Fusion immunogens targeting clostridioides difficile infection

Abstract

The present disclosure relates to methods, compositions, kits, and expression systems to treat or prevent Clostridioides difficile infection or recurrence.

Description

[0001] Docket No. 11001-247W01

[0002] FUSION IMMUNOGENS TARGETING Clostridioides difficile INFECTION CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U. S. Provisional Patent Application No. 63 / 777,056 filed on March 25, 2025, the disclosure of which is expressly incorporated by reference herein in their entireties.

[0004] REFERENCE TO SEQUENCE LISTING

[0005] The sequence listing submitted on, as an. XML entitled “11001-247W01_ST26.xml” created on March 22, 2026, and having a file size of 57,344 bytes is hereby incorporated by¬ reference pursuant to 37 C. F. R. § 1.52(e)(5).

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under grant R01AI132711 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] FIELD

[0009] Disclosed herein are methods and compositions for treating and preventing Clostridioides difficile infection.

[0010] BACKGROUND

[0011] Clostridioides difficile ( C. difficile) is a Gram-positive, spore-forming enteric pathogen responsible for a significant public health burden due to its ability to cause intestinal disorders, including inflammation and diarrhea. These symptoms primarily result from the bacterium’s production of toxins, which disrupt the intestinal epithelial barrier and trigger severe inflammatory responses. C. difficile infection (GDI) is a leading cause of healthcare-associated infections globally, with increased prevalence, morbidity, and mortality rates, particularly in hospitalized and immunocompromised patients.

[0012] Standard treatment options for GDI primarily involve antibiotics such as metronidazole, vancomycin, and fidaxomicin. However, these antibiotics have significant limitations. Although they can effectively reduce the bacterial load, they also disrupt the gut microbiota, increasing the risk of recurrent infections. GDI recurrence rates remain high, with approximately 20-30% of patients experiencing a relapse after their first episode, and the riskDocket No. 11001-247W01

[0013] increases with subsequent infections. Additionally, the emergence of hypervirulent C. difficile strains has further complicated treatment outcomes, contributing to higher recurrence rates, more severe disease presentations, and increased mortality.

[0014] Given the limitations of antibiotic therapies and the high recurrence rates associated with GDI, there is a critical need for alternative preventive strategies. Vaccination represents a promising approach to address these challenges by inducing protective immunity against C. difficile, thereby reducing both primary infections and recurrences. Despite efforts to develop vaccines targeting C. difficile toxins, clinical trials have yet to yield an approved vaccine. A more comprehensive approach is needed to improve vaccine efficacy.

[0015] While previous research has primarily focused on toxin-based vaccines, there remains a gap in understanding other potential C. difficile-based antigens as vaccine targets. There is limited information on different C. difficile strains and their potential to elicit a robust and protective immune response. Additionally, the protective efficacy of C. difficile-based antigen immunization in animal models has not been fully elucidated.

[0016] Therefore, there is a need for vaccines against GDI, especially ones that have a potential to provide long-lasting immunity against GDI, reduce recurrence rates, and alleviate the global burden of this infection. Such vaccines can complement or in some cases replace existing toxin- targeted vaccines, offering a broader and more effective preventive strategy against GDI. New methods and compositions for treating and preventing C. difficile infection are also needed. The compositions, methods, and vaccines disclosed herein address these and other needs.

[0017] SUMMARY

[0018] Disclosed herein are methods and compositions of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing C. difficile infection in a subject in need thereof.

[0019] In one example, disclosed herein is a fusion immunogen, comprising: a first segment comprising a full length Clostridioides difficile exosporium cysteine-rich protein (CdeC) polypeptide or at least one CdeC immunogenic fragment; a second segment comprising a full length Clostridioides difficile exosporium cysteine -rich protein M (CdeM) polypeptide or at least one CdeM immunogenic fragment; and a linker between the first segment and the second segment, wherein the fusion immunogen elicits an immune response against one or both of CdeC and CdeM polypeptides.

[0020] In some examples, the fusion immunogen comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a sequence having at least 95%, 98%, 99%’ or 99.8% identity thereto.Docket No. 11001-247W01

[0021] In some examples, the at least one CdeC immunogenic fragment or the at least one CdeM immunogenic fragment is selected from R20291, RT244, RT019, RT027, RT012, RT125, or RT106 ribotypes of C. difficile.

[0022] In some examples, the at least one CdeC immunogenic fragment is about 6 to about 50 amino acids in length.

[0023] In some examples, the at least one CdeC immunogenic fragment comprises an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 7, or a sequence having at least 90% identity thereto.

[0024] In some examples, the at least one CdeM immunogenic fragment is about 6 to about 50 amino acids in length.

[0025] In some examples, the at least one CdeM immunogenic fragment comprises an amino acid sequence as set forth in SEQ ID NO: 5, or a sequence having at least 90% identity thereto.

[0026] In some examples, the at least one CdeC immunogenic fragment or the at least one CdeM immunogenic fragment comprises a B cell epitope, a CD4+ T cell epitope, a CD8+ T cell epitope or a combination thereof.

[0027] In some examples, the linker is a flexible linker, a rigid linker, a protease-cleavable linker, or a peptide spacer.

[0028] In some examples, the linker comprises a glycine -rich linker, a serine-rich linker, a G4S linker, a GPGPG linker, a KK linker, or a ggtggcggt linker.

[0029] In some examples, the fusion immunogen further comprises a heterologous signal peptide, a carrier domain, a scaffold domain, a secretion sequence, a cell wall anchoring domain, or a surface display domain.

[0030] In some examples, the cell wall anchoring domain comprises an LPXTG motif¬ containing anchoring sequence.

[0031] In some examples, the fusion immunogen further comprises an adjuvant peptide, a mucosal targeting peptide, or an immunostimulatory domain.

[0032] In some examples, the at least one CdeC immunogenic fragment comprises a CD8+ T cell epitope, wherein the CD8+ T cell epitope is selected from YTDEINSED (SEQ ID NO: 11), RNCETTFEF (SEQ ID NO: 12), LEDFDLDPL (SEQ ID NO: 13), CTEFVALAF (SEQ ID NO: 14), FVALAFPAV (SEQ ID NO: 15), AVRAGGGCK (SEQ ID NO: 16), RVDYVEFTF (SEQ ID NO: 17), LPADGRAVT (SEQ ID NO: 18), YELIIPNDI (SEQ ID NO: 19), RAVTLRQEY (SEQ ID NO: 20), CEPFYELII (SEQ ID NO: 21), or IQPRLVDTF (SEQ ID NO: 22).Docket No. 11001-247W01

[0033] In some examples, the at least one CdeC immunogenic fragment comprises a CD4+ T cell epitope, wherein the CD4+ T cell epitope is selected from NFSVSNAVPFAIEAN (SEQ ID NO: 23), CEPFYEEIIPNDIDE (SEQ ID NO: 24), DTMQFQTFTDATGPN (SEQ ID NO: 25), EEVYTDEINSEDMRG (SEQ ID NO: 26), EFTFNTLSAPICLPA (SEQ ID NO: 27), EVYTDEINSEDMRGF (SEQ ID NO: 28), MQFQTFTDATGPNGE (SEQ ID NO: 29), QRGLTVAVRNLVEEL (SEQ ID NO: 30), RNFSVSNAVPFAIEA (SEQ ID NO: 31), SNDGIVIDTGMTTLE (SEQ ID NO: 32), or VEFTFNTLSAPICLP (SEQ ID NO: 33). In some examples, the at least one CdeC immunogenic fragment comprises a B cell epitope, wherein the B cell epitope is selected from YVEFTFNTLSAPICLP (SEQ ID NO: 34), NFSVSNAVPFAIEANR (SEQ ID NO: 35), VGRNCETTFEFAVCGE (SEQ ID NO: 36), DMRGFKKSHHHNGCNT (SEQ ID NO: 37), PNPIQPRLVDTFSKVC (SEQ ID NO: 38), YTDEINSEDMRGFKKS (SEQ ID NO: 39), PSAGQASVTIEKICLS (SEQ ID NO: 40), EVFGSIPSAGQASVTI (SEQ ID NO: 41), or IVVLASPNPIQPRLVD (SEQ ID NO: 42).

[0034] In some examples, the at least one CdeM immunogenic fragment comprises a CD8+ T cell epitope, a CD4+ T cell epitope or a B cell epitope, wherein the CD8+ T cell epitope, the CD4+ T cell epitope or the B cell epitope, is selected from REREAREAF (SEQ ID NO: 43), ERFRREAEIREREAR (SEQ ID NO: 44), KYNYKGIEYLAEAAR (SEQ ID NO: 45), CERFRREAEIREAREA (SEQ ID NO: 46), NCERFRREAEIRERE (SEQ ID NO: 47), GMECEARRNGNNGGNN (SEQ ID NO: 48), EYEREAYDEDRERRGS (SEQ ID NO: 49), EAEIREREAREAFCES (SEQ ID NO: 50), or REAREAFCESSEKKKE (SEQ ID NO: 1).

[0035] In some examples, the fusion immunogen is selected from a CdeM-EF peptide comprising a sequence as set forth in SEQ ID NO: 5 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto; CdeC-EF-1 peptide comprising a sequence as set forth in SEQ ID NO: 6 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto; CdeC-EF-2 peptide comprising a sequence as set forth in SEQ ID NO: 7 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto; CdeCM-EF-1 peptide comprising a sequence as set forth in SEQ ID NO: 8 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto; CdeCM-EF-2 peptide comprising a sequence as set forth in SEQ ID NO: 9 or a sequence having at least 95%, 98%, 99% or 99.8% or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

[0036] In some examples, the first segment comprises a full length CdeC polypeptide comprising a sequence as set forth in SEQ ID NO: 4 or a sequence having at least 90% identity thereto.Docket No. 11001-247W01

[0037] In some examples, the full length CdeC polypeptide comprises at least one conserved region selected from a KKNKRR motif, a HUH motif, six NPC repeats, two CCRQGKGK repeats, or a CNECC motif.

[0038] In some examples, the second segment comprises a full length CdeM polypeptide comprising a sequence as set forth in SEQ ID NO: 3 or a sequence having at least 90% identity thereto.

[0039] In some examples, the full length CdeM polypeptide comprises at least one conserved region selected from RREA repeats, NGNNGGNNNNC repeats, or CNCCNCCRK repeats.

[0040] In some examples, one or more amino acids as set forth in SEQ ID NO: 4 or SEQ ID NO: 3 are substituted by a conservative substitution.

[0041] In one example, disclosed herein is a method of producing a fusion immunogen, comprising: cloning a polynucleotide encoding a fusion immunogen as set forth in SEQ ID NO: 1 into a bacterial plasmid; expressing the fusion immunogen in Escherichia coli BL21(DE3), wherein the fusion immunogen comprises an amino acid sequence as set forth in SEQ ID NO: 2; and purifying and obtaining the fusion immunogen.

[0042] In some examples, the bacterial plasmid comprises a pET vector, a pBAD vector, a pGEX vector, a pMAL vector, a pQE vector, a pTrc99A vector, or a pCDF vector.

[0043] In some examples, the bacterial plasmid is a pET28a vector.

[0044] In some examples, the fusion immunogen comprises a full-length CdeC, a full-length CdeM, at least one CdeC immunogenic fragment; or at least one CdeM immunogenic fragment.

[0045] In some examples, the full-length CdeC comprises an amino acid sequence as set forth in SEQ ID NO: 4.

[0046] In some examples, the full-length CdeM comprises an amino acid sequence as set forth in SEQ ID NO: 3.

[0047] In some examples, the full-length CdeC or the at least one CdeC immunogenic fragment is joined to the full-length CdeM or the at least one CdeM immunogenic fragment by a glycine rich linker.

[0048] In some examples, the fusion immunogen is purified by chromatography or western blotting.

[0049] In one example, disclosed herein is an expression vector comprising: a promoter operably linked to a polynucleotide encoding a fusion immunogen, wherein the fusion immunogen comprises a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment; and a vector backbone for expression in a bacterial host cell.Docket No. 11001-247W01

[0050] In some examples, the fusion immunogen comprises the full length CdeC polypeptide fused to the full length CdeM polypeptide.

[0051] In some examples, the fusion immunogen comprises a linker sequence fusing the full length CdeC polypeptide to the full length CdeM polypeptide.

[0052] In some examples, the linker sequence is glycine rich.

[0053] In some examples, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 1.

[0054] In some examples, the expression vector is configured for expression in Escherichia coli.

[0055] In some examples, the expression vector is pET28a or a derivative thereof.

[0056] In some examples, the expression vector further comprises a nucleotide sequence encoding a His tag at C-terminus of the fusion immunogen.

[0057] In one example, disclosed herein is a vaccine composition, comprising: a fusion immunogen, wherein the fusion immunogen comprises (i) a full length CdeC polypeptide or at least one CdeC immunogenic fragment and (ii) a full length CdeM polypeptide or at least one CdeM immunogenic fragment, wherein (i) and (ii) are fused directly or through a linker; and a pharmaceutically acceptable carrier, an adjuvant or a combination thereof.

[0058] In some examples, the fusion immunogen comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

[0059] In some examples, the vaccine composition elicits at least a B cell response, a CD4+ T cell response, including Thl, Th2, or Thl7, or a CD8+ T cell response.

[0060] In some examples, the adjuvant comprises alum, aluminum hydroxide or aluminum phosphate.

[0061] In some examples, the pharmaceutically acceptable carrier comprises a nanoparticle or a liposome.

[0062] In some examples, the vaccine composition further comprises at least one other pharmaceutically effective drug, wherein the at least one other pharmaceutically effective drug is an antibiotic.

[0063] In some examples, the antibiotic is selected from one or more metronidazole, amoxycillin, tetracycline, erythromycin, clarithromycin or tinidazole.

[0064] In some examples, the vaccine composition is formulated for parenteral, intraperitoneal, or intramuscular administration.Docket No. 11001-247W01

[0065] In some examples, the vaccine composition reduces one or more of spore colonization, TcdA toxin or TcdB toxin production, or spore burden in a subject.

[0066] In one example, disclosed herein is a method of treating Clostridioides difficile infection in a subject, comprising: administering to a subject an effective amount of a vaccine composition, wherein the vaccine composition comprises a fusion immunogen comprising a CdeC component and a CdeM component, wherein the CdeC component comprises an amino acid sequence of SEQ ID NO: 4 or an immunogenic fragment thereof, and wherein the CdeM component comprises an amino acid sequence of SEQ ID NO: 3 or an immunogenic fragment thereof.

[0067] In some examples, the fusion immunogen comprises an amino acid sequence as set forth in SEQ ID NO: 2.

[0068] In some examples, the vaccine composition further comprises an adjuvant.

[0069] In some examples, the vaccine composition is administered intraperitoneally or intramuscularly.

[0070] In some examples, the vaccine composition is administered in three doses.

[0071] In some examples, the three doses are administered at about two-week intervals. In some examples, each dose comprises about 10 pg of the fusion immunogen.

[0072] In some examples, the method reduces Clostridioides difficile spore colonization. In one example, disclosed herein is a bacterial expression vector for expression in Lactococcus lactis (L. lactis), comprising: a promoter operably linked to a polynucleotide encoding a fusion immunogen, wherein the fusion immunogen comprises a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment; and an air selection gene, wherein the air selection gene enables growth of an air-deficient L. lactis host cell.

[0073] In some examples, the promoter is a constitutive promoter.

[0074] In some examples, the constitutive promoter is PpepN.

[0075] In some examples, the bacterial expression vector is pNZ7025 or a derivative thereof. In some examples, the air selection gene encodes alanine racemase.

[0076] In some examples, the bacterial expression vector is configured for transformation into Lactococcus lactis NZ1 30.

[0077] In some examples, the bacterial expression vector carrying air selection gene enables L. lactis host cell to grow in absence of D-alanine.

[0078] In some examples, the bacterial expression vector further comprises a sequence encoding a C -terminal His tag fused to the fusion immunogen.Docket No. 11001-247W01

[0079] In some examples, the bacterial expression vector has constitutive expression of the fusion immunogen in vitro and in intestine.

[0080] In one example, disclosed herein is a method of generating a recombinant Lactococcus lactis vaccine expressing a Clostridioides difficile fusion immunogen, comprising: cloning a polynucleotide encoding a fusion immunogen into a bacterial expression vector; and expressing the fusion immunogen in Lactococcus lactis NZ1 30 host cell, thereby generating a recombinant Lactococcus lactis vaccine expressing the fusion immunogen.

[0081] In some examples, the bacterial expression vector is pNZ7025.

[0082] In some examples, the bacterial expression vector comprises an air selection gene. In some examples, the bacterial expression vector comprises a PpepN promoter operably linked to the polynucleotide encoding the fusion immunogen.

[0083] In some examples, the fusion immunogen further comprises a C-terminal His tag. In some examples, the Lactococcus lactis NZ1330 host cell is air-deficient and is incapable of growth in absence of D-alanine.

[0084] In one example, disclosed herein is an oral vaccine composition for inducing a mucosal immune response against C. difficile in a subject, comprising: a recombinant L. lactis cell comprising a polynucleotide encoding a fusion immunogen, wherein the fusion immunogen comprises a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment; and a pharmaceutically acceptable carrier, an adjuvant or a combination thereof.

[0085] In some examples, the adjuvant comprises alum, aluminum hydroxide or aluminum phosphate.

[0086] In some examples, the fusion immunogen comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

[0087] In some examples, the oral vaccine composition elicits at least a B cell response, a CD4+ T cell response, including Thl, Th2, or Thl7, or a CD8+ T cell response.

[0088] In some examples, the pharmaceutically acceptable carrier comprises a nanoparticle or a liposome.

[0089] In some examples, the recombinant L. lactis cell is Lactococcus lactis NZ1330 or a derivative thereof.

[0090] In some examples, the polynucleotide encoding the fusion immunogen is cloned on a bacterial expression vector.

[0091] In some examples, the bacterial expression vector comprises an air selection gene.Docket No. 11001-247W01

[0092] In some examples, the air selection gene encodes an alanine racemase.

[0093] In some examples, the recombinant L lactis cell constitutively expresses the fusion immunogen.

[0094] In some examples, the fusion immunogen further comprises a C-terminal His tag. In some examples, the oral vaccine composition comprises about 1010CFU of the recombinant L. lactis cell per dose.

[0095] In some examples, the oral vaccine composition is formulated for administration as a booster dose following one or more parenteral administrations of a vaccine composition comprising a fusion immunogen.

[0096] In some examples, the oral vaccine composition is formulated for simultaneous administration with one or more parenteral administrations of a vaccine composition comprising a fusion immunogen.

[0097] In some examples, the oral vaccine composition is capable of inducing a local mucosal immune response against C. difficile upon oral administration to the subject.

[0098] In one example, disclosed herein is a method of treating a subject against Closmdioides difficile infection, comprising: administering a first vaccine composition, wherein the first vaccine composition comprises a fusion immunogen comprising a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment, wherein the first vaccine composition further comprises a pharmaceutically acceptable carrier, an adjuvant or a combination thereof; and administering a second vaccine composition comprising a recombinant Lactococcus lactis cell comprising a polynucleotide encoding said fusion immunogen, wherein the second vaccine composition further comprises a pharmaceutically acceptable carrier, an adjuvant or a combination thereof.

[0099] In some examples, the first vaccine composition is administered intramuscularly, and the second vaccine composition is administered orally.

[0100] In some examples, the first vaccine composition is administered in two doses separated by about 2 weeks and the second vaccine composition is administered as a third booster dose.

[0101] In some examples, the first vaccine composition comprises about 10 pg of the fusion immunogen.

[0102] In some examples, the second vaccine composition comprises about 107CFU to about 1011CFU of the recombinant Lactococcus lactis cell.

[0103] In one example, disclosed herein is a method of treating a subject against Clostridioides difficile infection, comprising: orally administering to the subject a recombinant LactococcusDocket No. 11001-247W01

[0104] lactis cell comprising a bacterial expression vector encoding a fusion immunogen, wherein the fusion immunogen comprises an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0105] In some examples, the recombinant Lactococcus lactis cell comprises Lactococcus lactis NZ1330 cell strain.

[0106] In some examples, the bacterial expression vector is pNZ7025 or a derivative thereof. In some examples, the bacterial expression vector comprises an air gene for selection in an air-deficient L. lactis cell.

[0107] In some examples, the bacterial expression vector comprises a PpepN promoter operably linked to a polynucleotide encoding the fusion immunogen.

[0108] In some examples, the fusion immunogen further comprises a C-terminal His tag. In some examples, the recombinant Lactococcus lactis cell constitutively expresses the fusion immunogen.

[0109] In some examples, about 1010CFU of the recombinant Lactococcus lactis cell are administered to the subject.

[0110] In some examples, the oral administration induces a local mucosal immune response against C. difficile.

[0111] In some examples, the oral administration reduces C. difficile colonization following challenge.

[0112] In some examples, disclosed herein are isolated proteins comprising a spore exosporium layer proteins of one or more strains of Clostridioides difficile (C. difficile) (such as, for example, a RT027 strain, aRT078 strain, a RT017 strain, a RT012 strain, a RT003 strain, or aRT009), wherein the surface component comprises surface proteins (such as, for example, a cysteine-rich protein CdeC, or a cysteine -rich protein CdeM), colonization factors or a combination thereof. As disclosed herein, the one or more strains of C. difficile is selected from a group consisting of a RT027 strain, a RT078 strain, a RT017 strain, a RT012 strain, a RT003 strain, and a RT009 strain.

[0113] In some examples, the spore protein is a fusion spore protein (CdeCM). As disclosed herein, the CdeCM contains an amino acid sequence SEQ ID NO: 2, or a fragment thereof. In some examples, the CdeCM is encoded by a polynucleotide sequence SEQ ID NO: 1 or a sequence having at least 90% identity thereto.

[0114] In some examples, the CdeCM comprises a fragment CdeCM, wherein the fragment CdeCM comprises a sequence from about 20 amino acid number to about 200 amino acidDocket No. 11001-247W01

[0115] number of SEQ ID NO: 2 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

[0116] In some examples, disclosed herein are immunogenic compositions comprising a C. difficile CdeCM protein or a fragment thereof; and a pharmaceutically acceptable carrier (such as, for example, including but not limited to nanoparticle or liposome), an adjuvant (such as, for example, including but not limited to alum, aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, calcium phosphate hydroxide, Freund's complete adjuvant, Montanide®, Freund's incomplete adjuvant, isconis, iscom matrix, ISCOMATRIX™ adjuvant, MATRIX M™ adjuvant, MATRIX C™ adjuvant, MATRIX Q™ adjuvant, AbISCO™-100 adjuvant, AbISCO™-300 adjuvant, ISCOPREP™, an ISCOPREP™ derivative, adjuvant containing ISCOPREP™ or an ISCOPREP™ derivative, QS-21, a QS-21 derivative, and an adjuvant containing QS-21 or a QS21 derivative) or a combination thereof.

[0117] In some examples, the C. difficile CdeCM protein comprises a sequence as set forth in SEQ ID NO: 2 or a sequence having at least 95%’, 98%, 99% or 99.8%’ identity thereto.

[0118] In some examples, the immunogenic compositions of any preceding aspect elicit at least a B cell response, a CD4+ T cell response, including Thl, Th2, or Thl7, or a CD8+ T cell response.

[0119] In some examples, the immunogenic compositions of any preceding examples further comprises at least one other pharmaceutical product, wherein the at least one other pharmaceutical product is an antibiotic (such as, for example, including but not limited to metronidazole, amoxycillin, tetracycline, erythromycin, clarithromycin or tinidazole).

[0120] In some examples, disclosed herein is a method of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing C. difficile infection in a subject, comprising administering to the subject a therapeutically effective dose of a vaccine, wherein the vaccine comprises an isolated protein or a vector (such as, for example, including but not limited to a plasmid, an expression vector or a viral vector), wherein the isolated protein or the vector comprises a C. difficile CdeCM protein or a fragment thereof.

[0121] In some examples, the vaccine is administered intravenously, intramuscularly, intraperitoneally, intradermally, or subcutaneously to the subject.

[0122] In some examples, the C. difficile CdeCM protein comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, wherein the CdeCM protein is encoded by a polynucleotide, wherein the polynucleotide comprises a sequence as set forth in SEQ ID NO: 1 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.Docket No. 11001-247W01

[0123] BRIEF DESCRIPTION OF THE DRAWINGS

[0124] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several examples described below.

[0125] FIG. 1 shows the phylogeny of C. difficile CdeC.

[0126] FIG. 2 shows the phylogeny of C. difficile CdeM.

[0127] FIG. 3 shows the homology of C. difficile CdeC. The figure shows a comparative alignment of CdeC Homologs and mapped epitope conservation. This figure displays a multiple sequence alignment of 19 distinct CdeC protein variants from C. difficile clades 1, 2, 3, 4, 5, and C-III. The top sequence (Strain CD630) corresponds to the reference SEQ ID NO: 4. The alignment demonstrates the high level of conservation for the short peptide sequences provided in the sequence listing (SEQ ID NOs: 11-42). For example: N-Terminal Region: The " KKNKRR" (SEQ ID NO: 52) motif (blue box) and " HHH" motif (yellow box) encompass epitopes such as SEQ ID NO: 37 and 39, showing near-total identity across all 19 strains. Variable regions are areas marked with pink arrows show where the 19 sequences differ. Notably, the targeted epitopes in the sequence listing were strategically selected to avoid these variable sites, ensuring broad-spectrum efficacy. Structural motifs are the repetitive " NPC" (red), " CCRQGKGK" (SEQ ID NO: 53) (light green), and " CNECC" (SEQ ID NO: 54) (purple) regions represent the structural core of the protein. These correspond to the " Epitope Fusions" (SEQ ID NOs: 6-10), which concatenate these conserved domains into synthetic immunogens. The figure shows a total of 51 unique sequences categorized by their functional and structural roles in the study. The set begins with 4 full-length proteins and immunogens (SEQ ID NOs: 1-4), which serve as the primary biological templates, followed by 6 synthetic epitope fusions (SEQ ID NOs: 5-10) designed as chimeric constructs for enhanced delivery. The largest portion of the listing consists of specific antigenic determinants, including 32 CdeC epitopes (SEQ ID NOs: 11-42) and 9 CdeM epitopes (SEQ ID NOs: 43-51), spanning Cytotoxic T Lymphocyte (C'I'L), Helper T Lymphocyte (HTL), and B-cell (B) categories.

[0128] FIG. 4 shows the homology of C. difficile CdeM.

[0129] FIG. 5 shows the expression and purification of CdeCM. The gene sequences encoding CdeC and CdeM from R20291 were bridged with a linker (ggt ggc ggt), optimized, synthesized and cloned into pET28a. Fusion protein CdeCM (67 kDa) was expressed in E. coli BL21 (DE3) and purified by Ni-affinity chromatography and analyzed via SDS-PAGE.

[0130] FIG. 6 shows the immunizations via the intraperitoneal (i.p.) route showing induced CdeC and CdeM antibody responses. Group of 6 mice (n=12) were immunized 3 times at 12-day intervals with lOpg of CdeC and CdeM with alum as adjuvant. Sera and feces wereDocket No. 11001-247W01

[0131] collected, and CdeC and CdeM IgG and IgA titers were measured via standard ELISA. The data are presented as the mean + SEM and mean + SD. (*, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant).

[0132] FIG. 7 shows the immunizations via the intraperitoneal (i.p.) route showing induced anti-CdeC anti-CdeM antibody responses. Group of 6 mice (n=12) were immunized 3 times at 12-day intervals with lOpg of CdeCM with alum as adjuvant. Sera and feces were collected, and anti-CdeC and anti-CdeM IgG and IgA titers were measured via standard ELISA. The data are presented as the mean + SD. (*, p<0.05; **, p<0.01; ***, pcO. OOl; ****, p<0.0001; ns, not significant).

[0133] FIGS. 8A-8C show the immunizations of mice with CdeCM provide mice significant protection against infection with C. difficile strain R20291. Mice were challenged with C. difficile R20291 spores (106 / mouse) 14 days after the third immunization of groups of mice (n=10) with CdeCM (compare with CdeC and CdeM) at 10 pg / mouse / immunization or PBS in the presence of alum. Kaplan-Meier survival plots (A), mean relative weight of all surviving mice (up to the day of death) (B) of different groups, and frequency of diarrhea (C) are illustrated. Data were presented as mean relative weight ± standard error(* p<0.05).

[0134] FIGS. 9A-9C show the immunization of mice with CdeM, CdeC, and CdeCM decrease the C. difficile toxins in the feces after CDI. TcdA (A) or TcdB (B) levels in feces were determined by ELISA. (C) Fecal samples were collected for bacterial spore enumeration. Bars stand for means ±SD (*, p<0.05; **, p<0.01; ***, pcO. OOl; ****, pcO. OOOl; ns, not significant * p<0.05).

[0135] FIG. 10 shows the expression of CdeCM in L. lactis NZ1330. The gene sequences encoding CdeC and CdeM from R20291 were bridged with a linker (ggt ggc let ggt), optimized, synthesized, and cloned into vector pNZ7025 in L. lactis NZ1330. Expression of fusion protein CdeCM in L. lactis NZ1 30 was detected by western blot analysis of bacterial cell lysates using anti-His tag antibodies.

[0136] FIGS. 11A-11C show the immunizations with combined intramuscular injection of CdeCM and oral delivery of L. lactis (CdeCM) provide mice significant protection against CDI. Three groups of mice (n=10) were immunized with 10 pg of CdeCM adjuvanted with alum via intramuscular route for 3 times or with 10 pg of CdeCM adjuvated with alum via intramuscular route for twice, followed by the 3rd boosting immunization with L. lactis (CdeCM) (abbreviated as LAC) at 1010CFU / mouse via oral route, or PBS in the presence of alum. Immunizations were carried out at a two-week interval. Fourteen days after 3rd immunization,Docket No. 11001-247W01

[0137] mice were challenged with C. difficile R20291 spores (106 / mouse). Kaplan-Meier survival plots (A), mean relative weight of all surviving mice (up to the day of death) (B) of different groups, and frequency of diarrhea (C) are illustrated. Bars stand for means ± SD.

[0138] DETAILED DESCRIPTION

[0139] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular examples only and is not intended to be limiting.

[0140] Clostridium difficile more recently re-classified as Clostridioid.es difficile, is a Grampositive, sporogenic anaerobic bacterium that is the most common cause of antibiotic-associated diarrhea within healthcare systems of the developed world. The clinical manifestation of the infection is diarrhea and in severe cases can produce pseudomembranous colitis, toxic megacolon and death. Mortality of C. difficile infections (GDI) may reach up to 5% of GDI cases, but in several outbreaks, it has increased up to 20%. Conventional metronidazole and / or vancomycin treatment (depending on the severity of the symptoms) although resolve single episodes of GDI, exhibit high rates of recurrence of the infection after a first episode. The rate of recurrence of GDI of a first, second and third episode may reach up to 20%, 40% and 60%, respectively.

[0141] During the infection, C. difficile colonization leads to secretion of large toxins (TcdA and TcdB) that glycosylate intestinal epithelial cell proteins, induce massive inflammation of the gut epithelium, causing disease symptoms ranging from mild diarrhea to pseudomembranous colitis, toxic megacolon and even death. However, before C. difficile can colonize a susceptible host, the highly resistant and metabolically dormant spore must germinate in response to secondary bile salts present in high levels in the gastrointestinal tract of antibiotic-treated host. In addition to toxin-production during C. difficile colonization of the host, a subset of C. difficile vegetative cells initiates a sporulation program that culminates with the formation of metabolically dormant spores. These spores have intrinsic resistance properties enabling their survival to enzymatic degradation, phagocytic cells, and chemicals normally found in the host's gastrointestinal (GI) environment, enabling their persistence in the host's GI tract.Docket No. 11001-247W01

[0142] To persist in the host, C. difficile spores must interact with the host's colonic mucosa through specific interactions mediated by spore-ligand(s) molecules and host cellular receptors. In this context, as demonstrated in other spore-former species, the surface of C. difficile spores is likely to be the primary site of spore-host interactions that contributes to spore persistence. Consequently, there is keen interest in understanding fundamental aspects of the outermost exosporium layer of C. difficile spores. Notably, the exosporium layer of C. difficile spores differs from previously described outermost layers. For example, in contrast with the exosporium layer of spores of the Bacillus cereus group, where an interspace gap separates the exosporium from the spore coat, the exosporium of C. difficile spores is in direct contact with the spore coat layers in a similar fashion to the outer crust of Bacillus subtilis spores. Despite these differences with the outer layer of spores of other bacterial endospore formers, the exosporium layer of most C. difficile strains have hair-like extensions similarly as those observed in spores of the B. cereus group. However, in striking difference from other endospore formers, during the sporulation program, C. difficile forms spores with two distinctive exosporium morphotypes that arise from the same clonal sporulating culture, during either standard sporulation conditions (i.e., agar plates), or during biofilm development conditions. These exosporium morphotypes include: i) spores with a thick-exosporium layer, defined by an electron dense material surrounding the spore coats; and ii) a thin-exosporium layer, where the electron-dense material that surrounds the spore coat is notably thinner. In some examples, disclosed herein are isolated proteins comprising a spore exosporium layer proteins of one or more strain of Clostridioides difficile (C. difficile)(snch as, for example, a RT027 strain, a RT078 strain, a RT017 strain, a RT012 strain, a RT003 strain, or aRT009), wherein the surface component comprises surface proteins (such as, for example, a cysteine-rich protein CdeC, or a cysteine-rich protein CdeM), colonization factors or combination thereof. As disclosed herein, the one or more strains of C. difficile is selected from a group consisting of a RT027 strain, a RT078 strain, a RT017 strain, a RT012 strain, aRT003 strain, and a RT009 strain.

[0143] Terminology

[0144] Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicant desires that the following terms be given the particular definition as defined below.

[0145] As used herein, the article “a,” "an.” and "the” means “at least one,” unless the context in which the article is used clearly indicates otherwise.Docket No. 11001-247W01

[0146] “Administration” to a subject or “administering” includes any route of introducing or delivering to a subject an agent. Administration can be earned out by any suitable route, including oral, intravenous, intraperitoneal, intranasal, inhalation and the like. Administration includes seif-administration and the administration by another.

[0147] The terms "about" and "approximately" are defined as being '"close to" as understood by one of ordinary skill in the art. In one non-limiting example, the terms are defined to be within 10%. In another non-limiting example, the terms are defined to be within 5%’. In still another non-limiting example, the terms are defined to be within i %.

[0148] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various examples, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific examples and are also disclosed.

[0149] A "composition" is intended to include a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.

[0150] As used herein, the terms “determining,” “measuring,” and “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

[0151] By the term “effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect. The amount of beneficial agent that is “effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.

[0152] An “effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0153] As used herein the term “encoding” refers to the inherent property of specific sequences of nucleotides in a nucleic acid, to serve as templates for synthesis of other molecules havingDocket No. 11001-247W01

[0154] a defined sequence of nucleotides (i.e. rRNA, tRNA, other RNA molecules) or amino acids and the biological properties resulting therefrom.

[0155] The “fragments” or “functional fragments,” whether atached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the functional fragment must possess a bioactive property, such as antigen binding and antigen recognition.

[0156] The term "gene" or "gene sequence" refers to the coding sequence or control sequence, or fragments thereof. A gene may include any combination of coding sequence and control sequence, or fragments thereof. Thus, a "gene" as referred to herein may be all or part of a native gene. A polynucleotide sequence as referred to herein may be used interchangeably with the term "gene”, or may include any coding sequence, non-coding sequence or control sequence, fragments thereof, and combinations thereof. The term "gene" or "gene sequence" includes, for example, control sequences upstream of the coding sequence (for example, the ribosome binding site).

[0157] The term “isolating” as used herein refers to isolation from a biological sample, i.e., blood, plasma, tissues, exosomes, or cells. As used herein the term “isolated,” when used in the context of, e.g., a nucleic acid, refers to a nucleic acid of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the nucleic acid is associated with prior to purification.

[0158] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0159] The term “nucleic acid” refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0160] The term "oligonucleotide" denotes single- or double-stranded nucleotide multimers of from about 2 to up to about 100 nucleotides in length. Suitable oligonucleotides may beDocket No. 11001-247W01

[0161] prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Let., 22: 1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLS1PSTM technology. Wien oligonucleotides are referred to as "double-stranded," it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term "double-stranded," as used herein is also meant to refer to those forms which include such structural features as bulges and loops, described more fully in such biochemistry texts as Stryer, Biochemistry, Third Ed., (1988), incorporated herein by reference for all purposes.

[0162] The term "polynucleotide" refers to a single or double stranded polymer composed of nucleotide monomers.

[0163] The term "polypeptide" refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

[0164] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

[0165] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” 'This definition also refers to, or may be applied to, the complement of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) nucleotide sequence identityDocket No. 11001-247W01

[0166] is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0167] For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0168] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol.

[0169] 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih. ov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol.

[0170] 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivityDocket No. 11001-247W01

[0171] and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sei. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0172] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Nall. Acad. Set. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0173] As used herein, the term “pharmaceutically acceptable’’ component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When the term “pharmaceutically acceptable” is used to refer to an excipient, it is generally implied that the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U. S. Food and Drug Administration.

[0174] The term “subject” or “host” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. The subject can be either male or female.

[0175] A control sample or a reference sample as described herein can be a sample from a healthy subject or sample, a wild-type subject or sample, or from populations thereof. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject or a group of healthy subjects or a wild-type subject or sample. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.Docket No. 11001-247W01

[0176] The term “tissue” refers to a group or layer of similarly specialized cells which together perform certain special functions. The term “tissue” is intended to include, blood, blood preparations such as plasma and serum, bones, joints, muscles, smooth muscles, lung tissues, and organs.

[0177] As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder (e.g., a cancer), or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, prevention of the occurrence of symptoms and / or their underlying cause, and improvement or remediation of damage.

[0178] As used herein, a “therapeutically effective amount” of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result, and a “prophylactically effective amount” of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition (e.g. cancer). Therapeutically effective and prophylactically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject.

[0179] The term “therapeutically effective amount” can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and / or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.

[0180] “Fusion protein”, as used herein refers to a recombinant or engineered polypeptide comprising amino acid sequences derived from at least two distinct proteins, polypeptides, peptides, or fragments thereof, joined together in a single continuous molecule. The component sequences may be directly joined or connected by one or more linkers. In certain examples, the fusion protein comprises a CdeC sequence and a CdeM sequence. In certain examples, the fusion protein is CdeCM.

[0181] As used herein, the term “Fusion protein antigen” refers to a fusion protein that is capable of being recognized by the immune system of a subject. Such recognition may include binding by an antibody, a B cell receptor, or an antigen-specific T cell response followingDocket No. 11001-247W01

[0182] antigen processing and presentation. In certain examples, the fusion protein antigen comprises antigenic regions from CdeC and CdeM and is configured to induce or enhance an immune response against Clostridioides difficile.

[0183] As used herein, an “antigen” refers to a molecule, protein, polypeptide, peptide, fragment, variant, or construct that is capable of being recognized by the immune system of a subject. In some examples, CdeC, CdeM, and CdeCM each function as antigens. In some examples, an antigen is a spore surface-exposed protein or a recombinant construct comprising the same.

[0184] As used herein, an “immunogen” refers to a molecule, protein, polypeptide, fragment, or construct that is capable of eliciting an immune response in a subject. In some examples, CdeC, CdeM, CdeCM fusion protein, immune epitopes of CdeC or CdeM (for example, such as including but not limited to CdeC-CTL (as set forth in SEQ ID NOS: 11-22), CdeC-HTL (as set forth in SEQ ID NOS: 23-33), CdeC-B (as set forth in SEQ ID NOS: 34-42), CdeM-CTL (as set forth in SEQ ID NO: 43), CdeM-HTL (as set forth in SEQ ID NOS: 44-47), CdeM- B (as set forth in SEQ ID NOS: 48-51)), or immune fusion epitopes (for example, such as including but not limited to CdeC-EF-1 or CdeC-EF-2 (as set forth in SEQ ID NOS: 6-7), CdeM-EF (as set forth in SEQ ID NO: 5), CdeCM-EF-1 (as set forth in SEQ ID NO: 8), CdeCM-EF-2 (as set forth in SEQ ID NO: 9), CdeCM-EF-3 (as set forth in SEQ ID NO: 10)) are immunogens. In some examples, an immunogen induces IgG and / or IgA responses in serum, feces, mucosa, or combinations thereof. In some examples, an immunogen provides protective immunity against Clostridioides difficile infection and / or colonization.

[0185] As used herein, the term “Fusion immunogen” refers to a fusion protein or other recombinant construct comprising sequences from two or more antigens, wherein the construct is capable of eliciting an immune response in a subject. In certain examples, the fusion immunogen comprises a CdeC sequence and a CdeM sequence joined by a linker. In certain examples, the fusion immunogen is administered as a purified recombinant protein, is expressed by a microbial delivery vehicle, or both.

[0186] An “epitope” refers to a molecular region, sequence, motif, structural feature, or antigenic determinant that is recognized by a component of the immune system. An epitope may be linear or conformational. An epitope may be recognized directly by an antibody or B cell receptor, or may be presented as a processed peptide to a T cell receptor in the context of a major histocompatibility complex molecule.Docket No. 11001-247W01

[0187] As used herein, “immune epitopes” refers to one or more regions, motifs, peptide sequences, structural determinants, or surface-exposed portions of an antigen that are recognized by the immune system and / or contribute to the induction of an immune response.

[0188] In some examples, immune epitopes include one or more conserved or relatively conserved regions of CdeC and / or CdeM that are recognized following immunization. In some examples, immune epitopes may comprise B-cell epitopes, T-cell epitopes, conformational epitopes, linear epitopes, or combinations thereof. In some examples, immune epitopes are present in full-length CdeC, full-length CdeM, fragments thereof, variants thereof, or in the CdeCM fusion construct.

[0189] “Immunogenic epitope” refers to an epitope that is sufficient to induce a detectable immune response in a subject, either alone or when presented in association with a carrier, fusion partner, adjuvant, vector, or delivery system.

[0190] “Fusion immune epitope” refers to an immune epitope present within, incorporated into, or generated by a fusion construct. In certain examples, a fusion immune epitope comprises an epitope derived from a first antigen linked within the same recombinant molecule to an epitope or epitope-containing region derived from a second antigen. In certain examples, the fusion immune epitope is present within a fusion immunogen comprising CdeC and CdeM sequences (for example, such as including but not limited to CdeC-EF-1 or CdeC-EF-2 (as set forth in SEQ ID NOS: 6-7), CdeM-EF (as set forth in SEQ ID NO: 5), CdeCM-EF-1 (as set forth in SEQ ID NO: 8), CdeCM-EF-2 (as set forth in SEQ ID NO: 9), CdeCM-EF-3 (as set forth in SEQ ID NO: 10)).

[0191] As used herein, the term “antigenic fragment” refers to a portion of a protein or polypeptide that retains at least one epitope capable of specific immune recognition. In certain examples, an antigenic fragment of CdeC or CdeM comprises one or more conserved, surface- exposed, or immunoreactive regions.

[0192] “Immunogenic fragment” refers to a portion of a protein or polypeptide that retains the ability to induce an immune response in a subject. In certain examples, an immunogenic fragment comprises one or more immune epitopes from CdeC, CdeM, or both.

[0193] “Conserved epitope” refers to an epitope present in substantially similar form among two or more strains, isolates, ribotypes, sequence types, clades, or toxinotypes of a microorganism. In certain examples, conserved epitopes of CdeC and / or CdeM are useful for generating cross-protective immune responses against diverse C. difficile strains.Docket No. 11001-247W01

[0194] As used herein, a “chimeric antigen” refers to an antigen comprising portions derived from two or more different proteins, strains, species, or sources, combined into a single construct. In certain examples, a chimeric antigen is a fusion protein antigen.

[0195] As used herein, the term “linker” refers to one or more amino acids, codons, or nucleotide sequences positioned between two joined sequences in a fusion construct. A linker may provide spacing, flexibility, structural separation, improved folding, reduced steric hindrance, enhanced expression, or preservation of antigenicity. In certain examples, the linker comprises ggtggcggt or a sequence encoding glycine-containing residues.

[0196] An “epitope fusion” refers to a recombinant chimeric polypeptide comprising two or more antigenic peptide epitopes derived from one or more parent proteins, operably linked in a single contiguous amino acid sequence, optionally through peptide linkers, to enhance immunogenic presentation. The epitope fusion sequences, for example, including but are not limited to SEQ ID NOs: 5-10 are engineered chimeric immunogens comprising selected epitope-containing fragments of exosporium morphogenetic proteins CdeC, CdeM, or both, fused in tandem, optionally with linker sequences such as GPGPG, to generate a single immunogenic polypeptide. As used herein, epitope fusion refers to a synthetic fusion polypeptide comprising a plurality of contiguous epitope peptides selected from CdeC, CdeM, or a combination thereof, wherein the epitopes are joined directly and / or by linker peptides to form a non-native immunogen.

[0197] In some examples, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain examples of the present disclosure are to be understood as being modified in some instances by the term “about.” In some examples, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some examples, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular example. In some examples, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some examples of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some examples of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merelyDocket No. 11001-247W01

[0198] intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.

[0199] Throughout this application, various publications are referenced. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0200] Molecular Engineering

[0201] The term “transfection,” as used herein, refers to the process of introducing nucleic acids into cells by non-viral methods. The term “transduction,” as used herein, refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

[0202] The terms “heterologous DNA sequence”, “exogenous DNA segment”, or “heterologous nucleic acid,” as used herein, each refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[0203] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0204] An “expression vector,” otherwise known as an “expression construct,” is generally a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specificDocket No. 11001-247W01

[0205] gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and / or promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity, when necessary, through the use of an inducer, in some systems however the protein may be expressed constitutively. As described herein, Escherichia coli is used as the host for protein production, but other cell types may also be used.

[0206] In molecular biology, an “inducer” is a molecule that regulates gene expression. An inducer can function in two ways, such as:

[0207] (i) By disabling repressors. The gene is expressed because an inducer binds to the repressor. The binding of the inducer to the repressor prevents the repressor from binding to the operator. RNA polymerase can then begin to transcribe operon genes.

[0208] (ii) By binding to activators. Activators generally bind poorly to activator DNA sequences unless an inducer is present. An activator binds to an inducer and the complex binds to the activation sequence and activates target gene. Removing the inducer stops transcription. Because a small inducer molecule is required, the increased expression of the target gene is called induction.

[0209] Repressor proteins bind to the DNA strand and prevent RNA polymerase from being able to attach to the DNA and synthesize mRNA. Inducers bind to repressors, causing them to change shape and preventing them from binding to DNA. Therefore, they allow transcription, and thus gene expression, to take place.

[0210] For a gene to be expressed, its DNA sequence must be copied (in a process known as transcription) to make a smaller, mobile molecule called messenger RNA (mRNA), which carries the instructions for making a protein to the site where the protein is manufactured (in a process known as translation). Many different types of proteins can affect the level of gene expression by promoting or preventing transcription. In prokaryotes (such as bacteria), these proteins often act on a portion of DNA known as the operator at the beginning of the gene. The promoter is where RNA polymerase, the enzyme that copies the genetic sequence and synthesizes the mRNA, attaches to the DNA strand.Docket No. 11001-247W01

[0211] Some genes are modulated by activators, which have the opposite effect on gene expression as repressors. Inducers can also bind to activator proteins, allowing them to bind to the operator DNA where they promote RNA transcription. Ligands that bind to deactivate activator proteins are not, in the technical sense, classified as inducers, since they have the effect of preventing transcription.

[0212] A “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0213] A “ribosome binding site” or “(RBS)” refers to a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of translation. Generally, RBS refers to bacterial sequences, although internal ribosome entry sites (IRES) have been described in mRNAs of eukaryotic cells or viruses that infect eukaryotes. Ribosome recruitment in eukaryotes is generally mediated by the 5' cap present on eukaryotic mRNAs.

[0214] A “transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cell s are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN- 10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

[0215] The “transcription start site” or “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered.

[0216] 9Docket No. 11001-247W01

[0217] Downstream sequences (i.e., further protein encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.

[0218] “Operably linked’’ or “functionally linked’’ refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.

[0219] A “construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.

[0220] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'-untranslated region (3' UTR). Constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.

[0221] The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.

[0222] “Transformed,” “transgenic,” and “recombinant” refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or a plant into which a heterologous nucleic acidDocket No. 11001-247W01

[0223] molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term “untransformed” refers to normal cells that have not been through the transformation process.

[0224] “Wild-type” refers to a bacteria or organism found in nature without any known mutation. Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.

[0225] Nucleotide and / or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity=X / Y×100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A. For example, the percent identity can be at least 80% or about 80%, about 81%,Docket No. 11001-247W01

[0226] about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0227] Substitution refers to the replacement of one amino acid with another amino acid in a protein or the replacement of one nucleotide with another in DNA or RNA. Insertion refers to the insertion of one or more amino acids in a protein or the insertion of one or more nucleotides with another in DNA or RNA. Deletion refers to the deletion of one or more amino acids in a protein or the deletion of one or more nucleotides with another in DNA or RNA. Generally, substitutions, insertions, or deletions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); hydroxyl or sulfur / selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. An amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.

[0228] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-Docket No. 11001-247W01

[0229] mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transformed cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.

[0230] Exemplary nucleic acids that may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0231] Methods of down-regulation or silence genes are known in art. For example, expressed protein activity can be downregulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N. Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2.006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326-330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). TraitsDocket No. 11001-247W01

[0232] influential in defining optimal siRNA sequences include G / C content at the termini of the siRNAs, I'm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.

[0233] As would be apparent, the sequencing may be done using a next generation sequencing platform, e.g., Illumina's reversible terminator method, Roche's pyrosequencing method, Life Technologies' sequencing by ligation (the SOLiD platform) or Life Technologies' Ion Torrent platform, etc. Examples of such methods are described in the following references: Margulies et al (Nature 2005 437: 376-80); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9); Shendure (Science 2005 309: 1728); Imelfort et al (Brief Bioinform. 2009 10:609-18); Fox et al (Methods Mol Biol. 2009; 553:79-108); Appleby et al (Methods Mol Biol. 2009; 13:19-39) and Morozova (Genomics. 2.008 92:255-64), which are incorporated by reference for the general descriptions of the methods and the particular steps of the methods, including all starting products, reagents, and final products for each of the steps. In other examples, the sequencing may be done using nanopore sequencing (e.g. as described in Soni et al Clin Chem 53: 1996-2001 2007, or as described by Oxford Nanopore Technologies).

[0234] Vectors

[0235] In one example, disclosed herein is a bacterial expression vector for expression in Lactococcus lactis (L. lactis), comprising: a promoter operably linked to a polynucleotide encoding a fusion immunogen, wherein the fusion immunogen comprises a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment; and an air selection gene, wherein the air selection gene enables growth of an air-deficient L. lactis host cell.

[0236] As used herein, Lactococcus refers to a genus of Gram-positive, generally nonsporulating, lactic acid bacteria that are commonly used in food fermentation and, in recombinant biotechnology, as host cells for cloning, expression, secretion, and surface presentation of heterologous nucleic acids and proteins. In certain examples, Lactococcus comprises a food-grade or generally recognized as safe host suitable for production or delivery of antigens, immunogens, therapeutic proteins, enzymes, or surface-displayed constructs. Patent and scientific literature commonly identify Lactococcus lactis as a particularly useful recombinant host because it has been used for heterologous protein expression and secretion, including with the widely used Usp45 secretion signal and the ni sin-controlled NICE expression system.Docket No. 11001-247W01

[0237] Examples of Lactococcus include, but are not limited to, Lactococcus lactis, Lactococcus cremoris, Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus plantarum, Lactococcus piscium, and subspecies, strains, derivatives, mutants, recombinants, or engineered variants thereof. In some examples, the Lactococcus is Lactococcus lactis subsp. lactis or Lactococcus lactis subsp. cremoris. In some examples, the Lactococcus is a recombinant strain adapted for intracellular expression, secretion, cell wall anchoring, or surface display of a heterologous polypeptide. Literature describing Lactococcus expression systems specifically highlights L. lactis strains as suitable hosts for heterologous protein production and secretion.

[0238] In some examples, suitable Lactococcus options include host strains engineered or selected for inducible expression, constitutive expression, secretion, or mucosal delivery. Examples include, but are not limited to, NZ9000, NZ3900, MG1363, IL1403, KF147, or derivatives thereof. In some examples, the Lactococcus host is used with a nisin-inducible system, such as the NICE system, for regulated expression of an antigen or fusion immunogen. In some examples, the host is used with a secretion signal, such as the Usp45 signal peptide, to direct extracellular release of the expressed product. The NICE system and Usp45-based secretion are both well-established options in L. lactis expression platforms.

[0239] In some examples, suitable Lactococcus vector options include lactococcal plasmids or shuttle vectors configured for replication and expression in Lactococcus and, optionally, in E. coli for cloning. Examples include, but are not limited to, pNZ-series vectors such as pNZ8048 or pNZ8148, pIL253-derived vectors, pOri-derived shuttle vectors, pTRKH-type shuttle vectors, and related lactococcal expression plasmids. In some examples, such vectors comprise a nisin-inducible promoter, a constitutive promoter, a secretion signal, a selectable marker, a replication origin compatible with Lactococcus, or a fusion tag. pNZ8048-derived vectors are commonly associated with nisin-inducible Lactococcus expression, while pIL253-derived systems and related shuttle vectors are used for cloning and expression in Lactococcus In some examples, suitable Lac ococcus promoter options include inducible promoters and constitutive promoters functional in Lactococcus. Examples include, but are not limited to, the nisin-inducible PnisA promoter, promoters controlled through NisRK-regulated systems, the P32 promoter, the P23 promoter, and other lactococcal promoters operable in the selected host. In some examples, suitable secretion and localization options include the Usp45 signal peptide, propeptide-assisted secretion elements, cell wall anchoring domains, or surface display domains operable in Lactococcus. Usp45 is repeatedly described as a widely usedDocket No. 11001-247W01

[0240] secretion signal in L lactis, and the NICE system is a recognized inducible platform for lactococcal expression.

[0241] In the present application, Lactococcus may be used as a host cell, delivery vehicle, expression platform, secretion platform, or surface display platform for a fusion immunogen, a CdeC-derived polypeptide, a CdeM-derived polypeptide, an antigenic fragment, a carrier domain-containing construct, a scaffold-containing construct, or another recombinant polypeptide of interest. In some examples, the Lactococcus host is selected to provide food¬ grade production, mucosal administration, extracellular secretion, inducible expression, or surface-accessible presentation of the fusion immunogen

[0242] In some examples, the promoter is a constitutive promoter.

[0243] In some examples, the constitutive promoter is PpepN.

[0244] As used herein, a “constitutive promoter” refers to a promoter that drives transcription of an operably linked nucleic acid sequence in the absence of a specific externally added inducer, activator, or triggering condition. In certain examples, a constitutive promoter directs continuous or baseline expression of a gene in a host ceil under ordinary growth conditions. In some examples, the level of expression driven by the constitutive promoter may be relatively low, moderate, or high depending on the promoter sequence, the host organism, the copy number of the vector, and other regulatory features present in the construct. Unlike an inducible promoter, which is activated or repressed in response to a defined stimulus such as IPT'G, arabinose, tetracycline, heat shock, or another regulatory signal, a constitutive promoter is generally active without the need for deliberate induction.

[0245] Examples of constitutive promoters include, but are not limited to, a lac-derived constitutive promoter lacking effective repression, a trc promoter under constitutive conditions, a gapA promoter, a phosphoglycerate kinase promoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an elongation factor Tu promoter, a beta-lactamase promoter, a synthetic constitutive bacterial promoter, a Bacillus vegetative promoter, a lactococcal P23 promoter, a lactococcal P32 promoter, a ermE* promoter, a CMV promoter, an SV40 promoter, an EF-1 alpha promoter, a ubiquitin promoter, or a PGK promoter. In some examples, the constitutive promoter is selected for expression in a bacterial host. In other examples, the constitutive promoter is selected for expression in a yeast, fungal, insect, or mammalian host.

[0246] In some examples, a constitutive promoter is used to drive expression of a fusion immunogen, a CdeC-derived polypeptide, a CdeM-derived polypeptide, a carrier domaincontaining fusion protein, a scaffold-containing fusion protein, or a surface display construct without the need for addition of an inducer. In the present application, a constitutive promoterDocket No. 11001-247W01

[0247] may be used where continuous production of the encoded immunogen or fusion protein is desired during growth, propagation, secretion, or surface presentation in the selected host cell.

[0248] In some examples, the bacterial expression vector is pNZ7025 or a derivative thereof. In some examples, the air selection gene encodes alanine racemase.

[0249] In some examples, the bacterial expression vector is configured for transformation into Lactococcus lactis NZ1330.

[0250] In some examples, the bacterial expression vector carrying air selection gene enables L. lactis host cell to grow in absence of D-alanine.

[0251] In some examples, the bacterial expression vector further comprises a sequence encoding a C -terminal His tag fused to the fusion immunogen.

[0252] In some examples, the bacterial expression vector has constitutive expression of the fusion immunogen in vitro and in intestine.

[0253] Methods of making, generating or producing a vector, plasmid, host cell or vaccine composition

[0254] In one example, disclosed herein is a method of producing a fusion immunogen, comprising: cloning a polynucleotide encoding a fusion immunogen as set forth in SEQ ID NO: 1 into a bacterial plasmid; expressing the fusion immunogen in Escherichia coli BL21(DE3), wherein the fusion immunogen comprises an amino acid sequence as set forth in SEQ ID NO: 2; and purifying and obtaining the fusion immunogen.

[0255] In some examples, the bacterial plasmid comprises a pET vector, a pBAD vector, a pGEX vector, a pMAL vector, a pQE vector, a pTrc99A vector, or a pCDF vector.

[0256] In some examples, the bacterial plasmid is a pET28a vector.

[0257] In some examples, the fusion immunogen comprises a full-length CdeC, a full-length CdeM, at least one CdeC immunogenic fragment; or at least one CdeM immunogenic fragment.

[0258] In some examples, the full-length CdeC comprises an amino acid sequence as set forth in SEQ ID NO: 4.

[0259] In some examples, the full-length CdeM comprises an amino acid sequence as set forth in SEQ ID NO: 3.

[0260] In some examples, the full-length CdeC or the at least one CdeC immunogenic fragment is joined to the full-length CdeM or the at least one CdeM immunogenic fragment by a glycine rich linker.

[0261] In some examples, the fusion immunogen is purified by chromatography or western blotting.Docket No. 11001-247W01

[0262] In one example, disclosed herein is an expression vector comprising: a promoter operably linked to a polynucleotide encoding a fusion immunogen, wherein the fusion immunogen comprises a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment; and a vector backbone for expression in a bacterial host cell.

[0263] In some examples, the fusion immunogen comprises the full length CdeC polypeptide fused to the full length CdeM polypeptide.

[0264] In some examples, the fusion immunogen comprises a linker sequence fusing the full length CdeC polypeptide to the full length CdeM polypeptide. In some examples, the linker sequence is glycine rich.

[0265] In some examples, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 1. In some examples, the expression vector is configured for expression in Escherichia coli. In some examples, the expression vector is pET28a or a derivative thereof.

[0266] In some examples, the expression vector further comprises a nucleotide sequence encoding a His tag at C -terminus of the fusion immunogen.

[0267] In one example, disclosed herein is a method of generating a recombinant Lactococcus lactis vaccine expressing a Clostridioides difficile fusion immunogen, comprising: cloning a polynucleotide encoding a fusion immunogen into a bacterial expression vector; and expressing the fusion immunogen in Lactococcus lactis NZ1330 host cell, thereby generating a recombinant Lactococcus lactis vaccine expressing the fusion immunogen.

[0268] In some examples, the bacterial expression vector is pNZ7025.

[0269] In some examples, the bacterial expression vector comprises an air selection gene. In some examples, the bacterial expression vector comprises a PpepN promoter operably linked to the polynucleotide encoding the fusion immunogen.

[0270] In some examples, the fusion immunogen further comprises a C-terminal His tag. In some examples, the Lactococcus lactis NZ1330 host cell is air-deficient and is incapable of growth in absence of D-alanine.

[0271] In one example, disclosed herein is an oral vaccine composition for inducing a mucosal immune response against C. difficile in a subject, comprising: a recombinant L. lactis cell comprising a polynucleotide encoding a fusion immunogen, wherein the fusion immunogen comprises a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment; and a pharmaceutically acceptable carrier, an adjuvant or a combination thereof.Docket No. 11001-247W01

[0272] In some examples, the adjuvant comprises alum, aluminum hydroxide or aluminum phosphate.

[0273] In some examples, the fusion immunogen comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

[0274] In some examples, the oral vaccine composition elicits at least a B cell response, a CD4+ T cell response, including Thl, Th2, or Thl7, or a CD8+ T cell response.

[0275] In some examples, the pharmaceutically acceptable carrier comprises a nanoparticle or a liposome.

[0276] In some examples, the recombinant L. lactis cell is Lactococcus lactis NZ1330 or a derivative thereof.

[0277] In some examples, the polynucleotide encoding the fusion immunogen is cloned on a bacterial expression vector.

[0278] In some examples, the bacterial expression vector comprises an air selection gene. In some examples, the air selection gene encodes an alanine racemase.

[0279] In some examples, the recombinant L. lactis cell constitutively expresses the fusion immunogen.

[0280] In some examples, the fusion immunogen further comprises a C-terminal His tag. In some examples, the oral vaccine composition comprises about IO10CFU of the recombinant L. lactis cell per dose.

[0281] In some examples, the oral vaccine composition is formulated for administration as a booster dose following one or more parenteral administrations of a vaccine composition comprising a fusion immunogen.

[0282] In some examples, the oral vaccine composition is formulated for simultaneous administration with one or more parenteral administrations of a vaccine composition comprising a fusion immunogen.

[0283] In some examples, the oral vaccine composition is capable of inducing a local mucosal immune response against C. difficile upon oral administration to the subject.

[0284] In one example, disclosed herein is a method of making a fusion immunogen, the method comprising selecting at least one immunogenic epitope or fragment from CdeC, selecting at least one immunogenic epitope or fragment from CdeM, and joining the selected epitope or fragment from CdeC and the selected epitope or fragment from CdeM to form a fusion immunogen. In some examples, the selecting comprises in silico epitope prediction, immunoreactivity screening, structural analysis, sequence conservation analysis, or anyDocket No. 11001-247W01

[0285] combination thereof. In some examples, the joining comprises recombinant expression, peptide synthesis, chemical conjugation, or enzymatic ligation.

[0286] In one example, disclosed herein is a method of making a recombinant Lactococcus lactis (L. lactis) strain expressing a CdeC-CdeM fusion immunogen, the method comprising introducing into L. lactis a nucleic acid encoding the fusion immunogen and culturing the L. lactis under conditions permitting expression of the fusion immunogen. In some examples, the method further comprises selecting transformants or integrants that express the fusion immunogen. In some examples, the method further comprises formulating the recombinant L. lactis strain for oral admini stration.

[0287] In some examples, disclosed herein is a method of manufacturing an oral vaccine against C. difficile, wherein the method comprises providing a nucleic acid encoding a CdeC- CdeM fusion or an epitope fusion disclosed herein; cloning the nucleic acid into pNZ7025; transforming L. lactis NZ1330 with the pNZ7025 construct; and culturing the transformed cells under air-based selection conditions to obtain a recombinant oral vaccine strain. In some examples, the nucleic acid used in the method is codon-optimized for L. lactis. In some examples, the method comprises expression driven by promoter PpepN. In some examples, the method confirms the expression of the immunogen by western blot using anti-His antibodies. In some examples, the method comprises formulating a vaccine strain at 10zto 1011CPU per dose.

[0288] In some examples, disclosed herein is a method of manufacturing an immunogen against C. difficile comprises providing a nucleic acid encoding a CdeC-CdeM fusion or an epitope fusion disclosed herein; cloning the nucleic acid into an expression vector; expressing the immunogen in a host cell; and recovering the immunogen. In some examples, the method comprises expressing the immunogen in E. call BL21(DE3). In some examples, the method comprises cloning a nucleic acid encoding the immunogen into pET28a. In some examples, the method comprises purifying the immunogen by affinity chromatography. In some examples, the method comprises purifying the immunogen by Ni-affinity chromatography. In some examples, the method comprises producing an immunogen comprising a His tag.

[0289] Compositions

[0290] Clostridium difficile is the primary causative agent of antibiotic associated diarrhea. Increasing resistance to antibiotics in recent decades has resulted in a reduction in the efficacy of standard methods of treatment. This presents a clear need for a greater understanding of the bacterium, so that alternative methods of treating C. difficile infection (CDI) may be developed.Docket No. 11001-247W01

[0291] Clostridioides difficile (C.difficile) infection (CDI) is a bacterial infectious disease of the gastrointestinal tract caused by Clostridium difficile (C. difficile), a toxin-producing Grampositive anaerobic, spore- forming bacillus. As used herein, CDI includes recurrent CDI, which is defined as complete resolution of CDI while on appropriate therapy, followed by recurrence of CDI after treatment has been stopped. CDI is often associated with disorders of the gastrointestinal tract such as dysbiosis, Crohn's disease, ulcerative colitis, enteritis, irritable bowel syndrome, inflammatory bowel disease, diarrhea, antibiotic-associated diarrhea, and diverticular disease. In some examples, there are provided compositions and methods for prevention or treatment of disorders of the gastrointestinal tract associated with CDI such as, without limitation, dysbiosis, Crohn's disease, ulcerative colitis, enteritis, irritable bowel syndrome, inflammatory bowel disease, diarrhea, antibiotic-associated diarrhea, and diverticular disease.

[0292] In some examples, disclosed herein is an isolated protein comprising a spore surface component of one or more strain of Clostridioides difficile (C. difficile), wherein the spore surface component comprises spore surface proteins (CdeC or CdeM), colonization factors or a combination thereof. As disclosed herein, in some examples, the one or more strains of C. difficile include but not limited to a RT027 strain, a RT078 strain, a RT017 strain, a RT012 strain, a RT003 strain, and a RT009 strain. As used herein, the term "isolated" refers to a molecule that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other macromolecules (e.g., proteins, glycans) from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a protein or peptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A protein or peptide may also be rendered substantially free of naturally associated components by isolation, using purification or separation techniques well known in the art. Surface components from C. difficile used in compositions and methods described herein are generally provided in purified or substantially purified form, i.e., substantially free from other glycopeptides and polypeptides, particularly from host cell proteins or polypeptides. In some examples, the isolated protein is at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80%, at least about 90% pure, or at least about 95%’ pure (by weight).

[0293] As used herein, the term "antigen" refers to a substance that prompts the generation of antibodies and can cause an immune response. The terms "antigen" and "immunogen" are used interchangeably herein, although, in a strict sense, immunogens are substances that elicit aDocket No. 11001-247W01

[0294] response from the immune system, whereas antigens are defined as substances that bind to specific antibodies. An antigen or fragment thereof can be a molecule (i.e., an epitope) that makes contact with a particular antibody (i.e., elicit the immune response), wherein the antibody binds specifically to the antigen.

[0295] In some examples, “variants”, “analogs”, and “fragments” of CdeC, CdeM, or CdeCM refers to an amino acid sequence of the naturally occurring protein or peptide in which a small number of amino acids have been substituted, inserted, or deleted, and which retains the relevant biological activity or function of the starting protein. For example, in the case of an antigen for use in a vaccine, a variant may retain the immunogenic characteristics of the starting protein, sufficient for its intended use in inducing immunity. In the case of an antibody, a variant may retain the antigen-binding properties of the starting protein, sufficient for its intended use in binding specifically to antigen.

[0296] In some examples, a variant includes one or more conservative amino acid substitutions, one or more non-conservative amino acid substitutions, one or more deletions, and / or one or more insertions. A conservative substitution is one in which an amino acid residue is substituted by another amino acid residue having similar characteristics (e.g., charge or hydrophobicity ). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic -hydroxyl side chains: serine and threonine; 3) amide- containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Exemplary conservative amino acids substitution groups are valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine -valine, glutamate-aspartate, and asparagine¬ glutamine. Other conservative amino acid substitutions are known in the art and are included herein. Non-conservative substitutions, such as replacing a basic amino acid with a hydrophobic one, are also well-known in the art.

[0297] As used herein, an "analog" refers to an amino acid sequence of the naturally occurring protein in which one or more amino acids have been replaced by amino acid analogs. Nonlimiting examples of amino acid analogs include non-naturally occurring amino acids, synthetic amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems, polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-Docket No. 11001-247W01

[0298] naturally occurring. In some examples, analogs include modifications which increase glycoprotein or glycopeptide stability. In one example, an analog includes a beta amino acid, a gamma amino acid, or a D-amino acid.

[0299] A "fragment" refers to a portion of the starting molecule which retains the relevant biological activity or function (e.g, antigenicity, antigen-binding, immunogenicity) of the starting molecule.

[0300] A "biologically active" or "functional fragment”, fragment, variant, or analog generally retains biological activity or function of the starting molecule, sufficient for use in the present compositions and methods. Thus, a "biologically active" or "functional fragment”, fragment, variant, or analog may retain the binding specificity, the antigenicity, or the immunogenicity of the starting molecule. In some examples, a fragment, variant or analog has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity to the starting molecule (e.g., protein).

[0301] Disclosed herein, in some examples are bacterial expression vectors utilized for the recombinant production of fusion immunogens or epitope immunogen proteins within a prokaryotic host, such as Escherichia coli. In some examples, these vectors comprise engineered plasmids tailored for specific expression kinetics and purification requirements. For instance, some examples contemplate the use of pET series vectors, including pET28a, pET21a, and pET32, which utilize a strong T7 promoter for high-level protein synthesis in T7-polymerase-expressing strains like BL21(DE3). In some examples, regulated expression is achieved via pBAD vectors controlled by the araBAD promoter and induced by arabinose. To optimize protein processing, some examples include the use of pGEX vectors for the production of GST-tagged proteins, pMAL vectors for MBP-fusion proteins to improve solubility, and the pQE series for high-yield expression of His-tagged proteins suitable for Ni- NTA chromatography. Furthermore, some examples encompass the use of pTrc99A for trc promoter-driven expression and pCDF vectors, which possess unique origins of replication allowing for compatibility with other plasmids, such as those of the pET or pBR322 classes, to facilitate the co-expression of multiple genetic constructs within a single bacterial cell.

[0302] Variants, fragments, or analogs may also be modified at the N- and / or C-terminal ends to allow the polypeptide or fragment to be conformationally constrained and / or to allow coupling to a pharmaceutically acceptable carrier or an adjuvant. In some examples, disclosed herein are immunogenic compositions comprising a fusion immunogen CdeCM protein or a fragment thereof: and a pharmaceutically acceptable carrier, an adjuvant or a combination thereof. In some examples, the pharmaceutically acceptable carrier includes but is not limitedDocket No. 11001-247W01

[0303] to oil-in-water emulsions (e.g., MF59, AS03, AF03), TLR agonists (e.g., CpG oligodeoxynucleotides, monophosphoryl lipid A), saponin-based adjuvants (e.g., QS-21), cytokines (e.g., GM-CSF, IL-2, IL-12), lipid-based earners like liposomes, virosomes, and lipid nanoparticles (LNPs), polymer-based carriers such as PLGA nanoparticles, chitosan nanoparticles, and PEGylated polymers, viral vectors including adenovirus, lentivirus, and vesicular stomatitis virus (VSV), as well as stabilizers and preservatives like sucrose, trehalose, lactose, gelatin, albumin, thimerosal, and buffers such as phosphate-buffered saline (PBS) and Tris buffer, nanoparticle or liposome.

[0304] In some examples, disclosed herein is a vaccine composition comprising a recombinant immunogen, wherein the recombinant immunogen comprises (i) a CdeC polypeptide of Clostridioides difficile (C. difficile); (ii) a CdeM polypeptide of C. difficile; or (iii) a fusion polypeptide comprising a CdeC polypeptide operably linked to a CdeM polypeptide, wherein the composition induces an immune response against C. difficile spores. In some examples, the vaccine composition comprises a CdeC polypeptide, wherein the CdeC polypeptide comprises a full-length CdeC amino acid sequence. In some examples, the vaccine composition comprises a CdeC polypeptide, wherein the CdeC polypeptide is at least 90%, at least 91%, at least 95%, at least 98%, or 100% identical to a reference CdeC sequence from strain CD630. In some examples, the vaccine composition comprises a CdeC polypeptide, wherein the CdeC polypeptide comprises at least one conserved feature selected from: a KKNKRR motif, a HHH motif, six NPC repeats, two CCRQGKGK repeats, and a CNECC motif. In some examples, the vaccine composition comprises a CdeC polypeptide, wherein the CdeC polypeptide comprises a helical region corresponding to amino acids 134 to 365 of a reference CdeC sequence. In some examples, the vaccine composition comprises a CdeM polypeptide, wherein the CdeM polypeptide comprises a full-length CdeM amino acid sequence. In some examples, the vaccine composition comprises a CdeM polypeptide, wherein the CdeM polypeptide is at least 84%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to a reference CdeM sequence from strain CD630, excluding a strain-specific deletion variant corresponding to positions 98 to 137. In some examples, the vaccine composition comprises a CdeM polypeptide, wherein the CdeM polypeptide comprises at least one conserved feature selected from: RREA repeats, NGNNGGNNNNC repeats, and CNCCNCCRK repeats. In some examples, the vaccine composition comprises a fusion polypeptide, wherein the fusion polypeptide comprises full-length CdeC and full-length CdeM joined by a linker. In some examples, the vaccine composition comprises a recombinant immunogen comprising a fusion polypeptide, wherein the linker comprises ggtggcggt. In some examples, the vaccineDocket No. 11001-247W01

[0305] composition comprises a fusion polypeptide, wherein the fusion polypeptide has a molecular weight of about 67 kDa. In some examples, the vaccine composition comprises a fusion polypeptide, wherein the CdeC and CdeM sequences are derived from Clostridioides difficile (C. difficile) strain CD630. In some examples, the vaccine composition comprises a recombinant immunogen, wherein the immunogen is formulated with alum. In some examples, the vaccine composition is configured for intraperitoneal, intramuscular, oral, or combined intramuscular and oral administration. In some examples, the vaccine composition elicits serum IgG, serum IgA, fecal IgA, or a combination thereof against the immunogen. In some examples, the vaccine composition reduces one or more of C. difficile colonization, spore burden, fecal shedding, TcdA level, TcdB level, disease recurrence, and mortality after challenge. In some examples, the vaccine composition comprises an immunogen comprising a CdeC sequence present in strains representing at least 50 ribotypes and at least 6 clades of C. difficile. In some examples, the vaccine composition comprises an immunogen comprising a CdeC antigen encoded by substantially all tested strains and a CdeM antigen encoded by at least 95% of tested strains.

[0306] In some examples, disclosed herein is a fusion immunogen comprises: a first segment comprising a CdeC polypeptide or immunogenic fragment thereof; a second segment comprising a CdeM polypeptide or immunogenic fragment thereof; and an optional linker between the first segment and the second segment, wherein the fusion immunogen induces antibodies that bind C. difficile spores. In some examples, the fusion immunogen comprises an immunogenic fragment that is 6 to 50 amino acids in length. In some examples, the fusion immunogen comprises an immunogenic fragment that is 8 to 30 amino acids in length. In some examples, the fusion immunogen comprises an immunogenic fragment that is 10 to 25 amino acids in length. In some examples, the fusion immunogen comprises a linker selected from: a glycine -rich linker, a serine -rich linker, a G4S linker, a GPGPG linker, a KK linker, and a ggtggcggt linker. In some examples, the fusion immunogen comprises one or more CdeC cytotoxic T-cell epitopes selected from SEQ ID NOs: 11-22. In some examples, the fusion immunogen comprises one or more CdeC helper T-cell epitopes selected from SEQ ID NOs: 23-33. In some examples, the fusion immunogen comprises one or more CdeC B-cell epitopes selected from SEQ ID NOs: 34-42. In some examples, the fusion immunogen comprises one or more CdeM epitopes selected from SEQ ID NOs: 43 to 51. In some examples, the fusion immunogen comprises a sequence selected from: CdeM-EF, CdeC-EF-1, CdeC-EF-2, CdeCM- EF-1, CdeCM-EF-2, and CdeCM-EF-3. In some examples, the fusion immunogen comprises the sequence CdeCM-EF-1 as set forth on SEQ ID NO: 8. In some examples, the fusionDocket No. 11001-247W01

[0307] immunogen comprises the sequence CdeCM-EF-2 as set forth on SEQ ID NO: 9. In some examples, the fusion immunogen comprises the sequence CdeCM-EF-3 as set forth on SEQ ID NO: 10. In some examples, the fusion immunogen comprises one or more amino acids substituted by conservative substitution, wherein the immunogen retains immunogenicity against C. difficile spores. In some examples, the fusion immunogen comprises a conservative substitution selected from: D to E, E to D, N to Q, Q to N, K to R, R to K, S to T, T to S, V to I, I to V, L to I, I to L, F to Y, and Y to F. In some examples, the fusion immunogen has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to any one of the recited epitope fusion sequences.

[0308] In some examples, disclosed herein is a nucleic acid encoding any immunogen of any preceding aspect. In some examples, the nucleic acid encoding an immunogen is codon- optimized for expression in Escherichia coli or L. lactis. In some examples, the expression vector comprises a nucleic acid encoding any immunogen of any preceding example. In some examples, the expression vector is pET28a. In some examples, the expression vector is a pNZ7025 vector.

[0309] In some examples, disclosed herein is a recombinant host cell comprising a nucleic acid encoding any immunogen disclosed in any preceding example or an expression vector comprising the nucleic acid of any preceding example. In some examples, the recombinant host cell is Escherichia coli BL21(DE3). In some examples, the recombinant host cell is L. lactis NZ1330. In some examples, the recombinant host cell expresses an immunogen under control of promoter PpepN. In some examples, the recombinant host cell comprises an air-based selection system. In some examples, the immunogen expressed by a recombinant host cell further comprises a C-terminal His tag.

[0310] In some examples, disclosed herein is an oral vaccine composition comprising a recombinant Lactococcus lactis cell expressing a CdeC-CdeM fusion immunogen or an epitope fusion immunogen of any preceding aspect. In some examples, the immunogen is encoded on pNZ7025. In some examples, the oral vaccine composition is configured for secretion of the immunogen. In some examples, the oral vaccine composition is configured for cell-associated expression of the immunogen. In some examples, the oral vaccine composition comprises an immunogen fused to a Gram-positive cell wall anchor domain comprising an LPXTG motif. In some examples, the anchor domain is derived from a Gram-positive surface protein. In some examples, the oral vaccine composition comprises from 10 ’ to 10!lCFU per dose. In some examples, the oral vaccine composition comprises from 108to 1010CFU per dose. In some examples, the oral vaccine composition comprises about 1010CFU per dose.Docket No. 11001-247W01

[0311] In some examples, disclosed herein is a vaccine composition comprising an immunogen that binds antibodies raised against at least one of: a conserved CdeC region having at least 82% fully conserved positions across aligned CdeC sequences; or a conserved CdeM region having at least 84% fully conserved positions across aligned CdeM sequences after exclusion of a strain-specific truncation variant. In some examples, the vaccine composition comprises an immunogen that excludes a CdeM truncation corresponding to a deletion spanning positions 98 to 137 of a reference alignment. In some examples, the vaccine composition comprises an immunogen selected to preserve at least one conserved motif region of CdeC or CdeM identified in the specification. In some examples, the vaccine composition comprises an immunogen comprising an antigen sequence representative of a phylogenetic cluster associated with RT078 strains. In some examples, the vaccine composition comprises an immunogen comprising a consensus or cluster-representative sequence for a CdeC cluster, a CdeM cluster, or both. In some examples, the vaccine composition comprises a consensus or clusterrepresentative sequence selected from a cluster associated with clade 2, clade 5, or hypervirulent A+B+CDT+ strains.

[0312] In one example, disclosed herein is an isolated protein comprising a spore component of one or more ribotypes of C. difficile, wherein the spore component comprises spore surface layer proteins, spore colonization factors, or a combination thereof. In some examples, the one or more ribotypes of C. difficile are selected from R20291, RT244, RT019, RT027, RT012, RT125, or RT106 ribotypes. In some examples, the spore component is a spore surface layer protein comprising CdeC, CdeM, a functional variant thereof, a homologue thereof, or a combination thereof. In some examples, the CdeC comprises an amino acid sequence set forth in SEQ ID NO: 4, or a fragment thereof. In some examples, the CdeM comprises an amino acid sequence set forth in SEQ ID NO: 3, or a fragment thereof. In some examples, the CdeM comprises the amino acid sequence set forth in SEQ ID NO: 3, or a sequence having at least 90% identity thereto. In some examples, the CdeC comprises the amino acid sequence set forth in SEQ ID NO: 4, or a sequence having at least 90% identity thereto. In some examples, a combination of CdeC and CdeM comprises an amino acid sequence set forth in SEQ ID NO: 2, or a fragment thereof.

[0313] In one example, disclosed herein is an isolated protein comprising a spore exosporium layer protein, a functional variant thereof, or a fusion immunogen comprising at least one immunogenic epitope or immunogenic fragment of a C. difficile spore protein CdeC and at least one immunogenic epitope or immunogenic fragment of a C. difficile spore protein CdeM, wherein the fusion immunogen elicits an immune response against one or both of CdeC andDocket No. 11001-247W01

[0314] CdeM. In some examples, the at least one immunogenic epitope or immunogenic fragment of CdeC comprises aB cell epitope, aT cell epitope, or both, including in R20291, RT244, RT019, RT027, RT012, RT125, RT106AR-1075, C00009694, and C00006475. In some examples, the at least one immunogenic epitope or immunogenic fragment of CdeM comprises a B cell epitope, a T cell epitope, or both. In some examples, the fusion immunogen comprises a plurality of immunogenic epitopes derived from CdeC and a plurality of immunogenic epitopes derived from CdeM. In some examples, the CdeC-derived epitope or fragment and the CdeM-derived epitope or fragment are directly fused. In some examples, the CdeC-derived epitope or fragment and the CdeM-derived epitope or fragment are joined by a linker. In some examples, the linker of any preceding example is a flexible linker, a rigid linker, a protease-cleavable linker, or a peptide spacer. In some examples, the fusion immunogen further comprises a heterologous signal peptide, carrier domain, scaffold domain, secretion sequence, cell wall anchoring domain, or surface display domain. In some examples, the cell wall anchoring domain of any preceding example comprises an LPXTG motif-containing anchoring sequence. In some examples, the fusion immunogen is a chimeric polypeptide configured for mucosal delivery. In some examples, the fusion immunogen is configured to reduce C. difficile spore adherence, persistence, germination-associated colonization, vegetative outgrowth following spore exposure, or a combination thereof. In some examples, the fusion immunogen consists essentially of one or more CdeC-derived epitopes and one or more CdeM-derived epitopes. In some examples, the fusion immunogen further comprises an adjuvant peptide, a mucosal targeting peptide, or an immunostimulatory domain. In some examples, the fusion immunogen comprises full-length CdeC, full-length CdeM, an immunogenic fragment of CdeC, an immunogenic fragment of CdeM, or any combination thereof. In some examples, the fusion immunogen is designated CdeCM.

[0315] In one example, disclosed herein is an isolated nucleic acid encoding the fusion immunogen of any preceding example. In some examples, the nucleic acid is DNA, cDNA, RNA, mRNA, or a codon-optimized variant thereof. In some examples, the nucleic acid is operably linked to a promoter functional in a Gram-positive bacterium. In some examples, the promoter of any preceding example is constitutive, inducible, or environmentally regulated.

[0316] In one example, disclosed herein is an immunogenic composition comprising the fusion immunogen of any preceding example, the nucleic acid of any preceding example, the vector of any preceding example, the host cell of any preceding example, or the recombinant L lactis strain of any preceding example, and a pharmaceutically acceptable carrier, excipient, or diluent. In some examples, the composition is formulated for oral administration. In someDocket No. 11001-247W01

[0317] examples, the composition is formulated as a capsule, tablet, powder, suspension, sachet, gel, microsphere, or enteric dosage form. In some examples, the composition further comprises an adjuvant. In some examples, the adjuvant of any preceding example is a mucosal adjuvant. In some examples, the composition induces a systemic immune response, a mucosal immune response, or both. In some examples, the composition induces IgA, IgG, cellular immunity, or any combination thereof directed against CdeC, CdeM, or both.

[0318] In one example, disclosed herein is a vaccine composition comprising the recombinant Lactococcus lactis (L. lactis) strain of any preceding example and a pharmaceutically acceptable earner, wherein the vaccine composition is configured for oral administration to a subject in need thereof. In some examples, administration of the vaccine composition reduces gastrointestinal colonization by Clostridioides difficile (C. difficile) spores. In some examples, administration of the vaccine composition reduces the risk, severity, recurrence, or duration of C. difficile infection.

[0319] In one example, disclosed herein is an isolated immunogenic epitope of a C. difficile spore protein CdeC. In some examples, the epitope comprises a linear epitope, a conformational epitope mimic, or a combination thereof. In some examples, the epitope is capable of binding an antibody raised against C. difficile spores or of eliciting antibody production in a mammalian subject.

[0320] In one example, disclosed herein is an isolated immunogenic epitope of a C. difficile spore protein CdeM. In some examples, the epitope comprises a linear epitope, a conformational epitope mimic, or a combination thereof. In some examples, the epitope is capable of binding an antibody raised against C. difficile spores or of eliciting antibody production in a mammalian subject.

[0321] In one example, disclosed herein is an epitope fusion comprising a first immunogenic epitope derived from CdeC and a second immunogenic epitope derived from CdeM, wherein the first immunogenic epitope and the second immunogenic epitope are fused in a single molecular construct. In some examples, the first immunogenic epitope and the second immunogenic epitope are arranged in tandem. In some examples, the epitope fusion comprises two or more distinct epitopes from CdeC and one or more distinct epitopes from CdeM. In some examples, the epitope fusion further comprises a linker, scaffold, carrier, or targeting moiety. In some examples, the epitope fusion is a multi-epitope construct. In some examples, the epitope fusion is expressed by a probiotic or food-grade microorganism.Docket No. 11001-247W01

[0322] In one example, disclosed herein is a use of the fusion immunogen of any preceding example, the recombinant L. lactis strain of any preceding example, or the vaccine composition of any preceding example for inducing protective immunity against C. difficile.

[0323] In one example, disclosed herein is a use of the fusion immunogen of any preceding example, the recombinant L. lactis strain of any preceding example, or the vaccine composition of any preceding example in the manufacture of a medicament for preventing or reducing C. difficile colonization or infection.

[0324] In one example, disclosed herein is a fusion immunogen comprising a consensus sequence, variant sequence, conserved sequence, or allelic variant of CdeC and a consensus sequence, variant sequence, conserved sequence, or allelic variant of CdeM, wherein the fusion immunogen retains immunogenicity against C. difficile spores. In some examples, the immunogen comprises at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a reference CdeC -derived immunogenic region and a reference CdeM-derived immunogenic region.

[0325] In one example, disclosed herein is a recombinant microorganism other than L. lactis, the microorganism comprising a nucleic acid encoding the fusion immunogen of any preceding example, wherein the microorganism is suitable for mucosal delivery. In some examples, the microorganism is a Lactobacillus, Lactiplantibacillus, Lacticaseibacillus, Bifidobacterium, or Enterococcus species suitable for oral administration.

[0326] In one example, disclosed herein is a composition comprising a plurality of isolated immunogenic epitopes of CdeC and CdeM, wherein at least one CdeC epitope and at least one CdeM epitope are present in a fused arrangement. In some examples, the fused arrangement is configured to enhance breadth of immune recognition relative to an unfused single-epitope composition.

[0327] In one example, disclosed herein is a fusion immunogen, comprising: a first segment comprising a full length Clostridioides difficile exosporium cysteine-rich protein (CdeC) polypeptide or at least one CdeC immunogenic fragment; a second segment comprising a full length Clostridioides difficile exosporium cysteine -rich protein M (CdeM) polypeptide or at least one CdeM immunogenic fragment; and a linker between the first segment and the second segment, wherein the fusion immunogen elicits an immune response against one or both of CdeC and CdeM polypeptides.

[0328] In some examples, the fusion immunogen comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a sequence having at least 95%, 98%, 99%’ or 99.8% identity thereto.Docket No. 11001-247W01

[0329] In some examples, the at least one CdeC immunogenic fragment or the at least one CdeM immunogenic fragment is selected from R20291, RT244, RT019, RT027, RT012, RT125, or RT106 ribotypes of C. difficile.

[0330] In some examples, the at least one CdeC immunogenic fragment is about 6 to about 50 amino acids in length.

[0331] In some examples, the at least one CdeC immunogenic fragment comprises an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 7, or a sequence having at least 90% identity thereto.

[0332] In some examples, the at least one CdeM immunogenic fragment is about 6 to about 50 amino acids in length.

[0333] In some examples, the at least one CdeM immunogenic fragment comprises an amino acid sequence as set forth in SEQ ID NO: 5, or a sequence having at least 90% identity thereto.

[0334] In some examples, the at least one CdeC immunogenic fragment or the at least one CdeM immunogenic fragment comprises a B cell epitope, a CD4+ T cell epitope, a CD8+ T cell epitope or a combination thereof.

[0335] In some examples, the linker is a flexible linker, a rigid linker, a protease-cleavable linker, or a peptide spacer.

[0336] In some examples, the linker comprises a glycine -rich linker, a serine-rich linker, a G4S linker, a GPGPG linker, a KK linker, or a ggtggcggt linker.

[0337] In some examples, the fusion immunogen further comprises a heterologous signal peptide, a carrier domain, a scaffold domain, a secretion sequence, a cell wall anchoring domain, or a surface display domain. In some examples, a heterologous signal peptide refers to a signal peptide that is not naturally associated with the antigen, immunogen, or polypeptide of interest, but is operably fused thereto to direct trafficking, secretion, translocation, or expression in a selected host system. In certain examples, the heterologous signal peptide is derived from a protein, organism, or expression system different from that of the fused antigenic component. Such a sequence may be included to improve secretion efficiency, processing, extracellular release, membrane translocation, or overall recombinant production of the fusion immunogen. Examples include, but are not limited to, an Ig kappa chain leader peptide, an IL-2 signal peptide, a tissue plasminogen activator signal peptide, a BM40 signal peptide, a PelB leader peptide, an OmpA signal peptide, a PhoA signal peptide, a DsbA signal peptide, a Usp45 secretion signal, a staphylococcal YSIRK-type signal peptide, a protein A signal peptide, or an alpha-mating factor leader sequence. In some examples, the heterologous signal peptide is positioned at the N-terminus of a CdeC-derived polypeptide, a CdeM-derivedDocket No. 11001-247W01

[0338] polypeptide, or another antigenic fragment to promote secretion or proper processing in a bacterial, yeast, or mammalian expression host. In the present application, a heterologous signal peptide may be used to direct the fusion immunogen into a secretion pathway or other cellular trafficking pathway to improve expression, localization, or presentation of the antigenic component.

[0339] A carrier domain refers to a polypeptide domain fused to an antigen, epitope, or immunogenic fragment in order to enhance stability, solubility, expression, immunogenicity, half-life, or delivery of the fused construct. In some examples, the carrier domain serves as a molecular support framework for a relatively small, poorly immunogenic, or otherwise unstable antigenic sequence. In other examples, the carrier domain facilitates improved folding, purification, multimerization, or immune recognition of the fusion immunogen. Examples include, but are not limited to, CRM197, tetanus toxoid or a fragment thereof, diphtheria toxoid or a fragment thereof, keyhole limpet hemocyanin, hepatitis B core antigen, an Fc domain, albumin, an albumin-binding domain, maltose-binding protein, glutathione S-transferase, thioredoxin, SUMO, NusA, ferritin, or a virus-like particle capsid protein. In some examples, a carrier domain is fused to a CdeC antigenic fragment or a CdeM antigenic fragment to enhance immunogenicity or improve recombinant production. In the present application, a carrier domain may be used to support the fusion immunogen structurally or functionally and to improve the ability of the antigenic component to elicit or participate in an immune response.

[0340] As used herein, in some examples “scaffold domain” refers to a structural domain or framework that presents, supports, or organizes one or more fused antigenic sequences in a defined conformation or spatial arrangement. In certain examples, the scaffold domain provides a stable architecture for insertion, fusion, display, or multimeric presentation of an antigenic moiety. In some examples, the scaffold domain promotes ordered presentation of epitopes, enhances structural stability, or enables assembly into higher-order particles or complexes. Examples include, but are not limited to, ferritin, lumazine synthase, a foldon-containing trimerization domain, a fibronectin type III domain, an ankyrin repeat scaffold, an antibody variable domain scaffold, a VH scaffold, a VHH scaffold, a nanobody scaffold, a tetraspanin¬ based scaffold, a viral capsid scaffold, a self-assembling nanoparticle scaffold, or a bacterial spore coat-derived scaffold. In some examples, a CdeC-derived sequence or a CdeM-derived sequence is fused to or inserted into a scaffold domain to provide multivalent or structurally constrained display. In the present application, a scaffold domain may be used to present the fusion immunogen in a stable and optionally multimeric format that enhances antigen display, accessibility, or immunological recognition.Docket No. 11001-247W01

[0341] As used herein, “secretion sequence” refers to a sequence element that directs a fused polypeptide into a secretion pathway, such that the polypeptide is transported across a membrane, exported from the cytoplasm, released into the periplasm, secreted extracellularly, or otherwise processed for extracellular or surface-associated localization. In certain examples, the secretion sequence comprises a classical signal peptide. In other examples, the secretion sequence comprises a leader sequence, transport signal, or other secretion-facilitating sequence recognized by a Sec pathway, Tat pathway, or another secretion system. Examples include, but are not limited to, a PelB secretion sequence, an OmpA secretion sequence, a PhoA secretion sequence, aDsbA secretion sequence, a Tor A leader sequence, a Usp45 secretion sequence, an alpha-factor secretion leader, a YSIRK-type secretion signal, a Gram-positive secretion leader, or another N -terminal secretory leader peptide. In some examples, the secretion sequence directs a CdeC-containing or CdeM-containing fusion protein for export to the extracellular environment or to the cell surface. In the present application, a secretion sequence may be used to facilitate secretion, extracellular release, or surface-accessible localization of the fusion immunogen.

[0342] A “cell wall anchoring domain” refers to a domain or sequence that mediates attachment of a fusion protein to the cell wall of a host cell. In certain examples, the cell wall anchoring domain is a Gram-positive bacterial anchoring sequence that permits covalent or non-covalent retention of the fusion immunogen at or within the cell wall. In some examples, the cell wall anchoring domain comprises a sorting signal recognized by a sortase enzyme, thereby allowing attachment to peptidoglycan or another cell wall component. Examples include, but are not limited to, an LPXTG motif-containing anchoring sequence, a protein A anchoring region, an M protein anchoring region, a fibronectin-binding protein anchoring region, a Cna-type anchoring region, a sortase-recognized cell wall sorting signal, a cell wall anchor comprising an LPXTG motif with an adjacent hydrophobic region and charged tail, a LysM-containing wall -binding region, a G W module-containing wall-binding domain, or a choline-binding wall-associated domain. In some examples, the cell wall anchoring domain is fused to a CdeC-derived or CdeM-derived immunogen to retain the fusion protein at the surface of a bacterial host cell. In the present application, a cell wall anchoring domain may be used to localize and retain the fusion immunogen at the cell wall for surface presentation, mucosal delivery, or immune exposure.

[0343] A “surface display domain” refers to a domain that enables presentation of a fused antigen, epitope, or polypeptide on the exterior surface of a cell, spore, vesicle, viral particle, or other biological structure. In certain examples, the surface display domain functions byDocket No. 11001-247W01

[0344] membrane insertion, wall anchoring, coat incorporation, outer membrane exposure, spore coat association, lipid anchoring, or fusion to a naturally surface-localized protein. In some examples, the surface display domain provides external accessibility of the fusion immunogen to a host immune system, receptor, ligand, or binding partner. Examples include, but are not limited to, an LPXTG-containing anchoring domain, an autotransporter domain, an AIDA-I domain, an intimin domain, an ice nucleation protein domain, an Lpp-OmpA display domain, an outer membrane protein display domain, a spore coat protein such as CotB, CotC, or CotG, a glycosylphosphatidylinositol anchor signal, a viral envelope transmembrane display domain, a membrane anchor peptide, or an extracellular vesicle display scaffold. In some examples, the surface display domain is used to present a C deC-derived antigen, a CdeM-derived antigen, or another Clostridioides difficile spore-associated antigen on the exterior of a host cell or particle. In the present application, a surface display domain may be used to expose the fusion immunogen at an outer surface such that the antigenic component is accessible for immune recognition, binding interactions, or vaccine delivery.

[0345] In some examples, the cell wall anchoring domain comprises an LPXTG motif¬ containing anchoring sequence.

[0346] In some examples, the fusion immunogen further comprises an adjuvant peptide, a mucosal targeting peptide, or an immunostimulatory domain.

[0347] In some examples, the at least one CdeC immunogenic fragment comprises a CD8+ T cell epitope, wherein the CD8+ T cell epitope is selected from YTDEINSED (SEQ ID NO: 11), RNCETTFEF (SEQ ID NO: 12), LEDFDLDPL (SEQ ID NO: 13), CTEFVALAF (SEQ ID NO: 14), FVALAFPAV (SEQ ID NO: 15), AVRAGGGCK (SEQ ID NO: 16), RVDYVEFTF (SEQ ID NO: 17), LPADGRAVT (SEQ ID NO: 18), YELIIPNDI (SEQ ID NO: 19), RAVTLRQEY (SEQ ID NO: 20), CEPFYELII (SEQ ID NO: 21), or IQPRLVDTF (SEQ ID NO: 22).

[0348] In some examples, the at least one CdeC immunogenic fragment comprises a CD4+ T cell epitope, wherein the CD4+ T cell epitope is selected from NFSVSNAVPFAIEAN (SEQ ID NO: 23), CEPFYELIIPNDIDL (SEQ ID NO: 24), DTMQFQTFTDATGPN (SEQ ID NO: 25), EEV YTDEINSEDMRG (SEQ ID NO: 26), EFTFNTLSAPICLPA (SEQ ID NO: 27), EVYTDEINSEDMRGF (SEQ ID NO: 28), MQFQTFTDATGPNGE (SEQ ID NO: 29), QRGLTVAVRNLVLEL (SEQ ID NO: 30), RNFSVSNAVPFAIEA (SEQ ID NO: 31), SNDGIVIDTGMTTLE (SEQ ID NO: 32), or VEFTFNTLSAPICLP (SEQ ID NO: 33).

[0349] In some examples, the at least one CdeC immunogenic fragment comprises a B cell epitope, wherein the B cell epitope is selected from YVEFTFNTLSAPICLP (SEQ ID NO: 34),Docket No. 11001-247W01

[0350] NFSVSNAVPFA1EANR (SEQ ID NO: 35), VGRNCETTFEFAVCGE (SEQ ID NO: 36), DMRGFKKSHHHNGCNT (SEQ ID NO: 37), PNPIQPRLVDTFSKVC (SEQ ID NO: 38), YTDEINSEDMRGFKKS (SEQ ID NO: 39), PSAGQASVTIEKICLS (SEQ ID NO: 40), EVFGSIPSAGQASVTI (SEQ ID NO: 41), or IVVLASPNPIQPRLVD (SEQ ID NO: 42).

[0351] In some examples, the at least one CdeM immunogenic fragment comprises a CD8+ T cell epitope, a CD4+ T cell epitope or a B cell epitope, wherein the CD8+ T cell epitope, the CD4+ T cell epitope or the B cell epitope, is selected from REREAREAF (SEQ ID NO: 43), ERERREAEIREREAR (SEQ ID NO: 44), KYNYKGIEYLAEAAR (SEQ ID NO: 45), CERFRREAEIREREA (SEQ ID NO: 46), NCERFRREAEIRERE (SEQ ID NO: 47), GMECEARRNGNNGGNN (SEQ ID NO: 48), EYEREAYDEDRERRGS (SEQ ID NO: 49), EAEIREREAREAFCES (SEQ ID NO: 50), or REAREAFCESSEKKKE (SEQ ID NO: 51).

[0352] In some examples, the fusion immunogen is selected from a CdeM-EF peptide comprising a sequence as set forth in SEQ ID NO: 5 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto; CdeC-EF-1 peptide comprising a sequence as set forth in SEQ ID NO: 6 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto; CdeC-EF-2 peptide comprising a sequence as set forth in SEQ ID NO: 7 or a sequence having at least 95%, 98%, 99% or 99.8%’ identity thereto; CdeCM-EF-1 peptide comprising a sequence as set forth in SEQ ID NO: 8 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto; CdeCM-EF-2 peptide comprising a sequence as set forth in SEQ ID NO: 9 or a sequence having at least 95%, 98%, 99% or 99.8% or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

[0353] In some examples, the first segment comprises a full length CdeC polypeptide comprising a sequence as set forth in SEQ ID NO: 4 or a sequence having at least 90% identity thereto.

[0354] In some examples, the full length CdeC polypeptide comprises at least one conserved region selected from a KKNKRR motif, a HHH motif, six NPC repeats, two CCRQGKGK repeats, or a CNECC motif.

[0355] In some examples, the second segment comprises a full length CdeM polypeptide comprising a sequence as set forth in SEQ ID NO: 3 or a sequence having at least 90% identity thereto.

[0356] In some examples, the full length CdeM polypeptide comprises at least one conserved region selected from RREA repeats, NGNNGGNNNNC repeats, or CNCCNCCRK repeats.

[0357] In some examples, one or more amino acids as set forth in SEQ ID NO: 4 or SEQ ID NO: 3 are substituted by a conservative substitution.Docket No. 11001-247W01

[0358] As used herein, a “vector” refers to a nucleic acid construct, such as a plasmid, phagemid, cosmid, artificial chromosome, or other recombinant polynucleotide molecule, that is capable of carrying, maintaining, replicating, and / or expressing a heterologous nucleic acid sequence in a host cell. In certain examples, the vector is a bacterial plasmid configured for cloning, propagation, transcription, translation, inducible expression, fusion protein production, secretion, periplasmic targeting, or affinity-based purification in a bacterial host, such as E. coli. Patent literature commonly lists bacterial expression vectors such as pBAD vector series, pET vector series, pGEX vector series, pMAL vectors, pQE vector series, pTrc99A, and pCDF vectors as representative plasmid backbones for recombinant production of heterologous proteins. Examples of bacterial plasmids include, but are not limited to, a pET vector, a pBAD vector, a pGEX vector, a pMAL vector, a pQE vector, a pTrc99A vector, or a pCDF vector. In some examples, the bacterial plasmid is a pET vector, such as pET-3 or pET21a, for high-level expression of a recombinant protein in a bacterial host. In some examples, the bacterial plasmid is a pBAD vector for regulated expression from an arabinose¬ responsive promoter. In some examples, the bacterial plasmid is a pGEX vector, such as pGEX-4T-1, for expression of a glutathione S-transferase fusion protein. In some examples, the bacterial plasmid is a pMAL vector, such as pMAL-c2, pMAL-p2, or pMAL-c2x, for expression of a maltose-binding protein fusion. In some examples, the bacterial plasmid is a pQE vector, such as pQE-30 or pQE80L, for histidine-tagged recombinant protein expression. In some examples, the bacterial plasmid is a pTrc99A vector for IPTG-inducible expression in bacteria. In some examples, the bacterial plasmid is a pCDF vector, such as pCDF- lb, for expression in bacterial systems, including systems using multiple compatible plasmids. These vector families are expressly identified in patent literature as exemplary bacterial vectors or expression vectors.

[0359] In the current application, the vector may be used to introduce a nucleic acid encoding a fusion immunogen, antigenic fragment, CdeC -derived polypeptide, CdeM-derived polypeptide, carrier domain-containing construct, scaffold-containing construct, or surface display construct into a bacterial host cell for cloning, propagation, and / or recombinant expression. In some examples, the vector is selected based on the intended mode of expression, including inducible cytoplasmic expression, secretion, fusion-tag expression, affinity purification, or co-expression with one or more additional polynucleotides.

[0360] In one example, disclosed herein is a vaccine composition, comprising: a fusion immunogen, wherein the fusion immunogen comprises (i) a full length CdeC polypeptide or at least one CdeC immunogenic fragment and (ii) a full length CdeM polypeptide or at least oneDocket No. 11001-247W01

[0361] CdeM immunogenic fragment, wherein (i) and (ii) are fused directly or through a linker; and a pharmaceutically acceptable carrier, an adjuvant or a combination thereof.

[0362] In some examples, the fusion immunogen comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

[0363] In some examples, the vaccine composition elicits at least a B cell response, a CD4+ T cell response, including Thl, Th2, or Thl7, or a CD8+ T cell response.

[0364] In some examples, the adjuvant comprises alum, aluminum hydroxide or aluminum phosphate.

[0365] In some examples, the pharmaceutically acceptable carrier comprises a nanoparticle or a liposome.

[0366] In some examples, the vaccine composition further comprises at least one other pharmaceutically effective drug, wherein the at least one other pharmaceutically effective drug is an antibiotic.

[0367] In some examples, the antibiotic is selected from one or more metronidazole, amoxycillin, tetracycline, erythromycin, clarithromycin or tinidazole.

[0368] In some examples, the vaccine composition is formulated for parenteral, intraperitoneal, or intramuscular administration.

[0369] In some examples, the vaccine composition reduces one or more of spore colonization, TcdA toxin or TcdB toxin production, or spore burden in a subject.

[0370] Adjuvants generally increase the specificity and / or the level of immune response. An adjuvant may thus reduce the quantity of antigen necessary to induce an immune response, and / or the frequency of injection necessary in order to generate a sufficient immune response to benefit the subject. Any compound or compounds that act to increase an immune response to an antigen and are suitable for use in a subject (e.g., pharmaceutically acceptable) may be used as an adjuvant in compositions, vaccines, and methods of the invention. In some examples, the adjuvant includes but not limited to alum, aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, calcium phosphate hydroxide, Freund's complete adjuvant, MONTANIDE™, Freund's incomplete adjuvant, iscoms, iscom matrix, ISCOMATRIX™ adjuvant, MATRIX M™ adjuvant, MATRIX C™ adjuvant, MATRIX Q™ adjuvant, AbISCO™-10() adjuvant, AbISCO™-3()0 adjuvant, ISCOPREP™, an ISCOPREP™ derivative, adjuvant containing ISCOPREP ™ or an ISCOPREP™ derivative, QS-21, a QS-21 derivative, and an adjuvant containing QS-21 or a QS21 derivative.Docket No. 11001-247W01

[0371] As disclosed herein, in some examples, the immunogenic compositions of any preceding aspect elicit an immune response include but not limited to at least a B cell response, a CD4+ T cell response, or a CD8+ T cell response. As used herein, the term "immune response" refers to the response of immune system cells to external or internal stimuli (e.g., antigens, cell surface receptors, cytokines, chemokines, and other cells) producing biochemical changes in the immune cells that result in immune cell migration, killing of target cells, phagocytosis, production of antibodies, production of soluble effectors of the immune response, and the like. An “immunogenic" molecule is one that is capable of producing an immune response in a subject after administration. As disclosed herein, in some examples, the CD4+ T cell response includes but is not limited to Thl, Th2, or Thl7 response. In some examples, a Thl response is associated with the production of cytokines such as interferon gamma (IFN-y), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-a); a Th2 response is associated with IL-4, IL-5, and IL-13; and a Thl 7 response is associated with IL-17, IL-21, and IL-22. By tailoring the immunogenic composition to favor one or more of these cytokine profiles, it is possible to optimize the nature and magnitude of the immune response, thereby enhancing protection against or clearance of the GDI.

[0372] As disclosed herein, in some examples, the immunogenic compositions of any preceding aspect, further comprises at least one other pharmaceutical product. In some examples, the at least one other pharmaceutical product includes but is not limited to standard antibiotics, fecal microbial transplantation, monoclonal antibodies (e.g., Bezlotoxumab and Actoxumab), SER-109 and RBX2660 (live biotherapeutic products derived from fecal microbiota), rifaximin, probiotics, immunotherapeutic products targeting toxins, or proton pump inhibitors. In some examples, the antibiotics include but are not limited to metronidazole, amoxycillin, tetracycline, erythromycin, clarithromycin or tinidazole.

[0373] In some examples, the vaccine of any preceding aspect further comprises a pharmaceutically acceptable carrier(such as, for example, including but not limited to nanoparticle or liposome), an adjuvant (such as, for example, including but not limited to alum, aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, calcium phosphate hydroxide, Freund's complete adjuvant, MONTANIDE™, Freund's incomplete adjuvant, iscoms, iscom matrix, ISCOMATRIX™ adjuvant, MATRIX M™ adjuvant, MATRIX C™ adjuvant, MATRIX Q™ adjuvant, AbISCO™-100 adjuvant, AbISCO™-3()() adjuvant, ISCOPREP™, an ISCOPREP™ derivative, adjuvant containing ISCOPREP™ or an ISCOPREP™ derivative, QS-21, a QS-21 derivative, and an adjuvant containing QS-21 or a QS21 derivative) or a combination thereof.Docket No. 11001-247W01

[0374] In some examples, disclosed herein is a vector comprising a polynucleotide sequence encoding a Clostridioides difficile (C. difficile) fusion immunogen CdeCM or epitope fusion immunogen or a fragment thereof. The polynucleotide sequence encoding the CdeCM or CdeCM-EF immunogen may be obtained from one or more C. difficile strains, including but not limited to RT027, RT078, RT017, RT012, RT003, or RT009. As disclosed herein, the polynucleotide sequence may comprise a sequence as set forth in SEQ ID NO: 1 or a sequence exhibiting at least 95%, 98%, 99%, or 99.8% identity thereto.

[0375] A “vector” refers to any vehicle that carries a polynucleotide into a cell for the expression of the polynucleotide in the cell. The vector may be, for example, a plasmid, a virus, a phage particle, or a nanoparticle. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself. In some examples, the vector is a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host cell. Such control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation. In some examples, the vector is a lipid nanoparticle. Lipid nanoparticles can be used to deliver mRNA to a host ceil for expression of the mRNA in the host cell.

[0376] In some examples, the expression vector comprises a plasmid or a virus or viral vector. A plasmid or a viral vector can be capable of extrachromosomal replication or, optionally, can integrate into the host genome. As used herein, the term "integrated" used in reference to an expression vector (e.g., a plasmid or viral vector) means the expression vector, or a portion thereof, is incorporated (physically inserted or ligated) into the chromosomal DNA of a host cell. As used herein, a “viral vector” refers to a virus-like particle containing genetic material which can be introduced into a eukaryotic cell without causing substantial pathogenic effects to the eukaryotic cell. A wide range of viruses or viral vectors can be used for transduction but should be compatible with the cell type the virus or viral vector are transduced into (e.g., low toxicity, capability to enter cells). Suitable viruses and viral vectors include adenovirus, lentivirus, retrovirus, among others. In some examples, the expression vector encoding a chimeric polypeptide is a naked DNA or is comprised in a nanoparticle (e.g., liposomal vesicle, porous silicon nanoparticle, gold-DNA conjugate particle, polyethyleneimine polymer particle, cationic peptides, etc.).Docket No. 11001-247W01

[0377] The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61: 1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61: 1226-1239 (1987); Zhang " Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell. Biol. 4: 1528-1533 (1984); Varga et al„ J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

[0378] Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred example of this type of vector is the P4.1 C vector produced by A vigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0379] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specificDocket No. 11001-247W01

[0380] expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.

[0381] Typically, the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site¬ specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United states Patent No. 6,261,834 is herein incorporated by reference for material related to the AAV vector.

[0382] In some examples, the vector disclosed herein may be a plasmid or an expression vector. As disclosed herein, the vector may be formulated with a pharmaceutically acceptable carrier, which may include nanoparticles or liposomes, thereby enhancing delivery and stability. Also disclosed herein, the vector may be incorporated into a viral vector system, such as an adenovirus vector, to facilitate gene delivery.

[0383] In some examples, disclosed herein is an immunogenic composition comprising a C. difficile spore surface protein (CdeC, CdeM, or fusion CdeCM) or a fragment thereof, in combination with a pharmaceutically acceptable carrier, an adjuvant, or a combination thereof. In some examples, the adjuvant may be selected from aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, calcium phosphate hydroxide, Freund's complete adjuvant, MONTAN1DE™, Freund's incomplete adjuvant, ISCOMS™, ISCOMATRIX™ adjuvant, MATRIX M™ adjuvant, MATRIX C™ adjuvant, MATRIX Q™ adjuvant, AbISCO™-100 adjuvant, AbISCO™-300 adjuvant, ISCOPREP™, QS-21, or derivatives thereof.

[0384] As disclosed herein, the immunogenic composition may be derived from C. difficile strains including RT027, RT078, RT017, RT012, RT003, or RT009. The spore surface protein (CdeC, CdeM, or fusion CdeCM) included in the composition may have a sequence as set forth in SEQ ID NO: 1 or exhibit at least 95%, 98%, 99%, or 99.8% identity to the reference sequence. The immunogenic composition is capable of eliciting at least a B cell response, a CD4+ T cell response — including Thl, Th2, or Thl7 responses — or a CD8+ T cell response. Furthermore, the pharmaceutically acceptable carrier may comprise nanoparticles or liposomes to improve immunogenicity.

[0385] In some examples, disclosed herein is a vaccine for preventing C. difficile infection, comprising an isolated spore surface protein (CdeC, CdeM, or fusion CdeCM), a vector as disclosed herein, or an immunogenic composition. In some examples, the vaccine induces an adaptive immune response against all strains of C. difficile. As disclosed herein, the vaccine may also be combined with at least one other pharmaceutical product, such as an antibiotic,Docket No. 11001-247W01

[0386] which may include metronidazole, amoxicillin, tetracycline, erythromycin, clarithromycin, or tinidazole.

[0387] A recombinant protein, or isolated protein, encoding contiguous immunodominant epitopes from C. difficile spore surface protein components may be made for use in the vaccine. The recombinant protein may serve as the active component in a vaccine, or recombinant DNA encoding the recombinant protein may be inserted into an appropriate expression system for the generation of a recombinant peptide vaccine in a suitable host.

[0388] Recombinant DNA encoding the recombinant protein can be generated by PCR amplification of the DNA encoding peptide regions of interest, incorporating cleavage sites for restriction endonucleases into the primers. The amplified fragments can thus be cleaved to generate compatible ends and spliced together to create a recombinant DNA.

[0389] Methods of treating / preventing against C. difficile infection

[0390] In some examples, disclosed herein is a method of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing C. difficile infection in a subject, comprising administering to the subject a therapeutically effective dose of a vaccine, wherein the vaccine comprises an isolated protein or a vector (such as, for example, including but not limited to a plasmid, an expression vector or a viral vector), wherein the isolated protein or the vector comprises a C. difficile spore surface protein (CdeC, CdeM, or CdeCM) or a fragment thereof.

[0391] In some examples, disclosed herein is a method of treating a subject with a C. difficile infection, comprising administering to the subject a therapeutically effective dose of an isolated spore surface protein (CdeC, CdeM, or CdeCM), a vector, an immunogenic composition, or a vaccine as disclosed herein. In some examples, the subject may receive at least one therapeutically effective dose. Additionally, the method may involve the co-administration of another pharmaceutical product, such as an antibiotic, selected from metronidazole, amoxicillin, tetracycline, erythromycin, clarithromycin, or tinidazole. The therapeutically effective dose may be administered via intravenous, intramuscular, intraperitoneal, intradermal, or subcutaneous routes.

[0392] In some examples, disclosed herein is a method of immunizing a subject against C. difficile infection by administering a therapeutically effective dose of the vaccine as disclosed herein. In some examples, at least one dose of the vaccine is administered, and the vaccine may also comprise another pharmaceutical product such as an antibiotic. The vaccine may be administered intravenously, intramuscularly, intraperitoneally, intradermally, or subcutaneously.Docket No. 11001-247W01

[0393] In some examples, the vaccine is a nucleotide- based vaccine (DNA and mRNA). DNA vaccination involves immunization with recombinant DNA encoding the antigen or epitope of interest, cloned in a vector which promotes high level expression in mammalian cells. Typically, the vector is a plasmid vector which is also replicated in a prokaryotic vector such as Escherichia coli, so that the DNA can be produced in quantity. Following immunization, the plasmid enters a host cell, where it remains in the nucleus, and directs synthesis of the recombinant polypeptide. The polypeptide stimulates the production of neutralizing antibodies, as well as activating cytotoxic T-cells.

[0394] In some examples, an effective amount of an immunogenic composition or vaccine comprising a protein contains about 0.05 to about 1500 pg protein, about 10 to about 1000 pg protein, about 30 to about 500 pg, or about 40 to about 300 pg protein, or any integer between those values. For example, a protein may be administered to a subject at a dose of about 0.1 pg to about 200 mg, e.g., from about 0.1 pg to about 5 pg, from about 5 pg to about 10 pg, from about 10 pg to about 25 pg, from about 25 pg to about 50 pg, from about 50 pg to about 100 pg, from about 100 pg to about 500 pg, from about 500 pg to about 1 mg, or from about 1 mg to about 2 mg, with optional boosters given at, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, two months, three months, 6 months and / or a year later.

[0395] In some examples, an effective amount of an isolated protein or immunogenic composition for passive or active immunization ranges from about 0.001 to about 30 mg / kg body weight, for example, about 0.01 to about 25 mg / kg body weight, about 0.1 to about 20 mg / kg body weight, about 1 to about 10 mg / kg, or about 10 mg / kg to about 20 mg / kg.

[0396] An isolated protein, immunogenic composition, vaccine, or vector may also be administered once per month, twice per month, three times per month, every other week (qow ), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (lid). For prophylactic purposes, the amount of peptide in each dose is selected as an amount which induces an immunoprotective response without significant adverse side effects in a typical vaccine. Following an initial vaccination, subjects may receive one or several booster immunizations adequately spaced.

[0397] In one example, disclosed herein is a method of treating Clostridioides difficile infection in a subject, comprising: administering to a subject an effective amount of a vaccine composition, wherein the vaccine composition comprises a fusion immunogen comprising a CdeC component and a CdeM component, wherein the CdeC component comprises an amino acid sequence of SEQ ID NO: 4 or an immunogenic fragment thereof, and wherein the CdeMDocket No. 11001-247W01

[0398] component comprises an amino acid sequence of SEQ ID NO: 3 or an immunogenic fragment thereof.

[0399] In some examples, the fusion immunogen comprises an amino acid sequence as set forth in SEQ ID NO: 2.

[0400] In some examples, the vaccine composition further comprises an adjuvant.

[0401] In some examples, the vaccine composition is administered intraperitoneally or intramuscularly.

[0402] In some examples, the vaccine composition is administered in three doses.

[0403] In some examples, the three doses are administered at about two-week intervals. In some examples, each dose comprises about 10 pg of the fusion immunogen.

[0404] In some examples, the method reduces Clostridioides difficile spore colonization. In one example, disclosed herein is a method of treating a subject against Clostridioides difficile infection, comprising: administering a first vaccine composition, wherein the first vaccine composition comprises a fusion immunogen comprising a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment, wherein the first vaccine composition further comprises a pharmaceutically acceptable carrier, an adjuvant or a combination thereof; and administering a second vaccine composition comprising a recombinant Lactococcus lactis cell comprising a polynucleotide encoding said fusion immunogen, wherein the second vaccine composition further comprises a pharmaceutically acceptable carrier, an adjuvant or a combination thereof.

[0405] In some examples, the first vaccine composition is administered intramuscularly, and the second vaccine composition is administered orally.

[0406] In some examples, the first vaccine composition is administered in two doses separated by about 2 weeks, and the second vaccine composition is administered as a third booster dose.

[0407] In some examples, the first vaccine composition comprises about 10 pg of the fusion immunogen.

[0408] In some examples, the second vaccine composition comprises about 10⁹ CFU to about 1010CFU of the recombinant Lactococcus lactis cell.

[0409] In one example, disclosed herein is a method of treating a subject against Clostridioides difficile infection, comprising: orally administering to the subject a recombinant Lactococcus lactis cell comprising a bacterial expression vector encoding a fusion immunogen, wherein the fusion immunogen comprises an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.Docket No. 11001-247W01

[0410] In some examples, the recombinant Lactococcus lactis cell comprises Lactococcus lactis NZ1330 cell strain.

[0411] In some examples, the bacterial expression vector is pNZ7025 or a derivative thereof. In some examples, the bacterial expression vector comprises an air gene for selection in an air-deficient L. lactis cell.

[0412] In some examples, the bacterial expression vector comprises a PpepN promoter operably linked to a polynucleotide encoding the fusion immunogen.

[0413] In some examples, the fusion immunogen further comprises a C-terminal His tag. In some examples, the recombinant Lactococcus lactis cell constitutively expresses the fusion immunogen.

[0414] In some examples, about IO10CFU of the recombinant Lactococcus lactis cell are administered to the subject.

[0415] In some examples, the oral administration induces a local mucosal immune response against C. difficile.

[0416] In some examples, the oral administration reduces C. difficile colonization following challenge.

[0417] In one example, disclosed herein is a method of treating or preventing C. difficile infection in a subject, comprising: administering to the subject a therapeutically effective dose of a vaccine, wherein the vaccine comprises an isolated protein or a vector, wherein the isolated protein or the vector comprises a CdeCM fusion immunogen or a fragment thereof. In some examples, the vaccine further comprises a pharmaceutically acceptable carrier, an adjuvant or a combination thereof. In some examples, the vaccine is administered intravenously, intramuscularly, intraperitoneally, intradermally, or subcutaneously to the subject. In some examples, the vector is a plasmid, an expression vector or a viral vector. In some examples, the CdeCM fusion immunogen comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. In some examples, the CdeCM fusion immunogen is encoded by a polynucleotide, wherein the polynucleotide comprises a sequence as set forth in SEQ ID NO: 1 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

[0418] In one example, disclosed herein is a method of treating a subject against Clostridioides difficile infection by a heterologous prime-boost regimen, comprising: administering intramuscularly to a subject a first vaccine composition comprising about 10 pg of a fusion immunogen, wherein the fusion immunogen comprises an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 and an adjuvant: and orally administering to the subject a second vaccine composition comprising about 107CFU to aboutDocket No. 11001-247W01

[0419] IO10CPU of a recombinant L. lactis cell expressing said fusion immunogen. In some examples, the first vaccine composition is administered in two doses prior to oral administration of the second vaccine composition. In some examples, the two doses of the first vaccine composition are separated by about two weeks. In some examples, the oral administration comprises a third booster immunization. In some examples, the recombinant Lactococcus lactis cell is Lactococcus lactis NZ1330 comprising pNZ7025 encoding the fusion immunogen. In some examples, the heterologous prime-boost regimen provides significant protection against challenge with Clostridioides difficile strain R2.0291. In some examples, the regimen induces both systemic and mucosal immune responses. In some examples, the regimen reduces one or more of Clostridioides difficile colonization, toxin level, or spore burden following challenge.

[0420] In one example, disclosed herein is a method of preventing or reducing C. difficile colonization in a subject, the method comprising orally administering to the subject an effective amount of the recombinant L. lactis strain of any preceding example or the vaccine composition of any preceding example. In some examples, the colonization is spore-mediated intestinal colonization. In some examples, the orally administered strain survives gastric transit and delivers the fusion immunogen to the intestinal mucosa.

[0421] In one example, disclosed herein is a method of preventing or reducing C. difficile infection in a subject, the method comprising administering an effective amount of the fusion immunogen of any preceding example or a composition comprising the same. In some examples, the infection is a primary infection. In some examples, the infection is a recurrent infection. In some examples, administration is prophylactic. In some examples, administration is therapeutic adjunctive administration.

[0422] Kits

[0423] In some examples, disclosed herein is a kit, wherein the kit comprises a first component comprising recombinant CdeCM protein with alum for intramuscular administration; and a second component comprising recombinant Lactococcus lactis (L. lactis) expressing CdeCM for oral administration. In some examples, the kit comprises about 10 tig of recombinant CdeCM in the first component per dose. In some examples, the kit comprises about 1010CFU in the second component per dose. In some examples, the kit is configured for a prime-boost regimen comprising two intramuscular administrations followed by one oral booster administration.

[0424] In one example, disclosed herein is a kit comprising the fusion immunogen of any preceding example, the recombinant L. lactis strain of any preceding example, theDocket No. 11001-247W01

[0425] immunogenic composition of any preceding example, the vaccine composition of any preceding example, the isolated epitope of any preceding example, or the epitope fusion of any preceding example, and instructions for use in inducing immunity against C. difficile. In some examples, the kit further comprises a booster dose, an adjuvant, a dosing device, a delivery vehicle, or a combination thereof.

[0426] EXAMPLES

[0427] The following examples are set forth below to illustrate the compounds, systems, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all examples of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0428] Example 1: CdeC and CdeM are widely encoded by C. difficile

[0429] CdeC sequences were detected in all 150 strains in the database (Table 1), which represent a total of 52 ribotypes (RTs) and sequence types (STs), six clades, and five toxin genotypes. CdeM was found in 147 / 150 strains, with strains TW11-RT078 (RT078), HGP05 (RT125), and MA_2 (ST340) being the only strains that did not encode detectable CdeM. In the context of vaccinating against the CdeM and CdeC proteins of C. difficile, this result indicates that strains from a diverse array of RTs, STs, toxinotypes, and clades are likely to encode for these proteins. Although a larger-scale search could uncover additional strains of C. difficile that lack CdeC and / or CdeM, the initial analysis indicates that these proteins could serve as an abundant vaccine target against C. difficile.

[0430] Table 1. C. difficile strains for analysis. Toxin genotypes are reported based on either prior literature source or by BLAST analysis. 35 / 150

[0431] Ribotype Strain Toxins Database Accession number /

[0432] Barcode

[0433] RT001 CD105HS12 A+B+CDT- GenBank NZ. FMLV00000000.1

[0434] DSM 1296T A+B+CDT- GenBank CP011968.1 C00000193 A+B+CDT- EnteroBase CLO„AA3424AA RT002 S-0253 A+B+CDT- GenBank CP076401, CP076402

[0435] AR- 1070 A+B+CDT- EnteroBase CLO_CA3086AA CD105HS7 A+B+CDT- GenBank NZJFMLZOOOOOOOO.1

[0436]

[0437] Docket No. 11001-247W01

[0438] AR- 1074 A+B+CDT- GenBank SAMN 13029155 AR- 1084 A+B+CDT- GenBank SAMN 13029165 RT003 VP1 10463 A+B+CDT- EnteroBase CLO_AA6882AA C00000562 A+B+CDT- EnteroBase CLO_AA3453AA CD-15-01031 A+B-FCDT- EnteroBase CLO.. BA9410AA RT005 CD105HS10 AH-B-FCDT" GenBank NZ FMLY00000000.1 (ST6) C00002831 A+B+CDT- EnteroBase CLO„AA2409AA

[0439] CD-16-00159 A+B+CDT- EnteroBase CLO_BA9401AA RT009 CD37 A-B-CDT- GenBank NZ_AHJJ00000000.1

[0440] Z31 A-B-CDT- GenBank CP013196

[0441] CD- 16-00526 A-B-CDT- EnteroBase CLO.. BA9556AA RT010 DSM 29688 A-B-CDT- GenBank CP019858.1

[0442] (ST15) CD105HS9 A-B-CDT- GenBank NZ_FMKY00000000.1

[0443] NTCD-E4 A-B-CDT- GenBank CPI 18392.1

[0444] RT012 CD630 A+B+CDT- GenBank AMI 80355.1

[0445] CD105HS1 A+B+CDT- GenBank NZ_FMLF00000000.1 6285 A+B+CDT- EnteroBase CLO_AA0541AA RT014 S-0352 AH-B-FCDT" GenBank CP076377

[0446] AR- 1081 A+B+CDT- EnteroBase CLO_CA3103AA CD105HS4 A+B+CDT- GenBank NZ_FMLB00000000.1 AR- 1090 A+B+CDT- EnteroBase CLO_CA3093AA AR- 1091 A+B+CDT- GenBank SAMN 13029172 RT015 173070 A-B+CDT- EnteroBase CLO.. CA0242AA

[0447] AR- 1069 A+B+CDT- EnteroBase CLO.. CA3097AA AR- 1086 A+B+CDT- EnteroBase SAMN13029167 RT017 M68 A-B+CDT- GenBank NC„017175.1

[0448] (ST37) DSM 29627 A-B+CDT- GenBank CP016102.1

[0449] Xy06 A-B+CDT- GenBank NZJANFNF000000000.

[0450] 1

[0451] CF5 A-B+CDT- GenBank NC_017173.1

[0452] TGH33 A-B+CDT- GenBank JAJNHC000000000

[0453]

[0454] Docket No. 11001-247W01

[0455] TGH51 A-B+CDT- GenBank NZ_JAJNIID000000000.

[0456] 1

[0457] 1470 A-B+CDT- GenBank NZ_OEZL00000000.1 RT018 CD 8- 15 A+B+CDT- GenBank LYDP00000000.1

[0458] (STI 7) IT118 A+B-FCDT- GenBank FAXM00000000

[0459] CD-16-00005 A+B+CDT- EnteroBase CLO_AA7146AA RT019 SMG-21-1765 A+B+CDT+ EnteroBase CLO_FA2592AA_AS (ST67) SMG-20-1235 A+B+CDT+ EnteroBase CLO_EA5278AA_AS C00002493 A+B+CDT+ EnteroBase CLO_AA4580AA_AS AR- 1075 A+B+CDT+ EnteroBase CLO_CA3081AA RT020 AR- 1073 AH-B-FCDT" EnteroBase CLO.. CA3083AA

[0460] (ST2) AR- 1080 AH-B-FCDT" EnteroBase CLO.. CA3104AA

[0461] AR- 1096 A+B+CDT- EnteroBase CLO_ CA3087AA RT023 SIRN_ST-001 A+B+CDT+ EnteroBase CLO_EA5979AA_AS CD305 A+B+CDT- GenBank NZJAWXRG00000000 0.1

[0462] CD-16-00530 A+B+CDT+ EnteroBase CLO_DA8015AA_AS CD-15-00694 A+B+CDT+ EnteroBase CLO„DA7541AA„AS DSM 102859 A+B+CDT+ GenBank NZ_CP020378.1 RT027 R20291 A+B+CDT+ GenBank FN545816.1

[0463] (STI) CD 196 A~FB 4" C DT + GenBank FN538970.1

[0464] TGH35 A+B+CDT+ GenBank JAJNGZOOOOOOOOO TGH64 A+B+CDT+ GenBank JAJNHA000000000 CD- 17-01474 A+B+CDT+ GenBank NZ_CP026591.1 DSM 27638 A+B+CDT+ GenBank CP011846.1

[0465] G46 A+B+CDT-I- GenBank NZ. CDND01000001.1 TMD0138 A+B+CDT+ GenBank WUUI00000000.1 (ST97)

[0466] AR- 1067 A4-B4-CDT+ EnteroBase CLO.. CA3109AA AR- 1071 A+B+CDT+ EnteroBase CLO_CA3085AA AR- 1072 A+B+CDT+ EnteroBase CLO_CA3084AA

[0467]

[0468] Docket No. 11001-247W01

[0469] AR- 1076 A+B+CDT+ GenBank NZJADKQV000000000.1

[0470] AR- 1092 A+B+CDT-I- EnteroBase CLO.. CA3091AA AR- 1095 A+B+CDT+ EnteroBase CLO_CA3088AA CD105HS8 A+B+CDT+ GenBank NZ_FMLN00000000.1 RT031 CD105HS19 A-B-CDT- GenBank NZ_FMLL00000000.1 (ST29) CD-15-00867 A-B-CDT- EnteroBase CLO_BA9951AA RT032 DSM 29637 A-B-CDT- GenBank CP016106.1

[0471] CD-15-01028 A-B-CDT- EnteroBase CLOJBA9453AA SMG-21-2010 A-B-CDT- EnteroBase CLO_CA7839AA RT033 OCD52 A-B-CDT+ EnteroBase CLO_BA8168AA_AS IS58 A-B-CDT+ EnteroBase CL0_AA9965AA_AS RPH0101 A-B-CDT+ EnteroBase CLO„BA3454AA_AS RT045 C00002490 A4-B4-CDT+ EnteroBase CLO.. BA6150AA AS CD-16-00514 A4-B4-CDT+ EnteroBase CLO.. DA7993AA.. AS SIRNJIG-021 A+B+CDT+ EnteroBase CLO_EA6022AA_AS RT046 SMG-21-1773 A+B+CDT- EnteroBase CLO_CA7611AA (ST35) SIRN_HT-039 A+B+CDT- EnteroBase CLO_CA2857AA

[0472] CD-15-00938 A+B+CDT- GenBank DAEMXFOOOOOOOOO.1 RT053 CD- 16-00430 A+B+CDT- GenBank CLOJBA9519AA (ST63) SIRN ST-026 AH-B-FCDT" EnteroBase CLO.. CA2833AA

[0473] SMG-22-1463 A+B+CDT- EnteroBase CLO_ CA9991AA RT054 AR- 1082 A+B+CDT- GenBank SAMN 13029163

[0474] VPI’_138 A+B+CDT- EnteroBase CLO_CA9950AA AR- 1088 A+B+CDT- GenBank SAMN 13029169 RT056 S-0942 A+B+CDT- GenBank CP076376

[0475] (ST34) AR- 1068 A+B+CDT- EnteroBase SAMN 13029149

[0476] AR- 1079 A+B+CDT- EnteroBase SAMN 13029160 RT059 8864 A-B+CDT- GenBank NZ_OEZE00000000.1 RT066 C08-686 A+B+CDT+ EnteroBase CLO_AA9950AA_AS CD- 16-00440 A+B+CDT+ EnteroBase CLO_DA7971AA_AS SMG-21-2330 A-4-B -FC DT + EnteroBase CLO.. FA3269AA AS

[0477]

[0478] Docket No. 11001-247W01

[0479] RT078 TW11-RT078 A+B+CDT+ GenBank CP035499.1

[0480] Ml 20 A+B+CDT+ GenBank CP068555.1

[0481] R2 A+B+CDT+ GenBank CP026614.2

[0482] SUC36 A-B+CDT+ GenBank NZ_OEZZ00000000.1 AR- 1077 A+B+CDT-I- EnteroBase CLO.. CA3107AA AR- 1083 A+B+CDT+ EnteroBase CLO_CA3101AA CD105HS27 A+B+CDT+ GenBank NZ„FRET00000000.1 CD105HS26 A+B+CDT+ GenBank NZ_FMLD00000000.1 RT084 DSM 28666 A-B-CDT- GenBank CP012321.1

[0483] CD-16-00174 A-B-CDT- EnteroBase CLO_BA9789AA CD-15-00944 A-B-CDT- EnteroBase CLO.. BA9899AA RT087 SIRN HA-012 AH-B-FCDT" EnteroBase CLO.. CA2788AA (ST46) SMG-20-1333 A+B+CDT- EnteroBase CLO„CA2625AA

[0484] CD-15-00010 A+B+CDT- EnteroBase CLO_AA6357AA RT106 TGH120 A+B+CDT- GenBank JAJNHB000000000

[0485] DH / NAP11 / 106 A+B+CDT- GenBank CP022524.1

[0486] / ST-42

[0487] C00000224 A+B+CDT+ EnteroBase CLO_AA4561AA_AS CD- 16-00068 A+B+CDT- EnteroBase CLO„DA8040AA_AS AR- 1078 AH-B-FCDT" EnteroBase CLO.. CA3106AA AR- 1085 AH-B-FCDT" EnteroBase CLO.. CA3099AA AR- 1087 A+B+CDT- EnteroBase CLO_CA3096AA AR- 1089 A+B+CDT- EnteroBase CLO_CA3094AA AR- 1093 A+B+CDT- EnteroBase CLO_CA3090AA RT125 HGP05 (ST848) A-B-CDT- GenBank CP 103977

[0488] C00006475 A-B-CDT- EnteroBase CLO.. AA4820AA RT126 6058625 A+B+CDT+ EnteroBase CLO_BA0391AA„AS CD-16-00082 A+B+CDT+ EnteroBase CLO_AA7075AA_AS DSM 29020 A+B+CDT+ GenBank CP012325.1

[0489] RT220 CD105HS22 A+B+CDT- GenBank NZ_FMLJ00000000.1 (ST3) CD105HS6 A+B+CDT- GenBank NZ_FMLI00000000.1

[0490] CD-16-00120 A+B-FCDT- EnteroBase CLO.. BA9841AA

[0491]

[0492] Docket No. 1 I001-247W01

[0493] RT244 C00009691 A+B+CDT+ EnteroBase CLO„AA0004AA_AS C00009694 A+B+CDT+ EnteroBase CLO_AA0003AA_AS C00009695 A+B+CDT+ EnteroBase CLO_AA0002AA_AS RT255 Mta-79 A+B+CDT- GenBank CP042267

[0494] (ST34)

[0495] RT871 LC693 A+B+CDT+ GenBank NCXL00000000.1

[0496] SLO 091 DSM 28669 A-B-CDT- GenBank CP012323

[0497] SLO101 ESI 30 A-B+CDT- GenBank NZ_OEZV00000000.1 SLO 235 DSM 29629 A-B-CDT- GenBank CP016104

[0498] SLO 237 DSM 28670 A-B-CDT- GenBank CP012312

[0499] SKO098 WA151 A-B+CDT- GenBank NZ_OEZY00000000.1 STI TGFI29 A+B+CDT+ GenBank JAPKMB 000000000.1

[0500] RO 104a A+B+CDT+ GenBank CP025044

[0501] ST2 W0022a A+B+CDT- GenBank CP025045

[0502] ST3 CCUG37785 A-B-CDT- GenBank NZJAGKRT000000000.

[0503] 1

[0504] ST8 W0003a A+B+CDT- GenBank CP025047.1

[0505] ST 11 TGH79 A+B+CDT+ GenBank NZJAPKMA000000000.1

[0506] 2301802 A+B+CDT+ GenBank JBEJVX000000000.1 ST 15 TGH132 A-B-CDT- GenBank JAPKMC000000000.1 ST23 KS145 A-B-CDT- GenBank JARJHOOOOOOOOOO.l ST35 TGII91 A+B+CDT- GenBank JAPKLZ000000000.1 ST42 W0023a A+B+CDT- GenBank CP025045.1

[0507] ST99 2301801 A+B+CDT+ GenBank JBEJVW000000000.1 ST 109 TGH114 A-B-CDT- GenBank JAPKMD000000000.1 ST340 MA_2 A-B-CDT- GenBank CP 129431.1

[0508]

[0509] Example 2: The relatedness of CdeC and CdeM correlates with C. difficile typing methods To evaluate how conserved CdeC and CdeM are across different subtypes of C. difficile, the phylogeny of CdeC and CdeM sequences from different RTs, STs, clades, and toxinotypes were investigated (FIG. 1 and FIG. 2). CdeC sequences could be sorted into 5 clusters and oneDocket No. 11001-247W01

[0510] singleton (FIG. 1), whereas CdeM sequences formed 5 clusters and two singletons (FIG. 2). Closely clustered sequences of CdeC and CdeM tended to come from a particular ribotype or group of ribotypes. It was found that, for all RTs or STs with at least two genomes in the dataset, a majority of each type’s CdeC and CdeM sequences could be observed in a specific cluster of the phylogenetic trees. To give an example, CdeC sequences from hypervirulent RT078 C. difficile strains were found in cluster D (FIG. 1), and CdeM sequences from RT078 C. difficile were found in cluster J (FIG. 2).

[0511] To a lesser extent, the relatedness of CdeC and CdeM also correlated with the clade of the source strain. For CdeC, Clade 2 strains in cluster C, clade 5 in cluster D, and clade C-III in cluster E (FIG. 1). CdeM showed a similar result with clade 4 being found in cluster II, clade 2 in cluster J, and clade 5 in cluster K (FIG. 2). However, these trends did not hold for all clades. Clade 1 sequences of CdeC could be found in cluster A and the adjacent cluster B, which itself also had a combination of clade 3 and clade 4 sequences (FIG. 1). CdeM sequences from clade 1 formed two distinct clusters G and I (FIG. 2).

[0512] Toxin genotype showed the poorest association with CdeC or CdeM relatedness in the phylogenetic trees. Clusters A (FIG. 1) and G (FIG. 2) both contained primarily A+B+CDT-strains, but this toxinotype could also be found in smaller quantities in other clusters. For A+B+CDT+ strains, clusters C and D (FIG. 1) as well as J and K (FIG. 2) were dominated by this toxin genotype. However, the fact that A+B+CDT+ strains can be found in two distinct clusters demonstrates that the toxin genotype itself is not the main factor determining how these sequences cluster. Lastly, non-toxigenic (A-B-CDT-) strains did not form a distinct cluster in the CdeC or CdeM trees. Therefore, it was concluded that toxin genotype similarities between strains does not strongly correlate with the similarity between their respective CdeC and CdeM sequences.

[0513] Example 3: CdeC is well-conserved between strains

[0514] To further investigate the similarities between CdeC sequences of various C. difficile strains. All non-identical sequences of CdeC were selected from the phylogenetic tree in FIG.

[0515] 1 and aligned using MUSCLE (FIG. 3). Across the 411 positions in the CdeC alignment (including gaps), 337 positions are identical between all examined strains (82%). Percentage identity between CdeC sequences ranged from 100% to 91.61%. The distribution of sequence variations and conserved features throughout the CdeC and CdeM proteins was examined. AlphaFold predictions of the structure of CdeC (AlphaFold accession AF-A0A3T1GBE1-F1-v4) suggest the presence of a helical domain (amino acids 134-365), although no conservedDocket No. 11001-247W01

[0516] domain has been experimentally identified yet. Interestingly, despite making up over half of the protein length, only 23 / 74 (31%) of non -identical sites located in this putative domain 1. Since most sequence variations are outside of this putative domain region, this adds evidence for a potential domain in this region, reflecting the greater AlphaFold prediction confidence in this region. The presence of a conserved domain structure could make CdeC a more reliable vaccine antigen that can be readily recognized by the immune system. Further research into the structure of CdeC could experimentally confirm a domain in this protein.

[0517] To better understand CdeC across different C. difficile strains, previous studies were used to help map out important features of these proteins. CdeC has previously been reported to exhibit, in order from N-terminus to C -terminus, a conserved “KKNKRR” motif, “HUH” motif”, six “NFC” repeats, two “CCRQGKGK” repeats, and a conserved cysteine-rich “CNECC” motif. These features, annotated in FIG. 3, were also observed in the dataset. While these features are largely intact between the sequences, the “NPC” repeats are less conserved in six strains. Four of these strains (6058625, ES130, M120, SIRN. JIG-021) show' additional inserts within the repeat region. CD- 15-00694 exhibits a five amino acid deletion (position 73- 77), and MA_2 has a two amino acid deletion at amino acids 69 and 70. Interestingly, most of these are located in cluster C or are otherwise A+B+CDT+, suggesting that this cluster and toxinotype may be more prone to encoding divergent CdeC sequences.

[0518] Another prior study compared the CdeC sequences of CD630 and R20291 and reported six important substitutions between the strains (FIG. 3, blue arrow's). The commonality of the occurrence of the reported substitutions was examined using more strains. At position 58, CD630 encodes D, while 5 / 19 (26%) of other strains exhibit the synonymous mutation to E as seen in R20291. Similarly, only 4 / 19 (21%) of strains exhibited the N (CD630) to K and G (CD630)to E substitutions at positions 89 and 91, with the same four strains, primarily Clade II strains, showing both substitutions (R20291, AR-1075, C00009694, C00006475). At position 95, a substitution of N to K was reported between CD630 and R20291, but only 3 / 19 (16%) of strains exhibited this substitution. Eight other strains showed a N (CD630) to II substitution instead, but both of these substitutions swap a neutral amino acid for a basic amino acid. The previously reported V to I substitution at position 176 was also not common in the dataset (4 / 19, or 21% of strains). Finally, the S to A substitution at position 235 was very common, with 17 / 19 (89%) of strains exhibiting this substitution. Overall, in the context of the previous comparison between CD630 and R20291 CdeC sequences, CdeC sequences tended to be more similar to CD630 at these sites than R20291. Moreover, these substitutions were not restricted to RT027 / Clade II strains. When performing the broader comparison, additionalDocket No. 11001-247W01

[0519] eleven positions with substitutions between strains were observed, with seven of these being non-synonymous substitutions (positions 12, 17, 46, 104, 181, 362, 403). This analysis shows that several additional sites of variation are present in CdeC that were not previously reported, and these sites could have broader implications on the effectiveness of neutralizing antibodies against CdeC.

[0520] Example 4: CdeM exhibits a few regions of high variation but otherwise is strongly conserved

[0521] Non-identical CdeM sequences from FIG. 2 were also aligned and the alignment of these sequences was visualized (FIG. 4) to better understand the differences between strains. Across the 167 amino acid positions of the CdeM alignment, only 101 positions were identical (60%). Pairwise comparisons between CdeM sequences yielded percentage identity values between 67.53% and 100%. However, this was primarily due to strain C00000224, whose CdeM sequence exhibited a 40bp deletion between positions 98 and 137. If this strain is removed from the analysis, minimum pairwise sequence similarity rises to 84.76%, and the percentage of identical positions in the alignment rises to 141 out of 167 (84%). No domains have been reported for CdeM, and AlphaFold does not provide any confident prediction of structural features with for CdeM (AlphaFold accession AF-A0A031WDH0-Fl-v4). Still, there are known conserved regions consisting of “RREA” repeats, “NGNNGGNNNNC” repeats, and “CNCCNCCRK” repeats that is confirmed in the analysis (FIG. 4). These regions were largely intact with the exception of a few mutations seen in some strains. For example, strain CD-16-00530 showed an extra CHK repeat at position 151. C0000244 showed a substitution from N to S within the conserved “NGNNGGNNNNC” repeats, but this is a synonymous amino acid change. Both of these strains are Clade II, A+B+CDT+ strains in cluster I, suggesting that this type of strain may be slightly more likely to diverge from other CdeM sequences. Finally, C00006475 showed 1 deletion within the “NGNNGGNNNNC” repeats as well as two non-synonymous substitutions within the “CNCCNCCRK” repeats, which makes sense given its divergence from other sequences on the phylogenetic tree in FIG.

[0522] 2. From positions 6 to 10, most strains encode a common “YANGGY” motif, but two strains, including the prototype strain CD630, encodes a lone cysteine followed by a 4 amino acid deletion. Between positions 51 and 56, there are a couple of sites with non-synonymous substitutions between C and G (51), G and E (55), and E and G (56). Finally, the aforementioned 40 amino acid deletion between 98 and 137 in strain C00000224 is the final feature which adds substantial variation between sequences.Docket No. 11001-247W01

[0523] Example 5: Construction of a novel fusion immunogen (CdeCM), targeting C. difficile spore colonization.

[0524] CdeC and CdeM, both of which are abundant Clostridioides difficile spore surface- exposed proteins. Previously, it was shown that immunizations of mice / hamsters with CdeC or CedM via intraperitoneal route provided animals effective protection against CDI. To generate a more potent antigen targeting C. difficile spore adhesion, a fusion containing full-length CdeC and CedM was constructed. The gene sequences encoding CdeC and CdeM from CD630 were bridged with a linker (ggt ggc ggt), optimized, synthesized and cloned into pET28a. Fusion protein CdeCM (67 kDa) was expressed in E. coll BL21 (DE3) and purified by Ni-affinity chromatography (FIG. 5).

[0525] Example 6: Immunization with CdeCM induces significant anti-CdeCM antibody responses and provides mice full protection against C. difficile infection

[0526] Firstly, immune responses of CdeCM immunization were evaluated in mice via intraperitoneal route (i.p). Immunizations of mice (n=12) with 10 pg CdeCM adjuvanted with alum via i.p for 3 times at a 2-week interval induced significant anti-CdeCM IgG / IgA antibody responses in both sera and feces (FIGS. 6 and 7) and provided mice full protection against challenge with a hypervirulent strain C. difficile R20291 (FIGS. 8A-8C). After C. difficile challenge, immunized group showed significantly decreased C. difficile TcdA / TcdB and spore levels in comparison with non-immunized control group (FIGS. 9A-9C).

[0527] Subsequently, immune responses of CdeCM immunization were evaluated in mice via intramuscular route (IM). Data showed that immunization of mice with 10 pg CdeCM adjuvanted with alum via i.m for 3 times at a 2-week interval provided mice full protection against challenge with C. difficile R2.0291 (FIGS. 11A-11C).

[0528] Example 7: Generation of a Lactococcus lactis strain expressing CdeCM.

[0529] Previously failed vaccine candidates, which were based on chemically inactivated TcdA / TcdB administered via IM route, a parenteral route might not be sufficient for an enteric mucosal pathogen; and mucosal immunization would be useful to protect the host against CDI, especially colonization. An effective vaccine should induce both systemic and local mucosal anti-toxin and anti-C. difficile colonization responses.

[0530] Lactococcus lactis expression system is a well-established probiotic system for protein expression in vitro and in the intestine. L. lactis has been widely used as a safe vaccine platformDocket No. 11001-247W01

[0531] for the delivery of heterologous antigens. A food-grade L lactis expression system is used herein, comprising the strain Lactococcus lactis NZ1330 and the expression plasmid pNZ7025 to successfully express CdeCM. The strain NZ1330 is made for food-grade expression system based on an air selection marker in contrast with previous reports that use antibiotic as selective markers. The air gene (an alanine racemase encoding gene) is deleted in this strain. Deletion of the air gene results in auxotrophy for the essential component D-alanine, therefore, L. lactis NZ1330 is unable to grow on media without D-alanine unless air is provided on a plasmid. The plasmid pNZ7025 carries the air gene for a / r-based selection. pNZ7025 is constructed for constitutive gene expression under the control of a strong promoter PpepN. To construct L. lactis strains expressing CdeCM, the gene sequence encoding CdeCM was optimized, synthesized and cloned into vector pNZ7025 in L. lactis NZ1330. Expression of CdeCM was detected by western blot analysis using anti-His tag antibodies (the His-tag sequence was added at the c-terminus of the CdeCM fusion sequence) (FIG. 10).

[0532] Example 8: Immunizations with combined intramuscular injection of CdeCM and oral delivery of L. lactis (CdeCM) provide mice significant protection against GDI Mice (n=10) were immunized with 10 pg of CdeCM adjuvanted with alum via i.m. twice a week interval followed by the 3rd boosting immunization via oral route with L. lactis (CdeCM) at 10i0CFU / mouse. Data showed combined intramuscular and oral immunizations provided mice significant protection against challenge C. difficile R20291 (FIGS. 11A-11C).

[0533] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0534] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred examples of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.Docket No. 11001 -247 WO1 SEQUENCES SEQ ID NO: 1 -CdeCM sequence (1806 bp) atgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcggcagccatatggctagcatgactggtggacagca aatgggtcgcggatcccaagattataaaaaaaataaaagaagaatgatgaatcagccaatgtctacaatgaatgaagaagaagtgtata cagatgaaataaattcagaagacatgagaggttttaaaaaatcacaccatcataatggatgtaatactgataataagtgtgagtgccatga tgattgcaatccatgcaacccatgtaatccatgtaaacctaacccatgcaatccatgcaaacctaatccatgtgatgacaattgtggatgc catgacaattgtaaaigtgatigtgaaccatgigaaaiggattcagatgaaigttttgaaaacaaaigtggaccagaatgctgtaatcctat atctccaagaaacttctctgtatcaaatgcagtgccattgcaatagaggctaatagaatatttgatactatgcaatccaaacatttacaga tgcaacaggaccaaatggagagccattaacttttgaaacagaagtagtagaagtatttggttcagttccaagtgcaggtcaagcaagtgt aactatagaaaaaatatgcttaagtaatgatggaatcgttatagacacaggaatgacaactttagaagatttcgatttagacccattagga gatatagtaggaagaaactgtgaaacaacttttgaattgcagttgtggagaaagaaactctgagtgctgtagacaaggaaaaggcaa atcagtagcttataaacaaagaggattaactgtagcagttcgtaatttagtactagagctaagaggtagatgtggatgtacagagttcgtt gcattagctttcccagcagttagagcaggaggtggatgtaagagaagagtgattatgtagaatttacttttaacacactttcagcaccaat atgcttgccagctgacggaagagctgtacttaagacaagaatatcaaactaacttaactgtagattgtataggaaaatctatattaaaatt agaatgcaacgaatgtgtgaaccttctatgaataattataccaaatgatatagattagtactttgcttacaagaaacagtagcacatta ataagtgaacaaatagtagtttagcatcaccaaatccaatccaaccaagacttgttgatactttctctaaagtatgtgatttttcgcaatgtg gacciaatcatggaagtggaaagccaagtigccacagaggtggcictggtgaaaaiaaaaaatgtattcagaagatggtatgaaaga ggagaatctacagctaaatggttccaaaatgatagagaagaatatgaaagagaagcatatgatgaagatagagaaagaagaggtca aactgtggatgttcagattcaggagaaaatagacctagaaactgtgaaagatttagaagagaagctgagataagagaaagagaagca agagaagcattctglgaatcttcagagaaaaagaaagaggcattagcatatgaatgtgaagctagaaaattaigggaagaagcagaaa aatactgggatgaatatcaaaatacaactataaaggaatcgaatattagcagaagctgctagatatttgatgaaggtatggaatgtga agctagaagaaatggaaataatggaggaaacaataataattgttgccataaatgccataaatgtaattgtaactgctgtagaaaataa SEQ ID NO: 2 - CdeCM immunogen amino acid sequence MGSSHHHHHHSSCiLVPRGSHMASMTGGQQMGRGSQDYKKNKRRMMNQPMSTMN EEEVYTDEINSEDMRGFKKSHIIHNGCNTDNKCECIIDDCNPCNPC’NPCKPNPCNPCK PNPCDDNCGCHDNCKCDCEPCEMDSDECFENKCGPECCNPISPRNFSVSNAVPFAIE ANRIFDTMQFQTFTDATGPNGEPLTFETEVVEVFGSVPSAGQASVTIEKICLSNDGIVI DTGMTTLEDFDLDPLGDIVGRNCETTFEFAVCGERNSECCRQGKGKSVAYKQRGLT VAVRNLVLELRGRCGCTEFVALAFPAVRAGGGCKRRVDYVEFTFNTLSAPICLPADG RAVTLRQEYQTNLTVDCIGKSILKLECNECCEPFYELIIPNDIDLVLCLQETVSTLISEQI VVI. ASPNPIQPRLVDTFSKVCDFSQCGPNIIGSGKPSCIIRGGSGENKKCYSEDWYER GESTAKWFQNDREEYEREAYDEDRERRGSNCGCSDSGENRPRNCERFRREAEIREREDocket No. 11001 -247 WO1 AREAFCESSEKKKEALAYECEARKLWEEAEKYWDEYSKYNYKGIEYLAEAARLFDE GMECEARRNGNNGGNNNNCCHKCHKCNCNCCRK

[0535] SEQ ID NO: 3 - exosporium morphogenetic protein CdeM

[0536] MENKKCYSEDWYERGESTAKWFQNDREEYEREAYDEDRERRGSNCGCSDSGENRP RNCERFRJREAEIREREAREAFCESSEKKKEALAYECEARKLWEEAEKYWDEYSKYN YKGIEYLAEAARLFDEGMECEARRNGNNGGNNNNCCHKCHKCNCNCCRK

[0537] SEQ ID NO: 4 - exosporium morphogenetic protein CdeC

[0538] MQDYKKNKRRMMNQPMSTMNEEEVYTDEINSEDMRGFKKSHHHNGCNTDNKCEC HDDCNPCNPCNPCKPNPCNPCKPNPCDDNCGCHDNCKCDCEPCEMDSDECFENKCG PECCNPISPRNFS VSNAVPFAIEANRIFD TMQFQTFTD ATGPNGEPLTFETEV VE VFGS VPS AGQ AS VTIEKICI. SNDGIV ID TGMTTI JEDFDI. DPI. GDI VORNCEITFEFAVCGERN SECCRQGKGKSVAYKQRGLTVAVRNLVLELRGRCGCTEFVALAFPAVRAGGGCKR RVDYVEFTFNTLSAPICLPADGRAVTLRQEYQTNLTVDCIGKSILKLECNECCEPFYE

[0539] LnPNDlDLVLCLQETVSTLISEQIVVLASPNPIQPRLVDTFSKVCDFSQCGPNHGSGKP SCH

[0540] SEQ ID NO: 5 -Epitope fusion for CdeM: (CdeM-EF)

[0541] REREAREAFKKERFRREAEIREREARKKKYNYKGIEYLAEAARGPGPGEYEREAYDE DRERRGSGPGPGEAEIREREAREAFCESGPGPGGMECEARRNGNNGGNN

[0542] SEQ ID NO: 6 -Epitope fusion for CdeC-1: (CdeC-EF-1)

[0543] LEDFDLDPLKKFV ALAFP A VKK A VR A GGGCKKKRVD Y VEFTFKKLPADGR AV IKK RAVTLRQEYKKNFSVSNAVPFAIEANKKNFSVSNAVPFAIEANRGPGPGVGRNCETT FEFAVCGEGPGPGYVEFTFNTLSAPICLP

[0544] SEQ ID NO: 7 -Epitope fusion for CdeC-2: (CdeC-EF-2)

[0545] LEDFDLDPLKKFV ALAFPAVKKAVRAGGGCKKKRVDYVEFTFKKLPADGRAVTKK RAVTLRQEYKKNFSVSNAVPFAIEANRGPGPGVGRNCETTFEFAVCGEGPGPGYVEF TFNTLSAPICLP

[0546] SEQ ID NO: 8- Epitope fusion for CdeCM-1: (CdeCM-EF-1)Docket No. 11001 -247 WO1 REREAREAFKKLEDFDLDPLKKFVALAFPAVKKAVRAGGGCKKKR VDYVEFTFKKL PADGRAVTKKRAVTLRQEYKKERFRREAEIREREARKKKYNYKGIEYLAEAARKKN FSVSNAVPFAIEANRGPGPGEYEREAYDEDRERRGSGPGPGEAEIREREAREAFCESG PGPGGMECEARRNGNNGGNNGPGPGVGRNCETIFEFAVCGEGPGPGYVEFTFNTLS APICLP

[0547] SEQ ID NO: 9 - Epitope fusion for CdeCM-2: (CdeCM-EF-2)

[0548] REREAREAFKKYTDEINSEDKKLEDFDLDPLKKRNCE1TFEFKKCTEFVALAFKKFV AL FPAVKKAVRAGGGCKKKRVDYVEFTFKKLPADGRAVTKKYELIIPNDIKKRAV TLRQEYKKCEPFYEIJIKKIQPRLVDTFKKERFRREAEIREREARKKKYNYKGIEYLAE AARKKNFSVSNAVPFAIEANRGPGPGEYEREAYDEDRERRGSGPGPGEAEIREREAR EAFCESGPGPGGMECEARRNGNNGGNNGPGPGNFSVSNAVPFAIEANRGPGPGVGR NCETTFEFAVCGEGPGPGYVEFTFNTLS APICLP

[0549] SEQ ID NO: 10 - Epitope fusion for CdeCM-3: (CdeCM-EF-3)

[0550] YTDEINSEDKKI. EDFDLDPLKKRNCE T1TEFKKCT EFVALAFKKF VAI AFPAVKKA V RAGGGCKKKRVDY VEFTFKKLPADGRAV TKKYEI JIPND1KKRAVTLRQEYKKCEPF YELIIKKIQPRLVDTFKKREREAREAFKKNFSVSNAVPFAIEANKKERFRREAEIRERE ARKKKYNYKGIEYLAEAARGPGPGNFSVSNAVPFAIEANRGPGPGVGRNCETTFEFA VCGEGPGPGYVEFTFNTLSAPICLPGPGPGEYEREAYDEDRERRGSGPGPGEAEIRER EAREAFCESGPGPGGMECEARRNGNNGGNN

[0551] SEQ ID NO: 11 - C-CTL-1 CdeC

[0552] YTDE1NSED

[0553] SEQ ID NO: 12 - C-CTL-2 CdeC

[0554] RNCETTFEF

[0555] SEQ ID NO: 13 - C-CTL-3 CdeC

[0556] LEDFDLDPL

[0557] SEQ ID NO:14 - C-CTL-4 CdeC

[0558] CTEFVALAFDocket No. 11001 -247 WO1 SEQ ID NO: 15 - C-CTL-5 CdeC

[0559] FVALAFPAV SEQ ID NO: 16 - C-CTL-6 CdeC

[0560] AVRAGGGCK SEQ ID NO: 17 - C-CTL-7 CdeC

[0561] RVDYVEFTF SEQ ID NO: 18 - C-CTL-8 CdeC

[0562] LPADGRAVT SEQ ID NO: 19 - C-CTL-9 CdeC

[0563] YELIIPNDI SEQ ID NO: 20 - C-CTL-10 CdeC

[0564] RAVTLRQEY SEQ ID NO: 21 - C-CTL-11 CdeC

[0565] CEPFYELII SEQ ID NO: 22 - C-CTL-12 CdeC

[0566] 1QPRLVDTF

[0567] SEQ ID NO: 23 - C-HTL-1 CdeC

[0568] NFSVSNAVPFAIEAN SEQ ID NO: 24 - C-HTL-2 CdeC

[0569] CEPFYELIIPNDIDL SEQ ID NO: 25 -C-HTL-3 CdeCDocket No. 11001 -247 WO1 DTMQFQTFTDATGPN SEQ ID NO: 26 -C-HTL-4 CdeC

[0570] EEVYTDEINSEDMRG SEQ ID NO: 27 -C-HTL-5 CdeC

[0571] EFTFNTLSAPICLPA SEQ ID NO: 28- C-HTL-6 CdeC

[0572] EVYTDEINSEDMRGF SEQ ID NO:29- C-HTL-7 CdeC

[0573] MQFQTFTDATGPNGE SEQ ID NO: 30 - C-HTL-8 CdeC

[0574] QRGLTVAVRNLVLEL SEQ ID NO: 31 - C-HTL-9 CdeC

[0575] RNFSVSNAVPFAIEA SEQ ID NO: 32 - C-HTL-10 CdeC

[0576] SNDGIVID TGMTTLE SEQ ID NO: 33 - C-HTL-11 CdeC

[0577] VEFTFNTLSAPICLP SEQ ID NO:34- C-B-l CdeC

[0578] YVEFTFNTLSAPICLP SEQ ID NO: 35 - C-B-2 CdeC

[0579] NFSVSNAVPFAIEANRDocket No. 11001 -247 WO1 SEQ ID NO: 36 - C-B-3 CdeC

[0580] VGRNCETI’FEFAVCGE

[0581] SEQ ID NO: 37- C-B-4 CdeC

[0582] DMRGFKKSHHHNGCNT SEQ ID NO: 38 - C-B-5 CdeC

[0583] PNPIQPRLVDTFSKVC SEQ ID NO: 39 - C-B-6 CdeC

[0584] YTDEINSEDMRGFKKS SEQ ID NO: 40 - C-B-7 CdeC

[0585] PSAGQASVTIEKICLS SEQ ID NO: 41 - C-B-8 CdeC

[0586] EVFGSIPSAGQASVTI SEQ ID NO: 42 - C-B-9 CdeC

[0587] IVVLASPNPIQPRLVD SEQ ID NO: 43 - M-CTL-1 (CTL epitope CdeM)

[0588] REREAREAF SEQ ID NO: 44 - M-HTL-1 CdeM

[0589] ERFRREAEIREREAR SEQ ID NO: 45 - M-HTL-2 CdeM

[0590] KYNYKGIEYLAEAAR SEQ ID NO: 46 - M-HTL-3 CdeMDocket No. 11001 -247 WO1 CERFRREAEIREREA SEQ ID NO: 47 - M-HTL-4 CdeM

[0591] NCERFRREAEIRERE SEQ ID NO: 48 - M-B-l CdeM

[0592] GMECEARRNGNNGGNN SEQ ID NO: 49 - M-B-2 CdeM

[0593] E YERE YDEDRERRGS SEQ ID NO: 50 - M-B-3 CdeM

[0594] EAEIREREAREAFCES SEQ ID NO: 51 - M-B-4 CdeM

[0595] REARS AFCESSEKKKE SEQ ID NO: 52 - CdeC polypeptide conserved region

[0596] KKNKRR SEQ ID NO: 53 - CdeC polypeptide conserved region-2

[0597] CCRQGKGK SEQ ID NO: 54 - CdeC polypeptide conserved region-3

[0598] CNECC SEQ ID NO: 55 - CdeM polypeptide conserved region

[0599] NGNNGGNNNNC SEQ ID NO: 56 - CdeM polypeptide conserved region-2

[0600] CNCCNCCRK

Claims

1. Docket No. 11001 -247 WO1 CLAIMSWhat is claimed is:

1. A fusion immunogen, comprising:a first segment comprising a full-length Clostridioides difficile exosporium cysteine-rich protein (CdeC) polypeptide or at least one CdeC immunogenic fragment;a second segment comprising a full-length Clostridioides difficile exosporium cysteine-rich protein M (CdeM) polypeptide or at least one CdeM immunogenic fragment; anda linker between the first segment and the second segment, wherein the fusion immunogen elicits an immune response against one or both of CdeC and CdeM polypeptides.

2. The fusion immunogen of claim 1, wherein the fusion immunogen comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

3. The fusion immunogen of any one of claim 1 or claim 2, wherein the at least one CdeC immunogenic fragment or the at least one CdeM immunogenic fragment is selected from R20291, RT244, RT019, RT027, RT012, RT125, or RT106 ribotypes of C. difficile.

4. The fusion immunogen of any one of claims 1-3, wherein the at least one CdeC immunogenic fragment is about 6 to about 50 amino acids in length.

5. The fusion immunogen of any one of claims 1-4, wherein the at least one CdeC immunogenic fragment comprises an amino acid sequence as set forth in SEQ ID NO: 6 or SEQ ID NO: 7, or a sequence having at least 90%’ identity thereto.

6. The fusion immunogen of any one of claims 1-3, wherein the at least one CdeM immunogenic fragment is about 6 to about 50 amino acids in length.

7. The fusion immunogen of any one of claims 1-6, wherein the at least one CdeM immunogenic fragment comprises an amino acid sequence as set forth in SEQ ID NO: 5, or a sequence having at least 90% identity thereto.Docket No. 11001 -247 WO1 8. The fusion immunogen of any one of claims 1-7, wherein the at least one CdeC immunogenic fragment or the at least one CdeM immunogenic fragment comprises a B cell epitope, a CD4+ T cell epitope, a CD8+ T cell epitope or a combination thereof.

9. The fusion immunogen of any one of claims 1-8, wherein the linker is a flexible linker, a rigid linker, a protease-cleavable linker, or a peptide spacer.

10. The fusion immunogen of any one of claims 1-9, wherein the linker comprises a glycine- rich linker, a serine -rich linker, a G4S linker, a GPGPG linker, a KK linker, or a ggtggcggt linker.

11. The fusion immunogen of any one of claims 1-10, wherein the fusion immunogen further comprises a heterologous signal peptide, a carrier domain, a scaffold domain, a secretion sequence, a cell wall anchoring domain, or a surface display domain.

12. The fusion immunogen of claim 11, wherein the cell wall anchoring domain comprises an LPXTG motif-containing anchoring sequence.

13. The fusion immunogen of any one of claims 1-12, wherein the fusion immunogen further comprises an adjuvant peptide, a mucosal targeting peptide, or an immunostimulatory domain.

14. The fusion immunogen of any one of claims 1-13, wherein the at least one CdeC immunogenic fragment comprises a CD8+ T cell epitope, wherein the CD8+ T cell epitope is selected from YTDEINSED (SEQ ID NO: 11), RNCETTFEF (SEQ ID NO: 12), LEDFDLDPL (SEQ ID NO: 13), CTEFVALAF (SEQ ID NO: 14), FVALAFPAV (SEQ ID NO: 15), AVRAGGGCK (SEQ ID NO: 16), RVDYVEFTF (SEQ ID NO: 17), LPADGRAVT (SEQ ID NO: 18), YELIIPNDI (SEQ ID NO: 19), RAVTLRQEY (SEQ ID NO: 20), CEPFYELII (SEQ ID NO: 21), or IQPRLVDTF (SEQ ID NO: 22).

15. The fusion immunogen of any one of claims 1-13, wherein the at least one CdeC immunogenic fragment comprises a CD4+ T cell epitope, wherein the CD4+ T cell epitope is selected from NFSVSNAVPFAIEAN (SEQ ID NO: 23), CEPFYELIIPNDIDL (SEQ ID NO: 24), DTMQFQTFTDATGPN (SEQ ID NO: 25), EEVYTDEINSEDMRG (SEQ ID NO: 26), EFTFNTLSAPICLPA (SEQ ID NO: 27), EVYTDEINSEDMRGF (SEQ ID NO: 28),Docket No. 11001 -247 WO1 MQFQTFTDATGPNGE (SEQ ID NO: 29), QRGLTVAVRNLVLEL (SEQ ID NO: 30), RNFSVSNAVPFAIEA (SEQ ID NO: 31), SNDG1V1DTGMTTLE (SEQ ID NO: 32), or VEFTFNTLSAPICLP (SEQ ID NO: 33).

16. The fusion immunogen of any one of claims 1-13, wherein the at least one CdeC immunogenic fragment comprises a B cell epitope, wherein the B cell epitope is selected from YVEFTFNTLSAPICLP (SEQ ID NO: 34), NFSVSNAVPFAIEANR (SEQ ID NO: 35),VGRNCETTFEFAVCGE (SEQ ID NO: 36), DMRGFKKSHHHNGCNT (SEQ ID NO: 37),PNP1QPRLVDTFSKVC (SEQ ID NO: 38), YTDEINSEDMRGFKKS (SEQ ID NO: 39), PSAGQASVTIEKICLS (SEQ ID NO: 40), EVFGSIPSAGQASVTI (SEQ ID NO: 41), or IVVLASPNPIQPRLVD (SEQ ID NO: 42).

17. The fusion immunogen of any one of claims 1-13, wherein the at least one CdeM immunogenic fragment comprises a CD8+ T cell epitope, a CD4+ T cell epitope or a B cell epitope, wherein the CD8+ T cell epitope, the CD4+ T cell epitope or the B cell epitope, is selected from REREAREAF (SEQ ID NO: 43), ERFRREAEIREREAR (SEQ ID NO: 44), KYNYKGIEYLAEAAR (SEQ ID NO: 45), CERFRREAEIREREA (SEQ ID NO: 46), NCERFRREAEIRERE (SEQ ID NO: 47), GMECEARRNGNNGGNN (SEQ ID NO: 48), EYEREAYDEDRERRGS (SEQ ID NO: 49), EAEIREREAREAFCES (SEQ ID NO: 50), or REAREAFCESSEKKKE (SEQ ID NO: 51).

18. The fusion immunogen of any one of claims 1-17, wherein the fusion immunogen is selected from a CdeM-EF peptide comprising a sequence as set forth in SEQ ID NO: 5 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto; CdeC-EF-1 peptide comprising a sequence as set forth in SEQ ID NO: 6 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto; CdeC-EF-2 peptide comprising a sequence as set forth in SEQ ID NO: 7 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto; CdeCM-EF-1 peptide comprising a sequence as set forth in SEQ ID NO: 8 or a sequence having at least 95 %, 98%, 99% or 99.8% identity thereto; CdeCM-EF-2 peptide comprising a sequence as set forth in SEQ ID NO: 9 or a sequence having at least 95%, 98%, 99% or 99.8% or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.Docket No. 11001 -247 WOI 19. The fusion immunogen of any one of claims 1-18, wherein the first segment comprises a full length CdeC polypeptide comprising a sequence as set forth in SEQ ID NO: 4 or a sequence having at least 90% identity thereto.

20. The fusion immunogen of claim 19, wherein the full length CdeC polypeptide comprises at least one conserved region selected from a KKNKRR motif, a HHH motif, six NPC repeats, two CCRQGKGK repeats, or a CNECC motif.

21. The fusion immunogen of any one of claims 1-18, wherein the second segment comprises a full length CdeM polypeptide comprising a sequence as set forth in SEQ ID NO: 3 or a sequence having at least 90% identity thereto.

22. The fusion immunogen of claim 21, wherein the full length CdeM polypeptide comprises at least one conserved region selected from RREA repeats, NGNNGGNNNNC repeats, or CNCCNCCRK repeats.

23. The fusion immunogen of any one of claims 19-22, wherein one or more amino acids as set forth in SEQ ID NO: 4 or SEQ ID NO: 3 are substituted by a conservative substitution.

24. A method of producing a fusion immunogen, comprising:cloning a polynucleotide encoding a fusion immunogen as set forth in SEQ ID NO: 1 into abacterial plasmid;expressing the fusion immunogen in Escherichia coli BL21(DE3), wherein the fusion immunogen comprises an amino acid sequence as set forth in SEQ ID NO: 2; andpurifying and obtaining the fusion immunogen.

25. The method of claim 24, wherein the bacterial plasmid comprises a pET vector, a pBAD vector, a pGEX vector, a pMAL vector, a pQE vector, a pTrc99 A vector, or a pCDF vector.

26. The method of any one of claim 24 or claim 25, wherein the bacterial plasmid is a pET28a vector.

27. The method of any one of claims 24-26, wherein the fusion immunogen comprises a full- length CdeC, a full-length CdeM, at least one CdeC immunogenic fragment; or at least one CdeM immunogenic fragment.Docket No. 11001 -247 WO1 28. The method of claim 27, wherein the full-length CdeC comprises an amino acid sequence as set forth in SEQ ID NO: 4.

29. The method of claim 27, wherein the full-length CdeM comprises an amino acid sequence as set forth in SEQ ID NO: 3.

30. The method of any one of claims 27-29, wherein the full-length CdeC or the at least one CdeC immunogenic fragment is joined to the full-length CdeM or the at least one CdeM immunogenic fragment by a glycine rich linker.

31. The method of any one of claims 24-30, wherein the fusion immunogen is purified by chromatography or western blotting.

32. An expression vector comprising:a promoter operably linked to a polynucleotide encoding a fusion immunogen, wherein the fusion immunogen comprises a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment; anda vector backbone for expression in a bacterial host cell.

33. The expression vector of claim 32, wherein the fusion immunogen comprises the full length CdeC polypeptide fused to the full length CdeM polypeptide.

34. The expression vector of any one of claim 32 or claim 33, wherein the fusion immunogen comprises a linker sequence fusing the full length CdeC polypeptide to the full length CdeM polypeptide.

35. The expression vector of claim 34, wherein the linker sequence is glycine rich.

36. The expression vector of any one of claims 32-35, wherein the polynucleotide comprises a sequence as set forth in SEQ ID NO: 1.

37. The expression vector of any one of claims 32-36, wherein the expression vector is configured for expression in Escherichia coli.

38. The expression vector of any one of claims 32-37, wherein the expression vector is pET28a or a derivative thereof.Docket No. 11001 -247 WO1 39. The expression vector of any one of claims 32-38, wherein the expression vector further comprises a nucleotide sequence encoding a His tag at C -terminus of the fusion immunogen.

40. A vaccine composition, comprising:the fusion immunogen of any one of claims 1-39; anda pharmaceutically acceptable carrier or an adjuvant.

41. A vaccine composition, comprising:a fusion immunogen, wherein the fusion immunogen comprises (i) a full length CdeC polypeptide or at least one CdeC immunogenic fragment and (ii) a full length CdeM polypeptide or at least one CdeM immunogenic fragment, wherein (i) and (ii) are fused directly or through a linker; anda pharmaceutically acceptable earner, an adjuvant or a combination thereof.

42. The vaccine composition of claim 41, wherein the fusion immunogen comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

43. The vaccine composition of any one of claim 41 or claim 42, wherein the vaccine composition elicits at least a B cell response, a CD4+ T cell response, including Thl, Th2, or Thl 7, or a CD8+ T cell response.

44. The vaccine composition of any one of claims 41-43, wherein the adjuvant comprises alum, aluminum hydroxide or aluminum phosphate.

45. The vaccine composition of any one of claims 41-43, wherein the pharmaceutically acceptable carrier comprises a nanoparticle or a liposome.

46. The vaccine composition of any one of claims 41-45, wherein the vaccine composition further comprises at least one other pharmaceutically effective drug, wherein the at least one other pharmaceutically effective drug is an antibiotic.

47. The vaccine composition of claim 46, wherein the antibiotic is selected from one or more metronidazole, amoxycillin, tetracycline, erythromycin, clarithromycin or tinidazole.Docket No. 11001 -247 WO1 48. The vaccine composition of any one of claims 41-44, wherein the vaccine composition is formulated for parenteral, intraperitoneal, or intramuscular administration.

49. The vaccine composition of any one of claims 41-48, wherein the vaccine composition reduces one or more of spore colonization, TcdA toxin or TcdB toxin production, or spore burden in a subject.

50. A method of treating Clostridioides difficile infection in a subject, comprising:administering to a subject an effective amount of a vaccine composition, wherein the vaccine composition comprises a fusion immunogen comprising a CdeC component and a CdeM component, wherein the CdeC component comprises an amino acid sequence of SEQ ID NO: 4 or an immunogenic fragment thereof, and wherein the CdeM component comprises an amino acid sequence of SEQ ID NO: 3 or an immunogenic fragment thereof.

51. The method of claim 50, wherein the fusion immunogen comprises an amino acid sequence of SEQ ID NO: 2.

52. The method of any one of claim 50 or claim 51, wherein the vaccine composition further comprises an adjuvant.

53. The method of any one of claims 50-52, wherein the vaccine composition is administered intraperitoneally or intramuscularly.

54. The method of any one of claims 50-53, wherein the vaccine composition is administered in three doses.

55. The method of claim 54, wherein the three doses are administered at about two-week intervals.

56. The method of any one of claims 50-55, wherein each dose comprises about 10 pg of the fusion immunogen.

57. The method of any one of claims 50-56, wherein the method reduces Clostridioides difficile spore colonization.

58. A bacterial expression vector for expression in Lactococcus lactis (L. lactis), comprising:Docket No. 11001 -247 WO1 a promoter operably linked to a polynucleotide encoding a fusion immunogen, wherein the fusion immunogen comprises a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment; andan air selection gene, wherein the air selection gene enables growth of an air¬ deficient L. lactis host cell.

59. The bacterial expression vector of claim 58, wherein the promoter is a constitutive promoter.

60. The bacterial expression vector of claim 59, wherein the constitutive promoter is PpepN.

61. The bacterial expression vector of any one of claims 58-60, wherein the bacterial expression vector is pNZ7025 or a derivative thereof.

62. The bacterial expression vector of any one of claims 58-61, wherein the air selection gene encodes alanine racemase.

63. The bacterial expression vector of any one of claims 58-61, wherein the bacterial expression vector is configured for transformation into Lactococcus lactis NZ1330.

64. The bacterial expression vector of any one of claims 58-63, wherein the bacterial expression vector carrying air selection gene enables L. lactis host cell to grow in absence of D-alanine.

65. The bacterial expression vector of any one of claims 58-64, wherein the bacterial expression vector further comprises a sequence encoding a C -terminal His tag fused to the fusion immunogen.

66. The bacterial expression vector of any one of claims 58-65, wherein the bacterial expression vector has constitutive expression of the fusion immunogen in vitro and in intestine.

67. A method of generating a recombinant Lactococcus lactis vaccine expressing a Clostridioides difficile fusion immunogen, comprising:cloning a polynucleotide encoding a fusion immunogen into a bacterial expression vector; andDocket No. 11001 -247 WO1 expressing the fusion immunogen in Lactococcus lactis NZ1330 host cell, thereby generating a recombinant Lactococcus lactis vaccine expressing the fusion immunogen.

68. The method of claim 67, wherein the bacterial expression vector is pNZ7025.

69. The method of claim 68, wherein the bacterial expression vector comprises an air selection gene.

70. The method of claim 69, wherein the bacterial expression vector comprises a PpepN promoter operably linked to the polynucleotide encoding the fusion immunogen.

71. The method of any one of claims 67-70, wherein the fusion immunogen further comprises a C-terminal His tag.

72. The method of any one of claims 67-71, wherein the Lactococcus lactis NZ1330 host cell is air-deficient and is incapable of growth in absence of D -alanine.

73. An oral vaccine composition for inducing a mucosal immune response against C. difficile in a subject, comprising:a recombinant L. lactis cell comprising a polynucleotide encoding a fusion immunogen, wherein the fusion immunogen comprises a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment; anda pharmaceutically acceptable carrier, an adjuvant or a combination thereof.

74. The oral vaccine composition of claim 73, wherein the adjuvant comprises alum, aluminum hydroxide or aluminum phosphate.

75. The oral vaccine composition of any one of claim 73 or claim 74, wherein the fusion immunogen comprises a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 or a sequence having at least 95%, 98%, 99% or 99.8% identity thereto.

76. The oral vaccine composition of any one of claims 73-75, wherein the oral vaccine composition elicits at least a B cell response, a CD4+ T cell response, including Thl, Th2, or Thl 7, or a CD8+ T cell response.Docket No. 11001 -247 WO1 77. The oral vaccine composition of any one of claims 73-75, wherein the pharmaceutically acceptable carrier comprises a nanoparticle or a liposome.

78. The oral vaccine composition of any one of claims 73-77, wherein the recombinant L. lactis cell is Lactococcus lactis NZ1330 or a derivative thereof.

79. The oral vaccine composition of any one of claims 73-78, wherein the polynucleotide encoding the fusion immunogen is cloned on a bacterial expression vector.

80. The oral vaccine composition of claim 79, wherein the bacterial expression vector comprises an air selection gene.

81. The oral vaccine composition of claim 80, wherein the air selection gene encodes an alanine racemase.

82. The oral vaccine composition of any one of claims 73-81, wherein the recombinant L. lactis cell constitutively expresses the fusion immunogen.

83. The oral vaccine composition any one of claims 73-82, wherein the fusion immunogen further comprises a C -terminal His tag.

84. The oral vaccine composition any one of claims 73-83, wherein the oral vaccine composition comprises about 10i0CFU of the recombinant L. lactis cell per dose.

85. The oral vaccine composition any one of claims 73-84, wherein the oral vaccine composition is formulated for administration as a booster dose following one or more parenteral administrations of a vaccine composition comprising a fusion immunogen.

86. The oral vaccine composition any one of claims 73-84, wherein the oral vaccine composition is formulated for simultaneous administration with one or more parenteral administrations of a vaccine composition comprising a fusion immunogen.

87. The oral vaccine composition any one of claims 73-86, wherein the oral vaccine composition is capable of inducing a local mucosal immune response against C. difficile upon oral administration to the subject.

88. A method of treating a subject against Clostridioides difficile infection, comprising:Docket No. 11001 -247 WO1 administering a first vaccine composition, wherein the first vaccine composition comprises a fusion immunogen comprising a full length CdeC polypeptide or at least one CdeC immunogenic fragment fused to a full length CdeM polypeptide or at least one CdeM immunogenic fragment, wherein the first vaccine composition further comprises a pharmaceutically acceptable carrier, an adjuvant or a combination thereof; andadministering a second vaccine composition comprising a recombinant Lactococcus lactis cell comprising a polynucleotide encoding said fusion immunogen, wherein the second vaccine composition further comprises a pharmaceutically acceptable carrier, an adjuvant or a combination thereof.

89. The method of claim 88, wherein the first vaccine composition is administered intramuscularly, and the second vaccine composition is administered orally.

90. The method of any one of claim 88 or claim 89, wherein the first vaccine composition is administered in two doses separated by about 2 weeks and the second vaccine composition is administered as a third booster dose.

91. The method of any one of claims 88-90, wherein the first vaccine composition comprises about 10 pg of the fusion immunogen.

92. The method of any one of claims 88-91, wherein the second vaccine composition comprises about 107CPU to about 1011CFU of the recombinant Lactococcus lactis cell.

93. A method of treating a subject against Clostridioides difficile infection, comprising:orally administering to the subject a recombinant Lactococcus lactis cell comprising a bacterial expression vector encoding a fusion immunogen, wherein the fusion immunogen comprises an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

94. The method of claim 93, wherein the recombinant Lactococcus lactis cell comprises Lactococcus lactis NZ1330 cell strain.

95. The method of any one of claim 93 or claim 94, wherein the bacterial expression vector is pNZ7025 or a derivative thereof.Docket No. 11001 -247 WO1 96. The method of any one of claim 93-95, wherein the bacterial expression vector comprises an air gene for selection in an air-deficient L. lactis cell.

97. The method of any one of claim 93-96, wherein the bacterial expression vector comprises a PpepN promoter operably linked to a polynucleotide encoding the fusion immunogen.

98. The method of any one of claim 93-97, wherein the fusion immunogen further comprises a C-terminal His tag.

99. The method of any one of claim 93-98, wherein the recombinant Lactococcus lactis cell constitutively expresses the fusion immunogen.

100. The method of any one of claim 93-99, wherein about IO10CFU of the recombinant Lactococcus lactis cell are administered to the subject.

101. The method of any one of claim 93-100, wherein oral administration induces a local mucosal immune response against C. difficile.

102. The method of any one of claim 93-101, wherein oral administration reduces C. difficile colonization following challenge.

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