thr

TAT

Tyr 475 1801 1849 1897 AAC AGC ATC ACC Asn Ser Ile Thr

ACC

Thr 480 GCG CTC TCC AAA Ala Leu Ser Lys

GTC

Val 485 CCT AAC GCG CAA Pro Asn Ala Gin AGC TTG Ser Leu 490 CAA AAC GTG Gin Asn Val GAG ACC AAT Glu Thr Asn 510

GTG

Val 495 AGC AAA AAG AAT Ser Lys Lys Asn

AAC

Asn 500 CCC TAT AGC CCT Pro Tyr Ser Pro CAA GGC ATA Gin Gly Ile 505 ATC CAA ACC Ile Gin Thr 1945 1993 TAC TAC CTC AAT Tyr Tyr Leu Asn

CAA

Gin 515 AAT TCT TAC AAC Asn Ser Tyr Asn ATC AAC Ile Asn 525 CAA GAA CTA GGG Gin Glu Leu Gly AAC CCC TTT AGG Asn Pro Phe Arg

AAA

Lys 535 GTG GGC ATC GTC Val Gly Ile Val

AAT

Asn 540 TCT CAA ACC AAC Ser Gin Thr Asn

AAT

Asn 545 GGT GCC ATG AAT Gly Ala Met Asn ATC GGC ATT CAG Ile Gly Ile Gin 2041 2089 2137 GGC TAT AAG CAA Gly Tyr Lys Gin TTT GGC CAA AAA Phe Gly Gin Lys

AGA

Arg 565 AAA TGG GGC GCT Lys Trp Gly Ala AGG TAT Arg Tyr 570 TAC GGC TTT Tyr Gly Phe AAC TCG GCT Asn Ser Ala 590

TTT

Phe 575 GAT TAC AAC CAT Asp Tyr Asn His

GCG

Ala 580 TTC ATC AAA TCC Phe Ile Lys Ser AGC TTT TTC Ser Phe Phe 585 GAC GCG CTT Asp Ala Leu 2185 2233 TCT GAC GTG TGG Ser Asp Val Trp TAT GGT TTT GGA Tyr Gly Phe Gly TAT AAC Tyr Asn 605 TTC ATC AAC GAT Phe Ile Asn Asp GCC ACC AAT TTC Ala Thr Asn Phe

TTA

Leu 615 GGC AAA AAC AAC Gly Lys Asn Asn 2281

AAG

Lys 620 CTT TCT TTG GGG CTT TTT GGC GGG ATT Leu Ser Leu Gly Leu Phe Gly Gly Ile 625 TTA GCG GGC ACT Leu Ala Gly Thr 2329 2377 TGG CTC AAT TCT GAG TAC GTG AAT TTA GCC ACC GTG AAT AAC GTC TAT Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 650 AAC GCT AAA Asn Ala Lys GTG AGG ATG Val Arg Met 670

ATG

Met 655 AAT GTG GCG AAT Asn Val Ala Asn CAA TTC TTA TTC Gin Phe Leu Phe AAT ATG GGA Asn Met Gly 665 GAT CAT GCA Asp His Ala 2425 2473 AAT TTA GCC AGA Asn Leu Ala Arg

TCC

Ser 675 AAG AAA AAA GGC Lys Lys Lys Gly GCT CAG Ala Gin 685 CAT GGG ATT GAG His Gly Ile Glu

TTA

Leu 690 GGG CTT AAA ATC Gly Leu Lys Ile

CCC

Pro 695 ACC ATC AAC ACG Thr Ile Asn Thr 2521 2569

AAC

Asn 700 TAT TAT TCC TTT Tyr Tyr Ser Phe GGG GCT GAA CTC Gly Ala Glu Leu

AAA

Lys 710 TAC AGA AGG CTC Tyr Arg Arg Leu AGC GTG TAT TTG Ser Val Tyr Leu

AAT

Asn 720 NAT GTG TTC GCT Xaa Val Phe Ala

TAC

Tyr 725 TAAGCTTTTT GTGAAACTCC 2619 CTTTTTAAGG GGTTTTTTTT TGAACTCTCT TTTAAATTCT TTTTAAGCTT TTTTTTGAAC TTTTTTTTGA ATTCTTTGTT TTTCGTTTTT AAACTCCCTT TTTTAAGGGA TTTCTTTTTT CTTTTTTTTA AACCCTCTTT TTTTAAGGGG TTTCTTTTTA TTTAAATTCT TTTTTTGGGG GTTTGATCTT TCTTTTTGCC

CTTTTTAAAG

TTTAAGCTTT

TGAACTCCCT

AAGCTTTTTT

AATCCCCACT

AGATTTCTTT

TTTTAAACCC

TTTTTGAACC

GAAGTCTTTT

ACTTTC

2679 2739 2799 2859 2915 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 745 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...20 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Lys Lys His Ile Leu Ser Leu Ala Leu -15 Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Gly Ser -10 Thr Ser Leu Val Leu Val Gly Tyr Ile Gin Ser Gin Gin 1 5 Ile Gly Glu Ala Ala Gin Met Val Thr Asn Thr Lys Gly 20 Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser SUBSTITUTE SHEET (RULE 26) WO 98/43479 WO 9843479PCTIUS98/06421 Thr Ala Lys Ala Gly Pro 125 Gly Al a Giu Cys Ala 205 Thr Thr Ala Thr Giy 285 Phe Glu As n Phe Asn 365 Asn Thr Leu Tyr Gly 445 Leu Ser Leu Val1 Ala Val1 As n 110 Gly Pro Tyr Leu Arg 190 Lys Gin Tyr Giu Gin 270 Gly Gin Ile Asn Ala 350 Leu Asn Asn Pro Val 430 Thr Val Ile Asn Arg Asn Gly Giy Gin Giy Gin Gly 175 Gin Asn Thr As n Gin 255 Cys Gin Gin Ile Leu 335 Gin Ser Val Phe Asn 415 Gly Gin Asn Thr Thr Giu Ser Leu Thr Asp Lys Ile 160 Gly Thr Gly Thr Asn 240 Leu Pro Pro Giu Ala 320 Asp Ser Giu Asn Val 400 Ala Glu Giu Phe Thr 480 Leu Ile 50 Asn Leu Pro Ala Trp, Asn Giu Ser Ser Thr 130 Ser Met 145 Ile Gin Asn Gly Ala Asp Ser Ser 210 Ala Thr 225 Asn Lys Leu Asn Leu Val Trp Giy 290 Phe Ser 305 Gin Ser Thr Giy Met Leu Gin Val 370 Glu Lys 385 Ser Aia Gly Val Thr Ile Gin Gin 450 Lys Ser 465 Ala Leu 35 Lys Leu Gly Ala Tyr Gin Thr Ile 100 Gly Pro 115 Gin Ile Ser Ile Gin Ala Thr Lys 180 Ile Asn 195 Ser Ser Thr Thr Ala Thr Gin Ala 260 Arg Ser 275 Leu Ser Gin Val Lys Ile Lys Pro 340 Lys Asn 355 Lys Lys Leu Ala Phe Leu Thr Ser 420 Ser Ala 435 Ile Gin Arg Tyr Ser Lys Ser Ser Ala 85 Gly Gly Thr Asp Leu 165 Val Giy Ser Gin Val1 245 Al a Thr Thr Thr Val1 325 Phe Ala Asn Gly Ala 405 As n Leu Gin Ser Val 485 Al a Thr 70 Val1 Tyr Ser Cys Giu 150 Lys Ser Gly Asn Asp 230 Lys Asn Asn Ser Ser 310 Ser Asn Gin Leu Phe 390 Ser Thr Thr Al a Glu 470 Pro Asp 55 Lys Phe Ala Val1 Asn 135 Phe Asn Val Val1 Gly 215 Gly Phe Ile Asn Gly 295 Met Glu Pro Ala Giu 375 Gly Cys Trp Asn Glu 455 Leu Asn Pro Asn Leu Val Ile 120 Arg Lys Gin Asn Tyr 200 Gly Val Asp Met Glu 280 Asn Ile Asn Tyr Gin 360 Val1 Lys Lys G ly Ser 440 Asn Gly Ala Ser Leu Ala Met 105 Phe Phe Lys Ser Tyr 185 Gin As n Thr Ile Gin 265 Asn Ala Lys Aila Thr 345 Ala Met Glu Asp Al a 425 Ile Ile Asn Gin Al a Ile Ile Cys Asn Giu Leu Gly 170 As n Phe Gly Ile Thr 250 Val Thr Cys Asn Gin 330 Asp Glu Lys Giu Gly 410 Gly Al a Ala Thr Ser 490 Ile Asn Giy Asp Asn Ala Gly Asn Asp Gin Ser Thr 140 Asn Giu 155 Phe Pro Tyr Giu Cys Lys Ser Ser 220 Thr Thr 235 Asn Asn Leu Asn Pro Gly Ser Ile 300 Ala Gin 315 Asn Gin Ala Ser Met Phe Asn Asn 380 Val Met 395 Gly Thr Cys Ala His Phe Asp Thr 460 Tyr Asn 475 Leu Gin SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gin Gly Ile Glu 495 500 505 Thr Asn Tyr Tyr Leu Asn Gin Asn Ser Tyr Asn Gin Ile Gin Thr Ile 510 515 520 Asn Gin Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly Ile Val Asn 525 530 535 540 Ser Gin Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Ile Gin Val Gly 545 550 555 Tyr Lys Gin Phe Phe Gly Gin Lys Arg Lys Trp Gly Ala Arg Tyr Tyr 560 565 570 Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn 575 580 585 Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr 590 595 600 Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys 605 610 615 620 Leu Ser Leu Gly Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp 625 630 635 Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn 640 645 650 Ala Lys Met Asn Val Ala Asn Phe Gin Phe Leu Phe Asn Met Gly Val 655 660 665 Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala 670 675 680 Gin His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn 685 690 695 700 Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser 705 710 715 Val Tyr Leu Asn Xaa Val Phe Ala Tyr 720 725 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 2603 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 210...2342 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 210...270 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATGACCTTTA TTGGTTTAAT ATTTGTTTAG AAATAACACA AAAACCTTTT TTTTTTTTTT SUBSTITUTE SHEET (RULE 26) PCTIUS98/06421 WO 98/43479 TGAAAGGGCA AAAACGCCTA ATTAATATCA AAATCCCATG AAGCTTGCGG TATGGTTTCA CCTAAAGACA

CACTTCCGCA

TCTTATTTCA AGTAATAAAA GGAGAAAAC ATG AAG AAA Met Lys Lys AATTTATACT AtATTAACGA AGATTTACTA

ACAATTTCAA

AAA TTT CTG TCA TTA Lys Phe Leu Ser Leu 120 180 233 ACC TTA GGT Thr Leu Gly -120 TCG CTT TTA GTT TCC GCT TTA AGC GCT GAA GAC AAC GGC Ser Leu Leu Val Ser Ala Leu Ser Ala Glu Asp Asn Gly

TTT

Phe TTT GTG AGT GCG GGC TAT CAA ATC GGT GAA TCC GCT CAA ATG Phe Val Ser Ala Gly Tyr Gin Ile Gly Glu Ser Ala Gin Met 329 AAA AAC ACT AAA Lys Asn Thr LYS ATT CAA GAT CTT Ile Gin Asp Leu

TCA

Ser 30 GAT AGC TAT GAA Asp Ser Tyr Glu AGA CTG Arg Leu AAC AAT CTT Asn Asn Leu TCC GCC GAC Ser Ala Asp

TTA

Leu ACG AGT TAT AGT GCC CTA AAC ACT CTT Thr Ser Tyr Ser Ala Leu Asn Thr Leu 45 ATT AGG GAG Ile Arg Gin TTG AAC GCT Leu Asn Ala CCC AAC GCT ATC Pro Asn Ala Ile

AAT

Asn 60 AAC GCA AGG GGC Asn Ala Arg Gly

AAT

Asn AGT GCG Ser Ala PAG AAT TTG ATC Lys Asn Leu Ile

AAT

Asn 75 GAT AAA AAG AAT Asp Lys Lys Asn

TCC

Ser B0 CCG GCG TAT CAA Pro Ala Tyr Gin 521 569

GCG

Ala GTG CTT TTA GCC Val Leu Leu Ala

TTG

Leu 90 AAT GCG GCA GCG Asn Ala Ala Ala

GGG

Gly TTG TGG CAA GTC Leu Trp Gin Val AGC TAT TCG ATC Ser Tyr Ser Ile

AGC

Se r 105 GTT TGT GGC CCT GGC TCT GAG AAA AAT Val Cys Gly Pro Gly Ser Asp Lys Asn 11.0 AAA AAT Lys Asn 115 GGG GGC GTC Gly Gly Val ATT GCT TGC Ile Ala Cys 135

CAA

Gin 120 ACC TTT GAA AAT Thr Phe G2.u Asn CCG TCA AAT GGG Pro Ser Asn Gly GGG ACT ACC Gly Thr Thr 130 GGT ATA TCC Gly Ile Ser GAT TCA TTT TAT Asp Ser Phe Tyr CCA GGA AAG TGG Pro Gly Lys Trp

AGC

Ser 145 ACT GAA Thr Giu iso AAT TAC GCA AAA Asn Tyr Ala Lys

ATC

Ile 155 AAT AAA GCC TAT CAA ATC ATC CAA AAG Asn Lys Ala Tyr Gin Ile Ile Gin Lys 2.60

GCT

Aila 165 TTT GGA GCA AGC Phe Gly Ala Ser GGG CA Gly Gin 170 GAT ATT CCT Asp Ile Pro TTA AGC GAG Leu Ser Asp ACC AAA Thr Lys 180 CAG CCA Gin Pro 2.95 809 GPA CTT PAT TTT GPA ATT AAA GGG APA AAA PAT GAT AGC GTC Giu Leu Asn Phe Giu Ile Lys Gly Lys Lys Asn Asp Ser Val SUBSTITUTE SHEET (RULE 26) WO 98/43479 GGA GAA AGA Gly Giu Arg AAG TGG GTG Lys Trp Val 215 PCTIUS98/06421

TGG

Trp 200 AAA TTC CCA TGG Lys Phe Pro Trp

ACT

Thr 205 AAT GGA AAA TTT Asn Giy Lys Phe GTT TCA GTC Val Ser Vai 210 ATC AAA GTG Ile Lys Val AAT GGG AAG TAT Asn Gly Lys Tyr

GAA

Giu 220 GAA ATT AAA GAA Glu Ile Lys Giu TCA AAT Ser Asn 230 AAC GCT CAA GAG Asn Ala Gin Giu

CTT

Leu 235 TTA AAA~ CAG GCT Leu Lys Gin Ala

AGC

Ser 240 ACT ATT TTA ACC Thr Ile Leu Thr

ACT

Thr 245 CTT AAT GAA GCA Leu Asn Giu Ala CCA TGG TTG AGT Pro Trp Leu Ser GGT GOT GCA GGC Gly Gly Ala Giy

AAT

Asn 260 1001 1049 1097 GTG GCC GGT GGC Val Ala Giy Gly

AAT

Asn 265 AGT TTA TOG GCC Ser Leu Trp Ala

OGA

Gly 270 ATA GAT AAA GOC Ile Asp Lys Giy GAC GGG Asp Oly 275 AGC GCA TGC Ser Ala Cys ATC AAA AAC Ile Lys Asn 295

GGG

Gly 280 ATT TTT AAA AAT Ile Phe Lys Asn

GAA

Giu 285 ATC AGC GCG ATT Ile Ser Ala Ile CAA GAC ATO Gin Asp Met 290 OTT ACC 0CC Val Thr Ala 1145 1193 GCT OAA ATA 0CC Ala Giu Ile Ala

OTA

Val1 300 GAG CAA TCC AAA Giu Gin Ser Lys

ATC

Ile 305 AAC GCG Asn Aia 310 CAA AAC CAG CAC Gin Asn Gin His

AAC

Asn 315 CTA GAC ACT GGG Leu Asp Thr Oly

AAA

Lys 320 OCA TTC AAC CCC Ala Phe Asn Pro

TAT

Tyr 325 AAA GAC GCC AAC Lys Asp Ala Asn GCC CAA AGC ATG Ala Gin Ser Met

TTC

Phe 335 OCT AAC GCT AGA Ala Asn Ala Arg 1241 1289 1337 CAA GCG GAG ATT Gin Ala Giu Ile

TTA

Leu 345 AAC CGC GCT CAA Asn Arg Ala Gin

GCA

Ala 350 GTG GTG AAG GAC Val Val Lys Asp TTT GAA Phe Giu AGA ATC CCT Arg Ile Pro AAG OGT AGC Lys Giy Ser 375

GCA

Al a 360 GCG TTC OTG AAA Ala Phe Val Lys

GAC

Asp 365 TCT TTA GGA GTA Ser Leu Gly Val TGC CAT GAA Cys His Olu 370 ACG OTT ACT Thr Val Thr 1385 1433 GAC GGC AAT CTC Asp Gly Asn Leu

CGT

Arg 380 GOC ACO CCA TCT Gly Thr Pro Ser

GGC

Oly 385 TCT AAC Ser Asn 390 ACT TOG GGA GCC Thr Trp Giy Ala

GC

Gly 395 TOC GCO TAT OTG Cys Ala Tyr Val

GGA

Gly 400 GAA ACC OTA ACO Oiu Thr Val Thr 1481

AAT

Asn 405 CTA AAA AAC AGC ATC GCT CAT TTT GOC GAC CAA GCO GAG CGA Leu Lys Asn Ser Ile Ala His Phe Oly Asp Gin Ala Oiu Arg 410 415

ATC

Ile 420 1529 1577 CAT AAT GCG CGA AAT CTC GCC TAC ACT TTA GCG AAT TTC AGC GOC CAG SUBSTITUTE SHEET (RULE 26) WO 98/43479 WO 9843479PCTIUS98/06421I His Asn Ala Arg Asn Leu Ala Tyr Thr 425 Leu 430 Ala Asn Phe Ser Gly Gin 435 TAC AAA AAG CTA GGC GAA CAC TAT Tyr Lys Lys Leu Gly Giu His Tyr 440

GAC

Asp 445 AGC ATC ACA GCG Ser Ile Thr Ala GCG CTC TCT Ala Leu Ser 450 AAA AAG ACT Lys Lys Thr 1625 AGC TTG CCT Ser Leu Pro 455 GAT GCG CAA TCT Asp Ala Gin Ser

TTA

Leu 460 CAA AAT GTG GTG Gin Asn Val Val

AGC

Ser 465 1673 AAC CCT Asn Pro 470 AAC AGC CCG CAA Asn Ser Pro Gin

GGC

Gly 475 ATA CAG GAT AAT Ile Gin Asp Asn

TAC

Tyr 480 TAC ATT GAC TCC Tyr Ile Asp Ser

AAC

Asn 485 ATC CAT TCT CAA Ile His Ser Gin CAA TCT AGG AGT Gin Ser Arg Ser

CAA

Gin 495 GAA CTC GGC AGT Giu Leu Gly Ser

AAC

Asn 500 1721 1769 1817 CCT TTC AGA CGC Pro Phe Arg Arg

GCC

Aila 505 GGG CTA ATC GCC Giy Leu Ile Ala TCT ACC ACC AAT Ser Thr Thr Asn AAC GGC Asn Giy 515 GCG ATG AAT Ala Met Asn AAA AAC AAA Lys Asn Lys 535

GGG

Giy 520 ATT GGC TTT CAA Ile Gly Phe Gin

GTG

Vai 525 GGC TAT AAG CAA Giy Tyr Lys Gin TTC TTT GG Phe Phe Gly 530 GAT TAC AAC Asp Tyr Asn 1865 1913 CGA TGG GGC GCG Arg Trp Gly Ala

AGA

Arg 540 TAC TAC GGC TTT Tyr Tyr Gly Phe

GTG

Val1 545 CAC ACC His Thr 550 TAT AAC AAG TCC Tyr Asn Lys Ser

CAA

Gin 555 TTT TTC AAC TCC Phe Phe Asn Ser

GAT

Asp 560 TCT GAT GTT TGG Ser Asp Val Trp

ACT

Thr 565 TAT GGC GTG GGG Tyr Gly Val Gly GAT TTG TTA GTG Asp Leu Leu Val

AAT

Asn 575 TTC ATC AAC GAT Phe Ile Asn Asp

AAA

Lys 580 1961 2009 2057 GCC ACT AAA CAC Ala Thr Lys His

AAT

Asn 585 AAA ATT TCT TTT Lys Ile Ser Phe

GGC

Gly 590 GCG TTT GGC GGT Ala Phe Gly Gly ATC CAA Ile Gin 595 CTA GCC GGG Leu Ala Gly GTG AAC AAT Val Asn Asn 615 ACT TCA Thr Ser 600 TGG CTT AAT Trp Leu Asn

TCT

Ser 605 CAG TAT GTG AAT Gin Tyr Val Asn TTA GCG AAT Leu Ala Asn 610 TTC CAA TTC Phe Gin Phe 2105 2153 TAT TAT AAA GCT Tyr Tyr Lys Ala

AAA

Lys 620 ATC AAC ACC TCT Ile Asn Thr Ser

AAC

Asn 625 TTA TTC Leu Phe 630 AAT CTG GOC TTA Asn Leu Gly Leu

AGG

Arg 635 ACC AAT CTC GCC Thr Asn Leu Ala AAT AAA AGA ATA Asn Lys Arg Ile 2201 GGC GCT GAT CAT AGC GCG CAA CAT GGC ATG GAA TTA GGC GTG AAG ATC Gly Ala Asp His Ser Ala Gin His Giy Met Giu Leu Giy Vai Lys Ile 2249 SUBSTITUTE SHEET (RULE WO 98/43479 99 645 650 655 CCC ACG ATC AAC ACA AAT TAC TAT TCT TTG CTA Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Leu Leu 665 670 TAC AGA AGG CTT TAT AGC GTG TAT CTC AAC TAT Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr 680 685 GCTTAAACTC CTTTTTAAAC TCCCTTTTTA GGGGGTTTAA TTTAGCTTTT TTTAATTTTT TCCACCAAAC AAAGTTTTTT AAAAATACTC AAAGGCGTTT TTTGCAATCT AAATAAAAAA ATTTTAAACC ACCCAAGCAA GAAACCCCAA ACATCTTTAG CAAAAAACGC CCCAAA INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 711 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...20 OTHER INFORMATION: PCT/US98/06421 660 GGC ACT ACC TTG CAA Gly Thr Thr Leu Gin 675 GTG TTT GCT TAC TAAAA Val Phe Ala Tyr 690 TCTTTTTAAC TGACTTTTCT GACTTCAAGC GTTAATCACA TTAGCGTTAT TCAAGCGATC CGTTCGCGCG CTCCACTAAC 2297 2347 2407 2467 2527 2587 2603 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Lys Lys Lys Phe Leu Ser Leu Thr Leu Ala Ile Leu Ala Asn Lys Ala Pro Val 125 Pro Leu Gly Ser Leu Ala Lys Ala Gly 110 Pro Gly Ser Glu Asp Asn Arg Asn Gly Ser Ser Lys Ala Ser Ser Thr Gly Ser Leu Asp Asn Trp Glu 1 Ala Tyr Leu Asn Pro Trp Lys Gly Ser -15 Asp Gin Glu Ile 50 Leu Ala Gin Asn Gly 130 Gly Asn Met Arg 35 Arg Asn Tyr Val Lys 115 Thr Ile Phe 5 Lys Asn Ser Ser Ala 85 Ser Gly Ile Thr Phe Asn Asn Ala Ala 70 Val Tyr Gly Ala Glu Gly -10 Val Thr Leu Asp 55 Lys Leu Ser Val Cys 135 Asn Ser Ser Lys Leu Pro Asn Leu Ile Gin 120 Asp Tyr Leu Gly Ile Ser Ala Ile Leu Val Phe Phe Lys Val Tyr Gin Tyr Ile Asn Asn Cys Glu Tyr Ile Ser Gin Asp Ser Asn Asp Ala Gly Asn Glu 140 Asn SUBSTITUTE SHEET (RULE 26) WO 98/43479 WO 9843479PCT[US98/06421 Lys Ile Lys Thr 205 Giu Lys Leu Ala Glu 285 Glu Asp Ser Gin Asp 365 Gly Ala Phe Thr Asp 445 Gin Gin Arg Ala Val 525 Tyr Phe Leu Al a Pro Lys 190 Asn lie Gin Ser Gly 270 Ile Gin Thr Met Ala 350 Ser Thr Tyr Gly Leu 430 Ser Asn Asp Ser Ala 510 Gly Tyr Asn Val Tyr Al a 175 Asn Gly Lye Ala Asn 255 Ile Ser Ser Gly Phe 335 Val Leu Pro Val1 Asp 415 Ala Ile Val1 Aen Gin 495 Ser Tyr Gly Ser Asn Gin 160 Leu Asp Lys GlU Ser 240 Gly Asp Ala Lys Lys 320 Al a Val1 Gly Ser Gly 400 Gin Asn Thr Val Tyr 480 Glu Thr Lys Phe Asp 560 Phe 145 Ile Ser Ser Phe Asp 225 Thr Gly Lys Ile Ile 305 Al a Asn Lys Val1 Gly 385 Glu Ala Phe Ala Ser 465 Tyr Leu Thr Gin Val1 545 Ser Ile Ile Asp Val Val1 210 Ile Ile Al a Gly Gin 290 Val1 Phe Ala Asp Cys 370 Thr Thr Glu Ser Al a 450 Lys Ile Giy Asn Phe 530 Asp Asp As n Gin Thr Gin 195 Ser Lys Leu Gly Asp 275 Asp Thr Asn Arg Phe 355 His Val1 Val Arg Gly 435 Leu Lye Asp Ser Aen 515 Phe Tyr Val1 Asp Ile 595 Lye Lys 180 Pro Val Val1 Thr Aen 260 Gly Met Al a Pro Ala 340 Glu Glu Thr Thr Ile 420 Gin Ser Thr Ser Asn 500 Gly Gly Asn Trp Lye 580 Gin Al a 165 Giu Gly Lye Ser Thr 245 Val1 Ser Ile Asn Tyr 325 Gin Arg Lye Ser Aen 405 His Tyr Ser Asn Asn 485 Pro Al a Lye His Thr 565 Ala Leu 150 Phe Leu Glu Trp, As n 230 Leu Ala Al a Lys Ala 310 Lye Ala Ile Gly Asn 390 Leu As n Lys Leu Pro 470 Ile Phe Met Asn Thr 550 Tyr Thr Ala Gly Asn Arg Val1 215 Aen Asn Gly Cys Aen 295 Gin Asp Giu Pro Ser 375 Thr Lys Al a Lye Pro 455 Asn His Arg Aen Lye 535 Tyr Gly Lye Gly Al a Phe Trp 200 As n Ala Glu Gly Gly 280 Al a As n Al a Ile Al a 360 Asp Trp Asn Arg Leu 440 Asp Ser Ser Arg Gly 520 Arg Asn Val1 Hise Thr 600 Ser Giu 185 Lye Gly Gin Ala Aen 265 Ile Glu Gin Aen Leu 345 Ala Gly Gly Ser Aen 425 Gly Ala Pro Gin Ala 505 Ile Trp Lye Gly Aen 585 Ser Gly 170 Ile Phe Lye Glu Cye 250 Ser Phe Ile His Phe 330 Aen Phe As n Ala Ile 410 Leu Giu Gin Gin Val 490 Gly Gly Gly Ser Ser 570 Lye Trp 155 Gin Lye Pro Tyr Leu 235 Pro Leu Lye Ala Asn 315 Ala Arg Val Leu Gly 395 Ala Ala His Ser Gly 475 Gin Leu Phe Al a Gin 555 Asp Ile Leu Asp Gly Trp Giu 220 Leu Trp Trp As n Val1 300 Leu Gin Ala Lye Arg 380 Cys His Tyr Tyr Leu 460 Ile Ser Ile Gin Arg 540 Phe Leu Ser Asn 575 Phe Gly Ala Phe Gly Gly 590 SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 Ser Gin Tyr Val Asn Leu Ala Asn Val 605 610 Asn Asn 615 Phe Asn Ile Asn Thr Ser Asn 625 Asn Phe Gin Phe Leu Tyr Tyr Lys Ala Lys 620 Leu Gly Leu Arg Thr 635 His Ser Ala Gin His Asn Leu Ala Gly Met Glu 655 Ser Leu Leu Lys Arg Ile 630 Gly Ala 645 Pro Thr Tyr Arg Asp Gly Val Lys Ile 660 Gin Ile Asn Thr 665 Arg Leu Tyr 680 650 Asn Tyr Tyr Ser Val Tyr Gly Thr Thr 670 Leu Asn 685 Leu 675 Tyr Tyr Val Phe Ala 690 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 2427 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 232...2247 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 232...292 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AAAACGCGCA GCAAAAAATC TCTGTTAAGC TTTTATCATT TCTAAAAACC CTTTCCAATA CCACCCAAAC AAACGCGCAA TTTCTCCAAA TGACAAAAAA AAAAAAAACG ATTTTATGCT TTACTTAATG TATAAAATCT CAATCACTCA ATTTAATTTC AAA ACC CTT TTA CTC TCT CTC TCT CTC TCT CTC Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu -10 AAC GCT GAA GAC AAC GGC TTT TTT ATC AGC GCG Asn Ala Glu Asp Asn Gly Phe Phe Ile Ser Ala 1 5 GAA GCC GCT CAA ATG GTG AAA AAC ACC GGC GAA Glu Ala Ala Gln Met Val Lys Asn Thr Gly Glu 20 25 AGCGTTCCAT TGAAACAAAA AAAATGCAAA AATTCTAAAT ACAATGCTTT TAATACATTC AAAGGATATT T ATG AAA Met Lys TCG TCA TCG CTT TTA Ser Ser Ser Leu Leu GGC TAT CAA ATC GGT Gly Tyr Gln Ile Gly TTG AAA AAA CTT TCA Leu Lys Lys Leu Ser 120 180 237 285 333 381 SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 GAC ACT TAT GAG Asp Thr Tyr Glu TTG AGC AAC CTT Leu Ser Asn Leu

TTA

Leu ACC AAT TTT AAC Thr Asn Phe Asn AAC CTC Asn Leu AAT CAA GCG Asn Gin Ala ATC GAT AAT Ile Asp Asn

GTA

Val ACG AAC GCG AGC Thr Asn Ala Ser

AGC

Ser 55 CCT TCA GAA ATC Pro Ser Glu Ile AAT GCC ACG Asn Ala Thr GAA AAA ACC Glu Lys Thr TTA AAA GCA AAC Leu Lys Ala Asn

ACG

Thr CAA GGG CTG ATT Gin Gly Leu Ile AAT TCC Asn Ser CCG GCG TAT CAA Pro Ala Tyr Gin GTG TAT TTG GCG Val Tyr Leu Ala

CTC

Leu AAT GCG GCG GTG Asn Ala Ala Val

GGG

Gly CTG TGG AAT GTG Leu Trp Asn Val

ATA

Ile 100 GCC TAT AAT GTC Ala Tyr Asn Val

CAA

Gin 105 TGC GGT CCT GGT Cys Gly Pro Gly

AAG

Lys 110 AGT GGG GAT CAA Ser Gly Asp Gin

AGC

Ser 115 GTA ATT TTT GAT Val Ile Phe Asp CAA CCA GGA CAT Gin Pro Gly His GAT TCA Asp Ser 125 AGA TCC ATT Arg Ser Ile CCT TTA TCC Pro Leu Ser 145

AAT

Asn 130 TGC AAT TTA ACC Cys Asn Leu Thr

GGT

Gly 135 TAT AAC AAC GGG GTT AGC GGC Tyr Asn Asn Gly Val Ser Gly 140 ATT GAC AAT TTT Ile Asp Asn Phe

AAA

Lys 150 ACG CTT AAT CAA Thr Leu Asn Gin TAT CAA ACT Tyr Gin Thr ATC CAA Ile Gin 160 CAA GCT TTA AAA Gin Ala Leu Lys GAT AGC GGA TTT Asp Ser Gly Phe

CCT

Pro 170 GTT TTG GAT AGT Val Leu Asp Ser

AAA

Lys 175

AAT

Asn GGA AAA CAA GTA Gly Lys Gin Val AAA AGT GAA ACT Lys Ser Glu Thr 195

ACT

Thr 180 ATA AAA ATA ACA Ile Lys Ile Thr

ACA

Thr 185 CAA ACT AAT GGA Gin Thr Asn Gly

GCT

Ala 190 ACT ACT ACT ACT Thr Thr Thr Thr

ACT

Thr 200 ACT ACT AAT GAC Thr Thr Asn Asp GCT CAA Ala Gin 205 ACC CTT TTG Thr Leu Leu TGC CCA TGG Cys Pro Trp 225

CAA

Gin 210 GAA GCC AGT AAA Glu Ala Ser Lys

ATG

Met 215 ATA AGC GTC CTC Ile Ser Val Leu ACT ACA AAC Thr Thr Asn 220 GCA CCG TGG Ala Pro Trp 957 1005 GTA AAT ACC GCT Val Asn Thr Ala

CAT

His 230 AAC TCA AAC GGG Asn Ser Asn Gly AAT TTA Asn Leu 240 AAT ACG ACA GGG Asn Thr Thr Gly

AAT

Asn 245 GTG TGT CAG GTT Val Cys Gin Val

TTT

Phe 250 GCC ACG GAG TTT Ala Thr Glu Phe 1053 AGC GCC GTT ACT AGC ATG ATC AAA AAC GCG CAA GAA ATC GTA ACG CAA 1101 SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 Ser 255 Ala Val Thr Ser Met 260 Ile Lys Asn Ala Gin 265 Glu Ile Val Thr Gin 270 GCT CAA AGC CTT Ala Gin Ser Leu

AAC

Asn 275 AAC CCG CAA AGC Asn Pro Gin Ser

AAT

Asn 280 CAA AAC GCG CCG Gin Asn Ala Pro AAA GAT Lys Asp 285 1149 TTC AAT CCT Phe Asn Pro AAT CAC GCG Asn His Ala 305

TAC

Tyr 290 ACC TCT GCT GAT Thr Ser Ala Asp

AGG

Arg 295 GCT TTC GCT CAA Ala Phe Ala Gin AAC ATG CTC Asn Met Leu 300 GAT CAA ATG Asp Gin Met 1197 1245 CAA GCG CAA GCC Gin Ala Gin Ala

AAG

Lys 310 ATG CTT GAA CTA Met Leu Glu Leu

GCC

Ala 315 AAA AAA Lys Lys 320 GAC CTT AAC ACT Asp Leu Asn Thr CCA AAA CAA TTT Pro Lys Gin Phe

ATC

Ile 330 ACA AAC TAC TTG Thr Asn Tyr Leu

GCA

Ala 335 GCT TGC CGC AAT Ala Cys Arg Asn

GGG

Gly 340 GGT GGG ACA TTA Gly Gly Thr Leu

CCT

Pro 345 GAT GCA GGG GTT Asp Ala Gly Val

ACT

Thr 350 1293 1341 1389 TCT AAC ACT TGG Ser Asn Thr Trp GCC GGT TGC GCC Ala Gly Cys Ala

TAT

Tyr 360 GTG GAA GAG ACG Val Glu Glu Thr ATA ACC Ile Thr 365 GCC CTA AAT Ala Leu Asn AAG CAA TCT Lys Gin Ser 385

AAC

Asn 370 AGC CTT GCG CAT Ser Leu Ala His

TTT

Phe 375 GGC ACT CAA GCC Gly Thr Gin Ala GAT CAA ATC Asp Gin Ile 380 AGA GGC AGC Arg Gly Ser 1437 1485 GAG TTG TTG GCG Glu Leu Leu Ala ACG ATA CTT GAT Thr Ile Leu Asp

TTT

Phe 395 CTT AAG Leu Lys 400 GAT TTA AAC AAC Asp Leu Asn Asn

ACT

Thr 405 TAT AAC AGC ATC ACC ACG ACC GCT TCA Tyr Asn Ser Ile Thr Thr Thr Ala Ser 410 1533

AAC

Asn 415 ACG CCC AAT TCC Thr Pro Asn Ser

CCA

Pro 420 TTC CTT AAA AAT Phe Leu Lys Asn

TTG

Leu 425 ATA AGC CAA TCC Ile Ser Gin Ser

ACT

Thr 430 AAC CCT AAT AAC Asn Pro Asn Asn

CCC

Pro 435 GGG GGC TTA CAG Gly Gly Leu Gin

GCC

Ala 440 GTT TAT CAA GTC Val Tyr Gin Val AAC CAA Asn Gin 445 CAT AAC His Asn 1581 1629 1677 AGC GCT TAT Ser Ala Tyr CCT TTC AGA Pro Phe Arg 465

TCG

Ser 450 CAA TTA TTA AGC Gin Leu Leu Ser

GCC

Ala 455 ACG CAA GAA TTA Thr Gin Glu Leu

GGG

Gly 460 CGC GTT GGC TTA Arg Val Gly Leu

ATC

Ile 470 AGC TCT CAA ACC Ser Ser Gin Thr AAC GGT GCG Asn Gly Ala 1725 ATG AAT GGG ATC GGC GTG CAA ATA GGG TAT AAA CAA TTT TTT GGT GAA Met Asn Gly Ile Gly Val Gin Ile Gly Tyr Lys Gin Phe Phe Gly Glu 1773 SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 480 AAA AGA Lys Arg 495 AGA TGG GGG Arg Trp Gly TTA AGG Leu Arg 500 490 TAT TAT GGT TTT TTT GAT TAC AAC Tyr Tyr Gly Phe Phe Asp Tyr Asn 505

CAT

His 510 1821 GCT TAT ATC AAA Ala Tyr Ile Lys AGC TTT TTC AAC Ser Phe Phe Asn

TCC

Ser 520 GCC TCT GAT GTG Ala Ser Asp Val TTC ACT Phe Thr 525 1869 TAT GGG GTA Tyr Gly Val ACC AAA AAC Thr Lys Asn 545

GGA

Gly 530 ACA GAT GTC CTC Thr Asp Val Leu AAC TTT ATC AAC Asn Phe Ile Asn GAT AAA GCC Asp Lys Ala 540 ATT GCG TTA Ile Ala Leu 1917 1965 AAT AAG ATT TCT Asn Lys Ile Ser

TTT

Phe 550 GGG GTG TTT GGG Gly Val Phe Gly GCT GGC Ala Gly 560 ACT TCG TGG CTT Thr Ser Trp Leu

AAT

Asn 565 TCT CAA TAC GTG Ser Gin Tyr Val

AAT

Asn 570 TTA GCG ACA TTC Leu Ala Thr Phe

AAT

Asn 575 AAT TTT TAC AGC Asn Phe Tyr Ser

GCT

Ala 580 AAA ATG AAT GTG Lys Met Asn Val

GCG

Ala 585 AAT TTC CAA TTC Asn Phe Gin Phe TTC AAC TTG GGC Phe Asn Leu Gly

TTG

Leu 595 AGA ATG AAT CTC Arg Met Asn Leu AAA AAC AAA AAG Lys Asn Lys Lys AAA GCG Lys Ala 605 2013 2061 2109 2157 2205 AGC GAT CAT Ser Asp His ACG ATC AAC Thr Ile Asn 625

GTA

Val 610 GCT CAG CAT GGC GTG GAA CTA GGC GTG Ala Gin His Gly Val Glu Leu Gly Val 615 AAG ATC CCT Lys Ile Pro 620 ACG AAT TAC TAT Thr Asn Tyr Tyr

TCT

Ser 630 TTG CTA GGC ACT CAA CTC CAA TAC Leu Leu Gly Thr Gin Leu Gin Tyr 635 CGC AGG Arg Arg 640 CTT TAT AGC GTG TAT TTG AAT TAT GTG TTT GCT TAC Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr TAATATCTG 2256

TCTTTTTGTG

TCTTTTTTGG

AAAATGCGTT

AAACTCCCTT TTTAAGGGAT TTTTTTTGAA GGGTCAAGCG TAAAATTCAC CCCTATCCCT TTATAACAAA ATAAGATCTA AAACAATAAA GCCTTTCTTT TTTTAAACCC TTAAGAAAAT AAAATAAAAG ACAAAAACCC A 2316 2376 2427 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 672 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 NAME/KEY: Signal Sequence LOCATION: 1...20 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Lys Thr Leu Leu Leu Ser Leu Ile Leu Asn Ala Lys Ala Gly Asp 125 Ser Gin Asp Gly Ala 205 Thr Pro Glu Thr Lys 285 Met Gin Tyr Val Ile Leu Asn Gly Glu Ser Asp Leu Asn Thr Ile Thr Asn Val Gly Lys Ser 110 Ser Arg Gly Pro Thr Ile Ser Lys 175 Ala Asn 190 Gin Thr Asn Cys Trp Asn Phe Ser 255 Gin Ala 270 Asp Phe Leu Asn Met Lys Leu Ala 335 Thr Ser 350 Thr Ala Ala Ala Thr Gin Asp Ser Leu Gly Ser Leu Gin 160 Gly Lys Leu Pro Leu 240 Ala Gin Asn His Lys 320 Ala Asn Leu -15 Glu Asp 1 Ala Gin Tyr Glu Ala Val 50 Asn Leu Pro Ala Trp Asn Asp Gin Ile Asn 130 Ser Ile 145 Gin Ala Lys Gin Ser Glu Leu Gin 210 Trp Val 225 Asn Thr Val Thr Ser Leu Pro Tyr 290 Ala Gin 305 Asp Leu Cys Arg Thr Trp Asn Asn Asn Met Asn 35 Thr Lys Tyr Val Ser 115 Cys Asp Leu Val Thr 195 Glu Asn Thr Ser Asn 275 Thr Ala Asn Asn Gly 355 Ser Gly Val 20 Leu Asn Ala Gin Ile 100 Val Asn Asn Lys Thr 180 Thr Ala Thr Gly Met 260 Asn Ser Gin Thr Gly 340 Ala Leu Leu Ser Leu -10 Phe Phe Ile 5 Lys Asn Thr Ser Asn Leu Ala Ser Ser 55 Asn Thr Gin 70 Ala Val Tyr 85 Ala Tyr Asn Ile Phe Asp Leu Thr Gly 135 Phe Lys Thr 150 Gin Asp Ser 165 Ile Lys Ile Thr Thr Thr Ser Lys Met 215 Ala His Asn 230 Asn Val Cys 245 Ile Lys Asn Pro Gin Ser Ala Asp Arg 295 Ala Lys Met 310 Ile Pro Lys 325 Gly Gly Thr Gly Cys Ala Ala His Phe Ser Ser Gly Leu Pro Gly Leu Val Gly 120 Tyr Leu Gly Thr Thr 200 Ile Ser Gin Ala Asn 280 Ala Leu Gin Leu Tyr 360 Gly Leu Ala Glu Thr Ser Leu Ala Gin 105 Gin Asn Asn Phe Thr 185 Thr Ser Asn Val Gin 265 Gin Phe Glu Phe Pro 345 Val Thr Ser Gly Leu Asn Glu Ile Leu Cys Pro Asn Gin Pro 170 Gin Thr Val Gly Phe 250 Glu Asn Ala Leu Ile 330 Asp Glu Gin Ser Tyr Lys Phe Ile Gly Asn Gly Gly Gly Ala 155 Val Thr Asn Leu Gly 235 Ala Ile Ala Gin Ala 315 Thr Ala Glu Ala Ser Gin Lys Asn Asn Glu Ala Pro His Val 140 Tyr Leu Asn Asp Thr 220 Ala Thr Val Pro Asn 300 Asp Asn Gly Thr Asp SUBSTITUTE SHEET (RULE 26) WO 98/43479 106 PCT/US98/06421 365 370 375 380 Gin Ile Lys Gin Ser Glu Leu Leu Ala Arg Thr Ile Leu Asp Phe Arg 385 390 395 Gly Ser Leu Lys Asp Leu Asn Asn Thr Tyr Asn Ser Ile Thr Thr Thr 400 405 410 Ala Ser Asn Thr Pro Asn Ser Pro Phe Leu Lys Asn Leu Ile Ser Gin 415 420 425 Ser Thr Asn Pro Asn Asn Pro Gly Gly Leu Gin Ala Val Tyr Gin Val 430 435 440 Asn Gin Ser Ala Tyr Ser Gin Leu Leu Ser Ala Thr Gin Glu Leu Gly 445 450 455 460 His Asn Pro Phe Arg Arg Val Gly Leu Ile Ser Ser Gin Thr Asn Asn 465 470 475 Gly Ala Met Asn Gly Ile Gly Val Gin Ile Gly Tyr Lys Gin Phe Phe 480 485 490 Gly Glu Lys Arg Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr 495 500 505 Asn His Ala Tyr Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val 510 515 520 Phe Thr Tyr Gly Val Gly Thr Asp Val Leu Tyr Asn Phe Ile Asn Asp 525 530 535 540 Lys Ala Thr Lys Asn Asn Lys Ile Ser Phe Gly Val Phe Gly Gly Ile 545 550 555 Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gin Tyr Val Asn Leu Ala 560 565 570 Thr Phe Asn Asn Phe Tyr Ser Ala Lys Met Asn Val Ala Asn Phe Gin 575 580 585 Phe Leu Phe Asn Leu Gly Leu Arg Met Asn Leu Ala Lys Asn Lys Lys 590 595 600 Lys Ala Ser Asp His Val Ala Gin His Gly Val Glu Leu Gly Val Lys 605 610 615 620 Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Gin Leu 625 630 635 Gin Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr 640 645 650 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 2429 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 205...2277 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 205...259 OTHER INFORMATION: SUBSTITUTE SHEET (RULE 26) WO 98/43479 107 PCT/US98/06421 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i1: TGAAAGAAGA CTGATTAGTC TTTCTTTTAG GGGCGATTCA AGCCTTAAAA GCCGGGTCAA AATCCCCATT TTTCCCAATT TTTACAAAAA AAAAAAAAAC AAAATCTCTA AAATTTAGAG CTAAAATTAG CCATAAAATT CCATTTATTO CTTATAATAT GAAGTTTCTT TGTATCAAAG AAAAATCTAT TAAAAGGAGA AAAC ATG AAA AAA TCC CTC TTA CTC TCT CTT Met Lys Lys Ser Leu Leu Leu Ser Leu 120 180 231 TCT CTC ATC GCT Ser Leu Ile Ala

TCC

Ser TTA TCA AGA OCT GAA GAT GAC GGA Leu Ser Arg Ala Giu Asp Asp Gly 1 TTT TAT ACG Phe Tyr Thr AAA AAC ACA Lys Asn Thr AGT GTG GGC Ser Val Gly TAT CAG ATC GGT Tyr Gin Ile Gly

GAA

Giu GCG GTC CAA CAA Ala Val Gin Gin GGA GCA Gly Ala TTG CAA AAT CTT Leu Gin Asn Leu

GCA

Ala 30 GAC AGA TAC GAT Asp Arg Tyr Asp

AAC

As n TTA AAC AAC CTT Leu Asn Asn Leu

TTA

Leu AAC CAA TAC AAT Asn Gin Tyr Asn TTA AAT TCC TTA Leu Asn Ser Leu

GTC

Val 50 AAT TTA 0CC AGC Asn Leu Ala Ser

ACG

Thr 375 423 471 CCO AOC GCG ATC Pro Ser Ala Ile

ACC

Thr GGT GCG ATT GAT Gly Ala Ile Asp

AAT

Asn 65 TTA AGC TCA AGC Leu Ser Ser Ser GCO ATT Aia Ile AAC CTC ACT Asn Leu Thr TTA OCG CTC Leu Aia Leu

AGC

Ser GCC ACC ACC ACT Aia Thr Thr Thr

TCC

Ser 80 CCC GCC TAT CAA Pro Aia Tyr Gin OCT GTG OCT Aia Val Aia GCC CTT TTT Ala Leu Phe 519 567 AAT GCC GCT OTG Asn Ala Ala Val

GGC

Gly ATO TGG CAA GTC Met Trp Gin Vai ATT GGC Ile Giy 105 TGT GOC CCT GGC Cys Gly Pro Giy

CCT

Pro 110 ACC AAT AAT CAA Thr Asn Asn Gin

AGC

Ser 115 TAT CAA TCG TTT Tyr Gin Ser Phe

GOT

Gly 120 AAC ACA CCA 0CC Asn Thr Pro Ala AAT 000 ACC ACC Asn Oly Thr Thr

ACC

Thr 130 ACT TGC AAT CAA Thr Cys Asn Gin TAT 000 ACA GOC Tyr Oly Thr Gly

CCT

Pro 140 AAT GOC ATC CTA TCT ATT OAT OAA TAC Asn Gly Ile Leu Ser Ile Asp Giu Tyr 145 CAA AAA Gin Lys 150 CTC AAC CAA Leu Asn Gin

OCT

Ala 155 TAT CAG ATC ATC Tyr Gin Ile Ile ACC OCT TTA AAC Thr Ala Leu Asn CAA AAT CAA Gin Asn Oin 165 759 G GOT OOG ATG CCT 0CC TTG AAT GAC ACC ACC AAA ACA 000 GTA OTC Gly Gly Gly Met Pro Ala Leu Asn Asp Thr Thr Lys Thr Gly Vai Vai 807 SUBSTITUTE SHEET (RULE WO 98/43479 108 PCT/US98/06421 AAC ATA Asn Ile 185 CAA CAA ACC AAT Gin Gin Thr Asn

TAT

Tyr 190 AGG ACC ACC ACA Arg Thr Thr Thr AAC AAT ATC ATA Asn Asn Ile Ile 855

GAG

Glu 200 CAT TAT TAT ACA His Tyr Tyr Thr

GAG

Glu 205 AAT GGG AAA GAG ATC CCA GTC TCT TAT Asn Gly Lys Glu Ile Pro Val Ser Tyr 210

TCA

Ser 215 903 951 GGC GGA TCA TCA Gly Gly Ser Ser

TTC

Phe 220 TCG CCT ACA ATA CAA TTG ACA TAC CAT Ser Pro Thr Ile Gin Leu Thr Tyr His 225 AAT AAC Asn Asn 230 GCT GAA AAC Ala Glu Asn ACT CAA AAG Thr Gin Lys 250 TTG CAA CAA GCC Leu Gin Gin Ala

GCC

Ala 240 ACT ATC ATG CAA GTC CTT ATT Thr Ile Met Gin Val Leu Ile 245 999 CCG CAT GTG CAA Pro His Val Gin

ACG

Thr 255 AGC AAT GGC GGT Ser Asn Gly Gly

AAA

Lys 260 GCG TGG GGG Ala Trp Gly 1047 TTG AGT Leu Ser 265 TCT ACG CCT GGG Ser Thr Pro Gly GTG ATG GAT ATT Val Met Asp Ile

TTT

Phe 275 GGT CCT TCT TTT Gly Pro Ser Phe

AAC

Asn 280 GCT ATT AAT GAG Ala Ile Asn Glu

ATG

Met 285 ATT AAA AAC GCT Ile Lys Asn Ala

CAA

Gin 290 ACA GCC CTA GCA Thr Ala Leu Ala

AAA

Lys 295 1095 1143 1191 1239 ACC CAA CAG CTT Thr Gin Gin Leu

AAC

Asn 300 GCT AAT GAA AAC Ala Asn Glu Asn CAA ATC ACG CAA Gin Ile Thr Gin CCC AAC Pro Asn 310 AAT TTC AAC CCC TAC ACC TCT AAA Asn Phe Asn Pro Tyr Thr Ser Lys 315

GAC

Asp 320 AAA GGG TTC GCT Lys Gly Phe Ala CAA GAA ATG Gin Glu Met 325 CTC AAT AGA Leu Asn Arg 330 GCT GAA GCT CAA Ala Glu Ala Gin

GCA

Ala 335 GAG ATT TTA AAT TTA GCT AAG CAA Glu Ile Leu Asn Leu Ala Lys Gin 340 1287 GTA GCG Val Ala 345 AAC AAT TTC CAC Asn Asn Phe His

AGC

Ser 350 ATT CAA GGG CCT ATT CAA GGG GAT TTA Ile Gin Gly Pro Ile Gin Gly Asp Leu 355 1335

GAA

Glu 360 GAA TGT AAA GCA Glu Cys Lys Ala

GGA

Gly 365 TCG GCT GGC GTG Ser Ala Gly Val

ATC

Ile 370 ACT AAT AAC ACT Thr Asn Asn Thr

TGG

Trp 375 1383 14-31 GGT TCA GGT TGC Gly Ser Gly Cys

GCG

Ala 380 TTT GTG AAA GAA Phe Val Lys Glu

ACT

Thr 385 TTA AAC TCT TTA Leu Asn Ser Leu GAG CAA Glu Gin 390 CAC ACC GCT His Thr Ala

TAT

Tyr 395 TAC GGC AAC CAG Tyr Gly Asn Gin

GTC

Val 400 AAT CAG GAT AGG Asn Gin Asp Arg GCT TTG GCT Ala Leu Ala 405 1479 SUBSTITUTE SHEET (RULE 26) WO 98/43479 CAA ACC ATT Gin Thr Ile 410 PCTIUS98/06421 TTG AAT TTT AAA Leu Asn Phe Lys

GAA

Glu 415 GCC CTT AAC ACC Ala Leu Asn Thr

CTG

Leu 420 AAT AAA GAC Asn Lys Asp 1527 TCA AAA Ser Lys 425 GCG ATC AAT AGC Ala Ile Asn Ser

GGT

Gly 430 ATC TCC AAC TTG CCT AAC GCT AAA TCT Ile Ser Asn Leu Pro Asn Ala Lys Ser 1575 1623

CTT

Leu 440 CAA AAC ATG ACG CAT GCC ACT CAA AAC Gin Asn Met Thr His Ala Thr Gin Asn 445

CCT

Pro 450 AAT TCC CCA GAA Asn Ser Pro Glu

GGT

Gly 455 CTG CTC ACT TAT Leu Leu Thr Tyr

TCT

Ser 460 TTG GAT TCA AGC Leu Asp Ser Ser

AAA

Lys 465 TAC AAC CAG CTC Tyr Asn Gin Leu CAA ACC Gin Thr 470 1671 ATC GCG CAA Ile Ala Gin GAC TTT CAA Asp Phe Gin 490

GAA

Glu 475 TTG GGC AAA AAC Leu Gly Lys Asn

CCT

Pro 480 TTC AGG CGC TTT Phe Arg Arg Phe GGC GTG ATT Gly Val Ile 485 GTG CAA GTG Val Gin Val 1719 1767 AAC AAC AAC GGC Asn Asn Asn Gly

GCA

Ala 495 ATG AAC GGG ATC Met Asn Gly Ile

GGC

Gly 500 GGT TAT Gly Tyr 505 AAA CAA TTC TTT Lys Gin Phe Phe

GGT

Gly 510 AAA AAA AGG AAT Lys Lys Arg Asn

TGG

Trp 515 GGG TTA AGG TAT Gly Leu Arg Tyr

TAT

Tyr 520 GGT TTC TTT GAT Gly Phe Phe Asp AAC CAT GCT TAT Asn His Ala Tyr AAA TCT AAT TTT Lys Ser Asn Phe 1815 1863 1911 AAC TCC GCT TCT Asn Ser Ala Ser

GAT

Asp 540 GTG TGG ACT TAT Val Trp Thr Tyr GTG GGT ATG GAC Val Gly Met Asp GCT CTC Ala Leu 550 TAT AAC TTC Tyr Asn Phe AAG CTT TCA Lys Leu Ser 570

ATC

Ile 555 AAC GAT AAA AAC Asn Asp Lys Asn

ACC

Thr 560 AAC TTT TTA GGC Asn Phe Leu Gly AAG AAC AAC Lys Asn Asn 565 GGG ACT TCG Gly Thr Ser 1959 2007 GTA GGG CTT TTT Val Gly Leu Phe GGC TTT GCG TTA Gly Phe Ala Leu TGG CTT Trp Leu 585 AAT TCC CAA CAA Asn Ser Gin Gin

GTG

Val 590 AAT TTG ACC ATG Asn Leu Thr Met

ATG

Met 595 AAT GGC ATT TAT Asn Gly Ile Tyr

AAC

Asn 600 GCT AAT GTC AGC Ala Asn Val Ser TCT AAC TTC CAA Ser Asn Phe Gin

TTT

Phe 610 TTG TTT GAT TTA Leu Phe Asp Leu 2055 2103 2151 TTG AGA ATG AAC Leu Arg Met Asn

CTC

Leu 620 GCT AGG CCT AAG Ala Arg Pro Lys

AAA

Lys 625 AAA GAC AGC GAT Lys Asp Ser Asp CAT GCC His Ala 630 GCT CAG CAT GGC ATT GAA CTA GGT TTT AAG ATC CCC ACG ATC AAC ACC 2199 SUBSTITUTE SHEET (RULE 26) WO 98/43479 110 PCT/US98/06421 Ala Gin His Gly Ile Glu Leu Gly Phe Lys Ile Pro Thr Ile Asn Thr 635 640 645 AAC TAT TAT TCT TTC ATG GGC GCT AAA CTA GAA TAC AGA AGG ATG TAT 2247 Asn Tyr Tyr Ser Phe Met Gly Ala Lys Leu Glu Tyr Arg Arg Met Tyr 650 655 660 AGC CTT TTT CTC AAT TAT GTG TTT GCT TAC TAAAAATTCT TTTTGAACCC CTC 2300 Ser Leu Phe Leu Asn Tyr Val Phe Ala Tyr 665 670 TTTTTTTGGG GGAGTGTTGC AAAAATGCCC CCCTATTTGC TTGTGAGTTT TGGTTAAAAT 2360 TTTAGTTACC CACGCTTAAA AAGCGCCAAG CCTTTTACAC ACAACTCCTT TAATTTTGTT 2420 TTTAAGAAA 2429 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 691 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...18 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Lys Lys Ser Leu Leu Leu Ser Leu Ser Leu Ile Ala Ser Leu Ser -10 Arg Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gin Ile Gly 1 5 Glu Ala Val Gin Gin Val Lys Asn Thr Gly Ala Leu Gin Asn Leu Ala 20 25 Asp Arg Tyr Asp Asn Leu Asn Asn Leu Leu Asn Gin Tyr Asn Tyr Leu 40 Asn Ser Leu Val Asn Leu Ala Ser Thr Pro Ser Ala Ile Thr Gly Ala 55 Ile Asp Asn Leu Ser Ser Ser Ala Ile Asn Leu Thr Ser Ala Thr Thr 70 Thr Ser Pro Ala Tyr Gin Ala Val Ala Leu Ala Leu Asn Ala Ala Val 85 Gly Met Trp Gin Val Ile Ala Leu Phe Ile Gly Cys Gly Pro Gly Pro 100 105 110 Thr Asn Asn Gin Ser Tyr Gin Ser Phe Gly Asn Thr Pro Ala Leu Asn 115 120 125 Gly Thr Thr Thr Thr Cys Asn Gin Ala Tyr Gly Thr Gly Pro Asn Gly 130 135 140 Ile Leu Ser Ile Asp Glu Tyr Gin Lys Leu Asn Gin Ala Tyr Gin Ile 145 150 155 SUBSTITUTE SHEET (RULE 26) WO 98/43479 WO 9843479PCT/US98/06421 Ile Asn 175 Arg Gly Thr Al a Thr 255 Val Lys Giu Lys Ala 335 Ilie Ala Lys Gin Giu 415 Ilie Thr Ser Asn Ala 495 Lys His Thr As n Gly 575 Asn Asn Gin 160 Asp Thr Lys Ile Al a 240 Ser Met Asn As n Asp 320 Giu Gin Gly Glu Val 400 Ala Ser Gin Ser Pro 480 Met Lys Ala Tyr Thr 560 Gly Leu Phe Thr Thr Thr Giu Gin 225 Thr Asn Asp Ala Ala 305 Lys Ile Gly Val1 Thr 385 As n Leu As n As n Lys 465 Phe Asn Arg Tyr Gly 545 Asn Phe Thr Gin Ala Thr Thr Ile 210 Leu Ile Gly Ile Gin 290 Gin Gly Leu Pro Ile 370 Leu Gin Asn Leu Pro 450 Tyr Arg Gly As n Ile 530 Vai Phe Ala Met Phe Leu Lys Gin 195 Pro Thr Met Gly Phe 275 Thr Ile Phe As n Ile 355 Thr As n Asp Thr Pro 435 Asn Asn Arg Ile Trp 515 Lys Gly Leu Leu Met .595 Leu Asn Thr 180 Asn Val1 Tyr Gin Lys 26b Gly Ala Thr Al a Leu 340 Gin As n Ser Arg Leu 420 Asn Ser Gin Phe Gly 500 Gly Ser Met Gly Al a 580 Asn Phe Gin 165 Gly Asn Ser His Val1 245 Al a Pro Leu Gin Gin 325 Ala Gly Asn Leu Ala 405 Asn Al a Pro Leu Gly 485 Val1 Leu Asn Asp Lys 565 Gly Gly Asp Asn Val1 Ile Tyr Asn 230 Leu Trp Ser Ala Pro 310 Glu Lys Asp Thr Glu 390 Leu Lys Lys Glu Gin 470 Val1 Gin Arg Phe Al a 550 Asn Thr Ile Leu Gin Val Ile Ser 215 Asn Ile Gly Phe Lys 295 Asn Met Gin Leu Trp 375 Gin Ala Asp Ser Gly 455 Thr Ile Val Tyr Phe 535 Leu Asn Ser Tyr Gly Gly Asn Giu 200 Gly Ala Thr Leu As n 280 Thr Asn Leu Val Giu 360 Gly His Gin Ser Leu 440 Leu Ilie Asp Gly Tyr 520 Asn Tyr Lys Trp Asn 600 Leu Gly Gly Met Pro Ala Leu Ile 185 His Gly Giu Gin Ser 265 Ala Gin Phe Asn Ala 345 Glu Ser Thr Thr Lys 425 Gin Leu Ala Phe Tyr 505 Gly Ser Asn Leu Leu 585 Ala Arg 170 Gin Tyr Ser Asn Lys 250 Ser Ile Gin Asn Arg 330 Asn Cys Gly Ala Ile 410 Ala Asn Thr Gin Gin 490 Lys Phe Al a Phe Ser 570 Asn Asn Met Gin Tyr Ser Leu 235 Pro Thr Asn Leu Pro 315 Ala Asn Lys Cys Tyr 395 Leu Ile Met Tyr Glu 475 Asn Gin Phe Ser Ile 555 Val1 Ser Val1 Asn Thr Thr Phe 220 Leu His Pro Glu As n 300 Tyr Giu Phe Ala Ala 380 Tyr Asn Asn Thr Ser 460 Leu Asn Phe Asp Asp 540 Asn Gly Gin Ser Leu Asn Glu 205 Ser Gin Val Gly Met 285 Al a Thr Al a His Gly 365 Phe Gly Phe Ser His 445 Leu Gly Asn Phe Tyr 525 Val1 Asp Leu Gin Thr 605 Al a Tyr 190 Asn Pro Gin Gin As n 270 Ile Asn Ser Gin Ser 350 Ser Vai Asn Lys Gly 430 Ala Asp Lys Gly Gly 510 As n Trp Lys Phe Val 590 Ser Arg SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 610 615 Pro Lys Lys Lys Asp Ser Asp His Ala 625 630 Gly Phe Lys Ile Pro Thr Ile Asn Thr 620 Ala Gin His Gly Ile Glu Leu 635 Asn Tyr Tyr Ser Phe Met Gly 650 Ser Leu Phe Leu Asn Tyr Val 665 670 640 Ala Lys Leu 655 Phe Ala Tyr 645 Arg Met Tyr Glu Tyr Arg 660 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 2270 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 130...2049 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 130...193 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATTGAGCGCA TCAAAACACC CTAAAACTTT TTTGAAATCC AATAAATTTA TGTTATAATT AAACGCATTG TAAATAAATT CTCATTTTGA TACATTTTTA CAATAAAACA TTACTTTAAG GAACATCTT ATG AAA AAA ACG AAA AAA ACG ATT CTG CTT TCT CTA ACT CTC Met Lys Lys Thr Lys Lys Thr Ile Leu Leu Ser Leu Thr Leu -15 GCG GCG TCA TTG CTC CAT GCT GAA GAC AAC GGC GTT TTT TTA AGC GTG Ala Ala Ser Leu Leu His Ala Glu Asp Asn Gly Val Phe Leu Ser Val 120 171 219 GGT TAT CAA ATC GeF GAA Gly.Tyr Gin Ile Gly Glu 15 GTG CAA AAA CTT TCA GAC Val Gin Lys Leu Ser Asp AAC GAT AAT GGC ACA AAC Asn Asp Asn Gly Thr Asn GCG GTT CAA AAA GTG AAA Ala Val Gin Lys Val Lys 20 AAC GCC GAC AAG Asn Ala Asp Lys ACT TAT GAA CAA TTA AGC CGG CTT TTA ACC Thr Tyr Glu Gin Leu Ser Arg Leu Leu Thr 315 363 TCA AAG ACA AGC GCG CAA Ser Lys Thr Ser Ala Gin 50 ATC AAC CAA GCG Ile Asn Gin Ala SUBSTITUTE SHEET (RULE 26) WO 98/43479 GTT AAT AAT Val Asn Asn PCTIUS98/06421 TTG AAC GAA CGC Leu Asn Glu Arg

GCA

Ala 65 AAA ACT TTA GCC Lys Thr Leu Ala GGT GGG ACA ACC Gly Gly Thr Thr AGA TCG GTG TTA Arg Ser Val Leu AAT TCC Asn Ser CCT GCC TAT Pro Ala Tyr CAA GCC Gln Ala 80 ACG CTT TTA GCG Thr Leu Leu Ala

TTG

Leu

GGG

Gly CTA TGG PAT AGC Leu Trp Asn Ser

ATG

Met 95 GGT TAT GCG GTC Gly Tyr Ala Val

ATA

Ile 100 TGC GGA GGT TAT Cys Gly Gly Tyr

ACC

Thr 105 PAA AGT CCA GGC GPA PAC PAT CPA AAA Lys Ser Pro Gly Glu Asn Asn Gln Lys 110 TTC CAC TAC ACC Phe His Tyr Thr GAT GAG Asp Glu 120 PAT GGC PAT Asn Gly Asn GGC ACT CAT Gly Thr His 140

GGC

Gly 125 ACT ACA ATC PAT Thr Thr Ile Asn

TGC

Cys 130 GGT GGG AGC ACA Gly Gly Ser Thr PAT AGT PAT Asn Ser Asn 135 GAC AAA PAT Asp Lys Asn AGT TCT AGT GGC ACA PAT ACA TTA AAA Ser Ser Ser Gly Thr Asn Thr Leu Lys

GCA

Ala 150 GTT TCT Val Ser 155 CTA TCT ATT GAG Leu Ser Ile Glu

CAA

Gln 160 TAT GPA PAA ATC Tyr Glu Lys Ile

CAT

His 165 GAA GCT TAT CAG Glu Ala Tyr Gln

ATT

Ile 170 CTT TCA AAA GCT Leu Ser Lys Ala

TTA

Leu 175 PAA CPA GCC GGG Lys Gln Ala Gly

CTT

Leu 180 GCT CCT TTA PAT Ala Pro Leu Asn

AGO

Ser 185 AAA GGG GPA PAG Lys Gly Glu Lys

TTA

Leu 190 GAA GCG CAT GTA Glu Ala His Val ACA TCA AAA CCA Thr Ser Lys Pro GAA PAT Glu Asn 200 PAT AGT CAA Asn Ser Gln GCG CPA PAT Ala Gln Asn 220

ACT

Thr 205 AAA ACG ACA ACT Lys Thr Thr Thr

TCT

Ser 210 GTT ATT GAT ACO Val Ile Asp Thr ACT PAT GAT Thr Asn Asp 215 ACC CTT AAA Thr Leu Lys CTT TTG ACT CAA Leu Leu Thr Gln CAA ACG ATT GTC Gln Thr Ile Val

PAT

Asn 230 GAT TAT Asp Tyr 235 TGC CCC ATG TTG Cys Pro Met Leu

ATA

Ile 240 GCG PAA TCT AGT Ala Lys Ser Ser

AGT

Ser 245 GAA AGT AGT GGC Glu Ser Ser Gly

GCA

Ala 250 GCT ACT ACA PAC Ala Thr Thr Asn CCT TCA TGG CAA Pro Ser Trp Gln

ACA

Thr 260 GCC GGT GGC GGC Ala Gly Gly Gly

AAA

Lys 265 939 987 1035 PAT TCA TGT GCG Asn Ser Cys Ala

ACT

Thr 270 TTT GGT GCG GAG Phe Gly Ala Glu

TTT

Phe 275 AGT GCC GCT TCA Ser Ala Ala Ser GAC ATG Asp Met 280 ATT PAT PAT GCG CPA AAA ATC GTT CPA GAA ACC CPA CPA CTC AGC GCC 1083 SUBSTITUTE SHEET (RULE 26) WO 98/43479 Ile Asn Asn AAC CAA CCA Asn Gin Pro 300 PCT/US98/06421 Ala 285 Gin Lys Ile Val Gin 290 Glu Thr Gin Gin Leu Ser Ala 295 CTT AAC ACC Leu Asn Thr AAA AAT ATC ACA Lys Asn Ile Thr CCC CAT AAT CTC Pro His Asn Leu

AAC

Asn 310 1131 CCT AGC Pro Ser 315 AGT CTT ACG GCT Ser Leu Thr Ala

TTA

Leu 320 GCT CAA AAA ATG CTC AAA AAT GCG CAA Ala Gin Lys Met Leu Lys Asn Ala Gin 325 1179

TCT

Ser 330 CAA GCA GAA ATT Gin Ala Giu Ile

TTA

Leu 335 AAA CTA GCC AAT Lys Leu Ala Asn

CAA

Gin 340 GTG GAG AGC GAT Val Glu Ser Asp

TTT

Phe 345 1227 1275 AAC AAA CTT TCT Asn Lys Leu Ser

TCA

Ser 350 GGC CAT CTT.AAA Gly His Leu Lys

GAC

Asp 355 TAC ATA GGG AAA Tyr Ile Gly Lys TGC GAT Cys Asp 360 GCG AGC GCT Ala Ser Ala AAT TGG GGG Asn Trp Gly 380

ATA

Ile 365 AGC AGT GCG AAT Ser Ser Ala Asn ACA ATG CAA AAT Thr Met Gin Asn CAA AAG AAC Gin Lys Asn 375 TCT TCA TTA Ser Ser Leu 1323 1371 AAC GGG TGT GCT Asn Giy Cys Ala

GGC

Gly 385 GTG GAA GAA ACT Val Giu Giu Thr

CTG

Leu 390 AAA ACA Lys Thr 395 AGT GCC GCT GAT Ser Ala Ala Asp

TTT

Phe 400 AAC AAC CAA ACG Asn Asn Gin Thr

CCA

Pro 405 CAA ATC AAT CAA Gin Ile Asn Gin

GCG

Ala 410 CAA AAC CTA GCC Gin Asn Leu Ala

AAC

Asn 415 ACC CTT ATT CAA Thr Leu Ile Gin

GAA

Glu 420 CTT GGC AAC AAC Leu Gly Asn Asn

CCT

Pro 425 1419 1467 1515 TTT AGG AAT ATG Phe Arg Asn Met

GGC

Gly 430 ATG ATC GCT TCT Met Ile Ala Ser

TCA

Ser 435 ACC ACG AAT AAC Thr Thr Asn Asn GGC GCC Gly Ala 440 TTG AAT GGC Leu Asn Gly AAG AAA AGA Lys Lys Arg 460

CTT

Leu 445 GGG GTG CAA GTG Gly Val Gin Val

GGT

Gly 450 TAT AAG CAA TTT Tyr Lys Gin Phe TTT GGG GAA Phe Gly Glu 455 TAC AAC CAC Tyr Asn His 1563 1611 TGG GGG TTA AGG Trp Gly Leu Arg TAT GGT TTC TTT Tyr Gly Phe Phe

GAT

Asp 470 GCC TAT Ala Tyr 475 ATC AAA TCC AAT Ile Lys Ser Asn

TTC

Phe 480 TTT AAC TCG GCT Phe Asn Ser Ala GAT GTG TGG ACT Asp Val Trp Thr 1659 1707

TAT

Tyr 490 GGG GTG GGC AGC Gly Val Gly Ser

GAT

Asp 495 TTA TTG TTT AAT Leu Leu Phe Asn ATC AAT CAT AAA Ile Asn Asp Lys

AAC

Asn 505 ACC AAC TTT TTA GGC AAG AAT AAC AAG ATT TCA GTG GGA TTT TTT GGA Thr Asn Phe Leu Gly Lys Asn Asn Lys 1le Ser Val Gly Phe Phe Gly 1755 SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 GGT ATC GCC Gly Ile Ala TTA AAA ACC Leu Lys Thr 540

TTA

Leu 525 GCA GGG ACT TCA Ala Gly Thr Ser

TGG

Trp 530 CTT AAT TCT CAA Leu Asn Ser Gin TTC GTG AAT Phe Val Asn 535 ACG GCT AAC Thr Ala Asn 1803 1851 ATC AGC AAT GTT Ile Ser Asn Val AGC GCT AAA GTG Ser Ala Lys Val TTC CAA Phe Gin 555 TTT TTA TTC AAT Phe Leu Phe Asn

TTG

Leu 560 GGC TTG AGA ACC Gly Leu Arg Thr

AAT

Asn 565 CTC GCT AGA CCT Leu Ala Arg Pro

AAG

Lys 570 AAA AAA GAT AGT Lys Lys Asp Ser CAT GCG GCT CAA His Ala Ala Gin GGC ATG GAA TTG Gly Met Glu Leu

GGC

Gly 585 1899 1947 1995 2043 GTG AAA ATC CCT Val Lys Ile Pro

ACC

Thr 590 ATT AAC ACG AAT Ile Asn Thr Asn

TAT

Tyr 595 TAT TCT TTT CTA Tyr Ser Phe Leu GAC ACT Asp Thr 600 AAA CTA GAA Lys Leu Glu

TAT

Tyr 605 CGA AGG CTT TAT Arg Arg Leu Tyr GTG TAT CTC AAT TAT GTG TTT Val Tyr Leu Asn Tyr Val Phe 615 GCC TAT Ala Tyr TAAAAACCCT CTTTTTAAAA AAGGGGGGGC TTTAAAAAAC CTCTAAAGAT AA 2101

AAATTTTCAA

CCATTTTTAA

GGGGCGTTTG

AAAACAATCA TTAAACCCTA AAAAAGAAAT TTTTCCATGG CAAACTCCTT TTTAGAATTT TTTTGCAACA ATCTTTTCGA AACTATCCAA

TTTAAGGTAT

ATCCCCATAA

CAAGCTTTA

AATGCTTTCG

TCGCTCTTAT

2161 2221 2270 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 640 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...21 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Met Lys Lys Thr Lys Lys Thr Ile Leu Leu -15 Ser Leu Leu His Ala Glu Asp Asn Gly Val Ser Leu Thr Leu Ala Ala Phe Leu Ser Val Gly Tyr SUBSTITUTE SHEET (RULE 26) WO 98/43479 116 PCTIUS98/06421 Gin Lys Asn Asn Pro Trp Pro Asn His 140 Leu Ser Glu Gin Asn 220 Cys Thr Cys Asn Pro 300 Ser Ala Leu Ala Gly 380 Ser Asn Asn Gly Ile Leu Gly Leu Aila Asn Gly Giy 125 Ser Ser Lys Lys Thr 205 Leu Pro Thr Al a Aia 285 Lys Leu Giu Ser Ile 365 Asn Aia Leu Met Leu 445 Gly Ser Thr Asn Tyr Ser Giu 110 Thr Ser Ile Aia Leu 190 Lys Leu Met Asn Thr 270 Gin Asn Thr Ile Ser 350 Ser Gly Ala Ala Gly 430 Gly Giu Asp Asn Giu Gin Met Asn Thr Ser Giu Leu 175 Giu Thr Thr Leu Aia 255 Phe Lys Ile Al a Leu 335 Gly Ser Cys Asp Asn 415 Met Val Aila Thr Ser Arg Ala Gly Asn Ile Gly Gin 160 Lys Al a Thr Gin Ile 240 Pro Gly Ile Thr Leu 320 Lys His Ala Ala Phe 400 Thr Ile Gin Val1 Tyr Lys Al a 65 Thr Tyr Gin Asn Thr 145 Tyr Gin His Thr Ala 225 Ala Ser Al a Val Gin 305 Ala Leu Leu Asn Gly 385 Asn Leu Ala Val Gin Giu Thr 50 Lys Leu Ala Lys Cys 130 Asn Giu Ala Val1 Ser 210 Gin Lys Trp Glu Gin 290 Pro Gin Ala Lys Met 370 Val Asn Ile Ser Gly 450 Lys Gin 35 Ser Thr Leu Val Asp 115 Gly Thr Lys Gly Thr 195 Val Thr Ser Gin Phe 275 Giu His Lys As n Asp 355 Thr Glu Gin Gin Ser 435 Tyr Val 20 Leu Ala Leu Ala Ile 100 Phe Gly Leu Ile Leu 180 Thr Ile Ile Ser Thr 260 Ser Thr As n Met Gin 340 Tyr Met Giu Thr Glu 420 Thr Lys Lys Ser Gin Ala Leu 85 Cys His Ser Lys His 165 Al a Ser Asp Val Ser 245 Al a Aila Gin Leu Leu 325 Val Ile Gin Thr Pro 405 Leu Thr Gin Asn Arg Ile Gly 70 Arg Giy Tyr Thr Ala 150 Glu Pro Lys Thr Asn 230 Glu Gly Ala Gin Asn 310 Lys Giu Gly Asn Leu 390 Gin Gly Asn Phe Al a Leu Asn Gly Ser Gly Thr Asn 135 Asp Ala Leu Pro Thr 215 Thr Ser Gly Ser Leu 295 Leu Asn Ser Lys Gin 375 Ser Ile Asn Asn Phe 455 Asp Leu Gin Thr Val1 Tyr Asp 120 Ser Lys Tyr As n Giu 200 Asn Leu Ser Gly Asp 280 Ser Asn Ala Asp Cys 360 Lys Ser Asn Asn Gly 440 Gly Lys Thr Ala Thr Leu Thr 105 Giu Asn Asn Gin Ser 185 Asn Asp Lys Gly Lys 265 Met Ala Thr Gin Phe 345 Asp Asn Leu Gin Pro 425 Al a Glu Val Asn Val Asn Gly Lys Asn Gly Val Ile 170 Lys Asn Ala Asp Ala 250 Asn Ile Asn Pro Ser 330 Asn Ala Asn Lys Al a 410 Phe Leu Lys Gin Asp As n Ser Leu Ser Gly Thr Ser 155 Leu Gly Ser Gin Tyr 235 Ala Ser Asn Gin Ser 315 Gin Lys Ser Trp Thr 395 Gin Arg Asn Lys SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 Arg 460 Ile Val Phe Ala Thr 540 Phe Lys Ile Glu Trp Lys Gly Leu Leu 525 Ile Leu Asp Pro Tyr Gly Ser Ser Gly 510 Ala Ser Phe Ser Thr 590 Arg Leu Arg Tyr Tyr Gly 465 Asn Asp 495 Lys Gly Asn Asn His 575 Ile Arg Phe 480 Leu Asn Thr Val Leu 560 His Asn Leu Phe Leu Asn Ser Tyr 545 Gly Ala Thr Tyr Asn Phe Lys Trp 530 Ser Leu Ala Asn Ser 610 Ser Asn Ile 515 Leu Ala Arg Gin Tyr 595 Val Phe Ala Phe 500 Ser Asn Lys Thr His 580 Tyr Tyr Phe Ser 485 Ile Val Ser Val Asn 565 Gly Ser Leu Asp 470 Asp Asn Gly Gin Asn 550 Leu Met Phe Asn Tyr Val Asp Phe Phe 535 Thr Ala Glu Leu Tyr 615 Asn Trp Lys Phe 520 Val Ala Arg Leu Asp 600 Val His Thr Asn 505 Gly Asn Asn Pro Gly 585 Thr Phe Ala Tyr 490 Thr Gly Leu Phe Lys 570 Val Lys Ala Tyr 475 Gly Asn Ile Lys Gin 555 Lys Lys Leu Tyr INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2248 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 173...2128 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 173...224 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID TGGTTTTATC GTTACAAAAT TCAACATTTC AAAGATAAAT AAGTTAAAAT ACCCCAAAAT CTTTTTTTTT TTTTTGAAAT CCAATCAATT TATAGTAAAA TTAGGTTCAT TGTAAATATA TTATCACTTC ATGATATTCT TACAACAAAA ACATTACTTT AAGGAACATT TT ATG AAA Met Lys AAG ACA ATT CTG CTC TCT CTC TCT GCT TCA TCG CTC TTG CAC GCT GAA Lys Thr Ile Leu Leu Ser Leu Ser Ala Ser Ser Leu Leu His Ala Glu -10 -5 1 GAC AAC GGC TTT TTT GTG AGC GCC GGC TAT CAA ATC GGC GAA GCG GTG 120 178 226 274 SUBSTITUTE SHEET (RULE 26) WO 98/43479 Asp Asn Gly CAA ATG GTC Gin Met Val PCT/US98/0642 I Phe Val Ser Ala Gly Tyr Gin Ile Gly Giu Ala Val GAA AAA TAC Giu Lys Tyr AAA AAC ACC GGT Lys Asn Thr Gly

GAA

Giu TTG AAA AAC TTG Leu Lys Asn Leu GAG CAA Giu Gin TTA AGC CAG TAT Leu Ser Gin Tyr

TTA

Leu 40 AAT CAA GTG GCT Asn Gin Val Ala

TCG

Ser TTG AAG CAA AGC Leu Lys Gin Ser

ATT

Ile CAA AAC GCC AAC Gin Asn Ala Asn ATT GAG CTG GTC Ile Glu Leu Val AGC TCT TTA AAC Ser Ser Leu Asn

TAT

Tyr TTA AAA AGC TTT Leu Lys Ser Phe

ACC

Thr AAC AAC AAC TAT Asn Asn Asn Tyr

AAC

Asn 75 AGC ACC ACC CAA Ser Thr Thr Gin TCG CCC Ser Pro ATC TTT AAT Ile Phe Asn AGT CTT TAT Ser Leu Tyr 100

CC

Aia GTG CAA GCC GTT Val Gin Ala Val ACT TCG GTA TTG Thr Ser Val Leu GGT TTT TGG Gly Phe Trp AAA AAG GTG Lys Lys Val GCG GGG AAT TAC Ala Gly Asn Tyr

TTC

Phe 105 ACT TTT TTT GTG Thr Phe Phe Val GGT CAT Gly Asp ACT CCC CAA CCC Ser Gly Gin Pro

GCT

Aia 120 AGT GTC CAG GGT Ser Vai Gin Giy

AAC

Asn 125 CCT CCT TTT AAA Pro Pro Phe Lys

ACG

Thr 130 ATT ATA GAG AAC TGC TCA GGA ATT GAA AAC TGC GCT ATC CAT Ile Ile Giu Asn Cys Ser Gly Ile Giu Asn Cys Ala Met Asp

CAA

Gin 145 ACC ACT TAT CAT Thr Thr Tyr Asp

AAG

Lys 150 ATG AAA AAA CTC Met Lys Lys Leu

GCT

Aila 155 GAA GAC CTC CAA Giu Asp Leu Gin GCG GCT Aia Aia 160 CAA ACA AAC Gin Thr Asn TGT GCT GCA Cys Ala Ala 180

TCT

Ser 165 CCC ACT AAA GGC Ala Thr Lys Gly

AAC

Asn 170 AAT CTT TGC OCT Asn Leu Cys Ala TTA TCC CCC Leu Ser Giy 175 ACC GTG AGC Thr Val Ser ACA GAC TCA ACA Thr Asp Ser Thr

TCA

Ser 185 AAC CCA CCA AAC Asn Pro Pro Asn AAC OCT Asn Ala 195 CTT AAT TTG GCG Leu Asn Leu Ala

CAA

Gin 200 CAG CTT ATG CAT Gin Leu Met Asp

TTA

Leu 205 ATC GCA AAC ACT Ile Ala Asn Thr

AAA

Lys 210 ACG GCT ATG ATG Thr Ala Met Met

TGG

Trp 215 AAA AAT ATC GTC Lys Asn Ile Val AGT GCC OTT TCA Ser Gly Val Ser

AAC

Asn 225 ACA TCC GGT GCT ATC ACA TCC ACT AAT TAC CCA ACG CAA TAC GCG OTG Thr Ser Oly Aia Ile Thr Ser Thr Asn Tyr Pro Thr Gin Tyr Ala Val SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 235 240 TTT AAC AAC Phe Asn Asn CTT TCT CAA Leu Ser Gin 260

ATT

Ile 245 AAG GCG ATG ATA Lys Ala Met Ile CCC ATT Pro Ile 250 TTG CAA CAA Leu Gin Gin GCG GTT ACG Ala Val Thr 255 GCT CAA GCC Ala Gin Ala 994 1042 AGC AAC CAC ACC Ser Asn His Thr

CTA

Leu 265 TCT GCT AGC TTG Ser Ala Ser Leu ACA GGA Thr Gly 275 TCT CAA ACA AAC Ser Gin Thr Asn

CCT

Pro 280 AAA TTC GCT AAA Lys Phe Ala Lys

GAC

Asp 285 ATC TAC ACT TTC Ile Tyr Thr Phe

GCT

Ala 290 CAA AAC CAA AAG Gin Asn Gin Lys

CAA

Gin 295 GTC ATC TCT TAC Val Ile Ser Tyr

GCT

Ala 300 CAA GAC ATT TTC Gin Asp Ile Phe

AAC

Asn 305 1090 1138 1186 CTC TTT AAT TCT Leu Phe Asn Ser CCT GCA GAG CAG Pro Ala Glu Gin

TAT

Tyr 315 AAG TAT CTA GAG Lys Tyr Leu Glu AAA GCT Lys Ala 320 TAC TTG AAA Tyr Leu Lys CAA GTG GTG Gin Val Val 340

ATA

Ile 325 CCC AAT GCG GGT Pro Asn Ala Gly

TCA

Ser 330 ACG CCT ACT AAC Thr Pro Thr Asn CCT TAC AGA Pro Tyr Arg 335 AAC AAT GTG Asn Asn Val 1234 1282 AAT TTA AAC CAA Asn Leu Asn Gin

GAA

Glu 345 GTT CAG ACG ATT Val Gin Thr Ile

AAA

Lys 350 AGT TAT Ser Tyr 355 TAT GGT AAC CGG Tyr Gly Asn Arg

GTG

Val 360 GAT GCG GCT TTA Asp Ala Ala Leu GTG GCT AGA GAT Val Ala Arg Asp

GTT

Val 370 TAT AAC CTA AAA Tyr Asn Leu Lys

TCC

Ser 375 AAT CAA GCA GAA Asn Gin Ala Glu GTA ACC GCC TAT Val Thr Ala Tyr 1330 1378 1426 1474 GAC GCT AAG ACT Asp Ala Lys Thr AGC GAA GAG ATT Ser Glu Glu lie

TCT

Ser 395 AAA CTC CCG CAC Lys Leu Pro His AAT CAA Asn Gin 400 GTC AAT ACA Val Asn Thr GCA GCA GGC Ala Ala Gly 420

AAA

Lys 405 GAC ATT GTT ACA Asp Ile Val Thr

CTA

Leu 410 CCT TAC GAT AAA AAC GCC CCA Pro Tyr Asp Lys Asn Ala Pro CAA TCC AAC TAC Gin Ser Asn Tyr

CAA

Gin 425 ATC AAC CCA GAG Ile Asn Pro Glu CAA TCC AAT Gin Ser Asn 1522 CTT AAC Leu Asn 435 CAA GCT TTA GCA GCG ATG AGC AAT AAC Gin Ala Leu Ala Ala Met Ser Asn Asn 440

CCC

Pro 445 TTT AAA AAA GTG Phe Lys Lys Val 1570

GGC

Gly 450 ATG ATC AGC TCT Met Ile Ser Ser

CAA

Gin 455 AAC AAT AAC GGC Asn Asn Asn Gly TTG AAC GGG CTT Leu Asn Gly Leu

GGC

Gly 465 1618 SUBSTITUTE SHEET (RULE 26) WO 98/43479 120 PCT/US98/06421 GTG CAA GTG GGT Val Gin Val Gly

TAT

Tyr 470 AAG CAA TTC TTT Lys Gin Phe Phe GAA AGC AAA AGA Glu Ser Lys Arg TGG GGG Trp Gly 480 1666 TTA AGG TAT Leu Arg Tyr AGC TTC TTT Ser Phe Phe 500

TAC

Tyr 485 GGA TTC TTT GAT Gly Phe Phe Asp

TAC

Tyr 490 AAC CAC GGC TAC Asn His Gly Tyr ATC AAA TCC Ile Lys Ser 495 GGT GGG AGC Gly Gly Ser 1714 1762 AAC TCT TCT TCT Asn Ser Ser Ser ATA TGG ACT TAT Ile Trp Thr Tyr

GGC

Gly 510 GAT TTG Asp Leu 515 TTA GTG AAT ATT Leu Val Asn Ile

ATC

Ile 520 AAC GAT AGC ATC Asn Asp Ser Ile ACA AGA AAG AAC Thr Arg Lys Asn 525 CTA GCA GGG ACT Leu Ala Gly Thr

AAC

Asn

ACA

Thr 545 CTC TCC GTG GGT Leu Ser Val Gly TTT GGA GGC ATC Phe Gly Gly Ile

CAA

Gin 540 1810 1858 1906 TGG CTT AAT TCT Trp Leu Asn Ser

CAA

Gin 550 TAC GTG AAT TTA Tyr Val Asn Leu GCG TTC AAT AAC Ala Phe Asn Asn CCT TAC Pro Tyr 560 AGC GCG AAA Ser Ala Lys TTG AGG ACG Leu Arg Thr 580

GTC

Val 565 AAT GCT ACC AAT Asn Ala Thr Asn

TTC

Phe 570 CAA TTC TTG TTC Gin Phe Leu Phe AAT CTC GGC Asn Leu Gly 575 GAA CAT TCC Glu His Ser 1954 2002 AAT CTC GCT ACA Asn Leu Ala Thr AGG AAA AAA GAC Arg Lys Lys Asp

AGC

Ser 590 GCG CAA Ala Gin 595 CAT GGC ATT GAA His Gly Ile Glu

TTG

Leu 600 GGT ATT AAA ATC Gly Ile Lys Ile ACC ATT ACC ACG Thr Ile Thr Thr

AAT

Asn 610 TAC TAT TCT TTT Tyr Tyr Ser Phe CTA GGC ACT CAA TTG Leu Gly Thr Gin Leu 615 TAT GTG TTC GCT TAC Tyr Val Phe Ala Tyr 635

CAA

Gin 620 TAC AGA AGG CTC Tyr Arg Arg Leu

TAT

Tyr 625 2050 2098 2151 AGC GTG TAT CTC Ser Val Tyr Leu

AAT

Asn 630 TGAGTGATTC AAGCTCTCTT CTT TAAGGGGGTT TAGAAAAATC GCAACGCCAA GCTTTTTATC AACTAACGGC GCGACAACAA ACCCTAACGC TACGCTC INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 652 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal GTTGGTGATA AAATCTACAA 2211 2248 SUBSTITUTE SHEET (RULE 26) WO 98/43479 121 PCT/US98/06421 (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...17 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Lys Lys Thr Ile Leu Leu Ser Leu Ser Ala Ser Ser Leu Leu His -10 Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gin Ile Gly Glu 1 5 10 Ala Val Gin Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu Asn Glu 25 Lys Tyr Glu Gin Leu Ser Gin Tyr Leu Asn Gin Val Ala Ser Leu Lys 40 Gin Ser Ile Gin Asn Ala Asn Asn Ile Glu Leu Val Asn Ser Ser Leu 55 Asn Tyr Leu Lys Ser Phe Thr Asn Asn Asn Tyr Asn Ser Thr Thr Gin 70 Ser Pro Ile Phe Asn Ala Val Gin Ala Val Ile Thr Ser Val Leu Gly 85 90 Phe Trp Ser Leu Tyr Ala Gly Asn Tyr Phe Thr Phe Phe Val Gly Lys 100 105 110 Lys Val Gly Asp Ser Gly Gin Pro Ala Ser Val Gin Gly Asn Pro Pro 115 120 125 Phe Lys Thr Ile Ile Glu Asn Cys Ser Gly Ile Glu Asn Cys Ala Met 130 135 140 Asp Gin Thr Thr Tyr Asp Lys Met Lys Lys Leu Ala Glu Asp Leu Gin 145 150 155 Ala Ala Gin Thr Asn Ser Ala Thr Lys Gly Asn Asn Leu Cys Ala Leu 160 165 170 175 Ser Gly Cys Ala Ala Thr Asp Ser Thr Ser Asn Pro Pro Asn Ser Thr 180 185 190 Val Ser Asn Ala Leu Asn Leu Ala Gin Gin Leu Met Asp Leu Ile Ala 195 200 205 Asn Thr Lys Thr Ala Met Met Trp Lys Asn Ile Val Ile Ser Gly Val 210 215 220 Ser Asn Thr Ser Gly Ala Ile Thr Ser Thr Asn Tyr Pro Thr Gin Tyr 225 230 235 Ala Val Phe Asn Asn Ile Lys Ala Met Ile Pro Ile Leu Gin Gin Ala 240 245 250 255 Val Thr Leu Ser Gin Ser Asn His Thr Leu Ser Ala Ser Leu Gin Ala 260 265 270 Gin Ala Thr Gly Ser Gin Thr Asn Pro Lys Phe Ala Lys Asp Ile Tyr 275 280 285 Thr Phe Ala Gin Asn Gin Lys Gin Val Ile Ser Tyr Ala Gin Asp Ile 290 295 300 Phe Asn Leu Phe Asn Ser Ile Pro Ala Glu Gin Tyr Lys Tyr Leu Glu 305 310 315 Lys Ala Tyr Leu Lys Ile Pro Asn Ala Gly Ser Thr Pro Thr Asn Pro 320 325 330 335 Tyr Arg Gin Val Val Asn Leu Asn Gin Glu Val Gin Thr Ile Lys Aso 340 345 350 Asn Val Ser Tyr Tyr Gly Asn Arg Val Asp Ala Ala Leu Ser Val Ala SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 Arg Tyr Asn 400 Ala Ser Lys Leu Trp 480 Lys Gly Asn Thr Pro 560 Leu His Thr Leu Asp Asn 385 Gin Pro Asn Val Gly 465 Gly Ser Ser Asn Thr 545 Tyr Gly Ser Thr Tyr 625 Val 370 Asp Val Ala Leu Gly 450 Val Leu Ser Asp Lys 530 Trp Ser Leu Ala Asn 610 Ser 355 Tyr Ala Asn Ala Asn 435 Met Gin Arg Phe Leu 515 Leu Leu Ala Arg Gin 595 Tyr Val Asn Lys Thr Gly 420 Gin Ile Val Tyr Phe 500 Leu Ser Asn Lys Thr 580 His Tyr Tyr Leu Thr Lys 405 Gin Ala Ser Gly Tyr 485 Asn Val Val Ser Val 565 Asn Gly Ser Leu Lys Ser 375 Leu Ser 390 Asp Ile Ser Asn Leu Ala Ser Gin 455 Tyr Lys 470 Gly Phe Ser Ser Asn Ile Gly Leu 535 Gin Tyr 550 Asn Ala Leu Ala Ile Glu Phe Leu 615 Asn Tyr 360 Asn Glu Val Tyr Ala 440 Asn Gin Phe Ser Ile 520 Phe Val Thr Thr Leu 600 Gly Val Gin Glu Thr Gin 425 Met Asn Phe Asp Asp 505 Asn Gly Asn Asn Ala 585 Gly Thr Phe Ala Glu Ile Ser 395 Leu Pro 410 Ile Asn Ser Asn Asn Gly Phe Gly 475 Tyr Asn 490 Ile Trp Asp Ser Gly Ile Leu Thr 555 Phe Gin 570 Arg Lys Ile Lys Gin Leu Ala Tyr 635 Ile 380 Lys Tyr Pro Asn Ala 460 Glu His Thr Ile Gin 540 Ala Phe Lys Ile Gin 620 365 Val Thr Leu Pro Asp Lys Glu Gin 430 Pro Phe 445 Leu Asn Ser Lys Gly Tyr Tyr Gly 510 Thr Arg 525 Leu Ala Phe Asn Leu Phe Asp Ser 590 Pro Thr 605 Tyr Arg Ala His Asn 415 Gin Lys Gly Arg Ile 495 Gly Lys Gly Asn Asn 575 Glu Ile Arg INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 2161 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 122...2056 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 122...179 OTHER INFORMATION: SUBSTITUTE SHEET (RULE 26) WO 98/43479 WO 9843479PCTIUS98/06421 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i7: CAAAAATCTT TTTTTTTTTT TTTTGAAATC CAATAAATTT ATGGTAAAGT TAAACATATT GTAAATAAAT TTTAATTTCT ATTCATGTTT ACAATAAAAA AATTACTTTA AGGAACATTT T ATG AAA AAG, ACA ATT CTA CTC TCT CTC TCT CTC TCG CTT TCA TCG CTC Met Lys Lys Thr Ile Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Leu 120 169 TTG CAC GCT GAA GAC Leu His Ala Giu Asp AAC GGC TTT TTT GTG AGC GCC Asn Gly Phe Phe Val Ser Ala TAT CAA ATC Tyr Gin Ile 217 265 GGC GAA Gly Giu CGG GTG CAA ATG Arg Val Gin Met

GTC

Val 20 AAA AAC ACC GGC Lys Asn Thr Gly

GAA

Glu TTG AAA AAC TTG Leu Lys Asn Leu

AAC

As n GAA AAA TAC GAG Giu Lys Tyr Giu TTA AGC CAA TCT Leu Ser Gin Ser

TTA

Leu 40 GCC CAA CTG GCT Ala Gin Leu Ala

TCG

Ser TTA AAA AAA AGC Leu Lys Lys Ser

ATT

Ile CAA ACG GCG AAC AAC ATT CAG GCT GTC AAC AAT Gin Thr Ala Asn Asn Ile Gin Ala Val Asn Asn OCT TTA AGC Ala Leu Ser GAA ACA TCG Glu Thr Ser

GAT

Asp TTA AAA AGC TTT Leu Lys Ser Phe AGT AAC AAC CAC Ser Asn Asn His ACA AAC AAA Thr Asn Lys ACT TCA GTA Thr Ser Val 409 457 CCC ATC TAC AAC Pro Ile Tyr Asn

ACC

Thr 85 GCG CAA GCT GTT Ala Gin Ala Val

ATC

Ile TTG GCT Leu Ala TTT TOG AGT CTT Phe Trp Ser Leu GCA GGG AAC GCT Ala Gly Asn Ala

ACC

Thr 105 AGT TTT CAT GTG Ser Phe His Val

ACC

Thr 110 GGT TTG AAT OAT Gly Leu Asn Asp

GGA

Gly 115 TCT AAT GCT CCT Ser Asn Ala Pro OGA AGA ATC CAT Oly Arg Ile His

CAA

Gin 125 505 553 601 OAT GGG AAC TGC Asp Gly Asn Cys

ACA

Thr 130 GGA TTA CAA CAA Gly Leu Gin Gin

TGT

Cys 135 TTT ATG AAT AAA Phe Met Asn Lys GAA ACT Giu Thr 140 TAT GAT AAA Tyr Asp Lys AAT CTC TGT Asn Leu Cys 160

ATG

Met 145 AAA GCG CTT GCC Lys Ala Leu Ala AAT CTC CAA AAA Asn Leu Gin Lys GCT CAA GGC Ala Gin Gly 155 AAT GGA AAC Asn Gly Asn 649 657 GCC TTA TCA GAA Ala Leu Ser Giu

TGC

Cys 165 CCT AGC GAT CAA Pro Ser Asp Gin

TTA

Leu 170 AAT GGA Asn Gly 175 AAC AAA ACT TCC Asn Lys Thr Ser

ATG

Met 180 ACT AAA GCT CTT Thr Lys Ala Leu

GAA

Glu 185 ACC GCG CAA CAG- Thr Ala Gin Gin SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421

CTT

Leu 190 ATG GAT TTA ATC Met Asp Leu Ile

GCA

Ala 195 AAC ACT AAA ACG GCT ATG ATG TGG AAA Asn Thr Lys Thr Ala Met Met Trp Lys 200

AAT

Asn 205 ATC GTC ATC GCA Ile Val Ile Ala GTT ACA AAC AGA Val Thr Asn Arg

CCC

Pro 215 GGT GGT GCT GGC Gly Gly Ala Gly GCT ATC Ala Ile 220 841 ACA TCC ACT Thr Ser Thr GCG ATG ATA Ala Met Ile 240

GGT

Gly 225 CCT GTA ACC GAC Pro Val Thr Asp

TAT

Tyr 230 GCG GTG TTT AAC Ala Val Phe Asn AAC ATT AAG Asn Ile Lys 235 CAA AGC AAC Gin Ser Asn CCC ATT TTG CAA Pro Ile Leu Gin

CAA

Gin 245 GCG GTT ACG CTT Ala Val Thr Leu

TCT

Ser 250 CAC ACC His Thr 255 CTA TCT GCT AGC Leu Ser Ala Ser

TTG

Leu 260 CAA GCT CAA GCC Gin Ala Gin Ala

ACA

Thr 265 GGA TCT CAA ACA Gly Ser Gin Thr

AAC

Asn 270 CCT AAA TTC GCT Pro Lys Phe Ala

AAA

Lys 275 GAC ATC TAC ACT TTC GCT CAA AAC CAA Asp Ile Tyr Thr Phe Ala Gin Asn Gin 280

AAG

Lys 285 1033 CAA GTC ATC TCT Gin Val Ile Ser

TAC

Tyr 290 GCT CAA GAC ATT Ala Gin Asp Ile

TTC

Phe 295 AAC CTC TTT AAT Asn Leu Phe Asn TCT ATC Ser Ile 300 1081 CCT GCA GAG Pro Ala Glu AAT GCG GGT Asn Ala Gly 320

CAG

Gin 305 TAT AAG TAT CTA Tyr Lys Tyr Leu

GAG

Glu 310 AAA GCT TAC TTG Lys Ala Tyr Leu AAA ATA CCC Lys Ile Pro 315 GTG AAT TTA Val Asn Leu 1129 1177 TCA ACG CCT ACT Ser Thr Pro Thr

AAC

Asn 325 CCT TAC AGA CAA Pro Tyr Arg Gin AAC CAA Asn Gin 335 GAA GTT CAG ACG Glu Val Gin Thr

ATT

Ile 340 AAA AAC AAT GTG Lys Asn Asn Val

AGT

Ser 345 TAT TAT GGT AAC Tyr Tyr Gly Asn

CGG

Arg 350 GTG GAT GCG GCT Val Asp Ala Ala

TTA

Leu 355 AGC GTG GCT AGA Ser Val Ala Arg GTT TAT AAC CTA Val Tyr Asn Leu 1225 1273 1321 TCC AAT CAA GCA Ser Asn Gin Ala ATC GTA ACC GCC Ile Val Thr Ala

TAT

Tyr 375 AAC GAC GCT AAG Asn Asp Ala Lys ACT TTG Thr Leu 380 AGC GAA GAG Ser Glu Glu ATT GTT ACA Ile Val Thr 400

ATT

Ile 385 TCT AAA CTC CCG Ser Lys Leu Pro AAT CAA GTC AAT Asn Gin Val Asn ACA AAA GAC Thr Lys Asp 395 1369 CTA CCT TAC GAT Leu Pro Tyr Asp AAA AAC Lys Asn 405 GCC CCA GCA GCA GGC CAA TCC Ala Pro Ala Ala Gly Gin Ser 410 1417 AAC TAC CAA ATC AAC CCA GAG CAG CAA TCC AAT CTT AAC CAA GCT TTA 1465 SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCTIUS98/06421 Asn Tyr 415 Gin Ile Asn Pro Glu 420 Gin Gin Ser Asn Leu 425 Asn Gin Ala Leu

GCA

Ala 430 GCG ATG AGC AAT Ala Met Ser Asn

AAC

Asn 435 CCC TTT AAA AAA GTG GGC ATG ATC AGC TCT Pro Phe Lys Lys Vai Gay Met Ile Ser Ser 440 445 1513 CAA AAC PAT AAC Gin Asn Asn Asn

GGC

Gly 450 GCT TTG AAC GGG Ala Leu Asn Gly

CTT

Leu 455 GGC GTG CPA GTG Gly Val Gin Val GGT TAT Gly Tyr 460 1561 PAG CPA TTC Lys Gin Phe TTC TTT GAT Phe Phe Asp 480

TTT

Phe 465 GGC GPA AGC AAA Gly Giu Ser Lys

AGA

Arg 470 TGG GGG TTA AGG Trp Giy Leu Arg TAT TAC GGA Tyr Tyr Gly 475 TTT PAC TCT Phe Asn Ser 1609 1657 TAC PAC CAC GGC Tyr Asn His Gly

TAC

Tyr 485 ATC AAA TCC AGC Ile Lys Ser Ser TCT TCT Ser Ser 495 GAT ATA TGG ACT Asp Ile Trp Thr

TAT

Tyr 500 GGC GGT GGG AGC Gly Gly Giy Ser

GAT

Asp 505 TTG TTA GTG PAT Leu Leu Val Asn

ATT

Ile 510 ATC PAC GAT AGC Ile Asn Asp Ser

ATC

Ile 515 ACA AGA PAG PAC Thr Arg Lys Asn AAG CTC TCC GTG Lys Leu Ser Vai 1705 1753 1801 CTT TTT GGA GGC Leu Phe Gay Gly

ATC

Ile 530 CAA CTA GCA GGG Gin Leu Ala Gly

ACT

Thr 535 ACA TGG CTT PAT Thr Trp Leu Asn TCT CAA Ser Gin 540 TAC GTG PAT Tyr Vai Asn GCT ACC PAT Ala Thr Asn 560

TTA

Leu 545 ACC GCG TTC PAT Thr Ala Phe Asn CCT TAC AGC GCG Pro Tyr Ser Ala AAA GTC PAT Lys Vai Asn 555 ACG PAT CTC Thr Asn Leu 1849 1897 TTC CPA TTC TTG Phe Gin Phe Leu

TTC

Phe 565 PAT CTC GGC TTG Asn Leu Gly Leu

AGG

Arg 570 GCT ACA Ala Thr 575 GCT AGG AAA AAA Ala Arg Lys Lys

GAC

Asp 580 AGC GPA CAT TCC Ser Giu His Ser CAA CAT GGC ATT Gin His Gly Ile

GAA

Glu 590 TTG GGT ATT AAA Leu Gly Ile Lys CCC ACC ATT ACC Pro Thr Ile Thr PAT TAC TAT TCT Asn Tyr Tyr Ser

TTT

Phe 605 1945 1993 2041 CTA GGC ACT CAA Leu Gly Thr Gin

TTG

Leu 610 CAA TAC AGA AGG Gin Tyr Arg Arg

CTC

Leu 615 TAT AGC GTG TAT Tyr Ser Val Tyr CTC PAT Leu Asn 620 TAT GTG TTC Tyr Val Phe GCT TAT Ala Tyr 625 TAAAAAATCT TCTTTTTAAA ATAGGGGGAG CTTCATCAAA T 2097 2157 2161 CTATTTTGAT AGTTATCPAT ATTTGATGPA PATAAAGTCA AAAACAAAAT PAACCAPATC

ACCC

SUBSTITUTE SHEET (RULE 26) WO 98/43479 126 PCT/US98/06421 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 645 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...19 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Lys Lys Thr Ile Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Leu -10 Leu His Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gin Ile 1 5 Gly Glu Arg Val Gin Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu 20 Asn Glu Lys Tyr Glu Gin Leu Ser Gin Ser Leu Ala Gin Leu Ala Ser 35 40 Leu Lys Lys Ser Ile Gin Thr Ala Asn Asn Ile Gin Ala Val Asn Asn 55 Ala Leu Ser Asp Leu Lys Ser Phe Ala Ser Asn Asn His Thr Asn Lys 70 Glu Thr Ser Pro Ile Tyr Asn Thr Ala Gin Ala Val Ile Thr Ser Val 85 Leu Ala Phe Trp Ser Leu Tyr Ala Gly Asn Ala Thr Ser Phe His Val 100 105 Thr Gly Leu Asn Asp Gly Ser Asn Ala Pro Leu Gly Arg Ile His Gin 110 115 120 125 Asp Gly Asn Cys Thr Gly Leu Gin Gin Cys Phe Met Asn Lys Glu Thr 130 135 140 Tyr Asp Lys Met Lys Ala Leu Ala Glu Asn Leu Gin Lys Ala Gin Gly 145 150 155 Asn Leu Cys Ala Leu Ser Glu Cys Pro Ser Asp Gin Leu Asn Gly Asn 160 165 170 Asn Gly Asn Lys Thr Ser Met Thr Lys Ala Leu Glu Thr Ala Gin Gin 175 180 185 Leu Met Asp Leu Ile Ala Asn Thr Lys Thr Ala Met Met Trp Lys Asn 190 195 200 205 Ile Val Ile Ala Gly Val Thr Asn Arg Pro Gly Gly Ala Gly Ala Ile 210 215 220 Thr Ser Thr Gly Pro Val Thr Asp Tyr Ala Val Phe Asn Asn Ile Lys 225 230 235 Ala Met Ile Pro Ile Leu Gin Gin Ala Val Thr Leu Ser Gin Ser Asn 240 245 250 His Thr Leu Ser Ala Ser Leu Gin Ala Gin Ala Thr Gly Ser Gin Thr 255 260 265 Asn Pro Lys Phe Ala Lys Asp Ile Tyr Thr Phe Ala Gin Asn Gin Lys SUBSTITUTE SHEET (RULE 26) WO 98/43479 PTU9/62 PCT/US98/06421 270 Gin Pro Asn Asn Arg 350 Ser Ser Ile Asn Ala 430 Gin Lys Phe Ser Ile 510 Leu Tyr Ala Aia Giu 590 Leu Tyr Val1 Aila Aia Gin 335 Val1 As n Giu Vai Tyr 415 Ala Asn Gin Phe Ser 495 Ile Phe Vali Thr Thr 575 Leu Gly Val Ile Glu Gly 320 Giu Asp Gin Glu Thr 400 Gin Met Asn Phe Asp 480 Asp Asn Gly Asn Asn 560 Ala Gly Thr Phe Ser Gin 305 Ser Val1 Ala Aia Ile 385 Leu le Ser Asn Phe 465 Tyr Ile Asp Gly Leu 545 Phe Arg Ile Gin Ala 625 Tyr 290 Tyr Thr Gin Ala Giu 370 Ser Pro Asn Asn Gly 450 Gly Asn Trp Ser Ile 530 Thr Gin Lys Lys Leu 610 Tyr 275 Al a Lys Pro Thr Leu 355 Ile Lys Tyr Pro Asn 435 Ala Giu His Thr Ile 515 Gin Ala Phe Lys Ile 595 Gin Gin Tyr Thr Ile 340 Ser Val1 Leu Asp Giu 420 Pro Leu Ser Gly Tyr 500 Thr Leu Phe Leu Asp 580 Pro Asp Ile Leu Giu 310 Asn Pro 325 Lys Asn Val Ala Thr Ala Pro His 390 Lys Asn 405 Gin Gin Phe Lys Asn Gly Lys Arg 470 Tyr Ile 485 Gly Giy Arg Lys Ala Gly Asn Asn 550 Phe Asn 565 Ser Giu Thr Ile Phe 295 Lys Tyr Asn Arg Tyr 375 As n Ala Ser Lys Leu 455 Trp Lys Gly Asn Thr 535 Pro Leu His Thr Leu 615 280 Asn Ala Arg Val Asp 360 Asn Gin Pro Asn Val 440 Giy Gly Ser Ser Asn 520 Thr Tyr Gly Ser Thr 600 Tyr Leu Tyr Gin Ser 345 Val1 Asp Val Al a Leu 425 Gly Val Leu Ser Asp 505 Lys Trp Ser Leu Al a 585 Asn Ser Phe Leu Val 330 Tyr Tyr Ala Asn Ala 410 Asn Met Gin Arg Phe 490 Leu Leu Leu Aila Arg 570 Gin Tyr Val Asn Lys 315 Val Tyr Asn Lys Thr 395 Giy Gin Ile Val1 Tyr 475 Phe Leu Ser Asn Lys 555 Thr His Tyr Tyr Ser 300 Ile As n Gly Leu Thr 380 Lys Gin Ala Ser Gly 460 Tyr Asn Vai Val1 Ser 540 Val1 Asr.

Giy Ser Leu 620 285 Ile Pro Leu Asn Lys 365 Leu Asp Ser Leu Ser 445 Tyr Giy Ser Asn Giy 525 Gin Asn Leu Ile Phe 605 Asn Tyr Arg Arg INFORMATION FOR SEQ ID NO:19: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1799 base pairs TYPE: nucleic acid STRANDEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: SUBSTITUTE SHEET (RULE 26) WO 98/43479 128 PCT[US98/06421 NAME/KEY: Coding Sequence LOCATION: 185...1633 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 185.. .233 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TACTCAAAAC ATTTTTCACT ATCAAAAACC TTTTTTTTAA ATCCAAAAAA AAAGCAAAAT TTCTTAATTT TTGCTCAATT TTATTAAAAA TTCAATAAAT TTATGGCACA ATTTAAACTT ATTGTAAATA AAGTTTCAAT TTGATACGAT TTTACAAACA AAACATTACT TTAAGGAACA TTTT ATG AAA AAA ACG ATT TTA CTT TCT CTT ATG GTT TCA TCG CTC CTC Met Lys Lys Thr Ile Leu Leu Ser Leu Met Val Ser Ser Leu Leu 120 180 229 GCT GAA Ala Glu 1 AAT GAC GGC Asn Asp Gly TTT ATG AGC GTG Phe Met Ser Vai TAT CAA ATC GGC Tyr Gin Ile Gly

GAA

Glu 277 325 GCG GTT CAA CAA Ala Val Gin Gin

GTG

Val AAA AAC ACC GGC Lys Asn Thr Gly

GAA

Glu 25 ATC CAA AAA GTC Ile Gin Lys Vai TCC AAC Ser Asn GCT TAC GAA Ala Tyr Glu CAA ACG GCC Gin Thr Ala

AAT

Asn TTG AAC AAT CTT Leu Asn Asn Leu

TTA

Leu 40 ACC CGC TAT AAC Thr Arg Tyr Asn GAA CTC AAA Glu Leu Lys ATT GAT AAT Ile Asp Asn 373 421 TCT AAC ACC AAT Ser Asn Thr Asn AGT ACC GCT CAA Ser Thr Ala Gin

GCG

Ala CTA AAA Leu Lys GAG AGC GCT AGC Glu Ser Ala Ser

CGA

Arg 70 TTG AAA ACG ACC Leu Lys Thr Thr

CCC

Pro AAT AGC GCT AAT Asn Ser Ala Asn

CAA

Gin GCC GTG TCT TCA Ala Val Ser Ser CTC AGC TCT GCG Leu Ser Ser Ala GCC ATG TGG CAA Ala Met Trp Gin

GTA

Val 469 517 565 613 ATA GTC TCT AAT Ile Val Ser Asn

TTA

Leu 100 GCC AAT AAC TCG Ala Asn Asn Ser

CTA

Leu 105 CCC ACT AGT GAA Pro Thr Ser Glu TAC AAC Tyr Asn 110 AAT AAA Asn Lys AAA ATC AAT Lys Ile Asn AAC AAT GAT Asn Asn Asp 130

GCG

Ala 115 ATT TCT CAA TCG Ile Ser Gin Ser

CTC

Leu 120 CAA AAC ACC CTA Gin Asn Thr Leu

GAA

Glu 125 CTT AAA ATT GAA Leu Lys Ile Glu GAC TAC GAC CAT Asp Tyr Asp His TTA ACT CAA Leu Thr Gin, SUBSTITUTE SHEET (RULE 26) WO 98/43479 129 PCTIUS98/0642 1 GCT AGC ACC ATT ATT AAT ACC CTT CAA AGC CAA Ala Ser Thr Ile Ile Asn Thr Leu Gin Ser Gin CCA GGC ATA GAC Pro Gly Ile Asp 709

GGA

Gly 160 GGC AAT GGC AAA Gly Asn Gly Lys

CCA

Pro 165 TGG GGC ATT AAT Trp Gly Ile Asp

GCA

Ala 170 AGC GGG AAC GCA Ser Gly Asn Ala

TGC

Cys 175 757 805 AAT ATT TTT GGC Asn Ile Phe Gly

AAC

Asn 180 ACC TTT AAC GCC Thr Phe Asn Ala

ATC

Ile 185 ACT AGC ATG ATA Thr Ser met Ile GAT AGC Asp Ser 190 GCT AAA AAA Ala Lys Lys AAC CAA CCA Asn Gin Pro 210 GCC GCA GAT GCC Ala Ala Asp Ala

CGA

Arg 200 AGA ACT GCC CCA Arg Thr Ala Pro GAA AGT CCA Giu Ser Pro 205 AAC CTT AAT Asn Leu Asn AGT GCG TTT AAC Ser Ala Phe Asn

AAC

Asn 215 GCT GAT TTC AAT Ala Asp Phe Asn

AAA

Lys 220 CAA GTC Gin Val 225 TCA AGC GTT ATT Ser Ser Val Ile

AAT

Asn 230 GAC ACG ATC TCT Asp Thr Ile Ser CTC AAA GGG GAC Leu Lys Gly Asp

AAT

Asn 240 TTA GCA ACC ATC Leu Ala Thr Ile AAC ACC CTT CAA Asn Thr Leu Gin

AAA

Lys 250 ACG CCC GAT TCT Thr Pro Asp Ser 949 997 1045 GGG TTT CAA AGT Gly Phe Gin Ser

TTG

Leu 260 GTG AGC CGA TCT Val Ser Arg Ser

AGC

Ser 265 TAT AGT TAT TCC Tyr Ser Tyr Ser CTC AAC Leu Asn 270 GAA ACC CAA Giu Thr Gin AAC CCT TTT Asn Pro Phe 290 TCT GAA TTC CAA Ser Glu Phe Gin

ACT

Thr 280 ACC ACC AAA GAG Thr Thr Lys Glu TTT GGC CAT Phe Gly His 285 AAT AAC GGA Asn Asn Gly 1093 1141 AGA AGC GTG GGT Arg Ser Val Gly

TTA

Leu 295 ATC AAC TCT CAA Ile Asn Ser Gin

AGC

Ser 300 GCG ATG Ala Met 305 AAT GGC GTG GGC Asn Gly Val Gly CAA TTA CCC TAT Gin Leu Gly Tyr CAA TTC TTT GGG Gin Phe Phe Gly

AAA

Lys AAT AAA TTT TTT Asn Lys Phe Phe

GGG

Gly 325 ATC CGT TAT TAT Ile Arg Tyr Tyr

GCC

Ala 330 TTT TTT GAT TAC Phe Phe Asp Tyr

AAC

Asn 335 1189 1237 1285 CAT GCC TAT ATC His Ala Tyr Ile

AAA

Lys 340 TCC AAC TTT TTC Ser Asn Phe Phe

AAC

As n 345 TCC OCT TCC AAT Ser Ala Ser Asn GTT TTC Val Phe 350 ACT TAT GGC Thr Tyr Gly

GCA

Ala 355 GGC AGT GAT CTT Gly Ser Asp Leu

TTA

Leu 360 TTG AAT TTC ATC Leu Asn Phe Ile AAT GGC GGA Asn Gly Gly 365 1333 TCC GAT AAA AAC CCC AAA GTC TCT TTT GGC ATT TTT GGA GGC ATC GCT 1381 SUBSTITUTE SHEET (RULE 26) WO 98/43479 Ser Asp Lys 370 PCT/US98/06421 Asn Arg Lys Val Ser 375 Phe Gly Ile Phe Gly 380 Gly Ile Ala CTA GCA Leu Ala 385 GGC ACG ACA TGG Gly Thr Thr Trp

CTT

Leu 390 AAT TCC CAA TTT Asn Ser Gin Phe

ATG

Met 395 AAT TTA AAA ACC Asn Leu Lys Thr

ACC

Thr 400 AAT AGC GCC TAC Asn Ser Ala Tyr

AGC

Ser 405 GCT AAG ATC AAC Ala Lys Ile Asn ACC AAT TTC CAA Thr Asn Phe Gin

TTC

Phe 415 1429 1477 1525 TTA TTC AAT ACT Leu Phe Asn Thr

GGT

Gly 420 TTA AGG CTT CAA Leu Arg Leu Gin

GGG

Gly 425 ATT CAC CAT GGC Ile His His Gly GTT GAA Val Glu 430 TTA GGC GTG AAA ATC CCC ACC ATC Leu Gly Val Lys Ile Pro Thr Ile 435

AAC

Asn 440 ACG AAT TAC TAT Thr Asn Tyr Tyr TCT TTC ATG Ser Phe Met 445 TTC AAT TAT Phe Asn Tyr 1573 GGC GCT AAA Gly Ala Lys 450 TTA GCA TAC CGA Leu Ala Tyr Arg

AGA

Arg 455 CTT TAT AGC GTG Leu Tyr Ser Val

TAT

Tyr 460 1621 GTT TTG GCC TAT Val Leu Ala Tyr 465 TGATATTGAA TCGGTTCTCA TTACTAATGA GGACAAAGCC AAACT 1678 1738 1798 1799 TTTTGGCTCT CAATGAATAA CGGCATCATT TTACTTGACT TTTTACAAAA AACACACTAA AATTTCTTTT TCTTTTTTGA GCGAAATTCC AGATTAGCTC AGCGGTAGAG TAGGCGGCTG

T

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 483 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...16 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Lys Thr Ile Leu Leu Ser Leu Met Val Ser -10 Glu Asn Asp Gly Val Phe Met Ser Val Gly Tyr Gin 1 5 10 Val Gin Gin Val Lys Asn Thr Gly Glu Ile Gin Lys 25 Ser Leu Leu Ala Ile Gly Glu Ala Val Ser Asn Ala SUBSTITUTE SHEET (RULE 26) WO 98/43479 WO 9843479PCTIUS98/06421 Tyr Thr Lys Ala Val Ile Asn Ser 145 Gly Ile Lys Gin Val 225 Leu Phe Thr Pro Met 305 As n Al a Tyr Asp Al a 385 As n Phe Gly Ala Leu 465 Glu Al a Giu Val Ser Asn Asp 130 Thr Asn Phe Lys Pro 210 Ser Al a Gin Gin Phe 290 Asn Lys Tyr Gly Lys 370 Gly Ser Asn Val Lys 450 Ala Asn Ser Ser Ser Asn Ala 115 Leu Ile Gly Gly Al a 195 Ser Ser Thr Ser Tyr 275 Arg Gly Phe Ile Al a 355 Asn Thr Al a Thr Lys 435 Leu Tyr Leu Asn Ala Ser Leu 100 Ile Lys Ile Lys Asn 180 Ala Ala Val1 Ile Leu 260 Ser Ser Val1 Phe Lys 340 Gly Arg Thr Tyr Gly 420 Ile Ala Asn Thr Ser Ala Al a Ser Ile As n Pro 165 Thr Ala Phe Ile Tyr 245 Val Glu Val1 Gly Gly 325 Ser Ser Lys Trp Ser 405 Leu Pro Tyr Asn Asn Arg 70 Leu Asn Gin Giu Thr 150 Trp Phe Asp As n Asn 230 Asn Ser Phe Gly Val1 310 Ile Asn Asp Val Leu 390 Al a Arg Thr Arg Leu Ser 55 Leu Ser Asn Ser Asn 135 Leu Gly Asn Ala Asn 215 Asp Thr Arg Gin Leu 295 Gin Arg Phe Leu Ser 375 Asn Lys Leu Ile Arg 455 Leu 40 Ser Lys Ser Ser Leu 120 Asp Gin Ile Ala Arg 200 Ala Thr Leu Ser Thr 280 Ile Leu Tyr Phe Leu 360 Phe Ser Ile Gin Asn 440 Leu Thr Thr Thr Ala Leu 105 Gin Tyr Ser Asn Ile 185 Arg Asp Ile Gin Ser 265 Thr Asn Gly Tyr As n 345 Leu Gly Gin Asn Gly 425 Thr Tyr Arg Ala Thr Val1 90 Pro Asn Asp Gin Ala 170 Thr Thr Phe Ser Lys 250 Tyr Thr Ser Tyr Al a 330 Ser Asn Ile Phe Asn 410 Ile Asn Ser Tyr Gin Pro 75 Al a Thr Thr His Cys 155 Ser Ser Al a Asn Tyr 235 Thr Ser Lys Gin Lys 315 Phe Al a Phe Phe Met 395 Thr His Tyr Val1 Asn Al a Asn Met Ser Leu Leu 140 Pro Gly Met Pro Lys 220 Leu Pro Tyr Giu Ser 300 Gin Phe Ser Ile Gly 380 Asn Asn His Tyr Tyr 460 Giu Ile Ser Trp, Glu Giu 125 Leu Gly Asn Ile Giu 205 Asn Lys Asp Ser Phe 285 Asn Phe Asp Asn Asn 365 Gly Leu Phe Giy Ser 445 Phe Leu Asp Ala Gin Tyr 110 Asn Thr Ile Ala Asp 190 Ser Leu Gly Ser Leu 270 Giy Asn Phe Tyr Val1 350 Giy Ile Lys Gin Val1 430 Phe Asn Lys Asn Asn Val1 Asn Lys Gin Asp Cys 175 Ser Pro Asn Asp Lys 255 Asn His Gly Gly Asn 335 Phe Gly Ala Thr Phe 415 Giu Met Tyr Gin Leu Gin Ile Lys Asn Al a Gly 160 Asn Ala Asn Gin Asn 240 Gly Giu As n Ala Lys 320 His Thr Ser Leu Thr 400 Leu Leu Giy Val SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 2338 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 146...2218 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 146...200 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: ACTTAAAATT GTTTTTTTTT TTTTTCAAAA TATAAATTTT AAGCCAAAAA TAAGCATTTT ATGGTAAAAT GGCGAACTTT CATAAACATG ACTATTATGG GAATGTCATG GGAATGTGAA GAAAAATCTA TTAAAA GGA GAA AAC ATG AAA AAA TCC CTC TTA CTC TCT CTT Met Lys Lys Ser Leu Leu Leu Ser Leu -18 -15 120 172 TCT CTC ATC GCT Ser Leu Ile Ala

TCC

Ser TTA TCA AGA GCT GAA GAT GAC GGA TTT TAT ACG Leu Ser Arg Ala Glu Asp Asp Gly Phe Tyr Thr 220 268 AGT GTG GGC TAT CAG ATC GGT Ser Val Gly Tyr Gln Ile Gly

GAA

Glu 15 GCG GTC CAA CAA GTG AAA AAC ACA Ala Val Gin Gin Val Lys Asn Thr GGA GCA Gly Ala TTG CAA AAT CTT Leu Gin Asn Leu

GCA

Ala 30 GAC AGA TAC GAT Asp Arg Tyr Asp

AAC

Asn TTA AAC AAC CTT Leu Asn Asn Leu

TTA

Leu AAC CAA TAC AAT Asn Gin Tyr Asn TTA AAT TCC TTA GTC AAT TTA GCC AGC Leu Asn Ser Leu Val Asn Leu Ala Ser 50

ACG

Thr CCG AGC GCG ATC Pro Ser Ala Ile

ACC

Thr GGT GCG ATT GAT Gly Ala Ile Asp

AAT

Asn 65 TTA AGC TCA AGC Leu Ser Ser Ser GCG ATT Ala Ile AAC CTC ACT Asn Leu Thr

AGC

Ser GCC ACC ACC ACT Ala Thr Thr Thr CCC GCC TAT CAA Pro Ala Tyr Gin GCT GTG GCT Ala Val Ala TTA GCG CTC AAT GCC GCT GTG GGC ATG TGG CAA GTC ATA GCC CTT TTT Leu Ala Leu Asn Ala Ala Val Gly Met Trp Gin Val Ile Ala Leu Phe SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 ATT GGC Ile Gly 105 TGT GGC CCT GGC Cys Gly Pro Gly

CCT

Pro 110 ACC AAT AAT CAA Thr Asn Asn Gin TAT CAA TCG TTT Tyr Gin Ser Phe

GGT

Gly 120 AAC ACA CCA GCC Asn Thr Pro Ala

CTT

Leu 125 AAT GGG ACC ACC Asn Gly Thr Thr

ACC

Thr 130 ACT TGC AAT CAA Thr Cys Asn Gin TAT GGG ACA GGC CCT AAT GGC ATC CTA Tyr Gly Thr Gly Pro Asn Gly Ile Leu 140

TCT

Ser 145 ATT GAT GAA TAC Ile Asp Glu Tyr CAA AAA Gin Lys 150 652 CTC AAC CAA Leu Asn Gin GGG GGT GGG Gly Gly Gly 170

GCT

Ala 155 TAT CAG ATC ATC Tyr Gin Ile Ile

CAA

Gin 160 ACC GCT TTA AAC Thr Ala Leu Asn CAA AAT CAA Gin Asn Gin 165 GGG GTA GTC Gly Val Val 700 748 ATG CCT GCC TTG Met Pro Ala Leu

AAT

Asn 175 GAC ACC ACC AAA Asp Thr Thr Lys

ACA

Thr 180 AAC ATA Asn Ile 185 CAA CAA ACC AAT Gin Gin Thr Asn AGG ACC ACC ACA Arg Thr Thr Thr AAC AAT ATC ATA Asn Asn Ile Ile 796 844

GAG

Glu 200 CAT TAT TAT ACA His Tyr Tyr Thr

GAG

Glu 205 AAT GGG AAA GAG Asn Gly Lys Glu CCA GTC TCT TAT TCA Pro Val Ser Tyr Ser GGC GGA TCA TCA Gly Gly Ser Ser

TTC

Phe 220 TCG CCT ACA ATA Ser Pro Thr Ile

CAA

Gin 225 TTG ACA TAC CAT Leu Thr Tyr His AAT AAC Asn Asn 230 892 GCT GAA AAC Ala Glu Asn ACT CAA AAG Thr Gin Lys 250

CTT

Leu 235 TTG CAA CAA GCC Leu Gin Gin Ala ACT ATC ATG CAA Thr Ile Met Gin GTC CTT ATT Val Leu Ile 245 GCG TGG GGG Ala Trp Gly 940 988 CCG CAT GTG CAA Pro His Val Gin

ACG

Thr 255 AGC AAT GGC GGT Ser Asn Gly Gly

AAA

Lys 260 TTG AGT Leu Ser 265 TCT ACG CCT GGG Ser Thr Pro Gly GTG ATG GAT ATT Val Met Asp Ile GGT CCT TCT TTT Gly Pro Ser Phe

AAC.

Asn 280 GCT ATT AAT GAG Ala Ile Asn Glu

ATG

Met 285 ATT AAA AAC GCT Ile Lys Asn Ala

CAA

Gin 290 ACA GCC CTA GCA Thr Ala Leu Ala 1036 1084 1132 ACC CAA CAG CTT Thr Gin Gin Leu AAC GCT Asn Ala 300 AAT GAA AAC Asn Glu Asn CAA ATC ACG CAA Gin Ile Thr Gin CCC AAC Pro Asn 310 AAT TTC AAC Asn Phe Asn

CCC

Pro 315 TAC ACC TCT AAA Tyr Thr Ser Lys GAC AAA Asp Lys 320 GGG TTC GCT Gly Phe Ala CAA GAA ATG- Gin Glu Met 325 1180 SUBSTITUTE SHEET (RULE 26) WO 98/43479 CTC AAT AGA Leu Asn Arg 330 PCT/US98/06421 GCT GAA GCT CAA Ala Glu Ala Gin GCA GAG ATT TTA AAT TTA GCT AAG CAA Ala Glu Ile Leu Asn Leu Ala Lys Gin 335 340 1228 GTA GCG Val Ala 345 AAC AAT TTC CAC Asn Asn Phe His

AGC

Ser 350 ATT CAA GGG CCT Ile Gln Gly Pro CAA GGG GAT TTA Gin Gly Asp Leu

GAA

Glu 360 GAA TGT AAA GCA Glu Cys Lys Ala

GGA

Gly 365 TCG GCT GGC GTG Ser Ala Gly Val

ATC

Ile 370 ACT AAT AAC ACT Thr Asn Asn Thr

TGG

Trp 375 1276 1324 1372 GGT TCA GGT TGC Gly Ser Gly Cys

GCG

Ala 380 TTT GTG AAA GAA Phe Val Lys Glu

ACT

Thr 385 TTA AAC TCT TTA Leu Asn Ser Leu GAG CAA Glu Gln 390 CAC ACC GCT His Thr Ala

TAT

Tyr 395 TAC GGC AAC CAG Tyr Gly Asn Gin

GTC

Val 400 AAT CAG GAT AGG Asn Gin Asp Arg GCT TTG GCT Ala Leu Ala 405 AAT AAA GAC Asn Lys Asp 1420 CAA ACC ATT TTG AAT TTT AAA Gin Thr Ile Leu Asn Phe Lys

GAA

Glu 415 GCC CTT AAC ACC Ala Leu Asn Thr TCA AAA Ser Lys 425 GCG ATC AAT AGC Ala Ile Asn Ser

GGT

Gly 430 ATC TCC AAC TTG Ile Ser Asn Leu

CCT

Pro 435 AAC GCT AAA TCT Asn Ala Lys Ser 1468 1516 1564 1612

CTT

Leu 440 CAA AAC ATG ACG Gin Asn Met Thr

CAT

His 445 GCC ACT CAA AAC Ala Thr Gin Asn

CCT

Pro 450 AAT TCC CCA GAA Asn Ser Pro Glu

GGT

Gly 455 CTG CTC ACT TAT Leu Leu Thr Tyr

TCT

Ser 460 TTG GAT TCA AGC Leu Asp Ser Ser

AAA

Lys 465 TAC AAC CAG CTC Tyr Asn Gin Leu CAA ACC Gin Thr 470 ATC GCG CAA Ile Ala Gin GAC TTT CAA Asp Phe Gin 490

GAA

Glu 475 TTG GGC AAA AAC Leu Gly Lys Asn

CCT

Pro 480 TTC AGG CGC TTT Phe Arg Arg Phe GGC GTG ATT Gly Val Ile 485 GTG CAA GTG Val Gin Val 1660 1708 AAC AAC AAC GGC Asn Asn Asn Gly

GCA

Ala 495 ATG AAC GGG ATC Met Asn Gly Ile

GGC

Gly 500 GGT TAT Gly Tyr 505 AAA CAA TTC TTT Lys Gin Phe Phe

GGT

Gly 510 AAA AAA AGG AAT Lys Lys Arg Asn GGG TTA AGG TAT Gly Leu Arg Tyr

TAT

Tyr 520 GGT TTC TTT GAT Gly Phe Phe Asp

TAT

Tyr 525 AAC CAT GCT TAT Asn His Ala Tyr

ATC

Ile 530 AAA TCT AAT TTT Lys Ser Asn Phe

TTC

Phe 535 1756 1804 1852 AAC TCC GCT TCT Asn Ser Ala Ser

GAT

Asp 540 GTG TGG ACT TAT Val Trp Thr Tyr

GGG

Gly 545 GTG GGT ATG GAC Val Gly Met Asp GCT CTC Ala Leu 550 TAT AAC TTC ATC AAC GAT AAA AAC ACC AAC TTT TTA GGC AAG AAC AAC 1900 SUBSTITUTE SHEET (RULE 26) WO 98/43479 Tyr Asn Phe AAG CTT TCA Lys Leu Ser 570 PCT/US98/06421 Asn Asp Lys Asn Asn Phe Leu Gly Lys Asn Asn 565 GGG ACT TCG Gly Thr Ser GTA GGG CTT TTT Val Gly Leu Phe

GGA

Gly 575 GGC TTT GCG TTA Gly Phe Ala Leu 1948 TGG CTT Trp Leu 585 AAT TCC CAA CAA Asn Ser Gin Gin

GTG

Val 590 AAT TTG ACC ATG Asn Leu Thr Met

ATG

Met 595 AAT GGC ATT TAT Asn Gly Ile Tyr

AAC

Asn 600 GCT AAT GTC AGC Ala Asn Val Ser TCT AAC TTC CAA Ser Asn Phe Gin TTG TTT GAT TTA Leu Phe Asp Leu 1996 2044 2092 TTG AGA ATG AAC Leu Arg Met Asn

CTC

Leu 620 GCT AGG CCT AAG Ala Arg Pro Lys

AAA

Lys 625 AAA GAC AGC GAT Lys Asp Ser Asp CAT GCC His Ala 630 GCT CAG CAT Ala Gin His AAC TAT TAT Asn Tyr Tyr 650

GGC

Gly 635 ATT GAA CTA GGT Ile Glu Leu Gly

TTT

Phe 640 AAG ATC CCC ACG Lys Ile Pro Thr ATC AAC ACC Ile Asn Thr 645 AGG ATG TAT Arg Met Tyr 2140 2188 TCT TTC ATG GGC Ser Phe Met Gly

GCT

Ala 655 AAA CTA GAA TAC Lys Leu Glu Tyr AGC CTT Ser Leu 665 TTT CTC AAT TAT Phe Leu Asn Tyr

GTG

Val 670 TTT GCT TAC Phe Ala Tyr TAAAAACTCT CTTTAAAAAA GGG 2241 GTTTGTTTAA AAACGCTTAA AAGCATTTTT AAAATTAAGC AGTAAAGAGC CTAGATAATC TCTTGCAACC GCTCTCAAGC GATAAAATTA AAGTGAT INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 691 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...18 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 2301 2338 Met Lys Lys Ser Leu Leu Leu Ser Leu Ser -18 -15 -10 Arg Ala Glu Asp Asp Gly Phe Tyr Thr Ser Leu Ile Ala Ser Leu Ser Val Gly Tyr Gin Ile Gly SUBSTITUTE SHEET (RULE 26) WO 98/43479 PTU9/62 PCT/US98/06421 Giu Asp Asn Ile Thr Gly Thr Gly Ile Ile Asn 175 Arg Gly Thr Ala Thr 255 Val1 Lys Giu Lys Ala 335 Ile Al a Lys Gin Giu 415 Ile Thr Ala Arg Ser Asp Ser Met As n Thr Leu Gin 160 Asp Thr Lys Ile Al a 240 Ser Met Asn Asn Asp 320 Giu Gin Gly Giu Val 400 Ala Ser Gin 1 Val Tyr Leu Asn Pro Trp Asn Thr Ser 145 Thr Thr Thr Giu Gin 225 Thr Asn Asp Ala Ala 305 Lys Ile Giy Val1 Thr 385 Asn Leu Asn Asn Gin Gin Val Lys Asn Asp Val Leu Aia Gin Gin Thr 130 Ile Aia Thr Thr Ile 210 Leu Ile Gly Ile Gin 290 Gin Giy Leu Pro Ile 370 Leu Gin Asn Leu Pro Asn Asn Ser Tyr Val1 Ser 115 Thr Asp Leu Lys Gin 195 Pro Thr Met Gly Phe 275 Thr Ile Phe Asn Ile 355 Thr Asn Asp Thr Pro 435 Asn 20 Leu Leu Ser Gin Ile 100 Tyr Cys Giu Asn Thr 180 As n Vai Tyr Gin Lys 260 Gly Ala Thr Aila Leu 340 Gin Asn Ser Arg Leu 420 Asn Ser Asn Aila Ser Al a 85 Al a Gin Asn Tyr Gin 165 Gly Asn Ser His Vai 245 Ala Pro Leu Gin Gin 325 Ala Gly Asn Leu Ala 405 Asn Ala Pro Asn Ser Al a 70 Val Leu Ser Gin Gin 150 Asn Val1 Ile Tyr Asn 230 Leu Trp Ser Al a Pro 310 Giu Lys Asp Thr Giu 390 Leu Lys Lys Giu Thr Leu Thr 55 Ile Ala Phe Phe Ala 135 Lys Gin Val Ile Ser 215 Asn Ile Gly Phe Lys 295 Asn Met Gin Leu Trp 375 Gin Ala Asp Ser Gly Giy Leu 40 Pro Asn Leu Ile Giy 120 Tyr Leu Gly As n Giu 200 Gly Ala Thr Leu Asn 280 Thr Asn Leu Vali Giu 360 Gly His Gin Ser Leu 440 Leu Al a 25 Asn Ser Leu Al a Gly 105 Asn Giy Asn Giy Ile 185 His Gly Giu Gin Ser 265 Aia Gin Phe Asn Ala 345 Giu Ser Thr Thr Lys 425 Gin Leu Leu Gin Ala Thr Leu Cys Thr Thr Gin Gly 170 Gin Tyr Ser Asn Lys 250 Ser Ile Gin Asn Arg 330 Asn Cys Gly Al a Ile 410 Al a Asn Thr Gin Tyr Ile Ser Asn Gly Pro Gly Al a 155 Met Gin Tyr Ser Leu 235 Pro Thr Asn Leu Pro 315 Ala Asn Lys Cys Tyr 395 Leu Ile Met Tyr Asn Leu Asn Tyr Thr Gly Ala Thr Ala Ala Pro Giy Ala Leu 125 Pro Asn 140 Tyr Gin Pro Ala Thr Asn Thr Glu 205 Phe Ser 220 Leu Gin His Val Pro Gly Giu Met 285 Asn Ala 300 Tyr Thr Giu Ala Phe His Ala Giy 365 Ala Phe 380 Tyr Gly Asn Phe Asn Ser Thr His 445 Ser Leu Ala Leu Ala Thr Val1 Pro 110 Asn Gly Ile Leu Tyr 190 As n Pro Gin Gin Asn 270 Ile Asn Ser Gin Ser 350 Ser Val1 Asn Lys Gly 430 Al a Asp SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 Ser Asn Ala 495 Lys His Thr Asn Gly 575 Asn Asn Pro Gly Ala 655 Phe Ser Lys 465 Pro Phe 480 Met Asn Lys Arg Ala Tyr Tyr Gly 545 Thr Asn 560 Gly Phe Leu Thr Phe Gin Lys Lys 625 Phe Lys 640 Lys Leu Ala Tyr 450 Tyr Arg Gly Asn Ile 530 Val Phe Ala Met Phe 610 Lys Ile Glu Asn Arg Ile Trp 515 Lys Gly Leu Leu Met 595 Leu Asp Pro Tyr Gin Phe Gly 500 Gly Ser Met Gly Ala 580 Asn Phe Ser Thr Arg 660 Leu Gly 485 Val Leu Asn Asp Lys 565 Gly Gly Asp Asp Ile 645 Arg Gin 470 Val Gin Arg Phe Ala 550 Asn Thr Ile Leu His 630 Asn Met 455 Thr Ile Val Tyr Phe 535 Leu Asn Ser Tyr Gly 615 Ala Thr Tyr Ile Asp Gly Tyr 520 Asn Tyr Lys Trp Asn 600 Leu Ala Asn Ser Ala Phe Tyr 505 Gly Ser Asn Leu Leu 585 Ala Arg Gin Tyr Leu 665 Gin Gin 490 Lys Phe Ala Phe Ser 570 Asn Asn Met His Tyr 650 Phe Glu 475 Asn Gin Phe Ser Ile 555 Val Ser Val Asn Gly 635 Ser Leu 460 Leu Asn Phe Asp Asp 540 Asn Gly Gin Ser Leu 620 Ile Phe Asn Gly Asn Phe Tyr 525 Val Asp Leu Gin Thr 605 Ala Glu Met Tyr Lys Gly Gly 510 Asn Trp Lys Phe Val 590 Ser Arg Leu Gly Val 670 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: TCAAGGAGAA AACATGAAAA AAACCC INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GAAGACGACG GCTTTTACAC AAGCGT SUBSTITUTE SHEET (RULE 26) WO 98/43479 138 PCT/US98/06421 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID AAAGCTTAGT AAGCGAACAC ATAA 24 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: AAGGAGAAAA AACATGAAAA AACACATCC 29 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GAAGACGACG GCTTTTACAC AAGCG INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: AACATTAGTA AGCGAACACA TAGTTC 26 INFORMATION FOR SEQ ID NO:29: SUBSTITUTE SHEET (RULE 26) WO 98/43479 139 SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: AAGGAGAAAA AACATGAAAA AACACATCC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID GAAGACGACG GCTTTTACAC AAGCGT INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: AAAAGCTTAG TAAGCGAACA CAT INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: AAGGAGAAAA CATGAAGAAA AAATTT INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: PCT/US98/06421 29 26 23 26 SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GAAGACAACG GCTTTTTTGT GAGTG INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: AGCTTTTAGT AAGCAAACAC ATAGT INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID AAGGATATTT ATGAAAAAAA CCCTT INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: GAAGACAACG GCTTTTTTAT CAGCG INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs SUBSTITUTE SHEET (RULE 26) WO 98/43479 141 PCT/US98/06421 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: GATATTAGTA AGCAAACACA TAATTC 26 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: AAGGAGAAAA CATGAAAAAA TCCCTCT 27 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GAAGATGACG GATTTTATAC GAGTGT 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID TTTTAGTAAG CAAACACATA ATTGAG 26 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid SUBSTITUTE SHEET (RULE 26) WO 98/43479 142 PCT/US98/06421 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: AAGGAACATC TTATGAAAAA AACG 24 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GAAGACAACG GCGTTTTTTT AAGCG INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: GGTTTTTAAT AGGCAAACAC ATAAT INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: AAGGAACATT TTATGAAAAA GACAAT 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single SUBSTITUTE SHEET (RULE 26) WO 98/43479 143 PCT/US98/06421 TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID GAAGACAACG GCTTTTTTGT GAGCG INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: TCACTCAGTA AGCGAACACA TAA 23 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: AAGGAACATT TTATGAAAAA GACAA INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GAAGACAACG GCTTTTTTGT GAGCG INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: TTTTAATAAG CGAACACATA AAAGAG 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID AAGGAACATT TTATGAAAAA AACGAT 26 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: GAAAATGACG GCGTTTTTAT GAGCG INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: ATATCAATAG GCCAAAACAT AATTGA 26 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA SUBSTITUTE SHEET (RULE 26) WO 98/43479 145 PCT/US98/06421 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: AAGGAGAAAA CATGAAAAAA TCCCTC 26 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: GAAGATGACG GATTTTATAC GAGTGT 26 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (xi) SEQUENCE DESCRIPTION: SEQ ID TTTTAGTAAG CAAACACATA ATTGAG 26 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CGCGGATCCG AATCCAATTT AATCCAAAAA GG 32 INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: SUBSTITUTE SHEET (RULE 26) WO 98/43479 146 PCT/US98/06421 CCGCTCGAGT TAAGTAAGCG AACACATATT CAA 33 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 Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gin Ile Gly Glu Ala 1 5 10 Ala Gin Met Val INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: CTGAATTCGA TTTCAAGGAG AAAACATGAA A 31 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CCGCTCGAGT TAGTAAGCGA ACACATAATT INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 98/43479 147 PCT/US98/06421 (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: CGCGGATCCG AATCCAATTT AATCCAAAAA GG 32 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: CCGCTCGAGT TAGTAAGCGA ACACATAGTT CAA 33 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: CGCGGATCCG AAGTTTCTTT GTATCAAAG 29 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: CCGCTCGAGT TAGTAAGCAA ACACATAATT GTG 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1149 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCTIUS98/06421 (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 106...1002 OTHER INFORMATION: NAME/KEY: Signal Sequence LOCATION: 106...166 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID TTACTCTTTA ATGTGAGTTT TCTGTGTCAT GATAGCTGAT TTTGTTTTAA ATTTGCTATA ATGTGAATTT AATGATGAAA ATTAGTTTAG AGTGGAGAAC ACACA ATG AAA AAA AAT Met Lys Lys Asn ATC TTA Ile Leu GCT AAG Ala Lys AAT TTA GCG TTA Asn Leu Ala Leu CCG GCT CAT AAC Pro Ala His Asn 5 GAT TCT TCA GCA Asp Ser Ser Ala

GTG

Val -10

GCA

Ala

GGC

Gly GGT GCG TTG AGC ACG Gly Ala Leu Ser Thr AAT AAC GCT ACG CAT Asn Asn Ala Thr His TCG TTT TTG ATG Ser Phe Leu Met AAC ACG AAA AAA Asn Thr Lys Lys GAT GGC AGA CCT Asp Gly Arg Pro 1

ACG

Thr

ACT

Thr 10 GTG TTA GCG Val Leu Ala ACA GTG Thr Val ATC ACT AAA Ile Thr Lys GAT TTT GAC Asp Phe Asp

AGC

Ser GAT TTT GAC Asp Phe Asp

ATG

Met 40

AAA

Lys 25

ATT

Ile AAG CAA CGA Lys Gin Arg

AAT

Asn

TTG

Leu CCT AAT TTT Pro Asn Phe ATT GAT CAA Ile Asp Gin AAG CTT AAA Lys Leu Lys GAA AAA GAA Glu Lys Glu GCT ATT Ala Ile

GCC

Ala

AAA

Lys 117 165 213 261 309 357 405 453 501 549 597 645 693 CGC ACC GCC Arg Thr Ala

GAC

Asp

CTT

Leu 70

TTT

Phe GAA AAT GAA Glu Asn Glu

GCT

Ala

GAA

Glu ACC GAG AAA Thr Glu Lys AGC ACT CCA Ser Thr Pro

GAA

Glu

TTT

Phe AAA GCG ATG Lys Ala Met

ATG

Met 90

CAG

Gin GCG GTT AAA Ala Val Lys AAA CAG Lys Gin GCT TTA GTG Ala Leu Val GTC CAA ATC Val Gin Ile 115 GAT CAG CTT Asp Gin Leu

GAA

Glu 100

CCA

Pro TGG GCT AAA Trp Ala Lys

AAA

Lys 105

CAA

Gin GCT GAA GAA Ala Glu Glu GAA AAA GAA Glu Lys Glu

ATG

Met 120

GAA

Glu GAT TTT TAC Asp Phe Tyr

AAC

Asn 125

CAT

His GTG AAA AAA Val Lys Lys 110 GCT AAC AAA Ala Asn Lys ATT TTA GTG Ile Leu Val TTT GTC AAG Phe Val Lys 130 AAA ACC Lvs Thr

CAA

Gin 135

AAA

Lys GCC CAT GCT Ala His Ala

AGG

Arg 140

GAG

Glu GAA GAT GAG Glu Asp Glu 145

CCA

Pro

GCT

Ala 150

GAA

Glu CGG ATT ATT Arg Ile Ile ATT GAC AAA Ile Asp Lys

CAG

Gin 160

GAT

Asp AAG GCT AAA Lys Ala Lys

AAA

Lys 165 GCT AAA TTC ATT Ala Lys Phe Ile 170 TTA GCC AAT Leu Ala Asn

CGG

Arg 175 SUBSTITUTE SHEET (RULE 26) WO 98/43479 ACG ATT GAT Thr Ile Asp AAA TTC CAA Lys Phe Gin 195 GCT TTA ACT Ala Leu Thr PCT/US98/06421

CCT

Pro 180

AAG

Lys AAC AGC AAG AAC Asn Ser Lys Asn CAA AAT GGC GGT Gin Asn Gly Gly AAC CAA ATG Asn Gin Met

GCT

Ala 200

ACT

Thr GAT TTT TCT Asp Phe Ser

AAA

Lys 205

AAA

Lys GAT TTG GGG Asp Leu Gly 190 GCC GCT TTC Ala Ala Phe ACA GAG TTT Thr Glu Phe CCT GGG GAT TAC Pro Gly Asp Tyr AAA ACC CCT Lys Thr Pro 210 GGT TAT Gly Tyr CAT ATT ATC His Ile Ile 225

ACT

Thr

TAT

Tyr 230

AAA

Lys 215

TTG

Leu ATT TCT AAA GAT Ile Ser Lys Asp

GTT

Val 220

AGC

Ser CCT GTA ACT Pro Val Thr

TAT

Tyr 240 741 789 837 885 933 981 1036 1096 1149 TAT GAA CAG Tyr Glu Gin

GCT

Ala 245

CGC

Arg CCT ACC ATT AAG Pro Thr Ile Lys ATG TTA CAA Met Leu Gin GAA AAG Glu Lys 255 AAG CAC Lys His CTT TTC CAA Leu Phe Gin GCT AAA ATT Ala Lys Ile 275

GAA

Glu 260

GTT

Val ATG AAT CAA CGC ATT GAG GAA CTA Met Asn Gin Arg Ile Glu Glu Leu

AGA

Arg 270 ATC AAC AAG Ile Asn Lys TAATTGATGA GGTGTTATCA TGTTAGTTAA AGGC AATGAAATTT TATTGAAAGC CCATAAAGAA GGTTATGGGG TGGGGGCGTT TAATTTCGTG AATTTTGAAA TGCTAAACGC TATTTTTGAA GCAGGAAATG AGGAAAATTC CCC INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 299 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Signal Sequence LOCATION: 1...20 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: Met Ser Lys Lys Asn Ile Leu Asn Leu Ala Leu -15 Phe Leu Met Ala Lys Pro Ala His Asn Val Gly -10 Ala Asn Ala Asn Leu Ile Leu Ser Thr Ala Thr His Ala Thr Val Lys Gin Arg Asn Thr Lys Lys Thr Thr Asp Ser 20 Asp Gly Arg Pro Ile Thr Lys Ser Asn Pro Asn Phe Asp Phe Asp Lys 50 Ser Ala Gly Val Asp Phe Asp Leu Lys Glu 55 Val Glu Lys Glu Ala Leu Ile Asp Gin Ala Thr Glu Lys Leu Asp Arg Thr Ala Glu Asn Glu Ala Lys Glu Ala Ser Thr Pro Glu Lys Ala Met Met SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 Val Glu Asn 125 His Ile Ala Gly Lys 205 Lys Pro Leu Leu Lys Val 110 Ala Ile Asp Asn Asp 190 Ala Thr Val Gin Arg 270 Lys Lys Asn Leu Lys Arg 175 Leu Ala Glu Thr Glu 255 Lys Gin Lys Lys Val Gin 160 Asp Gly Phe Phe Tyr 240 Lys His Ala Val Asp Lys 145 Pro Thr Lys Ala Gly 225 Thr Leu Ala Leu Gin Gin 130 Thr Lys Ile Phe Leu 210 Tyr Tyr Phe Lys Val Ile 115 Leu Glu Ala Asp Gin 195 Thr His Glu Gin Ile Glu 100 Pro Phe Asp Lys Pro 180 Lys Pro Ile Gin Glu 260 Val 85 Phe Glu Val Glu Lys 165 Asn Asn Gly Ile Ala 245 Arg Ile Trp Lys Lys Ala 150 Glu Ser Gin Asp Tyr 230 Lys Met Asn Ala Glu Gin 135 Lys Ala Lys Met Tyr 215 Leu Pro Asn Lys Lys Met 120 Glu Arg Lys Asn Ala 200 Thr Ile Thr Gin Lys 105 Gin Ala Ile Phe Ala 185 Pro Lys Ser Ile Arg 265 Gin Asp His Ile Ile 170 Gin Asp Thr Lys Lys 250 Ile Ala Phe Ala Ser 155 Glu Asn Phe Pro Asp 235 Gly Glu Glu Tyr Arg 140 Glu Leu Gly Ser Val 220 Ser Met Glu INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 1448 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA (ix) FEATURE: NAME/KEY: Coding Sequence LOCATION: 118...1314 OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: CTCTTGAATG GCGATAAGAC AAAAATGTCT TAAATTTTGT GGTAGCATTT AGGAATACTT AGGATTTTGT TTAGTATAAT TCTAAAATCC ATTTCAAAAA ATTAAGGAGA AATACAA ATG Met GCA AAA GAA AAG TTT AAC AGA ACT AAG CCG CAT GTT AAT ATT GGA ACC Ala Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn Ile Gly Thr 10 ATT GGG CAT GTA GAC CAT GGT AAA ACG ACT TTG AGT GCA GCG ATT TCA Ile Gly His Val Asp His Gly Lys Thr Thr Leu Ser Ala Ala Ile Ser 25 GCG GTG CTT TCT TTG AAA GGT CTT GCA GAA ATG AAA GAC TAT GAT AAT Ala Val Leu Ser Leu Lys Gly Leu Ala Glu Met Lys Asp Tyr Asp Asn 120 168 216 264 SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421

ATT

Ile

TCT

Ser

GAT

Asp AAC GCC CCT Asn Ala Pro

GAA

Glu 55 40

GAA

Glu AAA GAA AGA Lys Glu Arg

GGG

Gly 60

CAC

His

ATC

Ile ACT ATC GCT Thr Ile Ala

ACT

Thr CAC ATT GAA His Ile Glu

TAT

Tyr GAG ACT GAA AAC Glu Thr Glu Asn

AGA

Arg 75 TAT GCG CAT Tyr Ala His GTG GAT Val Asp TGC CCA GGA Cys Pro Gly CAA ATG GAC Gin Met Asp 100 CCT CAA ACT Pro Gin Thr

CAC

His

GGA

Gly

AGG

Arg GCT GAC TAT GTA Ala Asp Tyr Val GCG ATT TTG GTT Ala Ile Leu Val 105 GAG CAT ATC TTA Glu His Ile Leu

AAA

Lys 90

GTT

Val AAC ATG ATC ACC Asn Met Ile Thr TCT GCA GCT GAT Ser Ala Ala Asp GGT GCG GCG Gly Ala Ala GGC CCT ATG Gly Pro Met GGC GTG CCT Gly Val Pro 115 CAC ATC His Ile GTT GTT TTC Val ValPhe 130

TTG

Leu

TTA

Leu 135

GAA

Glu

AAA

Lys TTG TCT CGT Leu Ser Arg CAA GAC ATG Gin Asp Met

CAA

Gin 125

GTA

Val GAT GAC CAA Asp Asp Gin

GAA

Glu 145 TTA GAA CTT Leu Glu Leu

GTA

Val 150

GAT

Asp ATG GAA GTG Met Glu Val

CGC

Arg 155

GTA

Val 140

GAA

Glu TTG TTG AGC Leu Leu Ser GCG TAT Ala Tyr 160 GAA TTT CCT Glu Phe Pro GCT TTA GAA Ala Leu Glu 180 GTG CTT AAA Val Leu Lys GAC ACT CCT Asp Thr Pro

ATC

Ile 170

AAT

Asn GCG GGT TCA Ala Gly Ser GCA AAG GCT Ala Lys Ala

GGT

Gly 185

GTG

Val GTG GGT GAA Val Gly Glu

TGG

Trp 190

CCT

Pro GCT TTA AGA Ala Leu Arg 175 GGT GAA AAA Gly Glu Lys ACT CCA GAA Thr Pro Glu CTT ATG GCT Leu Met Ala 195 AGA GAC Arg Asp

GAA

Glu 200

TTC

Phe GAT GCC TAT Asp Ala Tyr

ATC

Ile 205

GAA

Glu 312 360 408 456 504 552 600 648 696 744 792 840 888 936 984 1032 1080 1128 1176 ACT GAA AAA Thr Glu Lys 210

ATT

Ile

ACT

Thr 215

ACT

Thr TTG ATG CCG Leu Met Pro

GTT

Val 220

AGG

Arg GAT GTG TTC Asp Val Phe

TCT

Ser 225 GCG GGT AGA Ala Gly Arg

GGG

Gly 230

GAT

Asp GTG GTT ACA Val Val Thr

GGT

Gly 235

GTT

Val ATT GAA AGA Ile Glu Arg GGC GTG Gly Val 240 GTG AAA GTA Val Lys Val AAA ACG ACT Lys Thr Thr 260 GGT GAA GCC Glv Glu Ala

GGC

Gly 245

GTA

Val GAA GTG GAA Glu Val Glu

ATC

Ile 250

ATG

Met GGT ATC AGA Gly Ile Arg ACC GGT GTA Thr Gly Val

GAA

Glu 265

GGC

Gly TTT AGG AAA Phe Arg Lys CCT ACA CAA Pro Thr Gin 255 TTG GAA AAA Leu Glu Lys ACT AAA AAA Thr Lys Lys GGC GAT AAT Gly Asp Asn 275 GAA GAA Glu Glu

GTG

Val 280

ATG

Met GTG CTT TTG Val Leu Leu

AGA

Arg 285

CCA

Pro GTG GAA CGC Val Glu Arg 290

CCG

Pro

GGT

Gly 295

GAG

Glu GTT CTA TGC Val Leu Cys

AAA

Lys 300

GTC

Val GGT TCT ATC Gly Ser Ile

ACT

Thr 305

GAA

Glu CAC AAG AAA His Lys Lys

TTT

Phe 310

ACT

Thr GGA GAA ATT Gly Glu Ile

TAT

Tyr 315

AAT

Asn CTT TCT AAA Leu Ser Lys

GAA

Glu 320 GGC GGG AGA Gly Gly Arg GTG CGC ACA Val Arg Thr

CAC

His 325

ACT

Thr CCA TTC TTC Pro Phe Phe TAC CGC CCG Tyr Arg Pro

CAA

Gin 335

GAA

Glu TTC TAT Phe Tyr GGC GTA Gly Val GAT GTG ACT GGC Asp Val Thr Gly ATC ACC CTT CCT Ile Thr Leu Pro SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 340 345 GAA ATG GTT ATG CCT GGC GAT AAT GTG AAA ATC Glu Met Val Met Pro Gly Asp Asn Val Lys Ile 355 360 AGC CCT GTT GCG TTA GAG TTG GGA ACT AAA TTT Ser Pro Val Ala Leu Glu Leu Gly Thr Lys Phe 370 375 380 GGT AGG ACC GTT GGT GCT GGT GTT GTG AGC AAT Gly Arg Thr Val Gly Ala Gly Val Val Ser Asn 390 395 CAAAAAGAGA GTTACCATAA AGGGTCATTA TGAAAGTTAA ATTGTGAAGA TATCAATTAC AGCACAACCA AGAACGCTAA

AGCTT

ACT GTA GAG TTG ATT Thr Val Glu Leu Ile 365 GCG ATT CGT GAA GGC Ala Ile Arg Glu Gly 385 ATT ATT GAA TAATATTAG Ile Ile Glu AATAGGGTTG AAGTGTTCTG AACTAACACT GAAAAACTGG 1224 1272 1323 1383 1443 1448 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 399 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: Met Ala Lys Glu Lys Phe Asn Arg Thr Lys 1 Thr Ser Asn Thr Asp Ala Met Pro Glu 145 Tyr Arg Lys Glu Ser 225 Ile Ala Ile Ser Cys Gin Pro His 130 Leu Glu Ala Val Arg 210 Ile Gly Val Asp His Pro Met Gin 115 Ile Leu Phe Leu Leu 195 Asp Ala His Leu Asn Ile Gly Asp 100 Thr Val Glu Pro Glu 180 Lys Thr Gly 5 Val Ser Ala Glu His Gly Arg Val Leu Gly 165 Glu Leu Glu Arg Asp Leu Pro Tyr 70 Ala Ala Glu Phe Val 150 Asp Ala Met Lys Gly 230 His Lys Glu 55 Glu Asp Ile His Leu 135 Glu Asp Lys Ala Thr 215 Thr Gly Gly 40 Glu Thr Tyr Leu Ile 120 Asn Met Thr Ala Glu 200 Phe Val Lys 25 Leu Lys Glu Val Val 105 Leu Lys Glu Pro Gly 185 Val Leu Val 10 Thr Ala Glu Asn Lys 90 Val Leu Gin Val Ile 170 Asn Asp Met Thr Pro Thr Glu Arg Arg 75 Asn Ser Ser Asp Arg 155 Val Val Ala Pro Gly 235 His Leu Met Gly His Met Ala Arg Met 140 Glu Ala Gly Tyr Val 220 Arg Val Ser Lys Ile Tyr Ile Ala Gin 125 Val Leu Gly Glu Ile 205 Glu Ile Asn Ala Asp Thr Ala Thr Asp 110 Val Asp Leu Ser Trp 190 Pro Asp Glu Ile Ala Tyr Ile His Gly Gly Gly Asp Ser Ala 175 Gly Thr Val Arg Gly Ile Asp Ala Val Ala Pro Val Gin Ala 160 Leu Glu Pro Phe Gly 240 SUBSTITUTE SHEET (RULE 26) WO 98/43479 153 PCT/US98/ Val Val Lys Val Gly Asp Glu Val Glu Ile Val Gly Ile Arg Pro Thr 245 250 255 Gin Lys Thr Thr Val Thr Gly Val Glu Met Phe Arg Lys Glu Leu Glu 260 265 270 Lys Gly Glu Ala Gly Asp Asn Val Gly Val Leu Leu Arg Gly Thr Lys 275 280 285 Lys Glu Glu Val Glu Arg Gly Met Val Leu Cys Lys Pro Gly Ser Ile 290 295 300 Thr Pro His Lys Lys Phe Glu Gly Glu Ile Tyr Val Leu Ser Lys Glu 305 310 315 320 Glu Gly Gly Arg His Thr Pro Phe Phe Thr Asn Tyr Arg Pro Gin Phe 325 330 335 Tyr Val Arg Thr Thr Asp Val Thr Gly Ser Ile Thr Leu Pro Glu Gly 340 345 350 Val Glu Met Val Met Pro Gly Asp Asn Val Lys Ile Thr Val Glu Leu 355 360 365 Ile Ser Pro Val Ala Leu Glu Leu Gly Thr Lys Phe Ala Ile Arg Glu 370 375 380 Gly Gly Arg Thr Val Gly Ala Gly Val Val Ser Asn Ile Ile Glu 385 390 395 INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: CGCGGATCCG AATGAAAAAA AATATCTTAA AT 32 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID CCGCTCGAGT TACTTGTTGA TAACAATTTT INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 06421 SUBSTITUTE SHEET (RULE 26) WO 98/43479 154 PCT/US98/06421 CGCGGATCCG AATGGCAAAA GAAAAGTTTA AC 32 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: CCGCTCGAGT TATTCAATAA TATTGCTCAC INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 22 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: Met Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn Ile Gly Thr 1 5 10 Ile Gly His Val Asp His INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: Ala His Asn Ala As Asn Ala Thr His Asn Thr Lys Lys 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 (xi) SEQUENCE DESCRIPTION: SEQ ID Lys Pro Ala His Asn Ala 1 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: Ile Asp Lys Gln Pro Lys Ala Lys Lys 1 INFORMATION FOR SEQ ID NO:77: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: Phe Trp Ala Lys Lys Gln Ala Glu 1 INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: GTGGAGAACA CACAATGAAA AAAAATATC 29 INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: SUBSTITUTE SHEET (RULE 26) WO 98/43479 156 PCT/US98/06421 GCTAATATTA TTCAATAATA TTGCTCACAA C 31 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID GGAGAAATAC AAATGGCAAA AGAAAAG 27 INFORMATION FOR SEQ ID NO:81: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: GCTAATATTA TTCAATAATA TTGCTCACAA C 31 INFORMATION FOR SEQ ID NO:82: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: CATAACGCAA ATAACGCTAC GCAT 24 INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: GGGAATTCAA AAAAACGAAA AAAACG 26 INFORMATION FOR SEQ ID NO:84: SEQUENCE CHARACTERISTICS: SUBSTITUTE SHEET (RULE 26) WO 98/43479 PCT/US98/06421 LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: CCCCTCGAGT TAATAGGCAA ACAC 24 SUBSTITUTE SHEET (RULE 26)

Claims (40)

1. An isolated polynucleotide that encodes: a polypeptide comprising an amino acid sequence that is homologous to the amino acid sequence of a Helicobacter membrane-associated polypeptide, wherein said amino acid sequence of said Helicobacter membrane-associated polypeptide is selected from the group consisting of the amino acid sequences as shown: -in SEQ ID NO:2, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 689 (GHPO 386); -in SEQ ID NO:4, beginning with an amino acid in any one of positions to 5, preferably in position -20 or position 1, and ending with an amino acid in position 713 (GHPO 789); -in SEQ ID NO:6, beginning with an amino acid in any one of positions 15 -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 725 (GHPO 1516); -in SEQ ID NO:8, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 691 (GHPO 1197); -in SEQ ID NO:10, beginning with an amino acid in any one of positions to 5, preferably in position -20 or position 1, and ending with an amino acid in position 652 (GHPO 1180); -in SEQ ID NO:12, beginning with an amino acid in any one of positions 18 to 5, preferably in position -18 or position 1, and ending with an amino acid 25 in position 673 (GHPO 896); -in SEQ D NO:14, beginning with an amino acid in any one ofpositions -in SEQ ID NO:14, beginning with an amino acid in any one ofpositions 159 -21 to 5, preferably in position -21 or position 1, and ending with an amino acid in position 619 (GHPO 711); -in SEQ ID NO: 16, beginning with an amino acid in any one of positions -17 to 5, preferably in position =U17 or positin 1, anu ending with an amino acid in position 635 (GHPO 190); -in SEQ ID NO: 18, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 626 (GHPO 185); -in SEQ ID NO:20, beginning with an amino acid in any one of positions -16 to 5, preferably in position -16 or position 1, and ending with an amino acid in position 467 (GHPO 1417); -in SEQ ID NO:22, beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1, and ending with an amino acid in position 673 (GHPO 1414); or (ii) a functional derivative of the polypeptide.

2. An isolated polynucleotide that encodes: a polypeptide comprising an amino acid sequence that is homologous to an amino acid sequence selected from the group consisting of the amino acid sequences as shown: -in SEQ ID NO:2, beginning with amino acid in position -19 and ending with an amino acid in position 689 (GHPO 386); S* *25

25. -in SEQ ID NO:4, beginning with an amino acid in position -20 and ending with an amino acid in position 713 (GHPO 789); -in SEQ ID NO:6, beginning with an amino acid in position -20 and ending with an amino acid in position 725 (GHPO 1516); -in SEQ ID NO:8, beginning with an amino acid in position -20 and ending with an amino acid in position 691 (GHPO 1197); -in SEQ ID NO: 10, beginning with an amino acid in position -20 and ending with an amino acid in position 652 (GHPO 1180); -in SEQ ID NO:12, beginning with an amino acid in position -18 and ending with an amino acid in position 673 (GHPO 896); -in SEQ ID NO: 14, beginning with an amino acid in position -21 and ending with an amino acid in position 619 (GHPO 711); -in SEQ ID NO:16, beginning with an amino acid in position -17 and ending with an amino acid in position 635 (GHPO 190); -in SEQ ID NO: 18, beginning with an amino acid in position -19 and ending with an amino acid in position 626 (GHPO 185); -in SEQ ID NO:20, beginning with an amino acid in position -16 and ending with an amino acid in position 467 (GHPO 1417); -in SEQ ID NO:22, beginningwith an amino acid in position -18 and ending with an amino acid in position 673 (GHPO 1414); or (ii) a functional derivative of the polypeptide. 161 3. The isolated polynucleotide of claim 1, which encodes the mature form of: a polypeptide comprising an amino acid sequence that is homologous to an amino acid sequence selected from the group consisting of the amino acid sequences as shown: -in SEQ ID NO:2, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 689 (GHPO 386); -in SEQ ID NO:4, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 713 (GHPO 789); -in SEQ ID NO:6, beginning with an amino acid in any one of positions to 5, preferably in position -20 or position 1, and ending with an amino acid in position 725 (GHPO 1516); -in SEQ ID NO:8, beginning with an amino acid in any one of positions to 5, preferably in position -20 or position 1, and ending with an amino acid in position 691 (GHPO 1197); SEQ ID NO:10, beginning with an amino acid in any one of positions to 5, preferably in position -20 or position 1, and ending with an amino acid in position 652 (GHPO 1180); -in SEQ ID NO: 12, beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1, and ending with an amino acid in position 673 (GHPO 896); :-in SEQ ID NO:14, beginning with an amino acid in any one of positions -21 to 5, preferably in position -21 or position 1, and ending with an amino acid in position 619 (GHPO 711); -in SEQ ID NO: 16, beginning with an amino acid in any one of positions 162 -17 to 5, preferably in position -17 or position 1, and ending with an amino acid in position 635 (GHPO 190); -in SEQ ID NO:18, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position 1, and ending with an amino acid in position 626 (GHPO 185); -in SEQ ID NO:20, beginning with an amino acid in any one of positions -16 to 5, preferably in position -16 or position 1, and ending with an amino acid in position 467 (GHPO 1417); -in SEQ ID NO:22, beginning with an amino acid in any one of positions -18 to 5, preferably in position -18 or position 1, and ending with an amino acid in position 673 (GHPO 1414); or (ii) a functional derivative of the polypeptide. 4. An isolated polynucleotide of any one of claims 1 to 3, wherein the polynucleotide is a DNA molecule. 5. An isolated polynucleotide according to claim 1, which is a DNA molecule that can be amplified and/or cloned by polymerase chain reaction from an Helicobacter genome, using either: *o go* H.\Pcabral\Keep\speci\68757.98.doc 17/05/02 163 -a 5'oligonucleotide primer having a sequence as shown in SEQ ID NO:23, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID (unprocessed GHPO 386); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:26, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:28 (unprocessed GHPO 789); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:29, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:31 (unprocessed GHPO 1516); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:32, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:34 (unprocessed GHPO 1197); a 5' oligonucleotide primer having a sequence as shown in SEQ ID and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:37 (unprocessed GHPO 1180); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:38, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:40 (unprocessed GHPO 896); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:41, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:43 (unprocessed GHPO 711); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:44, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:46 (unprocessed GHPO 190); 25 a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:47, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:49 (unprocessed GHPO 185); 164 a 5' oligonucleotide primer having a sequence as shown in SEQ ID and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:52 (unprocessed GHPO 1417); a 5' oligonucleotide primer having a sequence as shown in SEQ I NO:53, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID (unprocessed GHPO 1414); a 5' oligonucleotide primer comprising a sequence as shown in SEQ ID NO:78 and a 3' oligonucleotide primer comprising a sequence as shown in SEQ ID NO:79 (unprocessed GHPO 1360); a 5'oligonucleotide primer having a sequence as shown in SEQ ID NO:24, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID (mature GHPO 386); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:27, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:28 (mature GHPO 789); a 5' oligonucleotide primer having a sequence as shown in SEQ ID 9NO:30, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID *.NO:31 (mature GHPO 1516); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:33, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID NO:34 (mature GHPO 1197); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:36, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID *NO:37 (mature GHPO 1180); a 5' oligonucleotide primer having a sequence as shown in SEQ ID NO:39, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID (mature GHPO 896); 165 -a 5'oligonucleotide primer having a sequence as shown in SEQ ID NO:42, and a 3'oligonucleotide primer having a sequence as shown in SEQ ID NO:43 (mature GHPO 711); a 5'oligonucleotide primer having a sequence as shown in SEQ ID NO:45, and a 3'oligonucleotide primer having a sequence as shown in SEQ ID NO:46 (mature GHPO 190); a 5'oligonucleotide primer having a sequence as shown in SEQ ID NO:48, and a 3'oligonucleotide primer having a sequence as shown in SEQ ID NO:49 (mature GHPO 185); a 5'oligonucleotide primer having a sequence as shown in SEQ ID NO:51 and a 3'oligonucleotide primer having a sequence as shown in SEQ ID NO:52 (mature GHPO 1417); or a 5'oligonucleotide primer having a sequence as shown in SEQ ID NO:54, and a 3'oligonucleotide primer having a sequence as shown in SEQ ID NO:55 (mature GHPO 1414); *eee 6. An isolated DNA molecule of claim 5, which can be amplified and/or cloned by the polymerase chain reaction from a Helicobacter pylori genome. 7. An isolated polynucleotide according to claim 1, which is a DNA molecule that encodes the mature form or a derivative of a polypeptide encoded by the DNA molecule of claim 8. An isolated polynucleotide according to claim 1, which is a DNA molecule that encodes the mature form or a derivative of a polypeptide encoded by the DNA molecule of claim 6. H \Pcabral\Keep\speci\68757.98.doc 17/05/02 166 9. A compound, in a substantially purified form, that is the mature form or a derivative of a polypeptide comprising an amino acid sequence that is homologous to an amino acid sequence of a polypeptide associated with the Helicobacter membrane, which is selected from the group consisting of the amino acid sequences -in SEQ ID NO:2, beginning with amino acid in position -19 and ending with an amino acid in position 689 (GHPO 386); -in SEQ ID NO:4, beginning with an amino acid in position -20 and ending with an amino acid in position 713 (GHPO 789); -in SEQ ID NO:6, beginning with an amino acid in position -20 and ending with an amino acid in position 725 (GHPO 1516); -in SEQ ID NO:8, beginning with an amino acid in position -20 and ending with an amino acid in position 691 (GHPO 1197); -in SEQ ID NO: 10, beginning with an amino acid in position -20 and ending with an amino acid in position 652 (GHPO 1180); -in SEQ ID NO: 12, beginning with an amino acid in position -18 and ending with an amino acid in position 673 (GHPO 896); -in SEQ ID NO: 14, beginning with an amino acid in position -21 and ending with an amino acid in position 619 (GHPO 711); -in SEQ ID NO:16, beginning with an amino acid in position -17 and ending with an amino acid in position 635 (GHPO 190); S 30 -in SEQ ID NO:18, beginning with an amino acid in position -19 and ending with an amino acid in position 626 (GHPO 185); -in SEQ ID NO:20, beginning with an amino acid in position -16 and ending with an amino acid in position 467 (GHPO 1417); -in SEQ ID NO:22, beginning with an amino acid in position -18 and ending with an amino acid in position 673 (GHPO 1414); or 167 (ii) a functional derivative of said polypeptide. A compound according to claim 9, which is the mature form or a derivative of a polypeptide encoded by a DNA molecule of claim 11. A compound according to claim 9, which is the mature form or a derivative of a polypeptide encoded by a DNA molecule of claim 6. 12. A pharmaceutical composition for preventing or treating Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of a compound of any one of claims 9 to 11, and a pharmaceutically acceptable diluent or carrier. 13. A composition according to claim 12, further comprising an antibiotic, an antisecretory agent, a bismuth 20 salt, or a combination thereof. 14. A composition according to claim 13, wherein said antibiotic is selected from the group consisting of amoxicillin, clarithromycin, tetracycline, metronidizole, and erythromycin. A composition according to claim 13 or 14, wherein said bismuth salt is selected from the group **consisting of bismuth subcitrate and bismuth subsalicylate. *30 16. A composition according to any one of claims 13 to 15, wherein said antisecretory agent is a proton pump inhibitor. 17. A composition according to claim 16, wherein said proton pump inhibitor is selected from the group consisting of omeprazole, lansoprazole, and pantoprazole. H:\Pcabral\Keep\speci\68757.98.doc 17/05/02 168 18. A composition according to any one of claims 13 to 15, wherein said antisecretory agent is an H 2 -receptor antagonist. 19. A composition according to claim 18, wherein aid H 2 -receptor antagonist is selected from the group consisting of ranitidine, cimetidine, famotidine, nizatidine, and roxatidine. A composition according to any one of claims 13 to 15, wherein said antisecretory agent is a prostaglandin analog. 21. A composition according to claim 20, wherein said prostaglandin analog is misoprostil or enprostil. 22. A composition according to any one of claims 12 to 21, which further comprises a prophylactically or 20 therapeutically effective amount of a second Helicobacter polypeptide or a derivative thereof. 23. A composition according to claim 22, wherein the second Helicobacter polypeptide is a Helicobacter urease, a subunit, or a derivative thereof. 24. A composition according to any one of claims 12 to 23, further comprising an adjuvant. 25. A pharmaceutical composition for preventing or 0 treating Helicobacter infection in a mammal, said 0 composition comprising a prophylactically or i therapeutically effective amount of a polynucleotide of any one of claims 1 to 3, and a pharmaceutically acceptable carrier or diluent.

26. A pharmaceutical composition for preventing or H:\Pcabral\Keep\speci\68757.98.doc 17/05/02 169 treating Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of a polynucleotide of any one of claims 5 to 8, and a pharmaceutically acceptable carrier or diluent.

27. A composition comprising a viral vector, in the genome of which is inserted a DNA molecule according to claim 4, said DNA molecule being placed under conditions for expression in a mammalian cell and said viral vector being admixed with a physiologically acceptable diluent or carrier.

28. A composition according to claim 27, wherein said viral vector is a poxvirus.

29. A composition that comprises a bacterial vector comprising a DNA molecule of claim 4, said DNA molecule being placed under conditions for expression and said 20 bacterial vector being admixed with a physiologically acceptable diluent or carrier. so

30. A composition according to claim 29, wherein said vector is selected from the group consisting of Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bili6 de Calmette-Gu6rin, and Streptococcus.

31. A composition according to claim 25, wherein said polynucleotide is a DNA molecule that is inserted in a 30 plasmid that is unable to replicate and to substantially integrate in a mammalian genome and is placed under so.. conditions for expression in a mammalian cell. a

32. An expression cassette comprising a DNA molecule of claim 4, said DNA molecule being placed under conditions for expression in a procaryotic or eucaryotic cell. H:\Pcabral\Keep\gpeci\68757.98.doc 17/05/02 170

33. A process for producing a compound according to any one of claims 9 to 11, which comprises culturing a procaryotic or eucaryotic cell transformed or transfected with an expression cassette of claim 33, and recovering said compound from the cell culture.

34. A pharmaceutical composition for preventing or treating Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of an antibody that binds to the compound of any one of claims 9 to 11, and a pharmaceutically acceptable carrier or diluent. A method for preventing or treating Helicobacter infection in a mammal, comprising the step of administering a compound according to any one of claims 9 to 11, to a mammal in need of such treatment.

36. A method according to claim 35, further 20 comprising administering an antibiotic, an antisecretory agent, a bismuth salt, or a combination thereof.

37. A method according to claim 36, wherein said antibiotic is selected from the group consisting of amoxicillin, clarithromhycin, tetracycline, metronidizole, and erythromycin.

38. A method according to claim 36 or 37, wherein said bismuth salt is selected from the group consisting of bismuth subcitrate and bismuth subsalicylate.

39. A method according to any one of claims 36 to 38, wherein said antisecretory agent is a proton pump inhibitor. A method according to claim 36, wherein said proton pump inhibitor is selected from the group consisting H.\Pcabral\Keep\speci\68757.98.doc 17/05/02 171 of omeprazole, lansoprazole, and pantoprazole.

41. A method according to any one of claims 36 to 38, wherein said antisecretory agent is an H 2 -receptor antagonist.

42. A method according to claim 41, wherein said Hz- receptor antagonist is selected from the group consisting of ranitidine, cimetidine, famotidine, nizatidine, and roxatidine.

43. A method according to any one of claims 36 to 38, wherein said antisecretory agent is a prostaglandin analog.

44. A method according to claim 43, wherein said prostaglandin analog is misoprostil or enprostil. A method according to any one of claims 35 to 44, further comprising administering a prophylactically or therapeutically effective amount of a second Helicobacter 20 polypeptide or a derivative thereof. o

46. A method according to claim 45, wherein the second Helicobacter polypeptide is a Helicobacter urease, a subunit, or a derivative thereof.

47. A method according to any one of claims 36 to wherein an adjuvant or a pharmaceutically acceptable carrier is also administered. 30 48. A method for preventing or treating Helicobacter infection in a mammal, comprising the step of administering a polynucleotide of any one of claims 1 to 3, to a mammal in need of such treatment.

49. A method for preventing or treating Helicobacter infection in a mammal, comprising the step of administering a polynucleotide of any one of claims 5 to 8, to a mammal H:\Pcabral\Keep\speci\68757.98.doc 17/05/02 t 0 172 in need of such treatment. A method for preventing or treating Helicobacter infection in a mammal, comprising the step of administering an antibody that binds to the compound of anyone of claims 9 to 11 to a mammal in need of such treatment.

51. Use of a compound according to any one of claims 9 to 11, for the manufacture of a medicament for preventing or treating Helicobacter infection in a mammal.

52. Use according to claim 51, wherein said medicament further comprises an antibiotic, an antisecretory agent, a bismuth salt, or a combination thereof.

53. Use according to claim 52, wherein said antibiotic is selected from the group consisting of amoxicillin, clarithromycin, tetracycline, metronidizole, 20 and erythromycin.

54. Use according to claim 52 or 53, wherein said bismuth salt is selected from the group consisting of bismuth subcitrate and bismuth subsalicylate. Use according to any one of claims 52 to 54, wherein said antisecretory agent is a proton pump inhibitor. 30 56. Use according to claim 55, wherein said proton pump inhibitor is selected from the group consisting of omeprazole, lansoprazole, and pantoprazole.

57. Use according to any one of claims 52 to 54, wherein said antisecretory agent is an H 2 -receptor antagonist. H:\Pcabral\Keep\spec1\68757.98.doc 17/05/02 9I, 173

58. Use according to claim 57, wherein said H 2 receptor antagonist is selected from the group consisting of ranitidine, cimetidine, famotidine, nizatidine, and roxatidine.

59. Use according to claim 52, wherein said antisecretory agent is a prostaglandin analog. Use according to claim 59, wherein said prostalgandin analog is misoprostil or enprostil.

61. Use according to any one of claims 51 to wherein said medicament further comprises a prophylactically or therapeutically effective amount of a second Helicobacter polypeptide or a derivative thereof.

62. Use according to claim 61, wherein the second Helicobacter polypeptide is a Helicobacter urease, a subunit, or a derivative thereof.

63. Use according to any one of claims 51 to 62 wherein said medicament further comprises an adjuvant.

64. Use of a polynucleotide according to any one of claims 1 to 3, for the manufacture of a medicament for preventing or treating Helicobacter infection in a mammal.

65. Use of a polynucleotide according to any one of claims 5 to 8, for the manufacture of a medicament for 30 preventing or treating Helicobacter infection in a mammal.

66. Use of an antibody that binds to a compound according to any one of claims 9 to 11, for the manufacture of a medicament for preventing or treating Helicobacter infection in a mammal.

67. An isolated polynucleotide according to claim 1 H:\Pcabral\Keep\spec1i\8757.98.doc 17/05/02 4 I 174 or 2, substantially as herein described, with reference to the examples.

68. A compound according to claim 9, substantially as herein described, with reference to any one of the examples.

69. A pharmaceutical composition according to any one of claims 12, 25, 26 or 34, substantially as herein described, with reference to any one of the examples. A method according to any one of claims 35, 48, 49 or 50, substantially as herein described, with reference to any one of the examples.

71. Use according to any one of claims 51, 64, 65 or 66, substantially as herein described, with reference to any one of the examples. Dated this 17th day of May 2002 MERIEUX ORAVAX and HUMAN GENOME SCIENCES, INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia e e* .9. S. S S 55 \\melbfiles\home$\Pcabral\Keep\speci\68757.98.doc 17/05/02