CA2286893A1 - 76 kda, 32 kda, and 50 kda helicobacter polypeptides and corresponding polynucleotide molecules - Google Patents

Abstract

The invention provides 76 kDa, 32 kDa, and 50 kDa Helicobacter polypeptides, which can be used in vaccination methods for preventing or treating Helicobacter infection, and polynucleotides that encode these polypeptides.
The invention also provides diagnostic methods employing these polypeptides.

Description

76 kDa 32 kD~,, and 50 kDa HELICOBACTER POLYPEPTIDES AND
' ('ORRESPONDING PQLYNUCLEOTIDE 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 Helicobacter is a genus of spiral, gram-negative bacteria that colonize the gastrointestinal tracts of mammals. Several species colonize the 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 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 with histological evidence of gastritis and peptic ulceration. Indeed, H.
pylori 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,

-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 (Bolin et al., J. Clin. Microbiol. 33:381, 1995); a lactofernn 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 SO kDa polypeptide, designated GHPO 750, which can be used, e.g., in methods to prevent, treat, or diagnose Helicobacter infection. The polypeptides include those having the amino acid sequences

-3-shown 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 (i.e., 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 (i) 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, e.g., methods for preventing or treating Helicobacter infection) in combination with a diiuent 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 of polypeptides, or monospecific antibodies of the invention, and related pharmaceutical compositions; (iv) methods for detecting the presence of Helicobacter in biological samples, which can--involve the use of polynucleotide molecules, monospecific antibodies, or polypeptides of the invention; and (v) methods for purifying polypeptides of the invention by antibody-based affinity chromatography. -

-4-Brief Description of the Drawings Figure 1 is an alignment of the predicted amino acid sequences of GHPO 386 (SEQ ID N0:2), GHPO 789 (SEQ ID N0:4), and GHPO 1516 (SEQ ID N0:6), 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 N0:8), GHPO 1180 (SEQ ID NO:10), GHPO 896 (SEQ
ID N0:12), GHPO 711 (SEQ ID N0:14), GHPO 190 (SEQ ID N0:16), GHPO
185 (SEQ ID N0:18), GHPO 1417 (SEQ ID N0:20), and GHPO 1414 (SEQ
ID N0:22), 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 (i.e., as polypeptides that have been exported through class II or class III secretion

-5-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 71 l, GHPO 190, GHPO 185, GHPO 1417, GHPO 1414, GHPO 1360, and GHPO 750.
An isolated polynucleotide of the invention encodes:
{i) 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 N0:2, 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 N0:4, 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 N0:6, 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);

-6--in SEQ ID N0:8, beginning with an amino acid in any one of positions -20 to 5, preferably in position -20 or position l, 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 S 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 N0:12, 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 N0:14, 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 N0:16, beginning with an amino acid in any one of positions -17 to 5, preferably in position -17 or position l, and ending with an amino acid in position 635 (GHPO 190);
-in SEQ ID N0:18, beginning with an amino acid in any one of positions -19 to 5, preferably in position -19 or position l, and ending with an amino acid in position 626 (GHPO 185);
-in SEQ ID N0:20, 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 N0:22, beginning with an amino acid in any one of positions -18 to S, preferably in position -18 or position 1, and ending with an amino acid in position 673 (GHPO 1414);
-in SEQ ID N0:66, 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 _'7_ -in SEQ ID N0:68, 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-occurnng 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, e.g., a cloning event (amplification), is "isolated." Typically, an isolated DNA
molecule is free from DNA regions (e.g., 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 (e.g., 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 (anti-sense) strand. The sequences that encode polypeptides of the invention, as shown in SEQ ID NOs:2-22 (even numbers), 66, and 68, can be (a) the coding sequence as shown in SEQ ID NOs: l-21 (odd numbers), 65, and 67; (b) a ribonucleotide sequence derived by transcription of (a); or (c) 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:I-21 (odd numbers), 65, and 67. The polypeptides of the invention can be ones that are naturally secreted or excreted by, e.g., H. felis, H. mustelae, H. heilmanii, or H. pylori.

_g_ By "polypeptide" or "protein" is meant any chain of amino acids, regardless of length or post-translational modification (e.g., 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:I-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 75%, 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 90%, 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 aspartic 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 (e.g., 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 (i.e., 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 (i.e., 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:l-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 naturally-occurring 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, e.g., 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, e.g., 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 Urea subunits of H. pylori and H. fells 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. fells infection (Michetti et al., Gastroenterology 107:1002, 1994). In addition, it has beer~shown that the individual structural subunits of urease, UreA and Urea, 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 variants have been reported to be effective vaccine antigens, including, e.g., recombinant UreA + Urea apoenzyme expressed from pORV 142 (UreA and ' UreB sequences derived from H. pylori strain CPM630; Lee et al., J. Infect.
Dis.172:161, 1995); recombinant UreA + Urea apoenzyme expressed from pORV214 (UreA and Urea sequences differ from H. pylori strain CPM630 by one and two amino acid changes, respectively; Lee et al., supra, 1995); a UreA-giutathione-S-transferase fusion protein (UreA sequence from H. pylori strain ATCC 43504; Thomas et al., Acta Gastro-Enterologica Belgica 56:54, 1993); UreA + Urea holoenzyme purified from H. pylori strain NCTCl 1637 (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 Urea-MBP fusion protein (Urea from H. pylori strain 85P; Ferrero et al., supra); a UreA-MBP fusion protein (UreA from H. fells strain ATCC
49179; Ferrero et al., supra); a Urea-MBP fusion protein (Urea from H. fells strain ATCC 49179; Ferrero et al., supra}; and a 37 kDa fragment of Urea containing amino acids 220-569 (Dore-Davin et al., "A 37 kD fragment of Urea is sufficient to confer protection against Helicobacter fells infection in mice"). Finally, Thomas et al. (supra) showed that oral immunization of mice with crude sonicates of H. pylori protected mice from subsequent challenge with H. fells.
Polynucieotides, e.g., 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:l-21 (odd numbers), 65, and 67. Typically, a primer consists of 10 to 40, preferably 15 to 25 nucleotides. It can also be advantageous to select primers containing C and G nucleotides in proportions sufficient to ensure efficient hybridization, e.g., an amount of C and G nucleotides of at least 40%, 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 N0:2), including a signal peptide, are shown in SEQ ID N0:23 (matching at the 5' end) and in SEQ ID N0:25 {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
N0:2), lacking a signal peptide, are shown in SEQ ID N0:24 (matching at the 5' end) and in SEQ ID N0:25 (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 N0:66), are shown in SEQ ID N0:78 (matching at the S' end) and in SEQ ID N0:79 (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 N0:82 (matching at the 5' end of the coding sequence corresponding to the mature protein) and SEQ ID N0:79 (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:

(i) isolated polynucleotide molecules (e.g., DNA molecules) that can be amplified and/or cloned using the polymerase chain reaction from a Helicobacter, e.g., H. pylori, genome using either:
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:23, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:25 (unprocessed GHPO 386);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:26, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:28 (unprocessed GHPO 789);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:29, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:31 (unprocessed GHPO 1 S 16);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:32, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:34 (unprocessed GHPO 1197);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:35, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:37 (unprocessed GHPO 1180);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:38, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:40 (unprocessed GHPO 896);
a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:41, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
- N0:43 (unprocessed GHPO 711 );
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:44, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:46 (unprocessed GHPO 190); -- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:47, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:49 (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
N0:52 (unprocessed GHPO 1417);
a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:53, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO:55 (unprocessed GHPO 1414);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:78, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:79 (unprocessed GHPO 1360); or - a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:80, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:81 (GHPO 750); and (ii) isolated polynucleotide molecules (e.g., DNA molecules) that can be amplified and/or cloned by the polymerase chain reaction from a Helicobacter, e.g., H. pylori, genome using either:
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:24, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:25 (mature GHPO 386);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:27, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:28 (mature GHPO 789); - --- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:30, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:31 (mature GHPO 1516);

- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:33, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:34 (mature GHPO 1197);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
S N0:36, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:37 (mature GHPO 1180);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:39, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:40 (mature GHPO 896);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:42, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:43 (mature GHPO 711 );
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:45, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:46 (mature GHPO 190);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:48, and a 3' oligonucieotide primer having a sequence as shown in SEQ ID
N0:49 (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
N0:52 (mature GHPO 1417);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:54, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:55 (mature GHPO 1414); or - --- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:82, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
N0:79 (mature GHPO 1360).

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 5'-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 (e.g., 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 1 S invention include, e.g., 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 SO amino acids, more preferably at least 75 amino acids, and most preferably at least 100 amino acids in length.

Useful polypeptide derivatives, e.g., 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 (e.g., 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 amino 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, e.g., 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, e.g., 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, i.e., 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 inverrti-on fused to a polypeptide having adjuvant activity, such as, e.g., subunit B of either cholera toxin or E. toll heat-labile toxin. Several possibilities can be used 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-occurnng 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: l -21 (odd numbers), 65, and 67. Hybridization procedures are, e.g., 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 calculatian of the melting temperature (Tm), 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 + 0.5 x-{%
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 25°C, or, preferably, 30 to 40°C below the 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, (i) within 4-16 hours at 42°C, in 6 x SSC containing SO% formamide or (ii) within 4-16 hours at 65°C in an aqueous 6 x SSC
solution (1 M NaCI, 0.1 M sodium citrate (pH 7.0)). 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 (G+C) + 2 (A+T). 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 (i) 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 (i) an expression cassette containing a polynucleotide molecule of the invention placed under the control of elements (e.g., a promoter) required for expression; {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 (e.g., Saccharomyces cerevisiae or Pichia pastoris), mammalian cells (e.g., COS1, NIH3T3, or JEG3 cells), arthropods cells (e.g., Spodoptera frugiperda (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, e.g., 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, e.g., 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 is 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 (U.S. 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 (U.S. Patent No. 4,952,496); the OspA lipidation signal peptide; and RIpB
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 (e.g., 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 krrewn 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 can 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 aff nity-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 (i) 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 (e.g., 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, e.g., a protective or therapeutic immune response to Helicobacter; and (v) a method for preventing and/or treating a Helicobacter (e.g., 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, e.g., 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 ~e.g., ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via a parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route. Preferred routes depend 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 (e.g., 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, Tlibrio cholerae, Lactobacillus, Bacille bilie de Calmette-Guerin (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, e.g., 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 1 x 104 to about 1 x 10" , advantageously from about 1 x 10' to about 1 x 10' °, or, preferably, from about 1 x 10' to about 1x109 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 __ __ __ _~_.

chemical 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 attRSl 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, e.g., about 1 x 1 OS to about 1 x 109, preferably about 1x106 to about 1x108 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; Shigella flexneri); Medaglini et al. (Proc. Natl. Acad. Sci. USA
92:6868, 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 (i) a composition of matter containing a polynucleotide of the invention, together with a diluent or Garner; (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, e.g., a protective immune response to Helicobacter; and (iv) a method for preventing and/or treating a Helicobacter (e.g., H. pylori, H.
fells, 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, e.g., in a plasmid that is unable to replicate in mammalian cells and to substantially integrate into a mammalian genome. -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 (U.S. 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, e.g., 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 encoding 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 and/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, e.g., 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, e.g., (i) complemented with a chemical agent that modifies cellular permeability, such as bupivacaine (see, e.g.,

7), (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, LipofectinTM, which is also known as DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DOTAP (1,2-bis(oleyloxy)-3-(trimethylammonio)propane), DDAB
(dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine), and cholesterol derivatives. A description of these cationic lipids 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 transfection-facilitating compounds can be added to a formulation containing cationic liposomes. A number of them are described in, e.g., WO 93/18759,

8, WO 94/25608, and WO 95/2397. They include, e.g., 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 (e.g., 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 ~.g to about 1 mg, preferably, from about 10 ~,g to about 800 ~.g, and, more preferably, from about 25 ~.g to about 250 ~,g, can be administered to human adults. The administration can be achieved in a single dose or repeated at intervals.

9 PCT/US98/06421 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, e.g., 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, intradenmal, 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:l-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 -complementary or anti-sense sequence of any of SEQ ID NOs: l-21 (odd numbers), 65, and 67. Generally, probes are significantly shorter than the full-length sequences shown in any of SEQ ID NOs: l-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: l-21 (odd numbers), 65, and 67, or a sequence complementary to such sequences.
Probes can contain modified bases, such as inosine, methyl-5-deoxycytidine, deoxyuridine, dimethylamino-S-deoxyuridine, or diamino-2, 6-purine. 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, e.g., 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 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 40 nucleotides and are used to initiate enzymatic polymerization of DNA in an amplification process (e.g., 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 (i) 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 andlor identifying the presence of Helicobacter in a biological material, in which (a) a sample is recovered or derived from the biological material, (b) DNA or RNA
is extracted from the material and denatured, and {c) 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) a sample is recovered or derived from the biological material, (b) DNA is extracted therefrom, (c) the extracted DNA is contacted with at least one, or, preferably two, primers of the invention, and amplified by the polymerase chain reaction, and (d) an amplified DNA - --molecule is produced.
As mentioned above; polypeptides that can be produced by expression of the polynucieotides of the invention can be used as vaccine antigens: 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 (Towl~in 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-a-purified 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 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 ~,1 of a preparation of about 10 ~.g 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 20 (PBS/Tween buffer) and the wells are saturated with 250 ~,l 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 p,l dilutions are added to each well. The plate is incubated for 90 minutes at 37 ° C, 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 1.0 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 about 100 p,g/ml is serially diluted two-fold with 50 mM Tris-HCl (pH 7.5).
One hundred ~.1 of each dilution is applied to a filter, such as a 0.45 ~m 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-HCI (pH 7.5), 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°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, e.g., 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 (i) 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, e.g., a protective immune response to Helicobacter; and (iv) a method for preventing and/or treating a Helicobacter (e.g., H. pylori, H. felis, H. mustelae, or-H.

WO 981434'79 PCT/US98lU6421 heilmanii) 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 (e.g., ocular, intranasal, pulmonary, oral, gastric, intestinal, rectal, vaginal, or urinary tract) surface or via a parenteral (e.g., 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 w 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 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 (e.g., 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 (e.g., 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 ~,g 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 ~,g.
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, e.g., 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 (e.g., LT).
PoIypeptides and polypeptide derivatives of the invention can also be used as diagnostic reagents for detecting the presence of anti-Helicobacter antibodies, e.g., in blood samples. Such polypeptides can be about 5 to about 80, 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, e.g., 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 poiyclonal or monoclonal. Monospecific antibodies can be recombinant, e.g., chimeric (e.g., consisting of a variable region of murine origin and a human constant region), humanized (e.g., 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, e.g., 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 immunologicai assays, e.g., Western blot assays, dot blot assays, or ELISA (see, e.g., 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 (i) 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, e.g., 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 (e.g., 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 adsorption (i. e., 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, e.g., 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, e.g., 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, -preferably, equilibrated with the buffer used to dilute the biological sample, so that the polypeptide or polypeptide derivative of the invention {i.e., 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, e.g., guanidine HCI, or a buffer having high salt concentration (e.g., 3 M MgCl2). Eluted fractions are recovered and the presence of the antigen is detected, e.g., 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 (i) 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 (e.g., 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 immunization- --methods, the antibody is administered to a mucosal surface of a mammal, e.g., the gastric mucosa, e.g., orally or intragastrically, optionally, in the presence of a bicarbonate buffer. Alternatively, systemic administration, not requiring a bicarbonate buffer, can be earned 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, e.g., by measuring induction of a mucosal immune response or induction of protective and/or therapeutic immunity, using, e.g., the H.
fells mouse model and the procedures described by Lee et al. (Eur. J.
Gastroenterology & Hepatology 7:303, 1995) or Lee et al. (J. Infect. Dis.
172:161, 1995). Those skilled in the art will recognize that the H. fells 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 of Helicobacter 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, e.g., by Lee et al. (supra). Then, after an appropriate period of time, the bacterial load of the mucosa can be estimated by assessing 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, e.g., the cholera toxin (CT), the E. toll heat-labile toxin (LT), the Clostridium docile toxin A, the pertussis toxin (PT), 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, e.g., 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-1 I2-Asp mutants. Other adjuvants, such as the bacterial monophosphoryl lipid A (MPLA) of, e.g., E.
toll, 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.

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, e.g., water or a saline solution, such as PBS, optionally, including a bicarbonate salt, such as sodium bicarbonate, e.g., 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, e.g., macrolides, tetracyclines, ~i-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, e.g., H2-receptor antagonists (e.g., cimetidine, ranitidine, famotidine, nizatidine;
and roxatidine), proton pump inhibitors (e.g., omeprazole, lansoprazole, and pantoprazole), prostaglandin analogs (e.g., misoprostil and enprostil), and anticholinergic agents (e.g., pirenzepine, telenzepine, carbenoxolone, and proglumide); and bismuth salts, including colloidal bismuth subcitrate, tripotassium dicitrate bismuthate, bismuth subsalicylate, bicitropeptide, and pepto-bismol (see, e.g., Goodwin et al., Helicobacter pylori, Biology and Clinical Practice, CRC Press, Boca Raton, FL, pp 366-395, 1993; Physicians' S Desk Reference, 49"' edn., Medical Economics Data Production Company, Montvale, New Jersey, 1995). In addition, compounds containing more than one of the above-listed components coupled together, e.g., ranitidine coupled to bismuth subcitrate, can be used. The invention also includes compositions for carrying out these methods, i. e., 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 treatmentlimmunization 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, e.g., gastric and duodenal ulcers. a.
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 Helicobacter pylori strain ATCC number 43579 (American Type Culture Collection, Rockville, Maryland) by immunoaffinity-based 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 l, 5, or 25 ~.g of the purified 76 kDa proteins, purified GHPO 1360, or purified GHPO 750 in combination with 5 ~,g of the heat-labile enterotoxin (LT) of E. coli. Twenty five ~.g of recombinant urease, in combination with 5 ~g LT, was used as a positive control, and 5 ~g 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 (i.e., 1, 5, and 25 ~.g), 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 ~,g doses generally approaching those of mice immunized with 25 ~.g 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 kDa 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 2). 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 2). These results show that the purified 76 kDa proteins, GHPO 1360, and GHPO 750 are effective vaccine antigens for use in preventing Helicobacter infection.

Table 1 Prophylactic Immunization with PMsv Antigens as Oral Dose Response Against H.
pylori Challenge BALB/c mice Fisher's exactCFU/ml (1/4 Wilcoxon rank test antrum) sums # mice infectedinfection Mean t SD test status (based Antrum on quantitative CFU treatment Asso group (based on ratios, treatment v. LT only quantitative control Asso, 0.148 group v. LT (group 1 1 O.D. only ) Treatment cutoff) (group 11))p-value p-value S I ug 50 kDa 60% (6/10) 0.3034 30825 f 23210 0.1736 + LT

pg 50 kDa 40% (4/10) 0.0573 18910 t 16341 0.0588 + LT

25 pg 50 kDa 30% (3/10) 0.0198 22710 f 32397 0.0821 + LT

1 pg 32 kDa+ 50% (5/10) 0.1409 44225 f 87824 0.0756 LT

5 Etg 50 kDa I 0% ( 1 / 0.0011 11811 t l 15?90.0191 + LT I 0) 1 25 pg 50 kDa 0 (0/9) 0.0001 1608 t 23917 0.01 I 4 ~ + LT

25 wg rUre 0 (0/9) 0.0001 8208 t 8021 0.0179 + LT

LT 90% (9/10) - 107340 t 127949-90% (9/10) not determined46173 t 42325 0.2568 Table 2 Prophylactic Immunization with PMsv Antigens as Oral Dose Response Against H.
pylori Challenge BALB/c mice Fisher's exactCFU/ml (1/4 Wilcoxon rank test antrum) sums # mice infectedinfection Mean t SD test status (based Antrum on quantitative CFU treatment Asso group (based on ratios, treatment v. LT only quantitative control Asso, 0.148 group v. LT (group 1 1 O.D. only ) Treatment cutoff) (group 11 p-value )) p-value 1 pg 76 kDa 56% (5/9) 0.1409 39922 f 34708 0.2203 + LT

5 pg 76 kDa 8U% (415) 1 8802 t 7788 0.0864 + LT

25 pg 76 kDa 33% (3/9) 0.0198 9712 t 12183 0.0178 + LT

25 pg rUre 0 (0/9) 0.0001 8208 t 8021 0.017) + LT

LT 90% (9/lOj - 107340 t 127949-90% (9/10) not determined46173 f 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, e.g., genes encoding 76 kDa proteins, such as GHPO 386, GHPO 789, GHPO 1516, GHPO 1197, GHPO 1180, GHPO 896, GHPO 7I l, 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 1 S 16, 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 for 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 ltDa), GHPO 1360 (32 lzDa), 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 75 cm2 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 cm2 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°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 NaCI, 0.724 g disodium phosphate, and 0.210 g monopotassium phosphate for one liter (pH 7.2)) for a one liter culture. The - bacteria are then spun in a centrifuge again under the same conditions. - -The pellet (C 1 } is suspended in 1 % N-octyl-D-glucopyranoside (NOG; 30 ml/L; Sigma). The bacterial suspension is incubated for 1 hour at room temperature while stirring, spun in a centrifuge at 17,600 x g for 30 minutes at 4°C, and the pellet (C2) is recovered.
The supernatant (S2) is dialyzed against PBS overnight at 4°C
while stirring. The precipitate is recovered by centrifugation at 2,600 x g for 30 minutes at 4°C. The supernatant (S2d) is discarded and the pellet (Cs2d) is recovered and stored at -20°C.
The pellet (C2) is resuspended in 20 mM Tris-HCl buffer (pH 7.5) and 100 ~.M Pefabloc (Buffer A), 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°C, and then is spun in an ultracentrifuge at 210,000 x g for 30 minutes at 4°C. The supernatant (S3), 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°C. The supernatant (S4) is eliminated and the pellet (C4) is stored at -20°C. This pellet (C4) contains membrane proteins.
The pellet (C4) is washed in 50 mM NaCO, (pH 9.5) and 100 ~M
Pefabloc (buffer B). The suspension is spun in an ultracentrifuge at 210,000 x g for 30 minutes at 4°C. The supernatant (SS) is eliminated, and the pellet (CS) 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°C.

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 95°C. 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 8.8), 8 M urea, 10% SDS, 100 ~,M phenyl methyl sulfonyl fluoride (PMSF), and 10 wM Pefabloc (buffer C)).
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 75% methanol and 75% isopropanol, and then is spun in a centrifuge at 240,000 x g for 16 hours at 10°C.
Each pellet is resuspended in 2 ml of 10 mM NaPO, (pH 7.0) containing 1 M NaCI, 0.1 % Sarkosyl, 100 ~.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 are 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 ~,m), and stored at -20°C.
1.C. Purification of the 76 ltDa, 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 lcDa protein (GHPO 750), which are purified by preparative SDS-PAGE, is prepared by hyperimmunizing rabbits as follows. On day 0, a preparation containing 50 ~g 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 ~,g of the protein in incomplete Freund's adjuvant, and are sacrificed at day 60.
Complement is removed from the serum by heating for 30 minutes at 56°C.
The hyperimmune serum is then sterilized by filtration through a Millipore membrane (0.22 ~tm).
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 8.0). The column is washed with 10 column volumes of 100 mM Tris-HCl (pH 8.0), and then with 10 column volumes of 10 mM Tris-HCl (pH 8.0). IgGs are eluted in 0.1 M glycine buffer (pH 3.0), 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 IgG-containing fractions are pooled together and, if necessary, frozen at -70°C.

1.C.3. Preparation of the column An appropriate amount of CNBr-activated Sepharose 4B gel - (Pharmacia; reference: I 7-0430-O 1 ) is suspended in I 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 HCI. The total volume of 1 mM HCl that is used amounts to 200 ml/g of gel.
Purified IgGs are dialyzed for 4 hours at room temperature against 50 volumes of 500 mM sodium phosphate buffer (pH 7.5). The IgGs are then diluted to 3 mg/ml with the same buffer. IgGs are incubated with the gel overnight at 5~3°C while stirring. The gel is packed in a chromatography column and is washed with 2 column volumes of 500 mM phosphate buffer {pH 7.5). The gel is then transferred to a tube and is incubated with 100 mM
ethanolamine (pH 7.5), 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 7G kDa protein is adsorbed and eluted as follows. The membrane fraction Cs2d is suspended in 50 mM Tris-HCl (pH 8.0), 2 mM
EDTA, and then is filtered through a 0.45 ~m membrane. The supernatant is applied to the column, which is equilibrated with 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, at a flow rate of about 10 ml/hour. The column is washed with 20 column volumes of 50 mM Tris-HCI (pH 8.0), 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 2.5). The eluate is collected in 3 ml fractions, to each of which is added 150 ~,1 1 M
phosphate buffer (pH 8.0). The optical density of each fraction is measured at 280 nm, fractions containing the 76 kDa protein are pooled, and stored at -70°C.
Analysis by 10% SDS-PAGE reveals a single band at 76 kDa. N-terminal sequence was carried out on this purified 76 kDa preparation, and the sequence obtained is as follows: EDDGFYTSVGYQIGEAAQMV (SEQ ID
N0:58).
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 50 mM NaC03 (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°C. The 47 and 35 kDa proteins are insoluble in the NaC03 buffer and are eliminated in the pellet.
The supernatant is dialyzed against 50 mM Tris-HCL (pH 8.0), 2 mM EDTA, and then is filtered through a 0.45 ~m membrane. The filtered supernatant is applied to the column, which is equilibrated with 50 mM Tris-HCL (pH 8.0), 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 8.0), 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 2.5). The eluate is collected in 3 ml fractions, to each of which is added 150 ~1 1 M
phosphate buffer (pH 8.0). The optical density of each fraction is measured at 280 nm, and fractions containing the SO or 32 kDa protein are pooled and stored at -70°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 50 kDa protein preparation. The sequence found is as follows:
MKEKFNRTKPHVNIGTIGHVDH (SEQ ID N0:73). Similarly, N-terminal and internal sequencing is carried out with the purified 32 ltDa preparation.
The sequences found are as follows: AHNANNATHNTKK (SEQ ID N0:74) and KPAHNA (SEQ ID N0:75) (N-terminal), and IDKQPKAKK (SEQ ID
N0:76) and FWAKKQAE (SEQ ID N0:77) (internal).
1.D. Purification of the 76 IzDa protein from membrane fraction Cs2d and purification of the 32 kDa and 50 kDa proteins from membrane fraction 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 . 10 manufacturer's instructions (Pharmacia). The column is washed and equilibrated with buffer B, containing 50 mM NaCO, (pH 9.5), 100 ~,M
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 NaCI gradient is applied to the column. The fraction eluted between 0.35 and 0.45 M NaCI is further purified on a 10 ml S-Sepharose column (diameter: 1.5 cm; height:
5 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 ~,M 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 eluted with a 0-0.5 M NaCI gradient in acetate buffer. The fraction eluted at 0.15 M NaCl 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 NaCO, buffer (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°C. This allows the 32 and 35 kDa proteins to be separated, since the 35 kDa protein is insoluble in the NaC03 buffer.
The supernatant is dialyzed against 50 mM NaP04 buffer (pH 7.0), and then is applied to an SP-Sepharose column, which is equilibrated with the NaP04 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°C. This allows the 50 and 47 kDa proteins to be separated, since the 47 kDa protein is insoluble in the NaC03 buffer. The supernatant is dialyzed against 50 mM NaPO, buffer (pH 7.0).
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 p,M 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 0.1-0.5 M NaCl gradient in buffer B ( 10 fold VT), which is followed by washing in WO 98/43479 PCTlUS98/06421 buffer B containing 0.5, and then 1, M NaCI (2 fold V,.). 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 DEAF Sepharose column (diameter: 1.5 cm, height: 5 cm) is prepared according to the manufacturer's instructions (Pharmacia) {up to 10 mg proteinlml 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 NaCI gradient in buffer B ( 10 fold VT), followed by washing in buffer B, containing 1 M NaCI (2 fold VT). Fractions are recovered and analyzed by SDS-PAGE. The 50 kDa protein is found in the fractions eluted at 0.3-0.4 M NaCI.
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 containing 100,000 nucleotides, and a 17t" contig containing the remaining 76,000 nucleotides. A header was added to each of the 17 f les using the format: >hpg0.txt (representing contig 1), .hpgl.txt (representing contig 2), etc.
The resulting 17 files, named hpg0 through hpgl G, were then copied together to S form one file that represented the plus strand of the complete H. pylori genome.
The constructed database was given the designation "H." 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 "N."
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 "O." 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 ("H" and/or "N"), or a peptide sequence against the ORF
library ("O"). TFASTX was used to search a peptide sequence against all possible reading frames of a DNA database ("H" and/or "N" libraries). -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 I 5 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 (A), Serine (S), or Glycine (G) residue immediately after the core hydrophobic region. In the case of lipoproteins, a Cysteine (C) 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 gene-specific portion of the N-terminal primer is designed to start at the first codon beyond the cleavage site. In the case of lipoproteins, the 5'-end of the N-terminal 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 Helicobacter pylori Helicobacter pylori strain ORV2001, stored in LB medium containing 50% glycerol at -70°C, is grown on Colombia agar containing 7% sheep blood for 48 hours under microaerophilic conditions (8-10% C02, 5-7% 02,~and 85-87% Nz). Cells are harvested, washed with PBS (pH 7.2), and DNA is then extracted from the cells using the Rapid Prep Genomic DNA Isolation kit (Pharmacia Biotech). -WO 98!43479 PCTJUS98/06421 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 Polymerase Chain Reaction (PCR) using the following primers:
GHPO 386:
N-terminal primer:
5'-CTGAATTCGATTTCAAGGAGAAAACATGAAA-3' (SEQ ID N0:59);
and C-terminal primer:
5'-CCGCTCGAGTTAGTAAGCGAACACATAATT-3' (SEQ ID N0:60).
GHPO 789:
N-terminal primer:
5'-CGCGGATCCGAATCCAATTTAATCCAAAAAGG-3' (SEQ ID N0:61 );
and C-terminal primer:
5'-CCGCTCGAGTTAGTAAGCGAACACATAGTTCAA-3' (SEQ ID N0:62).
GHPO 1516:
N-terminal primer:
5'-CGCGGATCCGAATCCAATTTAATCCAAAAAGG-3' (SEQ ID N0:56);
and C-terminal primer:
5'-CCGCTCGAGTTAAGTAAGCGAACACATATTCAA-3' (SEQ ID N0:57).
GHPO 896:
N-terminal primer:
' S'-CGCGGATCCGAAGTTTCTTTGTATCAAAG-3' (SEQ ID N0:63); and C-terminal primer: -5'-CCGCTCGAGTTAGTAAGCAAACACATAATTGTG-3' (SEQ ID N0:64).
GHPO 1360:
N-terminal primer:
5'-CGCGGATCCGAATGAAAAAAAATATCTTAAAT-3' (SEQ ID N0:69);
and C-terminal primer:
5'-CCGCTCGAGTTACTTGTTGATAACAATTTT-3' (SEQ ID N0:70).
GHPO 750:
N-terminal primer:
S'-CGCGGATCCGAATGGCAAAAGAAAAGTTTAAC-3' (SEQ ID N0:71);
and C-terminal primer:
5'-CCGCTCGAGTTATTCAATAATATTGCTCAC-3' (SEQ ID N0:72).
GHPO 711:
N-terminal primer:
5'-GGGAATTCAAAAAAACGAAAAAAACG-3' (SEQ ID N0:83); and C-terminal primer:
5'-CCCCTCGAGTTAATAGGCAAACAC-3' (SEQ ID N0:84).
The N-terminal and C-terminal primers for each clone both include a 5' clamp and a restriction enzyme recognition sequence for cloning purposes (BamHI (GGATCC) and XhoI (CTCGAG) recognition sequences).
Amplification of gene-specific DNA is carried out using a heat-stable DNA Polymerase (e.g., Thermalase DNA Polymerase (Amresco)) according to the manufacturer's instructions. The reaction mixture, which is brought to a final volume of 100 ~1 with distilled water, is as follows:
dNTPs mix 200 ~M
lOx ThermoPol buffer 10 ~1 primers 300 nM each DNA template 50 ng DNA polymerise 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
polymerise (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 polymerise, 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 polymerise was used for clone GHPO 1516, instead of Taq DNA
polymerise, and the annealing temperature was 55 °C. For GHPO 1360 and GHPO 750, Thermaiase DNA polymerise 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 ° C for 2 minutes. Thirty cycles were carried out. For GHPO 711, Vent DNA polymerise was used. A denaturing step was carried out at 94°C for 30 seconds, followed by an annealing step at 50°C for 30 seconds, and an extension step at 72°C for 1 minute.
Twenty five cycles were carried out.

3.C. Transformation and selection of transformants A single PCR product is thus amplified and is then digested at 37°C for 2 hours with BamHI and XhoI concurrently in a 20 ~.l 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 ~,l) is carried out at i4°C overnight and then is used to transform 100 ~,l fresh E. toll XL1-blue competent cells (Novagen).
The cells are incubated on ice for 2 hours, then heat-shocked at 42 ° C for 30 seconds, and returned to ice for 90 seconds. The samples are then added to I 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 ~g/ml) at a IOx and neat dilution and incubated overnight at 37°C. The following day, colonies are picked onto secondary plates and incubated at 37°C
overnight.
Five colonies are picked into 3 ml LB broth supplemented with kanamycin (100 ~g/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. toll XLl-blue cells is used to transform competent BL21 (~,DE3) E. toll competent cells (Novagen; as described previously). Transformants ( 10) are picked onto selective kanamycin (50 ~,g/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 ~,g/ml of kanamycin in a 250 ml Erlenmeyer flask. The flask is incubated at 37°C for 2 hours or until the absorbance at 600 nm (OD~oo) reaches 0.4-1Ø The culture is stopped from growing by placing the flask at 4°C overnight. The following day, 10 ml of the overnight culture are used to inoculate 240 ml LB medium containing kanamycin (25 lzg/ml), with the initial OD~oo about 0.02-0.04. Four flasks are inoculated for each ORF.
The cells are grown to an ODboo of 1.0 (about 2 hours at 37°C), a 1 mi 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°C.
The final OD6oo is taken and the cells are harvested by centrifugation at 5,000 x g for 15 minutes at 4°C. The supernatant is discarded and the pellets are resuspended in 50 mM Tris-HCl (pH 8.0), 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°C.
3. E. Protein purification Pellets obtained from 3.D. are thawed and resuspended in 95 ml of SO
mM Tris-HCl (pH 8.0). Pefabloc and lysozyme are added to final concentrations of 100 ~,M and 100 ~g/ml, respectively. The mixture is homogenized with magnetic stirring at 5°C for 30 minutes. Benzonase (Merck) is added at a 1 U/ml final concentration, in the presence of 10 mM MgCl2, 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 (i.e., in the supernatant obtained in 3.E.) NaCI and imidazole are added to the supernatant to final concentrations of 50 mM Tris-HCl (pH 8.0), 0.5 M NaCI, and 10 mM
imidazole (buffer A). The mixture is filtered through a 0.45 ~.m 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-HCl (pH 8.0), 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 ~.m membrane, and stored at -45°C.
3.E.2. Insoluble fraction - --If the target protein is expressed in the insoluble fraction (pellets obtained from 3.E.), purification is conducted under denaturing conditions.
NaCI, imidazole, and urea are added to the resuspended pellet to final -concentrations of 50 mM Tris-HCl {pH 8.0), 0.5 M NaCI, 10 mM imidazole, and 6 M urea (buffer C). After complete solubilization, the mixture is filtered through a 0.45 ~m membrane and loaded onto an IMAC column.
The purification procedures on the IMAC column are the same as described in 3.E.1., except that 6 M urea is included in all buffers used and

10 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 ~.m membrane and stored at -45°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~ellet is extracted once more by repeating the preceding extraction step. The pellet is dissolved in 8 M urea, 50 mM Tris (pH 8.0). The urea-solubilized protein is diluted with an equal volume of 2 M arginine, -50 mM Tris (pH 8.0), and is dialyzed against 1 M arginine for 24-48 hours to remove the urea. The final dialysate is filtered through a 0.22 ~,m membrane and stored at -45°C. -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-HCl (pH 7.9) by microfluidization at a pressure of 15,000 psi and clarified by centrifugation at S 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 8.5). 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 7.0). 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 25 p.g of the purified recombinant protein, admixed with 1 ~,g 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 ORV2001 grown in liquid media (the cells are grown on agar plates, as described in 1.A., and, after harvest, the cells are resuspended in Brucella broth; the flasks are then incubated overnight at 37 ° C).
Fourteen days after challenge, the mice are sacrificed and their stomachs are removed. The amount of H. pylori is determined by measuring the urease activity in the stomach and by culture. -3.G. Production of monospecific polyclonal antibodies 3.G.1. Hyperimmune rabbit antiserum ' New Zealand rabbits are injected both subcutaneously and intramuscularly with 100 ~,g of a purified fusion polypeptide, as obtained in 3.E.1. or 3.E.2., 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 ~m membrane.
3.G.2. Mouse hyperimmune ascites fluid Ten mice are injected subcutaneously with 10-50 p.g of a purified fusion polypeptide, as obtained in 3.E.1. or 3.E.2., in the presence of Freund's complete adjuvant and in a volume of approximately 200 ~.1. 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 SO
~.g 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, 5th 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 His-tags Methods for amplification and cloning of DNA encoding the polypeptides of the invention as transcriptional fusions lacking His-tags are described as follows. Two PCR primers for each clone are designed based upon the sequences of the polynucleotides that encode them (SEQ ID NOs: l-21 (odd numbers), 65, and 67). These primers can be used to amplify DNA
encoding the polypeptides of the invention from any Helicobacter pylori 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 XhoI (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 PCRaproduct for each clone is amplified and can be cloned into BafnHI 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.

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 S.A. Purification of specific IgGs An immune serum, as prepared as is described in section 3.G., is applied to a protein A Sepharose Fast Flow column (Pharmacia) equilibrated in 100 mM Tris-HCl (pH 8.0). 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 S ml fractions to each of which is added 0.25 ml 1 M Tris-HCl (pH 8.0). 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 8.0), and, if necessary, stored frozen at -70°C.
S.B. 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-O 1 ) is suspended in 1 mM HCl buffer and washed using a buchner by adding small quantities of 1 mM HCl buffer. The total volume of buffer is 200 ml per gram of gel.

Purified IgG antibodies are dialyzed for 4 hours at 205 °C against 50 volumes of 500 mM sodium phosphate buffer (pH 7.5). 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 St3 °C. The gel is packed into a chromatography column and is washed with 2 column volumes of 500 mM phosphate buffer (pH 7.5), and 1 column volume of 50 mM sodium phosphate buffer, containing S00 mM NaCI (pH 7.5). 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.
S.C. Adsorption and elution of the antigen An antigen solution in 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, for example, the supernatant obtained in 3.E.1. or the solubilized pellet obtained in 3.E.2., after centrifugation and filtration through a 0.45 p.m membrane, is applied to a column equilibrated with 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, at a flow rate of about 10 ml/hour. The column is then washed with 20 volumes of SO mM Tris-HCl (pH 8.0), 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 2.5). The eluate is recovered in 3 ml fractions, to each of which is added 1 SO ~,l of 1 M sodium phosphate buffer (pH 8.0). Absorption is measured at 280 nm for each fraction; those fractions containing the antigen are pooled and stored at -20°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 of H. 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 50 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 tn diagnosis of H. pylori infection.

Other embodiments are within the following claims.

SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: MERIEUX ORAVAX SOCIETE EN NOM COLLECTIF
PASTEUR MERIEUX SERUMS ET VACCINS S.A., 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:
(A) ADDRESSEE: Clark & Elbing LLP
(B) STREET: 176 Federal Street (C) CITY: Boston (D) STATE: MA
(E) COUNTRY: USA
(F) ZIP: 02110 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US98/----(B) FILING DATE: 31-MAR-98 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/834,666 (B) FILING DATE: O1-APR-1997 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/831,310 (B) FILING DATE: O1-APR-1997 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Clark, Paul T.
(B) REGISTRATION NUMBER: 30,162 (C) REFERENCE/DOCKET NUMBER: 06132/037W01 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617-428-0200 (B) TELEFAX: 617-428-7045 (C) TELEX:
(2) INFORMATION FOR SEQ ID NO:1:
SUBSTITUTE SHEET (RULE 26) WO 98/43479 _ PCT/US98/06421 ~g_ (i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 2798 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii} MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 328...2451 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 328...385 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:

AAAGCATCAAAATCAAAAAAATGACAAAATTTTTAAGAAA ATGACAAAAA F~AAAAAAAAC240 ATG CTC

Met Lys Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Phe Leu Leu His Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met Val Lys Asn Thr Lys Gly Ile Gln Glu Leu Ser Asp Asn Tyr Glu Lys Leu Asn Asn Leu Leu Asn Asn Tyr Ser Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn Asp Ala Arg Asp Asn Leu Gly Ser Ser Ser Arg Asn Leu Leu Asp Val Lys Thr Asn Ser Pro Ala Tyr Gln Ala Val SUBSTITUTE SHEET (RULE 26) - 79- _ Leu Leu Ala Leu Asn Ala Ala Val Gly Leu Trp Gln Val Thr Ser Tyr Ala Phe Thr Ala Cys Gly Pro Gly Ser Asn Glu Asn Ala Asn Gly Gly Ile Gln Thr Phe Asn Asn Val Pro Gly Gln Asp Thr Thr Thr Ile Thr Cys Asn Ser Tyr Tyr Glu Pro Gly His Gly Gly Pro Ile Ser Thr Ala Asn Tyr Ala Lys Ile Asn Gln Ala Tyr Gln Ile Ile Gln Lys Ala Leu Thr Ala Asn Gly Ala Asn Gly Asp Gly Val Pro Val Leu Ser Asn Thr Thr Thr Lys Leu Asp Phe Thr Ile Asn Gly Asp Lys Arg Thr Gly Gly Lys Pro Asn Thr Pro Glu Lys Phe Pro Trp Ser Asp Gly Lys Tyr Ile His Thr Gln Trp Ile Asn Thr Ile Val Thr Pro Thr Glu Thr Asn Ile Asn Thr Glu Asn Asn Ala Gln Glu Leu Leu Lys Gln Ala Ser Ile Ile Ile Thr Thr Leu Asn Glu Ala Cys Pro Asn Phe Gln Asn Gly Gly Arg Ser Tyr Trp Gln Gly Ile Ser Gly Asn Gly Thr Met Cys Gly Met Phe Lys Asn Glu Ile Ser Ala Ile Gln Gly Met Ile Ala Asn Ala Gln Glu.___ Ala Val Ala Gln Ser Lys Ile Val Ser Glu Asn Ala Gln Asn Gln Asn Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp Ala Ser Phe SUBSTITUTE SHEET (RULE 26) WO 98/43479 _gp_ PCT/US98/06421 Ala Gln Ser Met Leu Lys Asn Ala Gln Ala Gln Ala Glu Ile Leu Asn Gln Ala Glu Gln Val Val Lys Asn Phe Glu Lys Ile Pro Thr Ala Phe GGG

Val SerAspSer LeuGly ValCysTyr GluValGln GlyGly GluArg Arg GlyThrAsn ProGly GlnValThr SerAsnThr TrpGly AlaGly Cys AlaTyrVal LysGln ThrIleThr AsnLeuAsp AsnSer IleAla His PheGlyThr GlnGlu GlnGlnIle GlnGlnAla GluAsn IleAla Asp ThrLeuVal AsnPhe LysSerArg TyrSerGlu LeuGly AsnThr Tyr AsnSerIle ThrThr AlaLeuSer LysValPro AsnAla GlnSer Leu GlnAsnVal ValSer LysLysAsn AsnProTyr SerPro GlnGly ACC

IleGlu ThrAsn TyrTyrLeu AsnGln AsnSerTyr AsnGlnIle Gln ThrIle AsnGln GluLeuGly ArgAsn ProPheArg LysValGly Ile ValAsn SerGln ThrAsnAsn GlyAla MetAsnGly IleGlyIle Gln ValGly TyrLys GlnPhePhe GlyGln LysArgLys TrpGlyAla Arg -TyrTyr GlyPhe PheAspTyr AsnHis AlaPheIle LysSerSer Phe SUBSTITUTE SHEET (RULE 26) _gl_ _ Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr GAGATTTCTT

(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENC~CHARACTERISTICS:
(A) LENGTH: 708 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 1...19 SUBSTITUTE SHEET (RULE 26) (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Lys Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Phe Leu Leu His Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met Val Lys Asn Thr Lys Gly Ile Gln Glu Leu Ser Asp Asn Tyr Glu Lys Leu Asn Asn Leu Leu Asn Asn Tyr Ser Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn Asp Ala Arg Asp Asn Leu Gly Ser Ser Ser Arg Asn Leu Leu Asp Val Lys Thr Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala Leu Asn Ala Ala Val Gly Leu Trp Gln Val Thr Ser Tyr Ala Phe Thr Ala Cys Gly Pro Gly Ser Asn Glu Asn Ala Asn Gly Gly Ile Gln Thr Phe Asn Asn Val Pro Gly Gln Asp Thr Thr Thr Ile Thr Cys Asn Ser Tyr Tyr Glu Pro Gly His Gly Gly Pro Ile Ser Thr Ala Asn Tyr Ala Lys Ile Asn Gln Ala Tyr Gln Ile Ile Gln Lys Ala Leu Thr Ala Asn Gly Ala Asn Gly Asp Gly Val Pro Val Leu Ser Asn Thr Thr Thr Lys Leu Asp Phe Thr Ile Asn Gly Asp Lys Arg Thr Gly Gly Lys Pro Asn Thr Pro Glu Lys Phe Pro Trp Ser Asp Gly Lys Tyr Ile His Thr Gln Trp Ile Asn Thr Ile Val Thr Pro Thr Glu Thr Asn Ile Asn Thr Glu Asn Asn Ala Gln Glu Leu Leu Lys Gln Ala Ser Ile Ile Ile Thr Thr Leu Asn Glu Ala Cys Pro Asn Phe Gln Asn Gly Gly Arg Ser Tyr Trp Gln Gly Ile Ser Gly Asn Gly Thr Met Cys Gly Met Phe Lys Asn Glu Ile Ser Ala Ile Gln Gly Met Ile Ala Asn Ala Gln Glu Ala Val Ala Gln Ser Lys Ile Val Ser Glu Asn Ala Gln Asn Gln Asn Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp Ala Ser Phe Ala Gln Ser Met Leu Lys Asn Ala Gln Ala Gln Ala Glu Ile Leu Asn Gln Ala Glu Gln Val Val Lys Asn Phe Glu Lys Ile Pro Thr Ala Phe Val Ser Asp Ser Leu Gly Val Cys Tyr Glu Val Gln Gly Gly Glu Arg Arg Gly Thr Asn Pro Gly Gln_ Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Lys Gln Thr SUBSTITUTE SHEET (RULE 26) Ile Thr Asn Leu Asp Asn Ser Ile Ala His Phe Gly Thr Gln Glu Gln Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr Leu Val Asn Phe Lys 5er Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gln Ser Leu Gln Asn Val Val Ser Lys Lys Asn Asri Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly Ile Val Asn Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Ile Gln Val Gly Tyr Lys Gln Phe Phe Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val G1y Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2699 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 199..,2397 (D) OTHER INFORMATION:
SUBSTITUTE SHEET (RULE 2B) (A) NAME/KEY: Signal Sequence (B) LOCATION: 199...259 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:

TCAAAATGACF~AAAAP,AAAC GCTTTATGCT ATAATACCCCAAATACATTCTAATAGCAAA120 ATC CTT GCT

Met Lys Lys His Ile Leu Ser Leu Leu Gly Ala Ser Leu Leu Val Ser Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln Gln Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn Ala Val Arg Glu Asn Leu Gly Ala Ser Ala Lys Asn Leu Ile Gly Asp Lys Ala Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala Ile Asn Ala Ala Val Gly Phe Trp Asn Val Val Gly Tyr Val Thr Gln Cys Gly Gly Asn Ala Asn Gly Gln Glu Ser Thr Ser Ser Thr 105 ~"" 110 115 Thr Ile Phe Asn Asn Glu Pro Gly Tyr Arg Ser Thr Ser Ile Thr Cys Ser Leu Asn Gly His Lys Pro Gly Tyr Tyr Gly Pro Met Ser Ile Glu Asn Phe Lys Lys Leu Asn Glu Ala Tyr Gln Ile Leu Gln Thr Ala Leu SUBSTITUTE SHEET (RULE 26) WO 98143479 _85_ PCT/(JS98/06421 Lys Asn Gly Leu Pro Ala Leu Lys Glu Asn Asn Gly Lys Val Ser Val Thr Tyr Thr Tyr Thr Cys Ser Gly Gln Gly Asn Asn Asn Cys Ser Pro Ser Val Asn Gly Thr Lys Thr Thr Thr Gln Thr Ile Asp Gly Lys Ser Val Thr Thr Thr Ile Ser Ser Lys Val Val Gly Ser Ile Ala Ser Gly Asn Thr Ser His Val Ile Thr Asn Lys Leu Asp Gly Val Pro Asp Ser Ala Gln Ala Leu Leu Ala Gln Ala Ser Thr Leu Ile Asn Thr Ile Asn Glu Ala Cys Pro Tyr Phe His Ala Thr Asn Ser Ser Glu Ala Asn Ala Pro Lys Phe Ser Thr Thr Thr Giy Lys Ile Cys Gly Ala Phe Ser Glu Glu Ile Ser Ala Ile Gln Lys Met Ile Thr Asp Ala Gln Glu Leu Val Asn Gln Thr Ser Val Ile Asn Ser Asn Glu Gln Ser Thr Pro Val Gly Asn Asn Asn Gly Lys Pro Phe Asn Pro Phe Thr Asp Ala Ser Phe Ala Gln Gly Met Leu Ala Asn Ala Ser Ala Gln Ala Lys Met Leu Asn Leu Ala His Gln Val Gly Gln Ala Ile Asn Pro Glu Asn Leu Ser Glu Asn TTT AAA AAT TTT GTT ACA GGC TTT TTA GCC ACA TGC AAT AAC AAA TCA_ 1431 Phe Lys Asn Phe Val Thr Gly Phe Leu Ala Thr Cys Asn Asn Lys Ser sues~TUTE sHesr ~RU~ 2s~

WO 98/43479 _86_ PCT/US98106421 Thr Ala Gly Thr Gly Gly Thr Gln Gly Ser Ala Pro Gly Thr Val Thr Thr Gln Thr Phe Ala Ser Gly Cys Ala Tyr Val Glu Gln Thr Leu Thr Asn Leu Gly Asn Ser Ile Ala His Phe Gly Thr Gln Glu Gln Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gln Ser Leu Gln Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly Ile Val Asn Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Ile Gln Val Gly Tyr Lys Gln Phe Phe Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Leu Gly Leu Phe Gly SUBSTITUTE SHEET (RULE 26) WO 98/43479 _g~_ PCTNS98/06421 Gly Ile Ala Leu Ala Giy Thr Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr CTCTTTTTAA ATTCTCTTTT TAAAGAGATTTCTTTTTTT'TAAGCTTTTTT TTGAATTCTT2509 (2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 733 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal ix) FEATC7RE
(A) NAME/KEY: Signal Sequence (B) LOCATION: 1...20 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Lys Lys His Ile Leu Ser Leu Ala Leu Gly Ser Leu Leu Val Ser Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln SUBSTITUTE SHEET (RULE 26) WO 98/43479 _8S_ PCT/US98/06421 Ile Gly Glu Ala Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln Gln Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn Ala Val Arg Glu Asn Leu Gly Ala Ser Ala Lys Asn Leu Ile Gly Asp Lys Ala Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala Ile Asn Ala Ala Val Gly Phe Trp Asn Val Val Gly Tyr Val Thr Gln Cys Gly Gly Asn Ala Asn Gly Gln Glu Ser Thr Ser Ser Thr Thr Ile Phe Asn Asn Glu Pro Gly Tyr Arg Ser Thr Ser Ile Thr Cys Ser Leu Asn Gly His Lys Pro Gly Tyr Tyr Gly Pro Met Ser Ile Glu Asn Phe Lys Lys Leu Asn Glu Ala Tyr Gln Ile Leu Gln Thr Ala Leu Lys Asn Gly Leu Pro Ala Leu Lys Glu Asn Asn Gly Lys Val Ser Val Thr Tyr Thr Tyr Thr Cys Ser Gly Gln Gly Asn Asn Asn Cys Ser Pro Ser Val Asn Gly Thr Lys Thr Thr Thr Gln Thr Ile Asp Gly Lys Ser Val Thr Thr Thr Ile Ser Ser Lys Val Val Gly Ser Ile~Ala Ser Gly Asn Thr Ser His Val Ile Thr Asn Lys Leu Asp Gly Val Pro Asp Ser Ala Gln Ala Leu Leu Ala Gln Ala Ser Thr Leu Ile Asn Thr Ile Asn Glu Ala Cys Pro Tyr Phe His Ala Thr Asn Ser Ser Glu Ala Asn Ala Pro Lys Phe Ser Thr Thr Thr Gly Lys Ile Cys Gly Ala Phe Ser Glu Glu Ile Ser Ala Ile Gln Lys Met Ile Thr Asp Ala Gln Glu Leu Val Asn Gln Thr Ser Val Ile Asn Ser Asn Glu Gln Ser Thr Pro Val Gly Asn Asn Asn Gly Lys Pro Phe Asn Pro Phe Thr Asp Ala Ser Phe Ala Gln Gly Met Leu Ala Asn Ala Ser Ala Gln Ala Lys Met Leu Asn Leu Ala His Gln Val Gly Gln Ala Ile Asn Pro Glu Asn Leu Ser Glu Asn Phe Lys Asn Phe Val Thr Gly Phe Leu Ala Thr Cys Asn Asn Lys Ser Thr Ala Gly Thr Gly Gly Thr Gln Gly Ser Ala Pro Gly Thr Val Thr Thr Gln Thr Phe Ala Ser Gly Cys Ala Tyr Val Glu Gln Thr Leu Thr Asn Leu Gly Asn Ser Ile Ala His Phe Gly Thr Gln Glu Gln Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala SUBSTITUTE SHEET (RULE 26) WO 98/43479 $g PCT/US98/06421 Gln Ser Leu Gln Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly Ile Val Asn Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Ile Gln Val Gly Tyr Lys Gln Phe Phe Gly Gln Lys Arg Lys Trp Gly ~ Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Leu Gly Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR 5EQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2915 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 365...2597 (D) OTHER INFORMATION:
{A) NAME/KEY: Signal Sequence ---{B) LOCATION: 365...425 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
SUBSTITUTE SHEET (RULE 26) AGAAATAAAC CGCTCATAAG GGGCAAACGC CCCAP.AAAAG CGATTTTTAA AGAGGTTACG 120 Met Lys Lys His Ile Leu Ser Leu Ala Leu Gly Ser Leu Leu Val Ser Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln Gln Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn Ala Val Arg Glu Asn Leu Gly Ala Ser Thr Lys Asn Leu Ile Gly Asp Lys Ala Asn Ser Pro Ala Tyr Gln Ala Val Phe Leu Ala Ile Asn Ala Ala Val Gly Leu Trp Asn Thr Ile Gly Tyr Ala Val Met Cys Gly Asn Gly Asn Gly Thr Glu Ser Gly Pro Gly Ser Val Ile Phe Asn Asp Gln Pro Gly Gln Asp Ser Thr Gln Ile Thr Cys Asn Arg Phe Glu Ser Thr Gly Pro Gly Lys Ser Met Ser Ile Asp Glu Phe Lys Lys Leu Asn Glu Ala Tyr Gln Ile Ile Gln Gln Ala Leu Lys Asn Gln Ser Gly Phe Pro Glu Leu Gly Gly Asn Gly Thr Lys Val Ser Val Asn Tyr Asn Tyr SUBSTITUTE SHEET (RULE 26) Glu Cys Arg Gln Thr Ala Asp Ile Asn Gly Gly Val Tyr Gln Phe Cys Lys Ala Lys Asn Gly Ser Ser Ser Ser Ser Asn Gly Gly Asn Gly Ser Ser Thr Gln Thr Thr Ala Thr Thr Thr Gln Asp Gly Val Thr Ile Thr Thr Thr Tyr Asn Asn Asn Lys Ala Thr Val Lys Phe Asp Ile Thr Asn Asn Ala Glu Gln Leu Leu Asn Gln Ala Ala Asn Ile Met Gln Val Leu Asn Thr Gln Cys Pro Leu Val Arg Ser Thr Asn Asn Glu Asn Thr Pro Gly Gly Gly Gln Pro Trp Gly Leu Ser Thr Ser Gly Asn Ala Cys Ser Ile Phe Gln Gln Glu Phe Ser Gln Val Thr Ser Met Ile Lys Asn Ala Gln Glu Ile Ile Ala Gln Ser Lys Ile Val Ser Glu Asn Ala Gln Asn Gln Asn Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp Ala Ser Phe Ala Gln Ser Met Leu Lys Asn Ala Gln Ala Gln Ala Glu Met Phe Asn Leu Ser Glu Gln Val Lys Lys Asn Leu Glu Val Met Lys Asn , Asn Asn Asn Val Asn Glu Lys Leu Ala Gly Phe Gly Lys Glu Glu Val--ATG ACC AAT TTT GTT AGC GCC TTT TTG GCA AGC TGC AAe~ GAT GGT GGC 1657 Met Thr Asn Phe Val Ser Ala Phe Leu Ala Ser Cys Lys Asp Gly Gly SUBSTITUTE SHEET (RULE 26) Thr Leu Pro Asn Ala Gly Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Gly Glu Thr Ile Ser Ala Leu Thr Asn Ser Ile Ala His Phe Gly Thr Gln Glu Gln Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gln Ser Leu Gln Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly Ile Val Asn Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Ile Gln Val Gly Tyr Lys Gln Phe Phe Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Leu Gly Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr SUBSTITUTE SHEET (RULE 26) Asn Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Xaa Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 745 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 1...20 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Lys Lys His Ile Leu Ser Leu Ala Leu Gly Ser Leu Leu Val Ser -Thr Leu Ser Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met Val Thr Asn Thr Lys Gly Ile Gln Gln_ Leu Ser Asp Asn Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Ser SUBSTITUTE SHEET (RULE 26) Thr Leu Asn Thr Leu Ile Lys Leu Ser Ala Asp Pro Ser Ala Ile Asn Ala Val Arg Glu Asn Leu Gly Ala Ser Thr Lys Asn Leu Ile Gly Asp Lys Ala Asn Ser Pro Ala Tyr Gln Ala Val Phe Leu Ala Ile Asn Ala Ala Val Gly Leu Trp Asn Thr Ile Gly Tyr Ala Val Met Cys Gly Asn Gly Asn Gly Thr Glu Ser Gly Pro Gly Ser Val Ile Phe Asn Asp Gln Pro Gly Gln Asp Ser Thr Gln Ile Thr Cys Asn Arg Phe Glu Ser Thr Gly Pro Gly Lys Ser Met Ser Ile Asp Glu Phe Lys Lys Leu Asn Glu Ala Tyr Gln Ile Ile Gln Gln Ala Leu Lys Asn Gln Ser Gly Phe Pro Glu Leu Gly Gly Asn Gly Thr Lys Val Ser Val Asn Tyr Asn Tyr Glu Cys Arg Gln Thr Ala Asp Ile Asn Gly Gly Val Tyr Gln Phe Cys Lys Ala Lys Asn Gly Ser Ser Ser Ser Ser Asn Gly Gly Asn Gly Ser Ser Thr Gln Thr Thr Ala Thr Thr Thr Gln Asp Gly Val Thr Ile Thr Thr Thr Tyr Asn Asn Asn Lys Ala Thr Val Lys Phe Asp Ile Thr Asn Asn Ala Glu Gln Leu Leu Asn Gln Ala Ala Asn Ile Met Gln Val Leu Asn Thr Gln Cys Pro Leu Val Arg Ser Thr Asn Asn Glu Asn Thr Pro Gly Gly Gly Gln Pro Trp Gly Leu Ser Thr Ser Gly Asn Ala Cys Ser Ile Phe Gln Gln Glu Phe Ser Gln Val Thr Ser Met Ile Lys Asn Ala Gln Glu Ile Ile Ala Gln Ser Lys Ile Val Ser Glu Asn Ala Gln Asn Gln Asn Asn Leu Asp Thr Gly Lys Pro Phe Asn Pro Tyr Thr Asp AIa Ser Phe Ala Gln Ser Met Leu Lys Asn Ala Gln Ala Gln Ala Glu Met Phe Asn Leu Ser Glu Gln Val Lys Lys Asn Leu GIu Val Met Lys Asn Asn Asn Asn Val Asn Glu Lys Leu Ala Gly Phe Gly Lys Glu Glu Val Met Thr Asn Phe Val Ser Ala Phe Leu Ala Ser Cys Lys Asp Gly Gly Thr Leu Pro Asn Ala Gly Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Gly Glu Thr Ile Ser Ala Leu Thr Asn Ser Ile Ala His Phe-Gly Thr Gln Glu Gln Gln Ile Gln Gln Ala Glu Asn Ile Ala Asp Thr Leu Val Asn Phe Lys Ser Arg Tyr Ser Glu Leu Gly Asn Thr Tyr Asn Ser Ile Thr Thr Ala Leu Ser Lys Val Pro Asn Ala Gln Ser Leu Gln SUBSTITUTE SHEET (RULE 26) Asn Val Val Ser Lys Lys Asn Asn Pro Tyr Ser Pro Gln Gly Ile Glu Thr Asn Tyr Tyr Leu Asn Gln Asn Ser Tyr Asn Gln Ile Gln Thr Ile Asn Gln Glu Leu Gly Arg Asn Pro Phe Arg Lys Val Gly Ile Val Asn Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Ile Gln Val Gly Tyr Lys Gln Phe Phe Gly Gln Lys Arg Lys Trp Gly Ala Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Phe Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Phe Gly Ala Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Leu Gly Leu Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Glu Tyr Val Asn Leu Ala Thr Val Asn Asn Val Tyr Asn Ala Lys Met Asn Va1 Ala Asn Phe Gln Phe Leu Phe Asn Met Gly Val Arg Met Asn Leu Ala Arg Ser Lys Lys Lys Gly Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Leu Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Glu Leu Lys Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Xaa Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2603 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 210...2342 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 210...270 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:

SU9STITUTE SHEET (RULE 26) Met Lys Lys Lys Phe Leu Ser Leu Thr Leu Gly Ser Leu Leu Val Ser Ala Leu Ser Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gln Ile Gly Glu Ser Ala Gln Met Val Lys Asn Thr Lys Gly Ile Gln Asp Leu Ser Asp Ser Tyr Glu Arg Leu Asn Asn Leu Leu Thr Ser Tyr Ser Aia Leu Asn Thr Leu Ile Arg Gln 5er Ala Asp Pro Asn Ala Ile Asn Asn Ala Arg Gly Asn Leu Asn Ala Ser Ala Lys Asn Leu Ile Asn Asp Lys Lys Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala Leu Asn Ala Ala Ala Gly Leu Trp Gln Val Met Ser Tyr Ser Ile Ser Val Cys Giy Pro Gly Ser Asp Lys Asn Lys Asn Gly Gly Val Gln Thr Phe Glu Asn Val Pro Ser Asn Gly Gly Thr Thr Ile Ala Cys Asp Ser Phe Tyr Glu Pro Gly Lys Trp Ser Gly Ile Ser Thr Glu Asn Tyr Ala Lys Ile Asn Lys Ala Tyr Gln Ile Ile Gln Lys Ala Phe Gly Ala Ser Gly Gln Asp Ile Pro Ala Leu Ser Asp Thr Lys Glu Leu Asn Phe Glu Ile Lys Gly Lys Lys Asn Asp Ser Val Gln Pro SUBSTITUTE SHEET (RULE 26) Gly Glu Arg Trp Lys Phe Pro Trp Thr Asn Gly Lys Phe Val Ser Val Lys Trp Val Asn Gly Lys Tyr Glu Glu Ile Lys Glu Asp Ile Lys Val Ser Asn Asn Ala Gln Glu Leu Leu Lys Gln Ala Ser Thr Ile Leu Thr Thr Leu Asn Glu Ala Cys Pro Trp Leu Ser Asn Gly Gly Ala Gly Asn Val Ala Gly Gly Asn Ser Leu Trp Ala Gly Ile Asp Lys Gly Asp Gly Ser Ala Cys Gly Ile Phe Lys Asn Glu Ile Ser Ala Ile Gln Asp Met Ile Lys Asn Ala Glu Ile Ala Val Glu Gln Ser Lys Ile Val Thr Ala Asn Ala Gln Asn Gln His Asn Leu Asp Thr Gly Lys Ala Phe Asn Pro Tyr Lys Asp Ala Asn Phe Ala Gln Ser Met Phe Ala Asn Ala Arg Ala Gln Ala Glu Ile Leu Asn Arg Ala Gln Ala Val Val Lys Asp Phe Glu Arg Ile Pro Ala Ala Phe Val Lys Asp Ser Leu Gly Val Cys His Glu Lys Gly Ser Asp Gly Asn Leu Arg Gly Thr Pro Ser Gly Thr Val Thr ' Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Gly Glu Thr Val Thr Asn Leu Lys Asn Ser Ile Ala His Phe Gly Asp Gln Ala Glu Arg Ile SUBSTITUTE SHEET (RULE 26) His Asn Ala Arg Asn Leu Ala Tyr Thr Leu Ala Asn~Phe Ser Gly Gln Tyr Lys Lys Leu Gly Glu His Tyr Asp Ser Ile Thr Ala Ala Leu Ser Ser Leu Pro Asp Ala Gln Ser Leu Gln Asn Val Val Ser Lys Lys Thr Asn Pro Asn Ser Pro Gln Gly Ile Gln Asp Asn Tyr Tyr Ile Asp Ser Asn Ile His Ser Gln Val Gln Ser Arg Ser Gln Glu Leu Gly Ser Asn Pro Phe Arg Arg Ala Gly Leu Ile Ala Ala Ser Thr Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Phe Gln Val Gly Tyr Lys Gln Phe Phe Gly Lys Asn Lys Arg Trp Gly Ala Arg Tyr Tyr Gly Phe Val Asp Tyr Asn His Thr Tyr Asn Lys Ser Gln Phe Phe Asn Ser Asp Ser Asp Val Trp Thr Tyr Gly Val Gly Ser Asp Leu Leu Val Asn Phe Ile Asn Asp Lys Ala Thr Lys His Asn Lys Ile Ser Phe Gly Ala Phe Gly Gly Ile Gln Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Tyr Val Asn Leu Ala Asn Val Asn Asn Tyr Tyr Lys Ala Lys Ile Asn Thr Ser Asn Phe Gln Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Arg Asn Lys Arg Ile Gly Ala Asp His Ser Ala Gln His Gly Met Glu Leu Gly Val Lys Ile SUBSTITUTE SHEET (RULE 26) Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Thr Leu Gln Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr ATTTTAAACC ACCCAAGCAAGAAACCCCAA ACATCTTTAGCGTTCGCGCGCTCCACTAAC258'7 (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 711 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 1...20 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
Met Lys Lys Lys Phe Leu Ser Leu Thr Leu Gly Ser Leu Leu Val Ser Ala Leu Ser Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gln Ile Gly Glu Ser Ala Gln Met Val Lys Asn Thr Lys Gly Ile Gln Asp Leu Ser Asp Ser Tyr Glu Arg Leu Asn Asn Leu Leu Thr Ser Tyr Ser Ala Leu Asn Thr Leu Ile Arg Gln Ser Ala Asp Pro Asn Ala Ile Asn Asn Ala Arg Gly Asn Leu Asn Ala Ser Ala Lys Asn Leu Ile Asn Asp Lys Lys Asn Ser Pro Ala Tyr Gln Ala Val Leu Leu Ala Leu Asn Ala ' 80 85 90 Ala Ala Gly Leu Trp Gln Val Met Ser Tyr Ser Ile Ser Val Cys Gly - Pro Gly Ser Asp Lys Asn Lys Asn Gly Gly Val Gln Thr Phe Glu Asn Val Pro Ser Asn Gly Gly Thr Thr Ile Ala Cys Asp Ser Phe Tyr Glu Pro Gly Lys Trp Ser Gly Ile Ser Thr Glu Asn Tyr Ala Lys Ile Asn SU85TtTUTE SHEET (RULE 26) Lys Ala Tyr Gln Ile Ile Gln Lys Ala Phe Gly Ala Ser Gly Gln Asp Ile Pro Ala Leu Ser Asp Thr Lys Glu Leu Asn Phe Glu Ile Lys Gly Lys Lys Asn Asp Ser Vai Gln Pro Gly Glu Arg Trp Lys Phe Pro Trp Thr Asn Gly Lys Phe Val Ser Val Lys Trp Val Asn Gly Lys Tyr Glu Glu Ile Lys Glu Asp Ile Lys Val Ser Asn Asn Ala Gln Glu Leu Leu 225 230 235 _ Lys Gln Ala Ser Thr Ile Leu Thr Thr Leu Asn Glu Ala Cys Pro Trp Leu Ser Asn Gly Gly Ala Gly Asn Val Ala Gly Gly Asn Ser Leu Trp Ala Gly Ile Asp Lys Gly Asp Gly Ser Ala Cys Gly Ile Phe Lys Asn Glu Ile Ser Ala Ile Gln Asp Met Ile Lys Asn Ala Glu Ile Ala Val Glu Gln Ser Lys Ile Val Thr Ala Asn Ala Gln Asn Gln His Asn Leu Asp Thr Gly Lys Ala Phe Asn Pro Tyr Lys Asp Ala Asn Phe Ala Gln Ser Met Phe Ala Asn Ala Arg Ala Gln Ala Glu Ile Leu Asn Arg Ala Gln Ala Val Val Lys Asp Phe Glu Arg Ile Pro Ala Ala Phe Val Lys Asp Ser Leu Gly Val Cys His Glu Lys Gly Ser Asp Gly Asn Leu Arg Gly Thr Pro Ser Gly Thr Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Gly Glu Thr Val Thr Asn Leu Lys Asn Ser Ile Ala His Phe Gly Asp Gln Ala GIu Arg Ile His Asn Ala Arg Asn Leu Ala Tyr Thr Leu Ala Asn Phe Ser Gly Gln Tyr Lys Lys Leu Gly Glu His Tyr Asp Ser Ile Thr Ala Ala Leu Ser Ser Leu Pro Asp Ala Gln Ser Leu Gln Asn Val Val Ser Lys Lys Thr Asn Pro Asn Ser Pro Gln Gly Ile Gln Asp Asn Tyr Tyr Ile Asp Ser Asn Ile His Ser Gln Val Gln Ser Arg Ser Gln Glu Leu Gly Ser Asn Pro Phe Arg Arg Ala Gly Leu Ile Ala Ala Ser Thr Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Phe Gln Val Gly Tyr Lys Gln Phe Phe Gly Lys Asn Lys Arg Trp Gly Ala Arg Tyr Tyr Gly Phe Val Asp Tyr Asn His Thr Tyr Asn Lys Ser Gln Phe Phe Asn Ser Asp Ser Asp Val Trp Thr Tyr Gly Val Gly Ser Asp Leu Leu Val Asn Phe Ile Asn Asp Lys Ala Thr Lys His Asn Lys Ile Ser Phe Gly Ala Phe Gly Gly Ile Gln Leu Ala Gly Thr Ser Trp Leu Asn SUBSTITUTE SHEET (RULE 26) Ser Gln Tyr Val Asn Leu Ala Asn Val Asn Asn Tyr Tyr Lys Ala Lys Ile Asn Thr Ser Asn Phe Gln Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Arg Asn Lys Arg Ile Gly A1a Asp His Ser Ala Gln His Gly Met Glu Leu Gly Val Lys Ile Pro Thr I1e Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Thr Leu Gln Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2427 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 232...2247 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 232...292 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:

TTTCTCCAAATGACAAi~AAA AAAAAAAACGATTTTATGCT ACAATGCTTT TAATACATTC1B0 AAA

Met Lys Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Ser Leu Leu Asn Ala Glu Asp Asn Gly Phe Phe Ile Ser Ala Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met Val Lys Asn Thr Gly Glu Leu Lys Lys Leu Ser SUBSTITUTE SHEET (RULE 26) Asp Thr Tyr Glu Asn Leu Ser Asn Leu Leu Thr Asn Phe Asn Asn Leu Asn Gln Ala Val Thr Asn Ala Ser Ser Pro Ser Glu Ile Asn Ala Thr Ile Asp Asn Leu Lys Ala Asn Thr Gln Gly Leu Ile Gly Glu Lys Thr Asn 5er Pro Ala Tyr Gln Ala Val Tyr Leu Ala Leu Asn Ala Ala Val Gly Leu Trp Asn Val Ile Ala Tyr Asn Val Gln Cys Gly Pro Gly Lys Ser Gly Asp Gln Ser Val Ile Phe Asp Gly Gln Pro Gly His Asp Ser Arg Ser Ile Asn Cys Asn Leu Thr Gly Tyr Asn Asn Gly Val Ser Gly Pro Leu Ser Ile Asp Asn Phe Lys Thr Leu Asn Gln Ala Tyr Gln Thr Ile Gln Gln Ala Leu Lys Gln Asp Ser Gly Phe Pro Val Leu Asp Ser Lys Gly Lys Gln Val Thr Ile Lys Ile Thr Thr Gln Thr Asn Gly Ala Asn Lys Ser Glu Thr Thr Thr Thr Thr Thr Thr Thr Asn Asp Ala Gln Thr Leu Leu Gln Glu Ala Ser Lys Met Ile Ser Val Leu Thr Thr Asn TGC CCA TGG GTA AAT ACC GCT CAT AAC TCA AAC GGG GGT GCA CCG TGG __ 1005 Cys Pro Trp Val Asn Thr Ala His Asn Ser Asn Gly Gly Ala Pro Trp Asn Leu Asn Thr Thr Gly Asn Val Cys Gln Val Phe Ala Thr Glu Phe SUBSTITUTE SHEET (RULE 26) WO 98/43479 103 . PCT/US98/06421 Ser Ala Val Thr Ser Met Ile Lys Asn Ala Gln Glu Ile Val 'Thr Gln Ala Gln Ser Leu Asn Asn Pro Gln Ser Asn Gln Asn Ala Pro Lys Asp Phe Asn Pro Tyr Thr Ser Ala Asp Arg Ala Phe Ala Gln Asn Met Leu Asn His Ala Gln Ala Gln Ala Lys Met Leu Glu Leu Ala Asp Gln Met Lys Lys Asp Leu Asn Thr Ile Pro Lys Gln Phe Ile Thr Asn Tyr Leu Ala Ala Cys Arg Asn Gly Gly Gly Thr Leu Pro Asp Ala Gly Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Glu Glu Thr Ile Thr Ala Leu Asn Asn Ser Leu Ala His Phe Gly Thr Gln Ala Asp Gln Ile Lys Gln Ser Glu Leu Leu Ala Arg Thr Ile Leu Asp Phe Arg Gly Ser Leu Lys Asp Leu Asn Asn Thr Tyr Asn Ser Ile Thr Thr Thr Ala Ser Asn Thr Pro Asn Ser Pro Phe Leu Lys Asn Leu Ile Ser Gln Ser Thr Asn Pro Asn Asn Pro Gly Gly Leu Gln Ala Val Tyr Gln Val Asn Gln Ser Ala Tyr Ser Gln Leu Leu Ser Ala Thr Gln Glu Leu Gly His Asn Pro Phe Arg Arg Val Gly Leu Ile Ser Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Val Gln Ile Gly Tyr Lys Gln Phe Phe Gly Glu SUBSTITUTE SHEET (RULE 26) Lys Arg Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Phe Thr Tyr Gly Val Gly Thr Asp Val Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Lys Asn Asn Lys Ile Ser Phe Gly Val Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Tyr Val Asn Leu Ala Thr Phe Asn Asn Phe Tyr Ser Ala Lys Met Asn Val Ala Asn Phe Gln Phe Leu Phe Asn Leu Gly Leu Arg Met Asn Leu Ala Lys Asn Lys Lys Lys Ala Ser Asp His Val Ala Gln His Gly Val Glu Leu Gly Val Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Gln Leu Gln Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID NO:10:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 672 amino acids (B) TYPE: amino acid -(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein _ (v) FRAGMENT TYPE: internal (ix) FEATURE:
SUBSTITUTE SHEET (RULE 26) (A) NAME/KEY: Signal Sequence (B) LOCATION: 1...20 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Lys Lys Thr Leu Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Ser Leu Leu Asn Ala Glu Asp Asn Gly Phe Phe Ile Ser Ala Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met Val Lys Asn Thr Gly Glu Leu Lys Lys Leu Ser Asp Thr Tyr Glu Asn Leu Ser Asn Leu Leu Thr Asn Phe Asn Asn Leu Asn Gln Ala Val Thr Asn Ala Ser Ser Pro Ser Glu Ile Asn Ala Thr Ile Asp Asn Leu Lys Ala Asn Thr Gln Gly Leu Ile Gly Glu Lys Thr Asn Ser Pro Ala Tyr Gln Ala Val Tyr Leu Ala Leu Asn Ala Ala Val Gly Leu Trp Asn Val Ile Ala Tyr Asn Val Gln Cys Gly Pro Gly Lys Ser Gly Asp Gln Ser Val Ile Phe Asp Gly Gln Pro Gly His Asp Ser Arg Ser Ile Asn Cys Asn Leu Thr Gly Tyr Asn Asn Gly Val Ser Gly Pro Leu Ser Ile Asp Asn Phe Lys Thr Leu Asn Gln Ala Tyr Gln Thr Ile Gln Gln Ala Leu Lys Gln Asp Ser Gly Phe Pro Val Leu Asp Ser Lys Gly Lys Gln Val Thr Ile Lys Ile Thr Thr Gln Thr Asn Gly Ala Asn Lys Ser Glu Thr Thr Thr Thr Thr Thr Thr Thr Asn Asp Ala Gln Thr Leu Leu Gln Glu Ala Ser Lys Met Ile Ser Val Leu Thr Thr Asn Cys Pro Trp Val Asn Thr Ala His Asn Ser Asn Gly Gly Ala Pro Trp Asn Leu Asn Thr Thr Gly Asn Val Cys Gln Val Phe Ala Thr Glu Phe Ser Ala Val Thr Ser Met Ile Lys Asn Ala Gln Glu Ile Val Thr Gln Ala Gln Ser Leu Asn Asn Pro Gln Ser Asn Gln Asn Ala Pro Lys Asp Phe Asn Pro Tyr Thr Ser Ala Asp Arg Ala Phe Ala Gln Asn Met Leu Asn His Ala Gln Ala Gln Ala Lys Met Leu Glu Leu Ala Asp Gln Met Lys Lys Asp Leu Asn Thr Ile Pro Lys Gln Phe Ile Thr Asn - Tyr Leu Ala Ala Cys Arg Asn Gly Gly Gly Thr Leu Pro Asp Ala Gly Val Thr Ser Asn Thr Trp Gly Ala Gly Cys Ala Tyr Val Glu Glu Thr Ile Thr Ala Leu Asn Asn Ser Leu Ala His Phe Gly Thr Gln Ala Asp SUBSTITUTE SHEET (RULE 26) Gln Ile Lys Gln Ser Glu Leu Leu Ala Arg Thr Ile Leu Asp Phe Arg Gly Ser Leu Lys Asp Leu Asn Asn Thr Tyr Asn Ser Ile Thr Thr Thr Ala Ser Asn Thr Pro Asn Ser Pro Phe Leu Lys Asn Leu Ile Ser Gln Ser Thr Asn Pro Asn Asn Pro Gly Gly Leu Gln Ala Val Tyr Gln Val Asn Gln Ser Ala Tyr Ser Gln Leu Leu Ser Ala Thr Gln Glu Leu Gly His Asn Pro Phe Arg Arg Val Gly Leu Ile Ser Ser Gln Thr Asn Asn Gly Ala Met Asn Gly Ile Gly Val Gln Ile Gly Tyr Lys Gln Phe Phe Gly Glu Lys Arg Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Ser Phe Phe Asn Ser Ala Ser Asp Val Phe Thr Tyr Gly Val Gly Thr Asp Val Leu Tyr Asn Phe Ile Asn Asp Lys Ala Thr Lys Asn Asn Lys Ile Ser Phe Gly Val Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Tyr Val Asn Leu Ala Thr Phe Asn Asn Phe Tyr Ser Ala Lys Met Asn VaI Ala Asn Phe Gln Phe Leu Phe Asn Leu Gly Leu Arg Met Asn Leu Ala Lys Asn Lys Lys Lys Ala Ser Asp His Val Ala Gln His Gly Val Glu Leu Gly Val Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Leu Leu Gly Thr Gln Leu Gln Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2429 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 205...2277 ___ (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 205...259 -(D) OTHER INFORMATION:
SUBSTITUTE SHEET (RULE 26) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:

TTA CTC

Met Lys Lys Ser Leu Leu Leu Ser Leu Ser Leu Ile Ala Ser Leu Ser Arg Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala Val Gln Gln Val Lys Asn Thr Gly Ala Leu Gln Asn Leu Ala Asp Arg Tyr Asp Asn Leu Asn Asn Leu Leu Asn Gln Tyr Asn Tyr Leu Asn Ser Leu Vai Asn Leu Ala Ser Thr Pro Ser Ala Ile Thr Gly Ala Ile Asp Asn Leu Ser Ser Ser Ala Ile Asn Leu Thr Ser Ala Thr Thr Thr Ser Pro Ala Tyr Gln Ala Val Ala Leu Ala Leu Asn Ala Ala Val Gly Met Trp Gln Val Ile Ala Leu Phe Ile Gly Cys Gly Pro Gly Pro Thr Asn Asn Gln Ser Tyr Gln Ser Phe Gly Asn Thr Pro Ala Leu Asn Gly Thr Thr Thr Thr Cys Asn Gln Ala Tyr Gly Thr Gly Pro Asn Gly Ile Leu Ser Ile Asp Glu Tyr Gln Lys Leu Asn Gln Ala Tyr Gln Ile Ile Gln Thr Ala Leu Asn Gln Asn Gln Gly Gly Gly Met Pro Ala Leu Asn Asp Thr Thr Lys Thr Gly Val Val SU9STITUTE SHEET (RULE 26) WO 98/43479 1 ~g PCT/OS98/06421 Asn Ile Gln Gln Thr Asn Tyr Arg Thr Thr Thr Gln Asn Asn Ile Ile Glu His Tyr Tyr Thr Glu Asn Gly Lys Glu Ile Pro Val Ser Tyr Ser Gly Gly Ser Ser Phe Ser Pro Thr Ile Gln Leu Thr Tyr His Asn Asn Ala Glu Asn Leu Leu Gln Gln Ala Ala Thr Ile Met Gln Val Leu Ile Thr Gln Lys Pro His Val Gln Thr Ser Asn Gly Gly Lys Ala Trp Gly Leu Ser Ser Thr Pro Gly Asn Val Met Asp Ile Phe Gly Pro Ser Phe Asn Ala Ile Asn Glu Met Ile Lys Asn Ala Gln Thr Ala Leu Ala Lys Thr Gln Gln Leu Asn Ala Asn Glu Asn Ala Gln Ile Thr Gln Pro Asn Asn Phe Asn Pro Tyr Thr Ser Lys Asp Lys Gly Phe Ala Gln Glu Met Leu Asn Arg Ala Glu Ala Gln Ala Glu Ile Leu Asn Leu Ala Lys Gln Val Ala Asn Asn Phe His Ser Ile Gln Gly Pro Ile Gln Gly Asp Leu G1u Glu Cys Lys Ala Gly Ser Ala Gly Val Ile Thr Asn Asn Thr Trp Gly Ser Gly Cys Ala Phe Val Lys Glu Thr Leu Asn Ser Leu Glu Gln His Thr Ala Tyr Tyr Gly Asn Gln Val Asn Gln Asp Arg Ala Leu Ala SUBSTITUTE SHEET (RULE 26) Gln Thr Ile Leu Asn Phe Lys Glu Ala Leu Asn Thr Leu Asn Lys Asp Ser Lys Ala Ile Asn Ser Gly Ile Ser Asn Leu Pro Asn Ala Lys Ser Leu Gln Asn Met Thr His Ala Thr Gln Asn Pro Asn Ser Pro Glu Gly Leu Leu Thr Tyr Ser Leu Asp Ser Ser Lys Tyr Asn Gln Leu Gln Thr Ile Ala Gln Glu Leu Gly Lys Asn Pro Phe Arg Arg Phe Gly Val Ile Asp Phe Gln Asn Asn Asn Gly Ala Met Asn Gly Ile Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Lys Lys Arg Asn Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Val Gly Met Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Asn Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Phe Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Gln Val Asn Leu Thr Met Met Asn Gly Ile Tyr Asn Ala Asn Val Ser Thr Ser Asn Phe Gln Phe Leu Phe Asp Leu Gly Leu Arg Met Asn Leu Ala Arg Pro Lys Lys Lys Asp Ser Asp His Ala SUBSTITUTE SHEET (RULE 26) Ala Gln His Gly Ile Glu Leu Gly Phe Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Lys Leu Glu Tyr Arg Arg Met Tyr Ser Leu Phe Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 691 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single {D) TOPOLOGY: linear {ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
{A) NAME/KEY: Signal Sequence (B) LOCATION: 1...18 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Lys Lys Ser Leu Leu Leu Ser Leu Ser Leu Ile Ala Ser Leu Ser Arg Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala Val Gln Gln Val Lys Asn Thr Gly Ala Leu Gln Asn Leu Ala Asp Arg Tyr Asp Asn Leu Asn Asn Leu Leu Asn Gln Tyr Asn Tyr Leu Asn Ser Leu Val Asn Leu Ala Ser Thr Pro Ser Ala Ile Thr Gly Ala Ile Asp Asn Leu Ser Ser Ser Ala Ile Asn Leu Thr Ser Ala Thr Thr Thr Ser Pro Ala Tyr Gln Ala Val Ala Leu Ala Leu Asn Ala Ala Val Gly Met Trp Gln Val Ile Ala Leu Phe Ile Gly Cys Gly Pro Gly Pro Thr Asn Asn Gln Ser Tyr Gln Ser Phe Gly Asn Thr Pro Ala Leu Asn Gly Thr Thr Thr Thr Cys Asn Gln Ala Tyr Gly Thr Gly Pro Asn Gly 130 135 140 _ Ile Leu Ser Ile Asp Glu Tyr Gln Lys Leu Asn Gln Ala Tyr Gln Ile SUBSTITUTE SHEET (RULE 26) r Ile Gln Thr Ala Leu Asn Gln Asn Gln Gly Gly Gly Met Pro Ala Leu Asn Asp Thr Thr Lys Thr Gly Val Val Asn Ile Gln Gln Thr Asn Tyr Arg Thr Thr Thr Gln Asn Asn Ile Ile Glu His Tyr Tyr Thr Glu Asn Gly Lys Glu Ile Pro Val Ser Tyr Ser Gly Gly Ser Ser Phe Ser Pro Thr Ile Gln Leu Thr Tyr His Asn Asn Ala Glu Asn Leu Leu Gln Gln Ala Ala Thr Ile Met Gln Val Leu Ile Thr Gln Lys Pro His Val Gln Thr Ser Asn Gly Gly Lys Ala Trp Gly Leu Ser Ser Thr Pro Gly Asn Val Met Asp Ile Phe Gly Pro Ser Phe Asn Ala Ile Asn Glu Met Ile Lys Asn Ala Gln Thr Ala Leu Ala Lys Thr Gln Gln Leu Asn Ala Asn Glu Asn Ala Gln Ile Thr Gln Pro Asn Asn Phe Asn Pro Tyr Thr Ser Lys Asp Lys Gly Phe Ala Gln Glu Met Leu Asn Arg Ala Glu Ala Gln Ala Glu Ile Leu Asn Leu Ala Lys Gln Val Ala Asn Asn Phe His Ser Ile Gln Gly Pro Ile Gln Gly Asp Leu Glu Glu Cys Lys Ala Gly Ser Ala Gly Val Ile Thr Asn Asn Thr Trp Gly Ser Gly Cys Ala Phe Val Lys Glu Thr Leu Asn 5er Leu Glu Gln His Thr Ala Tyr Tyr Gly Asn Gln Val Asn Gln Asp Arg Ala Leu Ala Gln Thr Ile Leu Asn Phe Lys Glu Ala Leu Asn Thr Leu Asn Lys Asp Ser Lys Ala Ile Asn Ser Gly Ile Ser Asn Leu Pro Asn Ala Lys Ser Leu Gln Asn Met Thr His Ala Thr Gln Asn Pro Asn Ser Pro Glu Gly Leu Leu Thr Tyr Ser Leu Asp Ser Ser Lys Tyr Asn Gln Leu Gln Thr Ile Ala Gln Glu Leu Gly Lys Asn Pro Phe Arg Arg Phe Gly Val Ile Asp Phe Gln Asn Asn Asn Gly Ala Met Asn Gly Ile Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Lys Lys Arg Asn Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Val Gly Met Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Asn Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe ~ Gly Gly Phe Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Gln Val Asn Leu Thr Met Met Asn Gly Ile Tyr Asn Ala Asn Val Ser Thr Ser_ Asn Phe Gln Phe Leu Phe Asp Leu Gly Leu Arg Met Asn Leu Ala Arg SU8ST1TUTE SHEET (RULE 26) Pro Lys Lys Lys Asp Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Phe Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Lys Leu Glu Tyr Arg Arg Met Tyr Ser Leu Phe Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2270 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 130...2049 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 130...193 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:

Met Lys Lys Thr Lys Lys Thr Ile Leu Leu Ser Leu Thr Leu Ala Ala Ser Leu Leu His Ala Glu Asp Asn Gly Val Phe Leu Ser Val Gly Tyr Gln Ile Gly Glu Ala Val Gln Lys Val Lys Asn Ala Asp Lys Val Gln Lys Leu Ser Asp Thr Tyr Glu Gln Leu Ser Arg Leu Leu Thr Asn Asp Asn Gly Thr Asn Ser Lys Thr Ser Ala Gln Ile Asn Gln Ala_ SUBSTITUTE SHEET (RULE 26) ACT

Val AsnAsn LeuAsn GluArgAla LysThrLeu AlaGly GlyThrThr Asn SerPro AlaTyr GlnAlaThr LeuLeuAla LeuArg SerValLeu Gly LeuTrp AsnSer MetGlyTyr AlaValIle CysGly GlyTyrThr Lys SerPro GlyGlu AsnAsnGln LysAspPhe HisTyr ThrAspGlu Asn GlyAsn GlyThr ThrIleAsn CysGlyGly SerThr AsnSerAsn Gly ThrHis SerSer SerGlyThr AsnThrLeu LysAla AspLysAsn Val SerLeu SerIle GluGlnTyr GluLysIle HisGlu AlaTyrGln Ile LeuSer LysAla LeuLysGln AlaGlyLeu AlaPro LeuAsnSer Lys GlyGlu LysLeu GluAlaHis ValThrThr SerLys ProGluAsn Asn SerGln ThrLys ThrThrThr SerValIle AspThr ThrAsnAsp Ala GlnAsn LeuLeu ThrGlnAla GlnThrIle ValAsn ThrLeuLys Asp TyrCys ProMet LeuIleAla LysSerSer SerGlu SerSerGly Ala AlaThr ThrAsn AlaProSer TrpGlnThr AlaGly GlyGly_ Lys Asn SerCys AlaThr PheGlyAla GluPheSer AlaAla SerAspMet SUBSTITUTE SHEET (RULE 26) Ile Asn Asn Ala Gln Lys Ile Val Gln Glu Thr Gln Gln Leu Ser Ala Asn Gln Pro Lys Asn Ile Thr Gln Pro His Asn Leu Asn Leu Asn Thr Pro Ser Ser Leu Thr Ala Leu Ala Gln Lys Met Leu Lys Asn Ala Gln Ser Gln Ala Glu Ile Leu Lys Leu Ala Asn Gln Val Glu Ser Asp Phe Asn Lys Leu Ser Ser Gly His Leu Lys Asp Tyr Ile Gly Lys Cys Asp Ala Ser Ala Ile Ser Ser Ala Asn Met Thr Met Gln Asn Gln Lys Asn Asn Trp Gly Asn Gly Cys Ala Gly Val Glu Glu Thr Leu Ser Ser Leu Lys Thr Ser Ala Ala Asp Phe Asn Asn Gln Thr Pro Gln Ile Asn Gln Ala Gln Asn Leu Ala Asn Thr Leu Ile Gln Glu Leu Gly Asn Asn Pro Phe Arg Asn Met Gly Met Ile Ala Ser Ser Thr Thr Asn Asn Gly Ala Leu Asn Gly Leu Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Glu Lys Lys Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Val Gly Ser Asp Leu Leu Phe Asn Phe Ile Asn Asp Lys Asn Thr Asn Phe Leu Gly Lys Asn Asn Lys Ile Ser Val Gly Phe Phe Gly SUBSTITUTE SHEET (RULE 26) Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Phe Val Asn Leu Lys Thr Ile Ser Asn Val Tyr Ser Ala Lys Val Asn Thr Ala Asn Phe GlnPhe LeuPheAsn LeuGly LeuArgThr AsnLeuAla ArgPro Lys LysLys AspSerHis HisAla AlaGlnHis GlyMetGlu LeuGly Val LysIle ProThrIle AsnThr AsnTyrTyr SerPheLeu AspThr Lys LeuGlu TyrArgArg LeuTyr SerValTyr LeuAsnTyr ValPhe GCC TATTAAAAACCCT TTTAAAAAAC CTCTAAAGAT

CTTTTTAAAA AA
AAGGGGGGGC

Ala Tyr (2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 640 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 1...21 (D) OTHER INFORMATTON:
' (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Lys Lys Thr Lys Lys Thr Ile Leu Leu Ser Leu Thr Leu Ala Ala Ser Leu Leu His Ala Glu Asp Asn Gly Val Phe Leu Ser Val Gly Tyr SUBSTITUTE SHEET (RULE 26) Gln Ile Gly Glu Ala Val Gln Lys Val Lys Asn Ala Asp Lys Val Gln Lys Leu Ser Asp Thr Tyr Glu Gln Leu Ser Arg Leu Leu Thr Asn Asp Asn Gly Thr Asn Ser Lys Thr Ser Ala Gln Ile Asn Gln Ala Val Asn Asn Leu Asn Glu Arg Ala Lys Thr Leu Ala Gly Gly Thr Thr Asn Ser Pro Ala Tyr Gln Ala Thr Leu Leu Ala Leu Arg Ser Val Leu Gly Leu Trp Asn Ser Met Gly Tyr Ala Val Ile Cys Gly Gly Tyr Thr Lys Ser Pro Gly Glu Asn Asn Gln Lys Asp Phe His Tyr Thr Asp Glu Asn Gly Asn Gly Thr Thr Ile Asn Cys Gly Gly Ser Thr Asn Ser Asn Gly Thr His Ser Ser Ser Gly Thr Asn Thr Leu Lys Ala Asp Lys Asn Val Ser Leu Ser Ile Glu Gln Tyr Glu Lys Ile His Glu Ala Tyr Gln Ile Leu Ser Lys Ala Leu Lys Gln Ala Gly Leu Ala Pro Leu Asn Ser Lys Gly Glu Lys Leu Glu Ala His Val Thr Thr Ser Lys Pro Glu Asn Asn Ser Gln Thr Lys Thr Thr Thr Ser Val Ile Asp Thr Thr Asn Asp Ala Gln Asn Leu Leu Thr Gln Ala Gln Thr Ile Val Asn Thr Leu Lys Asp Tyr Cys Pro Met Leu Ile Ala Lys Ser Ser Ser Glu Ser Ser Gly Ala Ala Thr Thr Asn Ala Pro Ser Trp Gln Thr Ala Gly Gly Gly Lys Asn Ser Cys Ala Thr Phe Gly Ala Glu Phe Ser Ala Ala Ser Asp Met Ile Asn Asn Ala Gln Lys Ile Val Gln Glu Thr Gln Gln Leu Ser Ala Asn Gln Pro Lys Asn Ile Thr Gln Pro His Asn Leu Asn Leu Asn Thr Pro Ser Ser Leu Thr Ala Leu Ala Gln Lys Met Leu Lys Asn Ala Gln Ser Gln Ala Glu Ile Leu Lys Leu Ala Asn Gln Val Glu Ser Asp Phe Asn Lys Leu Ser Ser Gly His Leu Lys Asp Tyr Ile Gly Lys Cys Asp Ala Ser Ala Ile Ser Ser Ala Asn Met Thr Met Gln Asn Gln Lys Asn Asn Trp Gly Asn Gly Cys Ala Gly Val Glu Glu Thr Leu Ser Ser Leu Lys Thr Ser Ala Ala Asp Phe Asn Asn Gln Thr Pro Gln Ile Asn Gln Ala Gln Asn Leu Ala Asn Thr Leu Ile Gln Glu Leu Gly Asn Asn Pro Phe Arg Asn Met Gly Met Ile Ala Ser Ser Thr Thr Asn Asn Gly Ala Leu Asn Gly Leu Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Glu Lys Lys SUBSTITUTE SHEET (RULE 26) WO 98/43479 j j ~ PCT/US98/06421 Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Val Gly Ser Asp Leu Leu Phe Asn Phe Ile Asn Asp Lys Asn Thr Asn Phe Leu Giy Lys Asn Asn Lys Ile Ser Val Gly Phe Phe Gly Gly Ile Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Phe Val Asn Leu Lys Thr Ile Ser Asn Val Tyr Ser Ala Lys Val Asn Thr Ala Asn Phe Gln Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Arg Pro Lys Lys Lys Asp Ser His His Ala Ala Gln His Gly Met Glu Leu Gly Val Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Leu Asp Thr Lys Leu Glu Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2248 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 173...2128 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 173...224 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:

Met Lys Lys Thr Ile Leu Leu Ser Leu Ser Ala Ser Ser Leu Leu His Ala Glu -15 -10 -5 1 _ SUBSTITUTE SHEET (RULE 26) Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gln Ile Gly Glu Ala Val Gln Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu Asn Glu Lys Tyr Glu Gln Leu Ser Gln Tyr Leu Asn Gln Val Ala Ser Leu Lys GIn Ser Ile Gln Asn Ala Asn Asn Ile Glu Leu Val Asn Ser Ser Leu Asn Tyr Leu Lys Ser Phe Thr Asn Asn Asn Tyr Asn Ser Thr Thr Gln Ser Pro Ile Phe Asn Ala Val Gln Ala Val Ile Thr Ser Val Leu Gly Phe Trp Ser Leu Tyr Ala Gly Asn Tyr Phe Thr Phe Phe Val Gly Lys Lys Val Gly Asp Ser Giy Gln Pro Ala Ser Val Gln Gly Asn Pro Pro Phe Lys Thr Ile Ile Glu Asn Cys Ser Gly Ile Glu Asn Cys Ala Met Asp Gln Thr Thr Tyr Asp Lys Met Lys Lys Leu Ala Glu Asp Leu Gln Ala Ala Gln Thr Asn Ser Ala Thr Lys GIy Asn Asn Leu Cys Ala Leu Ser Gly Cys Ala Ala Thr Asp Ser Thr Ser Asn Pro Pro Asn Ser Thr Val Ser Asn Ala Leu Asn Leu Ala Gln Gln Leu Met Asp Leu Ile Ala Asn Thr Lys Thr Ala Met Met Trp Lys Asn Ile Val Ile Ser Gly Val Ser Asn Thr Ser Gly Ala Ile Thr Ser Thr Asn Tyr Pro Thr Gln Tyr Ala Val SUBSTITUTE SHEET (RULE 26) Phe Asn Asn Ile Lys Ala Met Ile Pro Ile Leu Gln Gln Ala Val Thr Leu Ser Gln Ser Asn His Thr Leu Ser Ala Ser Leu Gln Ala Gln Ala Thr Gly Ser Gln Thr Asn Pro Lys Phe Ala Lys Asp Ile Tyr Thr Phe Ala Gln Asn Gln Lys Gln Val Ile Ser Tyr Ala Gln Asp Ile Phe Asn Leu Phe Asn Ser Ile Pro Ala Glu Gln Tyr Lys Tyr Leu Glu Lys Aia Tyr Leu Lys Ile Pro Asn Ala Gly Ser Thr Pro Thr Asn Pro Tyr Arg AAA
AAC

GlnVal ValAsn LeuAsnGln GluValGln ThrIleLys AsnAsn Val SerTyr TyrGly AsnArgVal AspAlaAla LeuSerVal AlaArg Asp ValTyr AsnLeu Lys5erAsn GlnAlaGlu IleValThr AlaTyr Asn AspAla LysThr LeuSerGlu GluIleSer LysLeuPro HisAsn Gln ValAsn ThrLys AspIleVal ThrLeuPro TyrAspLys AsnAla Pro w AlaAla GlyGln SerAsnTyr GlnIleAsn ProGluGln GlnSer Asn Leu Asn Gln Ala Leu Ala Ala Met Ser Asn Asn Pro Phe Lys Lys Val Gly Met Ile Ser Ser Gln Asn Asn Asn Gly Ala Leu Asn Gly Leu Gly SUBSTITUTE SHEET (RULE 26~

val Gln Val Gly Tyr Lys Gln Phe Phe Gly Glu Ser Lys Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Gly Tyr Ile Lys Ser Ser Phe Phe Asn Ser Ser Ser Asp Ile Trp Thr Tyr Gly Gly Gly Ser Asp Leu Leu Val Asn Ile Ile Asn Asp Ser Ile Thr Arg Lys Asn Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Ile Gln Leu Ala Gly Thr Thr Trp Leu Asn Ser Gln Tyr Val Asn Leu Thr Ala Phe Asn Asn Pro Tyr Ser Ala Lys Val Asn Ala Thr Asn Phe Gln Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Thr Ala Arg Lys Lys Asp Ser Glu His Ser Ala Gln His Gly Ile Glu Leu Gly Ile Lys Ile Pro Thr Ile Thr Thr Asn Tyr Tyr Ser Phe Leu Gly Thr Gln Leu Gln Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 652 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal SUBSTITUTE SHEET (RULE 26) (ix) FEATURE:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 1...17 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Lys Lys Thr Ile Leu Leu Ser Leu Ser Ala Ser Ser Leu Leu His Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gln Ile Gly Glu Ala Val Gln Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu Asn Glu Lys Tyr Glu Gln Leu Ser Gln Tyr Leu Asn Gln Val Ala Ser Leu Lys Gln Ser Ile Gln Asn Ala Asn Asn Ile Glu Leu Val Asn Ser Ser Leu Asn Tyr Leu Lys Ser Phe Thr Asn Asn Asn Tyr Asn Ser Thr Thr Gln Ser Pro Ile Phe Asn Ala Val Gln Ala Val Ile Thr Ser Val Leu Gly Phe Trp Ser Leu Tyr Ala Gly Asn Tyr Phe Thr Phe Phe Val Gly Lys Lys Val Gly Asp Ser Gly Gln Pro Ala Ser Val Gln Gly Asn Pro Pro Phe Lys Thr Ile Ile Glu Asn Cys Ser Gly Ile Glu Asn Cys Ala Met Asp Gln Thr Thr Tyr Asp Lys Met Lys Lys Leu Ala Glu Asp Leu Gln Ala Ala Gln Thr Asn Ser Ala Thr Lys Gly Asn Asn Leu Cys Ala Leu Ser Gly Cys Ala Ala Thr Asp Ser Thr Ser Asn Pro Pro Asn Ser Thr Val Ser Asn Ala Leu Asn Leu Ala Gln Gln Leu Met Asp Leu Ile Ala Asn Thr Lys Thr Ala Met Met Trp Lys Asn Ile Val Ile Ser Gly Val Ser Asn Thr Ser Gly Ala Ile Thr Ser Thr Asn Tyr Pro Thr Gln Tyr Ala Val Phe Asn Asn Ile Lys Ala Met Ile Pro Ile Leu Gln Gln Ala Val Thr Leu Ser Gln Ser Asn His Thr Leu Ser Ala Ser Leu Gln Ala 260 265 27p Gln Ala Thr Gly Ser Gln Thr Asn Pro Lys Phe Ala Lys Asp Ile Tyr Thr Phe Ala Gln Asn Gln Lys Gln Val Ile Ser Tyr Ala Gln Asp Ile ' 290 295 300 Phe Asn Leu Phe Asn Ser Ile Pro Ala Glu Gln Tyr Lys Tyr Leu Glu Lys Ala Tyr Leu Lys Ile Pro Asn Ala Gly Ser Thr Pro Thr Asn Pro Tyr Arg Gln Val Val Asn Leu Asn Gln Glu Val Gln Thr Ile Lys As~1 Asn Val Ser Tyr Tyr Gly Asn Arg Val Asp Ala Ala Leu Ser Val Ala SUBSTITUTE SHEET (RULE 26) Arg Asp Val Tyr Asn Leu Lys Ser Asn Gln Ala Glu Ile Val Thr Ala Tyr Asn Asp Ala Lys Thr Leu Ser Glu Glu Ile Ser Lys Leu Pro His Asn Gln Val Asn Thr Lys Asp Ile Val Thr Leu Pro Tyr Asp Lys Asn Ala Pro Ala Ala Gly Gln Ser Asn Tyr Gln Ile Asn Pro Glu Gln Gln Ser Asn Leu Asn Gln Ala Leu Ala Ala Met Ser Asn Asn Pro Phe Lys Lys Val Gly Met Ile Ser Ser Gln Asn Asn Asn Gly Ala Leu Asn Gly Leu Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Glu Ser Lys Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Gly Tyr Ile Lys Ser Ser Phe Phe Asn Ser Ser Ser Asp Ile Trp Thr Tyr Gly Gly Gly Ser Asp Leu Leu Val Asn Ile Ile Asn Asp Ser Ile Thr Arg Lys Asn Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Ile Gln Leu Ala Gly Thr Thr Trp Leu Asn Ser Gln Tyr Val Asn Leu Thr Ala Phe Asn Asn Pro Tyr Ser Ala Lys Val Asn Ala Thr Asn Phe Gln Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Thr Ala Arg Lys Lys Asp Ser Glu His Ser Ala Gln His Gly Ile Glu Leu Gly Ile Lys Ile Pro Thr Ile Thr Thr Asn Tyr Tyr Ser Phe Leu Gly Thr Gln Leu Gln Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2161 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
( ix ) FEATURE ~' (A) NAME/KEY: Coding Sequence (B) LOCATION: 122...2056 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 122...179 _ (D) OTHER INFORMATION:
SUBSTITUTE SHEET (RULE 26) (xi) SEQUENCE DESCRIPTION:
SEQ
ID
N0:17:

CAAAAATCTT TTTTTTTTTT C ATGGTAAAGT TAAACATATT

TTTTGAAAT CAATAAATTT

GTAAATAAAT TTTAATTTCT AATTACTTTA AGGAACATTT

ATTCATGTTT
ACAATAAAAA

A AAA T CTC TCG T CTC
AAG TCT CTT
ACA C T
ATT
CTA
C

M et ys ys le Leu eu er eu Ser eu er eu er er L L Thr L S L L S L S S Leu I

GAC GGC AGC

LeuHisAla GluAspAsn Phe PheVal AlaGlyTyr GlnIle Gly Ser GTC GGC

GlyGluArg ValGlnMet Lys AsnThr GluLeuLys AsnLeu Val Gly AACGAAAAA TACGAGCAA AGC CAATCT GCCCAACTG GCTTCG
TTA TTA

AsnGluLys TyrGluGln Ser GlnSer AlaGlnLeu AlaSer Leu Leu ACG ATT

LeuLysLys SerIleGln Ala AsnAsn GlnAlaVal AsnAsn Thr Ile AGC AAC

AlaLeuSer AspLeuLys Phe AlaSer AsnHisThr AsnLys Ser Asn AAC GCT

GluThrSer ProIleTyr Thr AlaGln ValIleThr SerVal Asn Ala TAT GCT

LeuAlaPhe TrpSerLeu Ala GlyAsn ThrSerPhe HisVal Tyr Ala TCT CTT

ThrGlyLeu AsnAspGly Asn AlaPro GlyArgIle HisGln Ser Leu TTA TTT

AspGlyAsn CysThrGly Gln GlnCys MetAsnLys GluThr Leu Phe CTT CTC

TyrAspLys MetLysAla Ala GluAsn GlnLysAla GlnGly Leu Leu Asn Leu Cys Ala Leu Ser Glu Cys Pro Ser Asp Gln Leu Asn Gly Asn AAT GGA AAC AAA ACT TCC ATG ACT AAA GCT CTT GAA ACC GCG CAA CAG_ 745 Asn Gly Asn Lys Thr Ser Met Thr Lys Ala Leu Glu Thr Ala Gln Gln SUBSTITUTE SHEET (RULE 26) Leu Met Asp Leu Ile Ala Asn Thr Lys Thr Ala Met Met Trp Lys Asn Ile Val Ile Ala Gly Val Thr Asn Arg Pro Gly Gly Ala Gly Ala Ile Thr Ser Thr Gly Pro Val Thr Asp Tyr Ala Val Phe Asn Asn Ile Lys Ala Met Ile Pro Ile Leu Gln Gln Ala Val Thr Leu Ser Gln Ser Asn His Thr Leu Ser Ala Ser Leu Gln Ala Gln Ala Thr Gly Ser Gln Thr Asn Pro Lys Phe Ala Lys Asp Ile Tyr Thr Phe Ala Gln Asn Gln Lys Gln Val Ile Ser Tyr Ala Gln Asp Ile Phe Asn Leu Phe Asn Ser Ile Pro Ala Glu Gln Tyr Lys Tyr Leu Glu Lys Ala Tyr Leu Lys Ile Pro Asn Ala Gly Ser Thr Pro Thr Asn Pro Tyr Arg Gln Val Val Asn Leu Asn Gln Glu Val Gln Thr Ile Lys Asn Asn Val Ser Tyr Tyr Gly Asn Arg Val Asp Ala Ala Leu Ser Val Ala Arg Asp Val Tyr Asn Leu Lys Ser Asn Gln Ala Glu Ile Val Thr Ala Tyr Asn Asp Ala Lys Thr Leu Ser Glu Glu Ile Ser Lys Leu Pro His Asn Gln Val Asn Thr Lys Asp Ile Val Thr Leu Pro Tyr Asp Lys Asn Ala Pro Ala Ala Gly Gln Ser 400 405 410 _ SUBSTITUTE SHEET (RULE 26) Asn Tyr Gln Ile Asn Pro Glu Gln Gln Ser Asn Leu Asn Gln Ala Leu Ala Ala MetSer AsnAsnPro PheLys LysValGly MetIleSer Ser Gln Asn AsnAsn GlyAlaLeu AsnGly LeuGlyVal GlnValGly Tyr Lys Gln PhePhe GlyGluSer LysArg TrpGlyLeu ArgTyrTyr Gly Phe Phe AspTyr AsnHisGly TyrIle LysSerSer PhePheAsn Ser Ser Ser AspIle TrpThrTyr GlyGly GlySerAsp LeuLeuVal Asn Ile Ile AsnAsp SerIleThr ArgLys AsnAsnLys LeuSerVal Gly Leu Phe GlyGly IleGlnLeu AlaGly ThrThrTrp LeuAsnSer Gln Tyr Val AsnLeu ThrAlaPhe AsnAsn ProTyrSer AlaLysVal Asn Ala Thr AsnPhe GlnPheLeu PheAsn LeuGlyLeu ArgThrAsn Leu Ala Thr AlaArg LysLysAsp SerGlu HisSerAla GlnHisGly Ile Glu Leu GlyIle LysIlePro ThrIle ThrThrAsn TyrTyrSer Phe Leu Gly ThrGln LeuGlnTyr ArgArg LeuTyrSer ValTyrLeu Asn ___ TAT GTG TTCGCT TATTAAAAAATCT CTTCATCAAA

TCTTTTTAAA T
ATAGGGGGAG

Tyr Val PheAla Tyr AGTTATCAAT AAACCAAATC
ATTTGATGAA
AATAAAG

SUBSTITUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 645 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 1...19 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Lys Lys Thr Ile Leu Leu Ser Leu Ser Leu Ser Leu Ser Ser Leu Leu His Ala Glu Asp Asn Gly Phe Phe Val Ser Ala Gly Tyr Gln Ile Gly Glu Arg Val Gln Met Val Lys Asn Thr Gly Glu Leu Lys Asn Leu Asn Glu Lys Tyr Glu Gln Leu Ser Gln Ser Leu Ala Gln Leu Ala Ser Leu Lys Lys Ser Ile Gln Thr Ala Asn Asn Ile Gln Ala Val Asn Asn Ala Leu Ser Asp Leu Lys Ser Phe Ala Ser Asn Asn His Thr Asn Lys Glu Thr Ser Pro Ile Tyr Asn Thr Ala Gln Ala Val Ile Thr Ser Val Leu Ala Phe Trp Ser Leu Tyr Ala Gly Asn Ala Thr Ser Phe His Val Thr Gly Leu Asn Asp Gly Ser Asn Ala Pro Leu Gly Arg Ile His Gln Asp Gly Asn Cys Thr Gly Leu Gln Gln Cys Phe Met Asn Lys Glu Thr Tyr Asp Lys Met Lys Ala Leu Ala Glu Asn Leu Gln Lys Ala Gln Gly Asn Leu Cys Ala Leu Ser Glu Cys Pro Ser Asp Gln Leu Asn Gly Asn Asn Gly Asn Lys Thr Ser Met Thr Lys Ala Leu Glu Thr Ala Gln Gln Leu Met Asp Leu Ile Ala Asn Thr Lys Thr Ala Met Met Trp Lys Asn Ile Val Ile Ala Gly Val Thr Asn Arg Pro Gly Gly Ala Gly Ala Ile__ Thr Ser Thr Gly Pro Val Thr Asp Tyr Ala Val Phe Asn Asn Ile Lys Ala Met Ile Pro Ile Leu Gln Gln Ala Val Thr Leu Ser Gln Ser Asn His Thr Leu Ser Ala Ser Leu Gln Ala Gln Ala Thr Gly Ser Gln Thr Asn Pro Lys Phe Ala Lys Asp Ile Tyr Thr Phe Ala Gln Asn Gln Lys SUBSTITUTE SHEET (RULE 26) 270 275 280 ~ 285 Gln Val Ile Ser Tyr Ala Gln Asp Ile Phe Asn Leu Phe Asn Ser Ile Pro Ala Glu Gln Tyr Lys Tyr Leu Glu Lys Ala Tyr Leu Lys Ile Pro Asn Ala Gly Ser Thr Pro Thr Asn Pro Tyr Arg Gln Val Val Asn Leu Asn Gln Glu Val Gln Thr Ile Lys Asn Asn Val Ser Tyr Tyr Gly Asn Arg Val Asp Ala Ala Leu Ser Val Ala Arg Asp Val Tyr Asn Leu Lys Ser Asn Gln Ala Glu Ile Val Thr Ala Tyr Asn Asp Ala Lys Thr Leu Ser Glu Glu Ile Ser Lys Leu Pro His Asn Gln Val Asn Thr Lys Asp Ile Val Thr Leu Pro Tyr Asp Lys Asn Ala Pro Ala Ala Gly Gln Ser Asn Tyr Gln Ile Asn Pro Glu Gln Gln Ser Asn Leu Asn Gln Ala Leu Ala Ala Met Ser Asn Asn Pro Phe Lys Lys Val Gly Met Ile Ser Ser Gln Asn Asn Asn Gly Ala Leu Asn Gly Leu Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Glu Ser Lys Arg Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Gly Tyr Ile Lys Ser Ser Phe Phe Asn Ser Ser Ser Asp Ile Trp Thr Tyr Gly Gly Gly Ser Asp Leu Leu Val Asn Ile Ile Asn Asp Ser Ile Thr Arg Lys Asn Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Ile Gln Leu Ala Gly Thr Thr Trp Leu Asn Ser Gln Tyr Val Asn Leu Thr Ala Phe Asn Asn Pro Tyr Ser Ala Lys Val Asn Ala Thr Asn Phe Gln Phe Leu Phe Asn Leu Gly Leu Arg Thr Asn Leu Ala Thr Ala Arg Lys Lys Asp Ser Glu His Ser Ala Gln His Gly Ile Glu Leu Gly Ile Lys Ile Pro Thr Ile Thr Thr Asn Tyr Tyr Ser Phe Leu Gly Thr Gln Leu Gln Tyr Arg Arg Leu Tyr Ser Val Tyr Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1799 base pairs (B) TYPE: nucleic acid -(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA _ (ix) FEATURE:
SUBSTITUTE SHEET (RULE 26) (A) NAME/KEY: Coding Sequence (B) LOCATION: 185...1633 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 185...233 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:

ACG ATT TTA CTT CTC

Met Lys Lys Thr Ile Leu Ser Leu Met Val Ser Ser Leu Leu Leu Ala Glu Asn Asp Gly Val Phe Met Ser Val Gly Tyr Gln Ile Gly Glu Ala Val Gln Gln Val Lys Asn Thr Gly Glu Ile Gln Lys Val Ser Asn Ala Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Asn Glu Leu Lys Gln Thr Ala Sex Asn Thr Asn Ser Ser Thr Ala Gln Ala Ile Asp Asn Leu Lys Glu Ser Ala Ser Arg Leu Lys Thr Thr Pro Asn Ser Ala Asn Gln Ala Val Ser Ser Ala Leu Ser Ser Ala Val Ala Met Trp Gln Val Ile Val Ser Asn Leu Ala Asn Asn Ser Leu Pro Thr Ser Glu Tyr Asn Lys Ile Asn Ala Ile Ser Gln Ser Leu Gln Asn Thr Leu Glu Asn Lys Asn Asn Asp Leu Lys Ile Glu Asn Asp Tyr Asp His Leu Leu Thr Gln_ SUBSTITUTE SHEET (RULE 26) AGC ATA

Ala SerThr IleIleAsn ThrLeuGln Gln CysProGly Asp Ser Ile AAT

Gly GlyAsn GlyLysPro TrpGlyIle AsnAla SerGlyAsn AlaCys Asn IlePhe GlyAsnThr PheAsnAla IleThr SerMetIle AspSer Ala LysLys AlaAlaAla AspAlaArg ArgThr AlaProGlu SerPro Asn GlnPro SerAlaPhe AsnAsnAla AspPhe AsnLysAsn LeuAsn Gln ValSer SerValIle AsnAspThr IleSer TyrLeuLys GlyAsp Asn LeuAla ThrIleTyr AsnThrLeu GlnLys ThrProAsp SerLys Gly PheGln SerLeuVal SerArgSer SerTyr SerTyrSer LeuAsn Glu ThrGln TyrSerGlu PheGlnThr ThrThr LysGluPhe GlyHis Asn ProPhe ArgSerVal GlyLeuIle AsnSer GlnSerAsn AsnGly Ala MetAsn GlyValGly ValGlnLeu GlyTyr LysGlnPhe PheGly Lys AsnLys PhePheGly IleArgTyr TyrAla PhePheAsp TyrAsn His AlaTyr IleLysSer AsnPhePhe AsnSer AlaSerAsn ValPhe Thr TyrGly AlaGlySer AspLeuLeu LeuAsn PheIleAsn GlyGly AAC

SU9STtTUTE SHEET (RULE 26) Ser Asp Lys Asn Arg Lys Val Ser Phe Gly Ile Phe Gly Gly Ile Ala Leu Ala Gly Thr Thr Trp Leu Asn Ser Gln Phe Met Asn Leu Lys Thr Thr Asn Ser Ala Tyr Ser Ala Lys Ile Asn Asn Thr Asn Phe Gln Phe Leu Phe Asn Thr Gly Leu Arg Leu Gln Gly Ile His His Gly Val Glu ACG AAT ATG

LeuGly Val Lys Ile Pro Thr Ile Asn Tyr Tyr Ser Phe Thr Asn Met TAT AGC TAT

GlyAla Lys Leu Ala Tyr Arg Arg Leu Val Tyr Phe Asn Tyr Ser Tyr TTACTAATGA GGACAAAGCC AAACT

ValLeu Ala Tyr CAATGAATAA
CGGCATCATT
TTACTTGACT

TCTTTTTTGA
GCGAAATTCC
AGATTAGCTC

(2) INFORMATION FOR SEQ ID N0:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 483 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:

(A) NAME/KEY: Signal Sequence (B) LOCATION: 1...16 (D) OTHER INFORMATION:

(xi) SEQUENCE DESCRIPTION:SEQID N0:20:

MetLys Thr IleLeu Leu LeuMetVal Ser Leu Leu Lys Ser Ser Ala GluAsn Gly ValPhe Met ValGlyTyr Ile Gly Glu Asp Ser Gln Ala ValGln Val LysAsn Thr GluIleGln Val Ser Asn Gln Gly Lys Ala SUBSTITUTE SHEET (RULE 26) Tyr Glu Asn Leu Asn Asn Leu Leu Thr Arg Tyr Asn Glu Leu Lys Gln Thr Ala Ser Asn Thr Asn Ser Ser Thr Ala Gln Ala Ile Asp Asn Leu Lys Glu Ser Ala Ser Arg Leu Lys Thr Thr Pro Asn Ser Ala Asn Gln Ala Val Ser Ser Ala Leu Ser Ser Ala Val Aia Met Trp Gln Val Ile . 85 90 95 Val Ser Asn Leu Ala Asn Asn Ser Leu Pro Thr Ser Glu Tyr Asn Lys Ile Asn Ala Ile Ser Gln Ser Leu Gln Asn Thr Leu Glu Asn Lys Asn Asn Asp Leu Lys Ile Glu Asn Asp Tyr Asp His Leu Leu Thr Gln Ala Ser Thr Ile Ile Asn Thr Leu Gln Ser Gln Cys Pro Gly Ile Asp Gly Gly Asn Gly Lys Pro Trp Gly Ile Asn Ala Ser Gly Asn Ala Cys Asn Ile Phe Gly Asn Thr Phe Asn Ala Ile Thr Ser Met Ile Asp Ser Ala Lys Lys Ala Ala Ala Asp Ala Arg Arg Thr Ala Pro Glu Ser Pro Asn Gln Pro Ser Ala Phe Asn Asn Ala Asp Phe Asn Lys Asn Leu Asn Gln Val Ser Ser Val Ile Asn Asp Thr Ile Ser Tyr Leu Lys Gly Asp Asn Leu Ala Thr Ile Tyr Asn Thr Leu Gln Lys Thr Pro Asp Ser Lys Gly Phe Gln Ser Leu Val Ser Arg Ser Ser Tyr Ser Tyr Ser Leu Asn Glu Thr Gln Tyr Ser Glu Phe Gln Thr Thr Thr Lys Glu Phe Gly His Asn Pro Phe Arg Ser Val Gly Leu Ile Asn Ser Gln Ser Asn Asn Gly Ala Met Asn Gly Val Gly Val Gln Leu Gly Tyr Lys Gln Phe Phe Gly Lys Asn Lys Phe Phe Gly Ile Arg Tyr Tyr Ala Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asn Val Phe Thr Tyr Gly Ala Gly Ser Asp Leu Leu Leu Asn Phe Ile Asn Gly Gly Ser Asp Lys Asn Arg Lys Val Ser Phe Gly Ile Phe Gly Gly Ile Ala Leu Ala Gly Thr Thr Trp Leu Asn Ser Gln Phe Met Asn Leu Lys Thr Thr Asn Ser Ala Tyr Ser Ala Lys Ile Asn Asn Thr Asn Phe Gln Phe Leu Phe Asn Thr Gly Leu Arg Leu Gln Gly Ile His His Gly Val Glu Leu ' 420 425 430 Gly Val Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Lys Leu Ala Tyr Arg Arg Leu Tyr Ser Val Tyr Phe Asn Tyr Val Leu Ala Tyr SUBSTITUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2338 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 146...2218 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 146...200 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:

Met Lys Lys Ser Leu Leu Leu Ser Leu Ser Leu Ile Ala Ser Leu Ser Arg Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala Val Gln Gln Val Lys Asn Thr Gly Ala Leu Gln Asn Leu Ala Asp Arg Tyr Asp Asn Leu Asn Asn Leu Leu Asn Gln Tyr Asn Tyr Leu Asn Ser Leu Val Asn Leu Ala Ser Thr Pro Ser Ala Ile Thr Gly Ala Ile Asp Asn Leu Ser Ser Ser Ala Ile Asn Leu Thr Ser Ala Thr Thr Thr Ser Pro Ala Tyr Gln Ala Val Ala Leu Ala Leu Asn Ala Ala Val Gly Met Trp Gln Val Ile Ala Leu Phe SUBSTITUTE SHEET (RULE 26) GGC CAA TCG

Ile Gly CysGly ProGlyPro ThrAsn AsnGlnSer TyrGlnSer Phe GGT AAC ACACCA GCCCTTAAT GGGACC ACCACCACT TGCAATCAA GCA

- Gly Asn ThrPro AlaLeuAsn GlyThr ThrThrThr CysAsnGln Ala Tyr Gly ThrGly ProAsnGly IleLeu SerIleAsp GluTyrGln Lys Leu Asn GlnAla TyrGlnIle IleGln ThrAlaLeu AsnGlnAsn Gln Gly Gly GlyMet ProAlaLeu AsnAsp ThrThrLys ThrGlyVal Val Asn Ile GlnGln ThrAsnTyr ArgThr ThrThrGln AsnAsnIle Ile Glu His TyrTyr ThrGluAsn GlyLys GluIlePro ValSerTyr Ser Gly Gly SerSer PheSerPro ThrIle GlnLeuThr TyrHisAsn Asn Ala Glu AsnLeu LeuGlnGln AlaAla ThrIleMet GlnValLeu Ile Thr Gln LysPro HisValGln ThrSer AsnGlyGly LysAlaTrp Gly Leu Ser SerThr ProGlyAsn ValMet AspIlePhe GlyProSer Phe Asn Ala IleAsn GluMetIle LysAsn AlaGlnThr AlaLeuAla Lys 280 285 290 2g5 Thr Gln GlnLeu AsnAlaAsn GluAsn AlaGlnIle ThrGlnPro Asn AAT TTC AACCCC TACACCTCT AAAGAC AAAGGGTTC GCTCAAGAA ATG_1180 Asn Phe AsnPro TyrThrSer LysAsp LysGlyPhe AlaGlnGlu Met SUBSTITUTE SHEET (RULE 26) Leu Asn Arg Ala Glu Ala Gln Ala Glu Ile Leu Asn Leu Ala Lys Gln Val Ala Asn Asn Phe His Ser Ile Gln Gly Pro Ile Gln Gly Asp Leu Glu Glu Cys Lys Ala Gly Ser Ala Gly Val Ile Thr Asn Asn Thr Trp Gly Ser Gly Cys Ala Phe Val Lys Glu Thr Leu Asn Ser Leu Glu Gln His Thr Ala Tyr Tyr Gly Asn Gln Val Asn Gln Asp Arg Ala Leu Ala Gln Thr Ile Leu Asn Phe Lys Glu Ala Leu Asn Thr Leu Asn Lys Asp Ser Lys Ala Ile Asn Ser Gly Ile Ser Asn Leu Pro Asn Ala Lys Ser Leu Gln Asn Met Thr His Ala Thr Gln Asn Pro Asn Ser Pro Glu Gly Leu Leu Thr Tyr Ser Leu Asp Ser Ser Lys Tyr Asn Gln Leu Gln Thr Ile Ala Gln Glu Leu Gly Lys Asn Pro Phe Arg Arg Phe Gly Val Ile Asp Phe Gln Asn Asn Asn Gly Ala Met Asn Gly Ile Gly Val Gln Va1 Gly Tyr Lys Gln Phe Phe Gly Lys Lys Arg Asn Trp Gly Leu Arg Tyr TAT GGT TTC TTT GAT TAT AAC CAT GCT TAT ATC AAA TCT AAT TTT TTC _ 1804 Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Val Gly Met Asp Ala Leu 540 545 550 _ SUBSTITUTE SHEET (RULE 26) WO 98!43479 135 PCT/US98/06421 Tyr Asn Phe Ile Asn Asp Lys Asn Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Phe Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Gln Val Asn Leu Thr Met Met Asn Gly Ile Tyr Asn Ala Asn Val Ser Thr Ser Asn Phe Gln Phe Leu Phe Asp Leu Gly Leu Arg Met Asn Leu Ala Arg Pro Lys Lys Lys Asp Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Phe Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Lys Leu Glu Tyr Arg Arg Met Tyr Ser Leu Phe Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 691 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 1...18 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Met Lys Lys Ser Leu Leu Leu Ser Leu Ser Leu Ile Ala Ser Leu Ser_ Arg Ala Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly SUBSTITUTE SHEET (RULE 26) Glu Ala Val Gln Gln Val Lys Asn Thr Gly Ala Leu Gln Asn Leu Ala Asp Arg Tyr Asp Asn Leu Asn Asn Leu Leu Asn Gln Tyr Asn Tyr Leu Asn Ser Leu Val Asn Leu Ala Ser Thr Pro Ser Ala Ile Thr Gly Ala Ile Asp Asn Leu Ser Ser Ser Ala Ile Asn Leu Thr Ser Ala Thr Thr Thr Ser Pro Ala Tyr Gln Ala Val Ala Leu Ala Leu Asn Ala Ala Val Gly Met Trp Gln Val Ile Ala Leu Phe Ile Gly Cys Gly Pro Gly Pro Thr Asn Asn Gln Ser Tyr Gln Ser Phe Gly Asn Thr Pro Ala Leu Asn Gly Thr Thr Thr Thr Cys Asn Gln Ala Tyr Gly Thr Gly Pro Asn Gly Ile Leu Ser Ile Asp Glu Tyr Gln Lys Leu Asn Gln Ala Tyr Gln Ile Ile Gln Thr Ala Leu Asn Gln Asn Gln Gly Gly Gly Met Pro Ala Leu Asn Asp Thr Thr Lys Thr Gly Val Val Asn Ile Gln Gln Thr Asn Tyr Arg Thr Thr Thr Gln Asn Asn Ile Ile Glu His Tyr Tyr Thr Glu Asn Gly Lys Glu Ile Pro Val Ser Tyr Ser Gly Gly Ser Ser Phe Ser Pro Thr Ile Gln Leu Thr Tyr His Asn Asn Ala Glu Asn Leu Leu Gln Gln Ala Ala Thr Ile Met Gln Val Leu Ile Thr Gln Lys Pro His Val Gln Thr Ser Asn Gly Gly Lys Ala Trp Gly Leu Ser Ser Thr Pro Gly Asn Val Met Asp Ile Phe Gly Pro Ser Phe Asn Ala Ile Asn Glu Met Ile Lys Asn Ala Gln Thr Ala Leu Ala Lys Thr Gln Gln Leu Asn Ala Asn Glu Asn Ala Gln Ile Thr Gln Pro Asn Asn Phe Asn Pro Tyr Thr Ser Lys Asp Lys Gly Phe Ala Gln Glu Met Leu Asn Arg Ala Glu Ala Gln Ala Glu Ile Leu Asn Leu Ala Lys Gln Val Ala Asn Asn Phe His Ser Ile Gln Gly Pro Ile Gln Gly Asp Leu Glu Glu Cys Lys Ala Gly Ser Ala Gly Val Ile Thr Asn Asn Thr Trp Gly Ser Gly Cys Ala Phe Val Lys Glu Thr Leu Asn Ser Leu Glu Gln His Thr Ala Tyr Tyr Gly Asn Gln Val Asn Gln Asp Arg Ala Leu Ala Gln Thr Ile Leu Asn Phe Lys ' Glu Ala Leu Asn Thr Leu Asn Lys Asp Ser Lys Ala Ile Asn Ser Gly Ile Ser Asn Leu Pro Asn Ala Lys Ser Leu Gln Asn Met Thr His Ala_ Thr Gln Asn Pro Asn Ser Pro Glu Gly Leu Leu Thr Tyr Ser Leu Asp SUBSTITUTE SHEET (RULE 26) Ser Ser Lys Tyr Asn Gln Leu Gln Thr Ile Ala Gln Glu Leu Gly Lys Asn Pro Phe Arg Arg Phe Gly Val Ile Asp Phe Gln Asn Asn Asn Gly Ala Met Asn Gly Ile Gly Val Gln Val Gly Tyr Lys Gln Phe Phe Gly Lys Lys Arg Asn Trp Gly Leu Arg Tyr Tyr Gly Phe Phe Asp Tyr Asn His Ala Tyr Ile Lys Ser Asn Phe Phe Asn Ser Ala Ser Asp Val Trp Thr Tyr Gly Val Gly Met Asp Ala Leu Tyr Asn Phe Ile Asn Asp Lys Asn Thr Asn Phe Leu Gly Lys Asn Asn Lys Leu Ser Val Gly Leu Phe Gly Gly Phe Ala Leu Ala Gly Thr Ser Trp Leu Asn Ser Gln Gln Val Asn Leu Thr Met Met Asn Gly Ile Tyr Asn Ala Asn Val Ser Thr Ser Asn Phe Gln Phe Leu Phe Asp Leu Gly Leu Arg Met Asn Leu Ala Arg Pro Lys Lys Lys Asp Ser Asp His Ala Ala Gln His Gly Ile Glu Leu Gly Phe Lys Ile Pro Thr Ile Asn Thr Asn Tyr Tyr Ser Phe Met Gly Ala Lys Leu Glu Tyr Arg Arg Met Tyr Ser Leu Phe Leu Asn Tyr Val Phe Ala Tyr (2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:

(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:

SUBSTITUTE SHEET (RULE 26) (2} INFORMATION FOR SEQ ID N0:25:

(i) SEQUENCE CHARACTERISTIC S:

(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:

(2) INFORMATION FOR SEQ ID N0:26:

(i) SEQUENCE CHARACTERISTIC S:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:

(2) INFORMATION FOR SEQ ID N0:27:

(i) SEQUENCE CHARACTERISTIC S:

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:

(2) INFORMATION FOR SEQ ID N0:28:

(i) SEQUENCE CHARACTERISTIC S:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:

AACATTAGTA AGCGAACACA TAGTTC _ 26 (2) INFORMATION FOR SEQ ID N0:29:

SUBSTITUTE SHEET (RULE 26) (i) SEQUENCE CHARACTERISTICS:

(Ay LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:

AAGGAGAAAA AACATGAAAA AACACATCC

29.

(2) INFORMATION FOR SEQ ID N0:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:

(2) INFORMATION FOR SEQ ID N0:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:

(2) INFORMATION FOR SEQ ID N0:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:

(2) INFORMATION FOR SEQ ID N0:33:

(i) SEQUENCE CHARACTERISTICS:

SUBSTITUTE SHEET (RULE 26) (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:

(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:

(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:

(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:

(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs SUBSTITUTE SHEET (RULE 26) (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:

(2) INFORMATION FOR SEQ ID N0:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C} STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:

(2) INFORMATION FOR SEQ ID N0:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs {B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:

(2) INFORMATION FOR SEQ ID N0:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

{xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:

(2) INFORMATION FOR SEQ ID N0:41:

(i) SEQUENCE CHARACTERISTICS: _ (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid SUBSTITUTE SHEET (RULE 26) (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:

(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:

(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:

(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:

(2) INFORMATION FOR SEQ ID N0:45:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single SUBSTITUTE SHEET (RULE 26) (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:

(2) INFORMATION FOR SEQ ID N0:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:

(2) INFORMATION FOR SEQ ID N0:47:

' (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:

(2) INFORMATION FOR SEQ ID N0:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:

(2) INFORMATION FOR SEQ ID N0:49: ' - (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid _ (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:

(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:

(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:

(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:

(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
SUBSTITUTE SHEET (RULE 26) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:

(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:

(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:

(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:

(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs __ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
SUBSTITUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID N0:58 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:58 Glu Asp Asp Gly Phe Tyr Thr Ser Val Gly Tyr Gln Ile Gly Glu Ala Ala Gln Met Val (2) INFORMATION FOR 5EQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:

(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUEN E DESCRIPTION: SEQ ID N0:60:

(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SUBSTITUTE SHEET (RULE 25) (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:

(2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STR.ANDEDNESS: single {D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:

(2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:

(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:

' (2) INFORMATION FOR SEQ ID N0:65:
(i) SEQUENCE CHARACTERISTICS:
- (A) LENGTH: 1149 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single _ (D) TOPOLOGY: linear SUBSTCTUTE SHEET (RULE 26) (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 106...1002 (D) OTHER INFORMATION:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 106...166 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:65:

ATGTGAGTTT ATTTGCTATA
TCTGTGTCAT
GATAGCTGAT

AATGATGAAA ATG
ATTAGTTTAG AAA
AAA
AAT

Met Lys Lys Asn IleLeuAsn LeuAla LeuValGly AlaLeuSer ThrSer PheLeuMet AlaLysPro AlaHis AsnAlaAsn AsnAlaThr HisAsn ThrLysLys ThrThrAsp SerSer AlaGlyVal LeuAlaThr ValAsp GlyArgPro IleThrLys SerAsp PheAspMet IleLysGln ArgAsn ProAsnPhe AspPheAsp LysLeu LysGluLys GluLysGlu AlaLeu IleAspGln AlaIleArg ThrAla LeuValGlu AsnGluAla LysThr GluLysLeu AspSerThr ProGlu PheLysAla MetMetGlu AlaVal LysLysGln AlaLeuVal GluPhe TrpAlaLys LysGlnAla GluGlu ValLysLys ValGlnIle ProGlu LysGluMet GlnAspPhe TyrAsn AlaAsnLys AspGlnLeu PheVal LysGlnGlu AlaHisAla ArgHis IleLeuVal LysThrGlu AspGlu AlaLysArg IleIleSer GluIle AspLysGln ProLysAla LysLys GluAlaLys PheIleGlu LeuAla AsnArgAsp SUBSTITUTE SHEET (RULE 26) Thr IleAsp ProAsn SerLysAsn AlaGln AsnGlyGly AspLeuGly Lys PheGln LysAsn GlnMetAla ProAsp PheSerLys AlaAlaPhe Ala LeuThr ProGly AspTyrThr LysThr ProValLys ThrGluPhe Gly TyrHis IleIle TyrLeuIle SerLys AspSerPro ValThrTyr Thr TyrGlu GlnAla LysProThr IleLys GlyMetLeu GlnGluLys Leu PheGln GluArg MetAsnGln ArgIle GluGluLeu ArgLysHis GGTGTTATCA
TGTTAGTTAA
AGGC

Ala LysIle ValIle AsnLys TATTGAAAGC TAATTTCGTG
CCATAAAGAA
GGTTATGGGG

TGCTAAACGC CCC
TATTTTTGAA
GCAGGAAATG

(2) INFORMATION FOR SEQ ID N0:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 299 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (ix) FEATURE:
(A) NAME/KEY: Signal Sequence (B) LOCATION: 1...20 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:66:
Met Lys Lys Asn Ile Leu Asn Leu Ala Leu Val Gly Ala Leu Ser Thr Ser Phe Leu Met Ala Lys Pro Ala His Asn Ala Asn Asn Ala Thr His Asn Thr Lys Lys Thr Thr Asp Ser Ser Ala Gly Val Leu Ala Thr Val Asp Gly Arg Pro Ile Thr Lys Ser Asp Phe Asp Met Ile Lys Gln Arg Asn Pro Asn Phe Asp Phe Asp Lys Leu Lys Glu Lys Glu Lys Glu Ala Leu Ile Asp Gln Ala Ile Arg Thr Ala Leu Val Glu Asn Glu Ala Lys_ Thr Glu Lys Leu Asp Ser Thr Pro Glu Phe Lys Ala Met Met Glu Ala SUBSTITUTE SHEET (RULE 26) Val Lys Lys Gln Ala Leu Val Glu Phe Trp Ala Lys Lys Gln Ala Glu Glu Val Lys Lys Val Gln Ile Pro Glu Lys Glu Met Gln Asp Phe Tyr Asn Ala Asn Lys Asp Gln Leu Phe Val Lys Gln Glu Ala His Ala Arg His IIe Leu Val Lys Thr Glu Asp Glu Ala Lys Arg Ile Ile Ser Glu Ile Asp Lys Gln Pro Lys Ala Lys Lys Glu Ala Lys Phe Ile Glu Leu Ala Asn Arg Asp Thr Ile Asp Pro Asn Ser Lys Asn Ala Gln Asn Gly Gly Asp Leu Gly Lys Phe Gln Lys Asn Gln Met Ala Pro Asp Phe Ser Lys Ala Ala Phe Ala Leu Thr Pro Gly Asp Tyr Thr Lys Thr Pro Val Lys Thr Glu Phe Gly Tyr His Ile Ile Tyr Leu Ile Ser Lys Asp Ser Pro Val Thr Tyr Thr Tyr Glu Gln Ala Lys Pro Thr Ile Lys Gly Met Leu Gln Glu Lys Leu Phe Gln Glu Arg Met Asn Gln Arg Ile Glu Glu Leu Arg Lys His Ala Lys Ile Val Ile Asn Lys (2) INFORMATION FOR SEQ ID N0:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1448 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Genomic DNA
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence (B) LOCATION: 118...1314 (D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:

GCGATAAGAC AGGAATACTT
AAAAATGTCT
TAAATTTTGT

TTAGTATAAT AATACAA
TCTAAAATCC ATG

Met __1 AAA AAG CAT AAT

Ala Glu PheAsnArg ThrLysPro Val IleGly Thr Lys Lys His Asn GGG GTA TTG GCA

Ile His AspHisGly LysThrThr Ser AlaIle Ser Gly Val Leu Ala GTG TCT ATG GAC

Ala Leu LeuLysGly LeuAlaGlu Lys TyrAsp Asn Val Ser Met Asp SUBSTITUTE SHEET (RULE 26) Ile AspAsn AlaProGlu GluLys GluArgGly IleThrIle AlaThr Ser HisIle GluTyrGlu ThrGlu AsnArgHis TyrAlaHis ValAsp Cys ProGly HisAlaAsp TyrVal LysAsnMet IleThrGly AlaAla Gln MetAsp GlyAlaIle LeuVal ValSerAla AlaAspGly ProMet Pro GlnThr ArgGluHis IleLeu LeuSerArg GlnValGly ValPro His IleVal ValPheLeu AsnLys GlnAspMet ValAspAsp GlnGlu Leu LeuGlu LeuValGlu MetGlu VaiArgGlu LeuLeuSer AlaTyr Glu PhePro GlyAspAsp ThrPro IleValAla GlySerAla LeuArg Ala LeuGlu GluAlaLys AlaGly AsnValGly GluTrpGly GluLys Val LeuLys LeuMetAla GluVal AspAlaTyr IleProThr ProGlu Arg AspThr GluLysThr PheLeu MetProVal GluAspVal PheSer Ile AlaGly ArgGlyThr ValVal ThrGlyArg IleGluArg GlyVal Val LysVal GlyAspGlu ValGlu IleValGly IleArgPro ThrGln Lys ThrThr ValThrGly ValGlu MetPheArg LysGluLeu GluLys Gly GluAla GlyAspAsn ValGly ValLeuLeu ArgGlyThr LysLys Glu GluVal GluArgGly MetVal LeuCysLys ProGlySer IleThr __ Pro HisLys LysPheGlu GlyGlu IleTyrVal LeuSerLys GluGlu Gly GlyArg HisThrPro PhePhe ThrAsnTyr ArgProGln PheTyr 325 330 335 _ Val ArgThr ThrAspVal ThrGly SerIleThr LeuProGlu GlyVal SUBSTITUTE SHEET (RULE 26) Glu Met Val Met Pro Gly Asp Asn Val Lys Ile Thr Val Glu Leu Ile AGC CCT GTT GCG TTA,GAG TTG GGA ACT AAA TTT GCG ATT CGT GAA GGC 1272 Ser Pro Val Ala Leu Glu Leu Gly Thr Lys Phe Ala Ile Arg Glu Gly Gly Arg Thr Val Gly Ala Gly Val Val Ser Asn Ile Ile Glu CAAAAAGAGA GTTACCATAA AGGGTCATTA TGAAAGTTAA AATAGGGTTG AAGTGTTCTG 1383 _ AGCTT

(2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 399 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
Met Ala Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn Ile Gly Thr Ile Gly His Val Asp His Giy Lys Thr Thr Leu Ser Ala Ala Ile Ser Ala Val Leu Ser Leu Lys Gly Leu Ala Glu Met Lys Asp Tyr Asp Asn Ile Asp Asn Ala Pro Glu Glu Lys Glu Arg Gly Ile Thr Ile Ala Thr Ser His Ile Glu Tyr Glu Thr Glu Asn Arg His Tyr Ala His Val Asp Cys Pro Gly His Ala Asp Tyr Val Lys Asn Met Ile Thr Gly Ala Ala Gln Met Asp Gly Ala Ile Leu Val Val Ser Ala Ala Asp Gly Pro Met Pro Gln Thr Arg Glu His Ile Leu Leu Ser Arg Gln Val Gly Val Pro His Ile Val Val Phe Leu Asn Lys Gln Asp Met Val Asp Asp Gln Glu Leu Leu Glu Leu Val Glu Met Glu Val Arg Glu Leu Leu Ser Ala Tyr Glu Phe Pro Gly Asp Asp Thr Pro Ile Val Ala Gly Ser Ala Leu Arg Ala Leu Glu Glu Ala Lys Ala Gly Asn Val Gly Glu Trp Gly Glu Lys Val Leu Lys Leu Met Ala Glu Val Asp Ala Tyr Ile Pro Thr Pro Glu Arg Asp Thr Glu Lys Thr Phe Leu Met Pro Val Glu Asp Val Phe Ser Ile Ala Gly Arg Gly Thr Val Val Thr Gly Arg Ile Glu Arg Gly SUBSTITUTE SHEET (RULE 26) Val Val Lys Val Gly Asp Glu Val Glu Ile Val Gly Ile Arg Pro Thr Gln Lys Thr Thr Val Thr Gly Val Glu Met Phe Arg Lys Glu Leu Glu Lys Gly Glu Ala Gly Asp Asn Val Gly Val Leu Leu Arg Gly Thr Lys Lys Glu Glu Val Glu Arg Gly Met Val Leu Cys Lys Pro Gly Ser Ile Thr Pro His Lys Lys Phe Glu Gly Glu Ile Tyr Val Leu Ser Lys Glu Glu Gly Gly Arg His Thr Pro Phe Phe Thr Asn Tyr Arg Pro Gln Phe Tyr Val Arg Thr Thr Asp Val Thr Gly Ser Ile Thr Leu Pro Glu Gly Val Glu Met Val Met Pro Gly Asp Asn Val Lys Ile Thr Val Glu Leu Ile Ser Pro Val Ala Leu Glu Leu Gly Thr Lys Phe Ala Ile Arg Glu Gly Gly Arg Thr Val Gly Ala Gly Val Val Ser Asn Ile Ile Glu (2) INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:

(2) INFORMATION FOR SEQ ID N0:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:

(2) INFORMATION FOR SEQ ID N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid ~ (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
SUBSTITUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID N0:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single _ (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:

(2) INFORMATION FOR SEQ ID N0:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:73:
Met Lys Glu Lys Phe Asn Arg Thr Lys Pro His Val Asn Ile Gly Thr Ile Gly His Val Asp His (2) INFORMATION FOR SEQ ID N0:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:74:
Ala His Asn Ala Asn Asn Ala Thr His Asn Thr Lys Lys (2) INFORMATION FOR SEQ ID N0:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide SUBSTITUTE SHEET (RULE 26) (xi) SEQUENCE DESCRIPTION: SEQ ID N0:75:
Lys Pro Ala His Asn Ala (2) INFORMATION FOR SEQ ID N0:76:
_ (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:76:
Ile Asp Lys Gln Pro Lys Ala Lys Lys (2) INFORMATION FOR SEQ ID N0:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:77:
Phe Trp Ala Lys Lys Gln Ala Glu (2) INFORMATION FOR SEQ ID N0:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:78:

(2) INFORMATION FOR SEQ ID N0:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:79:
SUBSTITUTE SHEET (RULE 26) (2) INFORMATION FOR SEQ ID N0:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:80:

(2) INFORMATION FOR SEQ ID N0:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:81:

(2) INFORMATION FOR SEQ ID N0:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:82:

(2) INFORMATION FOR SEQ ID N0:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:83:

(2) INFORMATION FOR SEQ ID N0:84: _ (i) SEQUENCE CHARACTERISTICS:
SUBSTITUTE SHEET (RULE 26) (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:84:

SUBSTITUTE SHEET (RULE 2B)

Claims (35)

What is claimed is:

1. An isolated polynucleotide that encodes:
(i) 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 -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);
-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 the positions from -20 to 5, preferably in position -20 or position 1, and ending with an amino acid in position 279 (GHPO 1360); and 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.

2. An isolated polynucleotide that encodes:
(i) 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);

-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, beginning with an amino acid in position -18 and ending with an amino acid in position 673 (GHPO 1414);
-in SEQ ID NO:66, beginning with an amino acid in position -20 and ending with an amino acid in position 279 (GHPO 1360); and - 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.

3. The isolated polynucleotide of claim 1, which encodes the mature form of:
(i) 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 -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 -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 - 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.

4. The isolated polynucleotide of claim 1, 2, or 3, wherein the polynucleotide is a DNA molecule.

5. The isolated polynucleotide of claim 1, which is a DNA molecule that can be amplified and/or cloned by polymerase chain reaction from-an Helicobacter 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
NO:25 (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
NO:35, 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);
- 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);

- 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
N0:52 (unprocessed GHPO 1417);
- a 5' oligonucleotide primer having a sequence as shown in SEQ ID
N0:53, and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO:55 (unprocessed GHPO 1414);
- a 5' oligonucleotide primer comprising a sequence as shown in SEQ ID
N0:78 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
NO:25 (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
NO: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
NO:40 (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
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);
- 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);
- a 5' oligonucleotide primer comprising a sequence as shown in SEQ ID
NO:80 and a 3' oligonucleotide primer having a sequence as shown in SEQ ID
NO:81 (GHPO 750); or - a 5' oligonucleotide primer comprising 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).

6. The isolated DNA molecule of claim 5, which can be amplified and/or cloned by the polymerase chain reaction from a Helicobacter pylori genome.

7. The isolated polynucleotide of 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 5.

8. The isolated polynucleotide of 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.

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 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);
-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 -1 g 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, beginning with an amino acid in position -18 and ending with an amino acid in position 673 (GHPO 1414);
- in SEQ ID NO:66, beginning with an amino acid in position -20 and ending with an amino acid in position 279 (GHPO 1360); and - 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 said polypeptide.

10. The compound of claim 9, which is the mature form or a derivative of a polypeptide encoded by a DNA molecule of claim 5.

11. The compound of 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 claim 9, 10, or 11 and a pharmaceutically acceptable diluent or carrier.

13. The composition of claim 12, further comprising an antibiotic, an antisecretory agent, a bismuth salt, or a combination thereof.

14. The composition of claim 13, wherein said antibiotic is selected from the group consisting of amoxicillin, clarithromycin, tetracycline, metronidizole, and erythromycin.

15. The composition of claim 13, wherein said bismuth salt is selected from the group consisting of bismuth subcitrate and bismuth subsalicylate.

16. The composition of claim 13, wherein said antisecretory agent is a proton pump inhibitor.

I7. The composition of claim 16, wherein said proton pump inhibitor is selected from the group consisting of omeprazole, lansoprazole, and pantoprazole.

18. The composition of claim 13, wherein said antisecretory agent is an H2-receptor antagonist.

19. The composition of claim 18, wherein said H2-receptor antagonist is selected from the group consisting of ranitidine, cimetidine, famotidine, nizatidine, and roxatidine.

20. The composition of claim 13, wherein said antisecretory agent is a prostaglandin analog.

21. The composition of claim 20, wherein said prostaglandin analog is misoprostil or enprostil.

22. The composition of claim 12, which further comprises a prophylactically or therapeutically effective amount of a second Helicobacter polypeptide or a derivative thereof.

23. The composition of claim 22, wherein the second Helicobacter polypeptide is a Helicobacter urease, a subunit, or a derivative thereof.

24. The composition of claim 12, further comprising an adjuvant.

25. A pharmaceutical composition for preventing or treating Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of a polynucleotide of claim 1, 2, or 3 and a pharmaceutically acceptable carrier or diluent.

26. A pharmaceutical composition for preventing or treating Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of a polynucleotide of claim 5, 6, or 7 and a pharmaceutically acceptable carrier or diluent.

27. A pharmaceutical composition for preventing or treating Helicobacter infection in a mammal, said composition comprising a prophylactically or therapeutically effective amount of a polynucleotide of claim 8 and a pharmaceutically acceptable carrier or diluent.

28. A composition comprising a viral vector, in the genome of which is inserted a DNA molecule of 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.

29. The composition of claim 28, wherein said viral vector is a poxvirus.

30. 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 bacterial vector being admixed with a physiologically acceptable diluent or carrier.

31. The composition of claim 30, wherein said vector is selected from the group consisting of Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille bilié de Calmette-Guerin, and Streptococcus.

32. The composition of claim 25, wherein said polynucleotide is a DNA
molecule that is inserted in a plasmid that is unable to replicate and to substantially integrate in a mammalian genome and is placed under conditions for expression in a mammalian cell.

33. 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.

34. A process for producing a compound of claim 9, 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.

35. 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 claim 9, 10, or 11 and a pharmaceutically acceptable carrier or diluent.