EPITOPES OF CLOSTRIDIUM DIFFICILE TOXINS A AND B AND USES THEREOF

Abstract

The present invention relates to a polypeptide comprising an epitope having a sequence homology of at least 75% to a sequence section of both Clostridium difficile toxin A and B. Moreover, the present invention refers to a vaccine comprising such polypeptide. The invention further relates to an antibody binding to Clostridium difficile toxins A and B and to a method for isolating and/or detecting such antibody and to uses of the polypeptides and antibodies.

Claims

1-16. (canceled)

17. A polypeptide comprising at least one epitope, wherein the epitope is at least eight consecutive amino acids in length and the epitope has a sequence homology of at least 75% to a sequence section of Clostridium difficile toxin A and a sequence homology of at least 75% to a sequence section of Clostridium difficile toxin B, and wherein the polypeptide has a length of 8 to 100 consecutive amino acid moieties, or an immunogenic peptidomimetic or retro-inverso polypeptide thereof.

18. The polypeptide of claim 17, wherein the polypeptide is obtained by means selected from the group consisting of polypeptide synthesis, gene technological means, and isolation of Clostridium difficile toxin A or Clostridium difficile toxin B and subsequent protein digestion.

19. The polypeptide of claim 17, wherein the epitope has a sequence homology of at least 75% to a sequence section located at the outer surface of Clostridium difficile toxin A and has a sequence homology of at least 75% to a sequence section located at the outer surface of Clostridium difficile toxin B.

20. The polypeptide of claim 17, wherein the epitope has or comprises an amino acid sequence selected from the group consisting of TABLE-US-00006 (SEQ ID NO: 3) ANQYEVRINSEGRX.sup.23ELLX.sup.1HSGX.sup.25WINKEEX.sup.26IX.sup.21; (SEQ ID NO: 4) GESX.sup.21X.sup.27VETEK; (SEQ ID NO: 5) X.sup.26X.sup.21KVQX.sup.21YAQLFSTGLNTI; (SEQ ID NO: 6) LX.sup.21PX.sup.21AGISAGIPSLVNNELX.sup.21L; (SEQ ID NO: 7) DDLVISEIDFNNNSI; (SEQ ID NO: 8) MEGGSGHTVTX.sup.1X.sup.25IDHFFSX.sup.26PSIX.sup.22; (SEQ ID NO: 9) PGLRSLENDGTX.sup.23LLD; and (SEQ ID NO: 10) AX.sup.21X.sup.25X.sup.24TIX.sup.25X.sup.21LPTX.sup.21X.sup.22EGX.sup.21PIX.sup.21X.sup.26TIX.sup.21DGX.sup.21 X.sup.22LGAAIKELX.sup.1X.sup.24X.sup.1X.sup.1DPLLX.sup.23X.sup.25EX.sup.21EAKX.sup.21GX.sup.21X.sup.21A X.sup.21NX.sup.21X.sup.22, wherein: X.sup.1 is any naturally occurring amino acid moiety or is a direct bond between the adjacent amino acid moieties; X.sup.21 is an amino acid moiety selected from the group consisting of G, A, V, P, L, I, M, W, and F; X.sup.22: is an amino acid moiety selected from the group consisting of S, T, Y, C, N, U, O, and Q; X.sup.23 is an amino acid moiety selected from the group consisting of K, R, and H; X.sup.24 is an acidic amino acid moiety selected from the group consisting of D and E; X.sup.25 is an amino acid moiety selected from the group consisting of S, T, Y, C, N, Q, K, R, H, U, O, D, and E; X.sup.26 is an amino acid moiety selected from the group consisting A, G, and S; and X.sup.27 is an amino acid moiety selected from the group consisting of Y, G, A, V, P, L, I, M, W, and F, or in an immunogenic peptidomimetic or retro-inverso polypeptide thereof.

21. The polypeptide of claim 17, wherein the polypeptide is immobilized on a solid support.

22. A vaccine comprising at least one polypeptide of claim 17 and at least one pharmaceutically acceptable carrier.

23. A method for preventing an individual from developing a Clostridium difficile infection, comprising administering the individual with a sufficient amount of the vaccine of claim 22.

24. An antibody or antibody fragment binding to Clostridium difficile toxin A with a dissociation constant Kd of less than 100 nM and to Clostridium difficile toxin B with a dissociation constant Kd of less than 100 nM.

25. A method for isolating and/or detecting an antibody or antibody fragment binding to Clostridium difficile toxins A and B from a fluid containing the antibody or antibody fragment, wherein the method comprises the following steps: (i) providing: the fluid containing the antibody or antibody fragment, and a polypeptide according to claim 17 immobilized on a solid support; (ii) contacting the fluid with the immobilized polypeptide and allowing the antibody or antibody fragment to bind to the immobilized polypeptide; and (iii) removing at least parts of the unbound fluid and optionally washing the solid support with a fluid not containing the containing the antibody or antibody fragment.

26. The method of claim 25, wherein the solid support is a solid phase of an affinity column.

27. The method of claim 25, wherein the fluid is a body fluid.

28. The method of claim 25, wherein the method further comprises a step of isolating or removing one or more antibody classes selected from the group consisting of IgG, IgM, IgD, IgE, and IgA.

29. The method of claim 25, wherein the method further comprises a step of detecting the bound antibody or antibody fragment.

30. A method for testing the ability of an antibody or antibody fragment for neutralizing the bioactivity of Clostridium difficile toxin A, Clostridium difficile toxin B or a combination of both, wherein the method comprises the following steps: (A) providing: adherent mammalian cells in a cell culture, Clostridium difficile toxin A, Clostridium difficile toxin B or both, and the antibody or antibody fragment; (B) contacting the Clostridium difficile toxin A, Clostridium difficile toxin B or combination of both and the antibody or antibody fragment with the adherent mammalian cells; (C) incubating the exposed mammalian cells for a time sufficient for detachment of cells of lower viability; and (D) detecting the degree of cell rounding, wherein the degree of cell rounding indicates the degree of remaining bioactivity of the Clostridium difficile toxin A, Clostridium difficile toxin B, or both.

31. A method for treating or preventing an individual suffering from a Clostridium difficile infection or being of risk of developing a Clostridium difficile infection, comprising administering the individual with a sufficient amount of the antibody or antibody fragment of claim 24.

32. A method for treating or preventing an individual suffering from a Clostridium difficile infection or being of risk of developing a Clostridium difficile infection, comprising administering the individual with a sufficient amount of an antibody or antibody fragment obtained from a method of claim 25.

33. The antibody or antibody fragment of claim 24, wherein the antibody or antibody fragment binds to an epitope with a dissociation constant Kd of less than 100 nM, wherein the epitope has a sequence homology of at least 75% to a sequence section of Clostridium difficile toxin A and a sequence homology of at least 75% to a sequence section of Clostridium difficile toxin B, and wherein the polypeptide has a length of 8 to 100 consecutive amino acid moieties, or an immunogenic peptidomimetic or retro-inverso polypeptide thereof.

34. The method of claim 25, wherein the antibody or antibody fragment has a dissociation constant Kd of less than 100 nM and to Clostridium difficile toxin B with a dissociation constant Kd of less than 100 nM.

35. The method of claim 26, wherein the method further comprises a step of eluting the antibody or antibody fragment from the affinity column.

36. The method of claim 27, wherein the fluid is a body fluid selected from the group consisting of blood plasma and a fraction of blood plasma.

37. The method of claim 27, wherein the method further comprises preparing of a fraction of blood plasma by a Cohn or Kistler-Nitschmann process.

38. The method of claim 29, wherein the step of detecting the bound antibody or antibody fragment comprises the following steps: (a) binding a secondary antibody selectively to the Fc part of the bound antibody or antibody fragment; and (b) detecting the secondary antibody.

39. The method of claim 38, wherein the secondary antibody is labeled with a detectable label or is conjugated to an enzyme that generates a detectable compound from a precursor.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0176] FIG. 1 and FIG. 2 show, visualized from different angles, the location of epitopes according to preferred embodiments (depicted in black) of the present invention at the outer surface of the glucosyltransferase-, cysteine-protease- and translocation-domain of Clostridium difficile toxin A (depicted in grey).

EXAMPLES

[0177] Polypeptides as described above, for instance polypeptides of SEQ ID NO: 2 to 10, are synthesized by means of solid phase peptide synthesis (SPPS) or obtained from recombinant expression.

Example I Positive Affinity Chromatography at an Epitope

[0178] A polypeptide as described above, for instance a polypeptide of SEQ ID NO: 2 to 10, is immobilized on an affinity chromatography matrix (affinity beads). This material is filled in an affinity chromatography column. The column is washed with a buffer (PBS). The column is contacted with the body fluid (blood serum or a fraction thereof).

[0179] The antibodies and antibody fragments specifically binding to the respective polypeptide comprising the epitope bind to their targets in the affinity column. The column is washed by a flow-through of buffer. Thereby unbound components including unbound antibodies and antibody fragments having no or merely a low affinity to the polypeptide are removed.

[0180] Subsequently, the specific antibodies and antibody fragments are eluted either by acetic buffers (e.g. 0.1 mol/L glycine/HCl pH 2.0) or by 3 mol/L KSCN, or by 8 mol/L urea. Thereby, the specifically binding antibodies are isolated.

Example II IgA Separation

[0181] IgA is isolated direct from plasma of from plasma fractions which are purified from IgG. When those fractions are used, the separation of the specific immunoglobulin classes is not needed. When the positive affinity chromatography is performed ahead of the separation of the immunoglobulin classes, the separation of them is performed in a second step.

[0182] An example for the separation of (hyperimmune) IgA includes subjecting a plasma pool to Cohn fractionation. This is followed by an IgG polishing step providing IgG and an IgA/IgM fraction. The IgA/IgM fraction is subjected to affinity chromatography as described above and provides (hyperimmune) IgA.

[0183] An alternative example for the separation of (hyperimmune) IgA includes subjecting an unfractioned plasma pool subjected to affinity chromatography as described above. This provides an immunoglobulin fraction (including IgG and IgA) and a residual blood fraction which is further subjected to Cohn fractionation. The immunoglobulin fraction (including IgG and IgA) is subjected to a second affinity chromatography separating IgG and IGA and provides (hyperimmune) IgA.

Example III Separation of Plasma Containing (Neutralizing) Antibodies Against Clostridium difficile Toxin a and/or Clostridium difficile Toxin B

[0184] The donations (plasma and or blood) are screened by commercially available ELISA. Here, the Clostridium difficile toxin A or B is insolubilized at a solid phase (e.g. microtiter plate) (Porstmann et al., “Enzyme immunoassay techniques. An overview”. Journal of Immunological Methods, 1992, 150:5-21). The specific antibodies of different immunoglobulin classes are separated by using different class-specific antibodies in a labelled form (e.g. anti-IgG-HRP, anti-IgA-HRP).

[0185] Due to the fact, that the immune reaction shows stable antibody responses even month and years after an infection, a quarterly or half-year screening of donors can be performed. The selected donations are pooled and used in the plasma fractionation for the separation of all other plasma proteins. The IgA containing fraction is used separately.

Example IV Enzyme-Linked Immunosorbent Assay (ELISA)

[0186] A polypeptide as described above, for instance a polypeptide of SEQ ID NO: 2 to 10, is immobilized on a bottom of a microtiter plate. The body fluid (blood fraction, blood plasma/serum) is contacted for several minutes. Then, the microtiter plate is washed with buffer (PBS) and contacted with an enzyme-labelled secondary antibody (e.g., conjugated with HRP). A substrate suitable to be converted into a detectable moiety by the enzyme is added and the staining of the microtiter plate is performed in a plate reader. This assay provides an antibody titer of the body fluid.

Example V Cell-Based Neutralization Assay

[0187] The ability of antibodies to neutralize the toxins is tested via the exposal of mammalian cells to one of the toxins (HT29 for Clostridium difficile toxin A and CHO for Clostridium difficile toxin B). The CHO cells are grown in DMEM/Ham's F12 and HT29 in McCoys 5A in in cell culture dishes and supplemented 20% fetal calf serum and glutamine. The toxins and the mono- or polyclonal antibodies or antiserum are incubated for 60 min at 37° C. On the first day, the cells are seeded in a 96 well cell culture plates and incubated overnight. Dilutions of antibodies and LCTs are done in the cell culture medium. The toxins concentrations for the assay are chosen in a way that just enough toxin is administered to induce complete rounding overnight. On the second day, the diluted antibodies are mixed with subsequent dilutions of the LCTs. After incubation (60 min at 37° C.) of the antibody-toxin mixes are added to the cells and cell rounding is observed after 20-24 hours. As control, toxin is added to the cells without antibodies.

[0188] The rounding is evaluated by microscopic analysis on the third day, using the following criteria:

(−) no cell rounding
(+) ≤10% cell rounding
(++) >10% cell rounding
(+++) 85-100% cell rounding.

Example VI Animal (Hamster) Model

[0189] The primary objective of such a study is to evaluate the dose of specific human IgA antibodies against Clostridium difficile toxin A and/or B in an oral therapy. In a three-armed feasibility animal study specific IgA against the Clostridium difficile toxins and standard therapy are compared. Any prolongation of life span is the primary measure the secondary is survival at day 24.

Study Design and Methodology:

[0190] IgA is prepared using common methods from plasma of normal healthy plasma donors. The donors have previously been screened first for the presence of specific antibodies against Clostridium difficile toxin A and B and antibodies against Clostridium difficile. IgA is enriched from those donations. Such IgA-enriched plasma-fraction is stored frozen until use in single does, each at 250 μL with 1 mg/mL. The IgAs is administered into Hamster free of Clostridium difficile (Checked by NAT at d−4 . . . d−6, at tgcBIOMICS GmbH)

Eight Male Animals are Used Per Group:

[0191] (A) Disease control group: remains completely untreated after Clindamycin spores application—time to death of hamsters is determined in this untreated control group. (B) Vancomycin-treatment group: 3-days of treatment with Vancomycin (10 mg/kg administered p.o. starting at day d+2), prolongation of life span is monitored. (C) Plasma-treatment group: treatment with Vancomycin (10 mg/kg, at days +2 till +4) is performed like for group (B), but is accompanied by treatment at days +3 till +6 with specific IgA-enriched plasma-concentrate which contains neutralizing antibodies against Clostridium difficile toxin A and B

[0192] Animals are housed in socially harmonious groups of four animals in individual ventilated cages with a surface area of 1500 cm.sup.2. Housing in groups becomes appropriate, if no animal is positive in the NAT for of C. diff genes. The general colonisation status of the animals is checked with the 24 animals in the forefront of starting the animal experiment.

[0193] To change the intestinal flora, the animal is treated at d0 with Clindamycin i.p. One day later (d0) the animal is challenged with 100 to 1000 spores of C. diff (strain: 630). Groups (B) and (C) receive a 3-day vancomycin treatment which reduces the bacterial load following infection. The IgA-enriched Plasma-treatment starts at d2 with 2 doses IgA for four days in total (d2: 2 doses enriched-IgA; d3: 2 doses enriched-IgA, d3: 2 doses enriched-IgA, d4: 2 dose2 enriched-IgA).

[0194] The animals are regularly observed for body temperature by infrared temperature measurement, stool consistency, clinical signs, and every 2.sup.nd day for their body weight. Stool samples are taken ahead of the experiment at d−4 . . . d−6 for C. diff-NAT-testing and at d+3; d+6 and d+10 to test for the presence of human IgA in stool and the detection of blood in the stool. Blood samples are taken at d−6 . . . d+7, d+14 . . . and at d+21 from orbital plexus for the detection of any specific antibodies induced in hamsters against both toxins and Clostridium difficile. The animals are euthanized when the weight loss exceeds 60%. The total observation time is 24 days. After necropsy, signs of inflammation and the length of the intestine have to be documented. Stool is collected of the individual animals. The animal receiving specific IgA against Clostridium difficile toxins A and B are protected and survive the observation period.