EP1000155A1 - Fragments immunogenes de toxine a de clostridium difficile - Google Patents

Fragments immunogenes de toxine a de clostridium difficile

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Publication number
EP1000155A1
EP1000155A1 EP98930905A EP98930905A EP1000155A1 EP 1000155 A1 EP1000155 A1 EP 1000155A1 EP 98930905 A EP98930905 A EP 98930905A EP 98930905 A EP98930905 A EP 98930905A EP 1000155 A1 EP1000155 A1 EP 1000155A1
Authority
EP
European Patent Office
Prior art keywords
toxin
molecule
die
difficile
sequence
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP98930905A
Other languages
German (de)
English (en)
Inventor
Stephen John Queen Mary & Westfield College WARD
Brendan William Queen Mary & Westfield Col WREN
Gordon Imperial College of Science DOUGAN
Gill-Imperial College of Science DOUCE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Imperial College of Science Technology and Medicine
Queen Mary University of London
Original Assignee
Imperial College of Science Technology and Medicine
Queen Mary and Westfiled College University of London
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Filing date
Publication date
Priority claimed from GBGB9713146.0A external-priority patent/GB9713146D0/en
Priority claimed from GBGB9800321.3A external-priority patent/GB9800321D0/en
Application filed by Imperial College of Science Technology and Medicine, Queen Mary and Westfiled College University of London filed Critical Imperial College of Science Technology and Medicine
Publication of EP1000155A1 publication Critical patent/EP1000155A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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)
    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to immunogenic fragments of Toxin A of
  • Clost ⁇ dium difficile methods for their preparation and their use as vaccines.
  • Clost ⁇ dium difficile is an important gastrointestinal pathogen causing infection in both hospitals and tertiary care centres. Disease generally occurs when the gastrointestinal flora of individuals is disrupted through the use of broad- spectrum antibiotics such as cephalosporins and ampicillin, which subsequently allows C. difficile to flourish within the intestine. The organism can form spores and contaminate the hands and clothing of health-care personnel quite readily, thereby posing a significant health care problem C. difficile causes a range of antibiotic associated disease, with symptoms ranging from mild diarrhoea to pseudomembraneous colitis which can be life threatening. Common pathological features of the disease include fluid accumulation, inflammation and necrosis of gastrointestinal mucosa. These pathological symptoms have been shown to be caused by two large proteinaceous toxins produced by C. difficile, termed toxin A and toxin B.
  • Toxin A is slightly larger than toxin B having molecular weights of 308 kDa and 270 kDa respectively.
  • Studies carried out in various animal models of C. difficile disease have shown toxin A to be the primary mediator of tissue damage within the intestine. Since toxin B has been shown to be an extremely potent cytotoxin against several cell lines in vitro, it is believed that toxin B acts after this initial toxin A-mediated damage, thus exacerbating the mucosal tissue damage.
  • toxin A and B A striking feature of both toxin A and B is the repetitive nature of the amino acid sequences located at the carboxyl terminus of the protein.
  • toxin A there are 38 tandem repeat sequences which are classified into class I or class II repeats based on their size.
  • the class II repeats are further subdivided into 4 groups on sequence homology. Work carried out with a recombinant peptide coding for 33 of the repeats showed the receptor-binding domain of the toxin to be located within this repeat region. Antiserum raised against this region was found to be able to neutralise the cytotoxic activity of toxin A (Lyerly et al Curr. Microbiol. 21 29-33 (1990)).
  • toxin A has been shown to be immunogenic in both animals and humans, the vast majority of animal vaccine studies performed to date have used parenteral routes of immunisation which generate a systemic anti-toxin response (Lyerly et al Curr. Microbiol. 21 29-33 (1990)). Subsequently, when these animals are challenged with intact C. difficile, only partial levels of protection are seen since toxin-mediated damage is allowed to occur at the gastrointestinal mucosa due to the absence of a relevant anti-toxin response initiated by the mucosal immune system. The majority of C. difficile vaccine studies have utilised chemically detoxified whole toxin as the immunogen.
  • toxin A has been shown to be an ideal vaccine candidate due to its immunogenicity and non-toxicity.
  • the protein encoding the entire repeat region is relatively large and therefore would be expected to be problematic with regard to retaining its structural integrity within recombinant expression systems. There is therefore a need to achieve a compromise between the requirement for immunogemcity and structural integrity of a protein to be used as pan of a vaccine.
  • International PCT Application WO96/12802 discloses fusion proteins comprising the C-te ⁇ ninus repeats of C. difficile.
  • constructs are shown to be capable of generating anti-toxin A antibodies upon administration to Syrian golden hamsters.
  • the whole C-terminal region i.e. comprising at least 36 repeats is described as necessary in order to efficiently prepare an agent against C. difficile. Sub-fragments of this region were shown not to be suitable as they were insoluble, unstable or failed to generate a suitable response.
  • the N-terminal part was shown to be the least essential and the C-terminal part as critical. There was no disclosure of a C-terminal sub- fragment that could efficiently generate antibodies against C. difficile toxin A.
  • a) comprises an amino acid sequence as shown in Figure 6 ;
  • b) has one or more amino acid substitutions, deletions or insertions relative to a sequence as defined in a) above;
  • d) comprises a multiple of a sequence as defined in a), b) or c); or
  • Molecules of the present invention may be in any appropriate form. They may be proteins, polypeptides, or peptides and may be fused to other moieties. As will be described below, the amino acid sequence shown in Figure 6 (SEQ ID No. 1 ) comprises 14 repeats from the C-terminal region of C. difficile toxin A. This sequence has been demonstrated to be superior to the whole C-terminal repeat region and other sub-fragments thereof in generating immunity to C. difficile.
  • the molecules of the present invention may be provided in substantially pure form.
  • a molecule of the present invention may be provided in a composition in which it is the predominant component present (i.e. it is present
  • a molecule within the scope of a) may consist of the pa ⁇ icular amino acid sequence given in Figure 6 , or may have an additional N- terminal and/or an additional C-terminal amino acid sequence.
  • Additional N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art. Additional sequences may be provided in order to alter the characteristics of a pa ⁇ icular polypeptide. This can be useful in improving expression or regulation of expression in pa ⁇ icular expression systems. For example, an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone Somatostatin by fusing it at its N-terminus to pan of the ⁇ -galactosidase enzyme (Itakwa et al., Science 198: 105-63 (1977)).
  • fusion protein may be provided in which a polypeptide is linked to a moiety capable of being isolated by affinity chromatography.
  • the moiety may be an antigen or an epitope and the affinity column may comprise immobilised antibodies or immobilised antibody fragments which bind to said antigen or epitope (desirably with a high degree of specificity).
  • the fusion protein can usually be eluted from the column by addition of an appropriate buffer.
  • the molecules of the present invention may be formulated as vaccines. Fusion proteins comprising these molecules can be prepared with immunogenic peptides from other sources.
  • An example of such a fusion protein comprises a molecule of the present invention and tetanus toxin, suitably the immunogenic fragment C of tetanus toxin (Khan et al PNAS USA 91 11261-11265 (1994)).
  • Other fusion proteins may comprise immunogenic peptides commonly used in vaccines against Haemophilus influenzae, Helicobacter pylori, diphtheria, cholera, whooping-cough or typhoid.
  • N-terminal or C-terminal sequences may, however, be present simply as a result of a pa ⁇ icular technique used to obtain a molecule of the present invention and need not provide any pa ⁇ icular advantageous characteristic to the molecule of the present invention. Such molecules are within the scope of the present invention.
  • the resultant molecule has at least a substantial propo ⁇ ion of the immunogenic activity of the molecules having the amino acid sequence shown in Figure 6.
  • the term "at least a substantial propo ⁇ ion of activity” when used herein means at least 50% of the activity of a given molecule (preferably at least 75% of said activity, more preferably at least 90% of said activity, and most preferably the same level of activity or a greater level of activity).
  • An example of a variant of the present invention is a molecule as defined in a) above, apa ⁇ from the substitution of one or more amino acids with one or more other amino acids.
  • the skilled person is aware that various amino acids have similar prope ⁇ ies.
  • One or more such amino acids of a molecule can often be substituted by one or more other such amino acids without eliminating a desired activirv of that molecule.
  • amino acids glycine, alanine, vaiine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains).
  • amino acids having aliphatic side chains amino acids having aliphatic side chains.
  • glycine and alanine are used to substimte for one another (since they have relatively sho ⁇ side chains) and that vaiine, leucine and isoleucine are used to substitute for one anodier (since they have larger aliphatic side chains which are hydrophobic).
  • amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains). Substitutions of this nature are often refe ⁇ ed to as "conservative" or "semi-conservative" amino acid substitutions.
  • Amino acid deletions or inse ⁇ ions may also be made relative to the amino acid sequence given in a) above.
  • amino acids which do not have a substantial effect on the activity of the polypeptide. or at least which do not eliminate such activity may be deleted.
  • Such deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced, e.g. dosage levels can be reduced.
  • Amino acid inse ⁇ ions relative to the sequence given in a) above can also be made. This may be done to alter the prope ⁇ ies of a molecule of the present invention (e.g. to assist in identification, purification or expression, as explained above in relation to fusion proteins). Amino acid changes relative to die sequence given in a) above can be made using any suitable technique e.g. by using site-directed mutagenesis.
  • amino acid substitutions or inse ⁇ ions within die scope of the present invention can be made using naturally occurring or non-natural amino acids, bom D- and L-amino acids.
  • Feature c) of the present invention therefore covers fragments of polypeptides a) or b) above, provided that such fragments have immunogenic activity. It is preferable that the fragments retain the N-terminal portion of SEQ ID No . 1.
  • the number of sequences is suitably enough to generate an immune reaction.
  • the number of multiples may be in the range of 1 to 10, generally 1 to 5 and preferably 2 to 4.
  • Preferred fragments are at least 10 amino acids long. They may be at least 20, at least 50 or at least 100 amino acids long.
  • the present invention includes molecules substantially homologous to those defined above.. Whatever amino acid changes are made (whether by means of substitution, inse ⁇ ion or deletion), preferred polypeptides of the present invention have at least 50% sequence identity with a polypeptide as defined in a) above more preferably the degree of sequence identity is at least 70% , 75 % or
  • the degree of amino acid sequence identity can be calculated using a program such as "bestfit” (Smith and Waterman, Advances in Applied Mathematics, 482- 489 (1981)) to find the best segment of similarity between any two sequences.
  • the alignment is based on maximising the score achieved using a matrix of amino acid similarities, such as that described by Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O.. Ed pp 353-358.
  • amino acid sequence where high degrees of sequence identity are present there will be relatively few differences in amino acid sequence. Thus for example they may be less than 20, less than 10, or even less than 5 differences.
  • Molecules in accordance with the present invention are also capable of eliciting an immune response in a mammal.
  • the immune response may be humoral (including cell-mediated) or innate, and found at bod systemic and mucosal sites (Immunology ed. Roitt et al, Gower Medical Publishing, London, Fifth edition, (1997)).
  • the animal may be a mammal, suitably a human or a non- human mammal, including dogs, cats, cows or bulls, sheep, horses, rabbits, llamas, rats or mice.
  • the animal may be a bird species, such as poultry, including chickens or turkeys.
  • a molecule as previously defined in the preparation of an agent for the prophylaxis or treatment of a C. difficile infection.
  • Therapeutic molecules of the present invention may be used in the treatment of a human or non-human animal suffering from infection with C. difficile.
  • the treatment may be prophylactic or may be in respect of an existing condition.
  • the molecules of the present invention may also be used in the manufacture of a medicament for the treatment of C. difficile infection. Where the medicament is to be used as a prophylactic, it may be conveniently formulated as a vaccine using vaccine preparations known in the a ⁇ .
  • Formulations for use as vaccines may be prepared in conjunction with several different adjuvants or live delivery vectors, e.g. attenuated live vectors, preferably rationally attenuated live vectors.
  • attenuated live vectors e.g. attenuated live vectors, preferably rationally attenuated live vectors.
  • Several bacterial toxins such as cholera toxin (CT) from Vibrio cholerae and heat-labile toxin from Escherichia coli have been shown to be efficient adjuvants when administered in small amounts at mucosal surfaces (Nedrud et al J. Immunol. 139 3484 (1987)),
  • the adjuvant is a heat-labile E-coh toxin.
  • Various antigens have also been delivered to die immune system encapsulated within biodegradable microspheres (Eldridge et al Curr. Top. Microbiol. Immunol. 146 59 (1989)) and phospholipid vesicles or liposomes (Ward et al Micro. Path. 21 499-512 (1996)). Rationally attenuated live vectors have been shown to be efficient at inducing protective immune responses against heterologous antigens expressed within the attenuated cell.
  • a suitable rationally attenuated live vector is Salmonella typhimurium BRD509 ( roA ⁇ roD) (Strugnell et al Infect Immun. 60 3994-4002 (1992)), BRD915 (Johnson et al Mol. Microb. 5 401-407 (1991)) or S. typhimurium BRD916 (btrA) (Tackett et al Infect Immun. 65 452- 456 ( 1 97)). It is preferable that the vector used is a Salmonella.
  • — — — such vectors also extends to the preparation of multivalent vaccines, for example, the single-dose tetanus vaccine (Chatfield et al Bio/Tech 10 888-892 (1992)).
  • the inclusion of more than one heterologous antigen within die same strain allows this principle to be extended even further (Khan et al Proc. Natl. Acad. Sci. USA 91 11261-11265 (1994)) and the recent development of safer attenuated S. ⁇ yphi strains means that this approach can be used in human trials (Tackett et al Infect. Immun. 65 452-456 (1997)).
  • This aspect of the present invention therefore extends to a vaccine comprising a fusion polypeptide based
  • a vaccine may be effective against one or more of C. difficile.
  • Clostridium tetani (tetanus), Salmonella typhi (typhoid).
  • the medicament will usually be supplied as pan of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient).
  • unit dosage form will generally be provided in a sealed container and may be provided as pa ⁇ of a kit.
  • a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.
  • the pharmaceutical composition may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), intragastric, vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route.
  • Such compositions may be prepared by any method known in die a ⁇ of phannacy, for example by admixing the active ingredient with the ca ⁇ ier(s) or excipient(s) under sterile conditions.
  • the molecules of the present invention need not be exclusively expressed by an attenuated Salmonella vector system and may be administered directly with a suitable adjuvant.
  • the molecules may be admixed with CT, LT or detoxified derivatives of these adjuvants (Douce et al Proc. Natl. Acad. Sci. USA 92 1644-1648 (1995)) and inoculated by any desired route described above. Inoculation via intranasal, vaginal or rectal routes may be conveniently employed to promote a mucosal immune response.
  • the molecules of the present invention can be admixed with Freund's adjuvant and injected subcutaneously, intraperitoneally, intramuscularly, intravenously or intradermally.
  • compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules: as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions).
  • Suitable excipients for tablets or hard gelatine capsules include lactose, maize starch or derivatives thereof, stearic acid or salts thereof.
  • Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.
  • excipients which may be used include for example water, polyols and sugars.
  • suspensions oils e.g. vegetable oils
  • oil-in-water or water in oil suspensions may be used.
  • compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3 (6) 318 (1986).
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • die compositions are preferably applied as a topical ointment or cream.
  • the active ingredient may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
  • Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
  • compositions adapted for rectal administration may be presented as suppositories or enemas.
  • compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a pa ⁇ icle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation dirough the nasal passage from a container of the powder held close up to the nose.
  • Suitable compositions wherein die carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.
  • compositions adapted for administration by inhalation include fine pa ⁇ icle dusts or mists which may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.
  • compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example.
  • compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use.
  • sterile liquid carried, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers. sweeteners, colourants, odourants, salts (molecules of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to d e molecule of die present invention.
  • molecules of the present invention can be used in diagnosis.
  • diagnosis in the diagnosis of C. difficile infection in a biological sample from an affected subject.
  • kit for the diagnosis of a C. difficile infection the kit comprising a molecule as defined above.
  • the method of diagnosis may comprise the use of a molecule of the present invention in an assay for the detection of circulating antibodies in an infected subject.
  • the means for detection may comprise the use of ELISA, fluorescence based or radioimmuno assay (RIA) techniques and die assay can be performed in a suitable biological sample, including blood, saliva, tears, urine, faeces, sweat, semen or milk.
  • RIA radioimmuno assay
  • the present invention therefore includes antibodies which bind to a molecule of die present invention.
  • Preferred antibodies bind specifically to molecules of the present invention so iat they can be used to purify such molecules.
  • the antibodies may be monoclonal or polyclonal.
  • Polyclonal antibodies can be raised by stimulating their production in a suitable animal host (e.g. a mouse, rat, guinea pig. rabbit, sheep, goat or monkey) when a molecule of the present invention is injected into the animal. If necessary an adjuvant may be administered together with a molecule of the present invention.
  • the antibodies can then be purified by virtue of their binding to a molecule of the present invention.
  • Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing myeloma cells and spleen cells which produce die desired antibody in order to form an immortal cell line. This is the well known Kohler & Milstein technique (Nature 256 52-55 (1975)).
  • the present invention includes derivatives thereof which are capable of binding to molecules of me present invention.
  • d e present invention includes antibody fragments and synthetic constructs.
  • Antibody fragments include, for example, Fab, F(ab') 2 and Fv fragments (see Roitt et al [supra]). Fv fragments can be modified to produce a syndietic construct known as a single chain Fv
  • (scFv) molecule This includes a peptide linker covalently joining V h and V, regions which contribute to the stability of the molecule.
  • CDR peptides include CDR peptides. These are synthetic peptides comprising antigen binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings which mimic the structure of a CDR loop and which include antigen-interactive side chains.
  • Syndietic constructs include chimaeric molecules.
  • humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention.
  • An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions.
  • Synthetic constructs also include molecules comprising a covalently linked moiety which provides the molecule widi some desirable property in addition to antigen binding.
  • the moiety may be a label (e.g. a fluorescent or radioactive label) or a pharmaceutically active agent.
  • the antibodies or derivatives thereof of the present invention have a wide variety of uses. They can be used in purification and/or identification of the molecules of the present invention. Thus diey may be used in diagnosis of a C. difficile infection in a biological sample from an affected subject. They can be provided in d e form of a kit for screening for the molecules of ie present invention.
  • iere is provided the use of an antibody as previously defined in the preparation of an agent for the prophylaxis or treatment of a C. difficile infection.
  • a mediod of diagnosing a Clostridium difficile infection comprising me step of contacting an optionally labelled antibody as previously defined with a biological sample.
  • the biological sample may be blood, saliva, tears, urine, faeces, sweat, semen or milk.
  • the method can also be used on samples of foodsmffs or other samples taken from the environment such as water to determine d e presence or otherwise of C. difficile.
  • a recombinant DNA construct comprising nucleotides
  • Suitable recombinant DNA constructs comprising such a sequence may be introduced into host cells to enable the expression of molecules of the present inventions using techniques known to the person skilled in die a ⁇ .
  • suitable vectors are pRSET-A or pTECH-1.
  • Recombinant DNA constructs prepared in accordance with the present invention include p5/6. The fragment expressed by p5/6 comprises 14 C-terminal toxin A repeats.
  • Molecules comprising amino acid sequences in accordance with the present application can be syndiesised by any convenient technique.
  • Peptides and polypeptides may be syndiesised by chemical routes using routine procedures.
  • the molecules may be synthesised by expressing a nucleotide sequence encoding d e peptides in a suitable host cell.
  • diere is provided a method for me preparation of a fragment of C. difficile Toxin A, comprising the step of expressing a nucleotide sequence encoding a molecule as previously defined.
  • d e nucleotide sequence will be incorporated into a vector appropriate for expression in the desired host cell.
  • Suitable expression systems include but are not limited to prokaryotes such as E. coli, S. typhi, or S. typhimurium, preferably an attenuated host cell.
  • expression may be in suitable eukaryotic systems including yeast such as S. cerevisiae, S. pombe, insect cells, plant cells such as Arabidopsis thaliana or tobacco, or mammalian cells such as COS, CHO, Vero or HeLa.
  • yeast such as S. cerevisiae, S. pombe
  • insect cells such as Arabidopsis thaliana or tobacco
  • mammalian cells such as COS, CHO, Vero or HeLa.
  • the choice of vector and appropriate promoter and regulatory sequences will generally depend on the expression system being used at d e time.
  • molecules of the present invention may be expressed in glycosylated or non-glycosvlated form.
  • Non-glycosvlated forms can be produced by expression in pro kary otic hosts, such as E. coli.
  • Polypeptides comprising N-terminal methionine may be produced using certain expression systems, whilst in odiers the mature polypeptide will lack this residue.
  • Preferred techniques for cloning, expressing and purifying a molecule of the present invention are summarised below.
  • nucleic acid molecules coding for molecules according to the present invention refe ⁇ ed to herein as "coding" nucleic acid molecules die present invention also includes nucleic acid molecules complementary thereto.
  • nucleic acid molecules complementary thereto For example, both strands of a double stranded nucleic acid molecule are included widiin the scope of ie present invention (whedier or not they are associated with one anodier).
  • mRNA molecules and complementary DNA molecules e.g. cDNA molecules.
  • nucleic acid molecules which can hybridise to any of the nucleic acid molecules discussed above are also covered by d e present invention. Such nucleic acid molecules are refe ⁇ ed to herein as "hybridising" nucleic acid molecules. Hybridising nucleic acid molecules can be useful as probes or primers, for example. Desirably such hybridising molecules are at least 10 nucleotides in length and preferably are at least 25 or at least 50 nucleotides in length. The hybridising nucleic acid molecules preferably hybridise to nucleic acids within the scope of a) or b) above specifically.
  • hybridising molecules will hybridise to such molecules under stringent hybridisation conditions.
  • stringent hybridisation conditions is where attempted hybridisation is carried out at a temperature of from about 35°C to about 65°C using a salt solution which is about 0.9 molar.
  • the skilled person will be able to vary such conditions as appropriate in order to take into account variables such as probe length, base composition, type of ions present, etc.
  • a hybridising nucleic acid molecule of die present invention may have a high degree of sequence identity along its lengdi with a nucleic acid molecule widiin the scope of a) or b) above (e.g. at least 50%, at least 75% or at least 90% sequence identity).
  • sequence identity e.g. at least 50%, at least 75% or at least 90% sequence identity.
  • nucleic acid molecules of the present invention may have one or more of the following characteristics:
  • a vaccine formulation comprising a recombinant DNA construct as previously defined, optionally togedier with one or more carriers or adjuvants.
  • Such vaccine formulations may also be employed in accordance with d e uses or methods described above.
  • the recombinant DNA construct may be suitably prepared using pRc/CMV (Invitrogen) for vaccines in accordance with this aspect of the invention.
  • DNA vaccines may be administered by any convenient route as described above but administration by subcutaneous or intramuscular injection may be prefe ⁇ ed (Wolff et al Science 247 1465 (1990)).
  • the molecules described above are capable, when co-expressed as a fusion protein or administered in a mixture together with a second peptide or protein, of increasing the antibody response to the co- administered peptide or protein.
  • a further embodiment of the preferred invention relates to the use of the molecules described above as an adjuvant in the administrations of peptides or proteins.
  • Prefe ⁇ ed aspects of the second and subsequent aspects of die present invention are as for the first aspect mutatis mutandis.
  • FIGURE 1 shows a map of Toxin A specific fragments amplified using PCR.
  • FIGURE 2 shows an SDS-PAGE analysis of attenuated S. typhimurium expressing Toxin A fragments from whole cell lysates expressing toxin A constructs under aerobic and anaerobic conditions.
  • FIGURE 3 shows ELISA titres of toxin specific antibody in the serum of BALB/c mice immunised intragastricallv with 2 doses of S. typhimurium
  • FIGURE 4 shows binding activity of Toxin fragments as measured by agglutination of rabbit erythrocytes.
  • FIGURE 5 shows the predicted amino acid sequence of PCR product fragment pTA2 resulting from expression of nucleotides 5983-6594 of the sequence of the Toxin A gene from C. difficile strain VPI 10463 (Dove et al Infection and Immunity 58 (2) 480-488 (1990)).
  • FIGURE 6 shows die predicted amino acid sequence of PCR product fragment p5/6 resulting from expression of nucleotides 7159-8118 of the sequence of the Toxin A gene from C. difficile strain VPI 10463 (Dove et al (1990)).
  • FIGURE 7 shows die predicted amino acid sequence of PCR product fragment p5/7 resulting from expression of nucleotides 6748-8118 of the sequence of the Toxin A gene from C. difficile strain VPI 10463 (Dove et al (1990)).
  • FIGURE 8 shows the predicted amino acid sequence of PCR product fragment p9/10 resulting from expression of nucleotides 5530-8115 of the sequence of the Toxin A gene from C. difficile strain VPI 10463 (Dove et ⁇ / (1990)).
  • FIGURE 9 shows the predicted N-terminal sequence of PCR product fragment p9/10 which was not encoded widiin vector pTECH-1.
  • FIGURE 10 shows the mean ELISA titres of toxin A specific total antibody found in serum of BALB/c mice immunised intragastrically with S. typhimurium BRD509 expressing 14 C. difficile toxin A repeats.
  • Samples were harvested before immunisation (day 0), 28 days after first dose (day 28), 35 days after the second dose (day 63), and 22 days after a subcutaneous boost witii 0.5 ⁇ g of purified p5/6 fragment C fusion protein (day 85).
  • E ⁇ or bars represent the standard e ⁇ or of the mean. Detection limit 1:50.
  • FIGURE 11 shows the mean ELISA titres of tetanus toxin-specific total antibody found in serum of BALB/c mice immunised intragastrically with S. typhimurium BRD509 expressing 14 C. difficile toxin A repeats. Samples were harvested before immunisation (day 0), 28 days after first dose (day 28), 35 days after the second dose (day 63), and 22 days after a subcutaneous boost with 0.5 ⁇ g of purified p5/6 fragment C fusion protein (day 85). E ⁇ or bars represent the standard e ⁇ or of the mean. Detection limit 1:50.
  • FIGURE 12 shows the mean ELISA titres of toxin A-specific IgA antibody found in gastric lavage samples of BALB/c mice immunised intragastrically with S. typhimurium BRD509 expressing 14 C. difficile toxin A repeats. Samples were harvested before immunisation (day 0), 28 days after first dose (day 28), and 22 days after a subcutaneous boost with 0.5 ⁇ g of purified p5/6-fragment C fusion protein (day 85). E ⁇ or bars represent the standard error of the mean. Detection limit 1:2.
  • FIGURE 13 shows die mean ELISA titres of tetanus toxin-specific IgA antibody found in gastric lavage samples of BALB/c mice immunised intragastrically with S. typhimurium BRD509 expressing 14 C. difficile toxin A repeats. Samples were harvested before immunisation (day 0), 28 days after first dose (day 28), and 22 days after a subcutaneous boost witii 0.5 ⁇ g of purified p5/6-fragment C fusion protein (day 85). E ⁇ or bars represent the standard error of the mean. Detection limit 1:2.
  • FIGURE 14 shows a plasmid map of vector pTECH-1.
  • FIGURE 15 shows a plasmid map of vector pRSET-A (Invitrogen) .
  • FIG. 16 SDS-PAGE analysis (10% polyacrylamide) of thyroglobulin affinity purified p56HIS (A) and p56TETC (B). Lane 1, cell lysates before affinity column; Lane 2, cell lysates after affinity column; Lane 3, purified protein collected from column (approximately 3 ⁇ g). Apparent molecular masses are shown in kDa. Pre-stained molecular weight markers are also shown (M).
  • FIG. 17. Immunoblot of affinity purified p56HIS (Lane 1) and p56TETC (Lane 2) against the toxin A specific monoclonal antibody PCG-4 (A), or anti-TT polyclonal antiserum (B). Apparent molecular weights are shown in kDa. Molecular weight markers are shown (M).
  • FIG. 18 Mean anti-toxin A total immunoglobulin responses in the serum of intransal immunised BALB/c mice. Antibody titres were measured by ELISA in serum taken after 1 dose (day 19), 2 doses (day 34) and 3 doses (day 47) of antigen. Mean titres are shown ⁇ SD from five mice. Individual pre-immune titres have been subtracted from each co ⁇ esponding mouse.
  • FIG. 19 Mucosal IgA responses against toxin A after 3 intranasal doses of antigen in nasal and pulmonary lavage. Responses show the variation in die immune responses between individual mice in each group. Bars represent mean antibody titres.
  • FIG. 20 Mean anti-TT total immunoglobulin responses in the serum of intransal immunised BALB/c mice. Antibody titres were measured by ELISA in serum taken after 1 dose (day 19), 2 doses (day 34) and 3 doses (day 47) of antigen. Mean titres are shown ⁇ SD from five mice. Individual pre-immune titres have been subtracted from each co ⁇ esponding mouse.
  • FIG. 21 Mucosal IgA responses against tetanus toxin after 3 intranasal doses of antigen in nasal and pulmonary lavage. Responses show the variation in the immune responses between individual mice in each group. Bars represent mean antibody titres.
  • Example 1 Amino acid composition of toxin A fragments The primer sequences used to generate die toxin A fragments were based on d e entire sequence of toxin A from C. difficile strain VPI 10463 published by Dove et al, 1990 Infection and Immunity, vol. 58(2), page 480-488. The predicted amino acid sequences shown in Figures 5, 6, 7, 8 and 9 are also from Dove et al, 1990.
  • die vector pTECH-1 was obtained from Medeva, Leatherhead.
  • fragments pTA2 8 repeats
  • p5/6 14 repeats
  • p5/7 20 repeats
  • the sequences shown for fragments pTA2 8 repeats
  • p5/6 14 repeats
  • p5/7 20 repeats
  • the sequences shown for fragments pTA2 8 repeats
  • p5/6 14 repeats
  • p5/7 20 repeats
  • the sequences shown for fragments pTA2 8 repeats
  • p5/6 14 repeats
  • p5/7 20 repeats
  • This PCR product contained the nucleotides 5983-6594 inclusively.
  • the predicted amino acid sequence encoded by this fragment is as shown in Figure 5.
  • This PCR product contained the nucleotides 7159-8118 inclusively.
  • the predicted amino acid sequence encoded by this fragment is as shown in Figure 6.
  • This PCR product contained die nucleotides 6748-8118 inclusive.
  • the predicted amino acid sequence encoded by tiiis fragment is as shown in Figure 7.
  • This PCR product contained me nucleotides 5530-8115 inclusive.
  • the predicted amino acid sequence encoded by tiiis fragment is as shown in Figure 8.
  • the fragment encoded widiin the pTECH-1 vector lacked the following sequence from the N-terminus as shown in Figure 9.
  • Example 2 Expression of toxin A fragments in E. coli utilising the polymerase chain reaction (PCR), four overlapping DNA fragments which spanned the entire C-terminal repeat region of toxin A were amplified from C. difficile strain VPI 10463 ( Figure 1). The toxin fragments encoded for 8, 14, 20 and 36 whole toxin A repeats and were labelled pTA2, p5/6, p5/7, and p9/10 respectively. These fragments were subsequently cloned into two expression vectors, pTECH-1 and pRSET-A and the integrity of the constructs was verified by di-deoxy terminal sequencing (ABI Prism). When expressed within E.
  • PCR polymerase chain reaction
  • Example 3 Expression of toxin A fragments in S. typhimurium The generation of a toxin-neutralising response at die gastrointestinal mucosa appears to be important in protecting against C. difficile disease.
  • the various pTECH-1 constructs containing the toxin fragments were introduced into an attenuated strain of Salmonella typhimurium The strain chosen contained an attenuating lesion within the htrA stress protem gene. Attenuated Salmonella were chosen as they have been shown to be an efficient mucosal delivery vehicle for other heterologous antigens.
  • the plasmid constructs pTA2, p5/6, p5/7 and p9/10 were introduced into the S. typhimurium htrA mutant stram (BRD915), a vaccine strain known to be efficacious against murme typhoid.
  • SDS-PAGE analysis showed that all 4 toxm constructs were only expressed when grown under these conditions in vitro ( Figure 2), and tiiat they reacted with monoclonal antibody PCG-4 in Western blots.
  • Group 3 S. typhimurium expressmg pTA2 (8 repeats)
  • Group 4 S. typhimurium expressmg p5/6 (14 repeats)
  • mice received 3-4 x 10 10 cfu per moculum. Two doses were given, the second after 28 days. Animals were terminated 56 days after the initial dose, and serum plus intestinal washes were collected from each animal. These samples were prepared to investigate the efficiency of the constructs in stimulating die immune system.
  • the anti-toxin A response elicited by die various fragments were evaluated by ELISA.
  • Wells of an EIA/RIA 96 well plate (Costar) were coated overnight with purified whole toxin A (0.05 ⁇ g/well) and blocked for 1 hour witii 1 % (w/v) BSA in PBS (Douce et al Proc. Natl. Acad. Sci. USA 92 1644-1648 (1995)). After washing 3 times with 0.1 % (v/v) Tween-20 in PBS, the wells were incubated for 2 hours with serum taken from each animal serially diluted five-fold in PBS.
  • Figure 3 depicts the mean anti-toxin A antibody titres elicited by all six groups of animals. It is clear mat die antibody levels induced by die toxin A constructs containing 8, 20 and 36 repeats were relatively low, and were not significantly higher than the background level obtained widi the control groups. However, the construct expressing the 14 carboxy-terminal repeats did give a positive antitoxin A response and elicited a mean anti-toxin A titre which was over 4-fold higher than that of background. Thus, it appears that a fragment expressing 14 toxin A repeats of which only 4 are of the IIB class may be optimum within this system with regard to generating an anti-toxin A response within serum. Studies are cu ⁇ ently in progress to evaluate the potency of this construct is stimulating die mucosal immune system.
  • Example 5 In vitro characterisation of Salmonella-expressed toxin A-fragment
  • Immunogenicity studies have identified a sho ⁇ protein encoding 4 class IIB repeats as being immunogenic when expressed widiin rationally attenuated S. typhimurium and given orally to mice. This is in contrast to fragments expressing 3, 7 and 12 IIB repeats which were not overtly immunogenic. Since the IIB repeats are known to bind to a characterised toxin A receptor, the toxin fragments were analysed for receptor-binding function in an established assay of toxin A-mediated binding. The toxin fragments were expressed from the pTECH-1 plasmid within S. typhimurium in vitro and die cells harvested. Sphaeroplasts were made from these cells and die soluble material released from the cell with 4 pulses of ultrasonic power.
  • a particular point of interest is mat the fragment containing 8 repeats gave no binding activity. Since the fragments comprising of 8 and 14 toxin repeats contain 3 and 4 class IIB repeats respectively, it appears that of die fragments expressed from the pTECH-I system, mat 4 class IIB repeats is the minimum requirement to promote binding to rabbit erythrocytes. Alternatively, it may be that die 14 toxin A repeats found in mis fragment generate a protein which is in an optimum configuration to promote receptor binding. In order to be an effective delivery system in vivo, the toxin A-expressing plasmids must be stable within the Salmonella in die absence of any antibiotic pressure.
  • Salmonella strain in the present study for delivering toxin A-fragment C fusions to the immune system a positive anti-toxin A serum response was seen with the fusion protein containing 14 toxin A repeats. This fusion also gave elevated levels of haemagglutination, implying mat immunogenicity may be correlated to efficient receptor binding. In an attempt to further increase die anti-toxin antibody response, these fusion proteins have also been delivered to the gastrointestinal mucosa within die ⁇ ro-mutated S. typhimurium strains which appear to be more proficient at mucosal delivery of antigen.
  • Example 6 Intragastric immunisation with S. typhimurium BRD509 (aroA aroD) expressing the p5/6 construct
  • each mouse received 1-2 x 10 10 cfu per inoculum. After 28 days, five animals from groups 2 and 3 were terminated, while the remaining animals in all 3 groups received a second dose. After 63 days, all animals were immunised subcutaneously witii 0.5 ⁇ g of the 14 repeat fragment which had been expressed from die pRSET-A vector in E. coli and affinity' purified. All animals were terminated 85 days after the initial dose. Serum plus intestinal washes were collected from each terminated animal.
  • Botii the anti-toxin A and die anti-tetanus toxin response elicited by die 14 repeats were evaluated by ELISA.
  • Figure 10 depicts die mean anti-toxin A titres found in die serum taken from all tiiree groups of animals. It is clear that even after one dose (day 28), animals which were immunised with S. typhimurium BRD509 expressing the 14 repeats elicited an anti-toxin A response, generating a mean serum titre approximately 8-fold higher than die control groups. This antibody response was boosted witii a second dose (day 63), increasing the mean fold-difference to over 200. A low dose of the 14 toxin A repeat protein was also administered witiiout the S. typhmurium BRD509 delivery system.
  • mice which had received two doses of the S. ryphimurium j)5/6 vaccine augmented tiieir antitoxin A response, producing levels of anti-toxin A antibody 7-fold higher than prior to the subcutaneous boost.
  • Example 7 Intra-naasal immunisation with S. typhimurium BRD509 expressing the p5/6 construct.
  • Salmonella typhimurium LB5010 (galE) and BRD915 (htrA) and plasmid pTECH-1 were kind gifts from Steve Chatfield. Medeva Vaccine Research Unit, Imperial College. London, U.K. E.coli BL21 (DE3) was obtained from Novagen. and plasmid pRSET-A was supplied by Invitrogen (De Schelp, The Netherlands). Bacteria were routinely cultivated in either Luria Broth (LB) or on LB agar with or without ampicillin (1 OO ⁇ g/ml).
  • Restriction enzymes and DNA ligase were purchased from Promega (Southampton, U.K.) and used according to the manufacturers instructions. DNA which had been subjected to restriction enzyme treatment was purified using either S-300 HR MicrospinTM columns (Pharmacia), or Prep-A-GeneTM purification resin (Bio Rad. Hemel Hempstead. U.K.). PCR was carried out using a Perkin Elmer 9600 cycle sequencer and Taq DNA Polymerase as described by the manufacturer (Appligene Oncor. U.K.). DNA cycle- sequencing was performed with an ABI PRISM® reaction kit (Perkin Elmer Applied Biosystems, Warrington, U.K.), and analysed with an Applied Biosystems 373 A DNA sequencer.
  • the amplified toxin A sequence was subcloned into the pTAg cloning vector (R&D Systems Europe Ltd, Abingdon, U.K.), excised with Xbal-Spel and inserted into Spel digested pTECH-1 downstream of the TETC encoding sequence (1994 Proc. Natl. Acad. Sci. USA. 91: 1 1261-11265) .
  • This construct was named p56TETC.
  • the entire toxin A sequence was first excised from the pTECH-1 vector with S el and Xbal and subcloned into ⁇ b ⁇ l-digested pUC18 (Promega).
  • Clones containing the toxin A fragment in the co ⁇ ect orientation were isolated, the toxin A sequence excised with BamUl and Hindlll and then inserted into similarly digested pRSET-A to create plasmid p56HIS. All recombinant plasmids were initially transformed into Epicurian coli® XL2- blue MRF " ultracompetent cells (Stratagene, Cambridge, U.K.), and e integrity of the cloning junctions confirmed by Dye Terminator cycle sequencing. Expression and affinity purification of recombinant toxin A C-terminal repeat proteins.
  • Plasmid p56TETC was first electroporated into S. typhimurium LB5010 using a Gene Pulser apparatus (Bio Rad) (1992 Vaccine. 10:53-60) and then introduced into S. typhimurium BRD915 (htrA) via P22 bacteriophage transduction (1993 Infect. Immun. 61 :5374-5380) .
  • S. typhimurium BRD915 were incubated at 37°C witii aeration in LB-ampicillin until early/mid log phase (OD600 0.3-0.5).
  • E.coli BL21(DE3) transformed witii p56HIS were grown in LB-ampicillin until mid log phase and expression of the plasmid encoded protein induced by the addition of isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and incubating for a further 5 h. After recombinant protein induction, both the S.
  • Both of the recombinant toxin A fragments were purified using Bovine thyroglobulin affinity chromatography using the method essentially described by Kirvan and Wilkins (1987 Infect. Immun. 55: 1873-1877) .
  • Soluble protein harvested as described above was diluted with an equal volume of TBS (0.05 M Tris. 0.15 M NaCl. pH 7.0) and cooled to 4°C.
  • the chilled material was then passed through 4 ml of pre-chilled Affi-Gel 15 resin (Bio Rad) which had been coupled to 140 mg of bovine thyroglobulin (Sigma). After 4 repeat applications of the cellular lysate at 4°C, the column was washed with at least 40 bed volumes of cold TBS.
  • Proteins were separated using 10% (w/v) polyacrylamide gels and the discontinuous buffer system of Laemmeli et al. (1970 Namre. 227:680-685) . Prior to SDS-PAGE analysis, the protein content of samples was determined using the BCA assay (Pierce &Warriner Ltd, U.K.). Proteins separated by SDS-PAGE were transferred to Hybond C nitrocellulose membrane (Amersham Life Science Ltd, U.K.) and processed as described ( Christodoulides, M.et al.. 1993 J. Gen. Microiol. 139:1729-1738) . Membranes were probed with either the monoclonal antibody PCG-4 specific for the C- terminal repeat region of toxin A, or a polyclonal rabbit anti-TETC antiserum.
  • Samples of protein were serially diluted 2-fold in PBS within a 96 well U- bottomed plate (Sterilin) and then reacted with 25 ⁇ l of a 2% (w/v) suspension of washed rabbit erythrocytes at 4 ⁇ C (TCS Microbiology, Botolph Claydon, U.K.) for 18 h.
  • Wells containing the highest dilution of protein able to promote 100%> agglutination of erythrocytes were taken as the endpoint, and each assay was performed in duplicate.
  • Bicester, U.K. were immunised intranasally with combinations of either 10 ⁇ g of p56HIS or p56TETC, 5 ⁇ g of TETC, and l ⁇ g of E.coli LT. Each inoculum was diluted to a final volume of 30 ⁇ l in PBS (pH 7.2), and 15 ⁇ l administered to each nostril of lightly anaesthetised animals (Halothane. Rhone Merieux). Mice were immunised with 3 identical doses administered on days 0, 20 and 35. Serum samples were collected from the tail vein of each animal 1 day prior to immunisation.
  • mice were exsanguinated on day 47 by cardiac puncture and nasal, lung and intestinal lavage carried out with 0.1% (w/v) BSA in PBS as described (Douce G et al., 1995 Proc. Natl. Acad. Sci. USA. 92:1644-1648) .
  • All samples were stored witii 1 mM PMSF to inhibit intestinal proteases.
  • Anti-toxin A and anti-tetanus toxoid (TT) antibody responses widiin individual serum samples were determined by ELISA. These assays were performed as described by Douce et al. (1997 Infect. Immun. 65:2821-2828) , using ELISA wells coated overnight at 4°C with either purified whole toxin A (0.15 ⁇ g/well in 0.1 M NaHCOs, pH 9.5 ) or formalin inactivated TT (0.5 ⁇ g/well in PBS, pH 7.2). ELISA titres were determined as the reciprocal of the highest serum dilution which gave an absorbance value of 0.5 units above the background for toxin A. and 0.3 units above the background for TT. All titres were standardised against either monoclonal antibody PCG-4, or polyclonal anti-TETC positive control antiserum.
  • the levels of both anti-toxin A and anti-TT specific IgA within the mucosal lavage samples were determined by ELISA using the same antigen coating concentration as above and the method of Douce et al. ( Douce G et al.,1995 Proc. Natl. Acad. Sci. USA. 92:1644-1648) . ELISA titres were calculated as the reciprocal of the highest dilution which gave an absorbance 0.2 units above the background.
  • the toxin A neutralising properties of both antiserum and mucosal lavage samples was determined using CHO-K1 cells (Tucker KD et al., 1990 J. Clin. Microbiol. 28:869-871) and thyroglobulin affinity-purified toxin A (Kirvan HC et al.. 1987 Infect. Immun. 55:1873-1877) .
  • CHO-K1 cells (passage number 28-38) were grown in Ham's F12 medium (Sigma) containing 10% (v/v) foetal bovine serum and 1 mM L-glutamine and supplemented with streptomycin/neomycin/penicillin (Sigma).
  • Freshly trypsinised CHO-K1 cells were seeded into 96 well trays (Corning Costar) at 3 x 10 ⁇ cells per well and allowed to recover for 24 h at 37°C in a 5%> CO2 atmosphere.
  • the test sample was serially diluted two-fold in growth medium, and allowed to react with toxin A (0.6 ⁇ g/ml final concentration) for 90 min at 37°C.
  • the toxin A-antiserum mixtures were then added to the CHO-K1 cells to give a final total volume of 100 ⁇ l for antiserum or 125 ⁇ l for lung lavage, and the cellular morphology noted after a 24 h incubation at 37°C and 5% CO2- The neutralising titre was taken as the highest dilution of sample to prevent 100% cellular rounding. All samples were tested in duplicate, and individual assays standardised against a positive control rabbit antiserum which had been raised against a conserved decapeptide located within the C- terminal region of toxin A (Wren BW et al., Infect. Immun. 59:3151-3155) .
  • the mucosal immunogenicity of the 14 C-terminal toxin A repeats was determined using purified recombinant proteins containing the toxin repeats. Proteins were generated by cloning a PCR fragment encoding the entire 14 C- terminal repeats of toxin A (aa 2387-2706 inclusive) into both pRSET-A and pTECH-1 vectors to produce two constructs p56HIS and p56TETC. Clone p56HIS in E.coli generated a recombinant 14 toxin A repeat protein with six adjacent histidine residues attached to the N-terminus. Construct p56TETC when expressed in S.
  • typhimurium produced a fusion between the 14 toxin A repeats and TETC of tetanus toxin.
  • SDS-PAGE analysis of E.coli cellular lysates expressing p56HIS showed the presence of an additional protein with a mean apparent molecular mass of 42 kDa (calculated from three separate gels) which co ⁇ esponded to the toxin A-polyhistidine fusion (Fig. 16A, lane 1).
  • Similar analysis of recombinant S. typhimurium lysates showed an additional band with a mean apparent molecular mass of 83 kDa which co ⁇ esponded to the toxin A-TETC fusion protein (Fig. 16B, lane 1) .
  • Both the p56HIS and p56TETC fusion proteins were successfully purified by utilising the inherent affinity the toxin A repeat region has for the trisaccharide Ga l-3, Gal ⁇ l-4, GlcNac present within bovine thyroglobulin.
  • SDS-PAGE analysis showed both the affinity purified p56HIS (Fig. 16A, lane 3) and p56TETC (Fig. 16B, lane 3) proteins to be relatively free from other E.coli or S. typhimurium proteins respectively.
  • Immunoblot analysis showed both proteins to be immunoreactive with monoclonal antibody PCG-4 which is specific for the toxin A repeat region (Fig. 17A).
  • the p56HIS purified material also appeared to contain a weaker immunoreactive 41 kDa protein.
  • This protein also reacted with a monoclonal antibody specific for the polyhistidine tag attached to the N-terminus of the toxin A repeats, indicating that limited proteolytic degradation had occu ⁇ ed at the C-terminus of the 42 kDa protein (data not shown).
  • the p56TETC protein also reacted with a polyclonal anti- TETC antiserum in immunoblot to generate a 83 kDa immunoreactive band plus several weaker bands of lower molecular weight (Fig. 17B). As these weaker bands did not react with the PCG-4 monoclonal antibody, limited proteolysis of TETC appeared to have occu ⁇ ed. Haemagglutination and cytotoxic properties of the 14 C-terminal toxin A repeats.
  • the toxin A receptor present on rabbit erythrocytes is also found on the surface of several other cell types such as CHO-K1 cells, thus allowing toxin A-mediated cytotoxicity to be quantified in vitro ( Katoh T et al., 1986 FEMS. Microbiol. Lett. 34:241-244) . Cytotoxicity was tested by incubation of both p56HIS and p56TETC with monolayers of CHO-K1 cells. Even when 10 pM of purified protein was tested no cytotoxic activity was observed, thus confirming the 14 C-terminal repeats to be non-toxigenic (data not shown). In contrast. 0.2 pM of whole toxin A was sufficient to promote cytotoxicity.
  • mice immunised i.n. with toxin A repeat proteins were immunised i.n. with 3 doses of either the p56HIS or p56TETC affinity purified C-terminal toxin A fragments as described.
  • LT was also co-administered with the proteins as a mucosal adjuvant.
  • Serum samples containing high titres of LT-specific antibody were unable to cross-react with toxin A in ELISA (data not shown).
  • Toxin A neutralisation with serum and mucosal antibodies harvested from i.n. immunised mice.
  • Toxin A exhibits a cytotoxic effect on CHO-K1 cells in vitro which can be neutralised by specific antibodies (f atoh T et al., 1986 FEMS. Microbiol. Lett. 34:241-244 and Wren SB et al., 1991 Infect. Immun. 59:3151-3155) . Therefore, serum and mucosal lavage samples were tested for the ability to neutralise the cytotoxic activity of whole toxin A. Serum harvested from all five mice immunised with p56HIS were unable to neutralise toxin A (Table 2). However, serum from one out of the five mice which received p56TETC was able to neutralise toxin A.
  • Toxin A neutralising properties of both serum and mucosal antibodies harvested after 3 intranasal doses of antigen. Toxin-neutralising titres were scored as the highest dilution of antiserum or mucosal lavage to promote 100%) neutralisation of toxin A (60 ng/well) as measured against CHO-Kl cells in vitro. Mean neutralising titres ⁇ SD for five mice are shown, with each assay being performed in duplicate.
  • Lung lavage samples were also tested for toxin A neutralising activity since these samples had been shown to contain high levels of toxin A-specific IgA by ELISA. Although the level of neutralisation was lower than that seen with the co ⁇ esponding antiserum, single mice from the p56TETC + LT immunised group did generate sufficient levels of IgA at the lung mucosa to neutralise toxin A (Table 2). However, the highest neutralisation titre was seen with lavage sample taken from p56HIS + LT immunised mice, with two out of five samples possessing toxin A neutralising activity.
  • the level of anti-TT serum antibodies was also determined (Fig. 20).
  • the control mice immunised witii TETC alone generated levels of anti-TT in the blood which were higher than expected.
  • the mean antibody titres induced by p56TETC after 3 i.n. doses were similar to those obtained with TETC alone (P > 0.05), when p56HIS was co-administered with TETC.
  • the mean titres of anti-TT antibody were found to be approximately 4 fold higher after both 2 and 3 i.n. doses than the titres seen with TETC only.
  • mice immunised i.n. with toxin A repeat proteins In contrast to the serum responses, mice immunised with TETC alone induced very poor immune responses at both the nose and lung mucosal surfaces (Fig. 21). However, mice immunised with either p56HIS + TETC + LT or p56TETC +LT induced strong anti-TT responses in the nose and lungs. These titres were very consistent within the group of 5 mice. Interestingly both nose and lung lavage samples taken from mice immunised with p56HIS + TETC were also shown to contain very high levels of anti-TT specific IgA.

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Abstract

La présente invention concerne la région C-terminale, à 14 répétitions, de la toxine A de Clostridium difficile efficace pour générer des anticorps dirigés contre la toxine A.
EP98930905A 1997-06-20 1998-06-19 Fragments immunogenes de toxine a de clostridium difficile Withdrawn EP1000155A1 (fr)

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EP2502998A3 (fr) * 1999-04-09 2013-02-27 Intercell USA, Inc. Vaccin à toxine recombinante A/B contre Clostridium difficile
WO2006121422A2 (fr) 2004-02-06 2006-11-16 University Of Massachusetts Anticorps diriges contre des toxines de clostridium difficile et utilisations de ces derniers
GB0422003D0 (en) * 2004-10-05 2004-11-03 Univ Glasgow Inhibitory analogues to block receptors for microbial pathogenic determinants
CN102365097A (zh) 2009-02-20 2012-02-29 卫生防护机构 艰难梭菌毒素的抗体
GB0921288D0 (en) 2009-12-04 2010-01-20 Health Prot Agency Therapies for preventing or suppressing clostridium difficile infection
GB201016742D0 (en) 2010-10-05 2010-11-17 Health Prot Agency Clostridium difficile antigens
EP4365196A3 (fr) 2011-04-22 2024-08-07 Wyeth LLC Compositions concernant une toxine mutante de clostridium difficile et procédés associés
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AU8118598A (en) 1999-01-04
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