AU2521600A - Treatment for verotoxin-producing escherichia coli - Google Patents

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ORIGINAL

Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Ophidian Pharmaceuticals, Inc.

5445 East Cheryl Parkway Madison Wisconsin 53711 United States of America Sean B. Carroll, Douglas C. Stafford and Nisha V.

Padhye Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Treatment for Verotoxin-producing Escherichia coli The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c TREATMENT FOR VEROTOXIN-PRODUCING ESCHERICHIA COLI FIELD OF THE INVENTION The present invention relates to antitoxin therapy for humans and other animals, and diagnostic assays to detecttoxins. Antitoxins which neutralize the pathologic effects of Escherichia coli toxins, such as verotoxin are provided.

n A rKGROUND OF THE INVENTION A. Escherichia coli as a Pathogenic Organism Escherichia coli is the organism most commonly isolated in clinical microbiology laboratories, as it is usually present as normal .flora. in the intestines of humans and other animals. However. it is an important cause of intestinal, as well as extraintestinal infections.

For example. in a 1984 su: y of nosocomial infections in the United States. E. coli was associated with 30.7% of the urinary tract infections. 11.5% of the surgical wound infections.

6.4% of the lower respiratory tract infections. 10.5% of the primary bacteremia cases. 7.0% of the cutaneous infections, and 7.4% of the other infections Farmer and M.T. Kelly.

"Enterobacteriaceae." in Manual of Clinical Microbioloey, Balows et al.(eds). American Society for Microbiology, [1991], p. 365). Surveillance reports from England. Wales and Ireland for 1986 indicate that E. coli was responsible for 5.473 cases of bacteremia 20 (including blood, bone marrow, spleen and heart specimens): of these. 568 were fatal. For spinal fluid specimens, there were 58 cases. with 10 fatalities Farmer and M.T. Kelly.

"Enterobacteriaceae." in Manual of Clinical Microbiolouv. Balows et American Society for Microbiology, [1991], p. 366 There are no similar data for United States. as these are not reportable diseases in this country.

25 Studies in various countries have identified certain serotypes (based on both the O and H antigens) that are associated with the four major groups of E. coli recognized as enteric pathogens. Table 1 lists common serotypes included within these groups. The first group includes the classical enteropathogenic serotypes the next group includes those that produce heat-labile or heat-stable enterotoxins the third group includes the enteroinvasive strains ("EIEC") that mimic Shigella strains in their ability to invade and multiply within intestinal epithelial cells: and the fourth group includes strains and serotypes that cause hemorrhagic colitis or produce Shiga-like toxins (or verotoxins) ("VTEC" or "EHEC" [enterohemmorrhagic E. coli])..

i 'Table L.

Pathogenic E. coli Serot-yies Group Associated Serotypes Enterotoxigenic 06:H16. 08:NM; 08:H-9; 0110127, 015:Hl 1: 020:NM:. 025:NM; (ETEC) 025:H142: 027:H7; 027:H120: 063:H12: 07-8:HI I: 078:1-112; 085:H1--7:0114:H21; 0115:H121; 0126:H9:' I 28ac:117: 0128ac:H12; 0128ac:H2 1: 0148:H28: 0149:H4: 0159:H4: 0166:H127; and 0167:H15 Enteropathogenic 026:NM; 026:1111; 055:NM; 055:H6: 086:NM: 086:112;, (EPEC) 086:1134; 011llab:NM; 01 I lab:H2: 01*1lab:H12: 011 lab:H21: 01 14:112; 01 10:H6; 0125ac:H21:. 0127:NM:.0127116: 0127:119; 0128ab:112; 0142:H16; and 0158-13 Enter6invasive 028ac:NM, 029:NM; 0 1 12ac:NM: 0 1 15:NM: 0 124:NM; (ETEC) 0124:H7: 0124:1130; 0135:-NM: 0136:NM: 0143:NM: 0144:NM: 0164:NM; and 0167:NM.

Verotoxiin-Producing (VTE Ol0:NM. 02:HS; 02:117; 04:NM 04110 5:M 06:H 1: 0 18:NM; 0 18:117; 025:NM: 026:NM: 026:11 1: 026:H32: 038:1121; 039-H4; 045:H2: 050:H7: 055:H17: 055:H IO:_ 082:118.:08412; 091:NM;- 091I:H-21: 0103:12: -0111 :NM; 0l11:18; O111:H30; 0111:1134; 0113:147:0113:H121; 0 114:H48: 0115:1110; 0117:H4; 0118:H12; 0118:H-30: 0121:NM; 0121:1H19, 01,25:NM; 0125:H18; 0126:NM, 01261H8: 0128:NM. 0128:12 0128:H8: 0128:1112; 0128:1425, 0145:NM: 0125:1125: 0146:H121.

0153:H!25; _0157NM; 0157:H7: 0163:H]19: 0165:NM: 0165:19: and 0165:1125 B. Verotoxin Producing Strains of E. coli Although all of these disease-associated serotypes cause potentially life-threatening disease. E. coli 0157:117 and other verotoxin-producing strains have recently gained wvidespread public attention in the United States due to their recently reco,,nized.association with two serious extraintestinal diseases, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura Worldwide. E. coli 0 1 57:H7. and other verotoxinproducing E. coli (VIEC) are an increasingly important human- health problem. First identified as a cause of human illness in early 1982 following two outbreaks of food-related hemorrhagic colitis in Oregon and Michigan Karmali. "Infection bN Verocytotoxin- Producing Esc/ierichia coi," Clin. Microbiol. Rev., 2:15-38 [19891; and L. W. Riley. el al "Hemorrhagic colitis associated with a rare Escherichia coi serotype," New Eng. I. Med., 308: 681-685 119831), the reported incidence of VTEC-associated disease has risen steadily, with outbreaks occurring in the Canada. and Europe.

With increased surveillance. E. coli 0157:H7 has been recognized in other areas of the world including Mexico. China, Argentina. Belgium, and Thailand V. Padhye and M. P.

Doyle, "Escherichia coli 0157:H7: Epidemiology, pathogenesis and methods for detection in food." J. Food. Prot., 55: 555-565 [1992]; and P. M. Griffin and R. V. Tauxe. "The epidemiology of infections caused by Escherichia coli 0157:H7. other enterohemorrhagic E.

coli. and the associated hemolytic uremic syndrome." Epidemiol. Rev.. 13: 60 [1991]).

The disease attracted national attention -in the U.S. after a major outbreak in the Pacific Northwest that was associated with consumption of undercooked E. coli 0157:H7contaminated hamburgers. Over 700 hundred people fell ill (more than 170 were hospitalized) and four young children died Recer. "Expetts call for irradiation of meat to protect against food-borne. bacteria." Associated Press. 7/12/94 [1994]): Several outbreaks since then have underscored the potential severity and multiple mechanisms for transmission of VTEC-associated diseases Bielaszewski et al.. "Verotoxigenic (enterohaemorrhagic) Escherichia coil in infants and toddlers in Czechoslovakia," Infection 18: 352-356 [1990]; A.

Caprioli et al.. "Hemolytic-uremic syndrome and Vero cytotoxin-prodticing Escherichia coil infection in Italy. Infect. Dis.. 166: 184-158 [1992]; A. Caprioli. et al.. "Community-wide Outbreak of Hemolytic-Uremic Syndrome Associated with Non-0157 Verocytotoxin- .o 20 Producing Escherichia coli." J. Infect. Dis.. 169: 208-211 [1994]: N. Cimolai. "Low frequency of high level Shiga-like toxin production in enteropathogenic Escherichia coli serogroups." Eur. J. Pediatr.. 151: 147 [1992]: and R. Voelker.. "Panel calls E. coli screening inadequate." Escherichia coli 0157:H7-Panel sponsored by the American Gastroenterological Association Foundation in July 1994. Medical News Perspectives. J. Amer. Med. Assoc..

25 272: 501 [1994]).

While 0157:H7 is currently the predominant E. coli serotype associated with illness in North America, other serotypes (as shown in Table 1. and in particular 026:H11. O0113:H21, 091:H21 and O111:NM) also produce verotoxins which appear to be important in the pathogenesis of gastrointestinal manifestations and the hemolytic uremic syndrome M.

Griffin and R. V. Tauxe. "The epidemiology of infections caused by Escherichia coli 0157:H7. other enterohemorrhagic E. coli. and the associated hemolytic uremic syndrome," Epidemiol. Rev.. 13: 60 [1990]; M. M. Levine, et al.. "Antibodies to Shiga holotoxin and to two synthetic peptides of the B subunit in sera of patients with Shigella dysenteriae 1 -3dOysententl, J,,Clifii. MicrobioI.230V561 636&164 1 [1 9921J1ffid RS't D~i el'a.. "Pro~perties of Vero cytotoxin producing Esj-herikhi*'.coi.-iof iuan and- aninial'igin belon ging 6* serotypes ,other than! 0 15 7:H7i!_ Epidemniol. lifict~. O03---83-9-5 989 ):-'-Sin&ei'diranisrns with these serotypes have been shown tofeause illniess in-humans they may assume'! greater public health importance over time Griffin and R. V. Tauxe. 'The epidemiology of infections caused by Escherichia coi 01 57:H7. other enterohemorrhagic E. coi, and the associated hemolvtic uremhic syndrome," Epidemniol. Rev., 13: 60 [1990]).

Clinicians usually observe cases of hemolytic uremnic syndrome clustered in a geographic -region.. However, small outbreaks, are- likely to be missed because .many laboratories do not routinely screen: stool specimens for E. coi 0 15 7:H7. Many cases related to non-commercial food preparation also probably -go unrecognized. 'Nonetheless, E. coi 0 157:H7 is responsible for a large number of cases. as. more than 20.000 cases of- E coi 0157:H7 infection-are reported annual ly-in' the U.S..-with 4W00.deaths fromh HUS.

However. these- estimates were compiled -when only I I states mandated reporting of E. coi 01 57:H7. twenty-nine states have~recently-made coili 0157:47 infection a reportable disease Voelker. "Panel calls.EK <coli- screening inadequate:: Escherlchia -coiO 01 57:H7.

panel sponsored by the American Gastroenterological Association Foundation in July 1994.

Medical News Perspectives," J. Amner. Med. Assoc.. 272: 501 [1994]). Indeed. the Centers for Disease Control recently added E. coi 01 57:H7 to their list of reportable diseases ("Public Health Threats." Science 267:1427 [1995]):- Nature of Verotoxin-Induced Disease Risk factors for HUS progression following infection with E. coi 01 51:H7 include age (very young or elderly), bloody diarrhea, leukocvtosis. fever, large amounts of ingested pathogen, previous gastrectomy, and the use of antimicrobial agents (in particular.

trimethoprim-sulfamethoxazole)(A. A. Harris -el al. "Results. of a screening method used in a 12 month stool survey for Escherichia coi 0157:H7." J. Infect. Dis.. 152: 775-777 [1985]; and M. A. Karmali. "Infection by Verocytotoxin-producing Escherichia coi." Clin. Microbiol.

.Rev-, 2: 15-38 [1989]).

As indicated above. E. coli 0157:H7 is associated with significant morbidity andmortality. The spectrum of illness associated with E. coi 0 1 57:H7 infection includes asymptomnatic infection, mild uncomplicated diarrhea. hemporrhagic colitis. HUS. and TTP".

Hemorrhagic colitis (or "ischemic colitis") is a distinct, clinical syndrome characterized by -4suddrpst Fj qf- rminal C ap_- ,likened to, e. pain asc. ierih=ao6b~o, 0 a~~~ppqqdicitis--fbqpowed within:24 hours by~wateryi--diarrhea. En~t tw.dv ter-P ie cdiarrhea.turns; gro~ssly Xbloody,inapprox matly-,90%)of .patients iiddhiis-beerw-d tib&J as "all blood and no stool" H. Pai el "Sporadic cases of hemorrhagic colitis. associated with Escherichia coli 0157:H7," Ann. Intern. Med., 101: 738-742 [1984]; and R. S. Remis et at., "Sporadic cases of hemorrhagic colitis associated with. Escherichia coli 0157:H7," Ann.

Intern. Med.. 10 1: 73 )8-742 [1984]). Vomiting may occur. but there. is little or no fever. The timre from. ingestion to first loose stool ranges from 3-9 days twNith ameari of 4- days) L. W.

Rile. et al.. "Hemorrhagic colitis associated. with a rare Esdwerichia coli serotype.." New Eng.

J. Med.. 3)0 681-685 [1983]; and. Pudden et "Hemorrhagic colitis in a nursing home." Ontario Can. Dis. Weekly Rpt.. 11: 169-1.70 [1 9851),:and thp. duration of illness ranges generally. from 2-9 days (with a mean of 4 days).

-HUS is a life-threatening blood disorder that appears within 3-7 days following onset of diarrhea in 10-15% of patients..- Those younger than 10 years and. the elderly are, atparticular risk. Symptoms include renal glomerular damage. hemolytic anemia. (rupturing of .erythrocytes- as they pass through damaged. renal glomeruli). thrombocytopenia and acute kidney failure. Approximately 15% of patients with HUS die or suffer chronic renal failure.

Indeed.- HUS is a leading cause of renal failure in childhood (reviewed by. M.A. Karmali.

"Infetction by' Verocvtotoxin.-producing Escherichia coli." Clin. Microbiol. Rev.. 2: 15-38 [1989]). Currently, blood transfusion and dialysis 'are the only therapies for HUS.

TTP shares similar histopathologic findings with HUS. but usually results- in multiorgan microvascular thrombosis. Neurological signs and fever are more prominent in TP, compared with HUS. Generally occurring in -adults. TIP is characterized by .microang-opathic hemolytic anemia, 'profound thrombocytopenia. fluctuating neurologic signs.

fever 'and mild azotemia C. Kwaan. "Clinicopathological features of thrombotic thrombocvtopenic purpr" Semin. Hematol.. 24: 71-81 [1987]: and J. Machin- "Clinical .annotation: Thrombotic. thrombocytopenic purpura" Br. J. Hematol.. 56: 191-197 [1984]).

Patients often die from microthrombi in the brain. In one review of 271 cases, a rapidly progressive course was noted, with 75% of patients dying within.90.. days Amnorosi and J.E. Ultmarnn. "Thrombotic thrombocytopenic purpura: Report of 16 cases and review of the.

literature." Med.. 45:139-159 (1966).

Other diseases associated with E coli 0 157:H-7 infection include hemorrhagic cystitis and balantitis R. Grandsen el "Hemorrhagic cystitis and balantitis associated with.

verotoxin-producing Escherichia coli 0157:H7." Lancetii: 150F(1985])L conulsions. sepsis with other organisms and anemia C. Rowe et al. "Hemolytic anemia after' chldhood Escherichia coli 0157:H7 infection: Are females at increased risk?" Epidemioi 'infdt.. 106: 523-530 [1991]).

D. Mechanism of Pathogenesis Verotoxins are strongly linked to E. coli 0157:H7 pathogenesis. All clinical isolates of E. coli 0157:H7 have been shown to produce one or both verotoxins (VTI and VT2) (C.

A. Bopp e al., "Unusual Verotoxin-producing Escherichia coli associated with hemorrhagic colitis," J. Clin. Microbiol.. 25: 1486-1489 [1987]). Both of these toxins are cytotoxic to Vero (African green monkey kidney) and HeLa cells, and cause paralysis and death in mice D. O'Brien et al.. "Purification of Shigella dysenieriae I (Shiga) like toxin from Escherichia coli 0157:H7 strain associated with hemorrhagic colitis." Lancet ii: 573 [1983]).

These toxins are sometimes referred to in the literature as Shiga-like toxins I and II (SLT-I and SLT-II. respectively), due to their similarities with the toxins produced by Shigella..

Indeed, much of our understanding of E. coli VTs is based on information accumulated on Shiga toxins. Shiga toxin, first described in 1903, has been recognized as one of the most potent bacterial toxins for eukaryotic cells (reviewed by M.A. Karmali. "Infection by Verocytotoxin-producing Escherichia coli," Clin. Microbiol. Rev.. 2: 15-38 [1989]).

20 Hereinafter. the VT convention will be used; thus. VTI and VT2 correspond to SLT-I and SLT-II. respectively.

While the pathogenic mechanism of E. coli 0157:H7 infection is incompletely S* understood. it is believed that ingested organisms adhere to and colonize the intestinal mucosa. where toxins are released which cause endothelial cell damage and bloody diarrhea.

It is also postulated that hemorrhagic colitis progresses to HUS when verotoxins enter the bloodstream, damaging the endothelial cells of the microvasculature and triggering a cascade of events resulting in thrombus deposition in small vessels. These microthrombi occlude the microcapillaries of the kidneys (particularly in the glomeruli) and other organs. resulting in their failure J. Bymes and J. L. Moake. "TTP and HUS syndrome: Evolving concepts of pathogenesis and therapy." Clin. Hematol.. 15: 413-442 [1986]; and T. G. Cleary. "Cytotoxinproducing Escherichia coli and the hemolytic uremic syndrome." Pediatr. Clin. North Am..

35: 485-501 [1988]). Verotoxins entering the bloodstream may also result in direct kidney cytotoxicity.

;YT1 is immunologically and structurally indistinguishable from Shiga toxin produced Sby, Shigella dysenteriae O'Brien et al., "Purification of Shigella dvsenteriac 1 (Shiga) like toxin from Escherichia coli 0157:H7 strain associated with hemorrhagic colitis." Lancet ii: 573 [1983]). VTI and VT2 holotoxins each consist of one A and five B subunits (A.

Donohue-Rolfe et al.. "Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized PI glycoprotein and production of cross reactive monoclonal antibodies." Infect. Immun.. 57: 3888-3893 [1989]: and A. Donohue- Rolfe et al., "Simplified high yield purification of Shigella toxin and characterization of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies," J. Exp. Med.. 160: 1767-1781 [1984]). The toxic A subunit is enzymatically active, while the B subunit binds the holotoxin to the receptor on the.target eukarvotic cell.

Crystal structure analysis of Shiga holotoxin and VTI B subunit pentamers have shown that the. holotoxin assembles with the C-terminal end of the A subunit associating with.

and inserting within, a pentamer of B chains E. Stein et al.. "Crystal structure of the cellbinding B oligomer of verotoxin-l from E. coli." Nature 355: 748-750 [1992]: and M.E.

Fraser et al.. "Crystal structure of the holotoxin from Shigella dysenteriae at 2.5 A resolution." Struct. Biol.. 1:59-64 [1994] This conformation is consistent with the observation that a Cterminally truncated Al subunit of VTI is toxic (in a ribosomal inhibition assay). but cannot associate with B subunit pentamers R. Austin et al. "Evidence that the A, fragment of 20 Shiga-like toxin type I is required for holotoxin integrity." Infect. Immun.. 62: 1768 [1994]).

The Verotoxin A Subunit. Examination of the crystal structure of Shiga holotoxin indicates that the N-terminus of its A subunit is both surface-exposed and functionally Simportant. Removal of amino acid interval 3-18 of the A subunit completely abolished toxicity P. Perera et al.. "Mapping the minimal contiguous gene segment that encodes 25 functionally active Shiga-like toxin II," Infect. Immun.. 59: 829-835 [1991]) while removal of interval 25-44 retained toxicity but abolished its association with B subunit pentamers E.

Haddad et al.. "Minimum domain of the Shiga toxin A subunit required for enzymatic activity," J. Bacteriol.. 175: 4970-4978 [1993]). Deletion of the first 13 residues of the homologous ricin A subunit also abolished toxicity, while deletion of the first 9 residues did not J. May, et al.. "Ribosome inactivation by.ricin A chain: A sensitive method to assess the activity of wild-type and mutant polypeptides. EMBO 8: 301-308 [1989]).

The Verotoxin B Subunit. Studies of Shiga toxin B subunit suggest that neutralizing epitopes may also be present at both the N- and C-terminal regions of VTI and VT2 B (subunitsRolyclonal antibodies raised against -peptidesfrom-these regions (residues 5-18.

S.13-26,.7-266r,54-67 .and 57-67) show partial neutralization"of Shiga toxin (I.LHarari and R.

Arnon, "Carboxy-terminal peptides from the B subunit of Shiga toxin induce a local and parenteral protective effect," Mol. Immunol., 27: 613-621 [1990): and I. Harari et al., "Synthetic peptides of Shiga toxin B subunit induce antibodies which neutralize its biological activity," Infect. Immun., 56: 1618-1624 [1988]). Deletion of the last five amino acids of Shiga toxin B P. Jackson et al.. "Functional Analysis of the Shiga toxin and Shiga-like toxin Type II variant binding subunits by using site-directed mutagenesis." J. Bacteriol.. 172: 653-658 [1990]). or four amino acids of VT2 B P. Perera et at., i"Mapping the minimal contiguous gene segment that encodes functionally active Shiga-like toxin II." Infect. Immun..

59: 829-835 [1991]), eliminate toxin activity, while deletion of the last two amino acids of VT2 B subunit reduced cytotoxicity. In contrast. the addition of an 18 or 21 amino acid extension to the native C-terminus of the VT2 B subunit was presumably conformationally correct, as these proteins assembled cytotoxic holotoxin.

Various approaches to express recombinant verotoxins have included individual or coordinate expression of A and B subunits from high-copy number plasmids and expression with fusion partners E. Haddad et al., "Minimum domain of the Shiga toxin A subunit required for enzymatic activity." J. Bacteriol., 175: 4970-4978 J. E. Haddad. and M. P.

Jackson. "Identification of the Shiga toxin A-subunit residues required for holotoxin assembly." J. Bacteriol., 175: 7652-7657 [1993]: M. P. Jackson et al:. "Mutational analysis of the Shiga toxin and Shiga-like toxin II enzymatic subunits." J. Bacteriol.. 172: 3346-3350 oo [1990]: C. J. Hovde et al.. "Evidence that glutamic acid 167 is an active-site residue of Shigalike toxin Proc. Natl. Acad. Sci.. 85: 2568-2572 [1988]: R. L. Deresiewicz et al.. "The role of tvrosine- 14 in the enzymatic activity of the Shiga-like toxin I A-chain." Mol. Gen.

Genet.. 241: 467-473 [1993]; T. M. Zollman et al.. "Purification of Recombinant Shiga-like Toxin Type I A, Fragment from Escherichia coli." Protein Express.Purific.. 5: 291-295 [1994]; K. Ramotar. et al.. "Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron." Biochem 272: 805-811 [1990]: S. B.

Calderwood et al.. "A system for production and rapid purification of large amounts of the Shiga toxin/Shiga-like toxin I B subunit," Infect. Immun.. 58: 2977-2982 [1990]; D. W. K.

Acheson. et al.. "Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using trc or Iron-regulated promoter systems." Infect.

Immun. 61: 1098-1104 [1993]; M. P. Jackson et al.. "Nucleotide sequence analysis and :comparisonof the structural, genes for Shiga-like toxin, and-Shiga-like toxiri II encoded by 0 bacteriophages from Escherichiacoli 933, FEMS Microbiol. Lett.. 44: 109-114 [1987]; J. W.

.ewland-el at.. "Cloning of genes for production of Escherichia coli Shiga-like toxin type II." Infect. Immun, 55: 2675-2680 [1987]; and F. Gunzer and H. Karch. "Expression of A and B subunits of Shiga-like toxin II as fusions with glutathione S-transferase and their potential for use in seroepidemiology,". J. Clin. Microbiol.. 31: 2604-2610 [1993]; and D.W. Acheson et al.. "Expression and purification of Shiga-like toxin II B subunits." Inf. Immun.. 63:301-308 [1995] In one case. bench top fermentation techniques yielded 22 mg/liter of soluble .recombinantiprotein Acheson. et al.. "Comparison of Shiga-like toxin I B-subunit expression and localization in Escherichia coli and Vibrio cholerae by using Irc or Ironregulated promoter systems." Infect. Immun. 61: 1098-1104 [1993]). However, there have been no systematic approaches to identifying the appropriate spectrum of VT antigens.

preserving immunogen and immunoabsorbant antigenicity and scaling-up.

The receptor for VTI and VT2 is a globotriaosyl ceramide containing a galactose a-(l-4)-.galactose--(l-4) glucose ceramide (Gb3) A. Lingwood et al.. "Glycolipid binding of natural and recombinant Escherichia coli produced verotoxin in vitro." J. Biol.

Chem.. 262: 1779-1785 [1987]; and T. Wadell et al.. "Globotriaosyl ceramide is specifically recognized by the Escherichia coli.verocytotoxin Biochem. Biophys. Res. Commun.. 152: 674-679 [1987]). Gb3 is abundant in the cortex of the human kidney and is present in primary human endothelial cell, cultures. Hence, the identification of Gb3 as the funciional receptor for VTI and VT2 is consistent with their role in HLS pathogenesis. in which endothelial cells of the renal vasculature are the principal site of damage. Therefore. toxinmediated pathogenesis may follow a sequence of B subunit binding to Gb3 receptors on kidney cells, toxin internalization. enzymatic reduction of the A subunit to an Al fragment.

binding of the Al subunit to the 60S ribosomal subunit, inhibition of protein synthesis and cell death D. O'Brien ei al.. "Shiga and Shiga-like toxins. Microbial Rev.. 51: 206-220 [1987]).

The role of verotoxins in the pathogenesis ofE. coli O157:H7 infections has been further studied in animal models. Infection or toxin challenge of laboratory animals do not produce all the pathologies and symptoms of hemorrhagic colitis. HUS. and TTP which occur in humans. Glomerular damage is noticeably absent. Nonetheless, experiments using animal models implicate verotoxins as the direct cause of hemorrhagic colitis. microvascular damage leading to the failuie of kidneys and other organs and CNS neuropathies.

For example, Barrett,: et at delivered VT2 into .theperiiorieal cavityof rabbits using mini-osmotic pumps J. Barrett et al.: "Continuous peritoneal infusion of:shiga-like toxin II (SLTII) as a model for.SLT II-induced diseases," J.-Infect. Dis.:i 159: 774-777 [1989]). In three days, most animals receiving the toxin developed diarrhea with intestinal lesions resembling those seen in humans with hemorrhagic colitis. Although there was some evidence of renal dysfunction, none of the rabbits developed HUS: Beerv. el al. showed that VT2, when administered intraperitoneally or intravenously to adult mice. produces lesions of the kidneys and colon T. Beery et al.. "Cytotoxic activity of Escherichia coli 0157:H7 culture filtrate on the mouse colon and kidney," Curr. Microbiol., 11: 335-342 [1984]).

Histologic lesions in the kidney included accumulation of numerous exfoliated collecting tubules and marked intracellular vacuolation of proximal convoluted tubular cells.

Sj6gren et. al. studied the pathogenesis of an entero-adherent:strain of E. coli (RDEC- 1) lysogenized with a VTI-containing bacteriophage (VTI-producing. RDEC-I) Sj6gren et al., "Role of Shiga-like toxin I in bacterial enteritis: comparison between isogenic Escherichia coli strains induced in rabbits." Gastroenterol., 106: 306-317 [1994]). In this study, rabbits were challenged with RDEC-1 or VTI-producing RDEC-1 and studied for onset of disease. The VTI-producing variant induced a severe, non-invasive. entero-adherent infection in rabbits which was characterized by serious histological lesions with vascular changes, edema and severe epithelial inflammation. Importantly. vascular changes consistent with endothelial damage were seen in infected animals that was similar to intestinal microvascular changes in humans with E. coli 0157:H7 infection. Based on these observations. they concluded that VTI is an important virulence factor in enterohemorrhagic S" E. coli 0157:H7 infection.

Fuji ei. al. described a model in which mice were treated for three days with streptomycin followed by a simultaneous challenge of E. coli 0157:H7 orally, and mitomycin intraperitoneally Fuji et al.. "Direct evidence, of neuron impairment by oral infection with Verotoxin-producing Escherichia coli 0157:H7 in mitomycin-treated mice." Infect. Immun..

62: 3447-34453 [1994]). All of the animals died within four days. Immunoelectronmicroscopy strongly suggested that death was due to the toxic effects of VT2v (a structural variant of VT2), on both the endothelial cells and neurons in the central nervous system which resulted in fatal acute encephalopathy.

Wadolkowski el al. studied colonization of E. coli 0157:H7 in mice. Mice were treated with streptomycin and fed 10'° E. coli 0157:H7 A. Wadolkowski et al.. "Mouse 10 .model for colonization and disease caused by enterohemorrhagic Escherichia coli 0157:H7." Infect Immun.. 58: 2418-2445 [1990]. and Wadolkowski etab^*Acute renil tubular necrosis and death of mice.orally infected with Escherichia coli strains that produice Shigalike toxin Type II." Infect.. Immun.. 58: 3959-3965 [1990]). All of the mice died due to severe, disseminated, acute necrosis of proximal convoluted tubules. In mouse models.

glomerular damage was not observed, but toxic acute renal tubular necrosis was observed which is characteristic of some HUS patients. The failure of mice to show glomerular damage is thought to be due to the absence of a functional globotriaosyl ceramide receptor specific for Verotoxins in the glomeruli of the kidneys. Administration of VT2 subunitspecific monoclonal antibodies prior to infection prevented all pathology and death.

E. Current Therapeutic Approaches E. coli 0157:H7 disease is not adequately controlled by current therapy. Patient treatment is tailored to manage fluid and electrolyte disturbances, anemia. renal failure and hypertension. Although E. coli 0157:H7 is susceptible to common antibiotics. the role of antibiotics in the treatment of infection hasquestionable merit. In both retrospective and prospective studies. piophylaxis'or treatment with antibiotics such astrimethoprimsulfamethoxazole. there was. either no benefit or an increased risk of developing HUS N.

Bokete et al.. "Shiga-like toxin producing Escherichia coli in Seattle children: a prospective study." Gastroenterol.. 105: 1724-1731 [1993]; A. T. Pavia et al.. "Hemolytic uremicsyndrome during an 6utbreak of Escherichia coli.0157:H7 infections in institutions for mentally retarded persons: clinical and epidemiologic observations." J. Pedatr.. 116: 544-551 oooo [1990]: F. Proulx et al.. "Randomized. controlled trial of antibiotic therapy for Escherichia co l i 0157:H7 enteritis." J. Pediatr. 121: 299-303 [1992]: and A. L: Carter et al.. "A severe outbreak of Escherichia coli 0157:H7-associated hemorrhagic colitis in a nursing home." New Eng. J. Med., 317: 1496-1500 [1987]).

The mechanisms by which.antibiotics increase the risk of infection or related complications might involve enhancement of toxin production, release of toxins from killed organisms, or alteration of normal competing intestinal flora allowing for pathogen overgrowth A. Karmali. "Infection by Verocytotoxin-producing Escherichia coli." Clin.

Microbiol. Rev.. 2: 15-38 [1989]). A further concern in the use of antibiotics is the potential acquisition of antimicrobial resistance by E. coli 0157:H7 R. Dorn. "Review of foodbome outbreak of Escherichia coli 0157:H7 infection in the western United States." JAVMA 203: 1583-1587 [1993]).

-11 2 ,n-tadditjonx-,by t:hetime.isymptomsi Areiiserilous- 'enoifgh~ tocattract-iedidj idttinfiion. it is ,likelyht -yerotoxins are/,already' &htering'the sytjr&jkriulaiiin 6will! do io'sorl ;thcreafter..FA-,togh antimicrobials may'help4 to:' pr 'nt;pathol 'gY rsltnfomheain of toxijn on. the. bowel -lumensj-. -HowevQer' by 'the time symptoms7 of 'HU S have developed. the patient has. -ceased shedding_:organisms, ;mus antimicrobial%:treatment during H-US disease is .of less value. and often tcontraindicatc&. due~to the increased:ri' k of complications associated .with administration of antimicrobials to patients susceptible to development ofi HUS& Importantly. there is currently no antitoxin- conmmercially available for use in treating affected ~patients. What is needed is a means to blockithe prdgr~si'oii_'bof disease' wiihout-* thecomplications associated'with -antimicrobial, treatmnent.

DESCRIPTION OF. THE. DRAWINGS;.

Figurej -is-an SDS-PAGE of rVTl and rVT2.

Figure: 2 -shows H4PLC results -for rVT1 Land rVT2-.

Figure 3 shows rVTI- and rVT2, toxicity in Vero cell culture.- Fiue4 shows EIA.,reactivity of rVTI-and rVT2 antibodiesto rVT1.

Figure 5, shows, EIAreactivity ofW.VTl and- rVT2 Antibodies to rVT-2) Figure 6 shows Westerin 13iat reactiVityof rVTI and rVT2 antibodies to rVT's: Panel 6A contains preimmune. IgY; Panel 6B contains rVTIl IgY; and Panel 6C contains rVT2 IkY.

Figure 7 shows neutralization of rVTI c totoxiivinVr cls :Figure 8 shows neutralization of rVT2 cytotoxicitv in Vero cells.

0@Figure 9 shows renal sections from E. coli 01 57:H-7-infected mice treahted with IgY 25 Panel 9A shows a representative kidney section from a mouse treated with preimmune IgY, Panel 9B shows a representative kidney sections from a mouse treated with rVTI: and Panel. 9C shows a representative kidney section from a mouse treated with rV2IgY.

Figure 10 shows the fusion constructs of VT components and affinity tags.

12

DEFINITIONS

o .facilitateunderstanding of:the inventiorma numnber;of terms--e--'defiied below.

As used herein ,thee.tern,,neutralizingi' is:used inreference to'antitoxiti. p' icularly antitoxins comprising antibodies, which have the ability to prevent the'pathological actions of the toxin against which the antitoxin is directed.

As used herein, the term "overproducing" is used in reference to the production of toxin polypeptides in a host cell, and indicates-that the host cell is producing.more of the toxin by virtue of the introduction of nucleic acid sequences encoding the toxin polypeptide than.would be expressed by the host cell absent the introduction of these nucleic acid sequences. To allow ease of purification of toxin polypeptides produced in a host cell it is preferred that the host cell express or overproduce.the toxin polypeptide at a level greater than 1 mg/liter of host cell culture..

As used herein, the term "fusion protein" refers to a chimeric protein containing the protein of interest an E coli verotoxin and/or fragments thereof) joined to an exogenous protein fragment (the fusion partner which consists of anon-toxin protein): The fusion partner may enhance solubility of theE. coli protein as expressed in a host cell. may provide an "affinity tag" to allow purification of the recombinant fusion protein from the host cell or culture supematant, or both. If desired. the fusion protein may- be removed from the protein of interest toxin protein or fragments thereof) prior to immunization by a variety of enzymatic or chemical means known to the art.

As used herein, the term "affinity tag" refers to such structures as a "poly-histidine *o tract" or "poly-histidine tag," or any other structure or compound which facilitates the purification of a recombinant fusion protein from a host cell. host cell culture supernatant or both. As used herein. the term "flag tag" refers to short polvpeptide marker sequence useful 25 for recombinant protein identification and purification.

As used herein, the terms "poly-histidine tract" and "poly-histidine tag." when used in 5 reference to a fusion protein refers to the presence of two to ten histidine residues at either o the amino- or carboxy-terminus of a protein of interest. A poly-histidine tract of six to ten residues is preferred. The poly-histidine tract is also defined functionally as being a number 30 of consecutive histidine residues added to the protein- of interest which allows the affinity purification of the resulting. fusion protein on a nickel-chelate column.

As used herein, the term "chimeric protein" refers to two or more coding sequences obtained from different genes, that have been cloned together and that after translation, act as 13 a single polypeptide sequence. Chimeric proteins are also referred to as iybrid proeins." As used herein, the term -"chimeric protein" refers to coding sequences that are obtained from different species of organisms, as well as coding sequences that are obtained from the same species of organisms.

As used herein, the term "protein of interest" refers to the protein whose expression is desired within the fusion protein. In a fusion protein, the protein of interest will be joined or fused with another protein or protein domain, the fusion partner, to allow for enhanced stability of the protein of interest and/or ease of purification of the fusion protein.

As used herein, the term "maltose binding protein" and "MBP" refers to the maltose binding protein of E. coli. A portion of the maltose binding protein may be added to a protein of interest to generate a fusion protein; a portion of the maltose binding protein may merely enhance the solubility of the resulting fusion protein when expressed in a bacterial host. On the other hand. a portion of the maltose binding protein may allow affinity purification of the fusion protein on an amylose resin.

As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. For example, antitoxins are purified by removal of contaminating non-immunoglobulin proteins; they are also purified by the removal of substantially all immunoglobulin that does not bind toxin. The removal of nonimmunoglobulin proteins and/or the removal of immunoglobulins that do not bind toxin results in an increase in the percent of toxin-reactive immunoglobulins in the sample. In another example. recombinant toxin polypeptides are expressed in bacterial host cells and the toxin polypeptides are purified by the removal of host cell proteins: the percent of reco:nbinant toxin polypeptides is thereby increased in the sample.

S o The term "recombinant DNA molecule" as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.

The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule which is expressed from a recombinant DNA molecule.

SThe term "native protein" as used herein refers to a protein which is isolated from a natural source as opposed to the production of a protein by recombinant means.

As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

14- As used herein: "soluble" when in reference to a protein produced by recombinant DNA technology in a host cell, is a protein which exists in solution in the cytoplasm of the host.cell; if the protein contains a signal sequence, the soluble protein is exported to the periplasmic space in bacterial hosts and is secreted into the culture medium of eukarvotic cells capable of secretion or by bacterial hosts possessing the appropriate genes. In contrast, an insoluble protein is one which exists- in denatured form inside cytoplasmic granules (called an inclusion bodies) in the host cell. High level expression greater than I mg recombinant protein/liter of bacterial culture) of recombinant proteins often results in the expressed protein being found in inclusion bodies in the bacterial host cells. A soluble protein is a protein which is not found in an inclusion body inside the host cell or is found both in the cytoplasm and in inclusion bodies and in this case the protein may be present at high or low levels in the cytoplasm.

A distinction is drawn between a soluble protein a protein which when expressed in a host cell is produced in a soluble form) and a "solubilized" protein. An insoluble recombinant protein found inside an inclusion body may be solubilized rendered into a soluble form) by treating purified inclusion bodies with denaturants such as guanidine hydrochloride, urea or sodium dodecyl sulfate (SDS). These denaturants must then be removed from the solubilized protein preparation to allow the recovered protein to renature (refold). Not all proteins will refold into an active conformation after solubilization in a denaturant and removal of the denaturant. Many proteins precipitate upon removal of the denaturant. SDS may be used to solubilize inclusion bodies and will maintain the proteins in solution at low concentration. However. dialysis will noi always remove all of the SDS (SDS can form micelles which do not dialyze out): therefore. SDS-solubilized inclusion body protein is soluble but not refolded.

25 As used herein, the term "reporter reagent" or "reporter molecule" is used in reference to compounds which are capable of detecting the presence of antibody bound to antigen. For example, a reporter reagent may be a colorimetric substance which is attached to an .enzymatic substrate. Upon binding of antibody and antigen, the enzyme acts on its substrate and causes the production of a color. Other reporter reagents include, but are not limited to fluorogenic and radioactive compounds or molecules.

i As used herein the term "signal" is used in reference to the production of a sign that a reaction has occurred, for example, binding of antibody to antigen. It is contemplated that signals in the form of radioactivity, fluorogenic reactions. and enzymatic reactions will be used with the.present ,invention.,? The:signal may be asssssed quaititativelv as well as qualitatively.

Asused herein, the term "therapeutic amount" refers to that amount of antitoxin required to neutralize the pathologic.effects of E. coli toxin in a subject.

As used herein, the term "acute intoxication" is used in reference to cases of E. coli infection in which the patient is currently suffering from the effects of toxin E. coli verotoxins or enterotoxins). Signs and symptoms of intoxication with the toxin may be immediately apparent. Or. the determination of intoxication.may require additional testing, such as detection of toxin present in the patient's fecal material.

As used herein, the term "at risk" is used in references to individuals who have been exposed to E. coli and may suffer the symptoms associated with infection or disease with these organisms. especially due to the effects of verotoxins.

SUMMARY OF THE INVENTION The present invention relates to antitoxin therapy for humans and other animals.

Antitoxins which neutralize the pathologic effects of E. coli toxins are generated by immunization of avian hosts with recombinant toxin fragments. one embodiment, thepresent invention contemplates a.method-of treatment administering at least one antitoxin directed-against at least a portion of an Escherichia.coli verotoxin in-arnrqueous solution in therapeutic amount that is administrable to an intoxicated subject. It is contemplated that the intoxicated subject will be either an adult or a child.

In a preferred embodiment the E. coli verotoxin is recombinant. In one embodiment.

the antitoxin is an avian antitoxin. In an alternative embodiment, the recombinant E. coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion of the 25 Escherichia coli verotoxin VTI sequence. In one embodiment of the E. coli fusion protein, the fusion protein comprises a non-verotoxin protein sequence arid a portion of the Escherichia coli verotoxin VT2 sequence.

Various routes of administration, are contemplated for providing the E. coli antitoxin(s) to an affected individual, including but not limited to, parenteral as well as oral routes of administration. In a particularly preferred embodiment, the route of administration is parenteral.

The present invention also includes the embodiment of a method of prophylactic treatment in which an antitoxin directed againstat-east one E. coli verotoxin in an aqueous o* 16solutiol~inr-thecrapeutic.'anount. that ,is parenterally -administrable and is administered to at least.,on e subject,-at -risk of diarrhea]. disease. It one embodiment. the anthitoxinl is -parenterally administered..

In one embodiment, the subject is at risk of developing extra-intestinal complications of E. co/i infections, including but not limited to. hemolytic uremic syndrome. thrombotic .thrombocvtopenic purpura. etc.

The present invention also includes the embodiment of a composition -which comprises neutralizing antitoxin-directed against at-least--one E. coli verotoxin in an aqueous solution in the"aeui amoni In one particularly preferred embodiment. the E. co/i verotoxin i a recombinant toxi n. In an alternative embodiment. the recombinant E. co/i verotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion of the E. co/i verotroxin VTl sequence. In another embodiment- the recombinant E. co/i verotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion of the E. co/i v'erotoxin VT2 sequence. -In yet another embodiment, the composition of the antitoxin is directed against a portion of at least one Escherichia coi verotoxin. In one embodiment, the portion of Escherichia co/1i is selected from the. group consisting of subunit A and subunit B of VTI. In an alternative embodiment_ the portion of Escherichia co/i is selected from the group. consisting of subunit.A and subunit B of VT2. -Indeed- the, invention contemplates an antitoxin that is directed against a portion of at least one EschL'ricllia co/i verotoxin. In one embodiment, the anti.toxins-an avian. ants-xini The present invejition also comprises a method-o f-treatment of'enteric- bacterial **infections comprising administering an avian antitoxin directed against at. least one verotoxin produced by co/i in an aqueous solution in therapeutic amount. to at least one infected Pbject.. In one preferred-embodiment. the avian antitoxin is administered parenterally.

:25 In another embodiment.- the E co/i is selected from the group consisting of Eschcrichia co/i serotypes 0157:H7, Ql:NM: 02:H5: 02:H7: 04:NM: 04:1-110 05:H16: 06:HI: 018:NM; 018:H7, 025:NM; 026:NM: _026:HI 1: 026:H')27 038:H21; ~0'39:H4: 045:H-2: 050:H7: 055:H7; 055:HIO; 082:H8: 084:1-2: 091:NM: 091:H21-, *0103:H2: 011ll:NM: 011II:H8: 01-1l:H30:'O I:H34: 0113:1-7: 0113:H2]: 0114:H48; 01 iS:HIO: 01 17:H4: 0118:H12: 0118:H30O: 0121:NM: 0121:1-19: 0125:NM; 0125:H8: 0126:NM: 0126:H8: 0128:NM; 0128:H2: 0128:H8: 0128:1-12: 0128:H25: 0145:NM; 0125:H25: 0146:H21; 0153:H25: 0157:NM, 0163:1-19: 0165:NM; 0165:19: and 0165:HY5. In one embodiment, the antitoxin comprises antitoxin directed against at least one 17 Escherichia coli verotoxin.; In another embodimerit;the'antitoxin is.cross-reactswiftf at least one Escherichia coli verotoxin.- In, yet anotheriembodiment. the antitoxin .is reactive against toxins produced by members of the genus Shigella. including S. dvseneriae The present invention also contemplates uses for the toxin fragments in vaccines and diagnostic assays. The fragments may be used separately as purified, soluble antigens or.

alternatively, in mixtures or "cocktails." The present invention thus comprises a method for detecting Escherichia coli verotoxin in a sample in which a sample.an antitoxin raised against Escherichia coli verotoxin. and a reporter reagent capable of binding the antitoxin are provided. The antitoxin is added to the sample, so that the antitoxin binds to the E. coli verotoxin in the sample. In one embodiment, the antitoxin is an avian antitoxin; In an alternative embodiment the method further comprises the steps of washing unbound antitoxin from the sample, adding at least one reporter reagent to the sample. so that said- reporter reagent binds to any antitoxin that is bound, washing the unbound reporter reagent from the sample.and detecting the reporter reagent bound to the antitoxin bound to the Escherichia coli verotoxin. so that the verotoxin-i-deeetted. In one embodiment, the detecting is accomplished through any means, such as enzyme immunoassay. radioimmunoassay.

fluorescence immunoassay, flocculation, particle agglutination, and in sint chromogenic assay.

In one preferred embodiment, the sample is a biological sample. In an alternative preferred embodiment. the sample is an environmental sample.

DESCRIPTION OF THE INVENTION The present invention contemplates treating humans and other animals. intoxicated with at l:ast one bacterial toxin. It is contemplated that administration of antitoxin will be used to treat patients effected by or at risk of symptoms due to the action of bacterial toxins. It is 25 also contemplated that the antitoxin will be used in a diagnostic assay to detect the presence *of toxins in samples. The organisms. toxins and individual steps of the present invention are described separately below.

I. Antibodies Directed Against E_ coli and Associated Toxins A preferred .embodiment of the method of the present invention is directed toward obtaining antibodies against various E. coli serotypes, their toxins, enzymes or other metabolic by-products, cell wall components, or synthetic or recombinant versions of any of these compounds. It is contemplated that these antibodies will be produced by immunization 18 of humans or other animals.j It isnot:intended.that;the preseit invention be limited to an particular toxin or any. species of organism.,In- one embodiment, toxins from all E. coli serotypes are contemplated as immunogens. Examples of these toxins incliide the-verotoxins VTI and VT2.

It is not intended that antibodies produced against one toxin will only be used against that toxin. It is contemplated that antibodies directed against one toxin may be used as an effective therapeutic against one or more toxin(s) produced by other E. coli serotvpes. or other toxin producing organisms Shigella. Bacillus cereus. Staphvyococclus atreus.

Streptococcus mutans. Acinetobacter calcoacelicus. Pseudomonas aeruginosa. other Pseudomonas species. Vibrio species. Clostridium species. etc.). It is further contemplated that antibodies directed against the portion of the toxin which binds to mammalian membranes can also be used against other organisms. It is contemplated that these membrane binding domains are produced synthetically and used as immunogens.

II. Obtaining Antibodies In Non-Mammals A preferred embodiment of the method of the present invention for obtaining antibodies involves immunization. However.it is also contemplated that antibodies may be obtained from non-mammals without immunization. In the case where no immunization is contemplated, the present invention may use non-mammals with preexisting antibodies to toxins as well as non-mammals that have antibodies to whole organisms by virtue of reactions with the administered antigen. An example of the:latter involves immunization with synthetic peptides or recomibinant proteins sharing epitopes with whole organism components.

In a preferred embodiment, the method of the present invention contemplates immunizing non-mammals with bacterial toxin(s). It is not intended that the present invention be limited to any particular toxin. In one embodiment, toxins from all E. coli serotypes are contemplated as immunogens.

A particularly preferred embodiment involves the use of bacterial toxin protein or fragments of toxin proteins produced by molecular biological means recombinant toxin proteins). In a preferred embodiment, the immunogen comprises recombinant VTI and/or VT2.

When immunization is used. the preferred non-mammal is from the class Aves. All birds are contemplated duck. ostrich. emu. turkey, etc.). A preferred bird is a chicken.

Importantly. chicken antibody does not fix mammalian complement (See H.N. Benson el al..

*oo 19- SJ bnmtuno .l 8 61 6 4r1961],).q Thus[hickeni ntibodv will nofrftlnot cause a cdmplementdependent- reaction BenedictandrK.. iYamaga .fImiunotglobilins and Antibody .P,:oduction..inAvi.an:Specie." ini Gomfarativetmmzunolog (J:.'Marchaloni. ed.) pp 335- 375, Blackwell, Oxford [1966]). Thus, the preferred antitoxins of the present invention will not exhibit complement-related side effects observed with antitoxins presently known.

When birds are used, it is contemplated that the antibody will be obtained from either the bird serum or the egg. A preferred embodiment involves collection of the antibody from the egg. Laying hens transport immunoglobulin to the egg.yolk in concentrations equal to or exceeding that found in serum (See R. Patterson et al., J. Immunol. 89 272 (1.962); and S.B. Carroll and B.D. Stollar. J. Biol. Chem. 258:24 [1983]). In addition, the large volume of egg yolk produced.vastly exceeds the volume of serum that can be safelv obtained from the bird over any given time period: Finally. the antibody from eggs is more pure and more homogeneous: there is.far less non-immunoglobulin protein (as compared to serum) and only one class of immunoglobulin is transported to the yolk.

When considering immunization with toxins, one may consider modification of the toxins to reduce the toxicity... In this regard, it is not intended that the present invention be limited by immunization with modified toxin. Unmodified ("native") toxin is also contemplated as an immunogen.

.It is also not intended that the present invention be limited by the type of modification if modification is used. The present invention contemplates all types of toxin modification.

including chemical and heat treatment. of the toxin. In one embodiment. glutaraldehyde treatment of the toxin is contemplated. In an alternative embodiment, formaldehyde treatment of the toxin is contemplated.

It is not intended that the present invention be limited to a particular mode of 25 immunization: the present invention contemplates all modes of immunization. including subcutaneous, intramuscular. intraperitoneal, and intravenous or intravascular injection, as well as per os administration of immunogen.

The present invention further contemplates immunization with or without adjuvant. As used-herein. theterm "adjuvant" is defined as a substance known to increase the immune response to other antigens when administered with other antigens. If adjuvant is used, it is not intended that the present invention be limited to any particular type of adjuvant or that the same adjuvant once used. be used all the time. While the present invention contemplates all types of adjuvant. whether used separately or in combinations, the preferred use of adjuyantisthe-use -pofComplete.reundisiAdjuvantfollved-someiie later ih icomplete -Freunds.Adjuvant Theiinvention also contemplates the:tseiof ibw idjuvancommerciall available from RIBI. as well as Quil A adjuvant commercially available from Accurate Chemical and Scientific Corporation, and Gerbu adjuvant also commercially available.

(GmDP: C.C. Biotech Corp.).

When immunization is used. the present invention contemplates a wide variety of immunization schedules. In one embodiment, a chicken is administered toxin(s) on day zero and subsequently receives toxin(s) in intervals thereafter. It.is not intended that the present invention be limited by the particular intervals or doses. Similarly. it is not intended that the present invention be limited to any particular schedule for collecting antibody. The preferred collection time is sometime after day Where birds are used and collection of antibody is performed by collecting eggs. the eggs may be stored prior to processing for antibody; It is preferred that eggs be stored at 4°C for less than one year.

It is contemplated that chicken antibody produced in this manner can be bufferextracted and used analytically. While unpurified. this preparation can serve as a reference for activity of the antibody prior to further manipulations immunoaffinity purification).

III. Increasing The Effectiveness Of Antibodies When purification is used, the present invention contemplates purifying to increase the effectiveness of both non-mammalian antitoxins and mammalian antitoxins. Specifically; the present invention contemplates increasing the percent of toxin-reactive immunoglobulin. The preferred purification approach for avian antibody is polyethylene glycol (PEG) separation.

The present invention contemplates that avian antibody be initially purified using i 25 simple. inexpensive procedures. In one embodiment, chicken antibody from eggs is purified by extraction and precipitation with PEG. PEG purification.exploits the differential solubilitv of lipids (which are abundant in egg yolks) and yolk proteins in high concentrations of PEG 8000 (Po l son et al., Immunol. Comm. 9:495 [1980]). The technique is rapid. simple, and relatively inexpensive and yields an immunoglobulin fraction that is significantly more pure.

in terms of contaminating non-immunoglobulin proteins than the comparable ammonium sulfate fractions of mammalian sera and horse antibodies. The majority of the PEG is removed from the precipitated chicken immunoglobulin by treatment with ethanol. Indeed.

-21- PEG-purified antibody is sufficiently pure that the present:invention -contemplates the use of PEG-purified antitoxins in the passive immunization of intoxicated humans and animals.

IV. Treatment The present invention contemplates antitoxin therapy for humans and other animals intoxicated by bacterial toxins. A preferred method of treatment is by parenteral administration of antitoxin.

A. Dosage Of Antitoxin It was noted by way of background that a balance must be struck when administering currently available antitoxin which is usually produced in large animals such as horses: sufficient antitoxin must be administered to neutralize the toxin, but not so much antitoxin as to increase the risk of untoward side effects. These side effects are caused by: i) patient sensitivity to foreign horse) proteins; ii) anaphylactic or immunogenic properties of nonimmunoglobulin proteins: iii) the complement fixing properties of mammalian antibodies: and/or iv) the overall burden of foreign protein administered. It is extremely difficult to .strike this balance when, as noted above, the degree of intoxication (and hence the level of antitoxin therapy needed) can only be approximated.

The present invention contemplates significantly reducing side effects so that this balance is more easily achieved. Treatment according to the present invention contemplates reducing side effects by using PEG-purified antitoxin from birds.

In one embodiment, the treatment of the present invention contemplates the use of PEG-purified antitoxin from birds. The use of yolk-derived. PEG-purified antibody as .antitoxin allows for the administration of: 1) non (mammalian)-complement-fixing, avian 25 antibody; 2) a less heterogeneous mixture of non-immunoglobulin proteins: and 3) less total protein to deliver the equivalent weight of active antibody present in currently available antitoxins. The non-mammalian source of the antitoxin makes it useful for treating patients who are sensitive to horse or other mammalian sera.

As is true in cases of botulism, the degree of an individual's exposure to E. coli toxin and the prognosis are often difficult to assess, and depend upon a number of factors the quantity of contaminated food ingested, the toxigenicity and serotype of E. coli strain ingested, etc.). Thus. the clinical presentation of a patient is usually a more important consideration than a quantitative diagnostic test, for determination of dosage in antitoxin -22 I administration.. Indeed. for many toxin-associated diseases botulism, tetanus, diphtheria, etc.), there is no rapid, quantitative test to detect the presence of the toxin or organism.

Rather, these toxin-associated diseases are medical emergencies which mandate immediate treatment. Confirmation of the etiologic agent must not delay the institution of therapy, as the condition of an affected patient may rapidly deteriorate. In addition to the initial treatment with antitoxin, subsequent doses may be indicated, as the patient's disease progresses. The dosage and.timing of these subsequent doses is dependent upon the signs and symptoms of disease in each individual patient.

It is contemplated that the administration of antitoxin to an affected individual would involve an initial injection of an approximately 10 ml dose of immune globulin (with less than approximately 1 gram of total protein). In one preferred embodiment. it is contemplated that at least 50% of the initial injection comprises immune globulin. It is also contemplated that more purified immune globulin be used for treatment, wherein approximately 90% of the initial injection comprises immune globulin. When more purified immune globulin is used.

it is contemplated that the total protein will be less than approximately 100 milligrams. It is also contemplated that additional doses be given, depending upon the signs and symptoms associated with E. coli verotoxin disease progression.

B. Delivery Of Antitoxin Although it is not intended to limit the route of delivery, the present invention contemplates a method for antitoxin treatment of bacterial intoxication in which delivery of antitoxin is parenteral or oral.

In one embodiment, antitoxin is parenterally administered to a subject in an aqueous solution.' It is not intended that the parenteral administration be limited to a particular route.

25 Indeed. it is contemplated that all routes of parenteral administration will be used. In one embodiment. parenteral administration is accomplished via intramuscular injection. In an alternative embodiment. parenteral administration is accomplished via intravenous injection.

In another embodiment, antitoxin is delivered in a solid form tablets). In an alternative embodiment antitoxin is delivered in an aqueous solution.. When an aqueous 30 solution is used, the solution has sufficient ionic strength to solubilize antibody protein, yet is made palatable for oral administration. The delivery solution may also be buffered carbonate buffer. pH 9.5) which can neutralize stomach acids and stabilize the antibodies when the antibodies are administered orally. In one embodiment the delivery solution is an 23 23 aqueous. solution.i.n another embodiment the delivery'soluiiohnis 4 nutritionial Triihla.

.Preferably. the delivery.solution is infant or a dietaryvsupplement:formula Sii iilac® .Ensure,. and Enfamil®)..Yet another embodiment icontemplates the'delivery of lyophilized antibody encapsulated or microencapsulated inside acid-resistant compounds.

Methods of applying enteric coatings to pharmaceutical compounds are well known to the art (companies specializing in the coating of pharmaceutical compounds are available: for.

example. The Coating Place [Verona, WI] and AAI [Wilmington. Enteric coatings which are resistant to gastric fluid and whose release dissolution of the coating to release the pharmaceutical compound) is pH dependent are commercially available (for example, the polymethacrylates Eudragit® L and Eudragit® S [R6hm Tech Inc.. Malden. MA]).

Eudragit® S is soluble in intestinal fluid from pH 7.0; this coating can be used to microencapsulate lyophilized antitoxin antibodies and the particles are suspended in a solution having a pH above or below pH 7.0 for oral administration. The microparticles will remain intact and undissolved until they reached the intestines where the intestinal pH would cause them to dissolve thereby releasing the antitoxin.

The invention contemplates a method of treatment which can be administered for treatment of acute intoxication. In one embodiment- antitoxin is administered orally in either a delivery solution or in tablet form, in therapeutic dosage, to a subject intoxicated by the bacterial toxin which served as immunogen for the antitoxin. In another embodiment of treatment of acute intoxication, a therapeutic dosage of the antitoxin in a delivery solution. is parenterally administered; The invention also contemplates a method of treatment which can be administered prophylactically. In one embodiment- antitoxin is administered orally, in a delivery solution.

in therapeutic dosage. to a subject. to prevent intoxication of the subject by the bacterial toxin which served as immunogen for the production of antitoxin. In another embodiment.

antitoxin is administered orally in solid form such as tablets or as microencapsulated particles.

-Microencapsulation of lyophilized antibody using compounds such as Eudragit® (Rohm GmbH) or polyethylene glycol. which dissolve at a wide range of pH units, allows the oral o* administration of solid antitoxin in a liquid form a suspension) to recipients unable to S* 30 tolerate administration of tablets children or patients on feeding tubes). In one preferred embodiment the subject is a child. In another embodiment, antibody raised against whole i bacterial organism is administered orally to a subject in a delivery solution, in therapeutic 24 dosage.J n yet- another preferredkembodiment of prophylactic treatmenta a therapeutic dosage ,ofth. antitoxin in.a-delivery solution. is parenterally administered.

V. Multivalent Vaccines Against E coli Strains SThe invention contemplates the generation of multivalent vaccines for the protection of an -organism (particularly humans) against several E. coli strains. Of particular interest is a vaccine.which stimulates the production of a humoral immune response to E. coli 0157:H7.

026:H11, 0113:H21..O91:H21. and O111:NM, in humans. The antigens comprising the vaccine preparation iay be native or recombinantly produced toxin proteins from the E. coli serotypes listed above. When native toxin proteins are used as immunogens they are generally modified to reduce the toxicity. It is contemplated that glutaraldehyde-modified toxin proteins will be used. In an alternative embodiment, is formaldehyde-modified toxin proteins will be.:used.

The invention contemplates that recombinant E. coli verotoxin proteins be used in conjunction with either native toxins or toxoids from other organisms as antigens in a multivalent vaccine preparation. It is also contemplated that recombinant E. coli toxin proteins be used in the. multivalent vaccine preparation.

VI. Detection Of Toxin The invention contemplates detecting bacterial toxin in a sample. The term "sample" in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture microbiolocical cultures). On the other hand. it is meant to include both biological and environmental samples.

Biological samples may be animal, including human, fluid, solid stool) or tissue, 25 as well as liquid and solid food and feed products and ingredients such as dairy items.

vegetables. meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of common domestic animals, including but not limited, to bovines cattle), ovines sheep), caprines goats). porcines swine), equines horses), canines dogs), lagamorphs rabbits), and felines cats), etc. It is also 30 intended that samples may be obtained from feral or wild animals, including, but not limited to. such animals as ungulates deer).- bear. fish. lagamorphs. rodents. etc.

Environmental samples include environmental material such as surface matter. soil.

water and industrial samples, as well as samples obtained from food and dairy processing Sinstruments,.apparatus; equipment. utensils, disposableand non-disipsable items .These examples are not to be construed as limiting the -sample typedapplicable:to-the pteseint invention.

The invention contemplates detecting bacterial toxin by a competitive immunoassav method that utilizes recombinant toxin VTI and toxin VT2 proteins, antibodies raised against recombinant bacterial toxin proteins. A fixed amount of the recombinant toxin proteins are immobilized to a solid support a microtiter plate) followed by the addition of a biological sample suspected of containing a bacterial toxin.. The biological sample is first mixed with affinity-purified or PEG fractionated antibodies directed against the recombinant toxin protein. A reporter reagent is. then added which is capable of detecting the presence of antibody bound to the immobilized toxin protein.. The reporter substance may corimprise an antibody with binding specificity for the antitoxin: attached to a molecule which is used to identify the presence of the reporter substance. If toxin is present in the sample, this toxin will compete with the immobilized recombinant toxin protein for binding to the antirecombinant antibody thereby reducing the signal obtained following the addition of the reporter reagent. A control is:employed where the antibody is not mixed with the sample.

This gives the highest (or reference) signal.

The invention also contemplates detecting bacterial toxin by a "sandwich" immunoassay method that utilizes antibodies directed against recombinant bacterial toxin proteins. Affinity-purified antibodies directed against recombinant bacterial toxin proteins are immobilized to a solid support microtiter plates). Biological samples suspected of containing bacterial toxins are then added followed by a washing step to remove substantially all unbound antitoxin. The biological sample is next exposed to the reporter substance, which bincis to antitoxin and is then washed free of substantially all unbound reporter.substance.

25 The reporter substance may comprise an antibody with binding specificity for the antitoxin attached to a molecule which is used to identify the presence of the reporter substance.

Identification of the reporter substance in the biological tissue indicates the presence of the bacterial toxin.

It is also contemplated that bacterial toxin be detected by pouring liquids soups 30 and other fluid foods and feeds including nutritional supplements for humans and other animals) over immobilized antibody which is directed against the bacterial toxin. It is.

contemplated that the immobilized antibody will be present in or on such supports as cartridges, columns, beads. or any other solid support medium. In one embodiment, following 26- 26 O the exposure oftheiliquid to the immobilized antibody,:unbound txin: is substaii"iaily removed by washing: The liquid is then exposed to a reporter substance whichdetects the presence of bound toxin. In a preferred embodiment the reporter substance is an enzyme.

fluorescent dye, or radioactive compound attached to an antibody which is directed against the toxin in a "sandwich" immunoassay). It is also contemplated that the detection system will be developed as necessary the addition of enzyme substrate in enzyme systems: observation using fluorescent light for fluorescent dye systems: and quantitation of radioactivity for radioactive systems).

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

In the disclosure which follows, the following abbreviations apply: °C (degrees Centigrade): rpm (revolutions per minute); BSA (bovine serum albumin): ELISA (enzymelinked immunosorbent assay); IgG (immunoglobulin IgY (immunoglobulin

IP

(intraperitoneal): SC (subcutaneous)y HO (water): HCI (hydrochloric acid): LDoo (lethal dose for 100% of experimental animals): aa (amino acid): HPLC (high performance liquid chromatography): Kda (kilodaltons): gm (grams); pg (micrograms): mg (milligrams): ng (nanograms): l (microliters); mi (milliliters): mm (millimeters); nm (nanometers): pm (micrometer): M (molar); mM (millimolar); MW (molecular weight): sec (seconds): min(s) (minute/minutes): hr(s) (hour/hours): MgCI, (magnesium chloride): NaCI (sodium chloride): Na,CO, (sodium carbonate): OD, 80 (optical density at 280 nm); OD, (optical density at 600 r nn); PAGE (polyacrylamide gel electrophoresis); PBS [phosphate buffered saline (150 mM NaCI. 10 mM sodium phosphate buffer, pH PEG (polyethylene glycol): SDS (sodium dodecyl sulfate); Tris (tris(hvdroxymethyl)aminomethane): w/v (weight to volume); v/v (volume to volume); Amicon (Amicon, Inc.. Beverly, MA): Amresco (Amresco. Inc... Solon.

OH); ATCC (American Type Culture Collection, Rockville. MD); BBL (Baltimore Biologics Laboratory, (a division of Becton Dickinson). Cockeysville. MD); Becton Dickinson (Becton Dickinson Labware. Lincoln Park. NJ); BioRad (BioRad. Richmond, CA): Biotech (C-C Biotech Corp., Poway. CA). Charles River (Charles River Laboratories. Wilmington. MA); Falcon Baxter Healthcare Corp., McGaw Park, IL and Becton Dickinson); Fisher Biotech (Fisher Biotech. Springfield. NJ); GIBCO (Grand Island Biologic Company/BRL, Grand Island, NY); Mallinckrodt (a division of Baxter Healthcare Corp., McGaw Park, IL); -27- S!il s.

i (Mi l iPgre rp..MarlbbroughtMA)NewfEngland*Bilabib (New England Biolabs.

InfriBeverly,hMA);-Novagen (Novagen:4nc,Madison. Wl): Pharmacia (Pharmac'ia Inc., Pescatway,-NJ);Qgeiagen(Qiagen,Chatsworth, CA);'Showdex (Showa Denko-America. Inc..

New York. NY); Sigma (Sigma Chemical Co., St. Louis, MO); RIBI (RIBI Immunochemical Research Inc.. Hamilton. Accurate Chemical and Scientific Corp. (Accurate Chemical and Scientific Corp., Hicksville, NY); Kodak (Eastman-Kodak, Rochester. NY); and Stratagene (Stratagene. La Jolla. CA).

When a recombinant protein is described in the..specification it is referred to in a short-hand manner by the amino acids in the toxin sequence present in the recombinant protein rounded to the nearest 10. The specification gives detailed construction details for all recombinant proteins such that one skilled in the art will know precisely which amino acids are present in a given recombinant protein.

The first set of Examples (Examples 1-5) was designed to develop an antidote to E.

coli 0157:H7 verotoxins and evaluate its effectiveness in vitro and in viro. In the first experiments, high titer verotoxin antibodies were generated in laying hens hyperimmunized with chemically detoxified and/or native verotoxins. These Laying hens were immunized with either recombinant E. coli 0157:H7 VTI or VT2 (rVTI and rVT2) treated with glutaraldehyde and mixed with adjuvant.

Next toxin-reactive polyclonal antibodies were isolated by bulk fractionation from egg yolks pooled from hyperimmunized hens. Large.quantities of polyclonal antibodies (IgY) were harvested from resulting eggs using a two-step polyethylene glycol fractionation procedure.

Third. the immunoreactivity and yields of VT IgY were analyzed by analytical "immunochemical methods enzyme immunoassay. (EIA) and Western blotting). EIA and 25 Western blot analysis showed that the resulting egg preparations contained.high titer IgY that reacted with both the immunizing and the heterologous toxins rVTI IgY reacted against both rVTI and rVT2. and vice versa).

Fourth. VT neutralization potency was analyzed in vitro using a Vero cytotoxicity assay. Vero cytotoxicity of rVTI and rVT2 could be completely inhibited by VT IgY. These 0 antibodies also demonstrated substantial verotoxin cross-neutralization.

Fifth, the efficacy of passively administered avian verotoxin antibodies in preventing the lethal effects of verotoxin poisoning was assessed in a mouse disease model. Toxin neutralizing antibodies were administered by parenteral dosing regimens to assess the most -28effectiestrategy.fortherapeutic intervention:.itEfficacy -of vertoxrin antibodies was demonstrated using multiple murine disease models. In these experiments antibodies prevented both themorbidity and lethality- of homologous and'heterologous toxins using a toxin/antitoxin premix format; mice infected orally with a lethal-dbse of viable E. coli 0157:H7 were protected from both morbidity and lethality when treated parenterally four hours post-infection with either rVTI or rVT2 antibodies: and mice given a lethal dose of E.

coli 091:H21 (a particularly virulent strain which only produces VT2c. a VT2 structural variant) and treated parenterally up to 10 hours later with rVT IgY administered parenterally were protected from both morbidity and lethality.

EXAMPLE I TOXIN ANALYSIS AND IMMUNIZATION Purified recombinant E. coli 0157:H7 verotoxins. rVTl and rVT2. were obtained from Denka Sieken Co., Ltd. (Tokyo. Japan). Toxin genes were isolated, inserted into expression plasmids. and expressed-in E. coli. Recombinant proteins were then purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE Sephacryl and hydroxyapatite.

and gel filtration chromatography by the supplier. Upon receipt, toxins were analyzed to verify identity, purity and toxicity, as described below.

A. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

(SDS-PAGE).

Samples of each toxin (2 pg) were heat-denatured in a buffer containing SDS and pmercaptoethanol followed by electrophoresis on 10-20% gradient gels (Bio-Rad. Richmond.

CA). Resolved polypeptide bands were visualized using the silver stain procedure of C.R.

Merril. etral.. "Ultrasensitive stain for proteins in polyacrylamide gels shows regional 25 variation in cerebrospinal fluid proteins," Science 211: 1437-1438 (1981).

VTI and VT2 are each composed of subunit A and multiple copies of subunit B.

".Subunit A is often nicked into fragments Al and A2 which are linked by a disulfide bridge.

As shown in Figure 1. when separated by SDS-PAGE in the presence of P-mercaptoethanol.

rVTI resolved into 3 bands that corresponded to subunit A (-31 Kda). fragment Al (-27 Kda) 30 and a mixture of subunit B and fragment A2 Kda). SiYnilarly, rVT2 resolved into subunit A (-33 Kda). fragment Al (-27 Kda) and a mixture of subunit B and fragment A2 Kda) (Figure In this Figure, rVTI is in Lane 1, and rVT2 is in Lane 2: the positions of 29molecular:weight markers (Kda) are shown at the left. VT component. polv-eotides are identified at the right.

These results are consistent with previous reports of VTI-and VT2 purified from naturally occurring toxigenic strains V. Padhye et al., "Purification and Phvsicochemical Properties of a Unique Vero Cell Cytotoxin From Escherichia coli 0157:H7." Biochem.

Biophys. Res. Commun.. 139: 424-430 [1986]; and F. B. Kittel et al.. "Characterization and inactivation of verotoxin 1 produced by Escherichia coli 0157:H7." J. Agr. Food Chem.. 39: 141-145 [1991]).

B. High Performance Liquid Chromatography

(HPLC).

Chromatography was performed at room temperature (RT) under isocratic conditions using a Waters 510 HPLC pump. Eluted protein was measured using a Waters 490E programmable multi-wavelength detector (Millipore-Corp.. Milford. MA). The VT's were separated on an 8 x 300 mm (ID) Shodex KW803 column, using 10 mM sodium phosphate.

0.15 M NaCI, pH 7.4 (phosphate buffered saline [PBS]) as the mobile phase at a flow rate of I ml/min.

The purity of non-denatured rVT's was assessed by HPLC. As shown in the chromatographs in Figure 2, each toxin eluted at approximately 10 min. as a single absorbance peak at 280 nm. By integration of the area under each peak. the rVT's were shown to be >99% pure.

C. Vero Cell Cytotoxicity Assay.

Cytotoxic activity of rVTI and rVT2 was assessed using modified procedures of Padhye. et al. V. Padhye et al.. "Purification and Physicochemical Properties of a Unique 25 Vero Cell Cytotoxin From Escherichia coli 0157:H7." Biochem. Biophys. Res. Commun..

139: 424-430 [1986]), and McGee. et al.. A. McGee. et al., "Local induction of tumor necrosis factor as molecular mechanism of mucosal damage by gonococci." Microbial Pathogenesis 12: 333-341 [1992]). Microtiter plates (96 well, Falcon. Microtest III) were inoculated with approximately 1 x 10' Vero cells (ATCC, CCL81) per well (100 pi) and 30 incubated overnight at 37 0 C in the presence of 5% CO, to form Vero cell monolayers. rVTI and rVT2 solutions were serially diluted in Medium 199 supplemented with fetal bovine serum (Life Technologies. Grand Island. NY), added to each well of the microtiter plates and incubated at 37"C for 18-24 hrs. Adherent (viable) cells were stained with 0.2% crystal 30 violet (Mallinckrodt) in 2% ethanol. Excess siain was rinsed away and the stained cells were solubilized by adding 100 pi of 1% SDS to each well. Absorbance-of each well was measured at 570 nm, and the percent cytotoxicity of each test sample wascalculatedusing the following formula: Vero Cytotoxicity [1 (Absorbance Sample/Absorbance Control)] x 100 To determine whether the rVT's possessed potency equivalent to published cytotoxicity values, a Vero cell cytotoxicity assay was performed (Figure Between 0.01-10.000 pg of either rVTI or rVT2 was added to Vero cells. The amounts of rVT causing 50% cell death

(CD,

0 as calculated by second degree polynomial curve fitting were 0.97 pg and 1.5 pg. for rVTI and rVT2. respectively. These results are consistent with CD,, values reported previously for naturally occurring VTI and VT2 in the range 1-35 pg and 1-25 pg.

respectively Petric el al.. Purification and biological properties of Escherichia coli verocytotoxin." FEMS Microbiol. Lett.. 41: 63-68 [1987]: V. L. Tesh. et al.. "Comparison of relative toxicities of Shiga-Like toxins Type I and Type II for mice." Infect. Immun.. 61: 3392-3402 [1993]: N. Dickie et al.. "Purification of an Escherichia coli Serogroup 0157:H7 verotoxin and its detection in North American hemorrhagic colitis isolates." J. Clin.

Microbiol.. 27: 1973-1978 [1989]; and U. Kongmuang. et al.. "A simple method for purification, of Shiga or Shiga-Like toxin from Shigella dysenteriae and Escherichia coli 0157:H7 by immunoaffinity chromatography." FEMS Microbiol. Lett.. 48: 379-383 [1987]).

It has been observed that toxicity is lost with storage, explaining why higher amounts of toxin were used in the neutralization assays described below.

25 D. Mouse Lethal Dose Determination.

STo verify rVTI and rVT2 toxicity, male (20-22 g) CD-I mice were injected intraperitoneally with varying amounts of rVTI or rVT2 in 200 VL phosphate buffer. Doses were selected based on published LD.o values for VTI and VT2 in CD-I mice. To minimize the sacrifice of live animals, a full statistical toxin LDs was not determined. Mice were 30 observed for morbidity and mortality over 7-day period.

Further confirmation of rVT toxicity was obtained from mouse lethality experiments (Table Mice were injected intraperitoneally with varying amounts of either rVTI or rVT2 and observed 7 days for mortality. Within 72-120 hrs. post-injection, all of the mice died -31 ngrTl.,or, o.fgofrVr2i respectively Ji.This"lethality, 9tdy srVed as a verification of expected. oxicity but-notas: a statisticaF determinati'onoFLD'.-. Nonetheless, -these. results -ee osset ihtoxicity studiesowhicclvreportedLD values-in, CD- I mice of 0.4-2.0 jig for purified VTI and 0.001-i1.0 gg for purified VT-) L. Tesh.'>e, al..

"Comparison of relative toxicities of Shiga-Like toxins Type I and Type 11 for mice." Infect.

Iniun.. 61: 3 92 -3402 [1993], and A. D. O'Brien- and LaVeck. "Purification and characterization of S/iigella dvsenreriae I -like toxin produced by Esc/ierichia co/i." Infect.

Imimun. 40: 675-681 [1983]).

Table 2.

Lethality- of rVTI in CD-I Mice ngVT.I Injected .Survivors/Total Hours Post-Injection .7n'.2 100 .5/7 48 2 0/72± 7/7 4 7/772± 6/6 2 1.0 6/6 48± 2 6/6 .72± 2 32 Table 3.

Lethality of rVT2 in.CD-1 Mic' ng VT2 Injected Survivors/Total Hours P6st-Injtion 3/6 48 2 10 2/6 72 2 0/6 120 2 5/6 48 ±2 72 2 0/6 120 6/6 48 2 0.1 6/6 72 2 6/6 120 2 The recombinant toxins used in these studies thus appeared to contain protein components and toxicities consistent with literature reports for native toxins. Based on these structural and functional analyses, the rVT's were considered suitable as antigens to generate specific avian antibodies.

E. Antigen Preparation.

Lybphilized samples. rVTI and rVT2 were received and each was reconstituted with mL of deionized water to a final concentration of 100 pgiml in phosphate buffer. To form a toxoid, the solutions were then treated with 0.4% glutaraldehyde (Mallinckrodt) at 4"C overnight and stored at -20 0 C thereafter. When needed, toxoid was thawed and mixed 25 5:1 (volume:volume) with GERBU adjuvant C. Biotech Corporation. Poway. CA). White Leghorn laying hens were injected subcutaneously with 25 pg of either rVTI or rVT2 toxoid in adjuvant at 2-3 week intervals.

EXAMPLE 2 PEG EXTRACTION OF EGG YOLK ANTIBODY Hyperimmune eggs were collected after 3 immunizations with toxoid. Egg yolks were separated from whites, pooled according to their immunogen group and blended.with 4 volumes of 10 mM sodium phosphate. 150 mM NaCI. pH 7.4 (PBS). Polyethylene glycol 33 8000 (PEG) (Amresco, Solon, Ot was then added to a final concentration of 3.5% and the mixture centrifuged at,;l0,:,00.x;g for 10min: to'remove the precipitated lipid fraction. IaYrich sunernatantwas filtered through cheesecloth and PEG was again added to a final concentration of 12%. The solution was centrifuged as above and the resulting supernatant discarded. The IgY pellet was then dissolved in PBS to either the original (1X PEG IgY) or of the original (4X PEG IgY) yolk volume, filtered through a 0.45 u membrane and stored at 4°C.

EXAMPLE 3 ANTITOXIN IMMUNOASSAYS A. Enzyme Immunoassay

(EIA).

EIA was used to monitor antibody responses during the immunization course. Wells of 96-well Pro-Bind microtiter plates (Falcon, through Scientific Products. McGaw Park. IL) were each coated with 1 Vg of rVT's (not toxoid) in PBS overnight at 2-8 0 C. Wells were washed 3 times with PBS containing 0.05% Tween-20 (PBS-T) to remove unbound antigen.

and the remaining protein binding sites were blocked with PBS containing 1 mg/ml BSA for min.. at room temperature IgY, diluted in PBS. was then added to the wells and incubated for 1 hr. at 37 0 C. Wells were washed as before to remove unbound primary antibody and incubated for 1 hr. at 37°C. with alkaline phosphatase-conjugated rabbit-antichicken IgG (Sigma Chemical Company, St. Louis. MO) diluted 1:1000 in PBS-T. Wells were again washed and I mg/ml p-nitrophenyl phosphate (Sigma Chemical Company. St.

Louis. MO) in 50 mM Na,CO, 10 mM MgCI, pH 9.5 was added and allowed to incubate at RT. Phosphatase activity was detected by absorbance at 410 nm using a Dynatech MR700 25 microtiter plate reader.

*0 I Laying Leghorn hens were immunized as described above (Example 1. part using glutaraldehyde-treated rVT's. Following several immunizations, eggs were collected and IgY harvested by PEG fractionation. Figures 4 and 5 show rVTI or rVT2 specific antibody responses detected using EIA at dilutions of the original yolk IgY concentration of 1:30.000 30 and 1:6.000. respectively. IgY fractionated similarly from unimmunized hens preimmune antibody) did not react with either antigen at test dilutions above 1:50. Although these EIA results indicate significant antibody responses, prior experience with other toxin antigens has shown that optimization of immunization regimens, including increasing the amount of -34 O 34 antigen., can yield titers in excess of 1:100.000'(B. S.Thalley, et al:.'"Development of an Avian Antitoxin to.Type A Botulinum Neurotoxin." inBotUlinum and Tetanus Neurotoxins: Neurotransmission and Biomedical Aspects, B. R. DasGupta, [Plenum Press. New York, 1993] pp. 467-472). As may be expected due to their structural homology and consistent with previous reports V. V. Padhye et al.. "Production and characterization of monoclonal antibodies to verotoxins 1 and 2 from Escherichia coli 0157:H7." J. Agr. Food Chem.. 39: 141-145 [1989]; S. C. Head et al.. "Purification and characterization of verocytotoxin 2." FEMS Microbiol. Lett.. 51: 211-216 [1988]; and N. C. Strockbine el al.. "Characterization of Monoclonal Antibodies against Shiga-Like Toxin from Eschericia coli." Infect. Immun.. 695-700 [1985]). Figures 4 and 5 also demonstrate that antibodies generated against one toxin cross-reacted in vitro with the other toxin.

B. Western Blot Analysis.

Western blots (Figure 6) performed to determine the reactivity of rVT antibodies against constituent VT polypeptides showed that rVTI and rVT2 antibodies reacted with subunit A and fragment Al of either toxin, and with subunit B and fragment A2 of rVTI only. In this Figure. Panel A contains preimmune IgY. Panel B contains rVTI IgY. and Panel C contains rVT2 IgY. Lane 1 in each panel contains rVTl (2pg) and Lane 2 contains rVT2 (2 pg). Preimmune IgY was largely nonreactive to either rVT. Both rVT IgY 20 preparations, however, failed to react with subunit B and fragment A2 of rVT2.- Some explanations for this lack of measurable reactivity might include poor immunogenicitv.

denaturation of the immunogen during glutaraldehyde treatment, loss of conformational S• "epitopes due to detergent or reducing agent, or poor transfer to nitrocellulose.

To resolve the high and low molecular weight components. 2 pg each of rVTI and rVT2 were separated by SDS-PAGE (described above) and then transferred to nitrocellulose paper using the Milliblot-SDE system (Millipore. Medford. MA) according to the manufacturer's instructions. Paper strips were stained temporarily with Ponceau S (Sigma Chemical Company. St. Louis. MO) to visualize the polypeptides and then blocked overnight in PBS containing 5% dry milk. Each strip was agitated gently in IgY diluted in PBS-T for 2 hrs. at RT. Strips were each washed with three changes of PBS-T to remove unbound primary antibody and incubated for 2 hrs. at RT with goat anti-chicken alkaline phosphatase (Kirkegaard and Perry. Gaithersburg, MD) diluted 1:500 in PBS-T containing 1 mg/ml BSA.

The blots were washed as before and rinsed in 50 mM NaCO,, pH 9.5. Strips were 35 submnergedinalkaline-phosphatasesubstate 5-bromo-4hloro.irido yl-po ,tetrazoli l.(Kirkegaard andPerry) .until-sufficientsignal waspobserved? Color development ;wasstopped bv: flooding the blots-with water..

EXAMPLE 4 IN VITRO TOXIN NEUTRALIZATION: VERO CELL ASSAY JgY neutralizationaof rMTI and rVT2ws.was.asseed using the modified Vero cytotoxicity assay described above (Example 1. part Various concentrations of IgY.

Sdiluted in Medium 199 supplemented with 5% fetal bovine serum (GIBCO), were mixed with sufficient toxin to cause 50% cell death and allowed to incubate ai 37 0 C for 60 minutes.

These toxin/antibody mixtures were then added to Vero cell-coated microtiter plate wells according to the procedure described above (Example 1. part C).

.The toxin neutralization capacity of the rVT antibodies was analyzed first using a Vero cell toxicity assay. The-results in Figure 7 show that rVTI IgY neutralized completely the cytotoxic activity of rVTI at an endpoint dilution of 1/320. Furthermore. rVT2 IgY neutralized the heterologous rVTI toxin, but at a higher endpoint concentration.

In a similar experiment (see Figure rVTI and rVT2 antibodies were each able to neutralize rVT2 at equivalent endpoint dilutions. This strong cross-neutralization correlates with the observed strong cross-reactivity of VTI IgY with VT2 A seen on Western blots (Figure These results show that IgY antibodies are able to neutralize effectively VT cytotoxicity and that the antibodies can cross-neutralize structurally-related heterologous i" toxins.

EXAMPLE TOXIN NEUTRALIZATION: MOUSE ASSAYS A. Toxin Challenge Model.

IgY in PBS was premixed with a lethal dose of toxin (as determined above) and 30 injected intraperitoneally into male CD-1 (20-22 gm) mice. Mice were observed for a 7-day period for signs of intoxication such as ruffled fur. huddling and disinclination to move.

followed by hind leg paralysis, rapid breathing and death. Untreated, infected mice usually died within 12 hrs. after signs of severe illness within 48-72 hrs. post-injection).

-36 Onceit;was demonstrated.thatirYT:antibodies were-able to neutralize rVT tiotoxicity in vitro. protection experiments were-next: performed in iniceuFirst u animals were challenged with rVT premixed with rVT IgYto determine:whether toxin lethality could be neutralized under conditions optimal for antigen/antibody reaction. Tables 4 and 5 show that antibodies premixed with the homologous toxin (e.g.,-rVTI with rVTI IgY) prevented lethality of rVT.

Preimmune IgY was unable to neutralize either toxin in these studies.

Table 4 Neutralization of rVTI Using rVT IgY 100 ng rVT2 Premixed* Survivors/Total p Preimmune Antibody 0/12.

rVTI Antibody 12/12 0.001 rVT2 Antibody 12/12 0.001 *Toxin was pre-mixed with IgY and incubated for I hour at room temperature prior to.

administration.

Table Neutralization of rVT2 Using rVT IgY 20 10 ne rVTI Premixed* Survivors/Total p Preimmune Antibody 0/12.

rVTI Antibody 12/12 0.001 rVT2 Antibody 12/12 0.001 *Toxin was pre-mixed with IgY and incubated for I hour at room temperature prior to administration.

Antibodies premixed with the heterologous toxin rVT2 with rVTI IgY) also prevented lethality in vivo. These data are-in contrast to previous observations where rabbit polyclonal antibodies generated against either toxin were cross-reactive with the heterologous toxin by EIA and Western blot, but were unable to neutralize the heterologous toxin in either Vero cell cytotoxicity and mouse lethality assays C. Head. et al., "Serological differences between verocytotoxin 2 and Shiga-like toxin Lancet ii: 751 [1988]; S. C. Head et al., "Purification and characterization of verocytotoxin FEMS Microbiol. Lett.. 51: 211-216 -37- [1988]; N. C. Strockbine el al.. "Characterization of Monoclonal Antibodies against Shiga- Like Toxin from Escherichia coli." Infect Immun., 50: 695-700 [1985]: andV. V. Padhve et al., "Purification and Physicochemical Properties of a Unique Vero Cell Cytotoxin From Escherichia coli 0157:H7." Biochem. Biophys. Res. Commun.. 139: 424-430 [1986]).

However, Head et al.. showed that VT2 B-subunit specific monoclonal antibodies neutralized VTI weakly in a Vero cytotoxicity assay C. Head. et al.. "Serological differences between verocytotoxin 2 and Shiga-like toxin II," Lancet ii: 751 [1988]). In a report by Donohue-Rolfe. et al.. a VT2 B subunit-specific monoclonal antibody neutralized both VTI-and VT2 completely in a Hela cytotoxicity assay Donohue-Rolke et al., "Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized PI glycoprotein and production of cross reactive monoclonal antibodies." Infect. Immun.. 57: 3888-3893 [1989]).

These results showed for the first time complete cross-neutralization in Vero cell cytotoxicity and mouse lethality assays. revealing that VTI and VT2 do indeed share common neutralizing epitopes. These results may indicate that hens generate different antibody specificities as compared to mammals, and/or that differences in immunization methods might have maintained the immunogenicity of conformational epitopes necessary for crossneutralization. Nonetheless, this cross-neutralization suggests that IgY antibodies may contain the range of reactivities essential for an effective antitoxin.

B. Viable organism infection model.

Streptomycin-resistant E. coli 0157:H7 (strain 933 cu-rev) or E. cli 091:H21 (strain SB2F 1) (both kindly provided by Dr. Alison O'Brien, Dept. of Microbiology and Immunology.

S. Uniformed Services University of the Health Sciences. Bethesda. MD) were used in a murine infection model described by Wadolkowski, et al. A. Wadolkowski et al.. "Mouse model for colonization and disease caused by enterohemorrhagic Escherichia coli 0157:H7," Infect.

Immun., 58: 2438-2445 [1990]). Organisms were grown in Luria broth and incubated overnight at 37 0 C in an Environ Shaker (Lab Line, Melrose Park, IL) Maniatis et al..

Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. N. [1982]). Bacterial suspensions were centrifuged at 6700 x g for 5 minutes.

The resulting pellet was then washed twice with sterile PBS and resuspended in sterile sucrose. Five to 8 week-old male CD-I mice were provided drinking water containing mg/ml streptomycin sulfate ad libitum for 24 hrs. Food and water were then withheld for 38 another 16-18 hrs, after which mice were challenged orally with 10 1 streptomycin-resistant

E.

coli 0157:H7 or 091:H21. Mice were housed individually and permitted food and water containing 5 mg/ml streptomycin sulfate. IgY was injected intraperitoneally at varying times post-infection and animals observed for both morbidity and mortality for 10 days.

To monitor bacterial colonization in animals, 1 gram of feces was collected.

homogenized, and plated onto MacConkey agar medium (Difco Laboratories. Detroit. MI) containing 100 pg/ml streptomycin and incubated at 37 C as described by Wadolkowski. et al. A. Wadolkowski et al., "Mouse model for colonization and disease caused by enterohemorrhagic Eicherichia coli 0157:H7." Infect. Immun.. 58: 2438-2445 [1990]). The serotype of E. coli 0157:H7, 933 cu-rev excreted in feces was confirmed by slide agglutination with 0- and H-specific antisera (Difco Laboratories. Detroit. MI).

Kidneys were removed from experimental animals and fixed in 10% buffered neutral formalin. Sections of parafilm-embedded tissue were stained with hematoxylin and eosin (General Medical Laboratories, Madison. WI) and examined by light microscopy. All tissue sections were coded to avoid bias before microscopic examination to determine renal pathology.

The toxin neutralization ability of rVT IgY was further studied using a streptomycintreated CD-I mouse infection model. This model was chosen because it produces definitive systemic pathology and reproducible mortality.

20 In contrast to previous studies by Wadolkowski. et al. A. Wadolkowski et al.

"Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin Type II." Infect. immun.. 58: 3959-3965 [1990]). where mice were given subunit-specific monoclonal antibodies prior to infection, the mice in this study were inoculated orally with 2 x 10'0 viable E. coli 0157:H7 (strain 933 cu-rev) and treated with rVT IgY 4 hrs. following inoculation. Fecal cultures showed that 107-108 challenge organisms per gram of feces were shed throughout the course of the experiment, thus confirming that infection was established. Tables 6 and 7 show that animals treated with either rVTI or rVT2 IgY were protected from lethality caused by infection (p<0.01 and p<0.001. respectively) and that preimmune IgY failed to provide protection to the mice.

39oooo o o• o o 0 0 :Table6 Protection :of Mice From E. coli 0157:H7 With rVTI IgY IgY Treatment Survivors/Total P Morbidity/Total Preimmune Antibody 0/5 rVTI Antibody 9/10 0.01 1/10 *IgY was administered intraperitoneally 4 hours following infection, and once daily for days thereafter.

Table 7 Protection of Mice From E. coli 0157:H7 With rVT2 IgY IgY Treatment Survivors/Total p Morbidity/Total Preimmune Antibody 0/6 6/6 rVT2 Antibody 10/10 0.005 0/10 *IgY was administered intraperitoneally 4 hours following infection. and once daily for days thereafter.

Renal histopathology (see Figure 9) of the control (preimmune IgY) animals showed dilation, degeneration and renal tubular necrosis with no glomerular damage. This is 25 consistent with previous reports showing that renal tubular involvement occurs predominantly in this streptomycin-treated mouse infectivity model A. Wadolkowski et al.. "Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce Shiga-like toxin Type II." Infect. Immun., 58: 3959-3965 [1990]). Importantly, none of the survivors exhibited similar signs of morbidity though treated with IgY 4 hrs. after infection 30 (see Figure 9).

Furthermore. avian antibodies generated against rVTI were able to prevent both mortality and morbidity in a mouse model where VT2 alone is implicated in the pathogenesis and lethality of E. coli 0157:H7 strain 933 cu-rev A. Wadolkowski et "Acute renal tubular necrosis and death of mice orally infected with Escherichia coli strains that produce 35 Shiga-like toxin Type II." Infect. Immun., 58: 3959-3965 [1990]).

To assess the broader utility of the IgY verotoxin antibodies in treating VTECassociated disease, the mouse infectivity study was performed using a more virulent VTEC serotype known to produce VT2c-a structural variant of VT2-but not VTI W. Lindgren 40 et al..,"Virulence of enterohemorrhagic Escherichia coli 091:H21 clinical isolates in an orally infected mouse model," Infect. Immun., 61: 3832-3842 [1993]). Mice were inoculated orally with 5 x 109 E. coli 091:H21 (strain B2FI) and treated subsequently with IgY. Notably, the heterologous rVTI IgY protected strongly against the lethal effects of the VT2c structural variant, even when administered as long as 10 hrs.

following infection (Table Ten hours was the longest treatment window tested in this study. Only 1 of the 8 animals treated with rVTI IgY died (p and those that survived showed no overt signs of renal histopathology acute bilateral tubular necrosis). It can thus be concluded that: rTI.T gY completely neutralized,toxicity of VT2c. indicating its potential as a therapeutic for at least one other pathogenic VTEC.

Table 8 Protection of Mice From E coli 091:H21 With rVTI IgY IgY Treatment Survivors/Total P Morbidity/Total Preimmune Antibody 0/7 7/7 rVTI Antibody 7/8 0.02 1/8 *1gY w as administered intraperitoneally 10 hours following infection, and once daily for 8 days thereafter.

These Examples highlight several important findings supporting the feasibility of using verotoxin antitoxin. First, polyclonal IgY generated against either VTI or VT2 from E. coli 0157:H7. cross-reacted with and fully cross-neutralized the toxicity of the non-immunizing toxin both in vitro and in vivo. Second. recombinant toxins fully neutralized the toxicity of naturally-occurring toxins produced by E. coli 0157:H7 during the course of infection. Third.

antibodies generated against rVTI from E. coli 0157:H7 could prevent morbidity and mortality in mice infected orally with lethal doses of E. coli 091:H21, a particularly virulent strain which only produces VT2c. suggesting their utility in preventing systemic sequelae.

Because VTI is identical to Shiga-toxin D. O'Brien et al.. "Shiga and Shiga-like toxins.

Microbial Rev., 51: 206-220 [1987]), VT antibodies may also be useful in preventing complications stemming from Shigella dysenteriae infection. Finally, animals treated with VT 41 IgY were protected against both death and kidney damage when treated as long as 10 hrs.

after infection, supporting the hypothesis that a window for antitoxin intervention exists.

These studies strongly support the use of parenterally-administered. toxin-specific IgY as a antitoxin to prevent life-threatening complications.associated with E. coli 01S7:H7 and other VTEC infections. It is contemplated that this approach would be most useful in preventing HUS and other complications when administered after the onset of bloody diarrhea and before the presentation of systemic disease.

The VT IgY developed in these studies were shown to react with and neutralize both recombinant and naturally-occuring VT. The antibody titers as measured by EIA are indicative of reasonable antibody production in the hen. however much higher production levels can be obtained with larger immunizing doses.

The results from these Examples clearly demonstrate the feasibility and provide the experimental basis for development of an avian antidote for E. coli O157:H7 verotoxins suitable for use in humans. In contrast to previous reports showing that rabbit polyclonal VTI and VT2 antibodies cross-reacted, but did not cross-neutralize the heterologous toxin in Vero cytotoxicity or in mouse lethality studies V. V. Padhye et al.. "Production and characterization of monoclonal antibodies to verotoxins I and 2 from Escherichia coli 0157:H7." J. Agr. Food Chem.. 39: 141-145 [1989]; S. C. Head et al.. "Purification and characterization of verocytotoxin FEMS Microbiol. Lett.. 51: 211-216 [1988]: and N. C.

20 Strockbine et al. "Characterization of monoclonal antibodies against Shiga-like toxin from Escherichia coli." Infect. Immun.. 50: 695-700 [1985]). these data provide the first demonstration of cross-neutralization in vivo. Antibodies against one toxin neutralized completely the heterologous toxin in both Vero cytotoxicity and mouse lethality assays. Both .rVTI and rVT2 antibodies also prevented morbidity (as assessed by renal histopathology) and mortality in mice infected with lethal doses of E. coli 0157:H7 the etiologic, agent in of the documented cases of hemolytic uremic syndrome (HUS) in the U.S. M. Griffin and R. V. Tauxe. "The epidemiology of infections caused by Escherichia coli 0157:H7. other enterohemorrhagic E. coli. and the associated hemolytic uremic syndrome." Epidemiol. Rev..

13: 60 [1990]). With at least two other VTEC serotypes known to cause HUS. the finding that rVTI antibodies neutralized a VT2 variant produced by E coli 091:H21 suggests that avian polyclonal antibodies may provide an effective antidote against other verotoxinproducing E. coli. These data also show for the first time. that antibodies may be administered after infection and still protect against morbidity and mortality.

42 SEXAMPLE 6 EXPRESSION OF TOXIN GENES The previous Examples clearly showed that avian polyclonal antibodies to recombinant toxins protected animals infected with verotoxigenic E. coli. This Example includes expression of toxin genes (A and B subunits alone and together as whole toxins) in suitable prokaryotic expression systems to achieve high levels of VT antigen production.

The sequence of the toxin gene has been determined (see M.P. Jackson et al..

"Nucleotide sequence analysis and comparison of the structural genes for Shiga-like toxin I and Shiga-like toxin II encoded by bacteriophages from Escherichia coli 933." 44:109 [1987]). The coding regions of the A and B subunits of VT-I are listed in SEQ ID NOS:1 and 3, respectively. The corresponding amino acid sequence of the A and B subunits of the VT-I toxin are listed in SEQ ID NOS:2 and 4. respectively. The coding regions of the A and B subunits of VT-2 are listed in SEQ ID NOS:5 and 7. respectively. The corresponding amino acid sequence of the A and B subunits of the VT-2 toxin are listed in SEQ ID NOS:6 and 8. respectively. In addition. SEQ ID NOS:9 and 10 list the sequences which direct the expression of a poly-cistronic RNA capable of directing the translation of both the A and B subunits from the VT-I and VT-2 genes. respectively.

In choosing a strategy for recombinant VT antigen production, there are three primary technical factors to consider. First, the appropriate VT antigen components representing the .20 spectrum of toxin epitopes encountered in nature must be utilized. Second. the protein antigens must be expressed at sufficient levels and purity to enable immunization and largescale antibody purification. Third. the neutralizing epitopes must be preserved in the immunogen and immunoabsorbant. Approaches that offer the greatest promise for high level expression of periplasmically localized, native, affinity-tagged proteins were developed.

Figure 10 shows the fusion constructs of VT components and affinity tags.

A. Expression of affinity-tagged C-terminal constructs.

The VTI and VT2 A and B subunits (SEQ ID NOS:I. 3, 5 and 7) are cloned into the pET-23b vector (Novagen). This vector is designed to allow expression of native proteins containing C-terminal poly-His tags. The vector utilizes a strong T7 polymerase promoter to drive high level expression of target proteins. The methionine initiation codon is engineered to contain a unique Ndel restriction enzyme site (CATATG). The VTI and VT2 genes are engineered to convert the signal sequence methionine codon into a Ndel site utilizing PCR -43 mutagenesis. PCR primers were designed which contain the sequence GCCAT fused to the O first 20-24 bases of the genes.(starting at'the ATG start codon of the signal tag; SEQ ID -,NOS:12-19, see Table below). Upon PCR amplification, the 5' start codon of each gene is converted to an Ndel site, compatible with the pET-23 vector-encoded NdeI site, allowing cloning of the amplified genes into the vector without the addition of vector-encoded amino acids.

Primers containing the C-terminal 7 codons of each gene (21 bases) fused to the sequence CTCGAGCC were synthesized, in order to add a C-terminal poly-His tag to each genre. The underlined bases are an Xhol site, that is compatible with the Xhol site of the pET-23 vector. These primers precisely delete the native stop codons. and when cloned into the pET-23 vector, add a C-terminal extension of "LeuGluHisHisHisHisHisHis" (SEQ ID NO: 11). The following table lists the primer pairs are utilized to create PCR fragments containing the A and B subunits derived from VT-1 and VT-2 toxin genes suitable for insertion into the pET-23b vector.

Table 9 Primers Toxin Gene and Subunit N-terminal Primer C-terminal Primer VT-I Subunit A SEQ ID NO:12 SEQ ID NO:13 VT-I Subunit B SEQ ID NO:14 SEQ ID 20 VT-2 Subunit A SEQ ID NO:16 SEQ ID NO:17 S* VT-2 Subunit B SEQ ID NO:18 SEQ ID NO:19 VT-I Subunits A and B SEQ ID NO:12 SEQ ID VT-2 Subunits A and B SEQ ID NO:16 SEQ ID NO:19 Thus. utilizing PCR amplification with the above modified N- and C-terminal primers, i the A and B subunits of VTI and VT2 are expressed as proteins containing an 8 amino acid C-terminal extension bearing an poly-histidine affinity tag. The amino acid sequence of the histidine-tagged VT-1 A subunit produced by expression from the pET-23b vector is listed in

S

SEQ ID NO:21 (the associated DNA sequence is listed in SEQ ID NO:20): the amino acid sequence of the histidine-tagged VT-I B subunit is listed in SEQ ID NO:23 (the associated -44- DNAi equenceis listed in.SEQJD NO:22);.the amino acid sequence of the histidine-tagged VT-2.A.subunit.is listed in-SEQ ID NO:25 (the associated DNA sequence is listed in SEQ ID NO:24); the amino acid. sequence of the histidine-tagged VT-2 B subunit is listed in SEQ ID NO:27 (the associated DNA sequence is listed in SEQ ID NO:26).

Both subunits may be expressed from a single expression constructs by utilizing SEQ ID NOS:12 and 15 to prime synthesis of the VT-I toxin gene and SEQ ID NOS:16 and 19 to prime synthesis of the VT-2 toxin gene. The resulting PCR products are cleaved with Ndel and Ahol. as described for the cloning of the subunit genes into the pET-23b vector.

Expression bf the A: and B subunits from such an expression vector, results in the exprezsion of a native A subunit and a his-tagged B subunit. As the A and B subunits assemble into a complex, the presence of the his-tag on only the B subunit is sufficient to allow purification of the holotoxin on metal chelate columns as described below.

The proofreading Pfu polymerase (Stratagene) is utilized for PCR amplification to reduce the error rate during amplification. Genomic DNA from an E. coli 0157:H7 strain is utilized as template DNA. Following the PCR, the amplification products are digested with NdeI and Xhol and cloned into the pCR-Script SK cloning vehicle (Stratagene) to permit DNA sequence analysis of the amplified products. The DNA sequence analysis is performed to ensure that no base changes are introduced during amplification. Once the desired clones are identified by DNA sequencing, the inserts are then excised utilizing Ndel and Xhol. and cloned into a similarly cut pET-23b vector to create the expression constructs. According to the published sequences: neither the VTI nor VT2 genes contain either of these restriction sites.

7 The poly-His-tagged proteins produced by expression of the VT-I and VT-2 gene sequences in the pET-23b constructs are then purified by IMAC. This method uses metalchelate affinity chromatography to purify native or denatured proteins which have histidine tails (see K. J. Petty, "Metal-Chelate Affinity Chromatography." in Current Protocols in Molecular Biology, Supplement 24, Unit 10.11B [1993]).

B. Expression of Toxin Containing N-terminal Affinity Tags Two expression systems, pMal-p2 and pFLAG-l are utilized to attach an N-terminal affinity tag to the A subunits from the VT-I and VT-2 toxins.

MBP-tagged constructs. To construct A chains containing the maltose binding protein (MBP) at the N-terminus of the A subunit, PCR amplified gene products are cloned into the 45 PMal-p2 ector (New England Biolabs): as C-terminal. fusions to'a' periplasiicallv-secreted version of the MBP. The MBP selectively:binds to ainylose resins and seved as anaffinity -tag on the MBP/A subunit fusion .proteinri The pMal-p2 vector contains an engineered factor Xa cleavage site, which permits the removal of the affinity tag MBP) from the fusion protein after purification.

The MBP/A subunit fusions are generated as follows. The VTI and VT2 A subunits are PCR-amplified utilizing the following DNA primers. SEQ ID NOS:28-31 SEQ ID NOS:28 and 29 comprise the 5' and 3' primers, respectively, for the amplification of the VTI A subunit; SEQ ID NOS:30 and 31 comprise the 5' and 3' primers, respectively, for the amplification of the VT2 A subunit. In both cases, the 5' or N-terminal primer contains the sequence CGGAATTC fused to the first codon of the mature polypeptide (rather than the start of the signal peptide, since the MBP signal peptide is utilized). These 5' primers contain an engineered EcoRI site that is not contained internally in either gene. that is compatible with the EcoRI site of the pMal-p2 vector. The 3' or C-terminal primers incorporate an Xhol site as described above for the generation of the His-tagged toxins, but in this case. the primer is designed to include the natural termination codon of the A subunits.

The genes are amplified, cloned into pCR-Script SK, and sequenced as described above. The inserts are then excised with EcoRI and Xhol. and cloned into EcoRI/Sall-cleaved pMal-p2 vector (Sail and Xhol sites are compatible). This construct allows expression and 20 secretion of the VTI and VT2 A subunit genes as C-terminal fusions with MBP. The amino acid sequence of the MBP/VT-IA fusion protein is listed in SEQ ID NO:33 (the associated DNA sequence is listed in SEQ ID NO:32). The amino acid sequence of the MBP/VT-2A fusion protein is listed in SEQ ID NO:35 (the associated DNA sequence is listed in SEQ ID NO:34).

The resulting fusion proteins are then affinity purified on an amylose column and the bound fusion protein is eluted under mild conditions by competition with maltose. The MBP N-terminal-tagged A subunits are cleaved with factor Xa and the MBP is removed by chromatography on an amylose column. The resulting A subunits which contain a 4 amino acid N-terminal extension are then used as immunogens.

Flag tag constructs. In an alternative embodiment, the VTI and VT2 A subunit genes are engineered to contain the "flag tag" through the use of the pFLAG-I vector system.

The flag tag is located between the OmpA secretion signal sequence and the authentic N- -46 terminus of the target protein in the pFlag-1- vector.. :To construct N-terminal flag-tagged A chains, the EcoRI/Xhol A subunit PCR fragments (generated as described above for the MBP fusion proteins) are cloned into identically cleaved pFlag-I vector (Eastman-Kodak). to produce an expression construct utilizing the OmpA signal peptide for secretion of A subunit fusion proteins containing the flag peptide at the N-terminus. After secretion, the periplasmic protein contains the N-terminal 8 amino acid flag tag. followed by 4 vector-encoded amino acids fused to the recombinant A subunit. The amino acid sequence of the flag tag/VT-1 A subunit fusion protein is listed in SEQ ID NO:37 (the associated DNA sequence is listed in SEQ ID NO:36). The amino acid sequence of the flag tag/VT-2 A subunit fusion protein is listed in SEQ ID NO:39 (the associated DNA sequence is listed in SEQ ID NO:38).

The flag tag fusion proteins are then purified by immunoaffinity chromatography utilizing a calcium-dependent monoclonal antibody (Antiflag Ml: Eastman-Kodak). Mild elution of purified protein is achieved by chelating the calcium in the column buffer with ethylenediamine tetraacetic acid (EDTA).

C. Evaluation of fusion construct expression.

The fusion constructs described above are expressed in E. coli strain BL21. or T7 polymerase-containing derivatives BL21(DE3). BL21(DE3) pLysS. BL21(DE3)pLysE] (Novagen) for pET plasmids. and periplasmically-secreted recombinant protein purified by affinity chromatography. Recombinant proteins are analyzed for correct conformation by testing the following parameters: a) It is believed that the B subunit must associate into pentamers to be conformationally correct. This is assessed by reducing and native SDS-PAGE analyses of native and chemically-cross-linked proteins and sizing HPLC: b) It is believed that a properly folded A subunit is expected to retain its native enzymatic activity. This is tested by its capacity to inhibit protein synthesis in an in vitro toxicity assay; c) It is believed that in vitro toxicity of assembled recombinant holotoxin is compared to commercially available holotoxins to determine whether recombinant A and B subunits can assemble into functional holotoxin. The 47- ,purified' N-terminal-tagged-.A-';subunitsi(after cleavage and purification from *MBP. or. untreated! flag-tagged proteins) are combined in vitro with the corresponding A B chains,- and-:theirn toxicity; evaluated utilizing a quantitative microtiter cytotoxicity assay, such as thatdescribed by M.K. Gentry and M.

Dalrymple, "Quantitative Microtiter Ctotoxicitv Assay for Shigella Toxin." I.

Clin. Microbiol., 12:361-366 (1980).

48 SEQUENCE. LISTING GENERAL INFORMATION: APPLICANT: OPHIDIAN PHARMACEUTICALS, INC.

(ii) TITLE OF INVENTION: TREATMENT FOR VEROTOXIN-PRODUCING

E.

COLI

(iii) NUMBER OF SEQUENCES: 39 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: MEDLEN CARROLL STREET: 220 MONTGOMERY STREET, SUITE 2200 CITY: SAN FRANCISCO STATE: CALIFORNIA E--COOUNRY' UNITED STATES OF AMERICA ZIP: 94104 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

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REGISTRATION NUMBER: 32,837 REFERENCE/DOCKET NUMBER: OPHD-02171 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (415) 705-8410 TELEFAX: (415) 397-8338 S* INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 945 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..945 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATG AAA ATA ATT ATT TTT AGA GTG CTA ACT TTT TTC TTT GTT ATC TTT 48 Met Lys Ile Ile Ile Phe Arg Val Leu Thr Phe Phe Phe Val Ile Phe 1 5 10 TCA GTT AAT GTG GTG GCG AAG GAA TTT ACC TTA GAC TTC TCG ACT GCA 96 Ser Val Asn Val Val Ala Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala 25 AAG ACG TAT GTA GAT TCG CTG AAT GTC ATT CGC TCT GCA ATA GGT ACT 144 S Lys Thr Tyr Val Asp Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr 40 CCA TTA CAG ACT ATT TCA TCA GGA GGT ACG TCT TTA CTG ATG ATT GAT 192 Pro Leu Gin Thr Ile Ser Ser Gly Gly Thr Ser Leu Leu Met Ile Asp 55 49 AGT GGC TCA GGG GAT AAT rra TI-r GCA GTT GAT GTC AGA GGG ATA GAT 240 Ser Gly Ser Gly Asp Asn Leu Phe Ala Val Asp Val Arg Gly Ile Asp 70 75 GCA GAG GAA 000 COG 1TrT AAT AAT CTA CGG CTT ATT GTT GAA CGA AAT 288 Ala Giu Giu Gly Arg Phe Asn Asri Leu -Arg Leu Ile Val Glu Arg Asn 90 AAT TTA TAT GTG ACA OGA TTT GTI' AAC AGG ACA AAT AAT GTT TTT TAT 336 Asn Leu Tyr Val Thr Gly Phe Val Asn Arg Thr Asn Asn Val Phe Tyr 100 105 110 CGC TTT GCT GAT TTT TCA CAT GTT ACC TTT CCA GOT AcA ACA GCG OTT 384 Arg Phe Ala Asp Phe Ser His Val Thr Phe Pro Gly Thr Thr Ala Val 115 120 125 ACA TTG TCT GGT GAC AGT AGC TAT ACC ACO TTA CAG CGT GTT GCA GOG 432 Thr Leu Ser Gly Asp Ser Ser Tyr Thr Th-r Leu Gin Arg Val Ala Clv 130 135 140 ATC ACT CGT ACG GGG ATG CAG ATA AAT CGC CAT TCO TTC ACT ACT TCT- 480 Ile Ser Arg Thr Gly Met Gin Ile Asn Arg His Ser Leu Thr Thr Ser 145 150 155 160 TAT CTG GAT TTA ATO TCG CAT AGT OGA ACC TCA CTO ACG CAG TCT GTO 528 Tyr Leu Asp Leu met Ser His Ser Oly Thr Ser Leu Thr Gin Ser Val 165 170 175 OCA AGA OCO ATO TTA CGG rrT OTT ACT GTO ACA OCT GAA GCT TTA COT 576 Ala Arg Ala Met Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu-Arg 180 185 -190 TTTr COG CAA ATA CAO AGO GGA TTT COT ACA ACA CTO GAT OAT CTC AGT 624 Phe Arg Gin Ile Gin Arg Gly Phe Arg Thr Thr Leu Asp Asp Leu Ser 195 200 205 000 COT TCT TAT GTA ATO ACT OCT OAA CAT GTT CAT CTT ACA TTG AAC 672 Gly Arg Ser Tyr Val Met Thr Ala Olu Asp Val Asp Leu Thr Leu Asn 210 2 15 220 TOO OGA AGO TTG ACT AGC GTC CTG CCT GAC TAT CAT OGA CAA GAC TCT 720 Trp Gly Ara Leu Ser Ser Val Leu Pro Asp Tyr His Oly Gin Asp Ser 225 230 235 240 TT COT GTA GGA AGA ATT TCT TTT GGA AGC ATT AAT'GCA ATT CTO OGA 768 ::.Val Arg Val Oly Arg Ile Ser Phe Gly Ser Ile Asn Ala Ile Leu Gly 245 250 255 *AGC GTG OCA TTA ATA CTC AAT TOT CAT CAT CAT OCA TCO CGA OTT GCC 816 Ser, Val Ala Leu Ile Leu Asn Cys His His His Ala Ser Arg Val Ala 260 265 270 AGA ATO OCA TCT OAT GAO TTT CCT TCT ATO TOT CCC GCA OAT OGA AGA 864 Arg Met Ala Ser Asp Olu Phe Pro Ser Met Cys Pro Ala Asp Oly Arg 275 280 285 GTC COT COO ATT ACO CAC AAT AAA ATA TTO TOO OAT TCA TCC ACT CTG 912 Val Arg GC'lv Ilie Thr His Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu 290 295 300 COG OCA ATT CTG ATO CCC AGA ACT ATT AGC AGT 945 Oly Ala Ile Leu Met Arg Arg Thr Ile Ser Ser 305 310 315 INFOPR4ATION FOR SEQ ID NO:2:- SEQUENCE CHAPLACTERISTICS: LENGTH: 315 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECUL~e'TYPE: protein (xi) SEQUENCE DESCRIPTION:- SEQ ID NO:2: Met Lys Ile Ile Ile Phe Arg Val Leu Thr Phe Phe Phe Val Ile Phe Ser Val Asn Val Val Ala Lys Giu Phe Thr Leu Lys Pro Ser Ala Asn Arg Thr Ile 145 Tyr Ala Phe Gly Trp 225 Val I Ser N Th: Let Gi) Glu Leu Phe Leu 130 Ser Leu Arg Arg bArg 210 31y ~rg tal rTyr Gin Ser Giu Tyr Ala 115 Ser Arg Asp Ala Gin 195 Ser Arg Val C Val Thr Gly Gly Val 100 Asp Gly Thr Leu Me t 180 Ile ryr eu fly -Asp Ile Asp *Arg Thr Phe A-sp Gly Met 165 Leu Gin Val Ser ArgI 245 Se: Se~ Ast Phe G1y Ser Ser Met 150 Ser Arg Arg 41et 3er ~30 Ele a a "Leu Ser 55 Leu Asn Phe *His *Ser 135 Gin His Phe Giy Thr 215 ValI Ser AsnC Phe P Asn Val Val 120 Tyr Ile Ser Val Phe 200 Ala Leu Asn 105 Thr Thr Asn Gly Thr 185 Arg G1u Phe Ala Val Arg Ser Asp 75 Leu Thr Pro Leu Hi's 155 Ser Thr Thr Val Ile Asn Gly Gin 140 Ser Leu Ala Leu Asp 220 Val Asn Thr 125 Arg Leu Thr Glu Asp 205 Glu Val 110 Thr Val1 Thr Gin Ala 190 Asp Val. Arg Gly Ile Asp Phe Ser Ala Leu Leu.

Ser Thr Ala Ile Gly Thr Met Ile Asp Asp Asn Tyr Val1 Gly Ser 160 Val1 Arg Ser Leu Thr Leu Asn Ala Leu Ile Leu 260 a Ar§ Met Ala 275 Ser Asp Glu .eu Pro Asp Phe Gly Ser 250 ~ys His His 265 ~ro Ser Met so0 .ys Ile Leu hr Ile Ser 51 Tyr 235 Ile His Cys Trp S er 315 His Gly Asn Ala Ala Ser Pro Ala 285 Asp Ser 300 Gin Asp Ile Leu 255 Arg Val 270 Asp Gly Ser Thr Val Arg 290 Gly Ala 305 Gly Ile Thr His Asn 295 Ile Leu Met Arg Arg 310 2

L

T

INFORMA~TION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS:.- LENGTH: 267 base pairs TYPE: nucleic acid STRAlqDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .267 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 7-AT 3 AAA:-.AAA-.ELA ACTTA -LTTA-U:ATA .GCT-; GCA, -TCG- CTT TCA,-TTT TTT TCA GCA 48 Met Lys Lys Thr eU Leu Ile AMa Ala Ser Leu Ser Phe Phe Ser Ala 1 5 10 AGT GCG CTG GCG ACG CCT GAT TGT GTA ACT GGA AAG GTG GAG TAT ACA 96 Ser Ala LeU Ala Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr 25 AAA TAT AAT GAT GAC GAT ACC TTT ACA GT*T AAA GTG GGT GAT AAA GAA 144 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu 40 TTA TTT ACC AAC AGA TGG AAT CTT CAG TCT CTT CTT CTC AGT GCG CAA 192 Leu Phe Thr Asn Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Ala Gln 55 ATT ACG GGG ATG ACT GTA ACC ATT AAA ACT AAT GCC TGT CAT AAT GGA 240 Ile Thr Gly Met Thr Val Thr Ile Lys Thr Asn Ala Cys His Asn Gly 70 75 GGG GGA TTC AGC GAA GTT ATT TTT CGT 267 Gly Gly Phe Ser Glu Val Ile Phe Arg INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 89 amino acids TYPE: amino acid CD) TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Lys Lys Thr Leu Leu Ile Ala Ala Ser Leu Ser Phe Phe Ser Ala 1 5 10 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly Lys Val Glu Tyr Thr 25 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys Glu 40 Leu Phe Thr Asn Arg Trp Asn Leu Gln Ser Leu Leu Leu Ser Ala Gln 55 *Ile Thr Gly Met Thr Val Thr Ile Lys Thr Asn Ala Cys His Asn Gly *65 70 75 Gly Gly Phe Ser Glu Val Ile Phe Arg INFORMATION FOR SEQ ID 52 Wi SEQUIENCE CHARACTEISTICS: CA) LENGTH: .954S bige pa irs 0(B) TYPE: nucleic acid-- STRANDHDNESS: doiible TOPOLOGY-: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KE-Y: CDS LOCATION: 1..954 Cxi) SEQUENCE DESCRIPTION: SEQ ID ATG AAG TGT ATA TTA TTT AAA TGG GTA CTG T.GC CTG TTA CTG GGT TTT 48 Met Lys Cys Ile LeU Phe Lys Trp, V al Leu Cys Leu Leu Leu Gly Phe 10 TCT TCG GTA TCC TAT TCC COG GAG TTrT ACG ATA GAC TTT TCG ACC CAA 96 Ser Ser Val Ser Tyr Ser Arg Giu Phe Thr Ile Asp Phe Ser Thr Gin 25 CAA AGT TAT GTC TCT TCG TTA AAT AGT ATA CGG ACA GAG ATA TC!G ACC 144 Gin Ser Tyr Val Ser Ser Leu Asn Ser Ile Arg Thr Giu Ile Ser Thr .40 CCT CTT GAA CAT ATA TCT CAG GGG ACC ACA TCG GTG TCT GTT ATT AAC 192 Pro Leu Glu His Ile Ser Gin Gly Thr Thr Ser Vai Ser Val Ile Asn 55 CAC ACC CAC GGC AGT TAT TTT GCT GTG GAT ATA CGA GGG CTT GAT GTC 240.

His Thr His Gly Ser Tyjr Phe Ala Val Asp Ile Arg Giy Leu Asp Val 70 75 TAT CAG GCG CGT TTT GAC CAT CTT CGT CTG ATT ATT GAG CAA AAT AAT 288 Tyr Gin Ala Arg Phe Asp His Leu Axg Leu Ile Ile Giu Gin Asn Asn 90 *TTA TAT GTG GCA GGG TTC GTT AAT ACG GCA ACA AAT ACT TTC TAC CGT 336 *Leu Tyr Val Ala Gly Phe Val Asn Thr Alai Thr Asn Thr Phe Tyr Arg *100 105 110 TTT TCA GAT TTT ACA CAT ATA TCA GTG CCC GOT GTG ACA ACG OTT TCC 384 Phe Ser Asp Phe Thr His Ile Ser Val Pro Giy Val Thr Thr Val Ser '115 1 20 125 ATG ACA ACG GAC AGC AGT TAT ACC ACT CTG CAA CGT GTC GCA GCG CTG 432 **Met Thr Thr Asp Ser Ser Tyr Tbr Thr Leu Gin Arg Val Ala Ala Leu 130 135 140 GAA CGT TCC GGA ATG CAA ATC AGT CGT CAC TCA CTG GTT TCA TCA TAT 480 Glu Arg Ser Gly Met Gin Ile Ser Arg His Ser Leu Vai Ser Ser Tyr 145 150 155 160 0**CTG GCG TTA ATG GAG TTC AGT GGT AAT ACA ATG ACC AGA GAT GCA TCC 528 Leu Ala Leu Met Glu Phe Ser Gly Asn Thr Met Thr Arg Asp Ala Ser 165 170 175 *0*AGA GCA GTT CTG CGT 'TT GTC ACT GTC ACA GCA GAA GCC TTA CGC TTC 576 Arg-_Aja Val Leu Arg Phe Val Thr Val Thr Ala Glu Ala Leu Arg Phe 180 185 190 OAGG -CAG ATA CAG AGA GAA TTT CGT CAG GCA CTG TCT GAA ACT GCT CCT 624 Arg Gin Ile Gin Arg Giu Phe Arg Gin Ala Leu Ser Glu Thr Ala Pro 195 200 205 GTG TAT ACG ATG ACG CCG GGA GAC GTG GAC CTC ACT CTG AAC TGG GGG 672 Val Tyr Thr Met Thr Pro Gly Asp Val Asp Leu Thr Leu Asn Trp Giy 210 215 220 53

CGA

Arg 225

GTG

Val

GCC

Ala

GTG

Val

GTT

Val

TTT

Phe 305 (2) ATC AGC AAT GTG CTT CCG GAG TAT, .CGG'GGX GAG Ile Ser Asn Val Leu Pro Glu Tyr, Arg Giy'Giu 230 -'235- GGG AGA ATA TCC TTT MAT AAT ATA TCA GCG ATA Gly Arg Ile Ser Phe Asn Asn le Ser Ala Ile 245 250 GTT ATA CTG AAT TGC CAT CAT CAG GGG GCG CGT Val Ile Leu Asn Cys His His Gin Giy Ala Arg 260 265 MAT GAA GAG AGT CAA CCA GAM TGT CAG ATA ACT Asn Giu Glu Ser Gin Pro Giu Cys Gin Ile Thr 275 280 ATA AAA ATA AAC AAT ACA TI'A TdG GMA JGT MAT Ile Lys Ile Asn Asn Thr Leu Tx-p Giu Ser Asn 290 295 300 CTG MAC AGA MAG TCA CAG TTT TTA TAT ACA ACG Leu AsYI Arg Lys Ser Gin Phe Leu Tyr Thr Thr 310 315 INFORMATION FOR SEQ ID NO.-6: SEQUENCE CHARACTERISTICS: LENGTH: 318 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: GAT GGT GTC AGAt Asp G ly Val Arg 240 CTG GGG ACT GTG Leu Giy ThrVa 255 TCT GTT CGC GCC Ser Val Aro Ala 270 GGC GAC AGO CC>- Gly Asp Arg Pro 285 ACA OCT OCA GCG Thr Ala Ala Ala GOT AMA Gly Lys Met Lys Cys Ile Leu- Phe Lys Trp Val Leu Cys Leu Leu Leu *0 9 @0O* 6 .9 9 .9 *9 6* 9 6 *94 S 6* Ser Gin Pro His Tyr Leu Phe M(Ft Glu 145 Leu Ser Val Sex- 20 Ser Tyr Val 35 Leu Glu His Thr His Gly Gin Ala Arg Tyr Val Ala 100 Ser Asp Phe 115 Thr Thr Asn 130 Arg Ser Gly Ala Leu Met Glu Phe 25 Asn Ser 40 Oly Thr Ala Val Leu Arg Asn Thr 105 Ser Val 120 Thr Thr Ser Arg Gly Asn Phe Ser Glu Ile Ser Val Gly Leu Glu Gin Thr Phe 110 Thr Thr 125 Val Ala Val Ser Arg Asp Gly Phe Thr Gln Ser Thr Ile Asr.

Asp Val Asn Asn Tyr Arg Val Ser Ala Leu Ser Tvr 160 Ala Ser 175 9* 6

S

Arg Ala Val Leu Arg Phe Val Thr Val 185 Thr Ala Glu Ala Leu Arg Phe 190 54 Arg Gin Ile Gin Arg Giu Phe Arg Gin Ala Leul Ser Giu Thr Ala 195 200 205 Val Tyr Thr Met Thr Pro Gly Asp Val Asp Leu Thr Leu Asn Trp 210 215 220 Arg Ile Ser Asn Val Leu Pro Giu Tyr Arg Giy Giu Asp Gly Val 225 230 235 Val Gly Arg Ile Ser Phe Asn Asn Ile Ser Ala Ile Leu Gly Thr 245 250 255 Ala Val Ile Leu Asn Cys His His Gin Giy Ala Arg Ser Vai Arg 260 265 270 Val Asn Giu Giu Ser Gin Pro Giu Cys Gin Ile Thr Gly Asp Arg 275 280 285 Vai Ile Lys Ile Asn Asn Thr Leu Trp Giu Ser Asn Thr Ala Ala 290 295 300 Phe Leu Asn Arg Lys Ser Gin Phe Leu Tyr Thr Thr Giy Lys 305 310 315 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 267 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: iinear (ii) MOLECULE TYPE: DNA (genoniic) (ix) FEATURE: NAME/KEY: COS LOCATION: 1. .267 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATG AAG AAG ATG TTT ATG GCG GTT TTA TTT GCA TTA GCT TCT GTT I Met Lys Lys Met Phe Met Ala Val Leu Phe Ala Leu Aia Ser Val 1 S 10 GCA ATG GCG GCG GAT TGT GCT AAA GGT AAA ATT GAG TTT TCC AAG I Aia Met Aia Ala Asp Cys Ala Lys Gly Lys Ile Giu Phe Ser Lys 7 25 AAT GAG GAT GAC ACA TTT ACA GTG AAG GTT GAC GGG AAA GAA TAC 'I Asn Giu Asp Asp Thr Phe Thr Vai Lys Val Asp Gly Lys Giu Tyr T 40 ACC AGT CGC TGG AAT CTG CAA CCG TTA CTG CAA AGT GCT CAG TTG A Thr Ser Arg Trp Asn Leu Gin Pro Leu Leu Gin Ser Ala Gin Leu T s0 55 GGA ATG ACT GTC ACA ATC AAA TCC AGT ACC TGT GAA TCA GGC TCC G Gly Met Thr Val Thr Ile Lys Ser Ser Thr Cys Giu Ser Gly Ser G 70 TTT GCT GAA GTG CAG TTT AAT AAT GAC Phe Ala Giu Val Gin Phe Asn Asn Asp 240 55 0 INFORMATION -FOR -SEQ ID_ NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 89 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECUILE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Lys Met Phe Met Ala Val Leu Phe 1 5 10 NO:8: Ala Leu Ala Met Ala Ala Aso Cys Ala Lys Gly Lys Ile Giu 25.

Asn Giu Asp Asp Thr Phe 'Thr Val Lys Val Asp Gly 40 Thr Ser Arg Trp Asn Leu Gln Pro Leu Leu Gin Ser 55 60 Ala Ser Val Asn Phe Ser Lys Tyr Lys Glu Tyr Trp Ala Gln Leu Thr Gly Met Thr Val Thr Ile Lys Ser Ser Thr Cys Giu Ser Giy Ser Giy 70 75 Phe Ala Giu Val Gin Phe Asn Asn Asp INFORMATION FOR SEQ ID NO:9: Wi SEQUENCE CHARACTERISTICS: LENGTH: 1241 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genonic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

ATGAAAATAA

GTGGCGAAGG

GTCATTCGCT

CTGATGATTG

GCAGAGGAAG

ACAGGAT'rrG

ACCTTTCCAG

CGTGTTGCAG

TATCTGGATT

TTACGGTTTG

CGTACAACAC

CTTACATTGA

GTTCGTGTAG

ATACTGAATT

TTATTTTTAG-

AATTTACCTrl

CTGCAATAGG

ATAGTGGCTC

GGCGGTTTAA

TTAACAGGAC

GTACAACAGC

GGATCAGTCG

TAATGTCGCA

TTACTGTGAC

TGGATGATCT

ACTGGGGAAG

GAAGAATTTC

GTCATCATCA

AGTGCTAACT TTTTTCTTTG TTATCTTTTC

*AGACTTCTCC

TACTCCATTA

AGGGGATAAT

TAATCTACGG

AAATAATGTT

GGTTACATTG

TACGGGGATG

TAGTGGAACC

AGCTGAAGCT

CAGTGGGCGT

GTTGAGTAGC

TTTTGGAAGC

TGCATCGCGA

ACTGCAAAGA

CAGACTATTT

CGTATGTAGA

CATCAGGAGG

AGTTAATGTG

TTCGCTGAAT

TACGTCTTTA

AGGGATAGAT

TTGTTTGCAG TTGATGTCAG

CTTATTGTTG

TTTTATCGCT

TCTGGTGACA

CAGATAAATC

TCACTGACGC

TTACGTTTTC

TcTTATGTrAA

GTCCTGCCTG

ATTAATGCAA

GTTGCCAGAA

AACGAAATAA TTTATATGTG TTGCTGATTT

TTCACATGTT

GTAGCTATAC CACGTTACAG GCCATTCGTT GACTACTTCT AGTCTGTGGC AAGAGCGATG GGCAAATACA GAGGGGATTT TGACTGCTGA AGATGTTGAT ACTATCATGG ACAAGACTCT TTCTGGGAAG CGTGGCATTA TGGCATCTGA TGAGTTTCCT 120 180 240 300 360 420 480 540 600 660 720 780 840 900 TCTATGTGTC CGGCAGATGG AAGAGTCCGT GGGATTACGC ACAATAAAAT

ATTGTGGGAT

56 TCATCCACTC TGGGGGCAAT TCTGATGCGC AGAACTATTA- GCAGTTGAAC AGGGGGTAAA TAAAGGAGTT AAGCATGAAA AAAACATTAT TAATAGCTGC ATCGCTTTCA TTTTTTCAG CAAGTGCGCT GGCGACGCCT GATTGTGTAA CTGGAAAGGT GGAGTATACA AAATATAATG ATGACGATAC CTTTACAGTT AAAGTGGGTG ATAAAGAATT ATTTACCAAC AGATGGAATC TTCAGTCTCT TCTTCTCAGT GCGCAAATTA CGGGGATGAC TGTAACCATT AAAACTAATG CCTGTCATAA TGGAGGGGGA rTCAGCGAAG TTATTTTTCG T INFORMATION FOR SEQ ID NO:lO: SEQUENCE CHARACTERISTICS: LENGTH: 1235 base pairs TYPE: nucleic acid- STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEO ID ATGAAGTGTA TATTATTTAA ATGGGTACTG TGCCTGTTAC TGGGTTTTTC TTCGGTATCC

TATTCCCGGG

AGTATACGGA

TCTGTTATTA

TATCAGGCGC

GGGTTCGTTA.

GTGCCCGGTG

GTCGCAGCGC

CTGGCGTTA.A

CGTTTTGTCA

CAGGCACTGT

CTGAACTGGG

GTGGGGAGAA*

AATTGCCATC

TGTCAGATAA

ACAGCTGCAG

AGTTAAGCAT

TGGCGGCGGA

TTACAGTGAA

TGCAAAGTGC

AGTTTACGAT

CAGAGATATC

ACCACACCCA

GTTTTGACCA

ATACGGCAAC

TGACAACGGT

TGGAACGTTC

TGGAGTTCAG

CTGTCACAGC

CTGAAACTGC

GGCGAATCAG

TATCCT TTAA

ATCAGGGGGC

CTGGCGACAG

CGTTTCTGAA

GAAGAAGATG

TTGTGCTAAA

GGTTGACGGG

TCAGTTGACA

AGACTTTTCG

GACCCCTCTT

CGGCAGTTAT

TCTTCGTCTG

AAATACTTTC

TTCCATGACA

CGGAATGCAA

TGGTAATACA

AGAAGCCTTA

TCCTGTGTAT

CA.ATGTGCTT

TAATATATCA

GCGTTCTGTT

GCCTGTTATA

CAGAAAGTCA

TTTATGGCGG

GGTAAAAT1TG

AAAGAATACT

GGAATGACTG

ACCCA.ACAA-A

GAACATATAT

TTTGCTGTGG

ATTATTGAGC

TACCGTTTT

ACGGACAGCA

ATCAGTCGTC

ATGACCAGAG

CGCTTCAGGC

ACGATGACGC

CCGGAGTATC

GCCATACTOG

CGCGCCGTGA

AAAATAAACA

CAGTTTTTAT

TITTATTTGC

AGTTTCCAA

GGACCAGTCG

TCACAATCAA

GTTATGTCTC

CTCAGGGGAC

ATATACGAGG

AAAATAATTT

CAGATTTTAC

GTTATACCAC

ACTCACTGGT

ATGCATCCAG

AGATACAGAG

CGGGAGACGT

GGGGAGAGGA

GGACTGTGGC

ATGAAGAGAG

ATACATTATG

ATACAACGGG

ATTAGCTTCT

GTATAATGAG

CTGGA.ATCTG

ATCCAGTACC

TTCGTTAAAT

CACATCGGTG

GCTTGATGTC

ATATGTGGCA

ACATATATCA

TCTGCAACGT

TTCATCATAT

AGCAGTTCTG

AGAATTTCGT

GGACCTCACT

TGGTGTCAGA

CGTTATACTG

TC?.ACCAGAA

GGAAAGTAAT

TAAATAAAGG

GTTAATGCAA

GATGACACAT

CAACCGTTAC

TGTGAATCAG

960 1020 1080 1140 1200 1241 120 180 240 300 360 420 480 .540 600 660 720 780 840 900 960 1020 1080 1140 1200 1235 GCTCCGGATT TGCTGAAGTG CAGTTTAATA ATGAC INFORMATION FOR SEQ ID NO:11: SEQUENCE CMARACTERISTICS: LENGTH: 8 amino acids S7 TYPE: amino. acid. 9 STRANDEDNESS: unknown -TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Leu Glu His His His His His His 1 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GCCATATGAA AATAATTATT TTTAGAGTG 29 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GGCTCGAGAC TGCTAATAGT TCTGCGCAT 29 S(2) INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: S(A) LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: 'single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCCATATGAA AAAAACATTA TTAATAGC 28 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single S TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID GGCTCGAGAC GAAAAATAAC TTCGCTGAA 29 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: 58 LENGTH: '29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCCATATGAA GTGTATATTA TTTAAATGG 29 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GGCTCGAGTT TACCCGTTGT ATATAAAAAC INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CGCATATGAA GAAGATGTTT ATGGCG 26 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGCTCGAGGT CATTATTAAA CTGCACTTC 29 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 969 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..969 (xi) SEQUENCE DESCRIPTION: SEQ ID ATG AAA ATA ATT ATT TTT AGA GTG CTA ACT TTT TTC TTT GTT ATC TTT 48 59 0 Met Lys le, le Ile 1 TCA GTT AAT OTG GTG Ser Val Asn Val Val AAG ACG TAT GTA GAT Lys Thr Tyr Val Asp CCA TTA CAG ACT ATT Pro Leu Gin Thr Ile AGT GGC TCA 000 GAT Ser Gly Ser Gly Asp GCA GAG GAA 000 CG Ala Glu Glu Gly Arg AAT TTA TAT GTG ACA Asn LeuTyr Val Thr 100 CGC TTT GCT OAT TTT Arg Phe Ala-Asp Phe l'11 ACA rro TCT GOT GAC Thr Leu Ser Oly Asp 130 ATC AGT CGT ACG GGG Ile Ser Arg Thr Gly 145 TAT CTG GAT TTA ATG Tyr Leu Asp Leu Met 165- OCA AGA OCO ATG TTA Ala Arg Ala Met Leu 180 TTT CGG CAA ATA CAG Phe Arg Gin Ile Gin 195 000 COT TCT TAT OTA Oly Arg Ser Tyr Val 210 TOG GOA AGO TTG AGT Trp Gly Arg Leu Ser 225 OTT COT OTA OGA AGA2 Val Arg Val Giy Arg 245 AOC GTO OCA TTA ATA Ser Val Ala Leu Ile 260 AGA ATO OCA TCT OAT Arg Met Ala Ser Asp C 275 Phe Axg Val Leu -Thr .,-Phe:PRhe :Phe!2Vat-.Ii&. Phe OCG AAG GAA TTT ACC TTAGAC .TC; TCO 'ACT-CA Ala Lys Giu PIhe Thr Leu Asp Phe Ser Thr Ala 25-. 3 30. a a a a. a.

a

TCG

Ser

TCA

Ser

AAT

Asin 70

TTT

Phe

GGA

Gly

TCA

Ser

AGT

Ser

ATO

Met 150

TCO

Ser

CG

PArg PArg

P.TG

Me t k.GC Ser 230 Ile

'TG

,eu

;AG

;iu

CTC

Let

TC;

Ser 55

TTG

Leu

AAT

Asn Phe

CAT

His

AGC

Ser 135

CAG

Gin

CAT

His

TTT

Phe

OGA

Gly

ACT

Thr 215

OTC

Val

TCT

Ser

AAT

Asn

TTT

Phe

AAT

Asn

OGA

Gly

ITTT

Phu

AAT

Asn

OTT

Val

OTT

Val 120

TAT

Tyr

ATA

Ile

AOT

Ser

OTT

Val

TTT

Phe 200

OCT

Ala

CTO

Leu Trr Phe

TOT

Cys

CCT

Pro 280 -GTC ATT Val Ile GOT ACO TCT, TTA CTG ATG ATT OAT Oly Thr Ser Leu Leu Met Ile Asp OCA OTT GAT OTC;-*AOA 000 ATA OAT Ala VadJWsp Val Arg Gbly l' A-)sp 75 CTA COG CTT ATT OTT GAA COA.'AAT Leu Arg Leu Ile Val Oiu Arg Asn 90 AAC AGO ACA AAT- AAT OTT TTT TAT Asn Arg Thr Asn Asn Val Phe Tyr 105 110 ACC .TTT CCA. GOT ACA ACA OCO OTT Thr Phe Pro,. Oly Thx Thr Ala Val 125 ACC ACO TTA- CAG COT OTT OCA 000 Thr Thr Leu Gin Arg Val Ala Oly 140 AAT COC CAT TCG TTO ACT ACT TCT Asn Arg His Ser Leu Thr Thr Ser 155 160 GGA ACC TCA CTO ACO CAG TCT GTG Oly Thr Ser Leu Thr Gin Ser Vai 170 175 ACT OTO ACA OCT GAA GCt TTA COT Thr Val Thr Ala Giu Ala Leu Arg 185 190 CO?. ACA ACA CTG OAT OAT CTC AG? Arg Thr Thr Leu Asp Asp Leu Ser 205 OAA OAT OTT OAT CTT ACA TTO AAC Oiu Asp Val Asp Leu Thr Leu Asn 220 CCT GAC TAT CAT OGA CAA GAC TCT Pro Asp Tyr His Oly Gin Asp Ser 235 240 GGA AOC ATT AMT OCA ATT CTO GGA Oly Ser Ile Ass Ala Ile Leu Oly 250 255 CAT CAT CAT OCA TCG COA OTT 0CC His His His Ala Ser Arg Val Ala 265 270 TCT ATO TOT CCO OCA OAT OGA AGA Ser Met Cys Pro Ala Asp Oly Arg CGC TCT. OCA ATA GOT ACT Arg Ser Ala Ile Giy Thr 144 192 240 288 336 384 432 480 528 576 624 672 720 768 816 864 a. a a 285 60 GTC CGTcCGG ATT AC-CC ATA-,T TG7 G',GT TCAW- TCC-.!ACT CTG- Val Arg Gly Ile Thr His Asn Lys le Leu Trp Asp Ser Ser-Thr Leu 290 295 300 GGG GCA ATT CTG ATG CGC AGA ACT AT1T AGC AGT CTC GAG CAC CAC' CAC Gly Ala Ile Leu Met Arg Arg Thr le Ser Ser Leu Giu His His His 305 310 315 320 CAC CAC CAC His His His INFORMATION FOR SEQ ID NO:2i: SEQUENCE CHARACTERISTICS: LENGTH: 323 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE Met Lys Ile Ile Ile Ser Val Asn Val Val Lys Thr Tyr Val Asp Pro Leu Gin Thr Ile Ser Gly Ser Giy Asp 65 Ala Glu Giu Gly Arg Asn Leu Tyr Val Thr 100 Arg Phe Ala Asp Phe 115 Thr Leu Ser Giy Asp 130 Ile Ser Arg Thr Gly 145 Tyr Leu Asp Leu Met 165 Ala Arg 'Ala Met Leu 180 Phe Arg Gin Ile Gin 195 Gly Arg Ser Tyr Val 210 Trp Gly Arg Leu Ser 225 Val Arg Val Giy Arg 245 Ala Lys Ser Leu Ser Ser 55 Asn Leu 70 Phe Asn Glv Phe Ser His Ser Ser 135 Met Gin 150 Ser His Arg Phe Arg Gly Met Thr 215 Ser Val 230 Phe Thr Val Ile Gly Thr Ala Val Leu Arg 90 Asn Arg 105 Thr Phe Thr Thr Asn Arg Gly Thr 170 Thr Vai 185 Arg Thr Glu Asp Pro Asp DESCRIPTION: SEQ ID Phe Arg Val Leu Thr NO :21: Phe Phe Phe Leu Asp Phe Arg Ser Ala Ser Leu Leu Asp Vai Arg 75 Leu Ile Val Thr Asn Ash Pro Gly Thr Leu Gin Arg 140 His Ser Leu 155 Ser Leu Thr Thr Ala Giu Thr Leu Asp 205 Vai Asp Leu 220 Tyr His Gly 23 5 le Asn Ala Ile Phe .Thr Ala Gly Thr Ile Asp) Ile Asp) Arg Asn Phe Tyr Ala Val Ala Gly Thr Ser 160 Ser Val 175 Leu Arg Leu Ser Leu An Asp Ser 240 Leu Gly 255 Ile Ser Phe Gly Ser .250 61 0 Val Ala Leu Ile Leu Asn Cys His His His Ala Ser Arg Val -Ala 260 265 270 Met Ala Ser Asp Glu Phe Pro Ser Met Cys Pro Ala Asp Gly Arg 275 280 285 Arg Gly Ile Thr His Asn Lys Ile Leu Trp Asp Ser Ser Thr Leu 290 295 300 Ala Ile Leu Met Arg Arg Thr Ile Ser Ser Leu Glu His His His 310 315 320 His His INFORMATION FOR SEQ ID NO:22: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 294 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: CA) NAME/KEY: CDS LOCATION: 1._294 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ATG AAA AAA ACA TA TTA ATA GCT GCA TCG CTT TCA T?1T 9* a Met Lys Lys Thr 1 AGT GCG CTG GCG Ser Ala Leu Ala 20 AAA TAT AAT GAT Lys Tyr Asn Asp 35 TTA TTT ACC AAC Leu Phe Thr Asn ATT ACG dGG ATG Ile Thr Gly Met GGG GGA TTC AGC Gly Gly Phe Ser CAC TG His

INFORMATION

CCT GAT TGT Pro Asp Cys GAT ACC TTT Asp Thr Phe 40 TGG AAT CT? Trp Asn Leu 55 GTA ACC ATT Val Thr Ile 70 GTT AT? TT Val Ile Phe 10 GTA ACT GGA Val Thr Gly 25 ACA GTT AAA Thr Val Lys CAG TCT CT? Gin Ser Leu AAA ACT AAT Lys Thr Asn 75 CGT CTC GAG Arg Leu Glu AAG GTG Lys Val GTG GGT Val Gly CTT CTC Leu Leu GCC TGT Ala Cys CAC CAC His His Leu Ile Ala Ala Ser Leu Ser Phe TT TCA GCA Phe Ser Ala GAG TAT ACA Glu Tyr Thr GAT AAA GAA Asp Lys Glu AGT GCG CPA Ser Ala Gin CAT AAT GGA His Asn Gly CAC CAC CAC His His His 294 FOR SEQ ID NO:23: a.

a Ci) SEQUENCE CHARACTERISTICS: LENGTH: 91 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Met Lys Lvs Thr Leu Leu Ile Ala Ala Ser Leu Ser Phe Phe Ser Ala -62- 0 10 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly Lys Val Giu Tyr 2530 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys Val Gly Asp Lys 40 Leu Phe Thr Asn Arg Trp Asn Leu Gin Ser Leu Leu Leu Ser Ala 55 Ile Thr Gly Met Thr Val Thr Ile Lys Thr Asn Ala Cys His Asn 70 Gly Gly Phe Ser Giu Val Ile Phe Arg Leu Giu His His His His 90 INFORMATION FOR SEQ ID NO:24: Ci) SEQUENCE CHARACTERISTICS: LENGTH: 981 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .981 (xi) SEQUENCE DESCRIPTION: SEQ ID 140:24: ATG AAG TGT ATA TTA TT met Lys Cys Ile Leu Phe 1 5 TCT TCG OTA TCC TAT TCC Ser Ser Val Ser Tyr Ser 20 CAA AGT TAT GTC TCT TCG Gin Ser Tyr Val Ser Ser CCT =T GAA CAT ATA TCT Pro Leu Glu His Ile Ser CAC ACC CAC GGC AGT TAT His Thr His Gly Ser Tyr 70 TAT CAG GCG CGT TTT GAC Tyr Gin Ala Arg Phe Asp TTA TAT GTG GCA GGG TTC Leu Tyr Vai Ala Giy Phe 100 TTT TCA GAT TTT ACA CAT Phe Ser Asp Phe Thr His 115 ATG ACA ACG GAC AGC AGT Met Thr Thr Asp Ser Ser 130 AAA TGG GTA CTG TGC CTG TTA Lys Trp Val Leu Cys Leu Leu 10 CGG GAG TTT ACG ATA GAC TTT Arg Giu Phe Thr Ile Asp Phe 25 TTA AAT AGT ATA CGG ACA GAG Leu Asn Ser Ile Arg Thr Glu 40 CAG GGG ACC ACA TCG GTG TCT Gin Gly Thr Thr Ser Vai Ser 55 TTT GCT GTG GAT ATA CGA GG Phe Aia Vai Asp Ile Arg Gly 75 CAT CTT CGT CTG ATT ATT GAG His Leu Arg Leu Ile Ile Giu 90 OTT AAT ACO GCA ACA AAT ACT Val A-sn Thr Ala Thr Asn Thr 105 ATA TCA GTG CCC GOT GTG ACA Tle Ser Val Pro OWy Val Thr CTG GGT TTT Leu Giy Phe is TCO ACC CAA Ser Thr Gin ATA TCG ACC Ile Ser Thr OTT ATT AAC Vai Ile Asn CTT GAT GTC Leu Asp Val CAA AAT AAT Gin Asn Asn TTC TAC CGT Phe Tyr Ar~g 110 ACG GTT-TCC Thr Val Ser OCA GCO CTG Ala Ala Leu

ACC

Thr ACT CTG CAA CGT Thr Leu Gin Arg 140 125

GTC

Val 63 GAA COT TCC GGA ATO CAA ATC AGT' CGT CAC TCA CTG GTT: TCA TCA TAT Glu Arg Ser Gly Met Gin Ile Ser Arg His Ser Leu Val Ser Ser Tyr 145 150 -4.5 -160 CTG GCC TTA ATG GAG TTC Leu Ala Leu Met Giu Phe 165 AGA GCA GTT CTG CGT TTT Arg Ala Val Leu Arg Phe 180 AGG CAG ATA CAG AGA GAA Arg Gin Ile Gin Arg Giu 195 GTO TAT ACG ATG ACG CCG Val Tyr Thr Met Thr Pro 210 CGA ATC AGC AAT GTG CTT Arg Ile Ser Asn Val Leu 225 230 GTG GGAGA ATA TCC TTT Val Gly Arg Ile Ser Phe 245 GCC GTT ATA CTG AAT. TGC Ala Val Ile Leu Asn Cys 260 GTG AAT GAA GAG AGT CAA Val Asn Oiu Giu Ser Gin 275 ACT OCT AAT Ser Gly Asn.

GTC ACT GTC Val Thr Val 185 TTT COT CAG Phe Arg Gin 200 ACC AGA GAT Thr Arg Asp GAA GCC TTA Giu Ala Leu 190 TCT GAA ACT GCA TCC Ala -Ser 175 COC TTC Arg Phe GCT CCT Ala Leu Ser Giu Thr. Ala Pro

GGA

Gly 215

CG

Pro

AAT

Asn

CAT

His

CCA

Pro GAC OTG GAC CTC ACT Asp Val Asp Leu'Thr 220 GAG TAT COG GGA GAG Giu Tyr Arg Gly Giu 235 AAT ATA TCA OCG ATA Asn Ile Ser Ala Ile 250 CAT-CAG 000 GCG CGT His Gin Gly Ala Arg 265 GAA TOT CAG ATA ACT Giu Cys Gin Ile Thr 280 205 CTG AAC TOG 000 Leu Asn Trp Gly GAT GOT GTC AGA Asp Oly Vai Arg 240 CTG 000 ACT OTG Leu Gly Thr Val 255 TCT OTT*CGC 0CC Ser Vai Arg.Ala 270 GGC GAC AGO CCT Oly Asp Arg Pro 285 ACA OCT OCA OCO Thr Ala Ala Ala GGT AAA CTC GAG Gly Lys Leu Giu 320 480 528 576 624 672 720 768 816 864 912 960 981 6O S S

S

0@ S S S 4

*SSS

*4*S OTT ATA AAA ATA AAC AAT ACA TTA TOG GAA Val Ile Lys Ile Asn Asn Thr Leu Trp Oiu 290 295 TTT CTO AAC AGA AAG TCA CAG TTT TTA TAT Phe Leu Asn Arg Lys Ser Gin Phe Leu Tyr 305 310 CAC CAC CAC CAC CAC CAC TO His His His His His His 325 INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 326 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Me-t Lys Cys Ile Leu Phe Lys Trp Val Leu 1 5 10 Ser Ser Val Ser Tyr Ser Arg Oiu Phe Thr 25 Gin Ser Tyr Val Ser Ser Leu Asn Ser Ile 3S 40 Pro Leu Giu His Ile Ser Gin Oly Thr Thr s0 55 AGT AAT Ser Asn 300 A.CA ACO Thr Thr 315 .4 5 0

S

NO: Cys Leu Leu Leu Oly Phe Ile Asp Phe Ser Thr Gin k.rg Thr Giu Ile Ser Thr 3er Val Ser Val Ile Asn 64 His Thr His -Gly Ser Tyr 70 Phe Ala Val: Asp -l11 Arg Giy :Leu -AspVal 75 so- 8 Tyr Gin Ala Arg.

Leu Tyr Val Ala 100 Phe Ser Asp Phe 115 Met Thr Thr Asp 130 Glu Arg Ser Gly 145 Leu Ala Leu Met Arg Ala Val Leu 180 Arg Gin Ile Gin 195 Val.Tyr Thr Met 210 Arg Ile Ser Asn 225 Val Gly Arg Ile Ala Val Ile Leu 260 Val Asn Glu Glu 275 Val Ile Lys Ile 290 *Phe Leu Asn Arg 305 His His His His Phe Asp His Gly Phe Val Thr His Ile Ser Ser Tyr 135 Met Gin Ile 150 Giu Phe Ser 165 Arg Phe Val Arg Glu Phe Thr Pro Gly 215 Val Leu Pro 230 Ser Phe Asn 245 Asn Cys His Ser Gin Pro Asn Asn Thr 295 Lvs Ser Gin 310 His His 325 Leu Arg Leu 90 Asn Thr Ala 105 Ser Val Pro 120 Thr Thr Leu Ser Arg His Gly Asn Thr 170 Thr Val Thr 185 Arg Gin Ala 200 Asp Val Asp Glu Tyr Arg Asn Ile Ser 250 His Gin Gly 265' Glu Cys Gin 280 Leu Trp Glu Phe Leu Tyr Ile Ile Giu Gin Asn Thr 'Asn Thr Phe Tyr 110 Gly Val Thr Thr Val 125 Gin Arg Val Ala Ala 140 Ser Leu Val Ser Ser 155 met Thr Arg Asp Ala 175 Ala Giu Ala Leu Arg 190 Leu Ser Giu Thr. Ala 205 Leu Thr Leu Asn Trp, 220 Gly Glu Asp Gly Val 235 Ala Ile Leu Gly Thr 255 Ala Arg Ser Val Arg 270 Ile Thr Gly Asp Arg 285 Ser Asn Thr Ala Ala 300 Thr Thr Gly Lys Leu 315 Asn Arg Ser Leu Tyr 160 Ser Phe Pro Gly Arg 240 Val Ala Pro Ala Glu 320 :0.6.0 INFORMATION FOR SEQ ID, NO:26: Wi SEQUENCE CHARACTERISTICS: LENGTH: 294 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (genomic)

FEATURE:

NAME/KEY: CDS LOCATION: 1.-.294 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: ATG AAG AAG ATG TTT ATG GCG GTT TTA TTT GCA TTA GCT TCT GTT AAT Met Lys Lys Met Phe Met Ala Val Leu Phe Ala Leu Ala Ser Vai Asn 1 5 10 65 GCA ATG GCG.GCG -~GAT TGT-GCT :AAA',GGT .AAA Ala Met Ala Ala Asp eys Ala :Lys Gly Lys 25 AAT GAG GAT GAC ACA TTT ACA GTG--AAG GTT Asn Glu Asp Asp Thr Phe Thr Val Lys Val 40 ACC AGT CGC TGG AAT CTG CAA CCG TTA CTG Thr Ser Arg Trp Asn Leu Gin Pro Leu Leu 55 GGA ATG ACT GTC ACA ATC AAA TCC AGT ACC Gly Met Thr Val Thr Ile Lys Ser Ser Thr 70 TTT GCT GAA GTG CAG TTT AAT AAT GAC CTC Phe Ala Glu Val Gin Phe Asn Asn. Asp Leu 90 CAC TG His INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids TYPE: amino acid TOPOLOGY:. linear (i)MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Lye Met Phe Met Ala Val Leu Phe 1 .5 Ala Met Ala Ala.Asp Cys Ala Lye Gly Lys 20 Asn Giu Asp. Asp Thr Phe Thr Val Lys Val 40 Thr Ser Arg TrD Asn Leu Gin Pro Leu Leu 55 Gly Met Thr Val Thr Ile Lys Ser Ser Thr 70 Phe Ala Giu Val Gin Php Asn Asn Asp Leu His

ATT.

Ile

GAC

Asp

CAA

Gin

TGT

Cys 75

GAG

Glu GAG TTT- TCC AAG TAT Glu Phe Ser Lys Tyr GGG AAA GAA TAC TG Gly Lye Giu.Tyr Trp AGT OCT CAG TTG ACA Ser Ala Gin Leu Thr GAA TCA GGC TCC GGA Giu Ser Gly Ser Gly so CAC CAC CAC CAC CAC His His His His His 96 144 192 240 NO: :27: Ala Leu Ala Ile Glu Phe Asp Gly Lye Gin Ser Ala Cys Oiu Ser Giu His His INFORMATION FOR SEQ ID 110:28: SEQUENCE CHARACTERISTICS: LENGTH: 32 -base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID 110:28: CGGAATTCAA GGAATTTACC TTAGACTTCT CG INFORMATION FOR SEQ ID NO:29: 66 SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE:- nucleic- acid AC) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA* (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GGCTCGAGTC AACTGCTAAT AGTTCTGC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID CGGAATTCCG GGAGTTI'ACG ATAGACTTTT CO INFORMATION FOR SEQ ID N0:31: Wi SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GGCTCGAGTT ATTTACCCGT TGTATATAA INFORMATION FOR SEQ ID NO:32: Wi SEQUENCE CHARACTERISTICS: 'LENGTH: 2127 base pairs TYPE: nucleic acid STRANDED)NESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (geflomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1.-2127 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:- ATO AAA ATA AAA ACA GGT GCA CGC ATC CTC GCA TTA Met Lys Ile, Lys Thr Oly Ala Arg Ile Leu Ala Leu 1 510 ACG ATG ATG TTT TCC 0CC TCG OCT CTC 0CC AAA ATC Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile 20 25

TCC

Ser

OAA

Glu OCA TTA ACG Ala Leu Thr GAA GOT AAA Olu Oly Lys CTC OCT GAA Leu Ala Glu ACC OTT GAO Thr Val Olu CTO OTA ATC TOG ATT AAC GOC GAT AAA GOC TAT AAC GOT Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Oly 40 OTC GOT AAG AAA TTC GAO AAA OAT ACC OGA ATT AAA GTC Val Gly Lys Lys Phe Olu Lys Asp Thr Oly Ile Lys Val 67 CCG GAT AAA. CTG Pro Asp Lys Leu GAA GAG AAA TTC CCA CAG- GTT-GCG GCA ACT GGC Giu Giu Lys Phe Pro .Gln Val*-Ala Ala Thr Gly -75 GAT GGC Asp Gly GCT CAA Ala Gin GAC AAG Asp Lys CTG ATT Leu Ilie 130 AAA GAT Lys Asp 145 CTG GAT Leu Asp CTG CAA Leu Gin TAT GCG Tyr Ala GTG GAT Vai Asp 210 ATr AAA Ile Lys 225 GCT GCC Ala Ala GCA TGG Ala Trp CTG CCG Leu Pro AGC GCA Se r Ala 290 CCT GAC Pro Asp TCT GGC Ser Gly 100 CTG TAT Leu Tvr 115 GCT TAC Ala Tyr CTG CTG Leu Leu AAA GAA Lys Giu GAA CCG Glu Pro 180 TTC AAG Phe Lys 195 AAC GCT Asn Ala AAC AAA Asn Lys TrT AAT Phe Asn TCC AAC Ser Asn 260 ACC TTC Thr Phe 275 GGT ATT Gly Ile ATC rrC TGG GCA CAC GAC CGC Ile Phe Trp Ala His Asp Arg TTG GCT GAA ATC ACC CCG GAC Leu Ala Glu Ile Thr Pro Asp 105 TTT ACC TGG GAT GCC GTA CGT Phe Thr Trp Asp-ika Val Arg 120 ATC GCT GTT GAA GCG TTA TCG Ile Ala Val Glu Ala Leu Ser 135 140 AAC CCG CCA AAA ACC TGG GAA Asn Pro Pro Lys Thr Trp Giu 150 155.

AAA GCG AAA GGT AAG AGC GCG Lys Ala Lys Gly Lys Ser Ala 170 TTC ACC TGG CCG CTG* ATT GCT Phe.Thr Trp Pro LeuIle Ala 185 GAA AAC GGC AAG TAC GAC. ATT Glu Asn Gly Lys Tyr Asp Ile 200 GCG AAA GCG GGT CTG ACC TTC Ala Lys Ala Gly Leu Thr Phe 215 220 ATG AAT GCA GAC ACC GAT TAC Met Asn Ala Asp Thr Asp Tyr 230 235 GGC GAA ACA GCG ATG ACC ATC Gly Glu Thr Ala Met Thr Ile 250 GAC ACC AGCAAA GTG AAT TAT Asp Thr Ser Lys Val Asn Tyr 265 GGT CAA CCA TCC AAA CCG TTC Gly Gin Pro Ser Lys Pro Phe 280 GCC GCC ACT CCG AAC AAA GAG Ala Ala Ser Pro Asn Lys Giu 295 300 TTT GGT GGC TAC Phe Gly Gly Tyr AAA GCG TTC CAG Lys Ala Phe Gin 110 TAC AAC GGC AAG Tyr Asn Gly Lys 123 CTG ATT TAT AAC Leu Ile Tyr Asn GAG ATC CCG GCG Giu Ile Pro Ala 160 CTG ATG TTC AAC Leu Met Phe Asn 175 GCT GAC GGG GGT Ala Asp Gly Gly 190 AAA GAC GTG GGC Lys Asp.Val Gly 205 CTG GTT GAC CTG Leu Val Asp Leu TCC ATC GCA GAA Ser Ile Ala Giu 240 AAC GGC CCG TCC Asn Gly Pro Trp 255 GCT GTA ACG GTA Gly Val Thr Val 270 CTT GCC GTG CTG Val Cly Val Leu 285 CTG GCG AAA GAG Leu Ala Lys Glu 240 288 336 384 432 480 S28* 576 624 672 720 768 816 864 912 68 TTC CTC Phe Leu .:305 AAA GAC GAA AAC TAT cTG CTG ACT GAT- GAA Glu Asn Tyr Leu Leu Thr Asp Giu '310

GT

Gly 315 CTO GAA GCG *GTT AAT Leu Giu Ala Val Asn 320 TCT TAC GAG GAA GAG AAA CCG CTG.GOT GCC GTA GCG CTG AAG Lys Asp Lys Pro Leu Oly Ala Val Ala Leu Lys Ser Tyr Glu Glu 325S 330 335 TTG GCG AAA GAT CCA COT ATT GCC 0CC ACC ATG GAA AAC GCC CAG Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Giu Ass Ala Gin 340 345 350 GOT GAA ATC ATG CCG AAC ATC CCG CAG ATO TCC GCT TTC TGO TAT Oly Glu Ile Met Pro As Ile Pro Gin Met Ser Ala Phe Trp Tyr 355 360 365 i3T .,C1X CT~aC~GTG ATCAACGCTGCCAGC GOTiIGT CAG ACT O'FN.

Val Arg Thr Ala Val Ile Ass Ala Ala Ser Gly Arg Gin Thr Val 370 375 380 GAA GCC CTG AAA *GAC GCG CAG ACT TCG AOC TCG AAC AAC AAC AAC Giu Ala Leu Lys Asp Ala Gin Thr Ser Ser Ser Ass Asn Ass Asfl 385 390 395 AAC AAT AAC AAC AAC CTC 000 ATC GAG GGA AGG ATT TCA GAA TTC Asn Asn Asn Ass Ass Leu Gly Ile Olu Giy Arg Ile Ser Oiu Phe 405 410 .415 GAA TTT ACC TTA GAC TTC .TCG ACT GCA AAG ACG TAT GTA OAT TCG Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp Ser 420 425' 430 .AAT GTC ATT CGC TCT GCA ATA GGT ACT CCA TTA CAG ACT ATT TCA Asn Val Ile Arg'Ser Ala Ile Oly Thr Pro Leu Gin Thr Ile Ser 435 440 .445 GGA G't ACG TCT TTA CTO ATO ATT OAT AOT. GGC TCA 000 GAt AAT Gly Giy Thr Ser Leu Leu Met Ile Asp-Ser Gly Ser Gly Asp Ass 450 455 460 TTT OCA GTT OAT OTC AGA 000 ATA OAT OCA GAO OAA 000 CGG TTT Phe Ala Val Asp Val Arg Gly Ile Asp Ala Glu Giu Gly Arg Phe 465 470 475 AAT CTA CGG CTT ATT OTT GAA CGA AAT AAT TTA TAT GTG ACA GGA Ass Leu Arg Leu Ile Val Oiu Arg Asn Ass Leu Tyr Val Thr Gly 485 490 495 OTT AAC AGO ACA AAT AAT GTT TTT TAT CGC TTT OCT OAT TTT TCA Val Ass Arg Thr Ass Asn Vai Phe Tyr Arg Phe Ala Asp Phe Ser 500 505 510 OTT ACC TTT CCA OGT ACA ACA OCO GTT ACA TTG TCT 00T GAC AGT Val Thr Phe Pro Oly Thr Thr Ala Val Thr Leu Ser Gly Asp Ser 515 520 525 TAT ACC ACO TTA CAG COT GTT GCA 000 ATC AGT COT ACO 000 ATG Tyr Thr Thr Leu Gin Arg Val Ala Oly Ile Ser Arg Thr Oly Met 530 535 .540 ATA AAT COC CAT TCO TTG ACT ACT TCT TAT CTG GAT TTA ATO TCG Ile Ass Arg His Ser Leu Thr Thr Ser Tyr Leu Asp Leu Met Ser 54§ 550 555 AOT 00A ACC TCA CTO ACO CAG TCT OTO OCA AGA OCO ATO TTA CG Ser Oly Thr Ser Leu Thr Gin Ser Vai Ala Arg Ala Met Leu Arg 565 570 575 OTT ACT OTO ACA OCT GAA OCT TTA COT TTT COO CAA ATA CAG A00 Val Thr Val Thr Ala Olu Ala Leu Arg Phe Arg ls Ile Oin Arg Olu

AAA

Lys

GCC

Ala

OAT-

Asp

AAT

Asn 400

AAO

Lys

CTG

Leu

TCA

Ser

TTO

Leu

AAT

Ass 480 Phe

CAT

His

AOC

Se r

CAG

Gin

CAT

His 560 Phe

OGA

Gly 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 69 580 585.

TTT CGT ACA ACA CTG GAT GAT CTC AGT GGG CGT TOT TA Phe Arg Thr Thr Leu Asp Asp Leu Ser Gly Arg Ser Ty 595 600. 60 GOT GAA GAT GTT GAT OTT ACA TTG AAC TGG GGA AGG TTl Ala Giu Asp Val Asp Leu Thr Leu Asn Trp Gly Arg Le' 610 615 620 CTG OCT GAC TAT OAT GGA CAA GAO TOT GTT CGT GTA GG~ Leu Pro Asp Tyr His Gly Gin Asp Ser Val Axg Val Gi1 625 630- 635 TTT GGA AGO ATT AAT GOA ATT CTG GGA AGO GTG GOA TT2 Phe Gly Ser Ile Asn Ala Ile Leu Gly Ser Val Ala Let 645 650 TGT OAT CAT OAT GOA TCG CGA GTT GCC AGA ATG GOA TC1 Cys His His His Ala Ser Arg Val Ala Arg-Met Ala Sez 660 665 COT TOT ATG TGT COG GOA GAT GGA AGA GTO CGT GGG ATT Pro Ser Met Cys Pro- Ala Asp Gly Arg Val Arg Gly Ile 675 680 685 AAA ATA TTG TGG GAT TOA TOO ACT CTG GGG GCA ATT CTG Lys Ile Leu Trp Asp Ser Ser Thr Leu Gly Ala Ile Leu 690 695 700 ACT kTT AGO AGT TG.

Thr Ile Ser Ser 705 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 708 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE -TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NQ:33: Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser 1 5 10 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu 25 Leu Vai Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val s0 55 60 His Pro Asp Lys Leu Giu Glu Lys Phe Pro Gin Val Ala 70 75 AsIT Giy Pro Asp Ile Ile-Phe Trp Ala His Asp Arg Phe 90 Ala Gin Ser Gly Leu Leu Ala Giu Ile Thr Pro Asp Lys 100 105 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr.

115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu 590 TGTA ATG ACT rVal Met Thr

S

SAGT AGO GTO u Ser Ser Val A AGA ATT TOT y Arg Ile Ser 640 ~ATA OTG AAT i Ile Leu Asn 655 GAT GAG TTT Asp Giu Phe 670 ACG CAC A AT Thr His Asn ATG OGC AGA Met Arg Arg 3,824 1872 1920 1968 2016 2064 2112 2127 Ala Leu Thr Glu Gly Lye Leu Ala Glu Thr Val Giu Ala Thr Gly Gly Gly Tyr Ala Phe Gin 110 Asn Gly Lys Ile Tyr Asn 130 Asp Leu Leu Pro Asp Lys Glu Leu 165 Gin Giu Pro Tyr 180 Ala Phe Lys Tyr 195 Asp Asn Ala Gly 210 Lys Ass Lys His Ala Phe Asn Lys 245 Trp Ser Asn Ilie 260 Pro Thr Phe Lys 275 Ala Gly Ile Ass 290 Leu Giu Ass Tyr Asp Lys Pro Leu Asn 150 Lys Phe Giu Ala Met 230 Gly Asp Gly Ala Leu 310 Gly 135 Pro Ala Thr Ass Lys 215 Asn Glu Thr Gin Ala 295 Leu Ala Ile Ile Asn 375 Gin Gly Ser le Met 455 Giy Glu Pro Lys Thr a a Leu Ala Gly *Glu Val Arg 370 Glu Ala 385 Ass Ass Giu Phe Ass Val Gl-y Giy 450 Phe- Ala 465 Ass Leu Ass Ass Thr Leu 420 Ile Arg 435 Thr Ser Vai Asp Arg Leu 325 Pro Arg Pro Ass Val Ile Asp Ala 390 Ass Leu 405 Asp Phe Ser Ala Leu Leu Val Arg 470 Ile Val Gly Lys Ser 170 Pro Leu Ile 185 Lys Tyr Asp Gly Leu Thr Asp Thr Asp 235 Ala Met Thr 250 Lys Val Ass 265 Ser Lys Pro Pro Asn Lys Asp Giu Gly 315 Ala Leu Lys 330 Ala Thr Met 345 Gin Met Ser Ala Ser Gly Ser Ser Ser 395 Giu Gly Arg 410 Ala Lys Thr 425 Thr Pro Leu Asp Ser Gly Asp Ala Giu 475 Asn Ass Leu 490 Tyr Arg Phe 505 ,140- Glu Giu Ile Pro Ala Leu Met Phe 175 Ala Ala Asp Gly 190 Ile Lys Asp Val 205 Phe Leu Val Asp 220 Tyr Ser Ile Ala Ile Ass Gly Pro -255 Tyr Gly Vai Thr 270 Phe Val Gly Val 285 Glu Leu Ala Lys 300 Leu Giu Ala Val Ser Tyr Giu Giu 335 Glu Ass Ala Gin 350 Ala Phe Trp Tyr 365 Arg Gin Thr Val 380 Ass Ass Asn Ass Ile Ser Giu Phe 415 Tyr Val Asp Ser 430 Gin Thr Ile Ser 445 Ser Gly Asp Ass 460 Giu Gly Arg Phe Tyr Val Thr Giy 495 Ala Asp Phe Ser Ala 160 Asn Gly Gly Leu Giu 240 Trp Val Leu Giu Ass 320 Glu Lys Ala Asp Ass 400 Lys Leu Ser Leu Ass 480 Phe His a. a.

a 485 Val Ass Arg Thr Ass 500 Ass Val Phe 71 Val Thr Phe Pro Gly Thr -Thr Ala 515 520 Tyr Thr-Trhr Leu Gin Arg Val -Ala Val Thr Leu Sdr Gly Gly Ile Ser Arg thr Asp Ser Ser Gly Met Gin 530 .535 Ile 545 Ser Vai Phe Ala Leu 625 Phe Cys Pro Lys Thr 705 Asn Arg His Gly Thr Ser Thr Val Thr 580 Arg Thr Thr 595.

Giu Asp Vai 610 Pro Asp Tyr Gly.Ser Ile His His His 660 Ser Met Cys -675 Ile Leu Trp 690 Ile Ser Ser Ser Leu Thr Thr Ser Tyr Leu Asp 550 555 Leu Thr Gin Ser Val Ala Arg Ala 565 570 Ala Glu Ala Leu Arg Phe Arg Gin .585 Leu Asp Asp Leu Ser Gly Arg Ser Asp Leu Thr Leu Asn Trp Gly Arg 615 .620 His Gly Gin Asp Ser Val Arg Val 630 635 Asn Ala Ile Leu Gly Ser Val Ala 645 650 Ala Ser Arg Val Ala Arg Met Aia 665 Pro Ala Asp Gly Arg Val Arg Gly 680 Asp Ser Ser Thr Leu Gly Ala Ile 695 700 Leu Met Met Leu Ile Gin 590 Tyr Val 605 Leu Ser Gly Arg Leu Ile Ser Asp 670 Ile Thr 685 Leu Met Ser His 560 Arg Phe 575 Arg Gly Met Thr Ser Val Ile Ser -640 Leu Asn 655 Glu Phe His Asn Arg Arg INFORMATION FOR SEQ ID NO: 34: a.

a a a a a SEQUENCE CHARACTERISTICS: LENGTH: 2136 base pairs TYPE: nucleic acid STR.ANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1.-2136 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: ATG AAA ATA AAA ACA GGT GCA CGC ATC CTC GCA TTA TCC GCA TTA ACG Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 AM~ ATG ATG TTT TCC GCC TCG GCT CTC GCC AAA ATC GAA GAA GGT AAA Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Giu Glu Gly Lys 20 25 CTG GTA ATC TGG ATT AAC GGC GAT AAA GGC TAT AAC GGT CTC CCT GAA Leu Vai Ile Trp Ile Asn Giy Asp Lys Cly Tyr Asn Gly Leu Ala Glu 35 .40 45 GTC OCT AAG AAA TTC GAG AAA GAT ACC GGA ATT AAA GTC ACC CTT GAG Val Cly Lys Lys Phe Giu Lys Asp Thr Gly Ile Lys Val Thr Val Glu s0 55 a. a a a 72'- CAT CMCGOAT AAA CTG GAA GAG AAA TTC CCA CAG GTT GCG GCA ACT GGC His Pro Asp Lys Leu Giu Giu Lys Phe Pro Gin Val Ala Ala Thr Gly 70 75. GAT GGC CCT GAC ATT ATC TTC TOO GCA CAC Asp Gly Pro Asp Ile Ile Phe Tip Ala His 90 GCT CAA TCT GOC CTG TTG GCT GAA ATC ACC Ala Gin Ser Gly Leu Leu Ala Olu Ile Thr 100 105 GAC AAG CTG TAT CCG TTT ACC TOG OAT GCC Asp Lys Leu Tyr Pro Phe Thr Tip Asp Aia 115 120 CTG -ATE.-GCT -TAC:CCG:E.C GTOT'rGAAAZCG Leu Ile Ala Tyr Pro Ile Ala VaI.Glu Ala 130 135 AAA OAT CTG CTG CCG AAC CCG CCA AAA ACC Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr 145 150 CTG OAT AAA GAA CTG AAA GCG AAA GOT AAG Leu Asp Lys Giu Leu Lys Ala Lys Oly-Lys 165 170 CTG CAA GAA CCG TAC TTC ACC TGG CCG CTG Leu Gin Giu Pro Tvr Phe Thr Tip Pro Leu 180 165 TAT OCG TTC AAG TAT GAA AAC-GGC AAG TAC Tyr Ala Phe Lys Tyr Oiu Asn Gly Lys Tyr 195 200 GTG GAT AAC GCT GOC GCO AAA GCG GOT CTG Val Asp Asn Ala Gly Ala Lys Ala Oly Leu 210 215 ATT AAA AAC AA.A CAC ATG AAT GCA GAC ACC Ile Lys Asn Lys His Met Asn Ala Asp Thr 225 230 GCT GCC TTT AAT AA GGC GAA ACA GCG ATG Ala Ala Phe Asn Lys Giy Giu Thr Ala Met 245 250 GCA TGG TCC AAC ATC GAC ACC AGC AAA GTG Ala Tip Ser Asn Ile Asp Thr.Ser Lys Val 260 265 CTG CCG ACC TTC AAG GGT CAA CCA TCC AAA Leu Pro Thr Phe Lys Oly Gin Pro Ser Lys- 275 280 AOC GCA GOT ATT AAC GCC GCC ACT CCG AAC.

Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn 290 29 5 T'TC CTC GAA AAC TAT CTG CTG ACT OAT GAA Phe Leu Glu Asn Tvr Leu Leu Thr Asp Olu 305 310 AAA GAC AAA CCG CTG GOT CCC OTA GCG CTG Lys Asp Lys Pro Leu Cly Ala Val Ala Leu 325 330 TTC OCO AAA OAT CCA COT ATT GCC GCC ACC Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr GAC CGC TTT Asp Arg Pkie CCG GAC AAA Pro Asp Lys OTA COT TAC Val Arg Tyr 1.25 TTA TCQ CTG Leu Ser Leu 140 TGG OAA GAG Tip Glu Glu 155 AOC GC CTG Ser Ala Leu ATT GCT OCT Ile Ala Ala CAC ATT. AAA Asp Ile Lys 205 ACC TTC CTG Thr Phe Leu 220 OAT TAC TCC Asp Tyr Ser 235 ACC ATC AAC Thr Ile Asn AAT TAT GGT Asn Tyr Gly CCG TTC GTT Pro Phe Val 285 AAA GAG CTG Lys Oiu Leu 300 GOT CTG OAA Oly Leu Olu 315 AAG TCT TAC Lys Ser Tyr ATG GAA AAC Met Glu Asn COT GOC Gly Gly GCG TTC Ala Phe 110 AAC C Asn Cly ATE TAT Ile Tyr ATC CCC Ile Pro ATG TTC met Phe 175 GAC 000 Asp Oly 190 GAC OTO Asp Val OTT GAC Val Asp ATC OCA Ile Ala GOC CCO Oly Pro 255 OTA ACG Val Thr 270 GOC GTO Gly Val OCO AAA Ala Lys GCG OTT Ala Val GAG CAA Olu Clu 335 GCC CAG Ala Gin

TAC

Tyr

CAG

Gin

AAO

Lys

AAC

Aco Ala 160

AAC

Asn

GOT

Gly

GOC

Oly

CTG

Leu

GAA

Glu 240

TGG

Tip

OTA

Val Cro Leu

GAG

GlU

AAT

320

GAO

Giu

AAA

Lys 288 336 384 432 480 528 576 624 672 720 768 816 864 912 960 1008 1056 9 9* 9.e9 a 73 -340: GGT GAA ATC ATG CCG AAC ATC CCG Gly Giu Ile Met Pro Asn Ile Pro 35 360 GTG CGT Val Arg 370 GAA GCC Giu Ala 385 AAC AAT Asn Asn GAG TTT Giu Phe AAT AGT Asn Ser GGG ACC Gly Thr 450 GCT GTG Ala Val 465 CTT CGT Leu Arg AAT AG Asn Thr TCA GTG Ser Val ACC ACT- Thr Thr 530 AGT CGT Ser Arg 545 GGT AAT Gly Asn ACT GTC Thr Val CGT CAG Arg Gin GAG GTG Asp Val 610 ACT GCG GTG ATC Thr Ala Vai Ile CTG AAA GAC GCG Leu Lys Asp Ala 390 MAC AAC AAC CTC Asn Asn Asn Leu 405 ACG ATA GAC TTT Thr Ile Asp Phe 420 ATA CGG ACA GAG Ile Arg Thr Giu 435 ACA TCG GTG TCT Thr Ser Val Ser GAT ATA CGA GGG Asp Ile Arg Gly 470 CTS ATT AT? GAG Leu Ile Ilie Giu 485 OCA ACA AAT ACT Ala Thr Asn Thr 500 C-C GGT GTG ACA pro Gly Val Thr 515.

C73 CAA CGT GTC Leu Gin Arg Val GAC TCA GTG GTT His Ser Leu Val 550- ACA ATG ACC AGA Th-r Met Thr Arg 565 ACA GCA GAA GCC T'-r Ala Giu Ala 580 GCA CTG TCT GAA Ala Leu Ser Giu 595 GAC GTG ACT CTG Asp Leu Thr Leu AAC GC Asn Ala 375 GAG ACT Gin Thr GGG ATC Gly le TCG ACC Ser Thr ATA TCG Ile Ser 440 GTT AT? Val Ile 455 CTT GA? Leu Asp

CAA"AAT

Gin Asn rrC TAG Phe Tyr AGG GTT Thr Val 520 GCA GCG Ala Ala 535 TGA TCA Ser Ser GAT GGA Asp Ala TTA GGC Leu Arg ACT GCT Thr Ala 600 AAC TGG Asn Trp 615 345' 350 GAG ATG TCC GCT TTC TGG TAT GCC Gin Met Ser Ala Phe Trp Tyr Ala 365.

GCG AGC GGT GGT GAG ACT GTC GAT Ala Ser Gly Arg Gin Thr Val Asp 380 TCG AGG TCG AAC AAG AAC MAC A.AT Ser Ser Ser Asn Asn Asn Asn Asn .395 400 GAG GGA AGG AT? TCA GAA TTC CGG Giu Gly Arg Ile Ser Giu Phe Arg 41i0. 415 CAA CAA AGT TAT GTG TGT TCG TTA Gin Gin Ser Tyr Val Ser Ser Leif 425 430 ACC CC? CTT GAA CAT ATA TCT GAG Thr Pro Leu Giu His Ile Ser Gin 445 AAC GAG ACC GAG GGC AGT TAT TTT Asn His Thr His Gly Ser Tyr Phe 460 GTC TAT GAG GCG CGT TTT GAG CAT Val Tyr Gin Ala Arg Phe Asp His 475 480 MAT TTA TAT GTG GCA GGG TTC GTT Asn Leu Tyr Val Ala Gly Phe Val 490 495 COT TTT TCA GAT TTT ACA CAT ATA Arg Phe Ser Asp Phe Thr His Ile 505 510 TCG ATG ACA ACG GAG AGC ACT TAT Ser'Met Thr Thr Asp Ser Ser Tyr 525 CTG GAA GGT TCC GGA ATG CAA ATC Leu Giu Arg Ser Giy Met Gin Ile 540 TAT CTG GCG TTA ATG GAG TTC AGT Tyr Leu Ala Leu Met Giu Phe Ser 555. 560 TCC AGA GCA GTT CTG CGT TTT GTC Ser Arg Ala Vai Leu Arg Phe Val 570 575 TTG AGG CAG ATA GAG AGA GMA TTT Phe Arg Gin Ile Gin Arg Giu Phe 585 590 CCT GTG TAT ACG ATG ACG CCG GGA Pro Val Tyr Thr Met Thr Pro Gly 605 GGG CGA ATC AGC MAT GTG CTT CCG Gly Arg Ile Ser Asn Vai Leu Pro 620 1104 1152 1200 1248 1296 1344 1392 1440 1488 1536 1584 1632 1680 1728 1776 18 24 1872 74 GAG TAT CGG GGA *-GAG GAT. GGTr GTC AGA GTG GGG AGA. ATA TCC TTr Glu Tyr Arg Giy Giu Asp Gly Val Arg Val Gly Arg Ile Ser Phe 625 630 635 AAT ATA TCA GCG ATA CTG GGG ACT GTG GCC GTT ATA CTG AAT TGC Asn Ile Ser Aia Ile Leu Giy Thr Val Ala Vai Ile Leu Asn Cys 645 650 655 CAT CAG GGG GCG CGT TCT GTT CGC GCC GTG AAT GAA GAG AGT CAA His Gin Gly Ala Arg Ser Val Arg Ala Val Asn Glu Giu Ser Gin 660 665 670 GAA TGT CAG ATA ACT GGC GAC AGG CCT.GTT ATA AAA ATA AAC AAT Giu Cys Gin Ile Thr Gly Asp Arg Pro Vai Ile Lys Ile Asn Asn 675 680 685 TTA TGG GAA AGT AAT ACA GCT OCA GCG TTT CTG AMC AC3A AAG':tCA Leu Trp Giu Ser Asn Thr Ala Ala Ala Phe Leu Asn Arg Lys Ser 690 695 700 TTT TTA TAT ACA ACG GGT AAA TA Phe Leu Tyr Thr Thr Gly Lys 705 710 INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 711 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein

AAT

Asn 640

CAT

His

CCA

Pro

ACA_

Thr

CAG

Gin 1920 1968 2016 2064 2112 2136 9 @9*9*0 9 0* .9 9@ 9 9 9 9.

9 *9 9 *9* (xi) SEQUENCE Met Lys Ile Lys Thr Thr Met Met Phe Ser 20 Leu Vai Ile Trp Ile Val Giy Lys Lys Phe 50 His Pro Asp Lys Leu 65 Asp Gly Pro Asp Ile as Ala Gin Ser Gly Leu 100 Asp Lys Leu Tyr Pro 115 LeU-Ile Ala Tyr Pro 130 Lys Asp Leu Leu Pro 145 Leu Asp Lys Glu Leu 165 Leu Gin Glu Pro Tyr 180 DESCRIPTION: SEQ ID Gly Ala Arg Ile Leu Ala Leu Ser Ala Ser Gly Lys Glu Phe Ala Thr Ala 135 Pro Ala Thr Leu Ala Lys Lys Gly Tyr Thr Gly Ilie Phe Pro Gin 75 Ala His Asp 90 Ile Thr Pro 105 Asp Ala Val Giu Ala Leu Lys Thr Trp 155 Giy Lys Ser 170 Pro Leu Ile 185 Leu Thr Gly Lys Ala Giu Val Giu Thr Giy Gly Tyr Phe Gin Gly Lys Tyr Asn Pro Ala 160 Phe Asn 175 Gly Giy 99 99 S 0 0 75 r.Tyr Aia;Phe:,Lysa -Tyr -'Glu2Asn '-iy Lys: T-iyr As le Lys Asp Val Gly 5200- 205 Val Asp Asn Ala Gly Ala Lys Ala 210 .215 Ile Lys Asn Lys His Met 225 Ala Ala

J

4 eu.

Ser Phe 305 Lys Leu Gly Val Glu 385 Asn Glu Asn Gly Ala 465 Leu Asn Ser Thr Ser 545 Ala Trp Pro Ala 290 Leu Asp Ala Glu Arg 370 Ala Asn Phe Ser Thr 450 Val Arg Thr Val Thr 530 Arg Phe Ser Thr 275 Gly Glu Lys Lys Ile 355 Thr Leu Asn Thr Ile 435 Thr Asp Leu Ala Pro 515 Leu His Asn Asn 260 Phe Ile Asn Pro Asp 340 Met Ala Lys Asn Ile 420 Arg Ser Ile Ile Thr 500 Gly Gin Ser 230 Lys Gly 245 Ile Asp Lys Gly Asn Ala Tyr Leu 310 Leu Gly 325 Pro Arg Pro Asn- Val Ile Asp Ala 390 Asn Leu 405 Asp Phe Thr Glu Val Ser Arg Gly 470 Ile Glu 485 Asn Thr Val Thr Arg Val 2 Leu Val 550 Asn Glu Thr Gin Ala 295 Leu Ala Ile Ile Asn 375 Gin Gly Ser Ile Val 455 Leu Gin 2 Phe Thr ia 2 S35 Ser I Ala Thr Ser Pro 280 Ser Thr Val Ala Pro 360 Ala Thr Ile Thr Ser 440 Ile Asp Asn2 ryr 2 lal 520 Ua I ;er I Gl Asp Ala Lys 265 Ser Pro Asp Ala Ala 345 Gin Ala Ser Glu Gin 425 rhr Asn Val ksn krg 505 3er .eu ryr Leu Thr Phe 220 Thr Asp Tyr 235 Met Thr Ile 250 Val Asn Tyr Lys Pro Phe Asn Lys Glu 300 Glu Gly Leu 315 Leu Lys Ser 330 Thr Met Glu Met Ser Ala Ser Gly Arg 380 Ser Ser Asn 395 Gly Arg Ile 410 Gin Ser Tyr Pro Leu Glu His Thr His 460 Tyr Gin Ala 475 Leu Tyr Val 2 490 Phe Ser Asp Met Thr Thr 2 Glu Arg Ser 540 Leu Ala Leu I 555 Ser Asn Gly Val 285 Leu Glu Tyr Asn Phe 365 Gin Asn Ser Val His 445 Sly Arg Aia Phe %sp 525 ,ly 4et Ile Ala Gly Pro 255 Val Thr 270 Gly Val Ala Lys Ala Vai Glu Glu 335 Ala Gin 350 Trp Tyr Thr Val Asn Asn Glu Phe 415 Ser Ser 430 Ile Ser Ser Tyr Phe Asp I Gly Phe 495 Thr His 510 Ser Ser I Met Gin I Glu Phe S 5 Leu Val Asp Leu, Glu 240 Trp Val Leu Glu Asn 320 Glu Lys Ala Asp Asn 400 Arg Leu Gin Phe .is 180 lal Ile yr :le ;er S. *S Gly Asn Thr Met Thr Arg Asp Ala Ser Arg Ala Val Leu Arg Phe Val 76 565 '570 2 575 Thr Val Thr Ala Giu Ala Leu Arg Phe Arg Gin Ile Gin Arg (flu Phe -50.585' 590 Arg Gin Ala Leu Ser:Giu Thr Ala Pro Val Tyr Thr Met Thr Pro Giy 595 600 605 Asp. Val Asp Leu Thr Leu Asn Trp Giy Arg Ile Ser Asn Vai Leu Pro 610 615 620 Glu Tyr Arg Giy Glu Asp Giy Vai Axg Vai Gly Arg Ile Ser Phe Asn 625 630 635 640 Asn Ile Ser Ala Ile Leu Giy Thr Val Ala Vai Ile Leu Asn Cys His 645 650 655 His*Gin Gly Ala Arg Ser Vai Arg -la Vai Asn Glu Giu Ser Gin Pro 660 665 670 Glu Cys Gin Ile Thr.Gly Asp Arg Pro Val Ile Lys Ile Asn Asn Thr 675 680 685 Leu Trp Giu Ser Asn Thr Ala Aia Ala Phe Leu Asn Arg Lys Ser Gin 690 695 700 Phe Leu Tyr Thr Thr Gly Lys 705 710 INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 981 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..981 SEQUENCE DESCRIPTION: SEQ, ID NO:36: :::ATG AAA A AG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT TTC GCT 48 Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala :1 5 10 ACC GTE GCG CAA GCT GAC TAC AAG GAC GAC GAT GAC AAG AAG CTT GAA 96 *Thr Val Ala Gln Ala Asp Tyr Lys Asp Asp Asp Asp Lys Lys Leu Giu 25 TTC AAG GAA TTT ACC TTA GAC TTC TCG ACT GCA AAG ACG TAT GTA GAT 144 Phe Lys Glu Phe Thr Leu Asp Phe Ser Thr Ala Lys Thr Tyr Val Asp 40 *TCG CTG AAT GTC ATE CGC TCT-GCA ATA GGT ACT CCA TEA CAG ACT ATT 192 Ser Leu Asn Val Ile Arg Ser Ala Ile Gly Thr Pro Leu Gin Thr Ile 55 TCA TCA GGA GGT ACG TCT TEA CTG ATG ATE GAT AGT GGC TCA GGG GAT 240 Ser-Ser Giy Gly Thr Ser Leu Leu Met Ile Asp Ser Gly Ser Gly Asp 70 75 AAT TTG TTE GCA.GTT GAT GTC AGA GGG ATA GAT GCA GAG GAA GGG CGG 288 Asn Leu Phe Ala Val Asp Val Arg Gly Ile Asp Ala Giu Glu Gly Arg 90 TET AAT AAT CTA CGG CTT ATE GTT GAA CGA AAT AAT TEA TAT GTG ACA 336 -77- Phe Asn GGA TTN Gly Phe TCA CAT Ser His 130 AGT AGC .Leu Arg Leu 100 AAC AGG ACA Asn Arg Thr ACC TTT CCA Thr Phe Pro ACC ACG TTA Thr Thr Leu 150 AAT CGC CAT Asn Arg His 165 GGA ACC TCA G].y Thr Ser 180 ACT GTG ACA Thr Val Thr CGT ACA-ACA Arg Thr Thr GAA GAT GTT Giu Asp Vai 230 CCT GAC TAT Pro Asp Tyr 245 GGA AGC ATT Gly Ser Ile 260 CAT CAT CAT His His His TCT ATG TGT Ser Met Cys ATA TTG TGG Ile -Leu Trp 310 ATT AGC AGT Ile Ser Ser 325 Ile Val AAT AAT Asn Asn 120 GGT ACA Gly Thr 135 CAG CGT Gin Arg TCG TTG Ser Leu CTG ACG Leu Thr GCT GAA Ala Giu 200 CTG GAT Leu Asp 215 GAT CTT Asp Leu CAT GGA His Giy AAT GCA Asn Ala GCA TCG Ala Ser 280 CCG GCA Pro Ala Glu Arg Asn Asn 105 GTT TTT TAT CGC Val Phe Tyr Arg ACA GCG GTT ACA Thr Ala Val Thr 140 Leu Tyr Val Thr 110 TTT GCT GAT Trr Phe Ala Asp Phe 125.

TTG TCT GGT GAC Leu Ser Gly Asp GTT GCA GGG'ATC AGT CGT Val Ala Gly Ile Ser Arg 155 ACT ACT TCT TAT CTG GAT Thr Thr Ser Tyr Leu Asp 10 CAG TCT GTG GCA AGA GCG Gin Ser Val Ala Arg Ala 185 190 GCT TTA CGT TTT CGG CAA Ala Leu Arg Phe Arg Gin 205 GAT CTC AGT GGG CGT TCT Asp Leu Ser Gly Arg Ser 220 ACA rrG AAC TGG GGA AGG Thr Leu Asn Trp.Gly Arg 235 CAA GAC TCT GTT CGT GTA Gin Asp Ser Val Arg Val 250 ATT CTG GGA AGC GTG GCA Ile Leu Gly Ser Val Ala 265 270 CGA GTT GCC AGA ATG GCA Arg Val Ala Arg Met Ala 285 GAT GGA AGA GTC CGT GGG ACG GGG Thr Giv 160 TTA ATG-.

Leu Met 175 ATG TTA Met Leu ATA CAG Ile Gin TAT GTA Tyr Val TTG AGT Leu Ser 240 GGA AGA G *ly Arg 255 TTA ATA Leu Ile TCT GAT Ser Asp ATT ACG Asp Gly Arg Val 300 Arg Gly Ile Thr GCA ATT CTG ATG Ala Ile Leu Met TCA TCC ACT Ser Ser Thr CTG GGG Leu Gly 315.

912 960 981 INFORMATION FOR SEQ ID NO:37: Wi SEQUENCE CHARACTERISTICS: LENGTH: 326 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 78 Met Lys Lys Thr Ala Ile Ala 1 5 Ile Ala Val. Ala Leu Ala Giy..Phe- Ala 10 Tyr Lys Asp Asp Asp Phe Ser Thr Ser Ala Ile Gly Leu Leu Met Ile Val Arg Gly Ile Ile Val Giu Arg 105 Asn Asn Val Phe 120 Gly Thr Thr Ala 135 Gin Arg Val Aia Ser Leu Thr Thr 170 Leu Thr Gin Ser 185 Ala Giu Ala Leu 200 Leu Asp Asp Leu 215 Asp Leu Thr Lfeu His Gly Gin Asp 250 Asn Ala Ile Leu 265 Ala Ser Arg Val 280 Pro Ala Asp Gly 295 Asp Lys Lys Thr Pro Leu Ser Gly Ala Giu Asn Leu Arg Phe 125 Thr Leu 140 Ile Ser Tyr Leu Ala Arg Phe Arg 205 Gly Arg 220 Trp Gly Val Arg Ser Val Arg Met 285 Val Arg 300 Lys Leu Giu Tyr Val Asp Gin Thr Ile Ser Gly Asp Giu Gly Arg Tyr Val Thr 110 Ala Asp Phe Ser Gly Asp Arg Thr Gly 160 Asp Leu Met 175 Ala Met Leu 190 Gin Ile Gin Ser Tyr Val Arg Leu Ser 240 Val Gly Arg 255 Ala Leu Ile 270 Ala Ser Asp Gly Ile Thr Leu Asn Giu Phe 290 His Asn Lys Ile Leu Trp, Asp'Ser Ser Thr LeU Gly Ala Ile Leu Met 30-5- 310 315 320 Arg Arg Thr Ile Ser Ser .325 INFORMATION FOR SEQ ID NO:38:.

SEQUENCE CHARACTERISTICS: LENGTH: 990 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 79 (ii) MOLECULE 'TYPE.:. DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .990 (xi) -SEQUENCE DESCRIPTION: SEQ:-ID 14:38: ATG AAA AAG ACA OCT ATC GCG ATT GCA GTG GCA CTG GCT GOT TTC GCT Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala LeU Ala Gly Phe Ala 1 5 10 ACC GTT GCG Thr Val Ala T-DC -CG -GAG Phe Arg Glu TCG TTA AAT Ser Leu Asn TCT CAG GG Ser Gin Gly TAT TTT OCT Tyr Phe Ala GAC CAT CTT Asp His Leu TTC OTt AAT Phe Val Asn 115 CAT ATA TCA His Ile Ser 130 AGT TAT ACC Ser Tyr Thr 145 CAA ATC AOT Gin Ile Ser TTC AGT GGT.

Phe Ser Gly TT GTC ACT Phe Vai Thr 195 GAA TTT CGT Giu-Phe Arg 210

CAA

Gin

*TT

Phe

AGT

Ser

ACC

Thr

GTG

Val

CGT

Arg 100

ACG

Thr

GTG

Val

ACT

Thr

CGT

Arg

AAT

Asn 180

GTC

Val

CAG

Gln GCT GAC TAC AAG GAC GAC Aia Asp Tyr Lys. Asp Asp 25 -ACG 'ATh GAC -'T'-;TCO-ACC Thr Ile Asp Phe Ser Thr 40 ATA COO ACA GAG ATA TCG Ile Arg Thr Giu Ile Ser 55 GAT GAC Asp Asi CAA CA Gin Gir ACC CCT Thr Prc 0* 0 *0 0.

ACA TCG Thr Ser 70 GAT ATA Asp Ile CTG ATT Leu le OCA ACA Ala Thr CCC GOT Pro Oly CTG CAA Leu Gin 150 CAC TCA His Ser 165 ACA ATO Thr Met ACA GCA Thr Ala GCA CTG Ala Leu GTG TCT OTT ATI' AAC CAC Val Ser Val Ile Asn His 7S CGA GG0 CTT GAT. GTC TAT Arg Gly Leu Asp Vai Tyr 90 ATT GAG CAA AAT AAT TTA Ile Giu Gin Asn Asn Leu 105 AAT ACT TTC TAC COT TTT Asn Thr Phe Tyr Arg Phe 120 GTG ACA ACO GTT TCC ATG Vai Thr Thr Val Ser Met 135 140 CGT GTC GCA GCG CTG GAA Arg Val Ala Ala Leu Giu 155 CTG GTT TCA TCA TAT CTG Leu Vai Ser Ser Tyr Leu 170 ACC AGA OAT GCA TCC AGA Thr Arg Asp Ala Ser Arg 185 GAA 0CC TTA CGC TTC AGO Glu Ala Leu Arg Phe Arg 200 TCT GAA ACT OCT CCT GTG Ser Giu Thr Ala Pro Val 215 220 ACT CTG AAC TOG GOG CGA Thr Leu Asn Trp Gly Arg 235 GAG OAT G GTC AGA GTO Glu Asp Oly Val Arg Val 80

AAG

)Lys i AGT 1Ser

CTT

Leu

ACC

Thr

CAG

*Gin

*TAT

Tyr

TCA

Ser 125

ACA

Thr

COT

Arg

GCG

Ala

GCA

Ala

CAG

Gin 205

TAT

Tyr

AAG

Lys

TAT

Tyr

GAA

Giu

CAC

His

GCG

Ala

OTO

Val 110

OAT

Asp

ACO

Thr

TCC

Ser

TTA

Leu

GTT

Val 190

P.TA

Ile

I.CG

rhr

CTT

Leu

*GTC

*Val

*CAT

His

GC

Gly

COT

Arg

OCA

*Ala

TTT.

Phe

GAC

Asp

OGA

Gly

ATO

Met 175

CTG

Leu

CAG

Gin ATG I Met I

GAA

Glu

TCT

Ser

ATA

Ile

AGT

Ser

TTI

Phe 000 Gly

ACA

Thr

AGC

Ser

A.TG

Me t 160

GAG

Glu

CGT

krg kGOA rg

~CG

'hr 96 144 192 240 286 336 384 432 480 528 576 624 672 *0 0 CCO GGA GAC OTO GAC CTC Gly Asp CCG GAO Pro Glu Asp Leu 230 COO GGA Arg Gly ATC AGC Ile Ser 000 AGA Gly Arg 768 245 250 255 AAT AAT ATA TCA GCG ATA Cro 000G ACT GTG GCC GTTTATA CTG AAT Asn Asn Ile Ser Ala Ile Leu Giy Thr Val Ala Val Ile Leu Asn 260 265 270 CAT CAT CAG 000 GCG CGT TCT GTT CGC GCC GTG AAT GAA GAG AGT His His Gin Gly Ala Arg Ser Val Arg Ala Val Asn Glu Giu Ser 275 280 285 CCA GAA TGT CAG ATA ACT GGC GAC AGG CCT GTT ATA AAA ATA AAC Pro Giu Cys Gin Ile Thr Gly Asp Arg Pro Val Ile Lys Ile Asn 290 295 300 ACA TTA TGG GAA AGT AAT ACA GCT GCA GCG TTT CTG ARC AGA AAG Thr Leu Trp Giu Ser Asn Thr Ala Aia Ala Phe Leu Asn Arg Lys- 310 315 320 CAG TTT TTA TAT ACA ACG GGT AAA TA Gin Phe Leu Tyr Thr Thr Gly Lys 325 330 INFORMATION FOR SEQ ID NO:39: Wi SEQUENCE CHARACTERISTICS- LENGTH: 329 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein a. a a.

a a a (xi) SEQUENCE Lys Lys Thr Ala Val Ala Gin Ala 20 Arg Giu Phe Thr Leu Asn Ser Ile s0 Gin Gly Thr Thr Phe Ala Val ASP His Leu-Arg Leu 100 Val Asn Thr Ala 115 Ile Ser Vai Pro 130 Tyr Thr Thr Leu Ile Ser Arg His 165 Ser Giy Asn Thr 180 Val Thr Val Thr DESCRIPTION: SEQ Ile Ala Ile Ala V ID NO: 39: Al Ala Leu Ala Gly 10 sp Asp Asp Lys Lys hr Gin Gin Ser Tyr er Thr Pro Leu Giu le Asn His'Thr His 75 sp Val Tyr Gin Ala 90 sn Asn Leu Tyr Val 110 yr Arg Phe Ser Asp 125 al Ser Met Thr Thr 140 la Leu Glu Arg Ser 155 er Tyr Leu Ala Leu 70 la Ser Arg Ala Val 190 rg Phe Arg Gin Ile Phe Ala Leu .Glu Val Ser His Ile Gly Ser Arg Phe Ala Gly Phe Thr Asp Ser Gly Met .160 Met Glu 175 Leu Arg Gln Arg a.

a a a 81 195 Giu Phe Arg Gin Ala Leu 210 Pro Gly Asp Val Asp Leu 225 230 Leu Pro Giu Tyr Arg Gly 245 Phe.Asn Asn Ile Ser Ala 260 Cys His His Gin Giy Ala 275 Gin*Pro Giu Cys Gin Ile 290 Asn Thr Leu Trp, Giu Ser 305 310 Ser Gin Phe Leu Tyr Thr 325 200 Giu Thr Leu Asn Asp Giy Leu Gly 265 Ser Vai 280 Giy Asp Thr Ala Gly Lys Ala Pro Val 22'0 Trp Gly Arg* 235.

Vai Arg Vai 250 Thr Val Mla Arg Ala Val 205 Tyr Thr Met Ile Ser Asn Gly Arg Ile 255 Vai Ile Leu 270 Asn Giu Giu 285 Ile Lys Ile- Leu Asn Arg Thr, Val 240 Ser Asn Ser.

Asn Lys 320 Arg Pro Aia Ala 315 -4 4**4 4 9.

.9 .9 4 4 4 4.

S.

4 82

Claims (13)

1. A method of treatment comprising: a) providing: i) antitoxin directed against at least a portion of an Escherichia coli verotoxin' in an aqueous solution in therapeutic amount that is-administrable, and ii) an intoxicated subject: and b) administering said antitoxin to said subject.

2. The method of Claim I wherein said Escherichia coli verotoxin is recombinant.

3. The method of Claim 1 wherein said antitoxin is an avian antitoxin.

4. The method of Claim 2 wherein said recombinant Escherichia coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion-of-the Escherichia coli verotoxin VTI sequence. The method of Claim 2 wherein said recombinant Escherichia coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion -ofthe Escherichia coli verotoxin VT2 sequence. 25 6. The method of Claim 1 wherein said subject is an adult.

7. The method of Claim 1 wherein said subject is a child.

8. The method of Claim i wherein said administering is parenteral.

9. The method of Claim I wherein said administering is oral. 83

10- A method of prophylactic treatment comprising: a) providing: i) an antitoxin directed against at least one Escherichia coli verotoxin in an aqueous solution in therapeutic amount that is parenterally administrable, and ii) at least one subject is at risk of diarrheal disease: and b) parenterally administering said antitoxin to said subject;

11. The method of Claim 10. wherein said subject-is at risk of developing extra- intestinal complications of Escherichia coli infection.

12. The method of Claim il1. wherein said extra-intestinal complication is hemolvtic uremic syndrome.

13. A composition comprising neutralizing antitoxin directed against at least one Escherichia coli verotoxin in an aqueous solution in therapeutic amounts.

14. The composition of Claim 13 wherein said Escherichia coli verotoxin is a recombinant toxin. The composition of Claim 14 wherein said recombinant Escherichia coli Sverotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion of the Escherichia coli verotoxin VTI sequence. 25 16. The composition of Claim 14 wherein said recombinant Escherichia coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and a portion of the. Escherichia coli verotoxin VT2 sequence. 1. 7. The composition of Claim 14 wherein said antitoxin is directed against a portion of at least one Escherichia coli verotoxin. 1 The composition of Claim 14 wherein said portion of Escherichia coli is selected from the group consisting of subunit A and subunit B of VTI.

84- 4 .The-composition of Claim 14 wherein said portionof Eseherichia coi is selected from the group consisting of subunit A and subunit B of VT2;. The composition of Claim 14 wherein said antitoxin is..directed-against a portion of at least one Escherichia coli verotoxin. 21. The composition of Claim 14 wherein said antitoxinis-an avian antitoxin. 22. A method of treatment of enteric bacterial infections compEsing: a) providingt i) an-avian, antitoxin directed- against at least- one verotoxin produced -by -Escherichia coi in an aqueous solution in therapeutic amount that is parenterally administrable. and ii) at least one infected subject: and. b) parenterally administering said avian antitoxin to said subject. 23. The method of Claim .18 wherein said Escherichia coi is selected from the group consisting of Escherichia coi serotypes 0157:H7. 01 :NM. 02:H5; 02:H7: 04:NM: 04:HI10: O5:NM; 05:H1I6:'06:H 1: 0 18:NM; 0 18:1-7; 025:NM: 026:NM: 026:HI11. 026:H32: 038:H21: 039:1-4: 045:H2. 050:1-7: 055:1-7: 053:1-10: 082:1-8: 084:H2: 091:NM: 091:H21: 0103:1-2: Oil INK: 0l11:H8. 011ll:H30: Oil1:H34: 0113:1-7: 0113:1-21: 0114:H48: 01 l5:Hl0: 0117:1-4: 0118:1-12: 0118:H30O: 0121:NM: 0121:H-19: 0125:NM: 0125:H8: 0126:NM: 012-6:H8: 0128:NM: 012.8:H2: 0128:H8: 0128:1-12: 250128:H-'5;: 0145:NM: 0125:1-25: 0146:H21: 0153:H25; 0157:NM: 0163:1-19: 0165:NM: 0165:19: and 0165:H25 24 The method of Claimr-22 -wherein said antitoxin comprises antitoxin directed against- at least one Escherichia coi verotoxin. 5. The method of Claim 22 wherein said antitoxin is cross-reactive wvith it least one Escherlchia coli v'erotoxin. The method of Claim 22 wherein said antitoxin is reactive against toxins produced by members of the genus Shigella.'' 27. The method of Claim 26. wherein said antitoxin is reactive against toxins produced by Shigella dysenteriae. 28. A method for detecting Escherichia coli verotoxin in a sample comprising: a) providing: i) a sample; ii) an antitoxin raised against Escherichia coli verotoxin; and iii) a reporter reagent capable of binding said antitoxin: and b) adding said antitoxin to said sample so that said antiioxin binds to the Escherichia coli verotoxin in said sample. 29. The method of Claim 28. wherein saidf-antitaxin i an .a antitoxin. The method of Claim 28. further comprising the steps of: c) washing said unbound antitoxin from said sample: d) adding said reporter reagent to said sample so that said reporter reagent binds to said bound antitoxin; e) washing said unbound reporter reagent from said sample: and f) detecting said reporter reagent bound to said antitoxin bound to the Escherichia coli verotoxin so that the verotoxin is detected. 25 3. The method of Claim 30 wherein said detecting is selected from the group consisting of enzyme irmmunoassay, radioimmunoassay, fluorescence immunoassay. flocculation. particle agglutination. and in situ chromogenic assay. 32. The method of Claim 30 wherein said sample is a biological sample. 33. The method of Claim 30 wherein said sample is an environmental sample. -86- I 34. A method of treatment comprising: providing: avian toxin-neutralising antitoxin raised against Escherichia coli verotoxin VT1 or VT2 in an aqueous solution in therapeutic amount that is administrable, and (ii) an intoxicated subject; and administering said avian antitoxin to said subject. The method of claim 34 wherein said Escherichia coli verotoxin is recombinant. 36. The method of claim 35 wherein said recombinant Escherichia coli verotoxin is a fusion 1o protein comprising a non-verotoxin protein sequence and an Escherichia coli verotoxin VT1 sequence. 37. The method of claim 35 wherein said recombinant Escherichia coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and an Escherichia coli verotoxin VT2 sequence. 38. The method of any one of claims 34 to 37 wherein said subject is an adult. 39. The method of any one of claims 34 to 37 wherein said subject is a child. 40. The method of any one of claims 34 to 39 wherein said administering is parenteral. 41. The method of any one of claims 34 to 39 wherein said administering is oral. 42. A method of prophylactic treatment, the method comprising: providing: an avian toxin-neutralising antitoxin raised against Escherichia coli verotoxin VT1 or VT2 in an aqueous solution in therapeutic amount that is parenterally administrable, and (ii) at least one subject is at risk of diarrhoeal disease; and parenterally administering said avian antitoxin to said subject. 43. The method of claim 42, wherein said subject is at risk of developing extra-intestinal S 25 complications of Escherichia coli infection. 44. The method of claim 43, wherein said extra-intestinal complication is haemolytic uraemic syndrome. 45. A composition comprising neutralising avian antitoxin raised against Escherichia coli verotoxin VT1 or VT2 in an aqueous solution in therapeutic amounts. 46. The composition of claim 45 wherein said Escherichia coli verotoxin is a recombinant toxin. 47. The composition of claim 46 wherein said recombinant Escherichia coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and an Escherichia coli verotoxin VT1 sequence. [R:\LIBAA]07977.doc:tab 48. The composition of claim 46 wherein said recombinant Escherichia coli verotoxin is a fusion protein comprising a non-verotoxin protein sequence and an Escherichia coli verotoxin VT2 sequence. 49. The composition of claim 46 wherein said antitoxin is directed against a portion of at least one Escherichia coli verotoxin. The composition of claim 46 wherein said portion of Escherichia coli is selected from the group consisting of subunit A and subunit B of VT1. 51. The composition of claim 46 wherein said portion of Escherichia coli is selected from the group consisting of subunit A and subunit B of VT2. 52. A method of treatment of enteric bacterial infections, the method comprising: providing: an avian toxin-neutralising antitoxin raised against Escherichia coli verotoxin VT1 or VT2 in an aqueous solution in therapeutic amount that is parenterally administrable, and (ii) at least one infected subject; and parenterally administering said avian antitoxin to said subject. 53. The method of claim 52 wherein said Escherichia coli is selected from the group consisting of Escherichia coli serotypes 0157:H7, 01:NM; 02:H5; 02:H7; 04:NM; 04:H10; 05:H16; 06:H1; 018:NM; 018:H7; 025:NM; 026:NM; 026:H11; 026:H32; 038:H21; 039:H4; 045:H2; 050:H7; 055:H7; 055:H10; 082:H8; 084:H2; 091:NM; 091:H21; 0103:H2; O111:NM; 0111:H8; 0111:H30; 0111:H34; 0113:H7; 0113:H21; 0114:H48; 0115:H10; 0117:H4; 0118:H12; 0118:H30; 0121:NM; 0121:H19; 0125:NM; 0125:H8; 0126:NM; 0126:H8, 0128:NM; 0128:H2; 0128:H8; 0128:H12; 0128:H25; 0145:NM; 0125:H25; 0146:H21; 0153:H25; 0157:NM; 0163:H19; 0165:NM; 0165:19; and 0165:H25. S 25 54. The method of claim 52 wherein said antitoxin is cross-reactive with at least one Escherichia coli verotoxin. The method of claim 54 wherein said antitoxin is reactive against toxins produced by members of the genus Shigella. 56. The method of claim 55 wherein said antitoxin is reactive against toxins produced by Shigella dysenteriae. 57. A method for detecting Escherichia coli verotoxin in a sample comprising: providing: a sample; (ii) an avian antitoxin raised against Escherichia coli verotoxin; and a reporter reagent capable of binding said antitoxin; and [R:\LIBAA107977.doc:tab adding said antitoxin to said sample so that said antitoxin binds to the Escherichia coli verotoxin in said sample; washing said unbound antitoxin from said sample; adding said reporter reagent to said sample so that said reporter reagent binds to said bound antitoxin; washing said unbound reporter reagent from said sample; and detecting said reporter reagent bound to said antitoxin bound to the Escherichia coli verotoxin so that the verotoxin is detected. 58. The method of claim 57 wherein said detecting is selected from the group consisting of enzyme immunoassay, radioimmunoassay, fluorescence immunoassay, flocculation, particle agglutination, and in situ chromogenic assay. 59. The method of claim 57 or claim 58 wherein said sample is a biological sample. The method of claim 57 or claim 58 wherein said sample is an environmental sample. 61. A composition comprising neutralising avian antitoxin raised against Escherichia coli verotoxin VT1 or VT2, the composition being substantially as hereinbefore described with reference to any one of the Examples. 62. A composition of any one of claims 45 to 51 or 61 when used to treat intoxication. 63. Use of a composition of any one of claims 45 to 51 or 61 in the manufacture of a medicament for treating intoxication. 64. A composition of any one of claims 45 to 51 or 61 when used for prophylactic treatment of diarrhoeal disease. Use of a composition of any one of claims 45 to 51 or 61 in the manufacture of a medicament for prophylactic treatment of diarrhoeal disease. 66. A composition of any one of claims 45 to 51 or 61 when used for the treatment of enteric bacterial infections. 67. Use of a composition of any one of claims 45 to 51 or 61 in the manufacture of a medicament for the treatment of enteric bacterial infections. Dated 3 April, 2000 Ophidian Pharmaceuticals, Inc. Patent Attorneys for the ApplicantlNominated Person SPRUSON FERGUSON [R:\LIBAA)07977.doc:tab