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WO2003013601A1 - Toxine liee au syndrome de la mort subite du nourrisson, et utilisations associees - Google Patents

Toxine liee au syndrome de la mort subite du nourrisson, et utilisations associees Download PDF

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Publication number
WO2003013601A1
WO2003013601A1 PCT/AU2002/001085 AU0201085W WO03013601A1 WO 2003013601 A1 WO2003013601 A1 WO 2003013601A1 AU 0201085 W AU0201085 W AU 0201085W WO 03013601 A1 WO03013601 A1 WO 03013601A1
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Prior art keywords
protein
sids
person
slds
curli
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PCT/AU2002/001085
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English (en)
Inventor
Paul N. Goldwater
Karl A. Bettelheim
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Womens and Childrens Hospital Adelaide
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Womens and Childrens Hospital Adelaide
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Publication of WO2003013601A1 publication Critical patent/WO2003013601A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K16/1232—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants

Definitions

  • the present invention relates to methods of detecting, screening and vaccination against Sudden Infant Death Syndrome.
  • SIDS Sudden Infant Death Syndrome
  • the object of this invention is to provide methods for diagnosing SLDS and vaccinating against SIDS that obviates or alleviates any one of the above problems, or at least to provide the public with a useful choice.
  • the present invention arises out of the inventors findings of a protein that is expressed by certain strains of E. coli and is proposed to be a potential causative factor in SLDS.
  • CsgA fimbrial curli protein
  • the curli protein can be produced in both soluble and polymerised (fimbrial) forms.
  • the invention could be said to reside in a method of diagnosing a person as SLDS by detecting the presence of curli protein in a biological sample taken from the person, the method including the steps of: taking the biological sample from the person, contacting the sample with an agent for specifically binding curli protein under conditions for formation of an agen protein complex, and assaying for the formation of the agent ⁇ rotein complex to detect the presence of the curli protein.
  • the invention could be said to reside in a method of diagnosing SLDS by detecting the presence of antibodies to curli protein, the method including the steps of: obtaining a sample of antibody containing fluid from a person, contacting the sample with a protein capable of reacting specifically with the antibody, and assaying for the formation of the protein: antibody complex to detect the presence of antibodies to the curli protein.
  • the invention may also be said to reside in a diagnostic kit containing one or more antibodies, or fragments thereof, specific for the curli protein, in conjunction with appropriate reagents.
  • the invention could be said to reside in a method of vaccinating a human against SIDS, the method including the step of administering a curli protein, or an immunogenically active derivative or variant thereof, to thereby induce an immune reaction.
  • the invention could be said to reside in a method of vaccinating a person against SIDS, the method including the step of administering a nucleic acid sequence that encodes a curli protein so that a host cell expresses the curli protein to thereby induce an immune reaction.
  • the invention may be said to reside in a composition for vaccinating a vertebrate against SLDS, the composition comprising curli protein, or immunologically active derivative thereof, and a pharmaceutically acceptable carrier.
  • the invention may also reside in a pharmaceutical preparation including the therapeutic agent, antibody or antibody fragment defined above in a pharmaceutically effective carrier.
  • a pharmaceutical preparation including the therapeutic agent, antibody or antibody fragment defined above in a pharmaceutically effective carrier.
  • the formulation and preparation of any of these pharmaceutical compositions using antibodies, antibody fragments or other compounds is well known to those skilled in the art of pharmaceutical formulation (see e.g. "Remington's Pharmaceutical Sciences", Sixteenth Edition, Mack Publishing Co, 1980).
  • SLDS serum 40
  • lane 4 SIDS serum (4) lane 5 molecular weight marker
  • lane 6 SIDS serum 56
  • lane 7 SIDS serum 60
  • lane 8 SIDS serum 50
  • lane 9 SIDS serum 55
  • lane 10 SIDS serum 62
  • lane 11 SIDS 51
  • lane 12 SIDS serum 63
  • lane 13 SIDS serum 52
  • lane 14 formic acid extracted soluble CsgA positive control E. coli strain DH1
  • lane 15 molecular weight marker The arrow represents 15-17 kDa
  • FIG. lb Western immunoblot (as performed in Figure 2a), lane 1 SLDS serum (29), lane 2 SIDS serum (30), lane 3 serum from a death resulting from Kawasaki disease (15), lane 4 serum from an asphyxial death , (16), lane 5 serum from a SIDS-like death & pathological findings in a case of Pierre Robin Syndrome (19), lane 6 serum from a case of a SLDS-like death & pathological findings in a case of arginosuccinosis (22), lane 7 serum from a case of primary pulmonary hypertension (a coliform was isolated from heart blood culture obtained at autopsy) (31), lane 8 molecular weight marker, lane 9 formic acid extracted soluble CsgA positive control (E. coli strain DH1), lane 10 formic acid extract from plate culture of negative control (E. coli strain H5).
  • the arrow represents 15-17 kDa
  • Figure 2 Representation of a PAGE of sera probed with anti csgA. Note the absence of the 15 to 17 kDA protein detected when probed with pre- immune rabbit serum.
  • One form of a first aspect the invention could be said to reside in a method of diagnosing a person for SLDS by detecting the presence of curli protein in a biological sample taken from the person, the method including the steps of: taking the biological sample from the person, contacting the sample with an agent for specifically binding curli protein under conditions for formation of an agent ⁇ rotein complex, and assaying for the formation of the agent:protein complex to detect the presence of the curli protein.
  • the method may be used pathologically to assist in determining if SLDS was a cause of death.
  • the method may be used as a pre- or antenatal screen to detect for susceptibility to SIDS.
  • the protein is the curli protein, CsgA.
  • the amino acid sequence of the soluble curli protein has been deduced (Normark et al, United States Patent No. 6,030,805).
  • the protein may be any immunologically active derivative of the above sequence, including a smaller peptide derived therefrom providing the smaller peptide retains at least one region that is responsible for specific immunological recognition. Whether a smaller peptide provides immunogenicity can be determined routinely by following the procedures outlined herein.
  • the method of the present invention encompasses diagnoses based on natural variants of the curli protein.
  • the agent for specifically binding curli protein may be any ligand having a high specificity for binding to the curli protein, including other peptides, proteins, organic molecules and inorganic molecules.
  • the agent is an antibody, or fragment thereof, to the curli protein.
  • Various procedures known in the art may be used for the production of such antibodies and fragments.
  • agent or antibody The specificity of the agent or antibody is to be understood in the context of requiring a positive result that is distinguishable from a negative result in an immulogically meaningful way.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983).
  • the antibody producing cells may be fused with a myeloma cell to produce a pool of hybridoma cells which can then be screened for cells that produce the monoclonal antibody.
  • Monoclonal antibody fragments may also be used in the above method.
  • the protein containing biological sample may be contacted with a fragment of a monoclonal antibody specific for the curli protein.
  • a fragment of a monoclonal antibody the term includes, but is not limited to, Fab, Fv and peptide fragments of the monoclonal antibody, and it may also include such fragments when made as part of a different, larger peptide or protein, which may be the product of a recombinant vector.
  • additional antibodies capable of binding to the curli protein may be produced in a two step procedure through the use of anti-idiotypic antibodies.
  • Such a method makes use of the fact that antibodies are themselves antigens, and that, therefore, it is possible to obtain an antibody which binds to a second antibody.
  • curli protein specific antibodies can be used to immunise an animal and the splenocytes of the animal are used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to block the curli protein specific antibody can be blocked by the protein.
  • Such antibodies comprise anti-idiotypic antibodies to the curli protein specific antibody.
  • the step of assaying for formation of the antibody:protein complex may include the step of separating the bound antibody :protein complex from unbound antibody.
  • the raised antibodies, or fragments thereof may be labelled as discussed below.
  • Any antibody :protein complex formed by contacting the sample with the antibody may be separated from unbound antibody using suitable techniques such as immunoprecipitation or techniques for separation based on size.
  • suitable techniques such as immunoprecipitation or techniques for separation based on size.
  • a mixture obtained after contacting the sample with antibody may be filtered through a suitable membrane so that antibody.protein complex is retained on the membrane and unbound antibody passes through the membrane.
  • the labelled antibody :protein complex can then be quantitatively assayed using standard techniques for the label used.
  • the antibodies may be used in an ELISA based assay to bind the curli protein in the sample, and a labelled antibody specific for another immuno-recognition site on the curli protein can be used to assay for bound protein.
  • the raised antibodies or fragments thereof may be attached or conjugated to a solid support. After contacting the raised antibodies or fragments thereof under conditions suitable for formation of an antibody :protein complex any component of the biological sample that is not bound to the antibodies or antibody fragments on the support may be washed or otherwise separated from the bound complex.
  • the assay for the foraiation of antibody :protein complex preferably involves adding a labelled antibody specific for the curli protein and assaying for the presence of the labelled antibody as discussed below.
  • These assays employ a wide variety of labels including radionuclides, enzymes, fluorescers, chemiluminescers, particles, ligands, enzyme substrates, enzyme cofactors, enzyme inhibitors, light emitter-quencher combinations and the like.
  • the labelled antibodies may be labelled with a radioisotope, which can then be determined by such means as the use of a gamma counter or a scintillation counter.
  • Another way in which the antibodies specific for antibodies can be detectably labelled is by linking to an enzyme.
  • This enzyme when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • enzymes which can be used to detectably label the antibody include malate dehydrogenase, staphylococcal nuclease, delta- V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose- Vl-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.
  • Avidin-biotin binding may be used to facilitate the enzyme labelling.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine.
  • the antibodies specific for antibodies can also be detectably labelled using fluorescent emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or emylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA emylenediaminetetraacetic acid
  • the antibodies specific for antibodies can also be detectably labelled by coupling it to a chemilummescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labelling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the antibodies specific for antibodies.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent antibody is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labelling are luciferin, luciferase and aequorin.
  • Another technique which may also result in greater sensitivity when used in conjunction with the present invention consists of coupling the antibodies specific for antibodies to low molecular weight haptens.
  • the haptens can then be specifically detected by means of a second reaction.
  • the sensitivity of the assay may be increased by use of amplification strategies including substrate cycling and enzyme channelling as described by Lindbladh et al. (1993).
  • tissue expression in tissues can be studied with classical immunohistological methods. In these, specific recognition is provided by the primary antibody but the secondary detection systems can utilise fluorescent, enzyme, or other conjugated secondary antibodies. As a result an irnmunologic.al staining of tissue section for pathological examination is obtained. Tissues can also be extracted, for example with urea and neutral detergent, for the liberation of the protein for Western-blot or dot/slot assay (Jalkanen et al, 1987).
  • the invention could be said to reside in a method of diagnosing SLDS by detecting the presence of antibodies to curli protein, the method including the steps of: obtaining a sample of antibody containing fluid from a person, contacting the sample with a protein capable of reacting specifically with the antibody, and assaying for the formation of the proteimantibody complex to detect the presence of antibodies to the curli protein.
  • the method may be used pathologically to assist in determining if SLDS was a cause of death, or as a screen to test for immunity to SIDS associated toxin and hence SLDS.
  • the antibody containing fluid may be any biological fluid that contains antibodies including, but not limited to, serum, plasma, whole blood, cerebro spinal fluid, amniotic fluid, and synovial fluid.
  • the protein In contacting the biological sample with the curli protein, the protein may be attached or conjugated to a carrier molecule or attached or conjugated to a solid support.
  • a solid support in the present invention means any solid material to which the curli protein can be complexed or attached. Examples of such solid supports include, but are not limited to, microtitre plates, petri dishes, bottles, slides, and other such containers made of plastic, glass, polyvinyl, polystyrene, and other solid materials which allow detection of labelled antibodies. Other suitable carriers for binding the curli protein exist or will be able to ascertained by routine experimentation.
  • any component of the biological sample that is not bound to the protein on the solid support may be washed or otherwise removed from the bound complex.
  • the curli protein, or derivative thereof is covalently or non-covalently bound to the surface of a microtitre well.
  • a serum sample suspected of containing antibody may then be added, unbound sample washed away and the level of antibodies bound in the protein: antibody complex assayed.
  • the assay for the formation of protei antibody complex preferably involves adding a compound that enables detection of the antibodies which are specifically bound to the protein.
  • These assays employ a wide variety of labels and provide for a varying range of sensitivity and susceptibility to interference.
  • Labels include radionuclides, enzymes, fluorescers, chemiluminescers, particles, ligands, enzyme substrates, enzyme cofactors, enzyme inhibitors, light emitter-quencher combinations and the like, as described in the first aspect of the invention.
  • the immunoassays may be homogenous or heterogenous, where the distinction relates to the use of a separation step for separating uncomplexed label from complexed label.
  • the assay is an ELISA and labelled anti- antibodies that are specific for the antibodies may be added to bind the antibodies, thereby allowing detection and quantification of the antibodies.
  • the antibodies being assayed may be members of any of the five major classes of antibodies and therefore the diagnostic method encompasses IgA, IgD, IgE, IgG and IgM antibodies and the labelled anti-antibodies for use in an ELISA may be IgA, IgD, IgE, IgG, and IgM anti-antibodies, respectively.
  • antibodies may be detected by binding with anti-antibodies specific for the antibodies, and then a second labelled antibody specific for the anti-antibody may be used to detect the anti-antibodies.
  • a second labelled antibody specific for the anti-antibody may be used to detect the anti-antibodies.
  • the antibodies may be detected with labelled rabbit anti-human IgG and then labelled anti-rabbit IgG may be added to bind and detect the anti-human IgG. This latter form of assay is believed to give better specificity in that it tends to give less false positives.
  • a protein: antibody complex may be used to detect the presence of antibody in the sample.
  • the antibody that forms part of the complex is labelled with a suitable label.
  • the invention may also be said to reside in a diagnostic kit containing one or more antibodies, or fragments thereof, specific for the curli protein, in conjunction with appropriate reagents.
  • the invention could be said to reside in a method of vaccinating a human against SIDS, the method including the step of administering a curli protein, or an immunogenically active derivative or variant thereof, to thereby induce an immune reaction.
  • a curli protein or an immunogenically active derivative or variant thereof
  • This might be especially useful in vaccinating a pregnant or prospectively pregnant female to potentially provide protection for the progeny.
  • a toxoided product This is attained by chemically altering the protein molecule by reaction with formaldehyde while retaining immunogenicity. This is a tried and true method used to inactivate viruses (e.g. Salk poliovirus vaccine) or bacterial toxin (e.g tetanus and diphtheria toxins).
  • viruses e.g. Salk poliovirus vaccine
  • bacterial toxin e.g tetanus and diphtheria toxins.
  • Other methods of reducing toxicity include creating antigenic polypeptide subunits of the protein and if necessary coupling the polypeptide to a suitable carrier protein.
  • the curli protein may form part of a larger protein by cross linking to a suitable earlier.
  • the carrier may be a carrier protein including but not limited to bovine serum albumin, tetanus toxoid, cholera toxin and deactivated diptheria toxin.
  • the protein may be chemically coupled to the ⁇ -subunit of the heat labile (LT) enteroteoxigenic E. coli ( ⁇ T ⁇ C) toxin which has previously been used as a carrier protein for other bacterial epitopes.
  • the carrier may be an organic molecule, or a solid phase carrier.
  • the carrier is a Ni-NTA (nickel-nitriloacetic acid) agarose to which the protein is bound through a (Histidine)6-tag at either the N or the C terminus.
  • the protein may be displayed on bacteriophage particles.
  • nucleic acid vectors are used wherein an oligonucleotide encoding the protein is fused to a portion of a gene encoding the transmembrane portion of an integral protein.
  • the fusion protein Upon expression of the fusion protein it is embedded in the outer cell membrane with the curli protein portion of the fusion protein facing outward.
  • the protein is linked to a solid support which is the bacterial cell itself.
  • the protein is administered in pharmaceutical dosage form as a composition or formulation comprising an immunogenically effective amount of the protein.
  • the amount of protein administered will vary depending on the pharmacokinetic parameters, severity of the disease treated or immunogenic response desired. Doses may be set by a physician considering relevant factors including the age, weight and condition of the person including, in the case of immunogenic dosage forms, whether the person has been previously exposed to the microorganism responsible for the disease to be vaccinated against as well as the release characteristics of the protein. The person may be vaccinated directly according to the method of the invention, or alternatively a progeny may be passively vaccinated by maternal vaccination.
  • the protein has the sequence set out in United States Patent number 6030805 to Nomark et al.
  • the protein may be homologous to the above sequence.
  • a protein is considered homologous when it is immuno cross-reactive with antibodies specific for the protein. It will be recognised by those skilled in the art that some amino acid sequences within the protein can be varied without significant effect on the structure or function of the protein. Thus, for instance, it is anticipated that conservative amino acid substitutions still retain immuno cross reactivity and as such a neutral amino acid may be conservatively substituted with another neutral natural or non-natural amino acid, an acidic amino acid may be conservatively substituted with a natural or non- natural acidic amino acid, a hydrophilic amino acid may be substituted with another hydrophilic amino acid, and so on, provided that the immunological function of the protein is not altered by the substitution.
  • the homologous protein shares 50% homology with the above sequence, more preferably shares 70% homology, and most preferably shares 90% homology.
  • the curli protein is expressed on a bacterial cell surface.
  • Whole live, or killed bacteria may be delivered in an appropriate form per orally.
  • semi purified curli protein may be administered. It will be understood that the administration of live or killed bacteria or partially fractionated bacteria will be in a manner known in the art and is likely to be delivered in an appropriate excipient and may or may not require delivery in a protective capsule or matrix that resists degradation in the stomach of the individual to the be treated.
  • the bacterial for delivery should be a non-harmful bacterium.
  • the invention could be said to reside in a method of vaccinating a person against SLDS, the method including the step of administering a nucleic acid sequence that encodes a curli protein so that a host cell expresses the curli protein to thereby induce an immune reaction.
  • nucleic acid sequences that encode the amino acid sequences corresponding to the curli protein for example synthesis of oligonucleotides, PCR, transforming cells, constructing vectors, expression systems and the like are well known in the art and standard reference materials may be consulted for specific conditions and procedures (Sambrook et al, eds., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory Press, 1989).
  • the nucleic acid sequence may be any sequence disclosed in United States Patent No. 6,030,805 to Nomark et al., which is incorporated herein specifically for the purpose of providing nucleic acid sequences.
  • the nucleic acid sequence may encode a chimeric protein in which the curli protein is tagged into the protein, wherein the chimeric protein is expressed by a suitable host.
  • a mutagenised gene encoding the cl imeric protein and expressing the protein may be inserted into a suitable DNA vaccine vector such as pcDNA3 which is a mammalian expression vector often used in DNA vaccines.
  • the method may also include the step of assaying for the presence of antibodies to the curli protein to ensure that there is an immune reaction to the expressed protein.
  • the assay for antibodies may be any of the assays discussed with respect to the first and second aspects of this invention.
  • the invention may be said to reside in a composition for vaccinating a vertebrate against SLDS, the composition comprising curli protein, or immunologically active derivative thereof, and a pharmaceutically acceptable carrier.
  • composition may be added to the pharmaceutically acceptable carrier as will be apparent to those skilled in the art and as set out in "Remington's Pharmaceutical Sciences", Sixteenth Edition, Mack Publishing Co, 1980, and include water and other polar substances, including lower molecular weight alkanes, polyalkanols such as ethylene glycol, polyethylene glycol and propylene glycol as well as non-polar carriers.
  • the method of administering the vaccine may vary and could include intravenous, buccal, oral, transdermal and nasal as well as intradermal, intramuscular or subcutaneous administration.
  • Other compounds capable of binding the curli protein may be isolated by screening for binding thereto. For example, a scramble of randomly synthesised compounds could be passed through a solid matrix to which the curli protein is bound. Following washing the strongly binding compounds remain and can be eluted and characterised using standard techniques.
  • the screening may also be a competitive binding screen used to identify compounds that bind the curli protein in preference to a monoclonal antibody specific for the curli protein.
  • the nature of the compounds obtained by screening is not limited and may include, but is not limited to, peptides, oligonucleotides, amino acids, nucleic acids or sugars.
  • the methods used for the binding assay can be any one of the many common techniques known to those skilled in the art. Such methods may include affinity selection chiOmatography, ultrafiltration assays, the scintillation proximity assay, interfacial optical techniques, the quartz crystal microbalance, the jet ring cell, interferometric assays using porous silicon to immobilise the receptor. Reference to such techniques can be found in Woodbury et al. , 1999.
  • a scramble of randomly synthesised oligonucleotides could be passed through a solid matrix to which the curli protein is bound. Following washing the strongly binding oligonucleotides remain and can be eluted under different conditions (salt, pH etc). The sequence can be determined by PCR and tested for inhibition of curli protein.
  • variable region of the respective monoclonal antibody may be cloned and be made part of a hybrid protein with properties appropriate for the therapeutic purposes of the respective agent.
  • the monoclonal antibody may be "humanised” by recombining nucleic acid encoding the variable region of the monoclonal antibody with nucleic acid encoding non- variable regions of human origin in an appropriate expression vector.
  • the invention may also reside in a pharmaceutical preparation including the therapeutic agent, antibody or antibody fragment defined above in a pharmaceutically effective carrier.
  • a pharmaceutical preparation including the therapeutic agent, antibody or antibody fragment defined above in a pharmaceutically effective carrier.
  • the formulation and preparation of any of these pharmaceutical compositions using antibodies, antibody fragments or other compounds is well known to those skilled in the art of pharmaceutical formulation (see e.g. "Remington's Pharmaceutical Sciences", Sixteenth Edition, Mack Publishing Co, 1980).
  • Dot-immunoblots of SIDS and control sera were prepared by spotting 0.5 ⁇ L volumes of serum diluted in native sample buffer (NSB) onto nitocellulose membranes (0.45 ⁇ pore size) (Invitrogen). After fixing in transfer buffer containing 10% methanol and then blocking with 5% skim milk in tris buffered saline (0.05% tween) (TBST), the antigens were incubated overnight at room temperature with gentle rocking with a 1:2000 dilution (in TBST) of affinity-purified rabbit anti-CsgA (ZB-ABI) kindly provided Dr Zhoa Bian, Karolinska Institute, Sweden. Pre-immune rabbit antiserum at the same dilution was used as negative control.
  • coli O4:Hl 1:4096 coliform (unspecified) 1:64, coliform (unspecified) 1:512 and coliform (unspecified) 1:32,768. .
  • Forty-one sera, sterile at autopsy yielded the following respective titres: four (1:4), three (1:8), three (1:16), four (1:32), sixteen (1:64), four (1:128), three (1:256), and four (1:512).
  • Typical contaminants coagulase negative staphylococci, Micrococcus spp.
  • diphtheroids, etc. were found in seventeen heart blood cultures and their respective number and titre were as follows: one (1:4), one (1:8), four (1:16), one (1:32), two (1:64), four (1:512), two (1:4096) and two (1:32,786).
  • Heart blood cultures were not performed in four babies.
  • Age-matched control sera from 61 healthy babies were all negative for curli protein.
  • Fourteen sera obtained from non-SLDS deaths (n 21) tested negative including five with sterile heart blood cultures. Seven of the 21 sera from non-SIDS deaths gave positive results. Of the positive babies, one had a coliform, and one had a Gram positive coccus isolated from heart blood obtained at autopsy.
  • Curli protein is known to be immunogenic through the raising of antibody in rabbits. Given its evident immunogenicity, vaccine development would entail the three standard phases involving 1) immunogenicity studies and challenge/protection studies in an animal model; 2) definition of safety and side-effects in animals, and 3) human trials for safety, tolerability, immunogenicity and protective efficacy.
  • the target population is babies at risk for SLDS (mainly babies 2-4 months old, in lower socioeconomic group, born premature with low birth weight/gestational age, etc.).
  • SLDS babies at risk for SLDS
  • approaches to achieve immunity in this target population includes immunisation of potential mothers, pregnant women, or new born babies. Mothers would convey transplacental antibody in babies of full term gestational age. Premature babies ( ⁇ 34 weeks gestation) may have lower levels of maternally acquired antibody and may therefore have less passive immunity than full term infants. Immunisation of all babies at birth would provide the best opportunity for protection.
  • Diagnosis n mean M:F sex No. with No. coliform No. blood Curlin Reciprocal age (SD) ratio sterile bacteria in culture +ve in dot mean blood blood not immunoblot GMT culture culture done (SD)
  • Pre-natal risk factors for SIDS include intrauterine growth retardation/lower birth weight, multiple births (twin/triplet), shorter gestational age, maternal smoking (independent risk factor), and urinary infections (mainly E. coli) in pregnancy.
  • Neonatal and postneonatal risk factors for SLDS strongly suggest infection; these include illness (mainly respiratory viral [cough] or gastrointestinal [diarrhoea]), listlessness/droopiness, poor health in last week, hospital admissions, GP & outpatient attendances in the weeks prior to death, sweatier than usual (probable fever), decreased feeding (frequency & demand for food) in last days, peak age 2-4 months (corresponding with declining passive maternal immunity), , never breast-fed (but several studies have failed to show bottle feeding to be an independent risk factor), previously used mattress, poor socioeconomic conditions, ethnicity (genetic predisposition/susceptibility), and passive smoking.
  • a second, but most important route of toxin absorption would be via the lymphatic system and the thoracic duct.
  • Toxin would be delivered via the duct to the Innominate vein and thence to the right side of the heart.
  • the first organ exposed to toxin would be the lungs followed by the heart and the thymus. These are the organs in which classical pathological findings in SIDS are seen (petechiae, and wet/heavy lungs). Bacterial toxins/products can perturb basement membranes of small blood vessels leading to the small haemorrhages (petechiae) seen in SIDS.
  • the epidemiological risk factors for SLDS are consistent with an infectious aetiology and exposure to an infectious agent and/or a susceptibility to infection.
  • the toxicity of SLDS sera and the potentiating effects of virus infection and nicotine are consistent with a bacterial toxin causation.
  • Enteric toxigenic bacteria E. coli and/or Cl. perfringens and/or S. aureus
  • E. coli and/or Cl. perfringens and/or S. aureus are good candidates and are found in a large proportion of SLDS cases but to a much lesser extent in living and dead controls.
  • the consistent pathological findings suggest a specific toxin.
  • the prone position may enhance toxin delivery to the systemic circulation and the heart.
  • the above hypothesis also accommodates deaths occurring in the supine and lateral positions reinforcing the point that proneness is not a prerequisite for SIDS. Memory-monitored deaths indicate that toxaemic shock and cardiac standstill are the final events

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Abstract

L'invention concerne un procédé de diagnostic du syndrome de la mort subite du nourrisson (SMSN), qui fait appel à des procédés d'immunologie pour détecter la présence d'une protéine curli ou protéine réactive immuno-croisée ou d'un anticorps dirigé contre une protéine curli dans un échantillon biologique prélevé sur un individu. Le diagnostic du SMSN présente une importante ramification pour l'identification et le traitement d'individus à risque. L'invention concerne en outre un procédé de vaccination contre des organismes véhiculant la toxine curli en vue de produire une immunisation contre ladite toxine et, par conséquent, de réduire l'incidence dudit syndrome.
PCT/AU2002/001085 2001-08-10 2002-08-09 Toxine liee au syndrome de la mort subite du nourrisson, et utilisations associees Ceased WO2003013601A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPR6906A AUPR690601A0 (en) 2001-08-10 2001-08-10 Toxin associated with sudden infant death syndrome, and uses thereof
AUPR6906 2001-08-10

Publications (1)

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WO2003013601A1 true WO2003013601A1 (fr) 2003-02-20

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PCT/AU2002/001085 Ceased WO2003013601A1 (fr) 2001-08-10 2002-08-09 Toxine liee au syndrome de la mort subite du nourrisson, et utilisations associees

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AU (1) AUPR690601A0 (fr)
WO (1) WO2003013601A1 (fr)

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
CN112743395A (zh) * 2019-10-31 2021-05-04 昆山市孚特莱电子设备有限公司 曝光玻璃成品表面修复方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0342173A2 (fr) * 1988-05-06 1989-11-15 Staffan Normark Protéine liant la fibronectine et sa préparation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0342173A2 (fr) * 1988-05-06 1989-11-15 Staffan Normark Protéine liant la fibronectine et sa préparation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BETTELHEIM K. ET AL.: "Toxigenic escherichia coli associated with sudden infant death syndrome", SCAND. J. INFECT. DISEASE, vol. 22, no. 4, 1990, pages 467 - 476 *
BETTIOL S. ET AL.: "Bacterial flora of Tasmanian SIDS infants with special reference to pathogenic strains of escherichia coli", EPIDEMIOLOGY & INFECTION, vol. 112, no. 2, 1994, pages 275 - 284 *

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