WO1998011229A2 - Histidine-tagged shiga toxins, toxoids, and protein fusions with such toxins and toxoids, methods for the purification and preparation thereof - Google Patents

Histidine-tagged shiga toxins, toxoids, and protein fusions with such toxins and toxoids, methods for the purification and preparation thereof Download PDF

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WO1998011229A2
WO1998011229A2 PCT/US1997/015836 US9715836W WO9811229A2 WO 1998011229 A2 WO1998011229 A2 WO 1998011229A2 US 9715836 W US9715836 W US 9715836W WO 9811229 A2 WO9811229 A2 WO 9811229A2
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shiga
toxin
toxins
toxoids
shiga toxin
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PCT/US1997/015836
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French (fr)
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WO1998011229A3 (en
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Alison D. O'brien
Clare K. Schmitt
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Henry M. Jackson Foundation For The Advancement Of Military Medicine
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Priority to EP97939845A priority Critical patent/EP0929679A2/en
Priority to CA002265887A priority patent/CA2265887A1/en
Priority to AU41845/97A priority patent/AU4184597A/en
Priority to JP10513748A priority patent/JP2001500730A/en
Publication of WO1998011229A2 publication Critical patent/WO1998011229A2/en
Publication of WO1998011229A3 publication Critical patent/WO1998011229A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/25—Shigella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to a family of multi-unit bacterial proteins that are associated with hemorrhagic colitis and the life-threatening sequela, hemolytic uremic syndrome. These proteins, defined as members of the "Shiga toxin family,” have been tagged with histidine residues.
  • the invention further relates to a non-toxinogenic but immunoreactive form of histidine-tagged Shiga toxins, or toxoids.
  • the invention relates to fusion proteins obtained by combining histidine-tagged Shiga toxins or toxoids with other proteins. Histidine tagging greatly facilitates purification of Shiga toxins, and the invention also relates to methods for purifying such toxins.
  • the invention further relates to using the histidine-tagged Shiga toxoids or fusion proteins of Shiga toxoids as antigens for generating an immune response against infection or transmission by bacteria expressing Shiga toxin. It also relates to antibodies to Shiga toxins, toxoids, or Shiga toxin/toxoid fusion proteins, both monoclonal and polyclonal, and their use in treating, diagnosing, and preventing of disease and infections by pathogenic E. coli. Finally, the invention relates to preparing the Shiga toxins, toxoids, and fusion proteins. BACKGROUND OF THE INVENTION
  • EHEC Enterohemorrhagic Escherichia coli
  • HC bloody diarrhea
  • HUS hemolytic uremic syndrome
  • HC and HUS are transmitted by the ingestion of contaminated food, particularly undercooked beef products, such as hamburger.
  • contaminated food particularly undercooked beef products, such as hamburger.
  • undercooked beef products such as hamburger.
  • HC and HUS appear to be mediated by the toxin produced by EHEC and Shigella dysenteriae (for review see O'Brien and Holmes, Microbiol. Rev., 51: 206-220 (1987)).
  • Shiga toxins (alternatively, “verotoxins”) have cytotoxic, neurotoxic, and enterotoxic activity (Strockbine, ⁇ . et al., "Two toxin- converting phages from Escheria coli 0157:H7 strain 933 encode antigenically distinct toxins with similar biological activities," Infect. Immun., 53:135-140 (1986)). Based on their immunological cross-reactivity, the Shiga toxins have been divided into two groups. (Strockbine et al., supra).
  • Stxl Shiga toxin type 1
  • Stx2 Shiga toxin type 2
  • Stx2e Stx2e
  • Stx2c Stx2vha
  • Stx2vhb Stx2vhb
  • Shiga toxin encompasses Shiga toxin and any other toxins in the Stxl or Stx2 group.
  • Stx will refer to the protein designation, and the abbreviation “stx” to the gene designation.
  • the A subunit gene encodes the enzymatically active subunit.
  • the A subunit polypeptide has two functional domains, Al and A2, which are linked by a disulfide bond.
  • the Al portion is an N-glycosidase that acts on the 28S rRNA subunit of eukaryotic ribosomes to inhibit protein synthesis.
  • Talina, S. et al., "Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28S RNA when microinjected into Xenopus oocytes," J. Biol.
  • the A2 fragment is required for the binding of 5 B subunit polypeptides.
  • the pentamer of B subunits is responsible for binding to a receptor on eukaryotic cells.
  • a polypeptide containing the entire A subunit and B subunit pentameter is referred to as a Shiga holotoxin.
  • progress in the search for such agents has been hampered by the lack of a fast and simple method for purifying Shiga toxins. Therefore, the need exists for such a fast and simple method.
  • the need also exists for such a method that allows for large-scale production of Shiga toxoids and fusion proteins of Shiga toxins and toxoids.
  • Such a method should simplify obtaining antibodies against Shiga toxin and vaccines against HC and HUS using Shiga toxoids and fusions of Shiga toxoids.
  • the present invention describes the isolation and purification of biologically and immunologically active histidine-tagged Shiga toxins (His-tagged), a toxin associated with HC and the potentially life-threatening disease HUS transmitted by strains of pathogenic bacteria.
  • His-tagged histidine-tagged Shiga toxins
  • HUS potentially life-threatening disease
  • One aspect of the invention is obtaining and using Shiga toxoids that are immunoreactive but not toxic.
  • the invention describes using such obtained 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 of combining beneficial properties of each protein, resulting, for example, in improved protein 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 antibodies can 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 more detailed description provided below, to be read in conjunction with the accompanying drawings.
  • Figure 1 depicts the protein structure of Shiga toxin genes.
  • Figure 2 depicts the predicted amino acid sequence for the mature A subunit and the unprocessed B subunit of Stxl. (Calderwood et al., Proc. Natl. Acad. Sci. USA, 84: 4364-4368 (1987); DeGrandis et al., J. BacterioL, 169:4313-4319(1987)).
  • Figure 3 depicts the predicted amino acid sequence for the mature A subunit and the unprocessed B subunit of Stx2. (Jackson et al., FEMS Lett., 44:109-1 14 (1987)).
  • Figure 4 depicts the predicted DNA sequence for stxl and DNA upstream of that sequence.
  • Figure 5 depicts the predicted DNA sequence for stx2 and DNA upstream of that sequence. (Jackson et al., FEMS Lett., 44: 109-1 14 (1987)).
  • Figure 6 depicts the approximately 1200 base pair fragments of stxl produced by PCR amplification.
  • Figures 6a-c depict the fragments used to make plasmids pQHI, pQHEl, and p7HI, respectively.
  • Nucleotides in lower case represent non-toxin sequences in the primers and/or base changes.
  • Figure 7 depicts the approximately 1200 base pair fragments of stx2 produced by PCR amplification.
  • Figures 7a and 7b depict the fragments used to make plasmids pQHI and pQHEII, respectively.
  • Nucleotides in lower case represent non-toxin sequences in the primers and/or base changes.
  • Figure 8 depicts the plasmid pQHI, encoding the His-Stx 1 fusion and driven by the T5 promoter.
  • Figure 9 depicts the plasmid pQHII, encoding the His-Stx 2 fusion and driven by the T5 promoter.
  • Figure 10 depicts the plasmid pQHEI, encoding the His-Enterokinase site-Stxl fusion and driven by the T5 promoter.
  • Figure 11 depicts the plasmid pQHEII, encoding the His-Enterokinase site-Stx 2 fusion and driven by the T5 promoter.
  • Figure 12 depicts the plasmid pQHIIvhb, encoding the His-Stx 2 fusion and driven by the T5 promoter.
  • Figure 13 depicts the plasmid pQHEIIvhb, encoding the His-Enterokinase site- Stx 2 fusion and driven by the T5 promoter.
  • Figure 14 depicts the plasmid p7HI, encoding the His-Stx 1 fusion and driven by the PT7 promoter.
  • Figure 15 depicts the plasmid p7HII, encoding the His-Stx 2 fusion and driven by the PT7 promoter.
  • Figure 16 depicts the expression of His-Stx fusion proteins according to the invention.
  • An object of the invention is to purify large quantities of Shiga toxins that retain their biological and immunological properties.
  • a Shiga toxin gene was cloned into a Histidine-tag expression vector, expressed, and purified.
  • An additional object of the invention is to obtain antigens specific to Shiga toxoids.
  • a toxin that is non-toxinogenic but immunoreactive for generating an immune response against Shiga toxins.
  • Another object of the invention is the creation of antibodies against Shiga toxins or toxoids for treating, diagnosing, or preventing disease and infections by pathogenic bacteria.
  • the his-tagged Shiga toxins or toxoids described above can be used for these purposes.
  • the size of the his-tagged Shiga toxin to be used may be varied according to the specific purpose for the Shiga toxin. For example, if the purpose is fusing the his-tagged Shiga toxin or toxoid with one or more proteins, a smaller fragment might be selected to enhance stability of the combined 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 the available restriction sites, in light of the materials and methods known to those skilled in the art.
  • His-Shiga toxin or “His-tagged Shiga Toxin” refers to the fragment of about 372-377 amino acids comprising the A and B subunits of any of the Shiga toxin family members fused with a histidine tag. Smaller fragments that retain biological and/or immunological function are also included. Biological function is measured by, for example, cytotoxicity to Vero cells, as described in Example III.A. Immunological function may also be tested by, for example, neutralization by specific antisera, as described in Example III.B.
  • a preferred embodiment of the invention is a His-Tagged Shiga holotoxin, containing 1 A subunit and 5 B subunits.
  • the tag consists of six histidine residues.
  • the most preferred embodiment is a His 6 -Tagged Shiga holotoxin.
  • One of the objects of the present invention is to administer His-Shiga toxoids to protect against illness or disease caused by EHEC or Shigella dysenteria type I, such as HC and HUS. The object is achieved through the stimulation of immune response directed against Shiga toxins.
  • immunizing or “immunization” is used in the application.
  • the degree of protection achieved by such immunization will vary with the degree of homology between Shiga toxins and the His-Shiga toxoids, as well as other factors, such as unique attributes of the patient or the species treated.
  • immunization is not limited to avoiding infection altogether; it also includes decreasing the severity of the infection, as measured by the following indicators: reduced incidence of death, HUS, or permanent kidney damage; decreased levels of toxin; reduced fluid loss; or other indicators of illness regularly used by those skilled in the relevant art.
  • His-Tagged Shiga toxins have the biological and immunological properties of Shiga toxins, they may be used for any application appropriate for Shiga toxins.
  • Stxl can be used to treat bone marrow cells from mice with human B-cell lymphomas. The Shiga toxin bound to the receptor on the lymphoma cell and the toxin killed the cancer cell. (LaCasse et al., Blood 88:1551(1996)).
  • His-Shiga toxins or fusions could be used for the same purpose and in the same manner. Isolating and Purifying His-Tagged Shiga Toxin
  • the standard protocol for purification of Shiga toxin comprising A and B subunits uses biochemical techniques.
  • the standard protocol was developed by O'Brien et al. (O'Brien et al., Infect. Immun. 40:675 (1983); O'Brien et al., Infect. Immun., 30: 170( 1980)).
  • the method employs four purification steps: 1 ) ammonium sulfate precipitation; 2) DEAE Sepharose column chromotography; 3) chromatofocusing; and 4) antibody affinity chromotography.
  • This method has the advantages of employing publicly available materials, being capable of purifying all Shiga toxins, and being capable of purifying Shiga toxins for human use. Its disadvantage is that the minimum time required for this test is three weeks.
  • the Shiga toxin gene or portions of the Shiga toxin gene have been cloned and expressed in bacteria and purified.
  • Downes et al., Infect. Immun. 56:1929 (1988) expressed the stx2 gene in bacteria and purified Stx2.
  • the purification methods following expression were essentially those of the standard method or hydatid cyst method and, therefore, had the same disadvantages.
  • 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 believed that a His-Shiga toxin fusion would have lost cytotoxicity, because the skilled artisan would have expected that the attachment of a His-Tag to the amino acid terminus of a toxin would destroy its activity. Moreover, the multi-unit toxin would have been expected to be more susceptible to losing toxicity upon fusion with additional amino acids, since it is known that the toxin must retain its conformation for enzymatic activity and for binding of the B subunits to cell receptors, and the addition of amino acids would have been expected to destroy proper conformation.
  • the His-Stx fusion clones were generated by PCR amplification of stx 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 to place histidine residues at the amino acid terminus of the toxins and place the constructs under the control of either an IPTG-inducible promoter (pQE vectors) (Qiagen, Inc., 9600 DeSoto Avenue, Chatsworth, CA 9131 1 , 1 -800-362-7737) or a T7 promoter (pt7-7) (Tabor et al., Proc. Natl. Acad. Sci. 82: 1074 (1985)).
  • pQE vectors IPTG-inducible promoter
  • pt7-7-7 T7 promoter
  • Bacterial strains and plasmids The bacterial strains and plasmids used in this study are shown in Table 1. Table 1. Bacterial strains and plasmids used in this study.
  • the Shiga toxin genes are cloned using polymerase chain reaction (PCR), a standard technique in the art. Primers were designed to amplify the stx toxin operons beginning at the first codon of the mature A subunit gene and ending downstream of the termination codon of the B subunit gene and created using standard techniques. The primers contained recognition sequences to generate unique restriction sites at the ends of the toxin operon. In a preferred embodiment, the 5' primers also contained sequences to encode the recognition sequence of the protease enterokinase to allow for removal of the histidine residues. The primers used are shown in Table 2. TABLE 2. Primers used.
  • Sequences encoding stx toxins were amplified from toxin clones using a PCR kit (GeneAmp kit, Perkin-Elmer Cetus, Norwalk, CT), which was used according to the manufacturer's instructions.
  • the resulting stx PCR products were approximately 1200 bp are shown in Figures 6a, 6b, 6c, 7a, and 7b.
  • the DNA products contain the coding sequences for the mature A subunit and the unprocessed B subunit.
  • Plasmid DNA was isolated by the method of
  • PCR Reactions and ligations were summarized in Table 3. TABLE 3: PCR Reactions and ligations.
  • Plasmid pJES120 was the template with primers IIEC and IIH3 in a PCR reaction for amplification of the stx2 operon.
  • the resulting PCR product started with the first codon of the mature A subunit gene, extended through the A subunit gene, the complete B subunit gene, and ended just downstream of the terminate codon of the B subunit gene ( Figure 6b).
  • the PCR product was digested with the restriction endonucleases Bam HI and Hind III, as was the vector plasmid pQE30.
  • the vector pQE30 was chosen because ligation of the PCR product with pQE30 at the Ba Hl sites would result in an in-frame protein fusion of the 6 histidine residues, the enterokinase cleavage site, additional amino acids, and the +1 residue of the mature A subunit.
  • the digested PCR product was ligated into the digested vector pQE30.
  • the ligation reaction was transformed into strain XL 1 -Blue and plated on agar that contained ampicillin. Colonies were screened for the presence of a plasmid that contained an approximately 1200 bp BamHI/HindHI DNA insert.
  • His-Shiga toxin was purified under nondenaturing conditions because of the multi-subunit nature of the Shiga toxins.
  • the strain was streaked onto a selective agar plate and incubated at 37°C for 18-24 hrs. A 20 ml overnight culture was then prepared from a colony. The saturated culture was then diluted 1/50 into one liter of L broth with antibiotics and the culture was grown at 37°C until it reached an O.D. 60 ⁇ of 0.7-0.9. IPTG (2mM final concentration) was then added to induce expression of the His 6 tagged toxin and the culture was grown for an additional 5 hrs. Cells were pelleted and the pellet was kept at -70°C overnight.
  • the pellet was resuspended in sonication buffer (50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, 20 mM imidazole, 30 ⁇ g/ml PMSF), and the cells were sonicated to release toxin.
  • sonication buffer 50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, 20 mM imidazole, 30 ⁇ g/ml PMSF
  • the cells were sonicated to release toxin.
  • the cells were treated with polymixin-B (2 mg/ml final concentration) for 3 hrs at 4°C.
  • the extracts were clarified by centrifugation and filtered through a milipore 0.45 ⁇ m filter.
  • Ni-NTA nickel-nitrilotriacetic acid ligand
  • Fractions were tested for cytotoxicity on Vero cells (as explained in Example III) and were subjected to SDS-PAGE and silver stain. Fractions that were highly cytotoxic and relatively clean were pooled and dialyzed against sonication buffer. This pool was then placed onto a Ni-NTA spin column (Qiagen) to further purify the His 6 - toxin and the resulting two fractions were dialyzed against PBS. A final cytotoxicity assay and BCA protein assay were performed for the determination of the specific activity of the purified toxin.
  • the protocol described above is modification of the non-denaturing protocol described by Qiagen to purify His-tagged proteins.
  • the toxin that eluted contained many contaminants.
  • modifications were made. Specifically, Tween-20 was added to the wash buffer, and the pH of the wash buffer was adjusted to 8. Also, a final Ni-NTA spin column was added.
  • This one-step His-affinity method for purifying His-Shiga toxin by an Ni-NTA column has several advantages over existing methods, as summarized in Table 4. TABLE 4. Comparison of Toxin Purification Techniques
  • the Ni-NTA one-step method is superior because of its relative speed and simplicity. It requires a minimum of one week as opposed to a minimum of two or more weeks. Moreover, all of the materials are readily available, the method is not limited to Shiga toxins that bind Plgp, and the products are suitable for use in humans.
  • the Shiga toxin obtained by the method has many uses.
  • the His- Shiga toxin may be used as a positive control antigen in a Shiga toxin detection kit. Such kits will use a purified His-Shiga toxin as positive indicator for the toxin in a sample. Other uses are detailed in the Examples below.
  • Example III Verifying Biological and Immunological Activity of His-Shiga Toxins
  • A. Vero Cell Cytotoxicity Assay The cytotoxicity of His-Shiga toxins obtained according to the methods described in Examples 1 and II was verified by determining their cytotoxicity for Vcro cells. Cytotoxicity assays on strains that expressed His-Shiga toxins were done essentially as described by Gentry and Dalrymple, J Clin. Microbiol, 12: 361-366 (1980). Briefly, cultures induced for the expression of His-Shiga toxins were disrupted by sonic lysis and clarified by centrifugation. The extracts were serially diluted in tissue culture medium (Dulbecco modified Eagle medium containing 10% fetal calf serum, 0.8 mM glutamine, 500 U of penicillin G per ml. and 500 mg of streptomycin per ml).
  • tissue culture medium Dulbecco modified Eagle medium containing 10% fetal calf serum, 0.8 mM glutamine, 500 U of penicillin G per ml. and 500 mg of streptomycin per ml.
  • microtiter plate wells containing about 10 4 Vero cells were added to microtiter plate wells containing about 10 4 Vero cells in 100 ⁇ l of medium.
  • the tissue culture cells were incubated at 37°C in 5% CO 2 for 48 hours and then fixed and stained with crystal violet. The intensity of color of the fixed and stained cells was measured with a Titertek reader at 620 nm.
  • His-Shiga toxins obtained according to the methods described in Examples I and II were tested for antisera neutralization. Neutralization of cytoxic activity was described in great detail in Schmitt et al., Infect, and Immun., 59:1065-1073 (1991).
  • the His-Shiga toxin could be fused with another protein of interest. These methods include chemical and genetic methods, as in cloning and expressing a fusion protein, although one skilled other methods arc readily apparent to one skilled in the art. (D.V. Goeddell, Meth Enzymol. Vol. 185(1990); Itakura, Science 198:1056 (1977)). For example, if a combination vaccine for immunization against Shiga toxin and another toxin (protein X) is desired, then these two toxins can be fused into a single protein. This can be achieved by first cloning the codons for the histidine residues in frame to the coding region of protein X.
  • the fragment containing His-Protein X is then subcloned in-frame of the Shiga toxin operon.
  • the fragment is subcloned in-frame to the A2-B portions of the Shiga toxin operon.
  • the resulting His-Protein X-A2-B5 fusion would ideally result in immunization against Shiga toxin and protein X.
  • Haptens and antigens may derive from but are not limited to bacteria, rickettsiae, fungi, viruses, parasites, drugs, or chemicals. They may include, for example, small molecules such as peptides, oligosaccarides, and toxins. Certain antimicrobial drugs, chemotherapeutic drugs having the capacity of being absorbed into the intestine may also be coupled to Shiga toxin for targeted delivery, since the B subunit pentamer binds to receptors in the intestine. Conjugation methods are well known in the art.
  • Conjugation may be achieved by genetically fusing His-Shiga toxoids by standard molecular techniques or by conjugation to a polysaccharide. Methods of conjugation include those outlined in M. Brunswick et al., J. Immunol, 140:3364 (1988) and Chemistry of Protein Conjugates and Crosslinking, CRC Press, Boston (1991). Coupling of Shiga toxids to other proteins or polysaccharides would prevent disease from additional pathogens.
  • a form of Shiga toxin that is immunoreactive but not toxinogenic is needed for immunization in animals.
  • Such a His-Shiga toxoid can be generated using chemical or genetic methods.
  • the chemical method involves treating the His-Shiga toxin with either formaldehyde or glutaraldehyde, as described by Perera et al., JClin. Microbiol. 26:2127 (1988)). Briefly, samples of toxin containing 100 ⁇ g of protein are treated for 3 days at 37°C with 0.1 M Na 2 HPO 4 (pH 8.0) containing 1% formaldehyde, and the residual formaldehyde is removed by dialysis against phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • a toxoid may be produced by site-directed mutagenesis. as described in Gordon et al., Infect. Immun. 60:485 (1992); Hovde et al., Proc. N ⁇ tl. Ac ⁇ d. Sci. 85:2568 (1988); Jackson et al., J. B ⁇ cteriol. 172: 3346-3350 (1990).
  • Several methods and kits exist for site-directed mutagenesis of a gene employs the Bio-Rad Muta-Gene in vitro mutagenesis kit. Oligonucleotides can be designed and synthesized which alter specific condons in the toxin genes. Uracil- incorporated, single-stranded target plasmid DNA will be mutagenized according to the directions supplied by the manufacturer of the mutagenesis kit. The nucleotide changes are then confirmed by DNA sequence analysis.
  • a subunit targets are the residues El 67 and E170.
  • the Shiga toxoid resulting from this mutation has been used for vaccinating pigs. (Gordon et al., supra).
  • Antisera specific for Shiga toxins are required to treat and prevent potentially- deadly infections by EHEC and Shigella dysenteriae type I. Specifically, once a child becomes infected, the child and his or her family members or children in his or her day care group can receive anti His-Shiga toxin sera to achieve a protective immune response.
  • a protective immune response is one that elicits sufficient antibody to permit a patient to avoid infection, decrease the significance or severity of an infection, or decrease the ability of bacteria to colonize the gastrointestinal tract.
  • His-Shiga toxoid obtained by the methods described in Example VI, can be administered to a mammal, such as a horse intraperitoneally.
  • a mammal such as a horse intraperitoneally.
  • the horse is used to produce serum against botulism toxin for administration to humans, Hibbs et al., Clin. Infect Dis., 23:337-40 (1996), and the horse would be a preferred method for producing shiga toxin antiserum.
  • the serum of the immunized horse or other mammal
  • a large amount of serum can be quickly made using this method.
  • patients must first be screened for an immune reaction to horse serum.
  • a small amount of horse serum would be subcutaneously injected, and the patient would be monitored for a reaction.
  • Such methods for administering horse antiserum against toxins to humans are well known to the skilled artisan. Hibbs et al., supra; Dehesa and Possani, Toxicon, 32: 1015-1018(1994); Gilan ct al., Toxicon, 27:1 105-1 1 12 (1989).
  • the His-Shiga toxoid can be administered to human volunteers, either intraperitoneally or orally.
  • the plasma from these volunteers is then isolated, and the human anli-His-Shiga toxin serum can be administered to patients. No threat of serum sickness arises from this method.
  • Human hyperimmune globulin to Hemophilus influenzae b, Streptococcus pneumoniae, and Neisseria meningitidis has previously been prepared by others (Siber et al., Infect, and Immun., 45: 248-254 (1984)).
  • B. Vaccines against Shiga Toxins An embodiment of the invention is vaccines against Shiga toxin infection.
  • these vaccines can include antibodies directed against His-Shiga toxin, obtained further described in Example VII.
  • these vaccines can be combination vaccines that comprise His-Shiga toxoid fused or conjugated with another protein, hapten, or antigen, as described in Example IV. These vaccines can be administered intraperitoneally or injectably by methods well known in the art.
  • a preferred method of administering His-Shiga toxin or toxoid and fusions thereof is by further conjugation to Synsorb® (SynSorb Biotech, Inc., 1204 Kensington Road, N.W., Calgary, Alberta, Canada, T2N3P5.)
  • Synosorb is a sand-like material 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 toxins and appears to be safe for human ingestion. (Armstrong et al., supra)
  • the Synsorb is bound to the B subunit pentamer via the B subunit pentamer-receptor reaction. Conjugation with Synsorb adds further stability.
  • Another embodiment of the invention involves the administration of nucleic acid vaccines. DNA encoding a His-Shiga toxoid is injected into a patient as naked
  • DNA 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 patient mounts an immune response against transiently expressed foreign antigens.
  • nucleic acid vaccines in general, are all nearing clinical trials. This approach to vaccines involves delivering the DNA encoding the desired antigen into the host by inserting the gene into a nonreplicating plasmid vector (Marwick, C.
  • His-Shiga antibodies polyclonal and monoclonal, can also be used in the treatment, diagnosis, and prevention of infections related to Shiga toxins. Because of their increased specificity, monoclonal antibodies are preferred. His-Shiga toxin antibodies can be administered to humans or other mammals to achieve a protective immune response, for treatment or prophylaxis. Antibodies, in a physiologically acceptable carrier, may be administered orally or intraperitoneally. For this purpose, monoclonal antibodies are preferred and humanized monoclonal antibodies are particularly preferred. Positive clinical responses in humans have been obtained with monoclonal antibodies, and one skilled in the art would know how to employ Shiga monoclonal antibodies in humans.
  • Another embodiment of the invention involves using antibodies to diagnose Shiga 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.
  • the antibody may be used to detect Shiga toxins in sample taken from cow, such as cow feces.
  • meat may be tested using the anti His-Shiga toxin antibody for detection.
  • a detection kit comprising the His-Shiga toxin antibody can be used for this purpose.
  • a sandwich Elisa can be used.
  • rabbit anti-His-Shiga toxin 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 as mouse ⁇ -goat antibody conjugated to horseradish peroxidase.
  • the antibody can be detected by standard methods.
  • primary Ab 50 ⁇ l test sera diluted in blocking solution for example, start with 1 :50 and do eleven 1 :2 dilutions, or start with 1 :50 and do eleven 1 :10 dilutions), incubate 2 h/RT.
  • secondary Ab goat horseradish-conjugated anti-mouse Ig, affinity purified (Boehringer Mannheim Corp., 91 15 Hague Rd., P.O. Box 50414, Indianapolis, IN. 46250,800-262-1640). Add secondary Ab diluted 1 :500 in blocking solution without azide. Incubate 1 h/RT.
  • a titer is defined as an absorbance value >0.2 units above that obtained for mouse preimmune sera.
  • Anti-Shiga toxin Abs obtained from animals may be used clinically if one changes the specificity of the antibody to human. Such techniques are well known to those 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-determining regions in a human antibody with those from a mouse,” Nature, 321 : 522-525 (1986); P. Carter et al., “Humanization of an anti-pl85 f ⁇ £R2 antibody for human cancer therapy," Proc. Nad. AcadSci. USA, 89: 4285-4289 (1992). Such antibodies may be given to the sibling of an infected patient to reduce the risk of infection of the sibling. C. Raising Monoclonal Antibodies to His-Shiga Toxin
  • Monoclonal antibodies directed against Shiga toxin are used to passively protect a patient against EHEC and Shigella dysenteriae type 1 infections. Monoclonal antibodies are generated from mouse cells, and the specificity of these antibodies are changed 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 regions in a human antibody with those from a mouse,” Nature, 321 : 522-525 (1986); P. Carter et al., “Humanization of an anti-pl ⁇ S” 1 2 antibody for human cancer therapy," Proc. Natl. AcadSci. USA, 89: 4285-4289 (1992). Monoclonal Abs represent a more "pure” antibody for administration to a patient.
  • mice Five 4- to 5-week old female BALB/cJ mice are prebled, and immunized intraperitoneally with 25 ⁇ g His- Shiga toxoid suspended in 100 ⁇ l of TiterMax. Mice are boosted twice in two week intervals, intraperitoneally with 25 ⁇ g His-Shiga toxoid suspended in 100 ⁇ l of TiterMax. Seven days after each boost, blood (-300 - 500 ⁇ l) is collected from the tail vein. Sera are assayed for the presence of anti-Shiga toxin antibody by ELISA (as described above).
  • mice producing high titers of anti-His Shiga toxin antibodies are boosted both intravenously and intraperitoneally with 25 ⁇ g of His-Shiga toxoid in 100 ⁇ l of PBS, sacrificed three days later, and sera collected.
  • Spleen cells are isolated and fused to Sp2/0-Ag mouse myeloma cells (ATCC #CRL1581 ) at a ratio of 10 spleen cells to 1 myeloma cell. Fused cells are distributed into microdilution plates, and culture supernatants arc assayed by ELISA after 3-4 weeks of culture for anti-His-Shiga toxin antibodies.

Abstract

The present invention describes the isolation and purification of biologically and immunologically active histidine-tagged Shiga toxins (His-tagged), a toxin associated with HC and the potentially life-threatening sequela HUS transmitted by strains of pathogenic bacteria. The present invention describes how his-tagging greatly simplifies and expedites purifying Shiga toxins, and describes an improved method for such purification. One aspect of the invention is obtaining and using Shiga toxoids that are immunoreactive but not toxic. Another aspect of the invention is obtaining and using fusion proteins of His-tagged Shiga toxins or toxoids. Yet another aspect of the invention is obtaining and using antibodies to His-gagged Shiga toxins, toxoids, or Shiga toxin/toxoid fusion proteins.

Description

Description
HISTIDINE-TAGGED SHIGA TOXINS, TOXOIDS, AND PROTEIN FUSIONS WITH SUCH TOXINS AND TOXOIDS, METHODS FOR THE PURIFICATION AND PREPARATION THEREOF
GOVERNMENT INTEREST
The invention described herein may be manufactured, licensed, and used for governmental purposes without payment of royalties to us thereon.
FIELD OF THE INVENTION The invention relates to a family of multi-unit bacterial proteins that are associated with hemorrhagic colitis and the life-threatening sequela, hemolytic uremic syndrome. These proteins, defined as members of the "Shiga toxin family," have been tagged with histidine residues. The invention further relates to a non-toxinogenic but immunoreactive form of histidine-tagged Shiga toxins, or toxoids. Moreover, the invention relates to fusion proteins obtained by combining histidine-tagged Shiga toxins or toxoids with other proteins. Histidine tagging greatly facilitates purification of Shiga toxins, and the invention also relates to methods for purifying such toxins. The invention further relates to using the histidine-tagged Shiga toxoids or fusion proteins of Shiga toxoids as antigens for generating an immune response against infection or transmission by bacteria expressing Shiga toxin. It also relates to antibodies to Shiga toxins, toxoids, or Shiga toxin/toxoid fusion proteins, both monoclonal and polyclonal, and their use in treating, diagnosing, and preventing of disease and infections by pathogenic E. coli. Finally, the invention relates to preparing the Shiga toxins, toxoids, and fusion proteins. BACKGROUND OF THE INVENTION
Enterohemorrhagic Escherichia coli (EHEC) are associated with food-borne outbreaks of bloody diarrhea or "hemorrhagic colitis" (HC) and the hemolytic uremic syndrome (HUS). (Spika, J. et al., "Hemolytic Uremic Syndrome and Diarrhea Associated with Escheria coli: 0157Η7 in a Day Care Center," J. Pediatr., 109: 287- 291 ( 1986); Remis, R., "Sporadic case of hemorrhagic colitis associated with Escheria coli 0157:H7," Ann. Intern. Med., 101 :624-626 (1984); "Riley, L. et al., "Hemorrhagic colitis associated with a rare Escheria coli serotype," N. Engl. J. Med., 308:681-685 (1983)). EHEC infection can be deadly and poses a significant threat to the young and the elderly, who are the most likely to develop serious complications from EHEC infections. Several outbreaks and sporadic cases of HC and HUS have occurred over the 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 Japan resulted in over 10,000 infected individuals and 8 deaths. Many Japanese children required hospitalization. Unfortunately, no cure or vaccine for HC and HUS is currently available.
Primarily, HC and HUS are transmitted by the ingestion of contaminated food, particularly undercooked beef products, such as hamburger. (Doyle et al., J. Appl. Environ. Microbiol. 53:2394 (1987); Samadpour et al., J. Appl. Environ. Microbiol. 60:1038 (1994)). With the prevalence of EHEC in cattle and the subjective nature of differentiating between cooked and undercooked hamburgers, a stop at a fast food restaurant or a family barbecue can result in tragedy. HC and HUS appear to be mediated by the toxin produced by EHEC and Shigella dysenteriae (for review see O'Brien and Holmes, Microbiol. Rev., 51: 206-220 (1987)). These bacteria produce a family of closely related cytotoxins that collectively will be called "Shiga toxins" for the purpose of this application. Shiga toxins (alternatively, "verotoxins") have cytotoxic, neurotoxic, and enterotoxic activity (Strockbine, Ν. et al., "Two toxin- converting phages from Escheria coli 0157:H7 strain 933 encode antigenically distinct toxins with similar biological activities," Infect. Immun., 53:135-140 (1986)). Based on their immunological cross-reactivity, the Shiga toxins have been divided into two groups. (Strockbine et al., supra). These groups have been designated 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," ASM News, 62:118-119 (1996)). The Stxl group contains the prototype Stxl toxin from 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 and Immunology, 61 :3832 (1993); Schmitt, C. et al., "Two copies of Shiga-like toxin II- related genes common in enterohemorrhagic Escheria coli strains are responsible for the antigenic heterogeneity of the 0157:H strain E325II," Infect. Immun., 59: 1065-1073 (1991); Marques, L. et al., "Escheria coli strains isolated from pigs with edema disease produce a variant of Shiga-like toxin II," FEMS Lett., 44:33-38 (1987)).
For the purposes of this application the term "Shiga toxin" encompasses Shiga toxin and any other toxins in the Stxl or Stx2 group. The abbreviation "Stx" will refer to the protein designation, and the abbreviation "stx" to the gene designation.
These Shiga toxins do share similar genetic and protein organization, as set forth in Figure 1. The A subunit gene encodes the enzymatically active subunit. The A subunit polypeptide has two functional domains, Al and A2, which are linked by a disulfide bond. The Al portion is an N-glycosidase that acts on the 28S rRNA subunit of eukaryotic ribosomes to inhibit protein synthesis. (Saxena, S. et al., "Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28S RNA when microinjected into Xenopus oocytes," J. Biol. Chem., 264:596-601 (1989)). The A2 fragment is required for the binding of 5 B subunit polypeptides. The pentamer of B subunits is responsible for binding to a receptor on eukaryotic cells. A polypeptide containing the entire A subunit and B subunit pentameter is referred to as a Shiga holotoxin. Despite this knowledge about the toxin components, there is no known cure or vaccine for HC or HUS.
The need exists for therapeutic agents for the treatment and prevention of HC and HUS. However, progress in the search for such agents has been hampered by the lack of a fast and simple method for purifying Shiga toxins. Therefore, the need exists for such a fast and simple method. Moreover, the need exists for such a method that further allows for large-scale production of Shiga toxins while retaining their biological and immunological activity. The need also exists for such a method that allows for large-scale production of Shiga toxoids and fusion proteins of Shiga toxins and toxoids. Such a method should simplify obtaining antibodies against Shiga toxin and vaccines against HC and HUS using Shiga toxoids and fusions of Shiga toxoids.
SUMMARY OF THE INVENTION The present invention describes the isolation and purification of biologically and immunologically active histidine-tagged Shiga toxins (His-tagged), a toxin associated with HC and the potentially life-threatening disease HUS transmitted by strains of pathogenic bacteria. The present invention describes how his-tagging greatly simplifies and expedites purifying Shiga toxins, and describes an improved method for such purification.
One aspect of the invention is obtaining and using Shiga toxoids that are immunoreactive but not toxic. For example, the invention describes using such obtained 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 of combining beneficial properties of each protein, resulting, for example, in improved protein 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 antibodies can 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 more detailed description provided below, to be read in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the protein structure of Shiga toxin genes. Figure 2 depicts the predicted amino acid sequence for the mature A subunit and the unprocessed B subunit of Stxl. (Calderwood et al., Proc. Natl. Acad. Sci. USA, 84: 4364-4368 (1987); DeGrandis et al., J. BacterioL, 169:4313-4319(1987)). Figure 3 depicts the predicted amino acid sequence for the mature A subunit and the unprocessed B subunit of Stx2. (Jackson et al., FEMS Lett., 44:109-1 14 (1987)).
Figure 4 depicts the predicted DNA sequence for stxl and DNA upstream of that 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 of that sequence. (Jackson et al., FEMS Lett., 44: 109-1 14 (1987)).
Figure 6 depicts the approximately 1200 base pair fragments of stxl produced by PCR amplification. Figures 6a-c depict the fragments used to make plasmids pQHI, pQHEl, and p7HI, respectively. Nucleotides in lower case represent non-toxin sequences in the primers and/or base changes.
Figure 7 depicts the approximately 1200 base pair fragments of stx2 produced by PCR amplification. Figures 7a and 7b depict the fragments used to make plasmids pQHI and pQHEII, respectively. Nucleotides in lower case represent non-toxin sequences in the primers and/or base changes.
Figure 8 depicts the plasmid pQHI, encoding the His-Stx 1 fusion and driven by the T5 promoter.
Figure 9 depicts the plasmid pQHII, encoding the His-Stx 2 fusion and driven by the T5 promoter.
Figure 10 depicts the plasmid pQHEI, encoding the His-Enterokinase site-Stxl fusion and driven by the T5 promoter.
Figure 11 depicts the plasmid pQHEII, encoding the His-Enterokinase site-Stx 2 fusion and driven by the T5 promoter. Figure 12 depicts the plasmid pQHIIvhb, encoding the His-Stx 2 fusion and driven by the T5 promoter.
Figure 13 depicts the plasmid pQHEIIvhb, encoding the His-Enterokinase site- Stx 2 fusion and driven by the T5 promoter. Figure 14 depicts the plasmid p7HI, encoding the His-Stx 1 fusion and driven by the PT7 promoter.
Figure 15 depicts the plasmid p7HII, encoding the His-Stx 2 fusion and driven by the PT7 promoter. Figure 16 depicts the expression of His-Stx fusion proteins according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
An object of the invention is to purify large quantities of Shiga toxins that retain their biological and immunological properties. To achieve this object, a Shiga toxin gene was cloned into a Histidine-tag expression vector, expressed, and purified.
An additional object of the invention is to obtain antigens specific to Shiga toxoids. a toxin that is non-toxinogenic but immunoreactive, for generating an immune response against Shiga toxins. Another object of the invention is the creation of antibodies against Shiga toxins or toxoids for treating, diagnosing, or preventing disease and infections by pathogenic bacteria. The his-tagged Shiga toxins or toxoids described above can be used for these purposes.
Those skilled in the art will also recognize that the size of the his-tagged Shiga toxin to be used may be varied according to the specific purpose for the Shiga toxin. For example, if the purpose is fusing the his-tagged Shiga toxin or toxoid with one or more proteins, a smaller fragment might be selected to enhance stability of the combined 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 the available restriction sites, in light of the materials and methods known to those skilled in the art. Consequently, the terms "His-Shiga toxin" or "His-tagged Shiga Toxin" refers to the fragment of about 372-377 amino acids comprising the A and B subunits of any of the Shiga toxin family members fused with a histidine tag. Smaller fragments that retain biological and/or immunological function are also included. Biological function is measured by, for example, cytotoxicity to Vero cells, as described in Example III.A. Immunological function may also be tested by, for example, neutralization by specific antisera, as described in Example III.B. A preferred embodiment of the invention is a His-Tagged Shiga holotoxin, containing 1 A subunit and 5 B subunits. In another preferred embodiment, the tag consists of six histidine residues. The most preferred embodiment is a His6-Tagged Shiga holotoxin. One of the objects of the present invention is to administer His-Shiga toxoids to protect against illness or disease caused by EHEC or Shigella dysenteria type I, such as HC and HUS. The object is achieved through the stimulation of immune response directed against Shiga toxins.
Consequently, the term "immunizing" or "immunization" is used in the application. The degree of protection achieved by such immunization will vary with the degree of homology between Shiga toxins and the His-Shiga toxoids, as well as other factors, such as unique attributes of the patient or the species treated. Moreover, immunization is not limited to avoiding infection altogether; it also includes decreasing the severity of the infection, as measured by the following indicators: reduced incidence of death, HUS, or permanent kidney damage; decreased levels of toxin; reduced fluid loss; or other indicators of illness regularly used by those skilled in the relevant art.
Unless specified otherwise, the uses and methods set forth herein are generally applicable to humans and animals. The term "patient" is used herein to mean both humans and animals, and "animals" is not limited to domesticated animals but also may include wildlife and laboratory animals.
Moreover, because His-Tagged Shiga toxins according to the invention have the biological and immunological properties of Shiga toxins, they may be used for any application appropriate for Shiga toxins. For example, it has been recently demonstrated that Stxl can be used to treat bone marrow cells from mice with human B-cell lymphomas. The Shiga toxin bound to the receptor on the lymphoma cell and the toxin killed the cancer cell. (LaCasse et al., Blood 88:1551(1996)). Thus, the skilled artisan would expect that His-Shiga toxins or fusions could be used for the same purpose and in the same manner. Isolating and Purifying His-Tagged Shiga Toxin
The standard protocol for purification of Shiga toxin comprising A and B subunits uses biochemical techniques. The standard protocol was developed by O'Brien et al. (O'Brien et al., Infect. Immun. 40:675 (1983); O'Brien et al., Infect. Immun., 30: 170( 1980)). The method employs four purification steps: 1 ) ammonium sulfate precipitation; 2) DEAE Sepharose column chromotography; 3) chromatofocusing; and 4) antibody affinity chromotography. This method has the advantages of employing publicly available materials, being capable of purifying all Shiga toxins, and being capable of purifying Shiga toxins for human use. Its disadvantage is that the minimum time required for this test is three weeks.
Another well-known method for purifying Shiga toxin from bacteria was developed by Keusch et al. (Donohuc-Rolfe et al., Infect. Immun. 57:3888 (1989); Acheson et al., Microb. Pathog. 14:57 (1993)). This method employs a Shiga toxin receptor analog. The receptor analog is the Pl glycoprotein (Plgp) from tapeworm hydatid cysts material (HCM) in sheep gut. This method contains three purification steps: 1) ammonium sulfate precipitation; 2) Blue sepharose chromotography; and 3) Plgp column chromatography. The Plgp must be prepared from the HCM. Though faster than the standard method, this method still requires a minimum of two or more weeks. The hydatid cyst material must be obtained from infected sheep and is not publicly available. The method has the additional disadvantages of being capable of use with only those Shiga toxins that bind Plgp and, because of possible contamination, it is not appropriate for obtaining Shiga toxoids for use in humans.
Using recombinant methods, the Shiga toxin gene or portions of the Shiga toxin gene have been cloned and expressed in bacteria and purified. Zollman et al., Prot. Expression Pur. 5:291 (1994), purified a recombinant Stxl Al fragment. Acheson et al., Infect. Immunol. 63:301(1995), expressed and purified the Stx2 B subunit. Downes et al., Infect. Immun. 56:1929 (1988), expressed the stx2 gene in bacteria and purified Stx2. However, the purification methods following expression 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 have developed a purification method based on the creation of a histidine-tagged Shiga toxin. Methods for histidine tagging are known in the art. For example, Fryxell et al., Biochem. Biophys. Res. Comm., 210:253-259 (1995), added a kemptide and histidine tag to the A chain of the eukaryotic toxin ricin, which was later associated with the B subunit. 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 a pentamer). Moreover, Fryxell et al. only expressed the A subunit with a His-Tag. In addition, Strauss et al., FEMS Microbiol. Lett., 127:249-254 (1995), have his-tagged the 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 toxin with a His-Tag, and the expressed fusion protein did not undergo multimerization. Finally, Terbush & Novick, J. Cell. BioL, 130:299-312 (1995), tagged the C-terminus of a multiunit yeast protein. This involves a eukaryotic rather than a prokaryotic system. Moreover, expressing a functionally active Shiga toxin requires retaining its multimer 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 believed that a His-Shiga toxin fusion would have lost cytotoxicity, because the skilled artisan would have expected that the attachment of a His-Tag to the amino acid terminus of a toxin would destroy its activity. Moreover, the multi-unit toxin would have been expected to be more susceptible to losing toxicity upon fusion with additional amino acids, since it is known that the toxin must retain its conformation for enzymatic activity and for binding of the B subunits to cell receptors, and the addition of amino acids would have been expected to destroy proper conformation. The skilled artisan would be aware that conformation and the charge of the molecule is critical to Shiga toxins. For example, the skilled artisan would know that deleting a few N -terminal amino acids from Stx2A destroyed enzymatic activity, as reported in Perera et al., Infect. Immunol, 59:829-835 (1991)). Similarly, altering the C-terminus of the B subunit affected toxicity. (Perera et al., supra). Additionally, Perera et al. suggested that the charge of the molecule plays an important role. The importance of preserving Shiga toxin conformation is further underscored by findings that the highly homologous Stxl A and Stx2B subunits cannot be combined to form an active toxin. (Weinstein et al., Infect. Immun., 57:3743-3750 (1989)). Based on this knowledge, the skilled artisan would have expected that tagging a Shiga holotoxin with histidine residues would have unfavorably affected conformation and charge of the toxin product.
Surprisingly, his-tagging of Shiga toxin comprising A and B subunits generated a functional Shiga toxin, which has similar specific activity to Shiga toxin purified by standard methods. Moreover, the His-Shiga toxins are neutralized by monoclonal antibodies specific for Shiga toxins. Example I describes how to create the His-Shiga toxin fusion protein.
The following examples are intended to illustrate the invention but not to limit it. The skilled artisan will understand from these examples that modifications can be made that are still within the scope of the invention. The scope of the invention is defined by the claims. Example I
A. Construction of Plasmid Encoding His-Tagged Shiga Toxin
The His-Stx fusion clones were generated by PCR amplification of stx 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 to place histidine residues at the amino acid terminus of the toxins and place the constructs under the control of either an IPTG-inducible promoter (pQE vectors) (Qiagen, Inc., 9600 DeSoto Avenue, Chatsworth, CA 9131 1 , 1 -800-362-7737) or a T7 promoter (pt7-7) (Tabor et al., Proc. Natl. Acad. Sci. 82: 1074 (1985)). The methods for obtaining His-Stx fusion clones are described in more detail below. 1. Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are shown in Table 1. Table 1. Bacterial strains and plasmids used in this study.
Strain or plasmid Characteristic(s) Source or reference
E. coli strains
DH5α Host strain for cloning BRL
XL 1 -Blue Host strain for cloning; lacl; Tc1 Stratagene
M15 Host strain for protein purification Qiagen
Plasmids pJΕS120 Encodes .vtx II toxin operon a pJN25 Encodes .ytje 1 toxin operon b pSQ543 Encodes stx Ilvhb operon c pQE30 Histidine fusion vector Qiagen pQE32 Histidine fusion vector Qiagen pREP4 lacl; Knr Qiagen pT7-7 T7 expression vector d pGPl-2 Encodes T7 RNA polymerase; KN1 d a = Lindgren et al., Infect. Immunol. 61 :3832 (1993). b= Newland et al., "Cloning of shiga-like toxin structural genes from a phage of
Escheria coli strain
933, in Advances in Research on Cholera and Diarheas (S. Kuwahara & N.F. Pierce eds. 1994). c= Lindgren et al., Infect. Immunol. 62:623 (1994). d= Tabor et al., Proc. Nat/. Acad. Sci. USA 82:1074 (1985).
2. Media and enzymes. Bacterial strains were grown in L broth (per liter: 10 g tryptone, 5 g yeast extract, 5 g ΝaCl). Kanamycin, tetracycline, and ampicillin (Sigma Chemical Co., St. Louis, MO.) were added to the medium at final concentrations of 25, 10, and 100 μg/ml (respectively) as needed. Restriction endonucleases, calf intestinal phosphatase, and ligase were from Boehringer Mannheim, Indianapolis, Ind., or U.S. Biochemicals Corporation, Cleveland, Ohio. Enzymes were used according to manufacturer's instructions.
3. Primers and PCR. The Shiga toxin genes are cloned using polymerase chain reaction (PCR), a standard technique in the art. Primers were designed to amplify the stx toxin operons beginning at the first codon of the mature A subunit gene and ending downstream of the termination codon of the B subunit gene and created using standard techniques. The primers contained recognition sequences to generate unique restriction sites at the ends of the toxin operon. In a preferred embodiment, the 5' primers also contained sequences to encode the recognition sequence of the protease enterokinase to allow for removal of the histidine residues. The primers used are shown in Table 2. TABLE 2. Primers used.
Primer Primer Sequent (5'-3') Restriction site
IIEC GCGGΛTCCGATGACGATGACΛAACGGGAGTTTACGATΛGACTT BamHl
IIBAM GCGGATCCGGGAGTTTACGA TAGACTT BamHl
MB CCACGAATAAGCTTATGCCTCA Hindlll
IBAM5 GCGGΛTCCAAGGAATTTACCTTAGACΪTC Bam} 11
I-.C5 ΩCΩGATCCGATGACGA ΓGACAAAAAGGAATΓTACCTTAGACTTC BamHl
IPST3 ATTTTCACTGCAGCTATTCTG PstI
SI.TIIH5 GCATΛTGCΛTCACCATCACCATCACCGGGAGTTTΛCGATAGΛC Ndel
SLTIH5 GCATATGCATCACCATCACCATCACAAGGAATTΓACC TTAGECTTC Ndel SLTLIC3 TAACATTTATCGATΛTCTCCGCCTG Clal
Sequences encoding stx toxins were amplified from toxin clones using a PCR kit (GeneAmp kit, Perkin-Elmer Cetus, Norwalk, CT), which was used according to the manufacturer's instructions. The resulting stx PCR products were approximately 1200 bp are shown in Figures 6a, 6b, 6c, 7a, and 7b. The DNA products contain the coding 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 of
Holmes and Quigley, Anal. Biochem., 114:193-197 (1981). Alternatively, plasmid DNA was purified using Qiagen columns (Qiagen Inc., Chatsworth, CA). PCR products were digested with restriction endonucleases and ligated into the pQE30/32 vectors (Qiagen, Inc.), or into the vector pT7-7. PCR reactions and ligations are summarized in Table 3. TABLE 3: PCR Reactions and ligations.
Plasmid Primer Resulting clone template pair Vector Cloning sites pJES120 IIEC + IIH3 pQE30 BamHI/HindHI pQHEII pJES120 IIBAM + IIH3 pQE32 BamHI/HindHI pQHIl pJN25 IEC5 + IPST3 pQE30 BamHI/PstI pQHEI pJN25 IBAM + IPST3 pQE30 BamHI/PstI PQHI pJES120 SLTIIH5 + IIH3 pT7-7 Ndel/Hindlll p7HIIa pJN25 SLTIH5 + SLTIC3 pT7-7 Ndel/Clal p7HI pSQ543 IIEC + IIH3 pQE30 BamHI/HindHI pQHEIIvhb" pSQ543 IIBAM + IIH3 pQE32 BamHI/HindHI pQHHvhb" a The construction of these plasmids is in progress.
To illustrate, the clone pQHEII was constructed as follows: Plasmid pJES120 was the template with primers IIEC and IIH3 in a PCR reaction for amplification of the stx2 operon. The resulting PCR product started with the first codon of the mature A subunit gene, extended through the A subunit gene, the complete B subunit gene, and ended just downstream of the terminate codon of the B subunit gene (Figure 6b). The PCR product was digested with the restriction endonucleases Bam HI and Hind III, as was the vector plasmid pQE30. The vector pQE30 was chosen because ligation of the PCR product with pQE30 at the Ba Hl sites would result in an in-frame protein fusion of the 6 histidine residues, the enterokinase cleavage site, additional amino acids, and the +1 residue of the mature A subunit. The digested PCR product was ligated into the digested vector pQE30. The ligation reaction was transformed into strain XL 1 -Blue and plated on agar that contained ampicillin. Colonies were screened for the presence of a plasmid that contained an approximately 1200 bp BamHI/HindHI DNA insert. Clones were confirmed by IPTG induction of toxin expression (Example II) with a subsequent test for cytoxicity on vero cells (Example III). Positive clones were then transformed into Ml 5(pREP4) for large scale production of toxin. Example II Large scale purification of His-Tagged Shiga Toxins
His-Shiga toxin was purified under nondenaturing conditions because of the multi-subunit nature of the Shiga toxins. The strain was streaked onto a selective agar plate and incubated at 37°C for 18-24 hrs. A 20 ml overnight culture was then prepared from a colony. The saturated culture was then diluted 1/50 into one liter of L broth with antibiotics and the culture was grown at 37°C until it reached an O.D.60ϋ of 0.7-0.9. IPTG (2mM final concentration) was then added to induce expression of the His6 tagged toxin and the culture was grown for an additional 5 hrs. Cells were pelleted and the pellet was kept at -70°C overnight. The pellet was resuspended in sonication buffer (50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, 20 mM imidazole, 30 μg/ml PMSF), and the cells were sonicated to release toxin. Alternatively, the cells were treated with polymixin-B (2 mg/ml final concentration) for 3 hrs at 4°C. The extracts were clarified by centrifugation and filtered through a milipore 0.45 μm filter.
The nickel-nitrilotriacetic acid ligand (Ni-NTA) gel was equilibrated with sonication buffer and the cell extract was added to the gel. Protein was allowed to bind for 1 hr at room temperature or at 4°C. The gel was washed with sonication buffer followed by wash buffer (50 mM sodium phosphate (pH 8.0), 300 mM sodium chloride, 20 mM imidazole, 30 μg/ml PMSF, 10% glycerol, 1% tween-20). Protein was eluted from the gel with a gradient of imidazole (0-500 mM in wash buffer without tween-20), and 1 ml fractions were collected.
Fractions were tested for cytotoxicity on Vero cells (as explained in Example III) and were subjected to SDS-PAGE and silver stain. Fractions that were highly cytotoxic and relatively clean were pooled and dialyzed against sonication buffer. This pool 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 final cytotoxicity assay and BCA protein assay were performed for the determination of the specific activity of the purified toxin.
The protocol described above is modification of the non-denaturing protocol described by Qiagen to purify His-tagged proteins. However, the toxin that eluted contained many contaminants. To achieve purer His-Shiga toxin, modifications were made. Specifically, Tween-20 was added to the wash buffer, and the pH of the wash buffer was adjusted to 8. Also, a final Ni-NTA spin column was added.
This one-step His-affinity method for purifying His-Shiga toxin by an Ni-NTA column has several advantages over existing methods, as summarized in Table 4. TABLE 4. Comparison of Toxin Purification Techniques
Purification Minimum time Steps1* Materials available Use for all Method required" Shiga toxins
Standard 3 weeks 4 yes yes
Hydatid 2 weeks + 3 no no
Cyst
His6 affinity 1 week 2 yes yes a Time from streaking the strain onto an agar plate. This does not include the preparation 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 purification of Plgp from hydatid cyst material and preparation of the column.
The Ni-NTA one-step method is superior because of its relative speed and simplicity. It requires a minimum of one week as opposed to a minimum of two or more weeks. Moreover, all of the materials are readily available, the method is not limited to Shiga toxins that bind Plgp, 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 purified His-Shiga toxin as positive indicator for the toxin in a sample. Other uses are detailed in the Examples below. Example III Verifying Biological and Immunological Activity of His-Shiga Toxins
A. Vero Cell Cytotoxicity Assay The cytotoxicity of His-Shiga toxins obtained according to the methods described in Examples 1 and II was verified by determining their cytotoxicity for Vcro cells. Cytotoxicity assays on strains that expressed His-Shiga toxins were done essentially as described by Gentry and Dalrymple, J Clin. Microbiol, 12: 361-366 (1980). Briefly, cultures induced for the expression of His-Shiga toxins were disrupted by sonic lysis and clarified by centrifugation. The extracts were serially diluted in tissue culture medium (Dulbecco modified Eagle medium containing 10% fetal calf serum, 0.8 mM glutamine, 500 U of penicillin G per ml. and 500 mg of streptomycin per ml). One hundred microliters of 10-fold dilutions of the lysates were added to microtiter plate wells containing about 104 Vero cells in 100 μl of medium. The tissue culture cells were incubated at 37°C in 5% CO2 for 48 hours and then fixed and stained with crystal violet. The intensity of color of the fixed and stained cells was measured with a Titertek reader at 620 nm.
B. Antisera Neutralization Assay
His-Shiga toxins obtained according to the methods described in Examples I and II were tested for antisera neutralization. Neutralization of cytoxic activity was described in great detail in Schmitt et al., Infect, and Immun., 59:1065-1073 (1991).
Briefly, lysates were incubated with serial dilutions of monoclonal or polyclonal antisera specific for Stxl or Stx2 at 37°C for 2 hours. One hundred microliters of the samples were then added to vero cells as described above. Percent neutralization was determined by the following formula:
{[A62o(toxin + antibody) - A620(toxin)]/A 620(untreated cells)} x 100. Example IV Constructing Fusions with His-Shiga Toxins and Other Proteins
Using methods well-known in the art, the His-Shiga toxin could be fused with another protein of interest. These methods include chemical and genetic methods, as in cloning and expressing a fusion protein, although one skilled other methods arc readily apparent to one skilled in the art. (D.V. Goeddell, Meth Enzymol. Vol. 185(1990); Itakura, Science 198:1056 (1977)). For example, if a combination vaccine for immunization against Shiga toxin and another toxin (protein X) is desired, then these two toxins can be fused into a single protein. This can be achieved by first cloning the codons for the histidine residues in frame to the coding region of protein X. The fragment containing His-Protein X is then subcloned in-frame of the Shiga toxin operon. In a preferred embodiment, the fragment is subcloned in-frame to the A2-B portions of the Shiga toxin operon. The resulting His-Protein X-A2-B5 fusion would ideally result in immunization against Shiga toxin and protein X.
One skilled in the art would recognize that various proteins from pathogens and haptens may be conjugated to a His-Shiga toxin. Haptens and antigens may derive from but are not limited to bacteria, rickettsiae, fungi, viruses, parasites, drugs, or chemicals. They may include, for example, small molecules such as peptides, oligosaccarides, and toxins. Certain antimicrobial drugs, chemotherapeutic drugs having the capacity of being absorbed into the intestine may also be coupled to Shiga toxin for targeted delivery, since the B subunit pentamer binds to receptors in the intestine. Conjugation methods are well known in the art. Exemplary methods are set forth in Goeddel, "Systems for Heterologous Gene Expression," Meth. EzymoL, 185 (1990), Itakura, "Expression in E. coli of a chemically synthesized gene for the hormone somatostatin," Science, 198:1056-1063 (1977), and Goeddel et al., "Expression of chemically synthesized genes for human insulin," Proc. Natl. Acad. Sci. USA, 281: 544-548(1979).
Conjugation may be achieved by genetically fusing His-Shiga toxoids by standard molecular techniques or by conjugation to a polysaccharide. Methods of conjugation include those outlined in M. Brunswick et al., J. Immunol, 140:3364 (1988) and Chemistry of Protein Conjugates and Crosslinking, CRC Press, Boston (1991). Coupling of Shiga toxids to other proteins or polysaccharides would prevent disease from additional pathogens. Example V His-Shiga Toxoids
A form of Shiga toxin that is immunoreactive but not toxinogenic is needed for immunization in animals. Such a His-Shiga toxoid can be generated using chemical or genetic methods. The chemical method involves treating the His-Shiga toxin with either formaldehyde or glutaraldehyde, as described by Perera et al., JClin. Microbiol. 26:2127 (1988)). Briefly, samples of toxin containing 100 μg of protein are treated for 3 days at 37°C with 0.1 M Na2HPO4 (pH 8.0) containing 1% formaldehyde, and the residual formaldehyde is removed by dialysis against phosphate-buffered saline (PBS). To prepare His-Shiga toxoid by treatment with glutaraldehyde, crude toxin samples containing 50 μg of protein are incubated at 37°C in 0.1 1% glutaraldehyde in 0.1 M Na2HP04 (pH 8.0) for 30 min. The toxoid is then tested on Vero cells, as described in Example III, for loss of cytotoxicity.
Genetically, a toxoid may be produced by site-directed mutagenesis. as described in Gordon et al., Infect. Immun. 60:485 (1992); Hovde et al., Proc. Nαtl. Acαd. Sci. 85:2568 (1988); Jackson et al., J. Bαcteriol. 172: 3346-3350 (1990). Several methods and kits exist for site-directed mutagenesis of a gene. One method employs the Bio-Rad Muta-Gene in vitro mutagenesis kit. Oligonucleotides can be designed and synthesized which alter specific condons in the toxin genes. Uracil- incorporated, single-stranded target plasmid DNA will be mutagenized according to the directions supplied by the manufacturer of the mutagenesis kit. The nucleotide changes are then confirmed by DNA sequence analysis.
In the His-shiga toxin, two or more amino acids essential for enzymatic activity should be altered. For example, A subunit targets are the residues El 67 and E170. The Shiga toxoid resulting from this mutation has been used for vaccinating pigs. (Gordon et al., supra). Example VI
Passive Immunity to Shiga Toxin Using His-Shiga Toxin and Toxoid A. Antisera Specific for His-Shiga Toxins Antisera specific for Shiga toxins are required to treat and prevent potentially- deadly infections by EHEC and Shigella dysenteriae type I. Specifically, once a child becomes infected, the child and his or her family members or children in his or her day care group can receive anti His-Shiga toxin sera to achieve a protective immune response. A protective immune response is one that elicits sufficient antibody to permit a patient to avoid infection, decrease the significance 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 mice results in resistance to normally lethal infection of EHEC. (Lindgren et al., Infect. Immun. 62:623(1994); Wadolowski et al., Infect. Immun. 58:3959). Thus, applicants believe that administering anti-Shiga toxin sera to humans and other mammals would result in a protective immune response against Shiga toxin infections.
Methods are well-known in the art for producing antisera for passive immunization. For example, His-Shiga toxoid, obtained by the methods described in Example VI, can be administered to a mammal, such as a horse intraperitoneally. Currently, the horse is used to produce serum against botulism toxin for administration to humans, Hibbs et al., Clin. Infect Dis., 23:337-40 (1996), and the horse would be a 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 tested for neutralizing the cytotoxicity of Shiga toxins. Advantageously, a large amount of serum can be quickly made using this method. However, patients must first be screened for an immune reaction to horse serum. For this purpose, a small amount of horse serum would be subcutaneously injected, and the patient would be monitored for a reaction. Such methods for administering horse antiserum against toxins to humans are well known to the skilled artisan. Hibbs et al., supra; Dehesa and Possani, Toxicon, 32: 1015-1018(1994); Gilan ct al., Toxicon, 27:1 105-1 1 12 (1989).
More preferably, the His-Shiga toxoid can be administered to human volunteers, either intraperitoneally or orally. The plasma from these volunteers is then isolated, and the human anli-His-Shiga toxin serum can be administered to patients. No threat of serum sickness arises from this method. Human hyperimmune globulin to Hemophilus influenzae b, Streptococcus pneumoniae, and Neisseria meningitidis has previously been prepared by others (Siber et al., Infect, and Immun., 45: 248-254 (1984)). B. Vaccines Against Shiga Toxins An embodiment of the invention is vaccines against Shiga toxin infection. For example, these vaccines can include antibodies directed against His-Shiga toxin, obtained further described in Example VII. Moreover, these vaccines can be combination vaccines that comprise His-Shiga toxoid fused or conjugated with another protein, hapten, or antigen, as described in Example IV. These vaccines can be administered intraperitoneally or injectably by methods well known in the art.
A preferred method of administering His-Shiga toxin or toxoid and fusions thereof is by further conjugation to Synsorb® (SynSorb Biotech, Inc., 1204 Kensington Road, N.W., Calgary, Alberta, Canada, T2N3P5.) Synosorb is a sand-like material 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 toxins and appears to be safe for human ingestion. (Armstrong et al., supra) The Synsorb is bound to the B subunit pentamer via the B subunit pentamer-receptor reaction. Conjugation with Synsorb adds further stability. Another embodiment of the invention involves the administration of nucleic acid vaccines. DNA encoding a His-Shiga toxoid is injected into a patient as naked
DNA, 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 patient mounts an immune response against transiently expressed foreign antigens.
Currently nucleic acid vaccines, in general, are all nearing clinical trials. This approach to vaccines involves delivering the DNA encoding the desired antigen into the host by inserting the gene into a nonreplicating plasmid vector (Marwick, C.
JAMA 273: 1403(1995); reviewed in Vogel, F.R. andN. Sarver, Clin Microbiol., Rev. 8:406 (1995)).
The first published demonstration of the protective efficacy of such a vaccine has shown that intramuscular injection of plasmid DNA encoding influenza A virus (A/PR/8/34) nucleoprotein (NP) elicited protective immune responses in BALB/c mice against a heterologous strain of influenza virus (A/HK/68) (Ulmer, J.B. et al. Science 259:1745(1993)). Immunized animals had reduced virus titers in their lungs, decreased weight loss, and increased survival compared with challenged control mice. Both NP-specific 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 complex class I-restricted peptide epitope.
Another study has shown that intramuscular injection of plasmid DNA encoding influenza virus A/PR/8/34 hemagglutinin resulted in the generation of neutralizing antibodies that protected mice against a heterologous lethal influenza virus challenge (Montgomery, D.L. et al. DNA Cell Biol, 12:777 (1993)). Example VII
His-Shiga Antibodies
His-Shiga antibodies, polyclonal and monoclonal, can also be used in the treatment, diagnosis, and prevention of infections related to Shiga toxins. Because of their increased specificity, monoclonal antibodies are preferred. His-Shiga toxin antibodies can be administered to humans or other mammals to achieve a protective immune response, for treatment or prophylaxis. Antibodies, in a physiologically acceptable carrier, may be administered orally or intraperitoneally. For this purpose, monoclonal antibodies are preferred and humanized monoclonal antibodies are particularly preferred. Positive clinical responses in humans have been obtained with monoclonal antibodies, and one skilled in the art would know how to employ Shiga monoclonal antibodies in humans. See Fagerberg et al., "Tumor Regression in Monoclonal Antibody-treated Patients Correlates with the Presence of Anti-idiotype- reactive T Lymphocytes," Cancer Research, 55:1824-27 (1995); "A Phase I Study of Human/Mouse Chimeric Anti-ganglioside GD2 Antibody chl4.18 in Patients with Neuroblastoma," Eur. J. Cancer, 2:261-267 (1995)).
Another embodiment of the invention involves using antibodies to diagnose Shiga 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. In addition, 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 toxin antibody for detection. A detection kit comprising the His-Shiga toxin antibody can be used for this purpose.
For example, a sandwich Elisa can be used. In this kit, rabbit anti-His-Shiga toxin 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 as mouse α-goat antibody conjugated to horseradish peroxidase. The antibody can be detected by standard methods.
His-Shiga toxin polyclonal antibodies and monoclonal antibodies are described below.
A. Making Polyclonal Antibodies
The technique of Harlow, E. and D. Lane (eds.), Antibodies- a Laboratory Manual, Cold Spring Harbor, New York (1988), may be followed. The general procedure is outlined herein. Take pre-bleeds of each mouse to be immunized: 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, and collect the serum (i.e., supernatant; red blood cells are the pellet). Store the serum at -20 °C. The sera obtained will be used as a negative control after the mice are immunized.
Inject a BALB/c mouse intraperitoneally with 25 μg of His-Shiga toxoid (using Titremax adjuvant, according to the instructions of the manufacturer (CytRyx Corp., 154 Technology Pkwy., Norcross, GA. 30092, 800-345-2987)). Wait 2 weeks, boost with 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, and collect the serum. Store the sera at -20°C. B. ELISA to test titer of Abs.
The technique of Harlow, E. and D. Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor, New York (1988), may be followed. The general procedure is outlined below:
(1) bind His-Shiga toxoid to plastic microtiter plates at 50 ng/well in PBS. Incubate 2h/RT (room temp) or overnight at 4°C.
(2) wash plate 2X with PBS. (3) block wells with 100 μl blocking solution [3% bovine serum albumin (Sigma Chemical, St. Louis, MO.), 0.02% sodium azide (Sigma) in PBS - store stock at 4°C] for 1 - 2 h at RT.
(4) wash plate 2X with PBS.
(5) primary Ab = 50 μl test sera diluted in blocking solution for example, start with 1 :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 Ig, affinity purified (Boehringer Mannheim Corp., 91 15 Hague Rd., P.O. Box 50414, Indianapolis, IN. 46250,800-262-1640). Add secondary Ab diluted 1 :500 in blocking solution without azide. Incubate 1 h/RT.
(8) wash 4X with PBS.
(9) add 100 μl TMB Peroxidase substrate to each well (prepared according to the instructions of the manufacturer, BioRad Labs, 3300 Regatta Blvd., Richmond, CA.
94804). Allow blue color to develop (no more than 10 min). Stop the reaction with 100 μl H2SO4. Read the plate at 450 nm.
A titer is defined as an absorbance value >0.2 units above that obtained for mouse preimmune sera. Anti-Shiga toxin Abs obtained from animals may be used clinically if one changes the specificity of the antibody to human. Such techniques are well known to those 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-determining regions in a human antibody with those from a mouse," Nature, 321 : 522-525 (1986); P. Carter et al., "Humanization of an anti-pl85fα£R2 antibody for human cancer therapy," Proc. Nad. AcadSci. USA, 89: 4285-4289 (1992). Such antibodies may be given to the sibling of an infected patient to reduce the risk of infection of the sibling. C. Raising Monoclonal Antibodies to His-Shiga Toxin
Monoclonal antibodies directed against Shiga toxin are used to passively protect a patient against EHEC and Shigella dysenteriae type 1 infections. Monoclonal antibodies are generated from mouse cells, and the specificity of these antibodies are changed 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 regions in a human antibody with those from a mouse," Nature, 321 : 522-525 (1986); P. Carter et al., "Humanization of an anti-plδS"1 2 antibody for human cancer therapy," Proc. Natl. AcadSci. USA, 89: 4285-4289 (1992). Monoclonal Abs represent a more "pure" antibody for administration to a patient.
The procedure outlined in Harlow, E. and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York (1988) is followed: Five 4- to 5-week old female BALB/cJ mice are prebled, and immunized intraperitoneally with 25 μg His- Shiga toxoid suspended in 100 μl of TiterMax. Mice are boosted twice in two week intervals, intraperitoneally with 25 μg His-Shiga toxoid suspended in 100 μl of TiterMax. Seven days after each boost, blood (-300 - 500 μl) is collected from the tail vein. Sera are assayed for the presence of anti-Shiga toxin antibody by ELISA (as described above).
Mice producing high titers of anti-His Shiga toxin antibodies are boosted both intravenously and intraperitoneally with 25 μg of His-Shiga toxoid in 100 μl of PBS, sacrificed three days later, and sera collected. Spleen cells are isolated and fused to Sp2/0-Ag mouse myeloma cells (ATCC #CRL1581 ) at a ratio of 10 spleen cells to 1 myeloma cell. Fused cells are distributed into microdilution plates, and culture supernatants arc assayed by ELISA after 3-4 weeks of culture for anti-His-Shiga toxin antibodies. Cultures positive for production of anti-His Shiga toxin antibodies are expanded and cloned twice by limiting dilution. The person skilled in the art would understand how to use and practice the invention based on the above disclosure. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

We claim:
1. A polypeptide comprising a Shiga toxin having a histidine tag.
2. A polypeptide comprising an immunoreactive but non-toxinogenic form of the polypeptide of claim 1.
3. A fusion protein comprising the polypeptide of claim 1 or 2 fused to a second polypeptide or a portion thereof.
4. A method for large-scale isolation and purification of Shiga toxin comprising the steps of: a) expressing Shiga toxin with a histidine tag in bacteria; and b) eluting cell extract containing histidine-tagged Shiga toxin over a nickel- nitrilotriacetic acid ligand (Ni-NTA) gel.
5. A method of providing passive immune protection comprising the step of administering antisera directed against the polypeptide of claim 2 to patients in need thereof.
6. A method of treating infections mediated by toxins of the Shiga toxin family comprising the step of administering antibodies against the polypeptide of claim 2 to patients in need thereof.
7. A vaccine comprising an antibody directed against the polypeptide of claim 2.
8. A vaccine comprising a nucleotide encoding the polypeptide of claim 2.
PCT/US1997/015836 1996-09-10 1997-09-09 Histidine-tagged shiga toxins, toxoids, and protein fusions with such toxins and toxoids, methods for the purification and preparation thereof WO1998011229A2 (en)

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EP0929679A2 (en) 1999-07-21
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AU4184597A (en) 1998-04-02
AR010218A1 (en) 2000-06-07
WO1998011229A3 (en) 1998-04-16

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