WO1994022474A1 - Procede de traitement du syndrome de kawasaki par modulation immunitaire - Google Patents

Procede de traitement du syndrome de kawasaki par modulation immunitaire Download PDF

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WO1994022474A1
WO1994022474A1 PCT/US1994/003719 US9403719W WO9422474A1 WO 1994022474 A1 WO1994022474 A1 WO 1994022474A1 US 9403719 W US9403719 W US 9403719W WO 9422474 A1 WO9422474 A1 WO 9422474A1
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superantigen
tsst
aureus
derivative
cell
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PCT/US1994/003719
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Brian Kotzin
Philippa Marrack
John Kappler
Donald Leung
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National Jewish Center For Immunology And Respiratory Medicine
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Priority to JP6522468A priority Critical patent/JPH08508734A/ja
Priority to AU65554/94A priority patent/AU6555494A/en
Publication of WO1994022474A1 publication Critical patent/WO1994022474A1/fr

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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
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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

Definitions

  • This invention relates to the field of immunology. More particularly, it relates to the determination of an etiological agent associated with Kawasaki Syndrome, which is also known as mucocutaneous lymph node syndrome, or Kawasaki Disease, and the ramifications which arise from this observation.
  • Kawasaki syndrome is an acute multi system vasculitis of unknown etiology. The disease primarily affects infants and young children, i.e.. aged sixteen or younger. See Kawasaki, Jpn. J. Allergol 16:178-222 (1967); Rauch et al., Pediatr. Infect. Dis. 4: 702-702 (1985). While KS does occur worldwide, it is most prevalent in Japan and in children of Japanese ancestry. Primary clinical manifestations include prolonged fever, bilateral non- exudative conjunctivitis, induration and erythema of extremities, inflammation of lips and oropharynx, polymorphous skin rash, and cervical lymphadenopathy. These indications are used in a clinical diagnosis of KS.
  • IVGG intravenously
  • KS has become one of the most common causes of acquired heart disease in children.
  • Recent studies have shown that when gamma globulin is administered intravenously ("IVGG") during the acute phase of the disease, coronary artery lesions, which otherwise develop in 15- 25% of patients, are significantly decreased.
  • IVGG intravenously
  • KS is characterized by an acute stage, as well as a convalescent stage.
  • the acute phase is characterized, inter alia, by marked immune activation.
  • Investigators have demonstrated, for example, increased number of circulating and infiltrating T cells bearing the HLA-DR activation antigen and elevated serum soluble IL-2 receptor levels. These phenomena are indicative of T-cell activation. See Leung et al.. J. Clin. Invest. 79: 468-472 (1987); Terai et al.. Hum. Pathol. 21: 231-234 (1990); Lan ⁇ et al.. J.
  • vascular endothelium in KS lesions, has been demonstrated to express cytokine- inducible leukocyte adhesion molecules known to be involved in localization of inflammatory cells. See Leung, supra. Patients with acute KS have been found to have cytotoxic antibodies against IL-l ⁇ , TNF- ⁇ and IFN- ⁇ stimulated endothelial cells, but not unstimulated cells. See Leung et al.. J. Clin. Invest. 77: 1428-35 (1986); Leung et al.. J. Exp. Med. 164: 1958-72 (1986) .
  • KS superantigens
  • SAgs superantigens
  • T cell repertoire a novel class of antigens which can have profound effects upon the T cell repertoire.
  • SAgs are stimulatory in their native conformation. Indeed, the mitogenic effects of SAgs are destroyed by conventional antigen processing (Fraser (1989) Nature 339:221) .
  • T cell antigen receptor TCR
  • MHC major histocompatibility complex
  • SAgs bind to lateral faces of both the TCR and MHC molecules, forming a bridge between these cell-surface molecules on apposing cells.
  • Selective activation of T cells by SAgs is determined primarily by the V ⁇ element of the TCR of the responding cells, independent of the specificity of that receptor for any particular MHC-processed antigen.
  • Acute exposure to these antigens either in vitro or in vivo often leads to massive activation of T cells bearing the appropriate V3 elements.
  • Chronic exposure to superantigens either by repeated injection, chronic infection, or in the case of retroviral superantigens, by the inheritance of an active provirus, generally leads to deletion of T cells bearing the appropriate V ⁇ elements subsequent to T cell activation.
  • All superantigens identified to date are proteins produced by bacteria, mycoplasma or viruses. Examples include the enterotoxins of Staphylococcus aureus. staphylococcal or streptococcal exotoxins, the Mycoplasma arthriditis mitogen (MAM) (reviewed in Marrack and Kappler (1990) Science 248:705; Herman et al. (1991) Ann. Rev. Immunol. .9:745; Cole and Atkin
  • Staphylococcus aureus is a common human pathogen that produces several enterotoxins, designated as SEA (staphylococcal enterotoxin A) through SEE (staphylococcal enterotoxin E) , which can be responsible for food poisoning and occasionally shock in humans (Marrack & Kappler (1990) supra; Bohack et al. (1990) Crit. Rev. Mocrobio. 117:251) .
  • Some S. aureus isolates also produce toxic shock syndrome toxin-1, which has been implicated in the majority of cases of human toxic shock syndrome, as well as the related exfoliative toxins (ExTF) , which are associated with scalded skin syndrome. Streptococcus pyrogenes.
  • streptococcus another common human pathogen of the skin and pharynx, also produces toxins with superantigenic properties (Abe et al. (1991) J. Immun. 146:3747) . These have been designated streptococcal erythrogenic toxins A-C (SPEA - SPEC) .
  • Staphylococcal enterotoxins and streptococcal erythrogenic toxins are potent inducers of IL-1 and TNF- ⁇ from monocytes (Fast et al.. (1989) Infect, and Immunity 52:291).
  • One way that activated T cells can be measured is by their ability to produce the cytokine IL-2.
  • Staphylococcal enterotoxin and SPE- mediated stimulation of monocytes is a consequence of the binding and transducing of a positive signal through class II major histocompatibility molecules on the monocyte cell surface.
  • these bacterial toxins are powerful stimulators of T cell proliferation in culture and do so by selectively stimulating T cells expressing particular V ⁇ gene segments (Marrack and Kappler (1990) supra.
  • T cells expressing particular V ⁇ gene segments
  • the majority of T cells stimulated by staphylococcal TSST-1 in vitro specifically express V ⁇ 2 .
  • peripheral blood T cells from patients who develop toxic shock syndrome demonstrate a remarkable expansion of circulating V/32 + T cells.
  • European Patent Publication 340,109 entitled Anti-T-cell receptor determinants as autoimmune disease treatment
  • U.S. Patent No. 4,550,086, issued October 29, 1985 to Reinherz et al. entitled Monoclonal antibodies that recognize human T cells
  • PCT Patent Application Publication WO 90/06758 describes a method for detecting specific V ⁇ regions associated with rheumatoid arthritis (RA) , specifically, V ⁇ 3, V ⁇ 9 and V?10, and for the treatment of RA with monoclonal antibodies which recognize V ⁇ 3 , V ⁇ 9 , and V310.
  • RA rheumatoid arthritis
  • the present invention is premised on the identification of a unique strain of toxic shock syndrome toxin-1 producing Staphylococcal aureus. which is associated with Kawasaki Syndrome. Also involved in the invention is a biologically pure culture of this unique strain of S___ aureus. The inventors herein show that Kawasaki
  • TSST-1 Streptococcal or Staphylococcal bacteria and their associated antigens, particularly TSST-1.
  • the present invention also includes therapeutic methodologies for treating and preventing Kawasaki Syndrome, based upon the finding that S. aureus is a causative agent.
  • the present invention includes a method for preventing the toxic effects of TSST-1 by treatment with a molecule, wherein said molecule interacts with the specific V ⁇ , V ⁇ 2 , element of the T cell receptors (TCR) , associated with Kawasaki Syndrome.
  • these molecules consist of mutated or modified derivatives of TSST-1. It has been found that the pathological condition known as Kawasaki Syndrome can be prevented or treated by administration of the mutant superantigen molecules of the present invention.
  • the molecules of this invention can function by leading to deletion or inactivation/desensitization of the V ⁇ regions associated with Kawasaki Syndrome, specifically, V ⁇ 2 .
  • FIGURE 1 depicts a Southern Blot of isolates from patients with Kawasaki Syndrome compared to controls.
  • FIGURE 2 depicts a Western Blot of isolates using anti- TSST antibodies.
  • FIGURE 3 depicts the SDS-PAGE analysis of the recombinant TSST-1 (24 kD) purified from E. coli.
  • FIGURE 4 depicts the nucleotide and amino acid sequence of the wild-type toxic shock syndrome toxin-1.
  • FIGURE 5 depicts the primers used in the mutagenesis of TSST-1. Each primer 1 top to 13 top was made so that at each position 99% of the molecules were the right nucleotide and 1% had a mixture of all of the different combinations.
  • FIGURE 6 depicts a schematic of the synthesis of the mutant TSST-1 molecules.
  • the present invention includes a method for diagnosing, preventing and treating Kawasaki Syndrome, originating from the isolation and identification of a unique strain of toxic shock syndrome toxin-1 producing Staphylococcal aureus, as a causative agent of Kawasaki Syndrome. Also involved in the invention is a biologically pure culture of this unique strain of S. aureus.
  • the methodology generally involves assaying a sample taken from a patient suspected of having KS, to determine at least one of (i) the presence of toxic shock syndrome toxin, (ii) the presence of white, toxic shock syndrome toxin producing S .
  • the method of this invention further involves preparing mutated superantigen molecules, specifically TSST-1 molecules, by methods known in the art and described herein, identifying antigen mutants unable to bind either MHC or TCR, and testing for the ability to protect against exposure to the non-mutated superantigen.
  • the molecules of the present invention may be effective in different ways in preventing or treating antigen-mediated or initiated diseases such as Kawasaki Syndrome. Some of the different ways in which the molecules of the present invention may be effective include modification of the T cell response and production of antibodies which provide protection against pathogens.
  • TSST-1 producing S_j_ aureus has been isolated and identified as a causative agent of KS (Examples 1-4) .
  • Mutants of TSST-1 were then prepared and purified as described in Examples 5-9 below.
  • the TSST-1 mutants were then tested for the presence of functional toxin by stimulation of murine T cell hybridomas bearing a/ ⁇ receptors with V015, using human DR-expressing cell lines as presenting cells (Example 7) .
  • the key event in an immune response is the interaction of MHC molecules with antigens to form a complex presented to T cells.
  • T cell response is quite specific, with only very limited subpopulations of T cells responding to specific complexes of antigen and MHC molecules.
  • the response generally requires interaction of most or all of the components of the T cell receptor.
  • the presented antigen need only interact with the V ⁇ element of the receptor, all other components are essentially irrelevant. This means that the antigen can, and does, react with a much greater array of T cells than is normally the case.
  • the molecules of this invention may interact with the V ⁇ elements of T cell receptors in a way which leads to modifications in the way T cells respond to a superantigen.
  • “Modifying T cell responsiveness” means that the mutant molecules are able to change the manner in which the subject's T cells respond when provoked by the administered molecule, or to an antigen administered concurrently, previously, or afterward. For example, it is believed that early in the development of T cells, certain subpopulations interact with presented antigens and are deleted.
  • the molecules of this invention can function in this manner, i.e., by leading to deletion or inactivation/desensitization of at least one or more subpopulations of T cells which present a particular V ⁇ element.
  • the molecules modify the T cell response without changing the B cell response that would normally occur in the subject under consideration.
  • This type of material is useful, for example, for providing passive immunity to a subject, or serving as a vaccine.
  • superantigen derivatives are no longer superantigenic, as they will not provoke a restricted T cell response, but will still serve as antigens in that they generate a B cell response.
  • the superantigen derivatives of the present invention are able to elicit normal antibody production against the superantigen protein.
  • the molecules of the present invention may also be seen as competitors for other antigens. If the molecules described herein interact with MHC elements otherwise required for generation of a full scale response to an antigen or superantigen, they may prevent or reduce the extent of that response.
  • the molecules of the present invention may also be viewed as "enhancers" in some instances, where an individual's T cell responsiveness is impaired or weakened by any of a number of causes. Via administration of the molecules encompassed by the present invention, the T cell populations of the individual can be greatly expanded.
  • modifying T cell responsiveness is always relative to a second element (e.g., an antigen), and always refers in particular to responsiveness of T cells presenting a particular V ⁇ element as part of their T cell receptors, other components of the receptors being essentially irrelevant.
  • a second element e.g., an antigen
  • the molecules of the present invention contain, at least, an amino acid sequence of sufficient size to bind to an MHC molecule.
  • the rest of the molecule may consist of amino acids, or may contain carbohydrate or lipid structures.
  • “Reducing responsiveness” is construed to also include deleting the portion of T cells expressing a particular V ⁇ element.
  • "Superantigen derivative” as used herein refers to a molecule whose structure, at the least, contains an amino acid sequence substantially identical to an amino acid sequence presented by a superantigen or portions of a superantigen required for binding to either the MHC or the T cell.
  • Modified superantigen derivative differs from “mutated” superantigen derivative (or fragment) .
  • modified superantigen is defined to refer to molecules which contain an amino acid sequence identical to an amino acid sequence of superantigen, but contain modifications not found in the superantigen molecule itself. For example, if a superantigen contains amino acids 1-250, a "modified” superantigen derivative may contain a sequence identical to amino acids 50-75, positioned in between stretches of amino acids not found in the native superantigen molecule. Additional modifications may include, for example, differing or absent glycosylation patterns, or glycosylation where there normally is none.
  • “Mutated” superantigen refer to structures where the actual amino acid sequence of the mutation has been altered relative to the native form of the molecule. For example, if a superantigen contains amino acids 1-250, a mutated superantigen may include amino acids 50-68 and 72-75 which are identical to the corresponding native sequence, but differ in amino acids 69-71. The difference may be one of "substitution” where different amino acids are used, “addition” where more amino acids are included so that the sequence is longer than the native form, or “deletion” where the amino acids are missing.
  • Vaccine refers to a formulation when administered to a subject provokes the same type of response typical of vaccines in general, e.g., active immunological prophylaxis. The vaccine may contain adjuvant, or other materials.
  • MHC molecules are available in a variety of "phenotypes", and different phenotypes are specific for various presented peptides and antigens.
  • HLA-DR is known to be associated with presentation of TSST-1.
  • different MHC phenotypes will be of value for different antigens, but determination of HLA phenotype and correlation to presentation of a particular antigen or antigen family is well within the skill of the artisan in this field.
  • this invention involves the modification of the T cell response via administration to a subject of a molecule which interacts with both an MHC molecule and at least one V ⁇ element on T cell receptors.
  • This interaction may affect the T cell response in any number of ways. Perhaps the most elementary manner of affecting the response is one where a molecule interacts with the MHC molecule, preventing the binding of other molecules to the MHC. If the competing molecule has been modified or does not naturally provoke proliferation of T cells, then there will be a lessening or elimination of the response because molecules such as normal antigens or superantigens cannot form the requisite complex with the MHC to generate a T cell proliferative response.
  • T cell response Another manner of modifying the T cell response is via “desensitizing”, “inactivating”, or “anergizing” the T cells.
  • This mechanism involves interaction of MHC molecule, antigen, and T cell receptor, with subsequent down regulation or inactivation of the T cells.
  • This mechanism is more common in mature subjects than the deletion phenomenon, which occurs in fetal subjects. The latter phenomenon is one where via interaction of the three units, various subpopulations of T cells are in fact removed from the organism.
  • the modification of the T cell response can also involve stimulation of T cell subpopulations.
  • Knowledge of the mechanisms described herein permits the artisan to administer to a subject a material which interacts with the MHC and a particular subpopulation of T cells, where proliferation of the T cell subpopulation results. This approach is particularly desirable in the treatment of conditions where a particular V ⁇ subpopulation or subpopulations are associated with a pathological condition, such as an autoimmune disease.
  • an immune response when fully considered, includes both a B cell and a T cell response.
  • One aspect of the invention contemplates the use of molecules which modify the T cell response without modifying the B cell response. Such materials are especially useful as vaccines, as discussed below.
  • the molecules of the invention are preferably, but not exclusively, superantigen derivatives. These derivatives may be modified or mutated, as discussed above. These, or any other molecules used herein, are administered in an amount sufficient to modify the T cell response in the manner described. The amount of material used will vary. depending on the actual material, the response desired, and the subject matter of the treatment.
  • the molecules may also serve as vaccines. These vaccines confer protective immunity on the subject in that they generate a B cell response without the full T cell response normally associated with the normal form of the molecule.
  • the material chosen for the vaccine is up to the artisan.
  • the vaccine may contain other materials which are normally found in vaccine compositions, including adjuvants, carriers, etc.
  • the mode of administration of the materials described herein may vary as well, including intravenous, intraperitoneal, and intramuscular injections, as well as all of the other standard methods for administering therapeutic agents to a subject.
  • the invention also discloses how to make particular mutants useful in the foregoing methodologies, including isolated nucleic acid sequences coding for mutants, cell lines transformed by these and the vectors and plasmids used therefor, as well as the isolated mutant molecules, including mutant superantigens.
  • Example 2 The bacteria isolated from the cultures were subjected to further examination (Example 2) . Each one produced 3.2 ug/ l of TSST-1, a quantity typical of most TSST associated S. aureus. All isolates were found to be tryptophan auxotrophs.
  • KS associated isolates were coagulase positive, and produced substantially less lipase, he olysis, and protease than did the cultures from skin infections and toxic, shock syndrome patients.
  • Table 2 summarizes the data on the bacterial isolates.
  • a set of experiments was then carried out in which the DNA of the TSST-1 secreting S. aureus of the cultures was probed (Example 3) .
  • the probe was the entire TSST-0 gene, i.e.. tstO, described by Lee et al. , J. Infect. Dis. 165: 1056-1063 (1992). This gene differs from gene tst which produces TSST-1, by only 14 nucleotides.
  • Examples 1-4 discloses that the S. aureus isolates found in the KS samples are clonally derived and that the virulence factors expressed by the organisms are such that they determine the host colonization niche. It had previously been shown, by Musser et al.. Proc. Natl. Acad. Sci. 87: 225-229 (1990) , that the majority of TSST S. aureus isolates are tryptophan auxotrophs, which have been clonally derived. This conclusion was based on electrophoretic analysis of bacterial enzymes. Analysis of the foregoing examples shows that all of the KS isolates are tryptophan auxotrophs, and demonstrates the predicted Southern hybridization patterns of tryptophan auxotrophs. The isolates also share a variety of other phenotypic characteristics.
  • TSST positive isolates of S . aureus differ from other S . . aureus isolates in that they not only produce TSST-1, but also other potential virulence factors in a pattern that predicts preferred site of attachment to the host.
  • TSST isolates from mucosal surfaces do not make as much lipase as isolates from penetrating skin infections (e.g., carbuncles). In skin infections, the production of lipase may be necessary for skin invasion.
  • Such isolates also tend to cause highly inflammatory lesions, while TSST isolates from mucous membranes typically cause little, if any inflammation.
  • KS isolates described named "Kawasaki Syndrome I" herein most closely resemble S. aureus mutants lacking a functional accessory gene regulator (agr) , the global regulator of virulence factor production in this strain (Recsel et al.. Mol. Gen. Genet. 202: 58-61 (1986)).
  • agr- mutants make only small amounts of lipase, hemolysin, and protease, and are white (Peng et al. , J. Bacteriol 170: 4365-4372 (1989)).
  • agr- mutants produce almost no TSST-1 (less than 0.1 ug/ml) , in contrast to the amounts produced by the KS related isolates. Further, the KS isolates are not agr- mutants.
  • the foregoing examples provide a new method for diagnosing Kawasaki syndrome.
  • the methodology involves assaying a sample taken from a patient suspected of having KS, to determine at least one of (i) the presence of toxic shock syndrome toxin, (ii) the presence of white, toxic shock syndrome toxin producing S. aureus in the culture, (iii) Streptococcus exotoxin B or C, or (iv) Streptococcus which produce either of the recited strepexotoxins. Any of these "markers" are indicative of KS in the subject.
  • S. aureus, toxic shock syndrome toxin or streptococcus are also indicative of other conditions.
  • KS i.e. , children, primarily children of oriental descent, especially Japanese, is not coextensive with the population prone to toxic shock syndrome.
  • KS is associated with several other diagnostic markers.
  • white S. aureus bacteria associated with the disorder all other pathological conditions where Staphylococcus is implicated involve standard, gold colored bacteria. Thus, white S. aureus is a specific marker for the disorder.
  • the manner in which the KS indicator is determined may vary, depending upon the wishes of the investigator.
  • immunoassays are preferred, such as the immunodiffusion assay discussed above. Any standard immunoassay using anti-toxin polyclonal or monoclonal antibodies may be used, including immunoblots, ELISAs, RIAs, sandwich assays, and so forth.
  • the targeted molecule may be TSST-1, SPE B, or SPE C. If culturing of a sample for the bacteria is desired, the sample can be cultured in any of the standard media used for culturing bacteria, such as the blood agar media discussed above. Visual inspection of the cultures for a white microorganism with phenotype and biochemical characteristics of S. aureus can then be carried out. Several of these characteristics are discussed above, but others will be familiar to the skilled artisan and need not be set forth herein.
  • a specific strain of TSST-1 producing S. aureus which meets the criteria set forth herein and cultured from samples taken from KS subjects was deposited at the American Type Culture Collection on March 24, 1993 and has been accorded Accession Number A.T.C.C. 55049.
  • This culture can be used, e.g.. as an immunogen for preparing strain specific antibodies, for nucleic acids to be used in probe assays, as well as for screening and/or development of potential therapeutic agents. Given the normal levels of toxin, but the low levels of other virulence factors, the organism is useful in further studies of the development of KS.
  • KS can also be diagnosed via carrying out a nucleic acid based assay, such as Southern blotting.
  • a nucleic acid based assay such as Southern blotting.
  • Other assays within this ambit include assaying with labelled probes, such as oligonucleotides which carry radiolabels, biotin, or other labels, polymerase chain reactions using oligonucleotides corresponding to the tst gene, and so forth.
  • kits include a support means for immobilizing the nucleic acids of the sample, such as nitrocellulose, and at least one probe for hybridizing to the target.
  • Other optional buffers, hybridization solutions, e.g. , SSC, wash buffers, and so forth may be included in the kit.
  • the kits may also contain a solid support, such as a membrane (e.g.. nitrocellulose) , a bead, sphere, test tube, rod, and so forth, to which a receptor such as an antibody or antibody fragment specific for the target molecule will bind.
  • kits can also include a second receptor, such as a labelled antibody or labelled binding antibody fragment.
  • the kits can be used for sandwich assays to detect toxins or bacteria presenting the toxins.
  • Kits for competitive assays are also envisioned.
  • Such kits include, e.g.. a solid phase to which a sample of the toxin to be detected is bound, as well as a portion of toxin specific antibody or antibody fragment.
  • the binding receptor portion of the kits may be presented in a separate portion within the kit, or may be already bound to the solid, phase bound toxin.
  • Such a system may be used in a displacement assay, e.g.
  • the essentially elements are a moiety capable of detecting an agent indicative of KS, and a solid phase to which the agent binds, directly or indirectly.
  • TSST-1 mutants were generated as described in Example 6. The method introduced random mutations in approximately 17 residues in the mature TSST-1 protein.
  • Initial identification of potential mutant TSST-ls tested the lysate from transformants for the presence of functional toxin by stimulation of murine T cell hybridomas bearing different V ⁇ elements in a human DR-expressing cell line.
  • mutated superantigens of the present invention may be used to selectively stimulate only some of the T cell populations stimulated by the wild-type superantigen.
  • Example 1 Identification of Bacterial Cultures from Kawasaki Patients.
  • Cultures were obtained from the pharynx, axilla, groin and rectum of sixteen patients with untreated, acute KS, and fifteen disease controls with fever and/or rash. The cultures were taken from all patients within 10 days of the onset of illness, and prior to initiation of intravenous gammaglobulin therapy. The criteria for diagnosing a subject as having acute KS were those of the American Heart Association Committee on Rheumatic Fever, JAMA 44: 1218-1219 (1990) . Cultures were obtained using cotton swabs at all four of the sites given above, and were then incubated overnight at 37°C.
  • Swabs from all sites were cultured on sheep blood agar plates, while swabs from the groin and rectum were also cultured on phenylethyl alcohol sheep blood agar. Both culture methodologies represent well known techniques. Bacterial cultures were identified, and all staphylococci and beta hemolytic streptococci isolated were screened for secretion of toxins by serial dilution double immunodiffusion, in accordance with Schlievert et_al. , J. Infect. Dis. 147: 236-242 (1983), the disclosure of which is incorporated by reference in its entirety.
  • Example 1 The bacteria isolated from the cultures in Example 1 were further examined. Each one produced 3.2 ug/ml of TSST-1, a quantity typical of most TSST associated S. aureus. The quantity of TSST-1 was determined as described in Example 1, while both lipase and hemolysis, presented in units per 10 8 bacteria, were determined in accordance with Schlievert et al. ,
  • Isolated organisms were grown on sheep blood agar plates, as is described in Example 1.
  • a disk of nitrocellulose paper was placed on top of the grown organisms, after which it was cultured for 24 hours on a blood agar plate. The disk was then removed, and Western Blotting procedure carried out.
  • the nitrocellulose was coated with 200 ml of 3% gelatin (3g in 100 ml of TBS buffer: 0.02 M Tris, 0.5 M NaCl in 4 liters of H 2 0, pH 7.5). This was then incubated for 30-45 minutes in 200 ml of 0.05% TBS/Tween (1 ml Tween/2 liters TBS), at 37°C.
  • the nitrocellose treated filter was with rabbit polyclonal anti-TSST at room temperature (25 ul) , in 50 ml of TBS Tween. This was followed by washing twice for five minutes (each wash) in TBS/Tween, and then followed by incubation for h hours with conjugates of anti-rabbit immunoglobulin and alkaline phosphatase (25 ul per filter) . This was followed by two washes in TBS/Tween, and two more washes in TBS, all for five minutes.
  • TSST-1 was overexpressed in E. coli as follows.
  • TSST- 1 * and TSST " isolates of the bacterium S_;_ aureus were obtained from Dr. James Todd of Children's Hospital in Denver.
  • the bacteria was cultured and the genomic DNA was isolated.
  • PCR was performed with primers Atop and Bbot ( Figure 5) .
  • the sequences of the primers were taken from a report published by Richard Novick and Patrick Schlivert (Blomster-Hautamaa et al. (1986) J. Biol. Chem. .261:15783).
  • the 5' primer was (SEQ ID N0:2): '-GGGGTACCCCGAAGGAGGAAAAAAATGGCTACAAACGATAATATAAAG-3'
  • This primer contains an Kpn I site which places the TSST-1 gene in-frame with the LacZ gene when cloned into plasmid pTZ18R (Pharmacia Fine Chemicals, Piscataway, NJ) .
  • This oligonucleotide primer also contains an S/D site which adds an GAG between the LacZ gene fragment and the beginning of the TSST-1 gene so that the TSST-1 gene could be moved easily to other plasmids carrying its own initiation GAG.
  • the 3' primer contained a Xba I site after the termination codon of the TSST-1 gene (SEQ ID N0:3): 5'-TGCTCTAGAGCATTAATTAATTTCTGCTTCTATAGTTTTTAT-3'
  • the PCR fragment was digested with Kpn I and Xba I and ligated into Kpn I/Xba I digested pTZl ⁇ R.
  • E. coli XLl-Blue (Stratagen, La Jolla, CA) was transformed with the plasmid.
  • the SDS-PAGE analysis if the recombinant TSST-1 (24kD) product is shown in Figure 3. This product specifically stimulated human V ⁇ 2 + T cells in a superantigenic fashion. It was functionally and immunogenically identical to purified TSST-1 obtained from commercial sources.
  • the PCR amplified gene was then sequenced ( Figure 4) .
  • the boxes show differences with the published sequence. The published sequence was incorrect and has now been corrected (Lee et al.
  • this gene has the same sequence as the wild type gene.
  • the TSST-1 gene from at least one S_ ⁇ . aureus culture grown from a KS patient has also been sequenced. This gene had the same sequence (results not shown) .
  • Figure 6 depicts a schematic of the synthesis of the mutant TSST-1 molecules.
  • the first PCR reaction uses 9top (mutagenic primer) and Bbot to amplify the region of the TSST-1 gene that lies between them (the primers themselves become incorporated into the product) .
  • the carrot symbol in 9top represents a nucleotide that does not match the wild type sequence.
  • 9top becomes incorporated into the PCR product, the mismatch is incorporated with it.
  • the mutagenic primer is 51 nucleotides long and encodes for 17 amino acids in the protein.
  • PCR 1 The product from PCR 1 is gel purified and used as a megaprimer for a second PCR reaction, this time paired with the primer Atop ( Figure 5) (Sarkar and Dommer (1990) BioTech. 8_:404) .
  • both primers are incorporated into the amplified DNA product, which is now a full length TSST-1 gene.
  • the many different DNA molecules formed by PCR 2 now contain altered nucleotides throughout the 9top region (amino acid residues 120-136) . These molecules were then cloned, tested, and sequenced (Examples 7 & 8) . Mutants in each library were screened for (1) presence of soluble protein, (2) presence of protein with native configuration; (3) inability to stimulate either mouse V315* cells or human peripheral blood cells.
  • Plasmid inserts were sequenced directly by the dideoxynucleotide method of Sanger et al. (1977) Proc. Natl. Acad. Sci. USA
  • Anti-TSST-1 Monoclonal Antibodies fmAbs 10 mAbs specific for at least five epitopes of TSST-1 were produced by standard methods from B10.Q( / 9BR) immunized multiple times with TSST-1. Two of these antibodies, 327H1 and 477L1, were used both for quantitation and immunoaffinity purification of TSST-1 and TSST-1 mutants. 327H1 was chosen because initial characterization showed that has a high affinity for TSST-1, bound equally well to all of the TSST-1 functional mutants, and it interacts with the TSST-1 toxin in the area where the MHC class II complex binds to the TSST-1.
  • 477L1 was chosen because initial characterization showed that has a high affinity for TSST-1, bound equally well to all of the TSST-1 functional mutants, and it interacts with the TSST-1 toxin in the area where the toxin interacts with the T cell receptor (data not shown) .
  • TSST-1 TSST-1 .
  • the amount of TSST-1 in preparations was determined by ELISA.
  • Microtiter wells were coated overnight with a solution of 6 ug/ml natural TSST-1 (Sigma Chemical Co., St. Louis, MO). The wells were then incubated with 25% FCS and washed throughly.
  • Various concentrations of known and unknown TSST-1 preparations were added to the wells as inhibitor followed by a constant amount of anti-TSST-1 antibody (polyclonal rabbit anti-TSST-1 (Toxin Technology, Madison, WI) in all experiments.
  • TSST- 1 Initial Screening of Potentially Mutant TSST- 1.
  • total lysates were prepared as described from individual transformants containing a potentially mutant TSST-1 gene. Aliquots of each lysate were tested for the presence of functional toxin by stimulation of urine T cell hybridomas bearing a/ ⁇ receptors with V315, using human DR-expressing cell lines as presenting cells. Lysates deficient in stimulating either of these hybridomas were assayed for the presence of TSST-1 protein to rule out mutations affecting the level or the full length of the TSST-1 produced.
  • each well of the second plate received 50 ul of HNM buffer (10 mM Hepes, pH 7.0, 30 mM NaCl, 5 mM MgCl 2 ) containing 3 mg/ml lysozyme and 300 ug/ml DNAse I.
  • HNM buffer 10 mM Hepes, pH 7.0, 30 mM NaCl, 5 mM MgCl 2
  • the plate was incubated at 37"C for 15 min, frozen, thawed three times, and centrifuged to pellet debris.
  • the supernatants were transferred to a new plate and tested for the presence of TSST-1 both by ELISA and T cell hybridoma stimulation. This method produced preparations containing 0.3 and 10 ub/ml of TSST-1.
  • transformants were recovered from the 96-well plate stored at -70°C. Bacteria from overnight cultures (1 vol) containing IPTG were collected by centrifugation, resuspended in a 1:10 vol of HNM buffer containing 1-2 mg/ml lysozyme and 10 ug/ml DNAse I, and frozen and thawed three times. The suspension was centrifuged at 15,000 g for 20 min to remove bacterial debris, and the supernatant was harvested and filtered (0.2 u) . The filtrate was passed through a column containing a 1:50 volume of Sepharose 4B beads coupled with 2-3 mg/ml of a mAb to TSST-1 (B344) .
  • the beads were washed thoroughly with PBS and the toxin was eluted with 0.1 M glycine-HCl (pH 2.7) and neutralized with 1 M Na 2 C0 3 .
  • the TSST-1 was concentrated to 1 mg/ml and its buffer changed to BSS using CentriconlO microconcentrators
  • TSST-1 and its mutants produced in this manner were > 95% pure as judged by SDS-PAGE.
  • Some of the TSST-1 mutants that have had their function altered are listed in Table 3. There are approximately 500 nonfunctional mutants- mutations spaced throughout the molecule, which are still being functionally characterized. The mutations described all involve a nucleotide substitution of A to G, or T to C, which would be predicted by the methodology used for their generation.
  • Example 9 In vitro T cell Stimulation Assay.
  • TSST-1 is a member of the "superantigen" protein family. These proteins are characterized by their potent ability to cause resting, inactive, T cells to begin widly proliferating. They also become larger and begin to do several metabolically interesting things. When this happens, the T cells are "activated”. One way that activated T cells can be measured is by their ability to produce the cytokine IL-2. Thus, the assay for superantigen activity utilized herein is an IL-2 assay. Mouse T cell hybridoma cells bearing V015 T cells receptors are mixed with different doses of candidate mutant TSST-1 proteins and LG-1 antigen-presenting cells. This assay takes place in a 96-well plastic dish. Four of the wells contain E.
  • the plate is cultured overnight. If the superantigen protein is active, the T cell hybridoma cells will make 11-2. On the following day, the culture supernatants are carefully removed from the plate and transferred to a new plate. These supernatants are then diluted 1:2 several times. An IL-2 dependent cell line is then added. The cells will die if IL-2 is not produced. After overnight culturing, the number of IL-2 dependent cells in each culture is measured. Such clones are then sequenced to determine the positions of the altered nucleotides in the gene and to identify the altered amino acid residue in the protein.
  • TSST-1 was determine as described supra, while both lipase and hemolysis are presented in units per 10 8 bacteria, determined in accordance with Schlievert et al. , Ann. Intern. Med. TABLE 3. TSST-1 residues affecting mouse/human T-cell stimulatory activity.
  • AAA AGC CAA CAT ACT AGC GAA GGA ACT TAT 80 lys ser gin his thr ser glu gly thr tyr
  • AGC CCC TTA AAG TAT TGG CCA AAG TTC GAT 120 ser pro leu lys tyr trp pro lys phe asp
  • CAGCTAACTG AAATACATGG ATTATATCGT TCAAGCGATA AAACGGGTGG 50 T 51

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Abstract

Cette invention se rapporte à différentes méthodes permettant de diagnostiquer le syndrome de Kawasaki. On a découvert que différentes bactéries, y compris le Staphylococcus aureus produisant TSST-1 et le stretocoque produisant SPEB et SPEC, indiquent cette pathologie. L'invention se rapporte également à un isolat de S. aureus impliqué dans le syndrome de Kawasaki, ainsi qu'à des procédés thérapeutiques permettant de prévenir et de traiter cet état.
PCT/US1994/003719 1993-04-05 1994-04-05 Procede de traitement du syndrome de kawasaki par modulation immunitaire WO1994022474A1 (fr)

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JP6522468A JPH08508734A (ja) 1993-04-05 1994-04-05 免疫調節による川崎症候群の処置方法
AU65554/94A AU6555494A (en) 1993-04-05 1994-04-05 Method for treating kawasaki syndrome via immune modulation

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996014744A1 (fr) * 1994-11-14 1996-05-23 National Jewish Center For Immunology And Respiratory Medicine Effets protecteurs de superantigenes mutants
US6391315B1 (en) * 1994-07-29 2002-05-21 Takahashi Hashimoto Vaccine for inhibiting and preventing induced staphylococcus infection, isolated antigens used therein, and isolated antibodies induced thereby
WO2003056015A1 (fr) * 2001-11-26 2003-07-10 U.S. Medical Research Institute Of Infectious Diseases Vaccins bacteriens a superantigenes
US7087235B2 (en) 1997-06-25 2006-08-08 The United States Of America As Represented By The Secretary Of The Army Fusion protein of streptococcal pyrogenic exotoxins

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4795037B2 (ja) * 2006-02-01 2011-10-19 財団法人 神戸市地域医療振興財団 川崎病の判定方法及びそのためのキット

Citations (1)

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Publication number Priority date Publication date Assignee Title
US4615884A (en) * 1985-04-09 1986-10-07 Vanderbilt University Method and vaccine for treatment of demyelinating diseases

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4615884A (en) * 1985-04-09 1986-10-07 Vanderbilt University Method and vaccine for treatment of demyelinating diseases

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
INFECTION AND IMMUNITY, Volume 53, No. 2, issued August 1986, SCOTT et al., "Immunological Protection of Rabbits Infected with Staphylococcus Aureus Isolates from Patients with Toxic Shock Syndrome", pages 441-444. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 89, isssued May 1992, ABE et al., "Selective Expansion of T Cell Expressing T-Cell Receptor Variable Regions VBeta2 and VBeta8 in Kawasaki Disease", pages 4066-4070. *
THE JOURNAL OF CLINICAL INVESTIGATION, Volume 91, No. 2, issued February 1993, TAKEI et al., "Intravenous Immunoglobulin Contains Specific Antibodies Inhibitory to Activation of T Cell by Staphylococcal Toxin Superantigens", pages 602-607. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6391315B1 (en) * 1994-07-29 2002-05-21 Takahashi Hashimoto Vaccine for inhibiting and preventing induced staphylococcus infection, isolated antigens used therein, and isolated antibodies induced thereby
WO1996014744A1 (fr) * 1994-11-14 1996-05-23 National Jewish Center For Immunology And Respiratory Medicine Effets protecteurs de superantigenes mutants
US7087235B2 (en) 1997-06-25 2006-08-08 The United States Of America As Represented By The Secretary Of The Army Fusion protein of streptococcal pyrogenic exotoxins
US7750132B2 (en) 1997-06-25 2010-07-06 The United States Of America As Represented By The Secretary Of The Navy Altered superantigen toxins
WO2003056015A1 (fr) * 2001-11-26 2003-07-10 U.S. Medical Research Institute Of Infectious Diseases Vaccins bacteriens a superantigenes

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