WO1994000487A1 - Peptide produisant une immunite protectrice contre le tetanos - Google Patents

Peptide produisant une immunite protectrice contre le tetanos Download PDF

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Publication number
WO1994000487A1
WO1994000487A1 PCT/US1993/006175 US9306175W WO9400487A1 WO 1994000487 A1 WO1994000487 A1 WO 1994000487A1 US 9306175 W US9306175 W US 9306175W WO 9400487 A1 WO9400487 A1 WO 9400487A1
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WO
WIPO (PCT)
Prior art keywords
toxin
peptide
tetanus
immunogen
epitope
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Application number
PCT/US1993/006175
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English (en)
Inventor
Jane L. Halpern
Original Assignee
The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services filed Critical The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services
Priority to AU45453/93A priority Critical patent/AU4545393A/en
Publication of WO1994000487A1 publication Critical patent/WO1994000487A1/fr

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Classifications

    • 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
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • 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
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1282—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Clostridium (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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • Tetanus is an acute infectious disease caused by the anaerobic bacteria Clostridiu tetani .
  • a potent protein neurotoxin elaborated by the bacteria causes the manifestations of the disease.
  • tetanus is characterized by neurological symptoms including convulsions and intermittent muscle spasms.
  • Lockjaw is a common name for the disease.
  • the neurotoxin inhibits neurotransmitter release by an undetermined mechanism of action believed to require at least three steps: 1) binding to a eukaryotic cell receptor, 2) translocation to the cytosol and 3) disruption of the secretory pathway.
  • Tetanus toxin binds to certain gangliosides present on neuronal cells, and some studies indicate that these gangliosides may act as the cellular receptor for tetanus toxin. It has also been proposed that tetanus toxin must interact with gangliosides plus an additional receptor in order for intoxication to proceed.
  • a vaccine based on the tetanus toxin causes the individual to produce antitoxin antibodies and thus provides active immunity.
  • tetanus toxoid causes the individual to produce antitoxin antibodies and thus provides active immunity.
  • -Survival of the patient after an active infection of tetanus does not cause immunity to further episodes of tetanus because the amount of neurotoxin sufficient to cause the disease is less than the amount of neurotoxin necessary to produce active immunity.
  • Anti-tetanus toxin immunoglobulins derived from humans are available to produce passive immunity. Equine tetanus antitoxin is also available, but it is less favored because of a shorter half-life and greater problems with hypersensitivity and serum sickness as compared to the human immunoglobulin.
  • tetanus Active immunization against tetanus, as well a number of other diseases, is routinely recommended for all members of the population. Typical immunization schedules begin with infants. The usual recommendation is four doses of tetanus toxoid vaccine by about 18 months of age with a fifth dose before entering school at about age 4 or 5. Normally, the vaccination is repeated about every 5 to 10 years thereafter depending on the individual's exposure to tetanus.
  • the current tetanus vaccine licensed for human and veterinary use consists of tetanus toxin which has been chemically inactivated and partially purified.
  • the toxin is treated with formaldehyde, but alternatively it may be treated with glutaraldehyde to become atoxic.
  • This product produces long lasting immunity, but is poorly characterized and contains impurities which can cause adverse side effects.
  • the toxin itself is innocuous when it has been treated in a form suitable for use in a vaccine, contaminants or other components of the vaccine may cause adverse reactions. Because the neurotoxin is purified from lysed Clostridium tetani cells, the process is time-consuming and involves some risk due to the production of the bacterial cultures which include a potent toxin.
  • Contaminants either from the lysed cells or from the procedure used to make the toxin atoxic can cause vaccination reactions in the individual patient.
  • the conventional tetanus vaccines must be kept under refrigeration. This causes difficulties in transportation and storage which may be significant problems in underdeveloped regions where the vaccine may be most urgent needed.
  • the overall death rate for tetanus was about 30%. Improved immunization status of the population can help decrease this mortality.
  • Tetanus toxin has 1315 amino acids and is synthesized by C. tetani as a single polypeptide chain that is proteolyzed to yield two fragments, the light chain (LC) derived from the amino terminus, and the heavy chain (HC) derived from the carboxyl terminus.
  • the heavy chain has a molecular weight of about 100,000 and the light chain of about 50,000. The two chains are linked by a single disulfide bond and noncovalent interactions.
  • the heavy chain can be readily proteolyzed by papain and other proteases at one additional site resulting in formation of two fragments: the light chain linked by a disulfide bond to approximately one-half of the heavy chain (L- HC n ) including the amino terminal, and the carboxyl terminal one-half of the heavy chain (H c ) or Fragment C.
  • L- HC n the heavy chain
  • H c carboxyl terminal one-half of the heavy chain
  • Fragment C Each of the three fragments of tetanus toxin appears to represent a functional domain necessary for different steps in intoxication.
  • the LC is responsible for intoxication of target cells, and the H cn fragment has been proposed to translocate tetanus toxin across membranes.
  • the H c fragment, or Fragment C is required for recognition of and binding to target cells. This fragment also retains the ability of intact tetanus toxin to undergo retrograde axonal transport.
  • the invention provides an immunogen against tetanus toxin comprising a peptide including linear tetanus-*-toxin- specific epitopes. These linear epitopes are derived from the carboxy terminus portion of the heavy chain C fragment of the toxin.
  • the immunogen is competent to induce a protective immune response to the toxin in a mammal.
  • the epitope is defined by the amino acid sequence DKILGCDWYFVPTDEGWTND (Seq. I.D. No. 1).
  • a cysteine can be optionally incorporated at the amino terminus for conjugation to a carrier protein.
  • the immunogen is preferably formulated into a vaccine adapted for administration to a mammal, especially a human.
  • the peptide may be conjugated to a carrier protein suitable for use in a vaccine.
  • the peptide is preferably produced by chemical synthesis, it could be produced by other means, such as recombinant techniques.
  • substantially equivalent peptides are included within the scope of the invention.
  • An example of a substantially equivalent peptide is one which is capable of binding to an antibody raised against Seq. I.D. No. 1 and which comprises neither nonlinear epitopes of the tetanus toxin nor epitopes which are tetanus-toxin-specific but not recognized by antisera raised against Seq. I.D. No. 1.
  • Another example is a peptide which induces an antibody competent to bind to the peptide described above and which comprises an antigenic single linear tetanus-toxin-specific epitope derived from the heavy chain C fragment of the toxin, but which does not induce an antibody which recognizes a conformational epitope on the tetanus toxin.
  • the invention includes a first peptide competent to bind to an antibody induced by a second peptide having Seq. I.D. No. 1, wherein the first peptide is not recognized by antibodies which recognize conformational epitopes of tetanus neurotoxin.
  • the invention also provides a variety of antibodies.
  • An example is an antibody competent to bind to a peptide comprising a linear, immunogenic, single tetanus-toxin-specific epitope derived from the heavy chain C fragment of the toxin but which does not induce an antibody which recognizes a conformational epitope on the tetanus toxin.
  • "Immunogenic" in this context means that the peptide is competent to induce a protective immune response to the toxin in a mammal.
  • An example of such a peptide is Seq. I.D. No. 1.
  • An antibody of the invention may be formulated into a therapeutic suitable for administration to a mammal, preferably a human. The antibody may be produced by a variety of procedures including monoclonal techniques.
  • a preferred method includes selecting an immunogen as defined above.
  • the epitope is derived from the heavy chain C fragment of the toxin.
  • the immunogen is competent to induce a protective immune response to the toxin in a mammal to which the immunogen has been administered.
  • the method further includes administering an effective amount of the immunogen to the animal.
  • the peptide of the selected immunogen preferably includes Seq. I.D. No. 1.
  • the vaccine may be administered parenterally, usually subcutaneously or intramuscularly. Additionally, the vaccine can be adapted for oral administration. In such a case, the immunogen of the invention could be a component of a recombinant vaccine.
  • Fig. 1 shows inhibition of labelled Fragment C binding by several antibodies.
  • a preferred embodiment provides an immunogen including a 20 amino acid peptide derived from tetanus toxin which, preferably conjugated to a carrier protein, induces protective immunity against tetanus toxin.
  • This peptide has potential use as a tetanus vaccine or vaccine component.
  • the peptides of the invention are significantly different from previous tetanus vaccines because they are well characterized molecules which are preferably chemically synthesized. Unlike the vaccines in present use, these peptides have potential use in recombinant vaccine vehicles, such as live oral vaccines.
  • a preferred peptide represents the final 20 carboxyl terminal amino acids of tetanus toxin and has the following amino acid sequence:
  • DKILGCDWYFVPTDEGWTND designated Seq. I.D. No. 1.
  • the peptide is conjugated to a cysteine at the amino terminus to facilitate conjugation to a hapten or carrier molecule such has a protein.
  • another preferred peptide is represented by the following amino acid sequence:
  • CDKILGCDWYFVPTDEGWTND designated Seq. I.D. No. 2.
  • the linear epitope(s) of the present invention is Seq. I.D. No. 1. It can be presented to an immune system in a variety of known forms, some which are described below.
  • the immunogen of this invention is directed to those having the epitope of Seq. I.D. No. 1 but not in combination with other native epitopes of the tetanus neurotoxin, especially conformational epitopes.
  • the antisera is absorbed with an excess of BSA conjugated to Seq. I.D. No. 1. Conjugation is done according to Proc . Natl. Acad . Sci . 78:3403-3407 (1981). The absorbed sera is thus used to test the capacity of the immunogens of this invention. Immunogens of this invention will not react with the crossreacted sera. Alternatively, one can use competitive immunoassays to establish whether immunogens carry native neurotoxin epitopes other than those of Seq. I.D. No. 1. A polyclonal antisera is raised against Seq. I.D. No. 1.
  • a second peptide representing a linear sequence of tetanus toxin, which does not comprise a nonlinear epitope of tetanus toxin, that inhibits the binding of this antisera to Seq. I.D. No. 1 in an ELISA or immunoprecipitation reaction is an immunogen of this invention.
  • the peptides of the invention can be synthesized in solid or liquid phase as is known in the art.
  • the peptide can be synthesized at different substitution levels.
  • the synthesis may follow a stepwise format or a coupling approach.
  • the stepwise method includes condensing an amino acid to the terminal amino group sequentially and individually.
  • the coupling, or segment condensation, approach involves coupling fragments divided into several groups to the terminal amino acid.
  • Synthetic methods include azide, chloride, acid anhydride, mixed anhydride, active ester, Woodward reagent K, and carbodiimidazole processes as well as oxidation-reduction and other processes. These processes apply to both solid and liquid phase synthesis.
  • the peptides are synthesized in the solid phase using a commercial polyacrylamide resin as an insoluble carrier.
  • a commercial polyacrylamide resin as an insoluble carrier.
  • the C terminal amino acid is bound to the insoluble carrier at its carboxyl group.
  • insoluble carriers include halo-genomethyl resins such as chloro ethyl-polystyrene-divinylbenezene polymer, bromomethyl resin, hydroxymethyl resins, phenol resins, tert-alkyloxy carbonylhydrazidated resins, and crosslinked poly-N-acrylyl- pyrrolidine resins or other equivalent resin supports.
  • the reactive functional groups in the amino acid side chains are protected using protector groups known in the art. Some examples are the trifluoro-acetyl group, the fluorenyl- methoxycarbonyl group (Fmoc) , butyl-ether and butyl-ester.
  • the protected amino acids are activated individually with symmetrical anhydrides or esters such as those of pentafluorophenol and p-nitrophenol or any other side-chain protective group.
  • a preferred system includes a polyamide resin or acidic resin and fluorenyl-methoxycarbony1 or tert- butyloxycarbonyl (Boc) as an ⁇ -amino group protector (the Fmoc- polyamide system) or equivalent system.
  • the peptide is removed from the resin, generally using an aqueous solution of trifluoroacetic acid (TFA) at room temperature (about 20-25°C) for sufficient time to remove certain of the protector groups, such as the butyl-ether and butyl-ester, but not the trifluoro-acetyl group.
  • TFA trifluoroacetic acid
  • HF hydrofluoric acid
  • TFMSA triflooromethone sulfonic acid
  • Boc Boc
  • the peptides are usually purified by a method such as gel filtration chromatography or high pressure liquid chromatography.
  • a method such as gel filtration chromatography or high pressure liquid chromatography.
  • an antigen can be conjugated to a suitable carrier, usually a protein molecule.
  • a suitable carrier usually a protein molecule.
  • This procedure has several facets. It can allow multiple copies of an antigen, such as a peptide, to be conjugated to a single larger carrier molecule.
  • the carrier may possess properties which facilitate transport, binding, absorption or transfer of the antigen.
  • Suitable carriers are the tetanus toxoid, the diphtheria toxoid, serum albumin and lamprey, or keyhole limpet, hemocyanin because they provide the resultant conjugate with minimum genetic restriction.
  • Conjugates including these universal carriers can function as T cell clone activators in individuals having very different gene sets.
  • the conjugation between a peptide and a carrier can be accomplished using one of the methods known in the art. Specifically, the conjugation can use bifunctional cross- linkers as binding agents as detailed, for example, by Means and Feeney, "A recent review of protein modification techniques" Bioconjugate Chem . 1:2-12 (1990).
  • the immunogen may be combined or mixed with various solutions and other compounds as is known in the art. For example, it may be administered in water, saline or buffered vehicles with or without various adjuvants or immunodiluting agents. Examples of such adjuvants or agents include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate
  • alum beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions, muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium parvu (Propionobacterium acnes) , Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin, liposomes, leva isole, DEAE-dextran, blocked copolymers or other synthetic adjuvants.
  • Such adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Michigan) .
  • Other suitable adjuvants are Amphigen (oil-in-water) , Alhydrogel (aluminum hydroxide) , or a mixture of Amphigen and Alhydrogel. Only aluminum is approved for human use.
  • the proportion of immunogen and adjuvant can be varied over a broad range so long as both are present in effective amounts.
  • aluminum hydroxide can be present in an amount of about 0.5% of the vaccine mixture (A1 2 0 3 basis) .
  • the amount of the immunogen can range from about 5 ⁇ g to about 100 ⁇ g protein per patient.
  • a preferable range is from about 20 ⁇ g to about 40 ⁇ g per dose.
  • a suitable dose size is about 0.5 ml. Accordingly, a dose for intramuscular injection, for example, would comprise 0.5 ml containing 20 ⁇ g of immunogen in admixture with 0.5% aluminum hydroxide.
  • the vaccines are formulated to contain a final concentration of immunogen in the range of from 0.2 to 200 ⁇ g/ml, preferably 5 to 50 ⁇ g/ml, most preferably 15 ⁇ g/ml.
  • the vaccine may be incorporated into a sterile container which is then sealed and stored at a low temperature, for example 4°C, or it may be freeze-dried. Lyophilization permits long-term storage in a stabilized form.
  • the vaccines may be administered by any conventional method for the administration of vaccines including oral and parenteral (e.g., subcutaneous or intramuscular) injection.
  • the treatment may consist of a single dose of vaccine or a plurality of doses over a period of time.
  • the immunogen of the invention can be combined with appropriate doses of compounds including other epitopes of the tetanus toxin.
  • the immunogen could be a component of a recombinant vaccine which could be adaptable for oral administration.
  • Vaccines of the invention may be combined with other vaccines for other diseases to produce multivalent vaccines.
  • a pharmaceutically effective amount of the immunogen can be employed with a pharmaceutically acceptable carrier such as a protein or diluent useful for the vaccination of mammals, particularly humans.
  • Other vaccines may be prepared according to methods well-known to those skilled in the art.
  • the epitopes are typically segments of amino acids which are a small portion of the whole protein.
  • Such derivatives may include peptide fragments, amino acid substitutions, amino acid deletions and amino acid additions within the natural amino acid sequence for the neurotoxin.
  • amino acid residues can be substituted with amino acids of similar size and polarity without an undue effect upon the biological activity of the protein. The following examples are for illustration only and are not intended to limit the invention in any way.
  • Example 1 Synthesis of peptide and KLH-conjugation
  • the published sequence of tetanus toxin (Eisel et al., EMBO Journal [1986] 5:2495-2502) was used to design a peptide with the sequence DKILGCDWYFVPTDEGWTND.
  • the peptide was synthesized on a Applied Biosystems 430A peptide synthesizer according to the manufacturer's instructions. This peptide corresponds to the carboxyl terminal 20 amino acids of tetanus toxin (amino acid residues 1219-1315 of the toxin and residues 438-458 of H c ) prepared in vitro .
  • a cysteine was incorporated at the amino terminus for conjugation to a carrier protein. After synthesis, the peptide was conjugated to
  • KLH Keyhole limpet hemocyanin
  • Sulfo-MBS cross-linker m- maleimidobenzoyl-N-hydroxysulfosuccinimide ester
  • Example 2 Anti-peptide-KLH production
  • the peptide-KLH conjugate was used to immunize rabbits by dissolving about 1 mg total protein in 1 ml of physiological saline, and emulsifying this with 1 ml of complete Freund's adjuvant. This mixture was injected into a rabbit at 4 subcutaneous sites. The rabbit was boosted with the same amount of peptide emulsified with incomplete Freund's adjuvant eight weeks later. Three weeks after boosting, approximately 25 ml of sera were collected from the rabbit via the ear vein.
  • Antibodies reactive with tetanus toxin were affinity purified by passing the sera over a tetanus toxoid-Sepharose affinity column constructed with tetanus toxoid and CNBr- activated Sepharose 4B (Pharmacia-LKB) according to the instructions of the manufacturer. Approximately 2.5 mg of tetanus toxoid was used per ml of activated gel. The affinity-purified antibodies were tested for tetanus toxin neutralizing potency in the mouse neutralization assay. The purified antibody was found to have a potency of 6.4 U/mg protein.
  • a peptide corresponding to amino acids 1219-1315 of tetanus toxin was synthesized as described in Example 1 and coupled to bovine serum albumin (BSA) as described in Example 1 for KLH conjugation.
  • BSA bovine serum albumin
  • a cysteine was incorporated at the amino terminus for conjugation to a carrier protein.
  • the peptide-BSA conjugate was prepared by dissolving 10 mg of bovine serum albumin (BSA) in 1.0 ml of 0.1 M NaH c 0 3 , pH 8.0, and transferring this mixture to a vial containing 5 mg of m-Maleimidobenzoyl-N-hydroxy sulfosuccinimide ester (Sulfo-MBS, Pierce, Rockford, IL) . This mixture was incubated at room temperature for 15 minutes, 10 mg of peptide were added, and the incubation continued, with stirring, for an additional 3 hours. The conjugate was dialyzed against PBS, and stored
  • Example 4 Anti-peptide-BSA antibody production A rabbit was immunized subcutaneously with -100 ⁇ g of peptide-BSA conjugate in complete Freund's adjuvant and boosted 3 and 6 weeks later with the same quantity in incomplete Freund's adjuvant. The rabbit was bled three weeks after the second boost, and antibodies specific for the immunizing peptide were purified by passing the serum over an affinity column as described in Example 2.
  • a polyclonal antibody against the H c fragment was produced and purified using methods similar to Example 4.
  • Monoclonal antibody 18.1.7 (MoAb 1817), which inhibits the binding of tetanus toxin and H c to gangliosides and neuronal cells, was produced as described by Kenimer, J. G. , et al., "Monoclonal antibodies as probes of tetanus toxin structure and function". Infect . Immun . 42:942-948 (1983).
  • Anti-1295-1315 also blocked the lethal effect of tetanus toxin in a mouse protection assay.
  • the anti-peptide antibody had a potency of -6.4U mg purified antibody.
  • a polyclonal antibody against H 0 purified by similar techniques had a potency of ⁇ 20U/mg purified antibody.
  • Anti-1295-1315 inhibited the binding of [ 125 I]-
  • Asp Lys lie Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly 1 5 10 15
  • MOLECULE TYPE DNA (genomic)
  • Cys Asp Lys lie Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu 1 5 10 15

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  • Genetics & Genomics (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

L'invention concerne un immunogène contre la toxine du tétanos comprenant un peptide possédant un déterminant antigénique unique, linéaire et spécifique à la toxine du tétanos. Le déterminant antigénique est dérivé du fragment C de chaîne lourde de la toxine. Dans un mode de réalisation préféré, l'immunogène comprend les 20 derniers acides aminés de la toxine, y compris le terminateur carboxy. Des anticorps, y compris des anticorps antipeptides, sont également décrits, ainsi que des procédés de vaccination.
PCT/US1993/006175 1992-06-30 1993-06-29 Peptide produisant une immunite protectrice contre le tetanos WO1994000487A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU45453/93A AU4545393A (en) 1992-06-30 1993-06-29 Peptide which produces protective immunity against tetanus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US90684192A 1992-06-30 1992-06-30
US07/906,841 1992-06-30

Publications (1)

Publication Number Publication Date
WO1994000487A1 true WO1994000487A1 (fr) 1994-01-06

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995032738A1 (fr) * 1994-05-31 1995-12-07 Allergan, Inc. Modification de toxines de clostridium utilisees comme proteines de transport
WO2001000839A1 (fr) * 1999-06-25 2001-01-04 Imperial College Of Science, Technology And Medicine Polypeptides de la toxine tetanique
US6372225B1 (en) 1996-03-23 2002-04-16 The Research Foundation Of Microbial Diseases Of Osaka University Tetanus toxin functional fragment antigen and tetanus vaccine
US7628992B1 (en) 1994-05-31 2009-12-08 Invent Dcu Limited Modification of clostridial toxins for use as transport proteins

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0209281A1 (fr) * 1985-06-28 1987-01-21 The Wellcome Foundation Limited Antigène cloné
EP0378881A1 (fr) * 1989-01-17 1990-07-25 ENIRICERCHE S.p.A. Peptides synthétiques et leur utilisation comme support universel pour la préparation de conjugués immunogènes convenant au développement de vaccins synthétiques
WO1990015871A1 (fr) * 1989-06-20 1990-12-27 The Wellcome Foundation Limited Expression du fragment c de toxine tetanique
EP0430645A2 (fr) * 1989-11-28 1991-06-05 The Wellcome Foundation Limited Vaccins

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0209281A1 (fr) * 1985-06-28 1987-01-21 The Wellcome Foundation Limited Antigène cloné
EP0378881A1 (fr) * 1989-01-17 1990-07-25 ENIRICERCHE S.p.A. Peptides synthétiques et leur utilisation comme support universel pour la préparation de conjugués immunogènes convenant au développement de vaccins synthétiques
WO1990015871A1 (fr) * 1989-06-20 1990-12-27 The Wellcome Foundation Limited Expression du fragment c de toxine tetanique
EP0430645A2 (fr) * 1989-11-28 1991-06-05 The Wellcome Foundation Limited Vaccins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 113 Columbus, Ohio, US; STIBITZ, SCOTT ET AL. 'Cloning and expression of DNA encoding tetanus-toxin fragment C' *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995032738A1 (fr) * 1994-05-31 1995-12-07 Allergan, Inc. Modification de toxines de clostridium utilisees comme proteines de transport
US6203794B1 (en) * 1994-05-31 2001-03-20 Allergan Sales, Inc. Modification of clostridial toxins for use as transport proteins
US7628992B1 (en) 1994-05-31 2009-12-08 Invent Dcu Limited Modification of clostridial toxins for use as transport proteins
US6372225B1 (en) 1996-03-23 2002-04-16 The Research Foundation Of Microbial Diseases Of Osaka University Tetanus toxin functional fragment antigen and tetanus vaccine
WO2001000839A1 (fr) * 1999-06-25 2001-01-04 Imperial College Of Science, Technology And Medicine Polypeptides de la toxine tetanique

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