Clostridium difficile vaccine composition

Abstract

Methods and compositions for treating or preventing C. difficile infection (CDI) via TcdB or TedA holotoxins. The compositions feature immunogens or binding agents, such as antibodies, nanobodies (VHHs), or single-domain antibodies (sdAbs), based on one or a combination of neutralizing epitopes of TcdB or TcdA. The immunogen inhibits the conformational change required for pore formation by TcdB at endosomal pH. Furthermore, the immunogen inhibits the movement of the scissile bond to the cleaved side of the CPD and the proper orientation of the GTD relative to the CPD, thereby inhibiting cleavage of the GTD required to activate the toxin. The present invention also describes vaccines for the treatment of CDI, e.g., vaccines targeting TcdB or TccLA.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 851,040, filed May 21, 2019, the specification of which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant Nos. R01AI125704 and R01AI139087 awarded by the National Institutes of Health, and Grant Nos. HDTRA1-16-C-0009 and HDTRA1-18-1-0035 awarded by the Department of Defense / Defense Threat Reduction Agency. The Government has certain rights in the invention.

[0003] Reference to sequence listing Applicant asserts that the information recorded in the form of Appendix C / ST.25 text file entitled UCI_19_16_PCT_Sequencing_Listing_ST25, submitted under Rule 13ter.1(a), is identical to the information forming part of the filed international application. The contents of the sequence listing are incorporated herein by reference in their entirety.

[0004] The present invention relates to neutralizing the holotoxin of Clostridium difficile, and more specifically to therapeutic compositions and methods for treating Clostridium difficile infection. [Background technology]

[0005] Clostridium difficile has been classified as one of the top three urgent antibiotic resistance threats by the Centers for Disease Control and Prevention (CDC), and C. difficile infection (CDI) is the most common cause of antibiotic-associated diarrhea and gastroenteritis-associated death in developed countries. CDI pathology is primarily mediated by two homologous exotoxins, TcdA and TcdB, which target and destroy the colonic epithelium, causing diarrhea and colitis. Although the relative roles of these two toxins in the pathogenesis of CDI are not fully understood, recent studies have shown that TcdB is more toxic than TcdA and is more important for inducing host inflammatory and innate immune responses.

[0006] TcdA (~308 kDa) and TcdB (~270 kDa) contain four functional domains, namely, an N-terminal glucosyltransferase domain (GTD), a cysteine ​​protease domain (CPD), a central transmembrane delivery and receptor binding domain (Delivery / RBD), and a C-terminal linked repeat oligopeptide (CROP) domain (Figure 1A). It is widely accepted that toxins bind to cell surface receptors via Delivery / RBD and CROP and enter cells via endocytosis. Endosome acidification induces a conformational change in the toxin, causing Delivery / RBD to form a pore, delivering the GTD and CPD across the endosomal membrane.

[0007] In the cytosol, the CPD is activated by eukaryotic-specific inositol hexakisphosphate (InsP6, also known as phytate) and subsequently undergoes autoproteolysis to release the GTD. The GTD then glucosylates small GTPases of the Rho family, such as Rho, Rac, and CDC42. Glucosylation of Rho proteins inhibits their function, leading to alterations in the actin cytoskeleton, cell rounding, and ultimately apoptotic cell death. Numerous structures have been reported for fragments of TcdA and TcdB, providing numerous insights into the function of these toxin domains. However, it remains unclear how the individual documents interact within the ultratertiary structure of the holotoxin and how the holotoxin dynamically responds in a precise stepwise manner to environmental and cellular cues, such as low pH and InsP6, that result in intoxication.

[0008] An anti-TcdB neutralizing antibody (bezlotoxumab) was recently approved by the US Food and Drug Administration (FDA) for the prevention of recurrent infection, as up to 35% of patients with CDI may relapse and many patients may require multiple treatments. However, this antibody is not indicated for the treatment or prevention of CDI. Summary of the Invention

[0009] The object of the present invention is to provide therapeutic compositions and methods that allow neutralization of the holotoxins of Clostridium difficile (i.e. TcdB or TcdA) as specified in the independent claims. Embodiments of the invention are set out in the dependent claims. The embodiments of the invention can be freely combined with one another if they are not mutually exclusive.

[0010] The present invention describes therapeutic compositions comprising one or more isolated polypeptides that neutralize the primary holotoxin (TcdB or TcdA) of C. difficile. In some embodiments, the isolated polypeptides are comprised of a group that binds to the holotoxin and inhibits its toxicity (function), thereby neutralizing the holotoxin.

[0011] Additionally, the invention may feature a method of neutralizing a primary holotoxin (TcdB or TcdA) of C. difficile. In some embodiments, the method includes producing an immunogen of the C. difficile holotoxin (TcdB or TcdA) and introducing the immunogen into a host to elicit an immune response against the immunogen. In another embodiment, the host produces antibodies specific to the holotoxin base on the immunogen.

[0012] Additionally, the invention may feature methods for designing and producing vaccines specific for the C. difficile holotoxin (TcdB or TcdA).

[0013] Without wishing to limit the invention to any theory or mechanism, it is believed that vaccines of the invention may be advantageous (e.g., compared to toxoid vaccines against CDI) because the immunogens of the invention are non-toxic, making them potentially safer; the immunogens of the invention can be produced in high yield and purity in E. coli, making them cheaper to produce, formulate, and store (whereas vaccine production can be difficult); the immunogens of the invention retain their native 3D structure (compared to the disrupted antigenic structure in toxoids), and therefore may be more efficient at eliciting an immune response as a vaccine; the immunogens of the invention are small and contain known neutralizing epitopes, and therefore may be more efficient at eliciting the production of neutralizing antibodies. Furthermore, these immunogens are smaller (compared to the entire holotoxin), and are directed to a more specific region of TcdB, which may result in a better immune response. The invention provides polypeptides that are smaller than the entire holotoxin, but larger than small (e.g., 15-mer) peptides: medium-sized peptides with well-defined 3D structures.

[0014] Any feature or combination of features described herein is included within the scope of the present invention, provided that the features included in any such combination are not mutually inconsistent, as would be apparent from the context, the specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

[0015] The features and advantages of the present invention will become apparent from a consideration of the following detailed description, taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0016] [Figure 1A] Figure 1A shows a schematic diagram of the full-length TcdB holotoxin, showing the domain organization of TcdB:GTD (red):CPD (purple);Delivery / RBD (yellow);CROP (blue) and the approximate VHH binding region.

[0017] [Figure 1B] Figure 1B shows a schematic of the 3D structure of TcdB holotoxin, omitting for clarity the three VHHs used to facilitate crystallization (GTD; CPD; Delivery / RBD; CROP).

[0018] [Figure 2A] Figure 2A shows a schematic diagram of the CROP domain showing the organization of the short repeat (SR, thin blue bar) and long repeat (LR, thick black bar). The dashed lines indicate the boundaries of the four CROP units (I-IV).

[0019] [Figure 2B] Figure 2B shows a close-up of the CROP domain, while the remainder of TcdB is in surface expression.

[0020] [Figure 2C] Figure 2C shows the superposition of four CROP units. The LR of each CROP unit induces a twist of approximately 132 to 146°.

[0021] [Figure 2D] FIG. 2D shows that the hinge region, which connects the CROP domain to the rest of the toxin, is located in the center of TcdB and is surrounded by the GTD, CPD, and delivery / RBD. [Figure 2E] FIG. 2E shows that the hinge region, which connects the CROP domain to the rest of the toxin, is located in the center of TcdB and is surrounded by the GTD, CPD, and delivery / RBD.

[0022] [Figure 3A] Figure 3A shows the curve-fit analysis of the SAXS study, indicating that the CROP domain undergoes pH-dependent conformational changes. The theoretical Kratky plot based on the structure of the TcdB holotoxin is nearly identical to the experimental scattering profile at pH 5.0 (upper panel) but differs from the scattering profile at pH 7.4 (lower panel).

[0023] [Figure 3B] FIG. 3B shows bridging peptides between different TcdB domains identified by XL-MS.

[0024] [Figure 3C] Figure 3C shows the XL-MS results, which suggest that TcdB can adopt a “closed” conformation at neutral pH, with the central part and C-terminal tip of the CROP domain migrating to within ∼30 Å of Delivery / RBD.

[0025] [Figure 3D] Figure 3D shows a model of the two restrained conformational states of the TcdB holotoxin, showing the acceptor dye of 7F bound to the GTD and the donor dye (hexagon) on B39 (star) bound to the CROP domain.

[0026] [Figure 3E]FIG. 3E shows population histograms of unaveraged FRET efficiencies from TcdB in complex with dye-labeled VHH at pH 5.0 (n=498) and pH 7.0 (n=594).

[0027] [Figure 4A] Figure 4A shows the pore-forming intermediate state of TcdB. 5D binds to Delivery / RBD and interacts directly with the pore-forming domain, shown as a ribbon model, and the rest of the toxin as a surface model.

[0028] [Figure 4B] FIG. 4B shows a representative 2Fo-Fc electron density map of a portion of the pore-forming region contoured at 1.0σ, which is overlaid with the final refined model.

[0029] [Figure 4C] Figure 4C shows an amino acid sequence alignment of the pore-forming regions between various members of the large Clostridial glucosylating toxin (LCGT) family. TcdB*, TcdB, and TcdB2 are produced by strains M68, VPI 10463, and BI / NAP1 / 027, respectively. The secondary structures of TcdB* and TcdA are shown at the top and bottom, respectively. Residues 1032-1047 of the TcdB holotoxin, which have no visible electron density, are indicated by an "x".

[0030] [Figure 4D] Figure 4D shows that TcdB at acidic pH (purple) and TcdA at neutral pH (orange) adopt dramatically different conformations in the pore-forming region. The two structures were superimposed based on the Delivery / RBD.

[0031] [Figure 4E] Figure 4E shows a calcein dye release assay. TcdB (0-25 nM) was tested in 50 mM calcein-loaded liposomes at pH 4.6 in the presence or absence of 5D or 7F.

[0032] [Figure 4F] FIG. 4F shows membrane depolarization assay. Liposomes were polarized at a positive internal voltage by adding valinomycin in the presence of a transmembrane KCl gradient. Membrane potential was measured using the voltage-sensitive fluorescent dye ANS (8-anilinonaphthalene-1-sulfonic acid). After 3 min, TcdB containing various concentrations of 5D or 7F was added. Data are presented as mean ± SEM, n=3.

[0033] [Figure 5A] FIG. 5A shows a schematic showing the locations of the β-flap, three-helical bundle (3-HB), and hinge in the primary sequence of TcdB.

[0034] [Figure 5B] Figure 5B shows the superposition of the apo CPD (grey coil) in the TcdB holotoxin with the CPD fragment bound to InsP6, where the zinc atoms of the apo CPD are shown as spheres and InsP6 is a stick model.

[0035] [Figure 5C] FIG. 5C shows that the β-flap, 3-HB, and hinge colocalize at the center of TcdB. [Figure 5D] FIG. 5D shows that the β-flap, 3-HB, and hinge colocalize at the center of TcdB.

[0036] [Figure 6] FIG. 6 shows the antibody titers of various nanobead subunit vaccines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Before the present compounds, compositions, and / or methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods or to particular compositions, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0038] 1-6, the present invention features therapeutic compositions and methods for neutralizing the primary holotoxins of C. difficile (i.e., TcdB and TcdA) to potentially treat and prevent CDI. Additionally, the present invention features methods for producing vaccines for the holotoxins of C. difficile.

[0039] As used herein, the sequence of TcdB from C. difficile is from the M68 strain (WP_003426838.1, see Table 1 below). All amino acid numbers refer to this sequence. [Table 1-1] [Table 1-2]

[0040] With reference to Table 1 and FIG. 1A, the TcdB holotoxin has an N-terminal glucosyltransferase domain (GTD) from amino acids 1 to 544, a cysteine ​​protease domain (CPD) from amino acids 545 to 841, a delivery domain / receptor binding domain (Delivery / RBD) from amino acids 842 to 1834, and a C-terminal linked repeat oligopeptide (CROP) domain from amino acids 1835 to 2367. In addition, there are three neutralizing epitopes: E3 (in the GTD, including amino acids 23 to 63); 7F (the C-terminus of the GTD immediately juxtaposed to the cleavage site encompassing amino acids 147 to 538); and 5D (part of the Delivery / RBD including amino acids 1105 to 1358). The regions included in the neutralizing epitopes are not linear in the primary amino acid sequence, but are clustered in 3D to form the epitope.

[0041] In some embodiments, the invention features therapeutic compositions that include one or more isolated polypeptides that neutralize a primary holotoxin of C. difficile. In some embodiments, the isolated polypeptides include a sequence that binds to the holotoxin and inhibits toxicity / function, thereby neutralizing the holotoxin. In some embodiments, the polypeptide sequences can be used as immunogens or targets for binding agents or other drugs.

[0042] Various immunogens of C. difficile TcdB were generated (see Table 2): TcdB-FL (full length TcdB); GTD (amino acids 1-543, SEQ ID NO:2), TD (amino acids 798-1805, sequence not shown); TD3 (amino acids 1286-1805, sequence not shown); CROP4 (amino acids 2235-2367, sequence not shown); and TD1 (amino acids 1072-1452, pore B epitope, SEQ ID NO:3). TD refers to the translocation domain.

[0043] Table 2 below lists non-limiting examples of polypeptide sequences that can be used as immunogens or as targets for binding agents or other drugs. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0044] Referring now to Table 2, the invention features isolated polypeptides including, but not limited to, the sequences listed therein. SEQ ID NO:2 refers to amino acids 1-543 of TcdB of C. difficile. SEQ ID NO:3 refers to amino acids 1072-1452 of TcdB of C. difficile, where amino acids 1072-1452 are part of the translocation domain required for pore formation. SEQ ID NO:5 refers to amino acids 1052-1472 of TcdB of C. difficile. SEQ ID NO:6 refers to amino acids 1022-1502 of TcdB of C. difficile. SEQ ID NO:7 refers to amino acids 1-533 of TcdB of C. difficile. SEQ ID NO:8 refers to amino acids 1-593 of TcdB of C. difficile. SEQ ID NO:9 refers to amino acids 1-573 of TcdB of C. difficile. SEQ ID NO: 10 refers to amino acids 1105-1358 of TcdB of C. difficile, the region encompassing the 5D epitope. SEQ ID NO: 11 refers to amino acids 23-63 of TcdB of C. difficile, the region encompassing the E3 epitope. SEQ ID NO: 12 refers to amino acids 147-538 of TcdB of C. difficile, the region encompassing the F7 epitope. SEQ ID NO: 13 refers to amino acids 1792-1845 of TcdB of C. difficile, which correspond to the hinge region. SEQ ID NO: 14 refers to amino acids 666-841 of TcdB of C. difficile, which correspond to the 3-HB region. SEQ ID NO: 15 refers to amino acids 741-841 of TcdB of C. difficile, which correspond to the beta flap region. SEQ ID NO: 16 refers to amino acids 1-541 of TcdA of C. difficile. SEQ ID NO: 17 refers to amino acids 1073-1452 of TcdA of C. difficile. SEQ ID NO: 18 refers to amino acids 22-62 of TcdA of C. difficile. SEQ ID NO: 19 refers to amino acids 146-536 of TcdA of C. difficile. SEQ ID NO: 20 refers to amino acids 1789-1840 of TcdA of C. difficile. SEQ ID NO: 21 refers to amino acids 664-842 of TcdA of C. difficile. SEQ ID NO: 22 refers to amino acids 743-842 of TcdA of C. difficile.

[0045] In some embodiments, the hinge epitope may be targeted. As used herein, the hinge epitope includes one, two, or all three of the hinge (amino acids 1792-1834), 3-HB (amino acids 766-841), and β-flap (amino acids 742-765). These three structural units are separated by amino acid sequence, but are clustered together in 3D.

[0046] In some embodiments, the isolated polypeptide comprises a peptide that is at least 50% identical to the sequence. In some embodiments, the isolated polypeptide comprises a peptide that is at least 60% identical to the sequence. In some embodiments, the isolated polypeptide comprises a peptide that is at least 75% identical to the sequence. In some embodiments, the isolated polypeptide comprises a peptide that is at least 90% identical to the sequence. In some embodiments, the isolated polypeptide comprises a peptide that is at least 98% identical to the sequence.

[0047] The invention also features an immunogen comprising at least one polypeptide according to the invention. In some embodiments, the immunogen is a bivalent immunogen specific for two polypeptides according to the invention. In some embodiments, the two polypeptides are mixed. In some embodiments, the two polypeptides are covalently linked. In some embodiments, the immunogen is a trivalent immunogen specific for three polypeptides according to the invention. In some embodiments, the three polypeptides are mixed. In some embodiments, the two or three polypeptides are covalently linked. In some embodiments, the immunogen is a tetravalent immunogen specific for four polypeptides according to the invention. In some embodiments, the four polypeptides are mixed. In some embodiments, the two, three, or four polypeptides are covalently linked.

[0048] In some embodiments, the invention features a method of neutralizing a primary holotoxin of C. difficile, in some embodiments, the method includes producing an immunogen of the C. difficile holotoxin and introducing the immunogen into a host to elicit an immune response against the immunogen, where the host produces antibodies specific for the holotoxin based on the immunogen.

[0049] As used herein, "immunogen" may refer to any compound capable of eliciting an immune response in a host. Non-limiting examples of immunogens include antigen-binding regions of heavy-chain only antibodies called binding agents, VHHs, or nanobodies (VHs). H ), an antibody, an antibody fragment, a small molecule, or a drug. Other suitable immunogens may also be considered. As used herein, a "host" may refer to a mammal, such as, but not limited to, a mouse or a human.

[0050] In some embodiments, the invention features methods for designing and producing vaccines specific for C. difficile holotoxins. In some embodiments, the vaccines may include, but are not limited to, any of the immunogens of the sequences listed in Table 2 above. In certain embodiments, the vaccines include immunogens or vaccines similar to the sequences listed above in Table 2, such as truncated versions, extended versions, or those that are homologous. The invention provides the first mouse CDI vaccine that uses the pore B epitope (SEQ ID NO:3). The invention is not limited to mouse vaccines, but includes other vaccines such as human.

[0051] The present invention also describes formulating antigens with a novel Toll-like receptor (TLR) triagonist adjuvant platform, which uses combinatorial chemistry to link three different TLR agonists together to form one adjuvant complex. The immunomodulatory activity of a panel of TLR triagonist adjuvants can be evaluated to find out whether they induce unique antigen-specific immune responses, for example, in vitro and / or in vivo. Top candidates may be evaluated to aid in the generation of effective vaccines.

[0052] The present invention also describes strategies for vaccine design and production. For example, the present invention describes a vaccine antigen (Ag) capture and in vivo delivery platform using an optimized microsphere capture system. Antigens can be attached to microspheres using tags or other chemical crosslinkers. For example, His-tagged proteins are expressed from a plasmid containing the antigen's sequence using an in vitro transcription translation (IVTT) system or in vivo (E. coli). Streptavidin-coated microspheres can be conjugated with a Tris-NTA biotin linker and used to capture the expressed protein in E. coli or from an IVTT reaction. The resulting Ag-bound microspheres are administered directly with or without a TLR agonist adjuvant to monitor the dynamics and isotype of antibody release. Ags were coated at a density of approximately 200,000 per bead. Immunogenicity studies revealed robust and durable Ag-specific responses. This demonstrates the isolation of specific proteins from complex mixtures by binding to microspheres, allowing direct immunogenicity testing to be performed in a high-throughput, scalable manner. The invention is not limited to this particular method, nor is the invention limited to the His tag.

[0053] Vaccine formulations were prepared according to Table 3. Mice were injected (SC) with the various formulations. Prime was on day 0 and Boost 1 on day 14, with 4 mice per group. Table 3 and Figure 6 show the measured midpoint titers. Ag stands for antigen alone, AV stands for Addavax, AV+TLR stands for Addavax, CpG, MPLA, TLR2,6. Figure 6 shows the antibody titers. Immunization with the non-toxic segment of C. difficile TcdB induces high antibody levels in mice. The antibody levels increase by more than 3 logs. The immune response is specific for the 381 amino acid immunogen (compared to the full-length toxin, 2367 amino acids). The antibodies induced against the 381 amino acid immunogen also react to the full-length toxin. [Table 3]

[0054] The present invention also describes methods for improving the anti-toxin activity of antibodies or binding agents, and for developing multi-domain antibodies or binding agents that simultaneously target multiple epitopes of interest (e.g., multiple neutralizing epitopes on the toxins herein).

[0055] The present invention describes targeting neutralizing epitopes to inactivate TcdB (e.g., with drugs, small molecules, binders, etc.) for the treatment of CDI. The present invention also describes the development of vaccines based on neutralizing epitopes. Immunogens or vaccines can inactivate the holotoxin, for example, by inhibiting the biological functions of individual domains that are prerequisites for its toxicity, or by promoting extracellular activation leading to its inactivation before it attacks the cell.

[0056] example The following are non-limiting examples of the present invention. It should be understood that the examples are not intended to limit the present invention in any manner. Equivalents or alternatives are within the scope of the present invention.

[0057] method TcdB produced by the M68 strain of C. difficile was used. The TcdB holotoxin and its GTD were expressed as previously described. Four VHHs (5D, E3, 7F, and B39), the GTD of TcdB produced by the VPI 10463 strain (residues 1 to 542, GTD VPI10463 (referred to as TcdB), and a truncated Delivery / RBD of TcdB (residues 1072–1433), TcdB 1072-1433 The genes encoding TD1 (residues 1072–1452 with a 10×His tag at the C-terminus) and TD2 (residues 1072–1452 with a 10×His tag at the C-terminus) were cloned into a modified pET28a vector in which a 6×His / SUMO (Saccharomyces cerevisiae Smt3p) tag was introduced at the N-terminus of all proteins. 1-1805 ) with SUMO tag and TcdB 1-1805 The mutants were cloned into a modified pET22b vector with twin-Strep tags introduced between the nucleotides and a C-terminal 6xHis tag. All mutants were generated by two-step PCR and verified by DNA sequencing.

[0058] 5D, E3, 7F, B39, GTD VPI10463 , TcdB 1-1805 , TcdB 1072-1433 , and TD1 were expressed in Escherichia coli BL21-Star(DE3) (Invitrogen). Bacteria were grown in LB medium containing kanamycin or ampicillin at 37°C. OD 600 When the ΔΨ reached approximately 0.8, the temperature was reduced to 16° C. Expression was induced with 1 mM IPTG (isopropyl-bD-thiogalactopyranoside) and continued overnight at 16° C. Cells were harvested by centrifugation and stored at −80° C. until use.

[0059] His6-tagged TcdB, GTD, and His6-SUMO-tagged 5D, E3, 7F, B39, GTD VPI10463 , TcdB 1-1805 , TcdB 1072-1433TD1 and TD2 were incubated with Ni in a buffer containing 50 mM Tris, pH 8.5, 400 mM NaCl, and 10 mM imidazole. 2+ -NTA (nitrilotriacetic acid, Qiagen) affinity resin. Proteins were eluted with high imidazole buffer (50 mM Tris, pH 8.5, 400 mM NaCl, and 300 mM imidazole) and dialyzed at 4°C against a buffer containing 20 mM Tris, pH 8.5, 1 mM TCEP, and 40 mM NaCl. 5D, E3, 7F, B39, GTD VPI10463 , TcdB 1072-1433 The His6-SUMO tags of TcdB, TcdB1, and TD1 were cleaved by SUMO protease. These proteins, as well as GTD with the TcdB holotoxin and uncleaved His tag, were further purified by MonoQ ion exchange chromatography (GE Healthcare) in a buffer containing 20 mM Tris, pH 8.5, and eluted with a NaCl gradient. 1-1805 was cleaved with SUMO protease and then further purified using streptavidin resin.

[0060] The TcdB-5D-E3-7F complex was assembled by mixing purified TcdB holotoxin with the three purified VHHs in a molar ratio of 1:2:2:2 for 2 h on ice. The complex was then purified by MonoQ ion exchange chromatography in 20 mM Tris, pH 8.5, followed by Superose 6 size exclusion chromatography (SEC; GE Healthcare) in 20 mM Tris, pH 8.5, 1 mM TCEP, and 40 mM NaCl. GTD-E3, GTD VPI10463 -7F, TcdB 1072-1433 The -5D complex was prepared by the purification of GTD, GTD VPI10463 and TcdB 1072-1433was prepared by mixing with E3, 7F and 5D, respectively, in a 1:2 molar ratio for 2 h on ice, followed by further purification using a MonoQ ion exchange column (20 mM Tris, pH 8.5) and Superdex-200 enhanced SEC (20 mM Tris, pH 8.5, 1 mM TCEP, and 40 mM NaCl). All protein complexes were concentrated to approximately 10 mg / ml and stored at -80°C until use.

[0061] Tandem online size-exclusion chromatography coupled with small-angle X-ray scattering (SEC-SAXS) experiments were performed at SSRL beamline 4-2 as previously described. Purified TcdB holotoxin was exchanged into buffer containing phosphate-buffered saline (PBS), pH 7.4, 5 mM DTT, or 20 mM sodium acetate, pH 5.0, 50 mM NaCl, 5 mM DTT, and then concentrated to 20 mg / ml. SEC-SAXS data were collected at pH 5.0 and 7.4 using a Superdex-200 Increase PC 3.2 / 300 column (GE Healthcare).

[0062] For DSSO cross-linking of TcdB, TcdB holotoxin (50 μL, 10 μM) in PBS buffer (pH 7.4) was reacted with DSSO at a molar ratio of 1:100 for 1 h at room temperature. The cross-linking reaction was quenched by adding a 50-fold excess of ammonium bicarbonate for 10 min, and the resulting product was subjected to enzymatic digestion using the FASP protocol. Briefly, the cross-linked protein was transferred to a Millipore Microcon™ Ultracel PL-30 (30 kDa filter), reduced / alkylated, and sequentially digested with Lys-C / trypsin as previously described. The resulting digest was desalted and fractionated by peptide SEC. Fractions containing cross-linked peptides were analyzed by subsequent MS analysis. n were collected for analysis. Three biological replicates were performed in this work.

[0063] LC-MS nThe analysis was performed online using a Thermo Scientific™ Dionex UltiMate 3000 system coupled with an Orbitrap Fusion Lumos™ mass spectrometer. Peptides were separated using a 50 cm x 75 μm Acclaim™ PepMap™ C18 column with a gradient of 1% to 25% ACN in 82 minutes at a flow rate of 300 nL / min. To maximize the identification of DSSO cross-linked peptides, two different types of acquisition methods were utilized.

[0064] For single molecule FRET analysis of TcdB, VHH-7F and B39 each contain a buried disulfide bond that renders the native cysteine ​​inaccessible for labeling. A cysteine ​​residue was introduced into the N-terminus of 7F (at position -1) or into the surface-exposed loop of B39 (G42C) by mutagenesis. Expression and purification of mutant VHHs were similar to the wild-type protein, except that 5 mM DTT was used in all buffers during purification. Purified 7F was labeled with an acceptor dye (Alexa-647 maleimide) and B39 was labeled with a donor dye (Alexa-555 maleimide) (Thermo Fisher Scientific). Labeling efficiency was determined to be >90% by UV-Vis spectroscopy. Purified 5D was biotinylated at pH 6.8 using EZ-Link NHS-PEG4-Biotin (Thermo Fisher Scientific) to preferentially label the N-terminal amine. TcdB holotoxin complexed with Alexa-647-labeled 7F, Alexa-555-labeled B39, and biotin-labeled 5D was further purified using Superose 6SEC to remove excess VHHs.

[0065] Prior to the addition of streptavidin, cleaned quartz slides were passivated with biotinylated bovine serum albumin, followed by a mixture of 2% Biolipidure 203 and 0.2% Biolipidure 206 (NOF America Corp.). After this treatment, preformed TcdB-3VHH complexes showed no nonspecific binding to the slides at concentrations orders of magnitude higher than the 100 pM concentration used to achieve single-molecule optical resolution.

[0066] At such low protein concentrations, non-covalently bound VHHs partially dissociate, so measurements had to be performed quickly and surface treatments had to be repeated seven times for each pH condition. Samples were imaged using a prism-based total internal reflection fluorescence microscope. Samples were excited with a laser diode at 637 nm for Alexa-647 (Coherent Inc., Santa Clara, CA) and a diode pumped solid-state laser at 532 nm for Alexa-555 (Laser Quantum USA, Fremont, CA). Emissions from donor and acceptor were separated using an Optosplit ratiometric image splitter (Cairn Research Ltd, Faversham, UK) equipped with a 645 nm dichroic mirror with a 585 / 70 bandpass filter for the donor channel and a 670 / 30 bandpass filter for the acceptor channel (IDEX Health&Science, Rochester, NY). Duplicate images were relayed to a single iXon DU-897 EMCCD camera (Andor Technologies, Belfast, UK) at a frame rate of 10 Hz.

[0067] Duplicate images were cross-correlated and the data processed with a home-written MATLAB script to extract time traces of diffraction-limited spots of intensity above the baseline. From the fluorescence intensity traces over time of the individual complexes, only complexes containing a single donor and acceptor dye that showed anti-correlated photobleaching relative to the baseline at a single time step were selected. The magnitude of the anti-correlated photobleaching events allowed a per-molecule gamma normalization to be performed, which allows the reporting of absolute FRET efficiencies. FRET efficiencies were compiled into histograms that were fitted to a Gaussian function.

[0068] To ensure that the changes in FRET were not the result of photophysical changes, the relative quantum yield and fluorescence anisotropy were measured for free dyes, dye-labeled VHHs, and individual dye-labeled VHHs complexed with TcdB. All measurements were performed at a dye concentration of 10 nM using the same buffers as for smFRET at pH 7 (50 mM Hepes, 100 mM NaCl, pH 7) and pH 5 (50 mM sodium acetate, 100 mM NaCl, pH 5).

[0069] Ensemble fluorescence was recorded on an ISS PC1 photon-counting spectrofluorometer using an excitation slit of 2.0 mm and an emission slit of 2.0 mm. Samples labeled with Alexa-555 and Alexa-647 were excited at 532 nm and 637 nm, respectively. The concentrations of samples used for fluorescence were determined from absorbance measurements using the same cuvette. Emission intensity was taken as the sum of a 20 nm window about the emission maximum. Relative quantum yields were calculated by normalizing the intensity to the emission of free dye at pH 7. Anisotropy measurements were collected with an excitation slit of 2.0 mm and an emission slit of 2.0 mm. Emission was recorded at 567 nm and 670 nm for the donor and acceptor, respectively. All measurements were performed in triplicate and reported as the mean and standard error.

[0070] Dynamic light scattering (DLS) was performed using a Zetasizer Nano S (Malvern Panalytical). TcdB was assayed at a concentration of 0.2 mg / ml in PBS buffer in a 200 μl volume cuvette at room temperature. Data was analyzed using Zetasizer version 7.13 software.

[0071] For calcein dye release assay, liposomes were prepared by extrusion using an Avanti Mini Extruder according to the manufacturer's protocol. Briefly, lipids (Avanti Polar Lipid) at the indicated molar ratios were mixed in chloroform, then dried under nitrogen gas and placed under vacuum overnight. The dried lipids were rehydrated and subjected to five rounds of freeze-thaw cycles. Unilamellar vesicles were prepared by extrusion through a 200 nm pore membrane using an Avanti Mini Extruder according to the manufacturer's instructions.

[0072] Dry lipids containing 55% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 15% 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and 30% cholesterol (10 mg / ml) were resuspended in 150 mM NaCl, 20 mM Hepes (pH 7.0), 1 mM EDTA, 50 mM calcein. Free calcein dye was separated from calcein-entrapped liposomes by desalting (Zeba). Fluorescence was measured in a Spectramax M2e cuvette module with excitation at 493 nm and emission at 525 nm. For the assay, liposomes were diluted in 150 mM NaCl, 20 mM sodium acetate (pH 4.6), 1 mM EDTA to a final concentration of 0.3 mM and incubated until the fluorescent signal stabilized. TcdB alone (0–25 nM) or TcdB preincubated with 5D or 7F at a molar ratio of TcdB:VHH = 1:2 was added, and the fluorescence intensity was recorded for 7 min. The reaction was stopped by adding 0.1% Trion X-100. The percentage of fluorescence change was calculated as (FF 開始時 ) / (F 終了時 -F 開始時The initial rate of calcein dye release was estimated from the slope of the linear portion of the curve. Experiments were repeated three times independently.

[0073] Membrane depolarization was measured as previously described with some modifications. Briefly, liposomes composed of 55% DOPC, 15% DOPS, and 30% cholesterol were prepared in 200 mM NaCl, 1 mM KCl, and 10 mM Hepes (pH 7.0). To create a trans-positive membrane potential (+135 mV), liposomes were diluted in 200 mM KCl, 1 mM NaCl, and 10 mM sodium acetate (pH 4.6) to a final concentration of 0.1 mM. Membrane potential was monitored using 12 μM ANS. Valinomycin was added at 0 s to a final concentration of 30 nM. At 180 s, 100 nM TcdB holotoxin alone or TcdB preincubated with 0.02–1 μM 5D or 1 μM 7F was added, and the fluorescence intensity at 490 nm was monitored for 7 min with an excitation of 380 nm. The reaction was stopped by adding 2 μM gramicidin from Bacillus anerinolyticus (Sigma-Aldrich). The fluorescence change relative to the maximum change in the presence of gramicidin was calculated as (FF 開始時 ) / (F 終了時 -F 開始時 The experiment was repeated three times independently.

[0074] TcdB autoprocessing assays were performed in 25 μl of 20 mM Tris-HCl, pH 8.0, containing 0.4 μM TcdB holotoxin or TcdB 1-1805 , InsP6 was included at the indicated concentrations with or without 7F (2 μM). The reaction mixture was incubated at 37 °C for 1 h and then boiled in SDS sample buffer for 5 min to quench the reaction. Samples were examined by 4-20% SDS-PAGE, and TcdB fragments were visualized by Coomassie blue staining.

[0075] Crystal structure of full-length TcdB Full-length TcdB holotoxin from the M68 strain of C. difficile was expressed in a well-validated Bacillus megaterium system and purified to high homogeneity. After extensive crystallization screening and optimization, as well as testing of numerous crystals at the synchrotron, the best X-ray diffraction data were collected at 3.87 Å resolution for crystals of a heterotetrameric complex composed of TcdB and three neutralizing VHHs (5D, E3, and 7F). The TcdB-VHH complex was crystallized at pH 5.2, the physiologically relevant pH in endosomes (Figure 1A, Figure 1B, and Table 2). The complete structure of TcdB holotoxin was constructed with the exception of two small regions (residues 944–949 and 1032–1047) that have no visible electron density due to high structural flexibility (Figure 1B).

[0076] The crystal structure shows that TcdB is composed of three major components: the GTD (residues 1–544) and CPD (residues 545–841) form a central element involving extensive interdomain interactions. The Delivery / RBD (residues 842–1834) forms an extension module, interacting with both the GTD and CPD on one side and pointing away from the GTD / CPD. The most striking finding is the elongated CROP domain (residues 1835–2367) that emerges from the junction of the CPD and Delivery / RBD and extends for ∼130 Å in the opposite direction to curve around the GTD like a hook (Figure 1B). The overall configuration of TcdB at endosomal pH differs from the structural models of TcdB and TcdA obtained from EM studies at neutral pH, in which the CROP interacts in parallel with the Delivery / RBD. Furthermore, the hydrophobic pore-forming region of TcdB (residues 957–1129 of Delivery / RBD) was observed in a conformation distinct from that seen in the TcdA fragment near neutral pH, which may represent a rarely seen pore-forming intermediate state of TcdB at endosomal pH that was “frozen” by a neutralizing antibody (5D).

[0077] Unique structure of the CROP domain The CROP of TcdB is composed of two types of repeat sequences, including 20 short repeats of 20–23 residues (termed SRs) and four long repeats of 30 residues (termed LRs) (Figure 2A). Each SR is composed of a β-hairpin followed by a flexible loop, and each LR has three β-strands that form a twisted antiparallel β-sheet together with the β-hairpin of the previous SR. The curvature of the CROP occurs because the straight rod-like segments of the β-solenoid composed of SRs are interrupted by interspersed LRs, inducing a twist of approximately 132–146° (Figure 2B, Figure 2C). Structurally, the CROP can be divided into four equivalent units (termed CROP I–IV), each composed of an SR1-SR2-SR3-LR-SR4-SR5 module (Figure 2C). Superposition of CROP I–IV yielded Cα root-mean-square deviations (rmsd) of approximately 0.9–2.6 Å.

[0078] Interestingly, an unrecognized SR module (residues 1815–1834) was identified at the C-terminus of Delivery / RBD, similar to all other SRs. This new SR forms a structurally distinct module (residues 1792–1834) (herein referred to as the “hinge”) together with a long upstream loop and a short α-helix to connect Delivery / RBD to the elongated CROP. Furthermore, the hinge directly interacts with the three-stranded β-sheet of the CPD (residues 742–765, referred to as the β-flap), which is important for CPD activation, as well as with a three-helical bundle (residues 766–841, referred to as the 3-HB) located in a cleft surrounded by the GTD, CPD, Delivery / RBD, and CROP (Figure 2D, Figure 2E). Due to its strategic location, this hinge is primed to mediate structural communication between all four domains of TcdB. The functional role of this hinge is supported by previous studies showing that deletion of this region dramatically reduced toxicity. Furthermore, hypervariable sequences near the hinge may contribute to differences in virulence and antigenicity exhibited by TcdB mutants generated by the hypervirulent C. difficile 027 ribotype and other hypovirulent strains.

[0079] TcdB exhibits distinct structures at neutral and acidic pH Because the structure of TcdB holotoxin was derived from crystals grown at acidic pH, its solution structure was further investigated using online size-exclusion chromatography coupled with SAXS (SEC-SAXS) at pH 5.0 and pH 7.4, respectively. Curve-fit analysis showed that the scattering profile calculated based on this crystal structure was nearly identical to the experimental scattering profile at pH 5.0, suggesting that the solution structure of TcdB is similar to the crystal structure at pH 5.0. However, discrepancies in the mid-angle (mid-angle q) region of the scattering profile between the experimental SAXS data at pH 7.4 and the calculated profile of the crystal structure suggest that TcdB adopts a different conformation at neutral pH (Figure 3A). Guinier and P(r) analyses showed similar R(r) at pH 5.0 and 7.4. g The D value was shown to be 233.0 Å at pH 5.0. max was longer than that at pH 7.4 (approximately 205.0 Å). max is comparable to that predicted from the crystal structure (~247 Å). However, the short D max is comparable to the value predicted for the TcdB core consisting of the GTD, CPD, and Delivery / RBD (~203 Å), suggesting that at pH 7.4, the elongated CROP may swing toward the TcdB core and adopt a more compact conformation.

[0080] To better characterize the conformation of CROP at pH 7.4, we used an XL-MS strategy to determine the interdomain interactions of TcdB using DSSO (disuccinimidyl sulfoxide), a sulfoxide-containing MS-cleavable crosslinker. A total of 87 crosslinks were identified, representing 27 interdomain and 60 intradomain interactions in TcdB at pH 7.4. Among them, 8 pairs, 4 pairs, and 8 pairs of unique crosslink peptides were identified between the GTD and CPD, the GTD and Delivery / RBD, and the CPD and Delivery / RBD, respectively (Figure 3B). When the XL-MS data were mapped to this crystal structure, almost all of these crosslinks met the distance cutoff of 30 Å, showing good correlation with the crystal structure of TcdB.

[0081] Interestingly, seven pairs of bridging peptides were identified between CROP and Delivery / RBD, which correspond to Cα-Cα distances ranging from 90 Å to 210 Å as measured in this crystal structure. This suggests that the CROP of TcdB may be much closer to Delivery / RBD at neutral pH than observed in this crystal structure. Specifically, the central part of CROP around residues K1965 and K1977, and the C-terminal tip of CROP around residues K2234 and K2249 should be able to move within about 30 Å of Delivery / RBD (Figure 3C). This new conformation is consistent with the D of TcdB at pH 7.4 obtained from SAXS studies. max and resembles the "closed" conformation of TcdA at neutral pH. Because XL-MS allows for the capture of dynamic and transient contacts in addition to stable structures, it remains unclear how much time TcdB spends in the "closed" TcdA-like conformation at neutral pH.

[0082] pH-dependent structural flexibility of CROP We next used smFRET to investigate the pH-dependent conformational changes of CROPs. smFRET is an established method for investigating protein structure and conformational changes and can distinguish individual species in heterogeneous or dynamic mixtures. Because TcdB has nine cysteine ​​residues, with C699 being essential for CPD function, we used three VHHs (7F, B39, and 5D) as molecular tools to label and capture TcdB rather than chemically labeling the toxin. Specifically, an acceptor dye (Alexa-647) was conjugated to a cysteine ​​residue introduced at the -1 position of 7F, which labels the core of the TcdB holotoxin. A donor dye (Alexa-555) was conjugated to B39, which specifically binds to CROPs IV (PDB code: 4NC2). Given the structure of the TcdB holotoxin, the distance between the two dyes is approximately 47 Å. Energy transfer between these two dye-labeled VHHs monitors the movement of CROPs (Figure 3D). Biotin-labeled 5D, which does not affect the conformational changes of TcdB based on ensemble FRET studies, was used for immunopull-down of TcdB onto passivated quartz microscope slides. The three VHHs were preassembled with TcdB, and the complex was purified by size-exclusion chromatography.

[0083] From the fluorescence intensity traces over time of individual heterotetrameric TcdB-VHH complexes, we selected only those complexes containing a single donor and acceptor dye that showed anti-correlated photobleaching relative to the baseline at a single time step. The magnitude of the anti-correlated photobleaching events was used to perform a per-molecule gamma normalization, which allows the reporting of absolute FRET efficiencies. The FRET efficiencies were compiled into a histogram, revealing a single FRET peak at both pH 5.0 and 7.0 (Figure 3E). The presence of a single peak is consistent with a static structure or dynamic averaging faster than 100 ms time binning, which cannot be distinguished with a single FRET pair.

[0084] A statistically significant difference in the average FRET efficiency was observed at pH 5.0 (0.532 ± 0.015) and pH 7.0 (0.484 ± 0.007), supporting the notion that TcdB exhibits a pH-dependent conformational change (Figure 3E). A simple calculation from the average FRET efficiency between dye-labeled VHHs at pH 5.0 yielded an estimated distance of 49.9 ± 0.05 Å, which is consistent with the crystal structure of the TcdB holotoxin at acidic pH (~47 Å). Similar results were observed at pH 5.5 and pH 5.25. At pH 7.0, the average FRET efficiency suggested that the distance between the labeled sites increased to 51.5 ± 0.05 Å. A single FRET pair is insufficient to position the CROP relative to the rest of TcdB, and changes in conformational dynamics affect the simple conversion from FRET to distance. This small increase in apparent average FRET was accompanied by a statistically significant 25% decrease in the distribution width at pH 7.0 (0.113 ± 0.002) compared to pH 5.0 (0.141 ± 0.026), consistent with an increased rate of conformational dynamics.

[0085] So far, two limiting structural states have been identified for TcdB: an “open” conformation at acidic pH supported by crystal structure, SAXS, and smFRET studies, and a “closed” conformation at neutral pH revealed by SAXS and XL-MS (Figure 3D). These data together suggest that CROP likely samples an ensemble of conformations associated with the core of TcdB at neutral pH, and such protein dynamics are not resolved by the 100 ms integration time of smFRET. The lack of stable contacts between CROP and the TcdB core, and potential structural rearrangements of the hinge connecting Delivery / RBD and CROP, could allow such conformational sampling.

[0086] Pore-forming intermediate state of TcdB at endosomal pH Delivery / RBD serves to shield the hydrophobic pore-forming region (residues 957–1129) predicted to be released upon endosomal acidification to form a pore that delivers the GTD and CPD to the cytosol. The pore-forming activity of TcdB also contributes to the cell necrosis observed in vitro. Structural comparison of the TcdB holotoxin at acidic pH and the TcdA fragment at neutral pH revealed dramatic differences in the homologous C-terminal portion of the pore-forming region (residues 1032–1134 in TcdB and 1033–1135 in TcdA) (Figure 4A,B). In TcdA, this region adopts a mixed α / β configuration, with the hydrophobic residues shielded by a continuous groove formed primarily by the β-sheet of Delivery / RBD (Figure 4C,D). However, in the acidic conformation of TcdB, no electron density was found for residues 1032-1047, likely due to high flexibility, indicating that these residues unfolded and dissociated from the toxin core at endosomal pH. Furthermore, TcdB residues corresponding to α2 in TcdA unfolded into a loop, and TcdB residues corresponding to β3 and part of α3 in TcdA assembled into a new helix occupying the same area as the original α3 in TcdA. Due to this transition, hydrophobic residues in TcdB (residues 1084-1094), which correspond to the C-terminal part of α3 in TcdA, bulged out as an extended loop. Interestingly, the conformational change did not extend to the region where TcdB is bound by 5D, which maintains a similar conformation to that observed in TcdA.

[0087] To further analyze the contribution of acidic pH and 5D to the conformational changes observed in the pore-forming region, we analyzed the Delivery / RBD fragment TcdB complexed with 5D at pH 8.5. 1072-1433 The crystal structure of TcdB was determined (Table 2). 1072-1433The pore-forming domain observed at pH 8.5 in T. elegans was found to adopt a neutral pH conformation like that of TcdA, thus suggesting that the novel conformation of the pore-forming domain observed in the TcdB holotoxin likely represents an intermediate state induced by endosomal pH.

[0088] Furthermore, we found that the binding mode of 5D to TcdB is nearly the same at pH 8.5 and 5.2, involving all three complementarity determining regions (CDRs) of 5D. The overall binding affinity of 5D is further strengthened by extensive polar and hydrophobic interactions involving TcdB residues outside the pore-forming region. Thus, 5D can modify the conformation of β4-β5 in TcdB, which prevents pH-induced conformational changes of the β4-β5-α4 module. Previous mutagenesis studies have shown that mutations introduced around the 5D binding site in TcdB effectively inhibit pore formation and cytotoxicity, and mutating L1107 (L1107K), the target of 5D, alone results in more than 1,000-fold reduced toxicity. These findings suggest that 5D likely inhibits the conformational changes required for pore formation by TcdB at endosomal pH.

[0089] To test this hypothesis, we investigated how 5D affects membrane insertion of TcdB using two complementary assays. By monitoring the ability of TcdB to permeabilize calcein-entrapped liposomes, we found that TcdB increased the rate of calcein release at pH 4.6 in a protein concentration-dependent manner (Figure 4E). Preincubating TcdB with 5D significantly reduced the rate of dye release by TcdB. As a control, 7F, which binds to GTD, did not affect dye release induced by TcdB. Further studying the effect of 5D or 7F on TcdB to dissipate valinomycin-induced membrane potential in liposomes, we found that 5D, but not 7F, reduced the ability of TcdB to depolarize membranes (Figure 4F).

[0090] Taken together, these findings suggest that 5D neutralizes TcdB by preventing the pore-forming region from completing the necessary pH-induced conformational changes. Notably, the pore-forming region recognized by 5D is highly conserved among a large family of clostridial glucosylating toxins (LCGTs), including TcdA and TcdB, C. novyi alpha toxin (Tcnα), C. sordellii lethal and hemorrhagic toxins (TcsL and TcsH), and C. perfringens toxin (TpeL) (Figure 4C). Thus, this portion of the pore-forming region represents an excellent target for the development of broad-spectrum vaccines and antibodies targeting TcdA, TcdB, other LCGTs, or other suitable targets.

[0091] Regulation of TcdB autoprocessing Activation of the CPD by InsP6 upon cell entry is a key step in regulating the pathology of TcdA and TcdB. Overall, the structures of the apo-CPD of the TcdB holotoxin and the InsP6-bound CPD fragment (PDB:3PEE) are highly similar (rmsd of approximately 1.1 Å), except for the β-flap (Figure 5A, Figure 5B). The structure of the apo-CPD is 542 SL 544 Based on the truncated sequence of TcdB holotoxin (PDB:3PA8), we compared the structures of the CPD fragment bound to InsP6 or a peptide inhibitor and found that the β-flap partially occupies the P1 substrate pocket of the CPD of TcdB holotoxin, preventing substrate binding. In the CPD fragment, InsP6 induces a rotation of the β-flap by approximately 90° (Figure 5B), which activates the CPD by properly ordering the active site and the substrate pocket. However, in the TcdB holotoxin, such a rotation of the β-flap is not possible, because it would otherwise sterically clash with the subsequent 3-HB (Figure 5D).

[0092] In addition to allosteric regulation by InsP6, several studies suggested that CROP also affects TcdB autoprocessing. The efficiency of InsP6-induced GTD cleavage was compared using the TcdB holotoxin and a truncated TcdB lacking the hinge and CROP (residues 1–1805). InsP6-induced GTD cleavage was mediated by TcdB 1-1805 In the absence of CROP, the TcdB fragment carrying the hinge (residues 1–1832) showed weaker InsP6-dependent GTD cleavage than that without the hinge (residues 1–1795). These data suggest that the hinge is involved in regulating TcdB self-processing. Notably, in the TcdB holotoxin, the hinge interacts with the β-flap and 3-HB to form the "heart" of TcdB, connecting all four domains (Figure 5C, Figure 5D). Because the β-flap and 3-HB are important in linking between InsP6 binding and CPD activation, the structural rearrangement of the hinge associated with the pH-dependent movement of CROP may contribute to regulating CPD function.

[0093] VHH 7F and E3 reveal two distinct neutralizing epitopes on the GTD Although 7F inhibits GTD cleavage, it does not directly interact with the CPD. Instead, 7F binds to the C-terminus of GTD and is immediately juxtaposed to the cleavage site (L544). In particular, the CDR3 of 7F binds to the α-helix upstream of the scissile bond (residues 525-539) and to the adjacent α-helix (residues 137-158) through extensive polar and hydrophobic interactions. Such interactions interfere with the movement of the scissile bond to the CPD cleavage site and with the proper orientation of the GTD relative to the CPD, thus inhibiting GTD cleavage.

[0094] By specifically targeting the GTD, E3 inhibits glucosylation of Rho and blocks the cytopathic effects of TcdB. In two crystal structures solved independently using a GTD fragment or TcdB holotoxin, E3 binds to the N-terminal four-helix bundle (residues 1–90) in a similar manner. More specifically, E3 recognizes the second and third helices (residues 21–64) of the GTD with extensive polar and hydrophobic interactions. Because no structure of the GTD-Rho complex has been reported, it is unclear how E3 affects GTD-Rho interaction or catalysis. Homologous four-helix bundles are also found in the glucosyltransferase domains of other LCGT members and may be involved in plasma membrane binding of the glucosyltransferase domain, suggesting that E3 may interfere with membrane binding of GTD. Thus, the structure of the GTD-E3 complex lays the foundation for further validation and exploitation of these mechanisms as novel strategies to counteract TcdB and potentially other LCGT members.

[0095] While preferred embodiments of the present invention have been shown and described, it will be readily apparent to one skilled in the art that modifications may be made without departing from the scope of the appended claims. Accordingly, the scope of the present invention should be limited only by the following claims. In some embodiments, the drawings presented in this patent application are drawn to scale, including angles, dimensional ratios, etc. In some embodiments, the figures are merely representative, and the claims are not limited by the dimensions of the figures. In some embodiments, the description of the invention set forth herein using the phrase "comprising" includes embodiments that may be described as "consisting essentially of" or "consisting of," and thus satisfies the written description requirement for claiming one or more embodiments of the invention using the phrase "consisting essentially of" or "consisting of."

[0096] The reference numbers appearing in the following claims are for ease of review of this patent application only, are exemplary, and are not intended to limit the scope of the claims to the specific features having corresponding reference numbers in the drawings. [Sequence List Free Text]

[0097] Sequence Listing 4 <223> TD1 immunogen (amino acids 1072-1452) + peptide linker + 10xHis tag Sequence Listing 4 <223> Peptide Linker Sequence Listing 4 <223> His Tag

Claims

1. 1. An immunogen comprising at least one isolated immunogenic polypeptide that is a fragment of the TcdB holotoxin of Clostridium difficile, wherein the amino acid sequence of the isolated immunogenic polypeptide is at least 98% identical to the sequence of SEQ ID NO:

3.

2. 10. The immunogen of claim 1 for use in neutralizing a holotoxin of Clostridium difficile, wherein the immunogen is introduced into a host to elicit an immune response against the immunogen, and the host produces antibodies specific to the holotoxin based on the immunogen.

3. A vaccine comprising the immunogen of claim 1 or 2.

4. 4. The vaccine of claim 3, which is specific for the holotoxin of Clostridium difficile.

5. 5. The vaccine of claim 3 or 4 which is a human vaccine.

6. A method for designing and producing a vaccine specific for a Clostridium difficile holotoxin, comprising: a) expressing a tagged immunogen comprising at least one polypeptide that is a fragment of the TcdB holotoxin of Clostridium difficile, wherein the amino acid sequence of said polypeptide is at least 98% identical to the sequence of SEQ ID NO:3; b) entrapment of said tagged immunogens in microspheres; A method comprising:

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