This is a Rule 62 continuation of application Ser. No. 08/239,910, filed May 9, 1994, now abandoned; which is a Rule 60 continuation of application Ser. No. 07/952,737, filed as PCT/GB91/00484, Mar. 28, 1991, now abandoned.

The present invention relates to attenuated microorganisms, to methods for their production, to their use in the immunoprophylaxis of an animal or a human, and to vaccines containing them.

The principle behind vaccination or immunoprophylaxis is to induce an immune response in an animal to a pathogenic organism by innoculation with an attenuated strain of the organism thus providing protection against subsequent challenge. In 1950 Bacon et al (Br.J.Exp.Path. 31, 714-724) demonstrated that certain auxotrophic mutants of S.typhi were attenuated in mice when compared to the parental strain. Certain of these auxotrophic mutants have been proposed as being suitable candidates for the basis of a whole cell vaccine. (See for example Hosieth and Stocker, Nature, 1981 241, 238-239, and European patent publication 322,237). In addition to mutations in an essential auxotrophic pathway, other loci have been identified where mutations result in attenuation of microorganisms. Examples of such loci include regulons that exert pleiotrophic effects, e.g., the cya/crp system (Roy Curtiss III et al, Vaccine 6, 155-160, 1988) and the ompR envZ system (Dorman et al, Infect.Immun. 57, 2136-2140, 1989) and the phoP system (Fields et al, Science 243, 1059-1062, 1989).

In many microorganisms, between one and two dozen proteins are produced in response to a range of different environmental stresses, such as high temperature, nutrient deprivation, toxic oxygen radicals and metabolic disruption. These represent part of the coordinated regulation of various different genes induced in response to the particular stress to which the microorganism is subjected. The family of major stress proteins (also known as heat shock proteins) is amongst the most highly conserved in nature. Substantial homology exists amongst members of this family isolated from E.coli, Drosophilia spp. and man (for a recent review see Neidhardt, G. C. & Van Bogelen, R. A. (1987) Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology. F. C. Neidhardt et al. eds. pp. 1334-1345. American Society for Microbiology, Washington D.C.). For example: Hsp90, Hsp70 and Hsp60 are heat shock proteins found in all prokaryotes and eukaryotes. Amino acid sequence comparison between Hsp90 from E.coli and that from man shows that approximately half the amino acid residues are identical. Other members of the stress protein family are GrpE, GroEL, DnaK, GroES, Lon and DnaJ.

The genes encoding the family of heat shock proteins are transcribed by RNA polymerase co-operating with the σ³² factor, the product of the rpoH gene (reviewed by Neidhardt, F. C. and van Bogelen, R. A, 1987. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Neidhardt, F. C. et al eds. pp. 1334-1345, American Society for Microbiology, Washington, D.C.). Recently, Lipinska et al (Nucleic.Acids.Res. 1988 21, 10053-10067) have described a heat shock protein in E.coli, referred to as HtrA, that appears to be σ³² -independent. Examination of the promoter region of the htrA gene shows DNA sequence homology with the P.3 promoter of the rpoH gene; a promoter known to be recognised by σ^E (σ²⁴) factor. This similarity suggests that the htrA promoter may also be recognised by the RNA polymerase-σ^E (σ²⁴)holoenzyme.

Phenotypically, in E.coli, a mutation in the htrA locus abolishes the ability of bacterium to survive at temperatures above 42° C. (Lipinska et al, 1989, J.Bacteriol, 171, 1574-1584). The gene maps at 4 min on the E.coli chromosome and encodes a protein with a relative molecular mass (Mr) of 51,163. This protein precursor undergoes N-terminal processing involving the removal of a signal peptide sequence (Lipinska et al, 1988, Nucleic.Acids.Res. 21, 10053-10067), to yield the mature form of the polypeptide upon secretion through the inner membrane of the bacterium. Independently, the htrA gene has been identified as degP by Strauch, K. L. and Beckwith, J. 1988 (Proc.Natl.Acad.Sci. USA 85, 1576-1580) who were examining E.coli mutants with decreased protease activity, degP mutants were isolated by TnphoA mutagenesis (Manoil, C. & Beckwith, J. 1985, Proc.Natl.Acad.Sci. USA 82, 8129-8133) and were recognised by the increased stability of a hybrid Tsr-phoA (Tsr-AP2) recombinant protein in a degP background (Strauch, K. L. and Beckwith, J. 1988. Proc.Natl. Acad.Sci. USA 85, 1576-1680). In E.coli the genes identified as degP and htrA appear to be identical and encode a protein that is a member of the `stress-response` family.

The present invention provides an attenuated microorganism for use in immunoprophylaxis in which the attenuation is brought about by the presence of a mutation in the DNA of the microorganism which encodes, or which regulates the expression of DNA encoding, a protein that is produced in response to environmental stress, the microorganism optionally being capable of expressing DNA encoding a heterologous antigen.

The microorganisms for use with the present invention are preferably bacteria especially Gram-negative bacteria which invade and grow within eucaryotic cells and colonise the muscosal surface. Examples of these include members of the genera Salmonella, Bordetella, Vibrio, Haemophilus and Escherichia. In particular the following species can be mentioned: S.typhi--the cause of human typhoid; S.typhimurium--the cause of salmonellosis in several animal species; S.enteritidis--a cause of food poisoning in humans; S.choleraesuis--the cause of salmonellosis in pigs; Bordetella pertussis--the cause of whooping cough; Haemophilus influenzae--a cause of meningitis; and Neisseria gonorrhoeae--the cause of gonorrhoea.

The mutation of the DNA is a non-reverting mutation, namely one which cannot be repaired in a single step. Genetic mutations of this sort include deletion, inversion, insertion or substitution mutations.

Deletion mutations can be generated using transposons. These are DNA sequences comprising from between 750 to thousands of base pairs which can integrate into the host's chromosomal DNA. The continuity of the DNA sequence of interest is thus disrupted with the loss of gene function. Transposons can be deleted from the host chromosomal DNA; most frequently excision is imprecise leading to a non-reverting mutation. Substitution or insertion mutations can arise by use of an inactivated DNA sequence carried on a vector which recombines with or crosses-over with the DNA sequence of interest in the host's chromosomal DNA with the consequent loss of gene function.

Examples of proteins that are produced in response to environmental stress include heat shock proteins (which are produced in response to a temperature increase above 42° C.); nutrient deprivation proteins (which are produced in response to levels of essential nutrients such as phosphates or nitrogen which are below that which the microorganism requires to survive); toxic stress proteins (which are produced in response to toxic compounds such as dyes, acids or possibly plant exudates); or metabolic disruption proteins (which are produced in response to fluctuations in for example ion levels affecting the microorganisms ability to osmoregulate, or vitamin or co-factor levels such as to disrupt metabolism).

Preferably a heat shock protein is the one encoded by the htrA gene as set out in FIG. 1. (SEQ ID No: 1) (also characterised as degP). Other proteins are encoded by genes known to be involved in the stress response such as grpE, groEL, (moPA), dnaK, groES, lon and dnaJ. There are many other proteins encoded by genes which are known to be induced in response to environmental stress (Ronson et al, Cell 49, 579-581). Amongst these the following can be mentioned: the ntrB/ntrC system of E.coli, which is induced in response to nitrogen deprivation and positively regulates glnA and nifLA (Buck et al., Nature 320, 374-378, 1986; Hirschman et al., Proc.Natl.Acad.Sci. USA, 82, 7525, 1985; Nixon et al., Proc.Natl.Acad.Sci. USA 83, 7850-7854, 1986, Reitzer and Magansanik, Cell, 45, 785, 1986); the phoR/phoB system of E.coli which is induced in response to phosphate deprivation (Makino et al., J.Mol.Biol. 192, 549-556, 1986b); the cpxA/sfrA system of E.coli which is induced in response to dyes and other toxic compounds (Albin et al., J.Biol.Chem. 261 4698, 1986; Drury et al., J.Biol.Chem. 260, 4236-4272, 1985). An analogous system in Rhizobium is dctB/dctD, which is responsive to 4C-discarboxylic acids (Ronson et al., J.Bacteriol. 169, 2424 and Cell 49, 579-581, 1987). A virulence system of this type has been described in Agrobacterium. This is the virA/virG system, which is induced in response to plant exudates (le Roux et al., EMBO J. 6, 849-856, 1987; Stachel and Zambryski., Am.J.Vet.Res. 45, 59-66, 1986; Winans et al., Proc.Natl. Acad.Sci. USA, 83, 8278, 1986). Similarly the bvgC-bvgA system in Bordetella pertussis (previously known as vir) regulates the production of virulence determinants in response to fluctuations in Mg2+ and nicotinic acid levels (Arico et al, 1989, Proc.Natl.Acad.Sci. USA 86, 6671-6675).

For use in the form of a live vaccine, it is clearly important that the attenuated microorganism of the present invention does not revert back to the virulent state. The probability of this happening with a mutation in a single DNA sequence is considered to be small. However, the risk of reversion occurring with a microorganism attenuated by the presence of mutations in each of two discrete DNA sequences, is considered to be insignificant. It is preferred therefore that the attenuation of the microorganism of the present invention is brought about by the presence of a mutation in the DNA sequence which encodes, or which regulates the expression of DNA encoding, a protein that is produced in response to environmental stress and by the presence of a mutation in a second DNA sequence. For bacteria, the second DNA sequence preferably encodes an enzyme involved in an essential auxotrophic pathway or is a sequence whose product controls the regulation of osmotically responsive genes, i.e. ompR, (Infect and Immun 1989 2136-2140). Most preferably, the mutation is in a DNA sequence involved in the aromatic amino acid biosynthetic pathway, more particularly the DNA sequences encoding aroA, aroC or aroD. (EP Publication Number 322237).

The attenuated microorganisms of the present invention are constructed by the introduction of a mutation into the DNA sequence by methods known to those skilled in the art (Maniatis, Molecular Cloning and Laboratory Manual, 1982). Non-reverting mutations can be generated by introducing a hybrid transposon TnphoA into, for example, S.typhimurium strains. TnphoA can generate enzymatically active protein fusions of alkaline phosphatase to periplasmic or membrane proteins. The TnphoA transposon carries a gene encoding kanamycin resistance. Transductants are selected that are kanamycin resistant by growing colonies on an appropriate selection medium.

Alternative methods include cloning the DNA sequence into a vector, eg. a plasmid or cosmid, inserting a selectable marker gene into the cloned DNA sequence, resulting in its inactivation. A plasmid carrying the inactivated DNA sequence and a different selectable marker can be introduced into the organism by known techniques (Maniatis, Molecular Cloning and Laboratory Manual, 1982). It is then possible by suitable selection to identify a mutant wherein the inactivated DNA sequence has recombined into the chromosome of the microorganism and the wild-type DNA sequence has been rendered non-functional in a process known as allelic exchange. In particular, the vector used is preferably unstable in the microorganism and will be spontaneously lost. The mutated DNA sequence on the plasmid and the wild-type DNA sequence may be exchanged by a genetic cross-over event. Additional methods eliminate the introduction of foreign DNA into vaccine strains at the site of mutations.

The invention therefore provides a process for the production of an attenuated microorganism according to the invention which comprises introduction of a mutation in the DNA sequence of the microorganism which encodes, or which regulates expression of a DNA sequence encoding, a protein that is produced in response to environmental stress, by either

a) transposon mutagenesis; or

b) transforming the microorganism with a vector incorporating a DNA sequence encoding, or regulating the expression of a DNA sequence encoding, a protein that is produced in response to environmental stress and which contains a non-reverting mutation; and screening to select the desired microorganisms.

The attenuated microorganism of the present invention is optionally capable of expressing a heterologous antigen. This expression is likely to be more favourable in htrA mutants because of the increased stability of recombinant antigens associated with the degP phenotype. Such antigens may be viral, bacterial, protozoal or of higher parasitic microorganisms. Such microorganisms may then form the basis of a bi- or multi-valent vaccine. Examples of useful antigens include E.coli heat labile toxin B subunit (LT-B), E.coli K88 antigens, FMDV (Foot and Mouth) peptides, Influenza viral proteins, P.69 protein from B.pertussis. Other antigens which could be usefully expressed would be those from Chlamydia, flukes, mycoplasma, roundworms, tapeworms, rabies virus and rotavirus.

A microorganism capable of expressing DNA encoding a heterologous antigen may be produced by transformation of the microorganism with an expression cassette. Expression cassettes will include DNA sequences, in addition to that coding for the heterologous antigen, which will encode transcriptional and translational initiation and termination sequences. The expression cassette may also include regulatory sequences. Such expression cassettes are well known in the art and it is well within the ability of the skilled man to construct them. The expression cassette may form part of a vector construct or a naturally occurring plasmid. An example of a genetically engineered attenuated Salmonella which is capable of expressing a heterologous antigen is described in EP publication 127,153. The expression cassette may also be engineered to allow the incorporation of the heterologous gene into the chromosome of the microorganism.

A further bivalent vaccine comprising an attenuated Salmonella typhi, capable of expressing the E.coli heat-labile enterotoxin subunit B is disclosed by Clements et al (Infection ad Immunity, 46, No.2. 1984, 564-569). Ty21a, an attenuated S.typhi strain, has been used to express other antigens such as the Shigella sonnei form I antigen (Formal et al., Infection and Immunity, 34, 746-750, 1981).

According to a further aspect of the invention there is provided a vaccine which comprises an effective amount of an attenuated microorganism, preferably a bacterium, as herein described and a pharmaceutically acceptable carrier.

The vaccine is advantageously presented in a lyophilised form, for example in a capsular form, for oral administration to a patient. Such capsules may be provided with an enteric coating comprising for example Eudragate "S" Eudragate "L" Cellulose acetate, cellulose pthalate or hydroxy propylmethyl cellulose. These capsules may be used as such, or alternatively, the lyophilised material may be reconstituted prior to administration, eg. as a suspension. Reconstitution is advantageously effected in a buffer at a suitable pH to ensure the viability of the organisms. In order to protect the attenuated bacteria and the vaccine from gastric acidity, a sodium bicarbonate preparation is advantageously administered before each administration of the vaccine. Alternatively, the vaccine may be prepared for parenteral administration, intranasal administration or intramammary.

The present invention also provides a method of prophylactic treatment of a host (particularly a human host) with an infection caused by a microorganism which comprises administering to said host an effective dose of a vaccine according to the invention. The dosage employed in such a method of treatment will be dependent on various clinical factors, including the size and weight of the host, the type of vaccine formulated. However, for attenuated S.typhi a dosage comprising the administration of 10⁹ to 10¹¹ S.typhi organisms per dose is generally convenient for a 70 kg adult human host.

The following examples provide experimental details in accordance with the present invention. It will be understood that these examples are not intended to limit the invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B. DNA sequence of the htrA gene and the amino acid sequence of the protein it encodes (SEQ ID NO:1 corresponds to the DNA sequence of FIG. 1, SEQ ID NO:2 corresponds to the lower of the two amino acid sequences in FIG. 1 and SEQ ID NO:3 corresponds to the upper of the amino acid sequences in FIG. 1).

FIG. 2. Sensitivity of S.typhimurium hrtA mutant 046 to temperatures above 42° C. and oxygen radicals

FIG. 3. In vivo kinetics of S.typhimurium strains harbouring a mutation in htrA (BRD726) and htrA aro mutations (BRD807).

EXAMPLE 1

Identification of the htrA gene in Salmonella typhimurium and generation of an htrA mutant.

TnphoA mutagenesis was used in the mouse virulent Salmonella typhimurium strain C5 (Miller et al, 1989, Infect.Immunol, 57, 2758-2763). Mutants were selected likely to harbour lesions in genes that have a signal peptide sequence, i.e. proteins likely to be targeted through a bacterial membrane. Isolation of the DNA flanking the TnphoA insertion identifies the gene that has been insertionally activated. This gene was isolated and its DNA sequence was determined by standard methods (see FIG. 1. SEQ ID No: 1) (Maniatis et al., 1982, In Molecular Cloning: A laboratory manual. Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y.; Sanger et al., 1977, Proc.Natl.Acad.Sci. USA 74, 5463-5467). Comparison of the translated protein sequence with sequences held in the EMBL Database showed surprisingly that it shared 88% homology with the sequence of the htrA product from E.coli (FIG. 1. SEQ ID No: 1).

EXAMPLE 2

Identification of htrA in S.typhimurium as a gene involved in the stress-response

E.Coli mutants harbouring lesions in the htrA gene are unable to grow at temperatures above 42° C. The S.typhimurium htrA mutant, 046, was tested for growth at elevated temperatures and was found to grow as well as the parent strain C5. However, when tested for sensitivity to oxygen radicals, the mutant 046 showed decreased resistance as compared with the parent C5 strain clearly indicating that the gene is responsible (at least in part) for this aspect of the stress response (see FIG. 2).

EXAMPLE 3

Comparison of attenuated Salmonella typhimurium strain 046 with virulent parent strain Salmonella typhimurium C5.

The attenuated strains were constructed using TnphoA transposon mutagenesis as described previously (Miller et al., 1989, Infect. Immun. 57, 2758-2763).

After oral administration the mutant strain 046 had a Log₁₀ LD₅₀ of greater than 9 cells as compared to the parental strain, C5, which has a Log₁₀ LD₅₀ of 6.38 cells. (All LD₅₀ were calculated after 28 days). Thus 046 is highly attenuated. After i.v. administration 046 had an i.v. Log₁₀ LD₅₀ of 5.13 cells compared to less than 10 cells for C5 and we again conclude that 046 is highly attenuated compared to C5.

EXAMPLE 4

Protection of mice after oral challenge.

Mice were immunised with 046 and challenged 28 days later with the virulent parental strain C5. Mice vaccinated with using 10¹⁰ cells of 046 showed excellent protection against challenge with C5. eleven weeks after vaccination. The Log₁₀ LD₅₀ in immunised animals was 9.64 cells compared with 6.6 cells for unimmunised controls. Thus, mice vaccinated orally with a single dose of 046 were well protected against virulent C5 challenge.

EXAMPLE 5

Construction of a defined S.typhimurium SL1344 htrA mutant

Sequence data facilitated the identification of suitable restriction endonuclease sites that could be used to introduce a deletion into the htrA gene. A 1.2 Kb deletion was introduced by digesting with EcoRV and religating. A drug resistant marker was also introduced into the gene (Kanamycin cassette, Pharmacia) by standard techniques to enable selection for the presence of the deleted gene. The plasmid harbouring the deleted htrA gene was introduced into a polA strain S.typhimurium (BRD207) in which the plasmid cannot replicate. The only way that kanamycin resistance can be maintained in the host is if there has been a recombination event between the S.typhimurium sequences on the vector and the homologous regions on the chromosome. Loss of ampicillin resistance while maintaining kanamycin resistance indicates a second homologous recombination event resulting in the replacement of the intact htrA gene with the deleted one. Colonies resistant to kanamycin were isolated and checked for ampicillin resistance. One colony that was kanamycin resistant and ampicillin sensitive was selected for further study and was designated BRD698 (deposited at PHLS, NCTC, 61 Colindale Avenue, London NW9 5HT under Accession No. NCTC 12457 on Mar. 22, 1991 in accordance with the terms of the Budapest Treaty).

A P22 lysate was prepared on this strain by standard techniques (Dougan et al, J.Infect.Dis. 158, 1329-1335, 1988) and used to infect SL1344. Kanamycin resistant colonies were isolated and checked for the presence of the deletion by Southern hybridisation. One strain, designated BRD726 (deposited at PHLS under Accession No. NCTC 12458 on Mar. 22, 1991 in accordance with the terms of the Budapest Treaty) was selected for further study.

EXAMPLE 6

Construction of an S.typhimurium SL1344 aroA htrA double mutant

The P22 lysate prepared on BRD698 was used to introduce the htrA deletion into an S.typhimurium SL1344 strain already harbouring a deletion in aroA. The method for introducing an aroA deletion has already been described by Dougan et al, J.Infect.Dis. 158, 1329-1335, 1988. One strain that was found to have deletions in both aroA and htrA was selected for further study and was designated BRD807, (deposited at PHLS under Accession No. NCTC 12459 on Mar. 22, 1991 in accordance with the terms of the Budapest Treaty).

EXAMPLE 7

Comparison of the attenuation of SL1344 htrA (BRD726) and SL1344 htrA and aroA (BRD807) with the virulent parent strain SL1344

After oral administration BRD726 and BRD807 had Log₁₀ LD₅₀ s of >10.0 cells compared to the virulent parent strain which has a Log₁₀ LD₅₀ of 6.8 cells*. Both strains were therefore highly attenuated compared to the virulent parent strain SL1344.

EXAMPLE 8

Assessment of oral vaccine potential of BRD726 and BRD807

BALB/c mice were orally immunised with approximately 10¹⁰ cells of BRD726 and BRD807 as previously described (Dougan et al, J.Infect.Dis. 158, 1329-1335, 1988) and challenged 4 and 10 weeks later with the virulent parent strain SL1344. LD₅₀ s were calculated by the method of Reed and Muench (Am.J.Hyg. 27, 493-497, 1934). All determinations were carried out at least twice. Mice vaccinated with BRD726 and BRD807 showed excellent protection against challenge with SL1344 at 4 weeks, the log₁₀ LD₅₀ s being >10.0 and 9.7 cells respectively. This compares with log 6.1 cells for unimmunised controls. At 10 weeks log₁₀ LD₅₀ s for BRD726 and BRD807 were 9.11 and 8.11 cells compared to 6.5 for SL1344. Thus the mice immunised with BRD726 had excellent long term immunity to virulent SL1344 challenge. This compares favourably with protection elicited by double aro mutants of SL1344 (Dougan et al, J.Infect.Dis. 158, 1329-1335, 1988). The long term protection afforded by vaccination with BRD807 is 46-fold better than unimmunised controls. Thus both BRD726 and BRD807 make good vaccine strains for BALB/c mice.

EXAMPLE 9

In vivo kinetics of BRD726 and BRD807 in BALB/c mice

The ability of BRD726 and BRD807 to grow in vivo after intravenous administration was assessed. Mice were infected with approximately 10⁵ organisms. Numbers of bacteria in livers and spleens were enumerated at different times during the infection up to 21 days. The results obtained are shown in FIG. 3. Neither BRD726 or BRD807 underwent an initial period of replication in murine tissues. The strains are cleared slowly from the organs and by day 21 BRD807 has almost cleared from the murine tissues while BRD726 is still persisting at low levels.

EXAMPLE 10

Formulation

An attenuated microorganism of the present invention is preferably presented in an oral tablet form.

______________________________________INGREDIENT              MC/TABLET______________________________________Core tablets1. Freeze-dried excipient carrier containing                   70.0  109 1010 attenuated bacteria.2. Silicon dioxide (AEROSIL 200)                   0.53. DIPAC (97% sucrose)  235.04. Cross-linked Cl      7.05. Microcrystalline Cellulose                   35.0  AVICEL PH1026. Magnesium Stearate   2.5Coating7. Opadry Enteric, OY-P-7156                   35.0  (Polyvinyl acetate phthalate +  Diethylphthate)                   385.0______________________________________

A carrier containing 5% sucrose, 1% sodium glutamate and 1% bacto casitone in an aqueous solvent is prepared. The organisms are suspended in this carrier and then subjected to freeze-drying.

The freeze-dried material is blended with Aerosil 200 and the blended mixture is sifted through a screen. The sifted powder is mixed with DIPAC (97% sucrose), KOLLIDON CL (poly (vinylpyrrolidone)), AVICEL PH102 (microcrystalline cellulose) and magnesium stearate in a blender. This blend is compressed into tablets for subsequent enteric coatings.

The skilled man will appreciate that many of the ingredients in this formulation could be replaced by functionally equivalent pharmaceutically acceptable excipients.

__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 3(2) INFORMATION FOR SEQ ID NO: 1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1980 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 395..1822(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:AAGCTTGTCGCTTAACGACTTTCGCGAGCTGGTGGAAAAAGAACGGTTGAAACGCTTCCC60CATAGAATCGCGCTTATTTCAGAAACTTTCTACGCGCCATCGTTTGGCCTACGTGGAAGT120CGTCAGTAAATTACCCACGGATTCGGCGGAGTACCCGGTACTGGAATATTATTATCGCTG180TCGGTTGATTCAGGATTATATCAGCGGGATGACTGACCTTTACGCATGGGATGAATATCG240GCGTTTGATGGCGGTCGAACAGTAAATGGACTTTTGTAAAGATGGACAATAAATTTTTAC300TTTTTCCAGAAACTTTATTCCGGAACTTCGCGTTATAAAATGAATCTGACGTACACAGCA360ATTTTGCGTTACCTGTTAATCGAGATTGAAACACATGAAAAAAACCACATTA412MetLysLysThrThrLeu15GCAATGAGTGCACTGGCTCTGAGTTTAGGTTTGGCATTGTCGCCTCTG460AlaMetSerAlaLeuAlaLeuSerLeuGlyLeuAlaLeuSerProLeu101520TCTGCCACGGCGGCTGAAACGTCCTCTTCAGCAATGACTGCCCAGCAG508SerAlaThrAlaAlaGluThrSerSerSerAlaMetThrAlaGlnGln253035ATGCCAAGCCTGGCACCGATGCTCGAAAAAGTGATGCCATCGGTGGTC556MetProSerLeuAlaProMetLeuGluLysValMetProSerValVal404550AGTATTAATGTTGAAGGTAGCACCACGGTGAATACGCCGCGTATGCCG604SerIleAsnValGluGlySerThrThrValAsnThrProArgMetPro55606570CGTAATTTCCAGCAGTTCTTTGGCGATGACTCCCCGTTCTGCCAGGAC652ArgAsnPheGlnGlnPhePheGlyAspAspSerProPheCysGlnAsp758085GGTTCTCCGTTCCAGAATTCTCCGTTCTGCCAGGGCGGCGGTAACGGC700GlySerProPheGlnAsnSerProPheCysGlnGlyGlyGlyAsnGly9095100GGCAACGGCGGTCAACAACAGAAATTCATGGCGCTGGGCTCCGGCGTA748GlyAsnGlyGlyGlnGlnGlnLysPheMetAlaLeuGlySerGlyVal105110115ATTATTGACGCCGCGAAGGGCTACGTCGTCACCAACAACCACGTGGTT796IleIleAspAlaAlaLysGlyTyrValValThrAsnAsnHisValVal120125130GATAACGCCAGCGTGATTAAAGTACAGCTTAGCGATGGGCGTAAATTC844AspAsnAlaSerValIleLysValGlnLeuSerAspGlyArgLysPhe135140145150GATGCTAAAGTGGTGGGCAAAGATCCGCGTTCTGATATCGCGCTGATT892AspAlaLysValValGlyLysAspProArgSerAspIleAlaLeuIle155160165CAAATTCAGAATCCGAAGAACCTGACGGCGATTAAGCTGGCGGACTCC940GlnIleGlnAsnProLysAsnLeuThrAlaIleLysLeuAlaAspSer170175180GACGCGCTGCGCGTGGGGGATTATACCGTCGCTATTGGTAACCCGTTT988AspAlaLeuArgValGlyAspTyrThrValAlaIleGlyAsnProPhe185190195GGTCTGGGCGAAACGGTGACGTCAGGTATCGTTTCGGCGCTGGGGCGT1036GlyLeuGlyGluThrValThrSerGlyIleValSerAlaLeuGlyArg200205210AGCGGCCTGAACGTAGAAAATTACGAGAACTTTATTCAGACCGACGCC1084SerGlyLeuAsnValGluAsnTyrGluAsnPheIleGlnThrAspAla215220225230GCGATTAACCGTGGTAACTCCGGCGGCGCGCTGGTGAACCTGAACGGT1132AlaIleAsnArgGlyAsnSerGlyGlyAlaLeuValAsnLeuAsnGly235240245GAGCTGATCGGTATTAACACCGCGATTCTGGCGCCGGACGGCGGCAAC1180GluLeuIleGlyIleAsnThrAlaIleLeuAlaProAspGlyGlyAsn250255260ATCGGTATCGGCTTCGCTATCCCCAGTAACATGGTGAAAAACCTGACG1228IleGlyIleGlyPheAlaIleProSerAsnMetValLysAsnLeuThr265270275TCGCAGATGGTGGAATACGGCCAGGTGAAACGCGGCGAACTGGGGATC1276SerGlnMetValGluTyrGlyGlnValLysArgGlyGluLeuGlyIle280285290ATGGGGACTGAGCTGAATTCCGAATTGGCGAAAGCGATGAAAGTCGAC1324MetGlyThrGluLeuAsnSerGluLeuAlaLysAlaMetLysValAsp295300305310GCCCAGCGAGGCGCGTTCGTCAGCCAGGTGATGCCGAATTCGTCCGCG1372AlaGlnArgGlyAlaPheValSerGlnValMetProAsnSerSerAla315320325GCGAAAGCGGGTATCAAAGCCGGGGATGTCATTACCTCGCTGAACGGT1420AlaLysAlaGlyIleLysAlaGlyAspValIleThrSerLeuAsnGly330335340AAACCGATCAGCAGCTTTGCGGCGCTGCGCGCTCAGGTCGGCACTATG1468LysProIleSerSerPheAlaAlaLeuArgAlaGlnValGlyThrMet345350355CCGGTCGGCAGCAAAATCAGCCTCGGTCTGCTGCGTGAAGGTAAAGCG1516ProValGlySerLysIleSerLeuGlyLeuLeuArgGluGlyLysAla360365370ATTACGGTGAATCTGGAACTGCAGCAGAGCAGCCAGAGTCAGGTTGAT1564IleThrValAsnLeuGluLeuGlnGlnSerSerGlnSerGlnValAsp375380385390TCCAGCACCATCTTCAGCGGGATTGAAGGCGCTGAAATGAGCAATAAA1612SerSerThrIlePheSerGlyIleGluGlyAlaGluMetSerAsnLys395400405GGCCAGGATAAAGGCGTTGTGGTGAGCAGCGTGAAAGCGAACTCACCC1660GlyGlnAspLysGlyValValValSerSerValLysAlaAsnSerPro410415420GCCGCGCAAATTGGCCTCAAAAAAGGCGATGTGATTATCGGCGCTAAC1708AlaAlaGlnIleGlyLeuLysLysGlyAspValIleIleGlyAlaAsn425430435CAGCAGCCGGTGAAAAATATCGCCGAGCTGCGTAAGATTCTCGACAGC1756GlnGlnProValLysAsnIleAlaGluLeuArgLysIleLeuAspSer440445450AAGCCGTCGGTTCTGGCGCTGAATATTCAGCGTGGTGATAGTTCTATT1804LysProSerValLeuAlaLeuAsnIleGlnArgGlyAspSerSerIle455460465470TATTTGCTGATGCAGTAATCACCTTTGTCCCCCTTCCGCCATGGAAGG1852TyrLeuLeuMetGln*475GGGCAACACTTTTCTGTGAAACCCCCCACAACTCCATACTTATTTGCACCGTTTTGTGCA1912TTTGCACAATGTCGAGACCTGTCATCTTCCTTATGCTTGTGCTCTGCTCACAGGAGGGAT1972TTATGGCT1980(2) INFORMATION FOR SEQ ID NO: 2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 475 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:MetLysLysThrThrLeuAlaMetSerAlaLeuAlaLeuSerLeuGly151015LeuAlaLeuSerProLeuSerAlaThrAlaAlaGluThrSerSerSer202530AlaMetThrAlaGlnGlnMetProSerLeuAlaProMetLeuGluLys354045ValMetProSerValValSerIleAsnValGluGlySerThrThrVal505560AsnThrProArgMetProArgAsnPheGlnGlnPhePheGlyAspAsp65707580SerProPheCysGlnAspGlySerProPheGlnAsnSerProPheCys859095GlnGlyGlyGlyAsnGlyGlyAsnGlyGlyGlnGlnGlnLysPheMet100105110AlaLeuGlySerGlyValIleIleAspAlaAlaLysGlyTyrValVal115120125ThrAsnAsnHisValValAspAsnAlaSerValIleLysValGlnLeu130135140SerAspGlyArgLysPheAspAlaLysValValGlyLysAspProArg145150155160SerAspIleAlaLeuIleGlnIleGlnAsnProLysAsnLeuThrAla165170175IleLysLeuAlaAspSerAspAlaLeuArgValGlyAspTyrThrVal180185190AlaIleGlyAsnProPheGlyLeuGlyGluThrValThrSerGlyIle195200205ValSerAlaLeuGlyArgSerGlyLeuAsnValGluAsnTyrGluAsn210215220PheIleGlnThrAspAlaAlaIleAsnArgGlyAsnSerGlyGlyAla225230235240LeuValAsnLeuAsnGlyGluLeuIleGlyIleAsnThrAlaIleLeu245250255AlaProAspGlyGlyAsnIleGlyIleGlyPheAlaIleProSerAsn260265270MetValLysAsnLeuThrSerGlnMetValGluTyrGlyGlnValLys275280285ArgGlyGluLeuGlyIleMetGlyThrGluLeuAsnSerGluLeuAla290295300LysAlaMetLysValAspAlaGlnArgGlyAlaPheValSerGlnVal305310315320MetProAsnSerSerAlaAlaLysAlaGlyIleLysAlaGlyAspVal325330335IleThrSerLeuAsnGlyLysProIleSerSerPheAlaAlaLeuArg340345350AlaGlnValGlyThrMetProValGlySerLysIleSerLeuGlyLeu355360365LeuArgGluGlyLysAlaIleThrValAsnLeuGluLeuGlnGlnSer370375380SerGlnSerGlnValAspSerSerThrIlePheSerGlyIleGluGly385390395400AlaGluMetSerAsnLysGlyGlnAspLysGlyValValValSerSer405410415ValLysAlaAsnSerProAlaAlaGlnIleGlyLeuLysLysGlyAsp420425430ValIleIleGlyAlaAsnGlnGlnProValLysAsnIleAlaGluLeu435440445ArgLysIleLeuAspSerLysProSerValLeuAlaLeuAsnIleGln450455460ArgGlyAspSerSerIleTyrLeuLeuMetGln465470475(2) INFORMATION FOR SEQ ID NO: 3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 492 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:MetLysLysThrThrLeuAlaLeuSerArgLeuAlaLeuSerLeuGly151015LeuAlaLeuSerProLeuSerAlaThrAlaAlaGluThrSerSerAla202530ThrMetThrAlaGlnGlnMetProSerLeuAlaProMetLeuGluLys354045ValMetProSerValValSerIleAsnValGluGlySerThrThrVal505560AsnThrProArgMetProArgAsnPheGlnGlnPhePheGlyAspAsp65707580SerProPheCysGlnGluGlySerProPheGlnSerSerProPheCys859095GlnGlyGlyGlnGlyGlyAsnGlyGlyGlyGlnGlnGlnLysPheMet100105110AlaLeuGlySerGlyValIleIleAspAlaAspLysGlyTyrValVal115120125ThrAsnAsnHisValValAspAsnAlaThrValIleLysValGlnLeu130135140SerAspGlyArgLysPheAspAlaLysMetValGlyLysAspProArg145150155160SerAspIleAlaLeuIleGlnIleGlnAsnProLysAsnLeuThrAla165170175IleLysMetAlaAspSerAspAlaLeuArgValGlyAspTyrThrVal180185190GlyIleGlyAsnProPheGlyLeuGlyGluThrValThrSerGlyIle195200205ValSerAlaLeuGlyArgSerGlyLeuAsnAlaGluAsnTyrGluAsn210215220PheIleGlnThrAspAlaAlaIleAsnArgGlyAsnSerGlyGlyAla225230235240LeuValAsnLeuAsnGlyGluLeuIleGlyIleAsnThrAlaIleLeu245250255AlaProAspGlyGlyAsnIleGlyIleGlyPheAlaIleProSerAsn260265270MetValLysAsnLeuThrSerGlnMetValGluTyrGlyGlnValLys275280285ArgGlyGluLeuGlyIleMetGlyThrGluLeuAsnSerGluLeuAla290295300LysAlaMetLysValAspAlaGlnArgGlyAlaPheValSerGlnVal305310315320LeuProAsnSerSerAlaAlaLysAlaGlyIleLysAlaGlyAspVal325330335IleThrSerLeuAsnGlyLysProIleSerSerPheAlaAlaLeuArg340345350AlaGlnValGlyThrMetProValGlySerLysLeuThrLeuGlyLeu355360365LeuArgAspGlyLysAsnValAsnValAsnLeuGluLeuGlnGlnSer370375380SerGlnAsnGlnValAspSerSerSerIlePheAsnGlyIleGluGly385390395400AlaGluMetSerAsnLysGlyLysAspAsnGlyValValValAsnAsn405410415ValLysThrGlyThrProAlaAlaGlnIleGlyLeuLysLysGlyAsp420425430ValIleIleGlyAlaAsnGlnGlnAlaValLysAsnIleAlaGluLeu435440445ArgLysValLeuAspSerLysProSerValLeuAlaLeuAsnIleGln450455460ArgGlyAspArgHisLeuProValAsnAlaValIleSerLeuAsnPro465470475480PheLeuLysThrGlyArgGlySerProTyrAsnLeu485490__________________________________________________________________________