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15202530W0 98/ 1 1229CA 02265887 1999-03-09PCT/US97I15836-1-DescriptionHISTIDINE-TAGGED SHIGA TOXINS, TOXOIDS, ANDPROTEIN FUSIONS WITH SUCH TOXINS AND TOXOIDS,METHODS FOR THE PURIFICATION AND PREPARATION THEREOF§iQVERNME[\_lT INTERESTThe invention described herein may be manufactured, licensed, and used forgovernmental purposes without payment of royalties to us thereon.FIELD OF THE INVENTIONThe invention relates to a family of multi-unit bacterial proteins that areassociated with hemorrhagic colitis and the lifeâthreatening sequela, hemolytic uremicsyndrome. These proteins, defined as members of the âSl1iga toxin family," have beentagged with histidine residues. The invention further relates to a non-toxinogenic butimmunoreactive form of histidineâtagged Shiga toxins, or toxoids. Moreover, theinvention relates to fusion proteins obtained by combining histidineâtagged Shigatoxins or toxoids with other proteins. Histidine tagging greatly facilitates puriï¬cationof Shiga toxins, and the invention also relates to methods for purifying such toxins.The invention further relates to using the histidineâtagged Shiga toxoids or fusionproteins of Shiga toxoids as antigens for generating an immune response againstinfection or transmission by bacteria expressing Shiga toxin. It also relates toantibodies to Shiga toxins, toxoids, or Shiga toxin/toxoid fusion proteins, bothmonoclonal and polyclonal, and their use in treating, diagnosing, and preventing ofdisease and infections by pathogenic E. coli. Finally, the invention relates to preparingthe Shiga toxins, toxoids, and fusion proteins.BACKGROUND OF THE INVEN I IONEnterohemorrhagic Escherichia coli (EHEC) are associated with food-bomeoutbreaks of bloody diarrhea or "hemorrhagic colitis" (HC) and the hemolytic uremicsyndrome (HUS). (Spika, J. et al., "Hemolytic Uremic Syndrome and DiarrheaAssociated with Escheria coli: 01 57:H7 in a Day Care Center," J. Pediatr., 109: 287-291 (1 986); Remis, R., "Sporadic case of hemorrhagic colitis associated with Escheriacoli 0157:H7," Ann. Intern. Med, 101 :624-626 (1984); "Riley, L. et al., "Hemorrhagic10152025W0 98/ 1 1229CA 02265887 1999-03-09PCT/US97I15836-2-colitis associated with a rare Escheria cali serotype," N. Engl. J Med., 308:681-685(1983)). EHEC infection can be deadly and poses a signiï¬cant threat to the young andthe elderly, who are the most likely to develop serious complications from EHECinfections. Several outbreaks and sporadic cases of HC and HUS have occurred overthe past few years, with the largest outbreak in United States in 1993. In that outbreak,over 500 cases of HC and HUS were traced to contaminated hamburgers from a Jack-in-the Box fast food restaurant. (Centers for Disease Control and Prevention, Morbid.Mortal. Weekly Rep., 42:258( 1993)). In July 1996, a large outbreak of EHEC in Japanresulted in over 10,000 infected individuals and 8 deaths. Many Japanese childrenrequired hospitalization. Unfortunately, no cure or vaccine for HC and HUS iscurrently available.Primarily, HC and HUS are transmitted by the ingestion of contaminated food,particularly undercooked beef products, such as hamburger. (Doyle et al., Appl.Environ. Microbial. 53:2394 (1987); Samadpour et al., J. Appl. Environ. Microbial.60:l038 (1994)). With the prevalence of EHEC in cattle and the subjective nature ofdifferentiating between cooked and undercooked hamburgers, a stop at a fast foodrestaurant or a family barbecue can result in tragedy. HC and HUS appear to bemediated by the toxin produced by EHEC and Shigella dysenteriae (for review seeO'Brien and Holmes, Microbial. Rev., 51: 206-220 (1987)). These bacteria producea family of closely related cytotoxins that collectively will be called "Shiga toxins" forthe purpose of this application. Shiga toxins (alternatively, âverotoxins") havecytotoxic, neurotoxic, and enterotoxic activity (Strockbine, N. et al., "Two toxin-converting phages from Escheria cali 01 57:H7 strain 933 encode antigenically distincttoxins with similar biological activities," Infect. Immun., 53:135-140 (1986)).Based on their immunological cross-reactivity, the Shiga toxins have beendivided into two groups. (Strockbine et al., supra). These groups have beendesignated Shiga toxin type 1 (Stxl) and Shiga toxin type 2 (Stx2). (Strockbine et al.,supra; Calderwood et al., "Proposed New Nomenclature for SLT (VT) Family," ASMNews, 62:l18â1l9 (1996)). The Stxl group contains the prototype Stxl toxin from10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97I 15836-3-EHEC as well as the Shiga toxin from Shigella dysenteriae type 1. In recent years,other types of toxins have been discovered and considered members of the Stx2 group.These are Stx2e, Stx2c, Stx2vha, and Stx2vhb. (Lindgren et al., Infection andImmunology, 6113832 (1993); Schmitt, C. et al., "Two copies of Shiga-like toxin II-related genes common in enterohemorrhagic Escheria coli strains are responsible forthe antigenic heterogeneity of the 0157:H strain E32511," Infect. Immun., 59: 1065-1073(1991); Marques, L. et al., "Escheria coli strains isolated from pigs with edema diseaseproduce a variant of Shiga-like toxin Il," FEMS Lett., 44:33-38 (1987)).For the purposes of this application the term "Shiga toxin" encompasses Shigatoxin and any other toxins in the Stxl or Stx2 group. The abbreviation "Stx" will referto the protein designation, and the abbreviation "Stx" to the gene designation.These Shiga toxins do share similar genetic and protein organization, as setforth in Figure l. The A subunit gene encodes the enzymatically active subunit. TheA subunit polypeptide has two functional domains, Al and A2, which are linked bya disulfide bond. The Al portion is an N-glycosidase that acts on the 28S rRNAsubunit of eukaryotic ribosomes to inhibit protein synthesis. (Saxena, S. et al., "Shigatoxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of28S RNA when microinjected into Xenopus ooeytes," J. Biol. Chem., 264:596-601(1989)). The A2 fragment is required for the binding of 5 B subunit polypeptides. Thepentamer of B subunits is responsible for binding to a receptor on eukaryotic cells. Apolypeptide containing the entire A subunit and B subunit pentameter is referred to asa Shiga holotoxin. Despite this knowledge about the toxin components, there is noknown cure or vaccine for HC or HUS.The need exists for therapeutic agents for the treatment and prevention of HCand HUS. However, progress in the search for such agents has been hampered by thelack of a fast and simple method for purifying Shiga toxins. Therefore, the need existsfor such a fast and simple method. Moreover, the need exists for such a method thatfurther allows for large-scale production of Shiga toxins while retaining theirbiological and immunological activity. The need also exists for such a method that10152025W0 98/ 11229CA 02265887 1999-03-09PCT/US97/15836-4-allows for large-scale production of Shiga toxoids and fusion proteins of Shiga toxinsand toxoids. Such a method should simplify obtaining antibodies against Shiga toxinand vaccines against HC and HUS using Shiga toxoids and fusions of Shiga toxoids.SUMMARY OF THE INVENTIONThe present invention describes the isolation and puriï¬cation of biologicallyand immunologically active histidineâtagged Shiga toxins (His-tagged), a toxinassociated with HC and the potentially life-threatening disease HUS transmitted bystrains of pathogenic bacteria. The present invention describes how his-tagging greatlysimplifies and expedites purifying Shiga toxins, and describes an improved method forsuch puriï¬cation.One aspect of the invention is obtaining and using Shiga toxoids that areimmunoreactive but not toxic. For example, the invention describes using suchobtained Shiga toxoids in vaccines against HC and HUS.Another aspect of the invention is obtaining and using fusion proteins of His-tagged Shiga toxins or toxoids. These fusion proteins have the advantage ofcombining beneï¬cial properties of each protein, resulting, for example, in improvedprotein stability or targeted delivery of a his-tagged Shiga therapeutic agent.Yet another aspect of the invention is obtaining and using antibodies to His-tagged Shiga toxins, toxoids, or Shiga toxin/toxoid fusion proteins. These antibodiescan be either monoclonal or polyclonal and have potential uses in treating, diagnosing,or preventing HC and HUS caused by EHEC or Shigella dysenteriae type 1 infections.Other aspects of the present invention will become apparent from the moredetailed description provided below, to be read in conjunction with the accompanyingdrawings.BRIEF DESQRIPTION OF THE DRAWINGSFigure 1 depicts the protein structure of Shiga toxin genes.Figure 2 depicts the predicted amino acid sequence for the mature A subunitand the unprocessed B subunit of Stxl. (Calderwood et al., Proc. Natl. Acad. Sci.USA, 84: 4364-4368 (1987); DeGrandis et al., J. Bacterial, 169:43l3-4319(l987)).10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97/ 15836-5-Figure 3 depicts the predicted amino acid sequence for the mature A subunitand the unprocessed B subunit of Stx2. (Jackson et al., FEMS Lett., 44:109-114(1987)).Figure 4 depicts the predicted DNA sequence for stxl and DNA upstream ofthat sequence. (Calderwood et al., Proc. Natl. Acad. Sci. USA, 84: 4364-4368 (1987);DeGrandis et al., J. Bacteriol., 169: 4313-4319 (1987)).Figure 5 depicts the predicted DNA sequence for stx2 and DNA upstream ofthat sequence. (Jackson et al._. FEMS Lett., 44: 109-114 (1987)).Figure 6 depicts the approximately 1200 base pair fragments of stx1 producedby PCR amplification. Figures 6aâc depict the fragments used to make plasmidspQHl, pQHEI, and p7HI, respectively. Nucleotides in lower case represent non-toxinsequences in the primers and/or base changes.Figure 7 depicts the approximately 1200 base pair fragments of stx2 producedby PCR ampliï¬cation. Figures 7a and 7b depict the fragments used to make plasmidspQHI and pQHEIl, respectively. Nucleotides in lower case represent non-toxinsequences in the primers and/or base changes.Figure 8 depicts the plasmid pQHI, encoding the His-Stx 1 fusion and drivenby the T5 promoter.Figure 9 depicts the plasmid pQHlI, encoding the His-Stx 2 fusion and drivenby the T5 promoter.Figure 10 depicts the plasmid pQHEI, encoding the His-Enterokinase site-Stxlfusion and driven by the T5 promoter.Figure 11 depicts the plasmid pQHEII, encoding the His-Enterokinase site-Stx2 fusion and driven by the T5 promoter.Figure 12 depicts the plasmid pQHIIvhb, encoding the His-Stx 2 fusion anddriven by the T5 promoter.Figure 13 depicts the plasmid pQHEllVhb, encoding the His-Enterokinase site-Stx 2 fusion and driven by the T5 promoter.10152025W0 98/1 1229CA 02265887 1999-03-09PCT/U S97/ 15836-6-Figure 14 depicts the plasmid p7HI, encoding the His-Stx 1 fusion and drivenby the PT7 promoter.Figure 15 depicts the plasmid p7HII, encoding the His-Stx 2 fusion and drivenby the PT7 promoter.Figure 16 depicts the expression of His-Stx fusion proteins according to theinvention.DETAILED DESCRIPTIQN OF THE INVENTIONAn object of the invention is to purify large quantities of Shiga toxins thatretain their biological and immunological properties. To achieve this object, a Shigatoxin gene was cloned into a Histidine-tag expression vector, expressed, and purified.An additional object of the invention is to obtain antigens speciï¬c to Shiga toxoids,a toxin that is nonâtoxinogenic but immunoreactive, for generating an immuneresponse against Shiga toxins. Another object of the invention is the creation ofantibodies against Shiga toxins or toxoids for treating, diagnosing, or preventingdisease and infections by pathogenic bacteria. The his-tagged Shiga toxins or toxoidsdescribed above can be used for these purposes.Those skilled in the art will also recognize that the size of the his-tagged Shigatoxin to be used may be varied according to the speciï¬c purpose for the Shiga toxin.For example, if the purpose is fusing the his-tagged Shiga toxin or toxoid with one ormore proteins, a smaller fragment might be selected to enhance stability of thecombined fusion product, although using a larger fragment is by no means precluded.The desired size of the His-Shiga toxin may also vary with the convenience of theavailable restriction sites, in light of the materials and methods known to those skilledin the art. Consequently, the terms "HisâShiga toxin" or "His-tagged Shiga Toxin"refers to the fragment of about 372-377 amino acids comprising the A and B subunitsof any of the Shiga toxin family members fused with a histidine tag. Smallerfragments that retain biological and/or immunological function are also included.Biological ï¬mction is measured by, for example, cytotoxicity to Vero cells, asdescribed in Example III.A. Immunological function may also be tested by, for10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97/ 15836-7-example, neutralization by speciï¬c antisera, as described in Example lII.B. Apreferred embodiment of the invention is a His-Tagged Shiga holotoxin, containing1 A subunit and 5 B subunits. In another preferred embodiment, the tag consists of sixhistidine residues. The most preferred embodiment is a Hisâ,-Tagged Shiga holotoxin.One of the objects of the present invention is to administer His-Shiga toxoidsto protect against illness or disease caused by EHEC or Shigella dysenteria type I, suchas HC and HUS. The object is achieved through the stimulation of immune responsedirected against Shiga toxins.Consequently, the term "immunizing" or "immunization" is used in theapplication. The degree of protection achieved by such immunization will vary withthe degree of homology between Shiga toxins and the His-Shiga toxoids, as well asother factors, such as unique attributes of the patient or the species treated. Moreover,immunization is not limited to avoiding infection altogether; it also includesdecreasing the severity of the infection, as measured by the following indicators:reduced incidence of death, HUS, or permanent kidney damage; decreased levels oftoxin; reduced ï¬uid loss; or other indicators of illness regularly used by those skilledin the relevant art.Unless specified otherwise, the uses and methods set forth herein are generallyapplicable to humans and animals. The term "patient" is used herein to mean bothhumans and animals, and "animals" is not limited to domesticated animals but alsomay include wildlife and laboratory animals.Moreover, because His.-Tagged Shiga toxins according to the invention havethe biological and immunological properties of Shiga toxins, they may be used for anyapplication appropriate for Shiga toxins. For example, it has been recentlydemonstrated that Stxl can be used to treat bone marrow cells from mice with humanB-cell lymphomas. The Shiga toxin bound to the receptor on the lymphoma cell andthe toxin killed the cancer cell. (LaCasse et al., Blood 88:1551(l996)). Thus, theskilled artisan would expect that His-Shiga toxins or fusions could be used for thesame purpose and in the same manner.10152025W0 98/11229CA 02265887 1999-03-09PCT/U S971 15836-3-Isolating and Purifying His-Tagged Shiga ToxinThe standard protocol for puriï¬cation of Shiga toxin comprising A and Bsubunits uses biochemical techniques. The standard protocol was developed byO'Brien et al. (O'Brien et al., Infect. Immun. 402675 (1983); O'Brien et al., Infect.Immun., 30:l70(l 980)). The method employs four puriï¬cation steps: 1) ammoniumsulfate 2) DEAE Sepharoseprecipitation; column chromotography; 3)chromatofocusing; and 4) antibody affinity chromotography. This method has theadvantages of employing publicly available materials, being capable of purifying allShiga toxins, and being capable of purifying Shiga toxins for human use. Itsdisadvantage is that the minimum time required for this test is three weeks.Another well-known method for purifying Shiga toxin from bacteria wasdeveloped by Keusch et al. (Donohue-Rolfe et al., Infect. Immun. 5723888 (1989);Acheson et al., Microb. Pathog. 14:57 (1993)). This method employs a Shiga toxinreceptor analog. The receptor analog is the Pl glycoprotein (Plgp) from tapewormhydatid cysts material (HCM) in sheep gut. This method contains three puriï¬cationsteps: 1) ammonium sulfate precipitation; 2) Blue sepharose chromotography; and 3)Plgp column chromatography. The Plgp must be prepared from the HCM. Thoughfaster than the standard method, this method still requires a minimum of two or moreweeks. The hydatid cyst material must be obtained from infected sheep and is notpublicly available. The method has the additional disadvantages of being capable ofuse with only those Shiga toxins that bind Plgp and, because of possiblecontamination, it is not appropriate for obtaining Shiga toxoids for use in humans.Using recombinant methods, the Shiga toxin gene or portions of the Shigatoxin gene have been cloned and expressed in bacteria and puriï¬ed. Zollman et al.,Prot. Expression Pur. 5:291 (1994), puriï¬ed a recombinant Stxl A1 fragment.Acheson et al., Infect. Immunol. 63:301(1995), expressed and puriï¬ed the Stx2 Bsubunit. Downes et al., Infect. Immun. 5621929 (1988), expressed the stx2 gene inbacteria and puriï¬ed Stx2. However, the puriï¬cation methods following expression10152025W0 98/1 1229CA 02265887 1999-03-09PCT/U S97l 15836-9-were essentially those of the standard method or hydatid cyst method and, therefore,had the same disadvantages.In the search for a method for purifying Shiga toxins, applicants havedeveloped a purification method based on the creation of a histidineâtagged Shigatoxin. Methods for histidine tagging are known in the art. For example, Fryxell et al.,Biochem. Biophys. Res. Comm., 2102253-259 (1995), added a kemptide and histidinetag to the A chain of the eukaryotic toxin ricin, which was later associated with the Bsubunit. The ricin toxin differs from Shiga toxins in origin (prokaryotic v. eukaryotic)and structure of the B subunit (the ricin B subunit is a single polypeptide, not apentamer). Moreover, Fryxell et al. only expressed the A subunit with a His-Tag. Inaddition, Strauss et al., FEMS Microbiol. Lett., 1272249-254 (1995), have his-taggedthe C-terminus of the cholera toxin B subunit, and expressed a his-tagged B subunit-IgA protease fusion protein. However, this did not involve expressing the entire toxinwith a His-Tag, and the expressed fusion protein did not undergo multimerization.Finally, Terbush & Novick, .1 Cell. Biol., 130:299â312 (1995), tagged the C-terminusof a multiunit yeast protein. This involves a eukaryotic rather than a prokaryoticsystem. Moreover, expressing a functionally active Shiga toxin requires retaining itsmultimer conformation, as well as its receptor binding and enzymatic activity.Although His-Tagging of proteins is known, it was not expected that His-Tagging of a Shiga toxin would be successful. The skilled artisan would have believedthat a His-Shiga toxin fusion would have lost cytotoxicity, because the skilled artisanwould have expected that the attachment of a His-Tag to the amino acid terminus ofa toxin would destroy its activity. Moreover, the multi-unit toxin would have beenexpected to be more susceptible to losing toxicity upon fusion with additional aminoacids, since it is known that the toxin must retain its conformation for enzymaticactivity and for binding of the B subunits to cell receptors, and the addition of aminoacids would have been expected to destroy proper conformation. The skilled artisanwould be aware that confonnation and the charge of the molecule is critical to Shigatoxins. For example, the skilled artisan would know that deleting a few N-terminall0152025W0 98/1 1229CA 02265887 1999-03-09PCT/U S97/ 15836-10-amino acids from Stx2A destroyed enzymatic activity, as reported in Perera et al.,Infect. Immun0l., 59:829-835 (1991)). Similarly, altering the C-terminus of the Bsubunit affected toxicity. (Perera et al., supra). Additionally, Perera et al. suggestedthat the charge of the molecule plays an important role. The importance of preservingShiga toxin conformation is further underscored by ï¬ndings that the highlyhomologous StxlA and Stx2B subunits cannot be combined to form an active toxin.(Weinstein et al., Irï¬zct. Immun, 57:3743-3750 (1989)). Based on this knowledge, theskilled artisan would have expected that tagging a Shiga holotoxin with histidineresidues would have unfavorably affected conformation and charge of the toxinproduct.Surprisingly, his-tagging of Shiga toxin comprising A and B subunits generateda functional Shiga toxin, which has similar speciï¬c activity to Shiga toxin puriï¬ed bystandard methods. Moreover, the His-Shiga toxins are neutralized by monoclonalantibodies speciï¬c for Shiga toxins. Example 1 describes how to create the His-Shigatoxin fusion protein.The following examples are intended to illustrate the invention but not to limitit. The skilled artisan will understand from these examples that modiï¬cations can bemade that are still within the scope of the invention. The scope of the invention isdeï¬ned by the claims.Example IA. Construction of Plasmid Encoding His-Tagged Shiga ToxinThe HisâStx fusion clones were generated by PCR amplification of six operons,restriction enzyme digestion of the PCR products, and ligation of the fragments in-frame into the appropriate vectors. The expression vectors and primers were used toplace histidine residues at the amino acid terminus of the toxins and place theconstructs under the control of either an IPTGâinducible promoter (pQE vectors)(Qiagen, Inc., 9600 DeSoto Avenue, Chatsworth, CA 91311, 1-800-362-7737) or a T7promoter Q3t7-7) (Tabor et al., Proc. Natl. Acad. Sci. 82: 1 074 (1985)). The methodsfor obtaining HisâStx fusion clones are described in more detail below.CA 02265887 1999-03-09W0 98/11229 PCT/US97l15836-11-1. Bacterial strains and plasmids. The bacterial strains and plasmidsused in this study are shown in Table 1.Table 1. Bacterial strains and plasmids used in this study.5 Strain or plasmid Characteristic(s) Source orreferenceE. coli strainsDH5oc Host strain for cloning BRLXL1-Blue Host strain for cloning; lacl; Tcâ StratageneM15 Host strain for protein puriï¬cation Qiagen10PlasmidspJES120 Encodes stx II toxin operon apJN 25 Encodes stx I toxin operon bpSQ543 Encodes stx IIvhb operon c15 pQE30 Histidine fusion vector QiagenpQE32 Histidine fusion vector QiagenpREP4 lacl; Knâ QiagenpT7-7 T7 expression vector dpGP1-2 Encodes T7 RNA polymerase; KNâ d20 a = Lindgren et al., Infect. Immunol. 61 :3832 (1993).b= Newland et al., "Cloning of shiga-like toxin structural genes from a phage ofEscheria coli strain933, in Advances in Research on Cholera and Diarheas (S. Kuwahara & N.F.Pierce eds. 1994).25 c= Lindgren et al., Infect. Immunol. 62:623 (1994).d= Tabor et al., Proc. Natl. Acad. Sci. USA 8221074 (1985).2. Media and enzymes. Bacterial strains were grown in L broth (perliter: 10 g tryptone, 5 g yeast extract, 5 g NaCl). Kanamycin, tetracycline, and30 ampicillin (Sigma Chemical Co., St. Louis, M0.) were added to the medium at ï¬nalconcentrations of 25, 10, and 100 ug/ml (respectively) as needed. RestrictionCA 02265887 1999-03-09WO 98111229 PCT/US97Il5836-12-endonucleases, calf intestinal phosphatase, and ligase were from BoehringerMannheim, Indianapolis, Ind., or U.S. Biochemicals Corporation, Cleveland, Ohio.Enzymes were used according to manufacturerâs instructions.3. Primers and PCR. The Shiga toxin genes are cloned using polymerase5 chain reaction (PCR), a standard technique in the art. Primers were designed toamplify the stx toxin operons beginning at the ï¬rst codon of the mature A subunit geneand ending downstream of the termination codon of the B subunit gene and createdusing standard techniques. The primers contained recognition sequences to generateunique restriction sites at the ends of the toxin operon. In a preferred embodiment, the10 5' primers also contained sequences to encode the recognition sequence of the proteaseenterokinase to allow for removal of the histidine residues. The primers used areshown in Table 2.TABLE 2. Primers used.15 Primer Primer Sequent (5'-3') Restrictionsitemac GCGGATCCGATGACGATGACAAACGGGAGTTTACGATAGACTT BamHlIIBAM GCGGATCCGGGAGTTTACGATAGACTT BamHlIIH3 CCACGAATAAGCTTATGCCTCA HindIII20 IBAM5 GCGGATCCAAGGAATTTACCTTAGACTTC BamHlIEC5 GCGGATCCGATGACGATGACAAAAAGGAATTTACCTTAGACTTC BamHlIPST3 ATTTTCACTGCAGCTATTCTG PstlSLTIIH5 GCATATGCATCACCATCACCATCACCGGGAGTTTACGATAGAC NdelSLTIH5 GCATATGCATCACCATCACCATCACAAGGAATTTACCTTAGECTTC Ndel25 SLTLIC3 TAACATTTATCGATATCTCCGCCTG ClalSequences encoding stx toxins were ampliï¬ed from toxin clones using a PCRkit (GeneAmp kit, Perkin-Elmer Cetus, Norwalk, CT), which was used according tothe manufacturer's instructions. The resulting stx PCR products were approximately1015202530W0 98/1 1229CA 02265887 1999-03-09PCT/US97l 15836-13-1200 bp are shown in Figures 6a, 6b, 6c, 7a, and 7b. The DNA products contain thecoding sequences for the mature A subunit and the unprocessed B subunit.Procedures for cloning are well known in the art and are described in Maniatis,Molecular Cloning: A Laboratory Manual (1982)).4. DNA manipulations. Plasmid DNA was isolated by the method ofHolmes and Quigley, Anal. Biochem., 1142193-197 (1981). Alternatively, plasmidDNA was purified using Qiagen columns (Qiagen Inc., Chatsworth, CA). PCRproducts were digested with restriction endonucleases and ligated into the pQE30/32vectors (Qiagen, Inc.), or into the vector pT7-7. PCR reactions and ligations aresummarized in Table 3.TABLE 3: PCR Reactions and ligations.Plasmid Primer Resulting clonetemplate pair Vector Cloning sitespJES 120 IIEC + IIH3 pQE30 BamHI/Hindlll pQHEIlpJESl2O IIBAM + IIH3 pQE32 BamHl/Hindlll pQHIlpJN25 IECS + IPST3 pQE30 BamHI/Pstl pQHEIpJN25 IBAM + lPST3 pQE30 BamHI/Pstl pQHIpJESl2O SLTIIH5 + IIH3 pT7-7 Ndel/Hindlll p7HIIâpJN25 SLTIH5 + SLTIC3 pT7-7 Ndel/Clal p7HlpSQ543 IIEC + IIH3 pQE30 BamHl/Hindlll pQHEIIvhbâpSQ543 [IBAM + IIH3 pQE32 BamHI/Hindlll pQHlIvhbââ The construction of these plasmids is in progress.To illustrate, the clone pQHEII was constructed as follows:Plasmid pJESl2O was the template with primers IIEC and IIH3 in a PCRreaction for ampliï¬cation of the stx2 operon. The resulting PCR product started withthe ï¬rst codon of the mature A subunit gene, extended through the A subunit gene, thecomplete B subunit gene, and ended just downstream of the terminate codon of the Bsubunit gene (Figure 6b). The PCR product was digested with the restrictionendonucleases Bam HI and Hind III, as was the vector plasmid pQE30. The vector10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97l15836-14-pQE30 was chosen because ligation of the PCR product with pQE30 at the BamHIsites would result in an in-frame protein fusion of the 6 histidine residues, theenterokinase cleavage site, additional amino acids, and the +1 residue of the matureA subunit. The digested PCR product was ligated into the digested vector pQE30.The ligation reaction was transformed into strain XL1-Blue and plated on agar thatcontained ampicillin. Colonies were screened for the presence of a plasmid thatcontained an approximately 1200 bp BamHI/HindIII DNA insert. Clones wereconï¬rmed by IPTG induction of toxin expression (Example II) with a subsequent testfor cytoxicity on vero cells (Example III). Positive clones were then transformed intoMl 5(pREP4) for large scale production of toxin.Example IILarge scale purification of His-Tagged Shiga ToxinsHis-Shiga toxin was puriï¬ed under nondenaturing conditionsibecause of themulti-subunit nature of the Shiga toxins. The strain was streaked onto a selective agarplate and incubated at 37°C for 18-24 hrs. A 20 ml overnight culture was thenprepared from a colony. The saturated culture was then diluted 1/50 into one liter ofL broth with antibiotics and the culture was grown at 37°C until it reached an O.D.600of 0.7-0.9. IPTG (2mM ï¬nal concentration) was then added to induce expression ofthe His; tagged toxin and the culture was grown for an additional 5 hrs. Cells werepelleted and the pellet was kept at -70°C overnight. The pellet was resuspended insonication buffer (50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, 20mM imidazole, 30 ug/ml PMSF), and the cells were sonicated to release toxin.Alternatively, the cells were treated with polymixin-B (2 mg/ml ï¬nal concentration)for 3 hrs at 4°C. The extracts were clariï¬ed by centrifugation and ï¬ltered through amilipore 0.45 pm ï¬lter.The nickel-nitrilotriacetic acid ligand (Ni-NTA) gel was equilibrated withsonication buffer and the cell extract was added to the gel. Protein was allowed to bindfor 1 hr at room temperature or at 4°C. The gel was washed with sonication bufferfollowed by wash buffer (50 mM sodium phosphate (pH 8.0), 300 mM sodium1015202530W0 98/1 1229CA 02265887 1999-03-09PCT/US97l15836-15-chloride, 20 mM imidazole, 30 ug/ml PMSF, 10% glycerol, 1% tweenâ20). Proteinwas eluted from the gel with a gradient of imidazole (0-500 mM in wash bufferwithout tween-20), and 1 ml fractions were collected.Fractions were tested for cytotoxicity on Vero cells (as explained in ExampleIII) and were subjected to SDS-PAGE and silver stain. Fractions that were highlycytotoxic and relatively clean were pooled and dialyzed against sonication buffer. Thispool was then placed onto a Ni-NTA spin column (Qiagen) to further purify the His6-toxin and the resulting two fractions were dialyzed against PBS. A ï¬nal cytotoxicityassay and BCA protein assay were performed for the determination of the speciï¬cactivity of the puriï¬ed toxin.The protocol described above is modiï¬cation of the non-denaturing protocoldescribed by Qiagen to purify His-tagged proteins. However, the toxin that elutedcontained many contaminants. To achieve purer His-Shiga toxin, modiï¬cations weremade. Speciï¬cally, Tween-20 was added to the wash buffer, and the pH of the washbuffer was adjusted to 8. Also, a ï¬nal Ni-NTA spin column was added.This one-step His-afï¬nity method for purifying His-Shiga toxin by an Ni-NTAcolumn has several advantages over existing methods, as summarized in Table 4.TABLE 4. Comparison of Toxin Puriï¬cation TechniquesPuriï¬cation Minimum time Stepsâ Materials available Use for allMethod requiredâ Shiga toxinsStandard 3 weeks 4 yes yesHydatid 2 weeks + 3 no noCystHisé afï¬nity 1 week 2 yes yes3 Time from streaking the strain onto an agar plate. This does not include thepreparation of Plgp from hydatid cyst material which takes a minimum of 1.5 weeks.b This does not include the multiple steps involved in the puriï¬cation of Plgp fromhydatid cyst material and preparation of the column.The Ni-NTA one-step method is superior because of its relative speed andsimplicity. It requires a minimum of one week as opposed to a minimum of two or10152025W0 98/ 11229CA 02265887 1999-03-09PCTIUS97/15836-16-more weeks. Moreover, all of the materials are readily available, the method is notlimited to Shiga toxins that bind Pl gp, and the products are suitable for use in humans.The Shiga toxin obtained by the method has many uses. For example, the His-Shiga toxin may be used as a positive control antigen in a Shiga toxin detection kit.Such kits will use a puriï¬ed His-Shiga toxin as positive indicator for the toxin in asample. Other uses are detailed in the Examples below.Example IIIVerifying Biological and Immunological Activity of His-Shiga ToxinsA. Vero Cell Cytotoxicity AssayThe cytotoxicity of His-Shiga toxins obtained according to the methodsdescribed in Examples 1 and II was veriï¬ed by determining their cytotoxicity for Verocells. Cytotoxicity assays on strains that expressed His-Shiga toxins were doneessentially as described by Gentry and Dalrymple, J Clin. Microbiol, 12: 361-366(1980). Brieï¬y, cultures induced for the expression of His-Shiga toxins were disruptedby sonic lysis and clariï¬ed by centrifugation. The extracts were serially diluted intissue culture medium (Dulbecco modiï¬ed Eagle medium containing 10% fetal calfserum, 0.8 mM glutamine, 500 U of penicillin G per ml, and 500 mg of streptomycinper ml). One hundred microliters of 10-fold dilutions of the lysates were added tomicrotiter plate wells containing about 104 Vero cells in 100 pl of medium. The tissueculture cells were incubated at 37°C in 5% CO2 for 48 hours and then ï¬xed and stainedwith crystal violet. The intensity of color of the ï¬xed and stained cells was measuredwith a Titertek reader at 620 nm.B. Antisera Neutralization AssayHis-Shiga toxins obtained according to the methods described in Examples Iand II were tested for antisera neutralization. Neutralization of cytoxic activity wasdescribed in great detail in Schmitt et al., Infect. and Immun., 59:1065-1073 (1991).Brieï¬y, lysates were incubated with serial dilutions of monoclonal or polyclonalantisera speciï¬c for Stxl or Stx2 at 37°C for 2 hours. One hundred microliters of the10152025W0 98/11229CA 02265887 1999-03-09PCT/US97I15836_17_samples were then added to vero cells as described above. Percent neutralization wasdetermined by the following formula:{ [A520(toxin + antibody) - A620(toxin)]/A 620(untreated cells)} x 100.Example IVConstructing Fusions with His-Shiga Toxins and Other ProteinsUsing methods well-known in the art, the His-Shiga toxin could be fused withanother protein of interest. These methods include chemical and genetic methods, asin cloning and expressing a fusion protein, although one skilled other methods arereadily apparent to one skilled in the art. (D.V. Goeddell, Meth. Enzymol. Vol.l85(1990); Itakura, Science 19821056 (1977)). For example, if a combination vaccinefor immunization against Shiga toxin and another toxin (protein X) is desired, thenthese two toxins can be fused into a single protein. This can be achieved by firstcloning the codons for the histidine residues in frame to the coding region of proteinX. The fragment containing His-Protein X is then subcloned in-frame of the Shigatoxin operon. In a preferred embodiment, the fragment is subcloned in-frame to theA2-B portions of the Shiga toxin operon. The resulting His-Protein X-A2-B5 fusionwould ideally result in immunization against Shiga toxin and protein X.One skilled in the art would recognize that various proteins from pathogens andhaptens may be conjugated to a His-Shiga toxin. Haptens and antigens may derivefrom but are not limited to bacteria, rickettsiae, fungi, viruses, parasites, drugs, orchemicals. They may include, for example, small molecules such as peptides,oligosaccarides, and toxins. Certain antimicrobial drugs, chemotherapeutic drugshaving the capacity of being absorbed into the intestine may also be coupled to Shigatoxin for targeted delivery, since the B subunit pentamer binds to receptors in theintestine. Conjugation methods are well known in the art. Exemplary methods are setforth in Goeddel, "Systems for Heterologous Gene Expression," Meth. Ezymol., 185(1990), Itakura, "Expression in E. callâ of a chemically synthesized gene for thehormone somatostatin," Science, 198:l056âl063 (1977), and Goeddel et a1.,10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97l15836-13-"Expression of chemically synthesized genes for human insulin," Proc. Natl. Acad.Sci. USA, 281: 544-548(l979).Conjugation may be achieved by genetically fusing His-Shiga toxoids bystandard molecular techniques or by conjugation to a polysaccharide. Methods ofconjugation include those outlined in M. Brunswick et al., J. Immunol, 14023364(1988) and Chemistry of Protein Conjugates and Crosslinking, CRC Press, Boston(1991). Coupling of Shiga toxids to other proteins or polysaccharides would preventdisease from additional pathogens.Example VHis-Shiga ToxoidsA form of Shiga toxin that is immunoreactive but not toxinogenic is needed forimmunization in animals. Such a His-Shiga toxoid can be generated using chemicalor genetic methods. The chemical method involves treating the His-Shiga toxin witheither formaldehyde or glutaraldehyde, as described by Perera et al., J Clin. Microbiol.2612127 (1988)). Brieï¬y, samples of toxin containing 100 pg of protein are treated for3 days at 37°C with 0.1 M Na2HPO,, (pH 8.0) containing 1% formaldehyde, and theresidual formaldehyde is removed by dialysis against phosphate-buffered saline (PBS).To prepare His-Shiga toxoid by treatment with glutaraldehyde, crude toxin samplescontaining 50 pg of protein are incubated at 37°C in 0.11% glutaraldehyde in 0.1 MNa3HP04 (pH 8.0) for 30 min. The toxoid is then tested on Vero cells, as described inExample III, for loss of cytotoxicity.Genetically, a toxoid may be produced by siteâdirected mutagenesis, asdescribed in Gordon et al., Infect. Immun. 602485 (1992); Hovde et al., Proc. Natl.Acad. Sci. 85:2568 (1988); Jackson et al., J. Bacteriol. 172: 3346-3350 (1990).Several methods and kits exist for siteâdirected mutagenesis of a gene. One methodemploys the Bio-Rad Muta-Gene in vitro mutagenesis kit. Oligonucleotides can bedesigned and synthesized which alter speciï¬c condons in the toxin genes. Uracil-incorporated, single-stranded target plasmid DNA will be mutagenized according to10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97/ 15836-19-the directions supplied by the manufacturer of the mutagenesis kit. The nucleotidechanges are then conï¬rmed by DNA sequence analysis.In the His-Shiga toxin, two or more amino acids essential for enzymatic activityshould be altered. For example, A subunit targets are the residues E167 and E170. TheShiga toxoid resulting from this mutation has been used for vaccinating pigs. (Gordonet al., supra).Example VIPassive Immunity to Shiga Toxin Using His-Shiga Toxin and ToxoidA. Antisera Specific for His-Shiga ToxinsAntisera speciï¬c for Shiga toxins are required to treat and prevent potentially-deadly infections by EHEC and Shigella dysenteriae type I. Speciï¬cally, once a childbecomes infected, the child and his or her family members or children in his or her daycare group can receive anti His-Shiga toxin sera to achieve a protective immuneresponse. A protective immune response is one that elicits sufï¬cient antibody topermit a patient to avoid infection, decrease the signiï¬cance or severity of an infection,or decrease the ability of bacteria to colonize the gastrointestinal tract.Animal studies have shown that administering anti-Shiga toxin sera to miceresults in resistance to normally lethal infection of EHEC. (Lindgren et al., Infect.Immun. 62:623(1994); Wadolowski et al., Infect. Immun. 5813959). Thus, applicantsbelieve that administering anti-Shiga toxin sera to humans and other mammals wouldresult in a protective immune response against Shiga toxin infections.Methods are wellâknown in the art for producing antisera for passiveimmunization. For example, His-Shiga toxoid, obtained by the methods described inExample VI, can be administered to a mammal, such as a horse intraperitoneally.Currently, the horse is used to produce serum against botulism toxin for administrationto humans, Hibbs et al., Clin. Infect Dis., 232337-40 (1996), and the horse would bea preferred method for producing shiga toxin antiserum. After several boosts with His-Shiga toxoid, the serum of the immunized horse (or other mammal) would be testedfor neutralizing the cytotoxicity of Shiga toxins. Advantageously, a large amount of10152025W0 98/11229CA 02265887 1999-03-09PCT/US97/15836-20-serum can be quickly made using this method. However, patients must first bescreened for an immune reaction to horse serum. For this purpose, a small amount ofhorse serum would be subcutaneously injected, and the patient would be monitored fora reaction. Such methods for administering horse antiserum against toxins to humansare well known to the skilled artisan. Hibbs et al., supra; Dehesa and Possani,Toxicon, 32: 1015-101 8(l 994); Gilan et al., Toxicon, 27:1 105-11 12 (1989).More preferably, the HisâShiga toxoid can be administered to humanvolunteers, either intraperitoneally or orally. The plasma from these volunteers is thenisolated, and the human anti-His-Shiga toxin serum can be administered to patients.No threat of serum sickness arises from this method. Human hyperimmune globulinto Hemophilus inï¬uenzae b, Streptococcus pneumoniae, and Neisseria meningitidishas previously been prepared by others (Siber et al., Infect. and Immun., 45: 248-254(1984)).B. Vaccines Against Shiga ToxinsAn embodiment of the invention is vaccines against Shiga toxin infection. Forexample, these vaccines can include antibodies directed against His-Shiga toxin,obtained further described in Example VII. Moreover, these vaccines can becombination vaccines that comprise His-Shiga toxoid fused or conjugated with anotherprotein, hapten, or antigen, as described in Example IV. These vaccines can beadministered intraperitoneally or injectably by methods well known in the art.A preferred method of administering HisâShiga toxin or toxoid and fusionsthereof is by further conjugation to Synsorb® (SynSorb Biotech, Inc., 1204Kensington Road, N.W., Calgary, Alberta, Canada, T2N3P5.) Synosorb is a sand-likematerial to which Shiga toxin receptor (Gb3) is covalently bound (Armstrong et al.,J. Infect. Dis., 171 : 1042 (1995)). This compound has been shown to bind Shiga toxinsand appears to be safe for human ingestion. (Armstrong et al., supra) The Synsorb isbound to the B subunit pentamer via the B subunit pentamer-receptor reaction.Conjugation with Synsorb adds further stability.10152025WO 98111229CA 02265887 1999-03-09PCT/US97l 15836-21-Another embodiment of the invention involves the administration of nucleicacid vaccines. DNA encoding a His-Shiga toxoid is injected into a patient as nakedDNA, or the DNA is delivered to the body by a carrier system such as retro viruses,adenoviruses, or other carriers known in the art. Following administration, the patientmounts an immune response against transiently expressed foreign antigens.Currently nucleic acid vaccines, in general, are all nearing clinical trials. Thisapproach to vaccines involves delivering the DNA encoding the desired antigen intothe host by inserting the gene into a nonreplicating plasmid vector (Marwick, C.JAMA 273: l403(1995); reviewed in Vogel, F.R. and N. Sarver, Clin. Microbiol, Rev.8:406 (1995)).The first published demonstration of the protective efï¬cacy of such a vaccinehas shown that intramuscular injection of plasmid DNA encoding inï¬uenza A virus(A/PR/8/34) nucleoprotein (NP) elicited protective immune responses in BALB/c miceagainst a heterologous strain of inï¬uenza virus (A/HK/68) (Ulmer, J .B. et al. Science259:] 745(l 993)). Immunized animals had reduced virus titers in their lungs,decreased weight loss, and increased survival compared with challenged control mice.Both NP-speciï¬c cytotoxic T lymphocytes (CTL's) and NP antibodies were generated.The NP antibodies were ineffective at conferring protection, but the CTL's killed virus-infected cells and cells pulsed with the appropriate major histocompatibility complexclass I-restricted peptide epitope.Another study has shown that intramuscular injection of plasmid DNAencoding inï¬uenza virus A/PR/8/ 34 hemagglutinin resulted in the generation ofneutralizing antibodies that protected mice against a heterologous lethal inï¬uenza viruschallenge (Montgomery, D.L. et al. DNA Cell Biol., l2:777 (1993)).Example VIIHis-Shiga AntibodiesHis-Shiga antibodies, polyclonal and monoclonal, can also be used in thetreatment, diagnosis, and prevention of infections related to Shiga toxins. Because oftheir increased speciï¬city, monoclonal antibodies are preferred. His-Shiga toxin10152025WO 98111229CA 02265887 1999-03-09PCT/US97/15836-22-antibodies can be administered to humans or other mammals to achieve a protectiveimmune response, for treatment or prophylaxis. Antibodies, in a physiologicallyacceptable carrier, may be administered orally or intraperitoneally. For this purpose,monoclonal antibodies are preferred and humanized monoclonal antibodies areparticularly preferred. Positive clinical responses in humans have been obtained withmonoclonal antibodies, and one skilled in the art would know how to employ Shigamonoclonal antibodies in humans. See Fagerberg et al., "Tumor Regression inMonoclonal Antibody-treated Patients Correlates with the Presence of Anti-idiotypeâreactive T Lymphocytes," Cancer Research, 55:1824â27 (1995); "A Phase I Study ofHuman/Mouse Chimeric Anti-ganglioside GD2 Antibody ch14.18 in Patients withNeuroblastoma," Eur. J. Cancer, 2:261-267 (1995)).Another embodiment of the invention involves using antibodies to diagnoseShiga toxin infections. The antibody, using well-known methods of immunoassaying,is brought into contact with a sample from a patient, such as a fecal sample. Inaddition, the antibody may be used to detect Shiga toxins in sample taken from cow,such as cow feces. Moreover, meat may be tested using the anti His-Shiga toxinantibody for detection. A detection kit comprising the His-Shiga toxin antibody canbe used for this purpose.For example, a sandwich Elisa can be used. In this kit, rabbit anti-His-Shigatoxin antibody can be used to capture toxin from a sample to be tested. Goat anti-His-Shiga toxin antibody can then be added followed by a secondary antibody such asmouse oz-goat antibody conjugated to horseradish peroxidase. The antibody can bedetected by standard methods.His-Shiga toxin polyclonal antibodies and monoclonal antibodies are describedbelow.A. Making Polyclonal AntibodiesThe technique of Harlow, E. and D. Lane (eds.), Antibodies- a LaboratoryManual, Cold Spring Harbor, New York (1988), may be followed. The generalprocedure is outlined herein. Take pre-bleeds of each mouse to be immunized: Bleed10152025W0 98/1 1229CA 02265887 1999-03-09PCT/U S97/ 15836-23-from the tail vein into an eppendorf tube. Incubate at 37°C for 30 min, stir gently witha sterile toothpick (to loosen the clot), store overnight at 4°C. In the morning, spin 10min/ 10,000 rpm in the microfuge, and collect the serum (i.e., supernatant; red bloodcells are the pellet). Store the serum at â20°C. The sera obtained will be used as anegative control after the mice are immunized.Inject a BALB/c mouse intraperitoneally with 25 pg of His-Shiga toxoid (usingTitremax adjuvant, according to the instructions of the manufacturer (CytRyx Corp.,154 Technology Pkwy., Norcross, GA. 30092, 800-345-2987)). Wait 2 weeks, boostwith an identical shot, wait 7 days and bleed from the tail vein into an eppendorf tube.Incubate at 37°C for 30 min, stir gently with a sterile toothpick (to loosen the clot),store overnight at 4°C. In the morning, spin 10 min/10.000 rpm in the microfuge, andcollect the serum. Store the sera at â20°C.B. ELISA to test titer of Abs.The technique of Harlow, E. and D. Lane (eds.), Antibodies: A LaboratoryManual, Cold Spring Harbor, New York (1988), may be followed. The generalprocedure is outlined below:(1) bind His-Shiga toxoid to plastic microtiter plates at 50 ng/well in PBS. Incubate2h/RT (room temp) or overnight at 4°C.(2) wash plate 2X with PBS.(3) block wells with 100 pl blocking solution [3% bovine serum albumin (SigmaChemical, St. Louis, MO.), 0.02% sodium azide (Sigma) in PBS - store stock at 4°C]forl-2hatRT.(4) wash plate 2X with PBS.(5) primary Ab = 50 pl test sera diluted in blocking solution for example, start with1:50 and do eleven 1:2 dilutions, or start with 1:50 and do eleven 1:10 dilutions),incubate 2 h/RT.(6) wash 4X with PBS.(7) secondary Ab = goat horseradish-conjugated anti-mouse lg, affinity puriï¬ed(Boehringer Mannheim Corp., 9115 Hague Rd., P.O. Box 50414, Indianapolis, IN.10152025W0 98/1 1229CA 02265887 1999-03-09PCT/US97/15836-24-46250,800-262-1640). Add secondary Ab diluted 1:500 in blocking solution withoutazide. Incubate 1 h/RT.(8) wash 4X with PBS.(9) add 100 pl TMB Peroxidase substrate to each well (prepared according to theinstructions of the manufacturer, BioRad Labs, 3300 Regatta Blvd., Richmond, CA.94804). Allow blue color to develop (no more than 10 min). Stop the reaction with100 pl HZSO4. Read the plate at 450 nm.A titer is deï¬ned as an absorbance value 20.2 units above that obtained formouse preimmune sera.Anti-Shiga toxin Abs obtained from animals may be used clinically if onechanges the speciï¬city of the antibody to htunan. Such techniques are well known tothose of ordinary skill in the art. G. Winter et al., "Man-made antibodies," Nature,349: 293-299 (1991); P.T. Jones et al., "Replacing the complementarity-deterrniningregions in a human antibody with those from a mouse," Nature, 321: 522-525 (1986);P. Carter et al., "Humanization of an anti-pl 85âER2 antibody for human cancertherapy," Proc. Natl. Acad Sci. USA, 89: 4285-4289 (1992). Such antibodies may begiven to the sibling of an infected patient to reduce the risk of infection of the sibling.C. Raising Monoclonal Antibodies to His-Shiga ToxinMonoclonal antibodies directed against Shiga toxin are used to passivelyprotect a patient against EHEC and Shigella dysenteriae type I infections. Monoclonalantibodies are generated from mouse cells, and the speciï¬city of these antibodies arechanged for use in humans. G. Winter et al., "Man-made antibodies," Nature, 349:293-299 (1991); P.T. Jones et al., "Replacing the complementarity-determining regionsin a human antibody with those from a mouse," Nature, 321:522-525 (1986); P. Carter et al., "Humanization of an antiâpl85"Eâ2 antibody for humancancer therapy," Proc. Natl. Acad Sci. USA, 89: 4285-4289 (1992). Monoclonal Absrepresent a more "pure" antibody for administration to a patient.The procedure outlined in Harlow, E. and D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor, New York (1988) is followed: Five 4- to 5-week oldl01520W0 98/1 1229CA 02265887 1999-03-09PCT/US97l15836-25-female BALB/cJ mice are prebled, and immunized intraperitoneally with 25 pg His-Shiga toxoid suspended in 100 pl of TiterMax. Mice are boosted twice in two weekintervals, intraperitoneally with 25 pg His-Shiga toxoid suspended in 100 pl ofTiterMax. Seven days after each boost, blood (~3 00 - 500 pl) is collected from the tailvein. Sera are assayed for the presence of antiâShiga toxin antibody by ELISA (asdescribed above).Mice producing high titers of anti-His Shiga toxin antibodies are boosted bothintravenously and intraperitoneally with 25 pg of His-Shiga toxoid in 100 pl of PBS,sacriï¬ced three days later, and sera collected. Spleen cells are isolated and fused toSp2/O-Ag mouse myeloma cells (ATCC #CRLl58l) at a ratio of 10 spleen cells to lmyeloma cell. Fused cells are distributed into microdilution plates, and culturesupematants are assayed by ELISA after 3-4 weeks of culture for anti-His-Shiga toxinantibodies. Cultures positive for production of anti-His Shiga toxin antibodies areexpanded and cloned twice by limiting dilution.The person skilled in the art would understand how to use and practice theinvention based on the above disclosure. Other embodiments of the invention will beapparent to those skilled in the art from consideration of the speciï¬cation and practiceof the invention disclosed herein. It is intended that the speciï¬cation and examples beconsidered as exemplary only, with the true scope and spirit of the invention beingindicated by the following claims.
