Escherichia coli compositions and methods thereof

Abstract

To provide compositions and methods of use thereof for producing recombinant adhesin proteins and for eliciting immune responses against E. coli serotypes.SOLUTION: In one aspect, the invention relates to a polypeptide derived from E. coli and a fragment thereof, including compositions and methods thereof. Also disclosed herein are compositions that include: a polypeptide derived from E. coli and a fragment thereof; and modified O-polysaccharide molecules derived from E. coli lipopolysaccharides and conjugates thereof. In a further aspect, disclosed herein are mammalian host cells that include a sequence encoding a polypeptide derived from E. coli or fragments thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 929,505, filed November 1, 2019, U.S. Provisional Application No. 63 / 045,038, filed June 26, 2020, and U.S. Provisional Application No. 63 / 081,629, filed September 22, 2020, the entire contents of each of the foregoing applications being incorporated herein by reference.

[0002] Sequence Listing Reference This application has been filed electronically via EFS-Web and contains a sequence listing that has been submitted electronically in .txt format. The .txt file contains a sequence listing entitled "PC072517_03_SEQ_List_ST25.txt," which was created on September 18, 2020, and is 152 KB in size. The sequence listing contained in this .txt file is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present invention relates to Escherichia coli compositions and methods. [Background technology]

[0004] The bacterial fimbrial adhesins FimH and FmlH recognize specific glycoproteins on host cells, enabling Escherichia coli to exploit the distinctive urinary tract microenvironment. FimH binds to mannosylated uroplakin receptors on the urothelium, while FmlH binds to galactose or N-acetylgalactosamine O-glycans attached to epithelial surface proteins in the kidney and inflamed bladder. FimH also mediates intestinal colonization by enterotoxigenic E. coli (ETEC) and multidrug-resistant invasive E. coli by binding to highly mannosylated proteins on the intestinal epithelium.

[0005] Full-length FimH consists of two domains: an N-terminal lectin domain and a C-terminal pyrin domain, connected by a short linker. The lectin domain of FimH contains a carbohydrate-recognition domain that is involved in binding to mannosylated uroplakin 1a on the surface of urothelial cells. The pyrin domain is anchored to the cilium core via the donor chain of the subsequent FimG subunit through a process called donor chain complementation.

[0006] The conformation and ligand-binding properties of the lectin domain of FimH are under allosteric control of the pyrin domain of FimH. In the resting state, the interaction of the two domains of full-length FimH results in a lectin domain with a shallow binding pocket and low affinity for monomannose (e.g., K). d Binding to a mannoside ligand induces a conformational change that results in a medium affinity state in which the lectin and pyrin domains remain in close proximity. However, upon shear stress, the lectin and pyrin domains separate, thereby reverting to a high affinity state (e.g., K d <1.2 μM).

[0007] Absent the negative allosteric regulation exerted by the pyrin domain, the isolated lectin domain of FimH is locked in a high-affinity state. Isolated recombinant lectin domains locked in a high-affinity state exhibit high stability. However, locking the adhesin in a low-binding conformation elicits the production of antibodies that inhibit adhesion. Therefore, there is interest in stabilizing the lectin domain in a low-affinity state.

[0008] Additionally, there is interest in methods for expressing FimH in high enough yields for product development. One factor hindering the development of FimH-containing compositions is the low yield achieved when expressing FimH in its native state in the periplasm of E. coli. Typical yields reported at the lab-bench scale are 3-5 mg / L for purified FimCH complex and 4-10 mg / L for FimH(LD), below the levels considered scalable for the production of clinical trial material. The in vivo conformation of FimH differs from that achieved by purified recombinant protein. In general, the native conformation of FimH is determined, at least in part, by the in vivo interaction of FimH with a periplasmic chaperone protein called FimC.

[0009] Recombinant production of FimH remains a challenge: protein expression and purification is not a routine process. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] U.S. Patent Application Publication No. 20200002727 [Patent Document 2] U.S. Patent Application Publication No. 2006 / 0228380 [Patent Document 3] U.S. Patent Application Publication No. 2007 / 0231340 [Patent Document 4] U.S. Patent Application Publication No. 2007 / 0184071 [Patent Document 5] U.S. Patent Application Publication No. 2007 / 0184072 [Patent Document 6] WO2006 / 110381 [Patent Document 7] WO2008 / 079653 [Patent Document 8] WO2008 / 143709 [Patent Document 9] U.S. Patent No. 9,517,274 [Patent Document 10] International Patent Application Publication No. 2014027302 [Patent Document 11] WO01 / 98334 [Patent Document 12] WO03 / 54007 [Patent Document 13] EP0594610B [Patent Document 14] US4709017 [Patent Document 15] US4950740 [Patent Document 16] US5917017 [Patent Document 17] US6455673 [Patent Document 18] US5843711 [Patent Document 19] WO04081515 [Patent Document 20] PCT / EP2005 / 010258 [Patent Document 21] WO00 / 37105 [Patent Document 22] WO00 / 39299 [Patent Document 23] WO2009 / 000826 [Patent Document 24] EP0372501 [Patent Document 25] EP0378881 [Patent Document 26] EP0427347 [Patent Document 27] WO93 / 17712 [Patent Document 28] WO94 / 03208 [Patent Document 29] WO98 / 58668 [Patent Document 30] EP0471177 [Patent Document 31] WO91 / 01146 [Patent Document 32] WO02 / 091998 [Patent Document 33] WO01 / 72337 [Patent Document 34] WO00 / 61761 [Patent Document 35] International Patent Application No. 2004 / 083251 [Patent Document 36] US5,614,382 [Patent Document 37] WO90 / 03184 [Patent Document 38] WO96 / 11711 [Patent Document 39] WO00 / 48630 [Patent Document 40] WO98 / 36772 [Patent Document 41] WO00 / 41720 [Patent Document 42] WO2006 / 134423 [Patent Document 43] WO2007 / 026190 [Patent Document 44] WO00 / 07621 [Patent Document 45] WO99 / 44636 [Patent Document 46] GB2220211 [Patent Document 47] EP0689454 [Patent Document 48] WO00 / 56358 [Patent Document 49] EP0835318 [Patent Document 50] EP0735898 [Patent Document 51] EP0761231 [Patent Document 52] WO99 / 52549 [Patent Document 53] WO01 / 21207 [Patent Document 54] WO01 / 21152 [Patent Document 55] WO00 / 62800 [Patent Document 56] WO00 / 23105 [Patent Document 57] WO99 / 11241 [Patent Document 58] WO98 / 57659 [Patent Document 59] U.S. Patent No. 6,194,388 [Patent Document 60] U.S. Patent No. 6,207,646 [Patent Document 61] U.S. Patent No. 6,214,806 [Patent Document 62] U.S. Patent No. 6,218,371 [Patent Document 63] U.S. Patent No. 6,239,116 [Patent Document 64] U.S. Patent No. 6,339,068 [Patent Document 65] WO2010 / 125480 [Patent Document 66] U.S. Patent Application No. 20200002727 [Non-patent literature]

[0011] [Non-Patent Document 1] Graham et al., J. Gen Virol., 36:59, 1977 [Non-patent document 2] Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980 [Non-patent document 3] Mather, Biol.Reprod., 23:243-251, 1980. [Non-patent document 4] Mather et al., Annals NYAcad. Sci., 383:44-68, 1982 [Non-Patent Document 5] Milstein et al., Nature, 537:3053, 1983 [Non-patent document 6] Uchida et al., J. Biol. Chem. 218; pp. 3838-3844, 1973 [Non-Patent Document 7] Nicholls and Youle, Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992 [Non-patent document 8] Kuo et al. (1995) Infect Immun 63;2706-13 [Non-Patent Document 9] Falugi et al. (2001) Eur J Immunol 31;3816-3824 [Non-Patent Document 10] Baraldoi et al. (2004) Infect Immun 72;4884-7 [Non-Patent Document 11] Uchida Cameron DM, RJ Collier.1987.J.Bacteriol.169:4967~4971 pages [Non-Patent Document 12] Uchida, T. et al., 1971, Nature New Biology 233:8-11 [Non-Patent Document 13] Sjolander et al. (1998) J. Leukocyte Biol. 64:713 Summary of the Invention [Means for solving the problem]

[0012] To meet these and other needs, the present invention relates to compositions and methods of use for producing recombinant adhesin proteins and for generating immune responses against E. coli serotypes.

[0013] In one aspect, the present invention relates to a recombinant mammalian cell comprising a polynucleotide encoding a polypeptide or fragment thereof derived from E. coli. In some embodiments, the polynucleotide encodes a polypeptide or fragment thereof derived from an E. coli fimbrial H (fimH) polypeptide. In some embodiments, the polypeptide or fragment thereof derived from E. coli FimH comprises a phenylalanine residue at the N-terminus of the polypeptide.

[0014] In one aspect, the present invention relates to a method for producing a polypeptide or fragment thereof derived from E. coli in a recombinant mammalian cell. The method includes expressing the polypeptide or fragment thereof by culturing the recombinant mammalian cell under suitable conditions, and recovering the polypeptide or fragment thereof. In some embodiments, the method further includes purifying the polypeptide or fragment thereof. In some embodiments, the yield of the polypeptide is at least 0.05 g / L. In some embodiments, the yield of the polypeptide is at least 0.10 g / L.

[0015] In one aspect, the present invention relates to a composition comprising a polypeptide having at least 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 99.9% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, or any combination thereof.

[0016] In another aspect, the invention relates to a composition comprising a polypeptide having at least n consecutive amino acids in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more). In some embodiments, the composition further comprises a saccharide selected from any one of formulas in Table 1, preferably Formula O1A, Formula O1B, Formula O2, Formula O6, and Formula O25B, where n is an integer between 1 and 100, preferably between 31 and 100. [Brief explanation of the drawings]

[0017] [Figure 1A-1] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1A-2] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1A-3] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1B] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1C] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1D] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1E-1] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1E-2] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1F-1] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1F-2] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1G] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1H-1] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1H-2] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 1H-3] 1A-1H are amino acid sequence diagrams, including the amino acid sequence of an exemplary polypeptide or fragment thereof from E. coli, and the amino acid sequence of an exemplary wzzB sequence. [Figure 2A] 2A-2T show maps of exemplary expression vectors. [Figure 2B] 2A-2T show maps of exemplary expression vectors. [Figure 2C] 2A-2T show maps of exemplary expression vectors. [Figure 2D] 2A-2T show maps of exemplary expression vectors. [Figure 2E] 2A-2T show maps of exemplary expression vectors. [Figure 2F] 2A-2T show maps of exemplary expression vectors. [Figure 2G] 2A-2T show maps of exemplary expression vectors. [Figure 2H] 2A-2T show maps of exemplary expression vectors. [Figure 2I] 2A-2T show maps of exemplary expression vectors. [Figure 2J] 2A-2T show maps of exemplary expression vectors. [Figure 2K] 2A-2T show maps of exemplary expression vectors. [Figure 2L] 2A-2T show maps of exemplary expression vectors. [Figure 2M] 2A-2T show maps of exemplary expression vectors. [Figure 2N] 2A-2T show maps of exemplary expression vectors. [Figure 2O] 2A-2T show maps of exemplary expression vectors. [Figure 2P] 2A-2T show maps of exemplary expression vectors. [Figure 2Q] 2A-2T show maps of exemplary expression vectors. [Figure 2R] 2A-2T show maps of exemplary expression vectors. [Figure 2S] 2A-2T show maps of exemplary expression vectors. [Figure 2T] 2A-2T show maps of exemplary expression vectors. [Figure 3] Figure 1 shows the results of expression and purification. [Figure 4] Figure 1 shows the results of expression and purification. [Figure 5] FIG. 1 shows the results of expression. [Figure 6A] 6A to 6C show the affinities, including SEC pools and yields, of pSB02083 and pSB02158. [Figure 6B] 6A to 6C show the affinities, including SEC pools and yields, of pSB02083 and pSB02158. [Figure 6C] 6A to 6C show the affinities, including SEC pools and yields, of pSB02083 and pSB02158. [Figure 7] FIG. 1 shows the results of expression of the pSB2198 FimH dscG locked mutant construct. [Figure 8] FIG. 1 shows the results of expression of wild-type pSB2307 FimH dscG. [Figure 9A] 9A-9C show the structures of O antigens synthesized by the polymerase-dependent pathway with four or fewer residues in the backbone. [Figure 9B] 9A-9C show the structures of O antigens synthesized by the polymerase-dependent pathway with four or fewer residues in the backbone. [Figure 9C] 9A-9C show the structures of O antigens synthesized by the polymerase-dependent pathway with four or fewer residues in the backbone. [Figure 10A] Figure 10A shows the structure of O antigens with five or six residues in the backbone synthesized by the polymerase-dependent pathway, and Figure 10B shows O antigens thought to be synthesized by the ABC transporter-dependent pathway. [Figure 10B] Figure 10A shows the structure of O antigens with five or six residues in the backbone synthesized by the polymerase-dependent pathway, and Figure 10B shows O antigens thought to be synthesized by the ABC transporter-dependent pathway. [Figure 11]FIG. 1 shows computational scanning of Phe1 mutagenesis with other amino acids with aliphatic hydrophobic side chains that may stabilize the FimH protein and allow mannose binding, such as Ile, Leu, and Val. [Figure 12A] 12A-12B show the pUC replicon plasmid, 500-700 copies per cell, chain length regulator (FIG. 12A), and the P15a replicon plasmid, 10-12 copies per cell, O-antigen operon (FIG. 12B). [Figure 12B] 12A-12B show the pUC replicon plasmid, 500-700 copies per cell, chain length regulator (FIG. 12A), and the P15a replicon plasmid, 10-12 copies per cell, O-antigen operon (FIG. 12B). [Figure 13A] Figures 13A-13B show modulation of O-antigen chain length in serotype O25a and O25b strains by plasmid-based expression of heterologous wzzB and fepE chain length regulators. Genetic complementation of LPS expression in plasmid transformants of the wzzB knockout strains O25K5H1 (O25a) and GAR2401 (O25b) is shown. The left side of Figure 13A shows the LPS profile of the plasmid transformant of O25a O25K5HΔwzzB, and the right side shows the similar profile of the O25b GAR 2401ΔwzzB transformant. Figure 13B shows an immunoblot of a replicate gel probed with O25-specific serum (Statens Serum Institute). Lanes 1-7 represent the O25a ΔwxxB (knockout) background, and lanes 8-15 represent the O25b 2401 ΔwzzB (knockout) background. [Figure 13B]Figures 13A-13B show modulation of O-antigen chain length in serotype O25a and O25b strains by plasmid-based expression of heterologous wzzB and fepE chain length regulators. Genetic complementation of LPS expression in plasmid transformants of the wzzB knockout strains O25K5H1 (O25a) and GAR2401 (O25b) is shown. The left side of Figure 13A shows the LPS profile of the plasmid transformant of O25a O25K5HΔwzzB, and the right side shows the similar profile of the O25b GAR 2401ΔwzzB transformant. Figure 13B shows an immunoblot of a replicate gel probed with O25-specific serum (Statens Serum Institute). Lanes 1-7 represent the O25a ΔwxxB (knockout) background, and lanes 8-15 represent the O25b 2401 ΔwzzB (knockout) background. [Figure 14] FIG. 1 shows the expression of long O antigens carried by E. coli and Salmonella fepE plasmids in host O25K5H1ΔwzzB. [Figure 15] FIG. 1 shows that expression of Salmonella fepE produces long-chain O-antigen LPS in diverse clinical isolates. [Figure 16] Figures 16A-16B show plasmid-mediated, arabinose-inducible expression of O25b long-chain O-antigen LPS in an O25b O-antigen knockout host strain. SPS PAGE results are shown in Figure 16A, and O25 immunoblot results are shown in Figure 16B. In both Figures 16A and 16B, lane 1 is clone 1 without arabinose, lane 2 is clone 1 with 0.2% arabinose, lane 3 is clone 9 without arabinose, lane 4 is clone 9 with 0.2% arabinose, lane 5 is an O55 E. coli LPS preparation, and lane 6 is an O111 E. coli LPS preparation. [Figure 17] FIG. 1 shows plasmid-mediated arabinose-inducible expression of long-chain O-antigen LPS in a common host strain. [Figure 18] FIG. 1 shows expression of O25 O-antigen LPS in Exploratory Bioprocess strains. [Figure 19A] Figures 19A-19B show the SEC profiles and characteristics of short O25b O antigens (Figure 19A, strain 1 O25b wild-type (wt) 2831) and long O25b O antigens (Figure 19B, strain 2 O25b 2401ΔwzzB / LT2 FepE) purified from strains GAR2831 and '2401ΔwzzB / fepE. [Figure 19B] Figures 19A-19B show the SEC profiles and characteristics of short O25b O antigens (Figure 19A, strain 1 O25b wild-type (wt) 2831) and long O25b O antigens (Figure 19B, strain 2 O25b 2401ΔwzzB / LT2 FepE) purified from strains GAR2831 and '2401ΔwzzB / fepE. [Figure 20A] 20A-20B show vaccination schedules for rabbits: (FIG. 20A) Information regarding vaccination schedule for rabbit study 1 VAC-2017-PRL-EC-0723; (FIG. 20B) vaccination schedule for rabbit study 2 VAC-2018-PRL-EC-077. [Figure 20B] 20A-20B show vaccination schedules for rabbits: (FIG. 20A) Information regarding vaccination schedule for rabbit study 1 VAC-2017-PRL-EC-0723; (FIG. 20B) vaccination schedule for rabbit study 2 VAC-2018-PRL-EC-077. [Figure 21A] Figures 21A to 21C show the IgG response of O25b glycoconjugates. -●- represents the results before blood collection, -■- represents the results from blood collection 1 (week 6), -▲- represents the results from blood collection 2 (week 8), and -◆- represents the results from blood collection 3 (week 12). Figure 21A shows the results from rabbit 1-3 (moderate activation), Figure 21B shows the results from rabbit 2-3 (low activation), and Figure 21C shows the results from rabbit 3-1 (high activation). [Figure 21B]Figures 21A to 21C show the IgG response of O25b glycoconjugates. -●- represents the results before blood collection, -■- represents the results from blood collection 1 (week 6), -▲- represents the results from blood collection 2 (week 8), and -◆- represents the results from blood collection 3 (week 12). Figure 21A shows the results from rabbit 1-3 (moderate activation), Figure 21B shows the results from rabbit 2-3 (low activation), and Figure 21C shows the results from rabbit 3-1 (high activation). [Figure 21C] Figures 21A to 21C show the IgG response of O25b glycoconjugates. -●- represents the results before blood collection, -■- represents the results from blood collection 1 (week 6), -▲- represents the results from blood collection 2 (week 8), and -◆- represents the results from blood collection 3 (week 12). Figure 21A shows the results from rabbit 1-3 (moderate activation), Figure 21B shows the results from rabbit 2-3 (low activation), and Figure 21C shows the results from rabbit 3-1 (high activation). [Figure 22A] Figures 22A-22F show the IgG response to the O25b long-chain O antigen glycoconjugate, i.e., the less activated O25b-CRM197 conjugate (Figures 22D-22F, -●- represents the results of rabbit 2-1 before bleeding and -■- represents the results of rabbit 2-1 antiserum at week 12) compared with the unconjugated polysaccharide, i.e., free O25b polysaccharide (Figures 22A-22C, -●- represents the results of rabbit A-1 before bleeding, -■- represents the results of rabbit A-1 antiserum at week 6, and -▲- represents the results of rabbit A-1 antiserum at week 8). Note that the MFI is plotted on a log scale to highlight the difference between pre-immune and immune antibodies in the MFI range below 1000. Figure 22A shows the results for rabbit A-1 (unconjugated polysaccharide), Figure 22B shows the results for rabbit A-3 (unconjugated polysaccharide), Figure 22C shows the results for rabbit A-4 (unconjugated polysaccharide), Figure 22D shows the results for rabbit 2-1 (low activation), Figure 22E shows the results for rabbit 2-2 (low activation), and Figure 22F shows the results for rabbit 2-3 (low activation). [Figure 22B]Figures 22A-22F show the IgG response to the O25b long-chain O antigen glycoconjugate, i.e., the less activated O25b-CRM197 conjugate (Figures 22D-22F, -●- represents the results of rabbit 2-1 before bleeding and -■- represents the results of rabbit 2-1 antiserum at week 12) compared with the unconjugated polysaccharide, i.e., free O25b polysaccharide (Figures 22A-22C, -●- represents the results of rabbit A-1 before bleeding, -■- represents the results of rabbit A-1 antiserum at week 6, and -▲- represents the results of rabbit A-1 antiserum at week 8). Note that the MFI is plotted on a log scale to highlight the difference between pre-immune and immune antibodies in the MFI range below 1000. Figure 22A shows the results for rabbit A-1 (unconjugated polysaccharide), Figure 22B shows the results for rabbit A-3 (unconjugated polysaccharide), Figure 22C shows the results for rabbit A-4 (unconjugated polysaccharide), Figure 22D shows the results for rabbit 2-1 (low activation), Figure 22E shows the results for rabbit 2-2 (low activation), and Figure 22F shows the results for rabbit 2-3 (low activation). [Figure 22C]Figures 22A-22F show the IgG response to the O25b long-chain O antigen glycoconjugate, i.e., the less activated O25b-CRM197 conjugate (Figures 22D-22F, -●- represents the results of rabbit 2-1 before bleeding and -■- represents the results of rabbit 2-1 antiserum at week 12) compared with the unconjugated polysaccharide, i.e., free O25b polysaccharide (Figures 22A-22C, -●- represents the results of rabbit A-1 before bleeding, -■- represents the results of rabbit A-1 antiserum at week 6, and -▲- represents the results of rabbit A-1 antiserum at week 8). Note that the MFI is plotted on a log scale to highlight the difference between pre-immune and immune antibodies in the MFI range below 1000. Figure 22A shows the results for rabbit A-1 (unconjugated polysaccharide), Figure 22B shows the results for rabbit A-3 (unconjugated polysaccharide), Figure 22C shows the results for rabbit A-4 (unconjugated polysaccharide), Figure 22D shows the results for rabbit 2-1 (low activation), Figure 22E shows the results for rabbit 2-2 (low activation), and Figure 22F shows the results for rabbit 2-3 (low activation). [Figure 22D]Figures 22A-22F show the IgG response to the O25b long-chain O antigen glycoconjugate, i.e., the less activated O25b-CRM197 conjugate (Figures 22D-22F, -●- represents the results of rabbit 2-1 before bleeding and -■- represents the results of rabbit 2-1 antiserum at week 12) compared with the unconjugated polysaccharide, i.e., free O25b polysaccharide (Figures 22A-22C, -●- represents the results of rabbit A-1 before bleeding, -■- represents the results of rabbit A-1 antiserum at week 6, and -▲- represents the results of rabbit A-1 antiserum at week 8). Note that the MFI is plotted on a log scale to highlight the difference between pre-immune and immune antibodies in the MFI range below 1000. Figure 22A shows the results for rabbit A-1 (unconjugated polysaccharide), Figure 22B shows the results for rabbit A-3 (unconjugated polysaccharide), Figure 22C shows the results for rabbit A-4 (unconjugated polysaccharide), Figure 22D shows the results for rabbit 2-1 (low activation), Figure 22E shows the results for rabbit 2-2 (low activation), and Figure 22F shows the results for rabbit 2-3 (low activation). [Figure 22E]Figures 22A-22F show the IgG response to the O25b long-chain O antigen glycoconjugate, i.e., the less activated O25b-CRM197 conjugate (Figures 22D-22F, -●- represents the results of rabbit 2-1 before bleeding and -■- represents the results of rabbit 2-1 antiserum at week 12) compared with the unconjugated polysaccharide, i.e., free O25b polysaccharide (Figures 22A-22C, -●- represents the results of rabbit A-1 before bleeding, -■- represents the results of rabbit A-1 antiserum at week 6, and -▲- represents the results of rabbit A-1 antiserum at week 8). Note that the MFI is plotted on a log scale to highlight the difference between pre-immune and immune antibodies in the MFI range below 1000. Figure 22A shows the results for rabbit A-1 (unconjugated polysaccharide), Figure 22B shows the results for rabbit A-3 (unconjugated polysaccharide), Figure 22C shows the results for rabbit A-4 (unconjugated polysaccharide), Figure 22D shows the results for rabbit 2-1 (low activation), Figure 22E shows the results for rabbit 2-2 (low activation), and Figure 22F shows the results for rabbit 2-3 (low activation). [Figure 22F]Figures 22A-22F show the IgG response to the O25b long-chain O antigen glycoconjugate, i.e., the less activated O25b-CRM197 conjugate (Figures 22D-22F, -●- represents the results of rabbit 2-1 before bleeding and -■- represents the results of rabbit 2-1 antiserum at week 12) compared with the unconjugated polysaccharide, i.e., free O25b polysaccharide (Figures 22A-22C, -●- represents the results of rabbit A-1 before bleeding, -■- represents the results of rabbit A-1 antiserum at week 6, and -▲- represents the results of rabbit A-1 antiserum at week 8). Note that the MFI is plotted on a log scale to highlight the difference between pre-immune and immune antibodies in the MFI range below 1000. Figure 22A shows the results for rabbit A-1 (unconjugated polysaccharide), Figure 22B shows the results for rabbit A-3 (unconjugated polysaccharide), Figure 22C shows the results for rabbit A-4 (unconjugated polysaccharide), Figure 22D shows the results for rabbit 2-1 (low activation), Figure 22E shows the results for rabbit 2-2 (low activation), and Figure 22F shows the results for rabbit 2-3 (low activation). [Figure 23A] Figures 23A to 23C show the surface expression of native O25b O antigen and long-chain O25b O antigen detected with O25b antiserum. Figure 23A shows the results, where -●- represents the results of O25b 2831 with PD3 antiserum, -■- represents the results of O25b 2831 wild-type with respect to before blood collection, -▲- represents the results of O25b 2831 / fepE with PD3 antiserum, and -▼- represents the results of O25b 2831 / fepE with respect to before blood collection. Figure 23B shows the results of O25b 2401 against PD3 antiserum (-●-), O25b 2401 against pre-blood collection (-▪-), O25b 2401 / fepE against PD3 antiserum (-▲-), and O25b 2401 / fepE against pre-blood collection (-▼-). Figure 23C shows the results of E. coli K12 against PD3 antiserum (-●-), and E. coli K12 against pre-blood collection (-▪-). [Figure 23B]Figures 23A to 23C show the surface expression of native O25b O antigen and long-chain O25b O antigen detected with O25b antiserum. Figure 23A shows the results, where -●- represents the results of O25b 2831 with PD3 antiserum, -■- represents the results of O25b 2831 wild-type with respect to before blood collection, -▲- represents the results of O25b 2831 / fepE with PD3 antiserum, and -▼- represents the results of O25b 2831 / fepE with respect to before blood collection. Figure 23B shows the results of O25b 2401 against PD3 antiserum (-●-), O25b 2401 against pre-blood collection (-▪-), O25b 2401 / fepE against PD3 antiserum (-▲-), and O25b 2401 / fepE against pre-blood collection (-▼-). Figure 23C shows the results of E. coli K12 against PD3 antiserum (-●-), and E. coli K12 against pre-blood collection (-▪-). [Figure 23C] Figures 23A to 23C show the surface expression of native O25b O antigen and long-chain O25b O antigen detected with O25b antiserum. Figure 23A shows the results, where -●- represents the results of O25b 2831 with PD3 antiserum, -■- represents the results of O25b 2831 wild-type with respect to before blood collection, -▲- represents the results of O25b 2831 / fepE with PD3 antiserum, and -▼- represents the results of O25b 2831 / fepE with respect to before blood collection. Figure 23B shows the results of O25b 2401 against PD3 antiserum (-●-), O25b 2401 against pre-blood collection (-▪-), O25b 2401 / fepE against PD3 antiserum (-▲-), and O25b 2401 / fepE against pre-blood collection (-▼-). Figure 23C shows the results of E. coli K12 against PD3 antiserum (-●-), and E. coli K12 against pre-blood collection (-▪-). [Figure 24]Figure 1 shows the generalized carbohydrate backbone of the outer core oligosaccharides of the five known chemotypes. All glycoses are in the α-anomeric configuration unless otherwise noted. The genes whose products catalyze the formation of each bond are indicated by dashed arrows. The asterisk denotes the residue on the core oligosaccharide at which O-antigen attachment occurs. [Figure 25] Figure 1 shows the lack of immunogenicity of unconjugated free O25b polysaccharide (dLIA). -●- represents the results of antiserum 4-1 at week 18 (week 1 = PD4), -■- represents the results of antiserum 4-2 at week 18 (week 1 = PD4), -▲- represents the results of antiserum 5-1 at week 18 (week 1 = PD4), -▼- represents the results of antiserum 5-2 at week 18 (week 1 = PD4), -*- represents the results of antiserum 6-1 at week 18 (week 1 = PD4), and -∧- represents the results of antiserum 6-2 at week 18 (week 1 = PD4). [Figure 26A] Figures 26A-26C are graphs showing the specificity of immune serum opsonophagocytic assay (OPA) titers of baby rabbit complement (BRC) rabbit O25b reductive amination chemistry (RAC) conjugate. Figure 26A shows the OPA titers of pre-immune serum (-●-) and week 13 post-immune serum (-■-) from rabbit 2-3. Figure 26B shows the OPA titers of pre-immune serum (-●-) and week 19 post-immune serum (-■-) from rabbit 1-2. Figure 26C shows the OPA titer specificity of rabbit 1-2 at week 19, in which the OPA activity of rabbit 1-2 immune serum was blocked by preincubation with 100 μg / mL of purified unconjugated O25b long-chain O antigen polysaccharide; -■- represents the results of rabbit 1-2 immune serum at week 19, and -▼- represents the results of rabbit 1-2 w / R1 long-chain O Ag at week 19. [Figure 26B]Figures 26A-26C are graphs showing the specificity of immune serum opsonophagocytic assay (OPA) titers of baby rabbit complement (BRC) rabbit O25b reductive amination chemistry (RAC) conjugate. Figure 26A shows the OPA titers of pre-immune serum (-●-) and week 13 post-immune serum (-■-) from rabbit 2-3. Figure 26B shows the OPA titers of pre-immune serum (-●-) and week 19 post-immune serum (-■-) from rabbit 1-2. Figure 26C shows the OPA titer specificity of rabbit 1-2 at week 19, in which the OPA activity of rabbit 1-2 immune serum was blocked by preincubation with 100 μg / mL of purified unconjugated O25b long-chain O antigen polysaccharide; -■- represents the results of rabbit 1-2 immune serum at week 19, and -▼- represents the results of rabbit 1-2 w / R1 long-chain O Ag at week 19. [Figure 26C] Figures 26A-26C are graphs showing the specificity of immune serum opsonophagocytic assay (OPA) titers of baby rabbit complement (BRC) rabbit O25b reductive amination chemistry (RAC) conjugate. Figure 26A shows the OPA titers of pre-immune serum (-●-) and week 13 post-immune serum (-■-) from rabbit 2-3. Figure 26B shows the OPA titers of pre-immune serum (-●-) and week 19 post-immune serum (-■-) from rabbit 1-2. Figure 26C shows the OPA titer specificity of rabbit 1-2 at week 19, in which the OPA activity of rabbit 1-2 immune serum was blocked by preincubation with 100 μg / mL of purified unconjugated O25b long-chain O antigen polysaccharide; -■- represents the results of rabbit 1-2 immune serum at week 19, and -▼- represents the results of rabbit 1-2 w / R1 long-chain O Ag at week 19. [Figure 27A] Figure 27A shows an example of an exemplary dosing schedule. Figures 27B and 27C are graphs showing O antigen O25b IgG levels generated by unconjugated O25b long chain O antigen polysaccharide (Figure 27B, O25b free polysaccharide (2 μg)) and the resulting O25b RAC / DMSO long chain O antigen glycoconjugate (Figure 27C, O25b-CRM197RAC long chain (2 μg)), where -...- (dotted line) represents naive CD1 O25b IgG levels. [Figure 27B] Figure 27A shows an example of an exemplary dosing schedule. Figures 27B and 27C are graphs showing O antigen O25b IgG levels generated by unconjugated O25b long chain O antigen polysaccharide (Figure 27B, O25b free polysaccharide (2 μg)) and the resulting O25b RAC / DMSO long chain O antigen glycoconjugate (Figure 27C, O25b-CRM197RAC long chain (2 μg)), where -...- (dotted line) represents naive CD1 O25b IgG levels. [Figure 27C] Figure 27A shows an example of an exemplary dosing schedule. Figures 27B and 27C are graphs showing O antigen O25b IgG levels generated by unconjugated O25b long chain O antigen polysaccharide (Figure 27B, O25b free polysaccharide (2 μg)) and the resulting O25b RAC / DMSO long chain O antigen glycoconjugate (Figure 27C, O25b-CRM197RAC long chain (2 μg)), where -...- (dotted line) represents naive CD1 O25b IgG levels. [Figure 28A] Figures 28A-28B are graphs showing OPA immunogenicity after 2 doses (Figure 28A) and 3 doses (Figure 28B) of RAC, eTEC O25b long-chain glycoconjugate, and single-terminal glycoconjugate; -○- represents results for 2 μg single-terminal short chain, -●- represents results for 2 μg single-terminal long chain, -▲- represents results for 2 μg RAC / DMSO long chain, -▼- represents results for 2 μg eTEC long chain, and * represents background control (n=20). †Responder rate is the percentage of mice with titers greater than twice the unvaccinated baseline. [Figure 28B]Figures 28A-28B are graphs showing OPA immunogenicity after 2 doses (Figure 28A) and 3 doses (Figure 28B) of RAC, eTEC O25b long-chain glycoconjugate, and single-terminal glycoconjugate; -○- represents results for 2 μg single-terminal short chain, -●- represents results for 2 μg single-terminal long chain, -▲- represents results for 2 μg RAC / DMSO long chain, -▼- represents results for 2 μg eTEC long chain, and * represents background control (n=20). †Responder rate is the percentage of mice with titers greater than twice the unvaccinated baseline. [Figure 29] Figure 1 shows the change in OPA immunogenicity and polysaccharide activation levels of eTEC chemistry. †Responder rate is the percentage of mice with titers greater than 2x the unvaccinated baseline. [Figure 30A] Figures 30A-30B show an example of an exemplary dosing schedule (Figure 30A) and a graph (Figure 30B) showing protection of mice immunized with E. coli eTEC conjugates against a lethal challenge with an O25b isolate. -◇- represents 17% eTEC long chain activation, -△- represents 10% eTEC long chain activation, -▽- represents 4% eTEC long chain activation, -□- represents O25b polysaccharide, and -○- represents unvaccinated controls. [Figure 30B] Figures 30A-30B show an example of an exemplary dosing schedule (Figure 30A) and a graph (Figure 30B) showing protection of mice immunized with E. coli eTEC conjugates against a lethal challenge with an O25b isolate. -◇- represents 17% eTEC long chain activation, -△- represents 10% eTEC long chain activation, -▽- represents 4% eTEC long chain activation, -□- represents O25b polysaccharide, and -○- represents unvaccinated controls. [Figure 31]31 is a schematic diagram showing an exemplary preparation of a single-terminated conjugate. The conjugation process involves selective activation of 2-keto-3-deoxyoctanoic acid (KDO) with a disulfide amine linker, followed by exposure of a thiol functional group. KDO is then conjugated to a bromo-activated CRM197 protein, as shown in FIG. 31 (Preparation of a Single-Terminated Conjugate). [Figure 32A] 32A-32B are exemplary process flow diagrams of the activation process (FIG. 32A) and conjugation process (FIG. 32B) used to prepare E. coli glycoconjugates with CRM197. [Figure 32B] 32A-32B are exemplary process flow diagrams of the activation process (FIG. 32A) and conjugation process (FIG. 32B) used to prepare E. coli glycoconjugates with CRM197.

[0018] Array Description SEQ ID NO: 1 shows the amino acid sequence of the wild-type type 1 fimbria D-mannose-specific adhesin [Escherichia coli FimH J96].

[0019] SEQ ID NO: 2 shows the amino acid sequence of a fragment of FimH (mature FimH protein) corresponding to amino acid (aa) residues 22 to 300 of SEQ ID NO: 1. SEQ ID NO: 3 shows the amino acid sequence of the FimH lectin domain.

[0020] SEQ ID NO: 4 shows the amino acid sequence of the FimH pilin domain. SEQ ID NO: 5 is the polypeptide derived from E. coli FimH (FimH mIgK signal peptide / F22..Q300 in pSB02198-pcDNA3.1(+) The amino acid sequence of J96 FimH N28S V48C L55C N91S N249Q / 7AA linker / FimG A1..K14 / GGHis8) is shown.

[0021] SEQ ID NO: 6 is the polypeptide derived from E. coli FimH (FimH mIgK signal peptide / F22..Q300 in pSB02307-pcDNA3.1(+) The amino acid sequence of J96 FimH N28S N91S N249Q / His8) is shown.

[0022] SEQ ID NO: 7 shows the amino acid sequence of a fragment of a polypeptide derived from E. coli FimH (pSB02083 FimH lectin domain wild-type construct). SEQ ID NO: 8 shows the amino acid sequence of a polypeptide fragment derived from E. coli FimH (pSB02158 FimH lectin domain-locked mutant).

[0023] SEQ ID NO: 9 shows the amino acid sequence of a polypeptide fragment (FimG A1..K14) derived from E. coli FimG. SEQ ID NO: 10 shows the amino acid sequence of a fragment of a polypeptide derived from E. coli FimC.

[0024] SEQ ID NO: 11 shows the amino acid sequence of the 4aa linker. SEQ ID NO: 12 shows the amino acid sequence of the 5aa linker. SEQ ID NO: 13 shows the amino acid sequence of the 6aa linker.

[0025] SEQ ID NO: 14 shows the amino acid sequence of the 7aa linker. SEQ ID NO: 15 shows the amino acid sequence of the 8aa linker. SEQ ID NO: 16 shows the amino acid sequence of the 9aa linker.

[0026] SEQ ID NO: 17 shows the amino acid sequence of the 10aa linker. SEQ ID NO: 18 shows the amino acid sequence of the FimH J96 signal sequence. SEQ ID NO: 19 shows the amino acid sequence of the signal peptide of SEQ ID NO: 5 (FimH mIgK signal peptide / F22..Q300 J96 FimH N28S V48C L55C N91S N249Q / 7AA linker / FimG A1..K14 / GGHis8 in pSB02198-pcDNA3.1(+)).

[0027] SEQ ID NO: 20 shows the amino acid sequence of the polypeptide derived from E. coli FimH according to SEQ ID NO: 5 (mature protein of FimH mIgK signal peptide / F22..Q300 J96 FimH N28S V48C L55C N91S N249Q / 7AA linker / FimG A1..K14 / GGHis8 in pSB02198-pcDNA3.1(+)).

[0028] SEQ ID NO: 21 shows the amino acid sequence of a polypeptide derived from E. coli FimG. SEQ ID NO: 22 shows the amino acid sequence of the signal peptide of SEQ ID NO: 6 (FimH mIgK signal peptide / F22..Q300 J96 FimH N28S N91S N249Q / His8 in pSB02307-pcDNA3.1(+)).

[0029] SEQ ID NO: 23 represents the polypeptide derived from E. coli FimH according to SEQ ID NO: 6 (FimH mIgK signal peptide / F22..Q300 in pcDNA3.1(+) The amino acid sequence of J96 FimH (mature protein of N28S N91S N249Q / His8) is shown.

[0030] SEQ ID NO: 24 shows the amino acid sequence of the polypeptide derived from E. coli FimH according to SEQ ID NO: 7 (mature protein of the pSB02083 FimH lectin domain wild-type construct).

[0031] SEQ ID NO: 25 shows the amino acid sequence of the His tag. SEQ ID NO: 26 shows the amino acid sequence of the polypeptide derived from E. coli FimH according to SEQ ID NO: 8 (mature protein of the pSB02158 FimH lectin domain-locked mutant).

[0032] SEQ ID NO: 27 shows the amino acid sequence of a polypeptide derived from E. coli FimH (pSB01878). SEQ ID NO: 28 is the amino acid sequence of the polypeptide (K12) from E. coli FimH. The acid sequence is shown.

[0033] SEQ ID NO: 29 shows the amino acid sequence of a polypeptide (UTI89) derived from E. coli FimH. SEQ ID NO: 30 shows the amino acid sequence of O25b 2401 WzzB.

[0034] SEQ ID NO: 31 shows the amino acid sequence of O25a:K5:H1 WzzB. SEQ ID NO: 32 shows the amino acid sequence of O25a ETEC ATCC WzzB. SEQ ID NO: 33 shows the amino acid sequence of K12 W3110 WzzB.

[0035] SEQ ID NO: 34 shows the amino acid sequence of Salmonella LT2 WzzB. SEQ ID NO: 35 shows the amino acid sequence of O25b 2401 FepE. SEQ ID NO: 36 shows the amino acid sequence of O25a:K5:H1 FepE.

[0036] SEQ ID NO: 37 shows the amino acid sequence of O25a ETEC ATCC FepE. SEQ ID NO: 38 shows the amino acid sequence of O157 FepE. SEQ ID NO: 39 shows the amino acid sequence of Salmonella LT2 FepE.

[0037] SEQ ID NO: 40 shows the primer sequence of LT2wzzB_S. SEQ ID NO: 41 shows the primer sequence of LT2wzzB_AS. SEQ ID NO: 42 shows the primer sequence of O25bFepE_S.

[0038] SEQ ID NO: 43 shows the primer sequence of O25bFepE_A. SEQ ID NO: 44 shows the primer sequence of wzzB P1_S. SEQ ID NO: 45 shows the primer sequence of wzzB P2_AS.

[0039] SEQ ID NO: 46 shows the primer sequence of wzzB P3_S. SEQ ID NO: 47 shows the primer sequence of wzzB P4_AS. SEQ ID NO: 48 shows the primer sequence of O157 FepE_S.

[0040] SEQ ID NO: 49 shows the primer sequence of O157 FepE_AS. SEQ ID NO: 50 shows the primer sequence of pBAD33_adaptor_S. SEQ ID NO: 51 shows the primer sequence of pBAD33_adaptor_AS.

[0041] SEQ ID NO: 52 shows the primer sequence of JUMPSTART_r. SEQ ID NO: 53 shows the primer sequence of gnd_f. SEQ ID NO: 54 shows the amino acid sequence of the mouse IgK signal sequence.

[0042] SEQ ID NO: 55 shows the amino acid sequence of the signal peptide of the human IgG receptor FcRn large subunit p51. SEQ ID NO: 56 shows the amino acid sequence of the signal peptide of the human IL10 protein.

[0043] SEQ ID NO: 57 shows the amino acid sequence of the human respiratory syncytial virus A (strain A2) fusion glycoprotein F0 signal peptide. SEQ ID NO: 58 shows the amino acid sequence of the influenza A hemagglutinin signal peptide.

[0044] SEQ ID NOs: 59-101 show the amino acid and nucleic acid sequences of nanostructure-associated polypeptides or fragments thereof. SEQ ID NOs: 102 to 109 are the signal peptides of various species used for signal peptide prediction. The sequence is shown by 1P 4.1 (DTU Bioinformatics). DETAILED DESCRIPTION OF THE INVENTION

[0045] The present inventors have overcome the challenges of producing polypeptides derived from E. coli adhesin proteins by using mammalian cells for expression. As illustrated throughout this disclosure and in the Examples section, it has been discovered that expression of recombinant polypeptides by mammalian cells consistently achieved higher yields compared to expression of the polypeptides in E. coli. Furthermore, the present inventors have surprisingly identified mutations and expression constructs that stabilize the recombinant polypeptides and fragments thereof in desired conformations.

[0046] Interfering with the primary steps of infection—bacterial attachment to host cell receptors and colonization of mucosal surfaces—is critical for preventing, treating, and / or reducing the likelihood of bacterial infection. Bacterial attachment can involve interactions between bacterial surface proteins, called adhesins, and host cell receptors. Previous preclinical studies using the FimH adhesin (derived from uropathogenic E. coli) have confirmed the development of antibodies against the adhesin. Advances in the identification, characterization, and isolation of adhesins are needed in efforts to prevent infectious diseases ranging from otitis media and dental caries to pneumonia and sepsis.

[0047] To produce adhesin proteins such as FimH and fragments thereof on a commercial scale, it is necessary to identify suitable constructs and suitable hosts so that the polypeptides and fragments thereof can be expressed in sufficient quantities, for extended periods of time, and in a preferred conformation. For example, in some embodiments, a preferred conformation of the recombinant polypeptide will have a low affinity for monomannose (e.g., K dIn some embodiments, the preferred conformation exhibits a high affinity for monomannose (e.g., K d <1.2μM).

[0048] Adhesin proteins from E. coli have been recombinantly expressed in E. coli cells; however, the yields were less than 10 mg / L. Purifying large amounts of pilus-associated adhesins can be difficult when produced in E. coli. Without being bound by theory or mechanism, it has been suggested that products expressed in E. coli may assume a conformation that is not optimal for generating an effective immune response in mammals.

[0049] In one aspect, the invention includes a recombinant mammalian cell comprising a polynucleotide sequence encoding a polypeptide or fragment thereof derived from a bacterial adhesin protein. In another aspect, the present invention includes a method for producing a polypeptide or fragment thereof in a mammalian cell, the method comprising: (i) expressing the polypeptide or fragment thereof by culturing mammalian cells under suitable conditions; and (ii) recovering the polypeptide or fragment thereof from the culture. The method may further comprise purifying the polypeptide or fragment thereof. Also disclosed herein are polypeptides or fragments thereof produced by the method.

[0050] In another aspect, the present invention includes a composition comprising a polypeptide or fragment thereof described herein. The composition may comprise a polypeptide or fragment thereof suitable for in vivo administration. For example, the polypeptide or fragment thereof in such a composition may have a purity of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by mass. The composition may further comprise an adjuvant.

[0051] In a further aspect, the invention includes compositions for use in eliciting an immune response against E. coli. Also disclosed are uses of the compositions described herein for eliciting an immune response against E. coli, and the use of the compositions described herein in the manufacture of a medicament for eliciting an immune response against E. coli.

[0052] Polypeptides and fragments thereof derived from IE coli In one aspect, disclosed herein is a mammalian cell comprising a polynucleotide encoding a polypeptide or fragment thereof derived from E. coli. As used herein, the term "derived from" refers to a polypeptide comprising the amino acid sequence of a FimH polypeptide or FimCH polypeptide complex described herein or a fragment thereof, altered by the introduction of amino acid substitutions, deletions, or additions. Preferably, the E. coli-derived polypeptide or fragment thereof comprises a sequence having at least 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 99.9% identity to the sequence of the corresponding wild-type E. coli FimH polypeptide or fragment. In some embodiments, the polypeptide or fragment thereof derived from E. coli has the same full-length amino acid sequence as the corresponding wild-type FimH polypeptide or FimCH polypeptide complex or fragment thereof.

[0053] A fragment should comprise at least n contiguous amino acids of the sequence, where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20, or more), depending on the particular sequence. Preferably, the fragment comprises an epitope of the sequence. In some embodiments, the fragment comprises an amino acid sequence of at least 50 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of a polypeptide derived from E. coli.

[0054] In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises one or more non-classical amino acids compared to the corresponding wild-type E. coli FimH polypeptide or fragment.

[0055] In some embodiments, the polypeptide or fragment thereof derived from E. coli has a similar or identical function to the corresponding wild-type FimH polypeptide or fragment thereof. In a preferred embodiment, the polypeptides or polypeptide complexes of the present invention or fragments thereof are isolated or purified.

[0056] In some embodiments, a polynucleotide encoding a polypeptide or fragment thereof derived from E. coli is integrated into the genomic DNA of a mammalian cell, and when cultured under suitable conditions, the polypeptide or fragment thereof derived from E. coli is expressed by the mammalian cell.

[0057] In a preferred embodiment, the E. coli derived polypeptide or fragment thereof is soluble. In some embodiments, the E. coli derived polypeptide or fragment thereof is secreted from mammalian host cells.

[0058] In some embodiments, the polypeptide or fragment thereof derived from E. coli may contain additional amino acid residues, such as an N-terminal or C-terminal extension. The polypeptide or fragment thereof may comprise one or more tags that can facilitate detection (e.g., an epitope tag for detection with a monoclonal antibody) and / or purification (e.g., a polyhistidine tag that allows purification with a nickel chelate resin). In some embodiments, the tag comprises an amino acid sequence selected from any one of SEQ ID NO: 21 and SEQ ID NO: 25. Such affinity purification tags are known in the art. Examples of affinity purification tags include, for example, His tags (e.g., hexahistidine capable of binding to metal ions), maltose binding protein (MBP) (e.g., capable of binding to amylose), glutathione S-transferase (GST) (e.g., capable of binding to glutathione), FLAG tags (e.g., capable of binding to anti-Flag antibodies), and Strep tags (e.g., capable of binding to streptavidin or a derivative thereof). In a preferred embodiment, the polypeptide or fragment thereof derived from E. coli does not contain additional amino acid residues, such as an N- or C-terminal extension. In some embodiments, the E. coli derived polypeptides or fragments thereof described herein do not comprise an exogenous tag sequence.

[0059] Although particular strains of E. coli may be referenced herein, it should be understood that polypeptides or fragments thereof derived from E. coli are not limited to any particular strain unless otherwise specified.

[0060] In some embodiments, a polypeptide or fragment thereof derived from E. coli FimH comprises a phenylalanine residue at the N-terminus of the polypeptide. In some embodiments, a polypeptide or fragment thereof derived from FimH comprises a phenylalanine residue within the first 20 residues of the N-terminus. Preferably, the phenylalanine residue is located at position 1 of the polypeptide. For example, in some embodiments, a polypeptide or fragment thereof derived from E. coli FimH does not comprise an additional glycine residue at the N-terminus of the polypeptide or fragment thereof derived from E. coli FimH.

[0061] In some embodiments, the phenylalanine residue at position 1 of wild-type mature E. coli FimH is substituted with an aliphatic hydrophobic amino acid, such as, for example, any one of Ile, Leu, and Val residues.

[0062] In some embodiments, a signal peptide may be used to express a polypeptide or fragment thereof from E. coli. Signal sequences and expression cassettes for producing proteins are known in the art. Leader peptides are generally 5 to 30 amino acids long and are usually present at the N-terminus of a newly synthesized polypeptide. Signal peptides generally contain a long stretch of hydrophobic amino acids that tend to form a single alpha-helix. Furthermore, many signal peptides begin with a short stretch of positively charged amino acids, which may help to properly shape the polypeptide during translocation. The end of a signal peptide usually contains a stretch of amino acids that is recognized and cleaved by a signal peptidase. The signal peptidase may cleave during translocation or after translocation is complete, resulting in a released signal peptide and mature protein. In some embodiments, the signal peptide comprises an amino acid sequence having at least 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%, 99.9%, or 100% identity to any one of SEQ ID NO:9, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:22.

[0063] In some embodiments, the E. coli derived polypeptides or fragments thereof described herein can comprise a cleavable linker. Such a linker can be, for example, a linker that can be cleaved. Addition of a factor capable of cleaving the linker allows the tag to be separated from the purified complex. Cleavable linkers are known in the art. Such linkers can be cleaved, for example, by irradiation of a photolabile bond or by acid-catalyzed hydrolysis. Another example of a cleavable linker is a polypeptide linker that incorporates a protease recognition site and can be cleaved by the addition of a suitable protease enzyme.

[0064] In some embodiments, a polypeptide or fragment thereof derived from E. coli contains a modification compared to the corresponding wild-type E. coli FimH polypeptide or fragment. The modification can include the covalent attachment of a molecule to the polypeptide. For example, such modifications can include glycosylation with known protecting / blocking groups, acetylation, pegylation, phosphorylation, amidation, derivatization, proteolytic cleavage, attachment to a cellular ligand or other protein, and the like. In some embodiments, a polypeptide or fragment thereof derived from E. coli contains a modification compared to the corresponding wild-type E. coli FimH polypeptide or fragment, such as by chemical modification using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In another embodiment, the modification can include the covalent attachment of a lipid molecule to the polypeptide. In some embodiments, the polypeptide does not contain a covalent attachment of a molecule to the polypeptide compared to the corresponding wild-type E. coli FimH polypeptide or fragment.

[0065] For example, proteins and polypeptides produced in cell culture can be glycoproteins containing covalently linked carbohydrate structures, including oligosaccharide chains. These oligosaccharide chains are attached to proteins via either N- or O-linkages. The oligosaccharide chains can constitute the majority of the glycoprotein's mass. Generally, N-linked oligosaccharides are attached to the amino group on the side chain of an asparagine residue in the target consensus sequence Asn-X-Ser / Thr (where X can be any amino acid except proline). In some embodiments, the glycosylation site comprises an amino acid sequence selected from any one of asparagine-glycine-threonine (NGT), asparagine-isoleucine-threonine (NIT), asparagine-glycine-serine (NGS), asparagine-serine-threonine (NST), and asparagine-threonine-serine (NTS). E. coli-derived polypeptides or fragments thereof produced in mammalian cells can be glycosylated. Glycosylation can occur at the N-linked glycosylation signal Asn-Xaa-Ser / Thr within the sequence of an E. coli-derived polypeptide or fragment thereof. "N-linked glycosylation" means that the carbohydrate moiety is attached to an asparagine residue within the polypeptide chain via GlcNAc. N-linked carbohydrates contain the common core structure Man1-6(Man1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-R, where R represents an asparagine residue in the resulting E. coli-derived polypeptide or fragment thereof.

[0066] In some embodiments, the glycosylation site in the E. coli derived polypeptide or fragment thereof is removed by mutation within the sequence of the E. coli derived polypeptide or fragment thereof. For example, in some embodiments, the Asn residue of the glycosylation motif (Asn-Xaa-Ser / Thr) may be mutated, preferably by substitution. In some embodiments, the substitution residue is selected from any one of Ser, Asp, Thr, and Gln.

[0067] In some embodiments, the Ser residue of the glycosylation motif may be mutated, preferably by substitution, hi some embodiments, the residue substitution is selected from any one of Asp, Thr, and Gln.

[0068] In some embodiments, the Thr residue of the glycosylation motif is preferably substituted. In some embodiments, the residue substitution is selected from any one of Ser, Asp, and Gln.

[0069] In some embodiments, glycosylation sites (e.g., Asn-Xaa-Ser / Thr) in a polypeptide or fragment thereof derived from E. coli are not removed or modified. In some embodiments, a compound that reduces or inhibits glycosylation may be added to the cell culture medium. In such embodiments, the polypeptide or protein contains at least one more non-glycosylated (i.e., aglycosylated) site, i.e., a completely unoccupied glycan site with no added carbohydrate moiety, or contains at least one less carbohydrate moiety at the same potential glycosylation site, compared to an otherwise identical polypeptide or protein produced by a cell under identical conditions except for the absence of the compound that inhibits glycosylation.Such compounds are known in the art and include tunicamycin, tunicamycin homologs, streptovirdin, mycospocidin, amphomycin, tsushimycin, antibiotic 24010, antibiotic MM19290, bacitracin, corynetoxin, showdomycin, duimycin, 1-deoxymannonojirimycin, deoxynojirimycin, N-methyl-1-deoxymannojirimycin, brefeldin A, glutamic acid ... Dose and mannose analogs, 2-deoxy-D-glucose, 2-deoxyglucose, D-(+)-mannose, D-(+) galactose, 2-deoxy-2-fluoro-D-glucose, 1,4-dideoxy-1,4-imino-D-mannitol (DIM), fluoroglucose, fluoromannose, UDP-2-deoxyglucose, GDP-2-deoxyglucose, hydroxymethylglutaryl-CoA reductase inhibitors, 25-hydroxycholesterol, The hydroxybenzoates may include, but are not limited to, hydroxycholesterol, swainsonine, cycloheximide, puromycin, actinomycin D, monensin, m-chlorocarbonyl-cyanide phenylhydrazone (CCCP), compactin, dolityl-phosphoryl-deoxyglucose, N-acetyl-D-glucosamine, hypoxanthine, thymidine, cholesterol, glucosamine, mannosamine, castanospermine, glutamine, bromoconduritol, conduritol epoxide and conduritol derivatives, glycosylmethyl-p-nitrophenyltriazene, β-hydroxynorvaline, threo-β-fluoroasparagine, D-(+)-gluconic acid δ-lactone, di(2-ethylhexyl) phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl ester of (diphenylmethyl)-phosphate, [2-(diphenylphosphinyloxy)ethyl]trimethylammonium iodide, iodoacetic acid, and / or fluoroacetic acid. Those of ordinary skill in the art will readily recognize or be able to determine, without undue experimentation, glycosylation inhibitors that may be used in accordance with the methods and compositions of the present invention.In such embodiments, glycosylation of a polypeptide or fragment thereof can be controlled without introducing amino acid mutations into the polypeptide or fragment thereof.

[0070] In some embodiments, the glycosylation level of a polypeptide or fragment thereof produced by a mammalian cell (e.g., the number of glycan sites occupied on the polypeptide or fragment thereof, the size and / or complexity of the glycoforms at those sites, etc.) is lower than the glycosylation level of a polypeptide or fragment thereof produced in an otherwise identical medium and under otherwise identical conditions that lacks such glycolysis-inhibiting compounds and / or mutations.

[0071] In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli does not contain a glycosylation site for an N-linked protein. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli does not contain at least one glycosylation site for an N-linked protein. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli does not contain at least one glycosylation site for an N-linked protein. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli does not contain any N-linked protein glycosylation sites. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli contains an N-linked protein glycosylation site. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli contains at most one N-linked protein glycosylation site. In some embodiments, the sequence of the polypeptide or fragment thereof derived from E. coli contains at most two N-linked protein glycosylation sites.

[0072] E. coli-derived polypeptides or fragments thereof expressed by different cell lines or in transgenic animals may have different glycan site occupancy, glycoforms, and / or glycosylation patterns compared to each other. In some embodiments, the present invention encompasses E. coli-derived polypeptides or fragments thereof produced in mammalian cells, regardless of the glycosylation, glycan occupancy, or glycoform pattern of the E. coli-derived polypeptides or fragments thereof.

[0073] In some embodiments, the polypeptide or fragment thereof derived from E. coli can be derived from an E. coli FimH polypeptide in which the amino acid residue at position 1 of the polypeptide is phenylalanine and not methionine, e.g., a polypeptide having the amino acid sequence of SEQ ID NO: 2. Preferably, the polypeptide derived from E. coli FimH comprises a phenylalanine at position 1 of the amino acid sequence of the polypeptide derived from E. coli. In another preferred embodiment, the polypeptide derived from E. coli FimH comprises the amino acid sequence of SEQ ID NO: 3, and preferably, the residue at position 1 of the amino acid sequence of the polypeptide derived from E. coli is a phenylalanine. In some embodiments, the polypeptide derived from E. coli or fragment thereof can comprise the amino acid sequence of SEQ ID NO: 4, which can be derived from an E. coli FimH polypeptide.

[0074] In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises an amino acid sequence having at least 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%, 99.9%, or 100% identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29. In some embodiments, the polypeptide or fragment thereof derived from E. coli may be derived from, for example, an E. coli FimG polypeptide having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the polypeptide or fragment thereof derived from E. coli may be derived from, for example, an E. coli FimC polypeptide having the amino acid sequence of SEQ ID NO: 10.

[0075] Polypeptides and fragments thereof derived from A. coli FimH In preferred embodiments, the polypeptide or fragment thereof is derived from E. coli FimH. In some embodiments, the polypeptide or fragment thereof comprises full-length E. coli FimH. Full-length FimH comprises two domains, an N-terminal lectin domain and a C-terminal pyrin domain, connected by a short linker. In some embodiments, full-length E. coli FimH comprises 279 amino acids, which includes the full-length mature E. coli FimH protein. In some embodiments, full-length E. coli FimH comprises 300 amino acids, which includes the full-length mature E. coli FimH protein and a 21-amino acid signal peptide sequence. The primary structure of the 300-amino acid wild-type FimH is highly conserved among E. coli strains.

[0076] An exemplary sequence of full-length E. coli FimH is SEQ ID NO: 1. The full-length FimH sequence includes the sequence of a lectin domain and the sequence of a pyrin domain. The lectin domain of FimH contains the carbohydrate recognition domain responsible for binding to mannosylated uroplakin 1a on the surface of urothelial cells. The pyrin domain is anchored to the core of the cilium via the donor strand of the subsequent FimG subunit through a process called donor strand complementation.

[0077] Starting from the N-terminus, the name of each domain of full-length FimH and exemplary amino acid sequences in brackets are shown, such as FimH lectin (SEQ ID NO: 2) and FimH pilin (SEQ ID NO: 3).

[0078] Other suitable polypeptides and fragments thereof derived from E. coli FimH include variants having various degrees of identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, for example, at least 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 99.9% identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29. In certain embodiments, the FimH variant protein (i) forms part of FimH-FimC, (ii) comprises at least one epitope of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29, and / or (iii) is capable of raising antibodies in vivo that immunologically cross-react with E. coli FimH.

[0079] In some embodiments, the composition comprises a polypeptide having at least n contiguous amino acids (where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more)) of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29. Preferably, the fragment comprises an epitope of the sequence. In some embodiments, the composition comprises a polypeptide having at least 50 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29.

[0080] In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO:1. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO: 2. In some embodiments, the composition comprises a polypeptide having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% identity to SEQ ID NO: 3. , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to SEQ ID NO:4. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO:4. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO:20. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO:23. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO:24. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO:26.In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO:28. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO:30.

[0081] Another example of a suitable polypeptide and fragment thereof derived from E. coli FimH described herein lacks the wild-type N-terminal signal sequence and is set forth as SEQ ID NO: 2, which corresponds to amino acid residues 22-300 of SEQ ID NO: 1. Another example of a FimH fragment includes the N-terminal signal sequence and the entire mature protein as set forth in SEQ ID NO: 1.

[0082] In some embodiments, a glycosylation site in a polypeptide or fragment thereof derived from E. coli is removed by a mutation in the sequence of the polypeptide or fragment thereof derived from E. coli. For example, in some embodiments, the Asn residue at position 7 (e.g., according to the numbering of SEQ ID NO: 2) of the mature E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the Asn residue at position 7 (e.g., according to the numbering of SEQ ID NO: 3) of the lectin domain of the E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the substituted residue is selected from any one of Ser, Asp, Thr, and Gln.

[0083] In some embodiments, the amino acid sequence at position 10 of the mature E. coli FimH polypeptide (e.g., In some embodiments, the Thr residue at position 7 (e.g., according to the numbering of SEQ ID NO: 2) of the lectin domain of the E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the Thr residue at position 7 (e.g., according to the numbering of SEQ ID NO: 3) of the lectin domain of the E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the substituted residue is selected from any one of Ser, Asp, and Gln.

[0084] In some embodiments, the Asn residue at position N235 (e.g., according to the numbering of SEQ ID NO: 2) of the mature E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the Asn residue at position N228 (e.g., according to the numbering of SEQ ID NO: 2) of the mature E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and Gln.

[0085] In some embodiments, the Asn residue at position 70 (e.g., according to the numbering of SEQ ID NO: 2) of the mature E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the Asn residue at position 70 (e.g., according to the numbering of SEQ ID NO: 3) of the lectin domain of the E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the residue substitution is selected from any one of Ser, Asp, Thr, and Gln.

[0086] In some embodiments, the Ser residue at position 72 (e.g., according to the numbering of SEQ ID NO: 2) of the mature E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the Ser residue at position 72 (e.g., according to the numbering of SEQ ID NO: 3) of the lectin domain of the E. coli FimH polypeptide may be mutated, preferably by substitution. In some embodiments, the substituted residue is selected from any one of Asp, Thr, and Gln.

[0087] The term "fragment," as used herein, in reference to a polypeptide, is defined as any distinct portion of a given polypeptide that is unique or characteristic of that polypeptide. This term, as used herein, also refers to any distinct portion of a given polypeptide that retains at least a portion of the activity of the full-length polypeptide. In certain embodiments, the portion of activity that is retained is at least 10% of the activity of the full-length polypeptide. In certain embodiments, the portion of activity that is retained is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the activity of the full-length polypeptide. In certain embodiments, the portion of activity that is retained is at least 95%, 96%, 97%, 98%, or 99% of the activity of the full-length polypeptide. In certain embodiments, the portion of activity that is retained is 100% or more of the activity of the full-length polypeptide. In some embodiments, a fragment comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more consecutive amino acids of the full-length polypeptide.

[0088] B. FimH and FimC complexes and their fragments In some embodiments, the polypeptide or fragment thereof derived from E. coli FimH is present as a complex with a polypeptide or fragment thereof derived from E. coli FimC. In preferred embodiments, the polypeptide or fragment thereof derived from E. coli FimH and the polypeptide or fragment thereof derived from E. coli FimC are present as a complex, preferably in a 1:1 ratio. Without being bound by theory or mechanism, it may be possible to purify the full-length FimH protein by stabilizing the full-length FimH in an active conformation by the periplasmic chaperone FimC. Thus, in some embodiments, the polypeptide or fragment thereof comprises full-length FimH and full-length FimC.

[0089] In some embodiments, the polypeptide or fragment thereof comprises a fragment of FimH and a fragment of FimC. In some embodiments, the polypeptide or fragment thereof comprises full-length FimH and a fragment of FimC. An exemplary sequence of E. coli FimC is set forth in SEQ ID NO: 10. In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises a complex-forming fragment of FimH.

[0090] A complexing fragment of FimH can be any portion of the FimH protein that retains the ability to form a complex with FimC or a fragment thereof. Suitable complexing fragments of FimH can be obtained or determined by standard assays known in the art, such as co-immunoprecipitation assays, cross-linking, or co-localization by fluorescent staining. SDS-PAGE or Western blots can also be used (e.g., by demonstrating the presence of a FimH fragment and FimC or a fragment thereof in a complex, as seen by gel electrophoresis). In certain embodiments, the complexing fragment of FimH (i) forms part of a FimH-FimC complex; (ii) comprises at least one epitope of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:29; and / or (iii) is capable of raising antibodies in vivo that immunologically cross-react with E. coli FimH.

[0091] In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises full-length FimH, wherein the FimH is not complexed with FimC. In further embodiments, the polypeptide or fragment thereof comprises a fragment of FimH, wherein the fragment is not complexed with FimC. In some embodiments, the polypeptide or fragment thereof derived from E. coli FimC comprises SEQ ID NO: 10. In some embodiments, the complexes can be expressed from the same plasmid, preferably under the control of separate promoters for each polypeptide or fragment thereof.

[0092] In some embodiments, a polypeptide or fragment thereof derived from E. coli FimH binds to a polypeptide or fragment thereof derived from E. coli FimC, which may be engineered into the structure of the polypeptide or fragment thereof derived from E. coli FimH. The portion of the FimC molecule that binds to FimH in the complex is referred to as the "donor strand," and the mechanism of formation of the native FimH structure with the strand of FimC that binds to FimH in the FimCH complex is known as "donor strand complementation."

[0093] In some embodiments, a polypeptide or fragment thereof derived from E. coli FimH can be expressed with a version of FimH complemented with an appropriate donor strand, where the amino acid sequence of FimC that interacts with FimH in the FimCH complex is itself engineered at the C-terminus of FimH to provide a native conformation without the need for the remainder of the FimC molecule. In some embodiments, a polypeptide or fragment thereof derived from E. coli FimH can be expressed in the form of a complex containing isolated domains, such as a lectin-binding domain and a pilin domain, which may be covalently or noncovalently linked together. For example, in some embodiments, the linking segment can comprise an amino acid sequence or other oligomeric structure, including a homopolymeric structure.

[0094] The methods and compositions of the present invention include the co-expression or combined formation of the complexes described herein of the polypeptides or fragments thereof derived from E. coli. It may include.

[0095] C. Lectin domains, pyrin domains, and their variants The conformation and ligand-binding properties of the lectin domain of FimH may be under allosteric control of the pyrin domain of FimH. In the resting state, the interaction of the two domains of full-length FimH results in a binding pocket with a low affinity for monomannose (e.g., K). d Binding to a mannoside ligand can induce a conformational change that results in a medium affinity state in which the lectin and pyrin domains remain in close proximity. However, upon shear stress, the lectin and pyrin domains separate and enter a high affinity state (e.g., K d <1.2 μM).

[0096] In the absence of the negative allosteric regulation exerted by the pyrin domain, the lectin domain of isolated FimH is able to bind to the lectin domain in a high-affinity state (e.g., K d <1.2 μM). Isolated recombinant lectin domains are locked into a high affinity state. However, it is possible to lock the adhesin into a low affinity conformation (e.g., K d Locking the lectin domain in a low affinity state (approximately 300 μM) induces the production of antibodies that inhibit adhesion. Therefore, there is interest in stabilizing the lectin domain in a low affinity state.

[0097] In some embodiments, the polypeptide or fragment thereof derived from E. coli comprises a lectin domain of E. coli FimH. Exemplary sequences of the lectin domain include any one of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, and SEQ ID NO:26. In some embodiments, the lectin domain of E. coli FimH comprises a cysteine ​​substitution. In preferred embodiments, the lectin domain of E. coli FimH comprises a cysteine ​​substitution within the first 50 amino acid residues of the lectin domain. In some embodiments, the lectin domain can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cysteine ​​substitutions. Preferably, the lectin domain comprises two cysteine ​​substitutions. See, e.g., pSB02158 and pSB02198.

[0098] Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants having various degrees of identity to SEQ ID NO:3, for example, at least 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 99.9% identity to the sequence set forth in SEQ ID NO:3. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO: 3. In some embodiments, the polypeptide derived from E. coli or fragment thereof comprises the pilin domain of E. coli FimH. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH pilin domain variants having various degrees of identity to SEQ ID NO:7, for example, at least 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 99.9% identity to the sequence set forth in SEQ ID NO:7. In some embodiments, the composition comprises a FimH pilin domain variant having at least 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 99.9% identity to the sequence set forth in SEQ ID NO:4. and polypeptides having 3%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants having various degrees of identity to SEQ ID NO:8, for example, at least 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 99.9% identity to the sequence set forth in SEQ ID NO:8. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO: 8. In some embodiments, the polypeptide derived from E. coli or fragment thereof comprises the pilin domain of E. coli FimH. Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH pilin domain variants having various degrees of identity to SEQ ID NO:24, for example, at least 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 99.9% identity to the sequence set forth in SEQ ID NO:24. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO:24.Other suitable polypeptides and fragments thereof derived from E. coli FimH include FimH lectin domain variants having varying degrees of identity to SEQ ID NO:26, for example, at least 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 99.9% identity to the sequence set forth in SEQ ID NO:26. In some embodiments, the composition comprises a polypeptide having at least 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 99.9% identity to SEQ ID NO:26.

[0099] In some embodiments, the composition comprises a polypeptide having at least n contiguous amino acids (where n is 7 or more (e.g., 8, 10, 12, 14, 16, 18, 20 or more)) of any one of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, and SEQ ID NO:26. Preferably, the fragment comprises an epitope of the sequence. In some embodiments, the composition comprises a polypeptide having at least 50 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of any one of SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, and SEQ ID NO:26.

[0100] The location and length of the lectin domain of E. coli FimH or its homologues or variants are shown in SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:24, and SEQ ID NO:10. The amino acid sequence of FimH can be predicted, for example, by aligning the amino acid sequence of FimH to SEQ ID NO: 1 and identifying sequences that align to residues 22 to 179 of SEQ ID NO: 1 based on pairwise alignments to any one of SEQ ID NO: 1 and SEQ ID NO: 26.

[0101] D. Wild-type N-terminal signal sequence In some embodiments, the native signal sequence at the N-terminus of full-length FimH is cleaved in the host cell, resulting in a mature FimH polypeptide. Thus, the FimH expressed by the host cell may lack the N-terminal signal sequence. In a preferred embodiment, the polypeptide or fragment thereof derived from E. coli may be encoded by a nucleotide sequence lacking the coding sequence for the native N-terminal signal sequence.

[0102] In some embodiments, the E. coli-derived polypeptide or fragment thereof comprises a FimH-FimC complex-forming fragment of FimH, an N-terminal signal sequence (e.g., residues 1-21 of SEQ ID NO: 1), or a combination thereof. The complex-forming fragment of FimH can be any portion of the FimH protein that retains the ability to form a complex with FimC.

[0103] In some embodiments, the polypeptide or fragment thereof derived from E. coli may lack 1 to 21 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid residues, or 1 to 21 residues, 1 to 20 residues, 1 to 15 residues, 1 to 10 residues, 2 to 20 residues, 2 to 15 residues, 2 to 10 residues, 5 to 20 residues, 5 to 15 residues, or 5 to 10 residues) at the N-terminus and / or C-terminus of a full-length FimH polypeptide, which may include the signal sequence, the lectin domain, and the pyrin domain.

[0104] II. Nucleic acids In one aspect, a nucleic acid encoding a polypeptide or fragment thereof derived from E. coli is disclosed. One or more nucleic acid constructs encoding the polypeptide or fragment thereof derived from E. coli can be used for genomic integration and subsequent expression of the polypeptide or fragment thereof derived from E. coli. For example, a single nucleic acid construct encoding the polypeptide or fragment thereof derived from E. coli can be introduced into a host cell. Alternatively, the coding sequence of the polypeptide or fragment thereof derived from E. coli can be carried on two or more nucleic acid constructs, which can then be introduced into a host cell simultaneously or sequentially.

[0105] For example, in one exemplary embodiment, a single nucleic acid construct encodes the lectin domain and the pyrin domain of E. coli FimH. In another exemplary embodiment, one nucleic acid construct encodes the lectin domain of E. coli FimH and a second nucleic acid construct encodes the pyrin domain. In some embodiments, genomic integration is achieved.

[0106] The nucleic acid construct may comprise genomic DNA or cDNA containing one or more introns. Some genes are more efficiently expressed when introns are present. In some embodiments, the nucleic acid sequence is suitable for expressing a foreign polypeptide in the mammalian cell.

[0107] In some embodiments, the nucleic acid encoding the polypeptide or fragment thereof is codon-optimized to increase expression levels in any particular cell. In some embodiments, the nucleic acid construct comprises a signal sequence encoding a peptide that directs secretion of the polypeptide or fragment thereof from E. coli. In some embodiments, the nucleic acid comprises the native signal sequence of the polypeptide derived from E. coli FimH. In some embodiments, where the polypeptide or fragment thereof derived from E. coli comprises an endogenous signal sequence, the nucleic acid sequence encoding the signal sequence may be codon-optimized to increase expression levels of the protein in a host cell.

[0108] In some embodiments, the signal sequence is any one of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 amino acids in length. In some embodiments, the signal sequence is 20 amino acids in length. In some embodiments, the signal sequence is 21 amino acids in length.

[0109] In some embodiments in which a polypeptide or fragment thereof includes a signal sequence, the endogenous signal sequence naturally associated with the polypeptide may be replaced with a signal sequence not associated with the wild-type polypeptide to enhance expression levels of the polypeptide or fragment in cultured cells. Thus, in some embodiments, the nucleic acid does not include the native signal sequence of a polypeptide or fragment thereof derived from E. coli. In some embodiments, the nucleic acid does not include the native signal sequence of a polypeptide derived from E. coli FimH. In some embodiments, a polypeptide or fragment thereof derived from E. coli can be expressed with a heterologous peptide, preferably a signal sequence or other peptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide or fragment thereof derived from E. coli (e.g., an IgK signal sequence), preferably a signal sequence or other peptide having a specific cleavage site at the N-terminus of the mature E. coli FimH protein. In a preferred embodiment, the specific cleavage site at the N-terminus of the mature protein E. coli FimH occurs immediately before the first phenylalanine residue of the mature E. coli FimH protein. The heterologous sequence selected preferably is one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell.

[0110] In preferred embodiments, the signal sequence is an IgK signal sequence. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 18. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 19. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 22. In preferred embodiments, the signal sequence is a mouse IgK signal sequence.

[0111] Suitable mammalian expression vectors for producing polypeptides or fragments thereof from E. coli are known in the art and some are commercially available, such as Invitrogen™'s pSecTag2 expression vector. An exemplary mouse Ig kappa signal peptide sequence comprises the sequence ETDTLLLWVLLLWVPGSTG (SEQ ID NO: 54). In some embodiments, the vector comprises Thermo Fisher's pBudCE4.1 mammalian expression vector. Further exemplary and suitable vectors include the pcDNA™ 3.1 mammalian expression vector (Thermo Fisher).

[0112] In some embodiments, the signal sequence does not include a hemagglutinin signal sequence. In some embodiments, the nucleic acid comprises a native signal sequence of a polypeptide or fragment thereof derived from E. coli. In some embodiments, the signal sequence is not an IgK signal sequence. In some embodiments, the signal sequence comprises a hemagglutinin signal sequence.

[0113] In one aspect, the present specification discloses a vector comprising a coding sequence of a polypeptide or a fragment thereof derived from E. coli. Exemplary vectors include plasmids that can replicate autonomously or in mammalian cells. Typical expression vectors contain suitable promoters, enhancers, and terminators that are useful for regulating the expression of the coding sequence in the expression construct. The vector may also contain a selection marker that provides a phenotypic trait for selection of transformed host cells (e.g., resistance to antibiotics such as ampicillin or neomycin).

[0114] Suitable promoters are known in the art. Exemplary promoters include, for example, CMV promoter, adenovirus, EF1a, GAPDH metallothionein promoter, SV-40 early promoter, SV-40 late promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, etc. The promoter may be constitutive or inducible. One or more vectors (e.g., one vector encoding all subunits or domains or fragments thereof, or multiple vectors encoding the subunits or domains or fragments thereof together) may be used.

[0115] Internal ribosome entry sites (IRES) and 2A peptide sequences may also be used. IRES and 2A peptides provide alternative approaches for simultaneous expression of multiple sequences. IRES is a nucleotide sequence that allows translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the process of synthesizing a larger protein. In eukaryotes, translation can usually only be initiated at the 5' end of an mRNA molecule. IRES elements allow the expression of multiple genes in a single transcript. IRES-based polycistronic vectors can express multiple proteins from a single transcript, reducing the chance of non-expressing clones escaping selection. 2A peptides allow the translation of multiple proteins in a single open reading frame into a polyprotein, which is subsequently cleaved into individual proteins via the ribosomal skipping mechanism. 2A peptides may further improve the balance of expression of multiple protein products. Exemplary IRES sequences include, for example, the EV71 IRES, EMCV IRES, and HCV IRES. Regarding genomic integration, integration can be site-specific or random. Site-specific recombination can be achieved by introducing a homologous sequence into the nucleic acid construct described herein. Such a homologous sequence substantially matches an endogenous sequence at a specific target site in the host genome. Alternatively, random integration can be used. In some cases, protein expression levels can vary depending on the integration site. Therefore, it may be desirable to select clones according to the expression level of the recombinant protein to identify clones that achieve the desired expression level.

[0116] Exemplary nucleic acid constructs are further illustrated in figures, such as any one of Figures 2A-2T. In one aspect, the nucleic acid sequence encodes an amino acid sequence having at least 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%, 99.9%, or 100% identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0117] III. Host cells In one aspect, the present invention relates to a mammalian host cell in which a sequence encoding an E. coli-derived polypeptide or fragment thereof is expressed. In one embodiment, the E. coli-derived polypeptide or fragment thereof is transiently expressed in the host cell. In another embodiment, the E. coli-derived polypeptide or fragment thereof is stably integrated into the genome of the host cell, and the host cell expresses the E. coli-derived polypeptide or fragment thereof when cultured under suitable conditions. In a preferred embodiment, the polynucleotide sequence is expressed with high efficiency and genome stability.

[0118] Suitable mammalian host cells are known in the art. Preferably, the host cell is suitable for producing proteins on an industrial scale. Exemplary mammalian host cells include Chinese hamster ovary (CHO) cells, COS cells (cell line derived from monkey kidney (African green monkey)), Vero cells, Hela cells, baby hamster kidney (BHK) cells, human embryonic kidney (HEK) cells, NS0 cells (mouse myeloma cell line), and C127 cells (non-tumorigenic mouse cell line), and any one of their derivatives. Further exemplary mammalian host cells include mouse Sertoli cells (TM4), buffalo rat liver cells (BRL 3A), mouse mammary carcinoma cells (MMT), rat hepatoma cells (HTC), mouse myeloma cells (NS0), mouse hybridoma cells (Sp2 / 0), mouse thymoma cells (EL4), Chinese hamster ovary (CHO) cells and CHO cell derivatives, mouse embryonic cells (NIH / 3T3, 3T3 Li), rat cardiomyocytes (H9c2), mouse myoblasts (C2C12), and mouse kidney cells (miMCD-3). Further examples of mammalian cell lines include NS0 / 1, Sp2 / 0, Hep G2, PER.C6, COS-7, TM4, CV1, VERO-76, MDCK, BRL 3A, W138, MMT 060562, TR1, MRC5, and FS4.

[0119] According to the present invention, any cells that are amenable to cell culture may be utilized. In some embodiments, the cells are mammalian cells. Non-limiting examples of mammalian cells that may be used according to the present invention include the BALB / c mouse myeloma line (NS0 / 1, ECACC number: 85110503), human retinoblastoma (PER.C6, CruCell, Leiden, The Netherlands), SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol., 36:59, 1977), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980), mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1 587), human cervical carcinoma cells (HeLa, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary carcinoma (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68, 1982), MRC 5 cells, FS4 cells, and human hepatoma line (Hep G2). In some preferred embodiments, the cells are CHO cells. In some preferred embodiments, the cells are GS cells.

[0120] Additionally, any number of commercially available and non-commercially available hybridoma cell lines may be utilized in accordance with the present invention. As used herein, the term "hybridoma" refers to a cell or progeny of cells resulting from the fusion of an immortalized cell with an antibody-producing cell. The term "hybridoma" refers to a descendant of a human myeloma cell line. The resulting hybridoma is an immortalized cell that produces an antibody. The individual cells used to create the hybridoma can be derived from any mammalian source, including, but not limited to, rats, pigs, rabbits, sheep, pigs, goats, and humans. In some embodiments, the hybridoma is a trioma cell line that results when the progeny of a heterohybrid myeloma fusion, which is the fusion product of a human cell and a mouse myeloma cell line, is subsequently fused with a plasma cell. In some embodiments, the hybridoma is any immortalized hybrid cell line that produces an antibody, such as a quadroma (see, e.g., Milstein et al., Nature, 537:3053, 1983). Those skilled in the art will understand that hybridoma cell lines may have different nutrient requirements and / or require different culture conditions for optimal growth and will be able to modify the conditions as needed.

[0121] In some embodiments, the cell comprises a first gene of interest, wherein the first gene of interest is integrated into a chromosome. In some embodiments, the first gene of interest comprises a reporter gene, a selection gene, a gene of interest (e.g., encoding a polypeptide or fragment thereof derived from E. coli), an auxiliary gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a difficult to express (DtE) protein.

[0122] In some embodiments, a first gene of interest is located between two different recombination target sites (RTSs) in site-specific integration (SSI) mammalian cells, and these two RTSs are integrated into the chromosome at the NL1 locus or the NL2 locus. For a description of the NL1 locus, NL2 locus, NL3 locus, NL4 locus, NL5 locus, and NL6 locus, see, e.g., U.S. Patent Application Publication No. 20200002727. In some embodiments, the first gene of interest is located at the NL1 locus. In some embodiments, the cell contains a second gene of interest, and the second gene of interest is integrated into the chromosome. In some embodiments, the second gene of interest comprises a reporter gene, a selection gene, a gene of therapeutic interest (e.g., a polypeptide or fragment thereof derived from E. coli), an auxiliary gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a DtE protein. In some embodiments, the second gene of interest is located between two RTSs. In some embodiments, the second gene of interest is located at the NL1 locus or the NL2 locus. In some embodiments, the first gene of interest is located at the NL1 locus and the second gene of interest is located at the NL2 locus. In some embodiments, the cell comprises a third gene of interest, wherein the third gene of interest is integrated into a chromosome. In some embodiments, the third gene of interest comprises a reporter gene, a selection gene, a gene of therapeutic interest (e.g., a polypeptide or fragment thereof derived from E. coli), an auxiliary gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a DtE protein. In some embodiments, the third gene of interest is located between two RTSs. In some embodiments, the third gene of interest is located at the NL1 locus or the NL2 locus. In some embodiments, the third gene of interest is located at a locus different from the NL1 locus and the NL2 locus. In some embodiments, the first gene of interest, the second gene of interest, and the third gene of interest are at three separate loci.In some embodiments, at least one of the first gene of interest, the second gene of interest, and the third gene of interest is at the NL1 locus, and at least one of the first gene of interest, the second gene of interest, and the third gene of interest is at the NL2 locus. In some embodiments, the cell comprises a site-specific recombinase gene. In some embodiments, the site-specific recombinase gene is integrated into a chromosome.

[0123] In some embodiments, the present disclosure provides a mammalian cell line comprising at least four different RTSs. The present invention provides a cell comprising: (a) at least two different RTSs integrated into a chromosome at the NL1 locus or the NL2 locus; (b) a first gene of interest located between the at least two RTSs of (a) and including a reporter gene, a gene encoding a DtE protein, an auxiliary gene, or a combination thereof; and (c) a second gene of interest integrated into a second chromosomal locus different from the locus of (a) and including a reporter gene, a gene encoding a DtE protein (e.g., a polypeptide derived from E. coli or a fragment thereof), an auxiliary gene, or a combination thereof. In some embodiments, the disclosure provides a mammalian cell comprising at least four different RTSs, the cell comprising: (a) at least two different RTSs integrated into a chromosome at the Fer1L4 locus; (b) at least two different RTSs integrated into a chromosome at the NL1 locus or the NL2 locus; (c) a first gene of interest integrated into a chromosome at the Fer1L4 locus, the first gene of interest comprising a reporter gene, a gene encoding a DtE protein, an auxiliary gene, or a combination thereof; and (d) a second gene of interest integrated into a chromosome at the NL1 locus or the NL2 locus of (b), the second gene of interest comprising a reporter gene, a gene encoding a DtE protein (e.g., a polypeptide derived from E. coli or a fragment thereof), an auxiliary gene, or a combination thereof.

[0124] In some embodiments, the disclosure provides a mammalian cell comprising at least six different RTSs, the cell comprising: (a) at least two different RTSs and a first gene of interest integrated into the chromosome at the Fer1L4 locus; (b) at least two different RTSs and a second gene of interest integrated into the chromosome at the NL1 locus; and (c) at least two different RTSs and a third gene of interest integrated into the chromosome at the NL2 locus.

[0125] As referred to herein, the terms "in operable combination," "in operable order," and "operably linked" refer to the linking of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and / or the synthesis of a desired protein molecule is produced. The term also refers to the linking of amino acid sequences in such a manner that a functional protein is produced. In some embodiments, a gene of interest is operably linked to a promoter, and the gene of interest is integrated into a chromosome of the host cell. In some embodiments, a gene of interest is operably linked to a heterologous promoter, and the gene of interest is integrated into a chromosome of the host cell. In some embodiments, an auxiliary gene is operably linked to a promoter, and the auxiliary gene is integrated into a chromosome of the host cell genome. In some embodiments, the auxiliary gene is operably linked to a heterologous promoter, and the auxiliary gene is integrated into a chromosome of the host cell genome. In some embodiments, a gene encoding a DtE protein is operably linked to a promoter, and the gene encoding a DtE protein is integrated into a chromosome of the host cell genome. In some embodiments, the gene encoding the DtE protein is operably linked to a heterologous promoter, and the gene encoding the DtE protein is integrated into a chromosome of the host cell genome. In some embodiments, the recombinase gene is operably linked to a promoter, and the recombinase gene is integrated into a chromosome of the host cell. In some embodiments, the recombinase gene is operably linked to a promoter, and the recombinase gene is not integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a heterologous promoter, and the recombinase gene is not integrated into a chromosome of the host cell genome. In some embodiments, the recombinase gene is operably linked to a heterologous promoter, and the recombinase gene is not integrated into a chromosome of the host cell genome.

[0126] As referred to herein, "chromosomally integrated" or "chromosomally integrated" means The term "integrated" means that the nucleic acid sequence is stably integrated into a chromosome of the host cell, e.g., a mammalian cell, i.e., the nucleic acid sequence is integrated into a chromosome of the genomic DNA (gDNA) of the host cell, e.g., a mammalian cell. In some embodiments, the chromosomally integrated nucleic acid sequence is stable. In some embodiments, the chromosomally integrated nucleic acid sequence is not located on a plasmid or vector. In some embodiments, the chromosomally integrated nucleic acid sequence is not excised. In some embodiments, the chromosomal integration is mediated by a clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) gene editing system (CRISPR / CAS).

[0127] In some embodiments, the host cells are suitable for growth in suspension culture. Suspension-competent host cells are typically monodispersed or grow in a loosely aggregated state without substantial aggregation. Suspension-competent host cells include cells that are suitable for suspension culture without adaptation or manipulation (e.g., hematopoietic cells, lymphoid cells) and cells that have been made suspension-competent by modification or adaptation of anchorage-dependent cells (e.g., epithelial cells, fibroblasts).

[0128] In some embodiments, the expression level or activity of the E. coli derived polypeptide or fragment thereof is increased by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 90-fold, or at least 100-fold compared to expression of the E. coli derived polypeptide or fragment thereof in a bacterial cell, such as an E. coli host cell.

[0129] The host cells described herein are suitable for large-scale cultivation. For example, cell cultures can be 10 L, 30 L, 50 L, 100 L, 150 L, 200 L, 300 L, 500 L, 1000 L, 2000 L, 3000 L, 4000 L, 5000 L, 10,000 L, or more. In some embodiments, the size of the cell culture is 10 L to 5000 L, 10 L to 10,000 L, 10 L to 20,000 L, 10 L to 50,000 L, 40 L to 50,000 L, 100 L to 50,000 L, 500 L to 50,000 L, 1000 L to 50,000 L, 2000 L to 50,000 L, 3000 L to 50,000 L, or more. L, 4000 L to 50,000 L, 4500 L to 50,000 L, 1000 L to 10,000 L, 1000 L to 20,000 L, 1000 L to 25,000 L, 1000 L to 30,000 L, 15 L to 2000 L, 40 L to 1000 L, 100 L to 500 L, 200 L to 400 L, or any integer between these values. Media components for cell culture are known in the art and can include, for example, buffer, amino acid content, vitamin content, salt content, mineral content, serum content, carbon source content, lipid content, nucleic acid content, hormone content, trace element content, ammonia content, cofactor content, indicator content, small molecule content, hydrolysate content, and enzyme modifier content.

[0130] As used herein, the terms "culture medium," "cell culture medium," and "culture medium" refer to a solution containing nutrients that nourish growing mammalian cells. Typically, such solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by cells for minimal growth and / or survival. Such solutions may also contain supplemental components, including, but not limited to, hormones and / or other growth factors, specific ions (e.g., sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), inorganic compounds (e.g., iron) present at high final concentrations, amino acids, lipids, and / or glucose or other energy sources, that enhance growth and / or survival beyond the minimal range. In some embodiments, the medium is advantageously adjusted to an optimal pH and salt concentration for cell survival and growth. In some embodiments, the medium is a feed medium that is added after the initiation of the cell culture.

[0131] In some embodiments, cells may be grown in one of a variety of chemically defined media in which the components of the medium are known and controlled. In some embodiments, cells may be grown in complex media in which not all components of the medium are known and / or controlled. Chemically defined growth media for mammalian cell culture have been extensively developed and published over the past several decades. All components of synthetic media are well characterized, and therefore synthetic media do not contain complex additives such as serum or hydrolysates. Early media formulations were developed to enable cell growth and maintenance of viability, with little or no consideration given to protein production. More recent media formulations have been developed with the express purpose of supporting cell cultures that produce highly productive recombinant proteins. Such media are preferred for use in the methods of the present invention. Such media generally contain large amounts of nutrients, particularly amino acids, to support cell growth and / or maintenance at high densities. If necessary, such media can be modified by one of skill in the art for use in the methods of the present invention. For example, one skilled in the art may reduce the amount of phenylalanine, tyrosine, tryptophan, and / or methionine in such media for use as basal or feed media in the methods disclosed herein.

[0132] Not all components of complex media are fully characterized; therefore, complex media may contain, among other things, simple and / or complex carbon sources, simple and / or complex nitrogen sources, and additives such as serum. In some embodiments, complex media suitable for the present invention contain additives such as hydrolysates in addition to the other components of synthetic media described herein. In some embodiments, synthetic media typically contain approximately 50 chemicals in water at known concentrations. Most of them also contain one or more well-characterized proteins such as insulin, IGF-1, transferrin, or BSA, while others do not require protein components and are therefore referred to as protein-free synthetic media. Typical chemical components of media fall into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and other categories that cannot be properly classified.

[0133] Cell culture media may optionally be supplemented with supplemental components. As used herein, the term "supplemental components" refers to components that enhance proliferation and / or survival beyond a minimal level, including, but not limited to, hormones and / or other growth factors, specific ions (e.g., sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, and / or glucose or other energy sources. In some embodiments, supplemental components may be added to the initial cell culture. In some embodiments, supplemental components may be added after the initiation of cell culture. Typically, trace elements refer to various inorganic salts present at submicromolar levels. For example, commonly included trace elements include zinc, selenium, copper, etc. In some embodiments, the initial cell culture medium may contain iron (ferrous iron or ferrous salts) as a trace element at micromolar concentrations. Among trace elements, manganese is also often included as a divalent cation (MnCl2 or MnSO4) in the nanomolar to micromolar concentration range. Many less common trace elements are usually added in nanomolar concentrations.

[0134] In some embodiments, the medium used in the methods of the present invention is suitable for cell cultures containing, for example, 1 x 10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL, or 5 x 10 8 Supports high cell densities such as cells / mL In some embodiments, the cell culture is a fed-batch culture of mammalian cells, preferably a fed-batch culture of CHO cells.

[0135] In some embodiments, the cell culture medium contains phenylalanine at a concentration of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains tyrosine at a concentration of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains tryptophan at a concentration of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains methionine at a concentration of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains leucine at a concentration of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains serine at a concentration of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains threonine at a concentration of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains glycine at a concentration of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains two of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains phenylalanine and tyrosine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains phenylalanine and tryptophan at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM.In some embodiments, the cell culture medium contains phenylalanine and methionine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains tyrosine and tryptophan at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains tyrosine and methionine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains tryptophan and methionine at concentrations of less than 2 mM, less than 1 mM, 0.1 to 2 mM, 0.1 to 1 mM, 0.5 to 1.5 mM, or 0.5 to 1 mM. In some embodiments, the cell culture medium contains three of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, 0.1 to 2 mM, 0.1 to 1 mM, 0.5 to 1.5 mM, or 0.5 to 1 mM. In some embodiments, the cell culture medium contains phenylalanine, tyrosine, and tryptophan at concentrations of less than 2 mM, less than 1 mM, 0.1 to 2 mM, 0.1 to 1 mM, 0.5 to 1.5 mM, or 0.5 to 1 mM. In some embodiments, the cell culture medium contains phenylalanine, tyrosine, and methionine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains phenylalanine, tryptophan, and methionine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains tyrosine, tryptophan, and methionine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In embodiments, the cell culture medium contains four of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, 0.1 to 2 mM, 0.1 to 1 mM, 0.5 to 1.5 mM, or 0.5 to 1 mM. In some embodiments, the cell culture medium contains phenylalanine, tyrosine, tryptophan, and methionine at concentrations of less than 2 mM, less than 1 mM, 0.1 to 2 mM, 0.1 to 1 mM, 0.5 to 1.5 mM, or 0.5 to 1 mM. In some embodiments, the cell culture medium contains five of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains six of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains seven of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium contains seven of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, 0.1-2 mM, 0.1-1 mM, 0.5-1.5 mM, or 0.5-1 mM. In some embodiments, the cell culture medium further comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at a concentration of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM.In some embodiments, the cell culture medium further comprises at least five of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at concentrations of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM. In some embodiments, the cell culture medium further comprises at least five of glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at concentrations of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM. In some embodiments, the cell culture medium further comprises at least one, two, three, four, five, six, seven, eight, or nine of valine, isoleucine, proline, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at a concentration of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM. In some embodiments, the cell culture medium further comprises at least five of valine, isoleucine, proline, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at a concentration of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM. In some embodiments, the cell culture medium further comprises valine, isoleucine, proline, lysine, arginine, histidine, aspartic acid, glutamic acid, and asparagine at concentrations of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably greater than 2 mM. In some embodiments, the cell culture medium comprises serine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM, or 20 mM, preferably greater than 10 mM. In some embodiments, the cell culture medium comprises valine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM, or 20 mM, preferably greater than 10 mM. In some embodiments, the cell culture medium comprises cysteine ​​at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM, or 20 mM, preferably greater than 10 mM.In some embodiments, the cell culture medium contains isoleucine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM, or 20 mM, preferably greater than 10 mM. In some embodiments, the cell culture medium is for use in the methods disclosed herein. In some embodiments, the cell culture medium is used as a basal medium in the methods disclosed herein. In some embodiments, the cell culture medium is used as a feed medium in the methods disclosed herein.

[0136] IV. Generation method In one aspect, the present invention includes a method for producing a polypeptide or fragment thereof derived from E. coli. The method includes expressing the polypeptide or fragment thereof by culturing mammalian cells under suitable conditions. The method may further include recovering the E. coli-derived polypeptide or fragment thereof from the culture. The method may further include purifying the E. coli-derived polypeptide or fragment thereof.

[0137] In some embodiments, the method produces the polypeptide or fragment thereof in a yield of between 0.1 g / L and 0.5 g / L. In some embodiments, cells may be grown in batch or fed-batch culture, in which the culture is terminated after sufficient polypeptide expression, and the expressed polypeptide is then recovered and optionally purified. In some embodiments, cells may be grown in perfusion culture, in which the culture is not terminated, but rather fresh nutrients and other components are periodically or continuously added to the culture, while the expressed polypeptide is periodically or continuously recovered.

[0138] In some embodiments, cells may be grown in small scale reaction vessels ranging in volume from a few milliliters to several liters, hi some embodiments, cells may be grown in large scale commercial bioreactors ranging in volume from at least about 1 liter to 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume between these values.

[0139] The temperature of the cell culture is selected primarily based on the temperature range in which the cell culture remains viable and produces high levels of polypeptides, the temperature range in which the production or accumulation of metabolic waste products is minimized, and / or any combination of these or other factors deemed important by the practitioner. As a non-limiting example, CHO cells grow well and produce high levels of proteins or polypeptides at approximately 37°C. Generally, most mammalian cells can grow well and / or produce high levels of proteins or polypeptides within a range of about 25°C to 42°C, although the methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells can grow well and / or produce high levels of proteins or polypeptides within a range of about 35°C to 40°C. In certain embodiments, the cell culture is grown at a temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C one or more times during the cell culture process.

[0140] As used herein, the terms "culture" and "cell culture" refer to a population of cells suspended in a medium under conditions suitable for the survival and / or growth of the cell population. As will be apparent to one of skill in the art, in some embodiments, these terms as used herein refer to a combination comprising a population of cells and the medium in which the population is suspended. In some embodiments, the cells of the cell culture comprise mammalian cells.

[0141] The present invention can be used with any cell culture method suitable for the desired process (e.g., production of a recombinant protein (e.g., an antibody)). As a non-limiting example, cells can be grown in batch or fed-batch culture, in which the culture is terminated after sufficient expression of the recombinant protein (e.g., an antibody) and the expressed protein (e.g., an antibody) is then harvested. Alternatively, as another non-limiting example, cells can be grown in batch refeed, in which the culture is not terminated but rather new nutrients and other components are periodically or continuously added to the culture while the expressed recombinant protein (e.g., an antibody) is periodically or continuously harvested. Other suitable methods (e.g., spin tube culture) are known in the art and can be used to practice the present invention.

[0142] In some embodiments, the cell culture suitable for the present invention is a fed-batch culture. As used herein, the term "fed-batch culture" refers to a cell culture method in which additional components are supplied to the culture one or more times after the start of the culture process. Such supplied components usually include nutrients for the cells that are depleted during the culture process. The fed-batch culture is usually stopped at some point, and the cells and / or components in the medium are recovered and optionally purified. In some embodiments, the fed-batch culture comprises a basal medium supplemented with a feed medium.

[0143] Cells may be grown in any convenient volume selected by the practitioner. For example, cells may be grown in small-scale reaction vessels ranging in volume from a few milliliters to several liters. Alternatively, cells may be grown in large-scale commercial bioreactors ranging in volume from at least about 1 liter to 10, 50, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,000, 15,000, 20,000, or 25,000 liters or more, or any volume between these values.

[0144] The temperature of the cell culture is selected primarily based on the temperature range in which the cell culture remains viable and in which high levels of a desired product (e.g., recombinant protein) are produced. Generally, most mammalian cells can grow well and produce a desired product (e.g., recombinant protein) within a range of about 25°C to 42°C, although the methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells can grow well and produce a desired product (e.g., recombinant protein or antibody) within a range of about 35°C to 40°C. In certain embodiments, cell cultures are grown at temperatures of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C at one or more times during the cell culture process. One of skill in the art would be able to select the appropriate temperature or temperatures at which to grow the cells depending on the particular needs of the cells and the particular production requirements of the practitioner. Cells may be grown for any length of time depending on the needs of the practitioner and the requirements of the cells themselves. In certain embodiments, the cells are grown at 37°C. In some embodiments, the cells are grown at 36.5°C.

[0145] In some embodiments, cells may be grown for a longer or shorter time during the initial growth stage (or growth stage), depending on the needs of the practitioner and the requirements of the cells themselves. In some embodiments, cells are grown for a time sufficient to achieve a predefined cell density. In some embodiments, cells are grown for a time sufficient to achieve a cell density that is a given percentage of the maximum cell density that the cells would ultimately reach if grown undisturbed. For example, the desired viable cell density may be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 ... The cells may be grown for a sufficient time to achieve 5, 80, 85, 90, 95, or 99 percent cell density. In some embodiments, the cells are grown to a point where the cell density does not increase by more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% per day of culture. In some embodiments, the cells are grown to a point where the cell density does not increase by more than 5% per day of culture.

[0146] In some embodiments, cells are grown for a predetermined period of time. For example, depending on the starting density of the cell culture, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, cells may be grown for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days, preferably 4-10 days. In some cases, cells may be grown for a month or more. Practitioners of the present invention will be able to select the duration of the initial growth phase depending on the protein production requirements and the needs of the cells themselves.

[0147] The cell culture may be agitated or shaken during the initial cultivation stage to increase oxygenation and nutrient distribution to the cells. In accordance with the present invention, one skilled in the art will appreciate that it may be beneficial to control or regulate certain internal conditions of the bioreactor during the initial growth stage, including, but not limited to, pH, temperature, oxygenation, etc.

[0148] At the end of the initial growth phase, a second set of culture conditions may be applied, and at least one of the culture conditions may be altered to induce a metabolic change in the culture. The metabolic change may be achieved, for example, by changing the temperature, pH, osmolality, or chemical inducer level of the cell culture. In a non-limiting embodiment, the culture conditions are altered by changing the temperature of the culture. However, as is known in the art, temperature alteration is not the only mechanism by which appropriate metabolic changes can be achieved. For example, such metabolic changes may also be achieved by altering other culture conditions, including, but not limited to, pH, osmolality, and sodium butyrate levels. The timing of altering the culture is determined by the practitioner of the present invention based on the protein production requirements or the needs of the cells themselves.

[0149] When the temperature of a culture is changed, the temperature change may be relatively gradual. For example, it may take several hours or days to complete the temperature change. Alternatively, the temperature change may be relatively abrupt. For example, the temperature change may be completed within a few hours. With appropriate production and control equipment, such as that standard in commercial large-scale production of polypeptides or proteins, the temperature change may even be completed within an hour.

[0150] In some embodiments, once the cell culture conditions have been altered as described above, the cell culture is maintained for the subsequent production stage under a second set of culture conditions that are conducive to cell culture survival and viability and suitable for expression of the desired polypeptide or protein at commercially reasonable levels.

[0151] As described above, the culture may be altered by changing one or more of several culture conditions, including, but not limited to, temperature, pH, osmolality, and sodium butyrate level. In some embodiments, the temperature of the culture is altered. According to this embodiment, in the subsequent production stage, the culture is maintained at a temperature or temperature range lower than the temperature or temperature range of the initial growth stage. As described above, multiple different temperature changes can be used to increase cell density or viability or to increase recombinant protein expression.

[0152] In some embodiments, the cells may be maintained in the subsequent production stage until a desired cell density or production titer is reached. In another embodiment of the present invention, the cells are grown for a predetermined period of time during the subsequent production stage. For example, depending on the density of the cell culture at the start of the subsequent growth stage, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, the cells may be grown for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or more. In some cases, the cells may be grown for a month or more. The practitioner of the present invention will be able to select the duration of the subsequent production stage depending on the production requirements of the polypeptide or protein and the needs of the cells themselves.

[0153] The cell culture may be agitated or shaken during the subsequent production stage to increase oxygenation and nutrient distribution to the cells. In accordance with the present invention, one skilled in the art will appreciate that it may be beneficial to control or regulate certain internal conditions of the bioreactor during the subsequent growth stage, including, but not limited to, pH, temperature, oxygenation, etc.

[0154] In some embodiments, the cells express a recombinant protein and the cell culture method of the invention comprises a growth step and a production step. In some embodiments, step (ii) of any of the methods disclosed herein is applied throughout the entire cell culture method. In some embodiments, step (ii) of any of the methods disclosed herein is applied as part of the cell culture method. In some embodiments, step (ii) is applied until a predetermined viable cell density is obtained.

[0155] In some embodiments, the cell culture method of the present invention comprises a growth phase and a production phase, and step (ii) is applied during the growth phase. In some embodiments, the cell culture method of the present invention comprises a growth phase and a production phase, and step (ii) is applied during part of the growth phase. In some embodiments, the cell culture method of the present invention comprises a growth phase and a production phase, and step (ii) is applied during both the growth phase and the production phase.

[0156] In step (ii) of any of the methods disclosed herein, the term "maintaining" can mean maintaining the amino acid or metabolite concentration below C1 or C2 throughout the entire culture process (until harvest) or throughout a portion of the culture process, such as during the growth phase, part of the growth phase, or until a predetermined cell density is achieved.

[0157] In some embodiments of any of the aforementioned methods, cell growth and / or productivity is increased compared to a control culture, which is identical except that it does not include step (ii).

[0158] In some embodiments of any of the above-described methods, the method is a method of improving cell growth. In some embodiments, the method is a method of improving cell growth at high cell densities in high density cell culture.

[0159] As used herein, high cell density refers to a cell density greater than 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7cells / mL, 1×10 8 cells / mL, or 5 x 10 8 cells / mL, preferably greater than 1 x 10 7 cells / mL, more preferably 5 x 10 7 Cell density refers to cells / mL.

[0160] In some embodiments, the methods of the present invention involve culturing cells at a cell density of 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL, or 5 x 10 8 In some embodiments, the methods of the present invention provide methods for improving cell growth in cell cultures where the maximum cell density is greater than 1 x 10 cells / mL. 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL, or 5 x 10 8 A method for improving cell growth in cell cultures at greater than 1000 cells / mL.

[0161] In some embodiments, cell proliferation is determined by viable cell density (VCD), maximum viable cell density, or integrated viable cell count (IVCC). In some embodiments, cell proliferation is determined by maximum viable cell density.

[0162] As used herein, the term "viable cell density" refers to the number of cells present in a given volume of culture medium. Viable cell density can be measured by any method known to those skilled in the art. Preferably, viable cell density is measured using an automated cell counter such as the Bioprofile Flex®. As used herein, the term "maximum cell density" refers to the maximum cell density achieved during cell culture. As used herein, the term "cell viability" refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. Those skilled in the art will understand that one of many methods for determining cell viability is encompassed by the present invention. For example, to determine cell viability, a dye (e.g., trypan blue) may be used, which does not pass through the membranes of living cells but can pass through the disrupted membranes of dead or dying cells.

[0163] As used herein, the term "integrated viable cell count (IVCC)" refers to the area under the viable cell density (VCD) curve. IVCC is calculated by the following formula: IVCC t+1 =IVCC t +(VCD t +VCD t+1 )*(Δt) / 2, where Δt is the time difference between time t and time t+1. t=0 can be considered negligible. t and VCDs t+1 is the viable cell density at time t and time t+1.

[0164] As used herein, the term "titer" refers to the total amount of recombinantly expressed protein produced by, for example, a cell culture in a given volume of medium. Titer is usually expressed in grams of protein per liter of medium.

[0165] In some embodiments, cell proliferation is increased by at least 5%, 10%, 15%, 20%, or 25% compared to a control culture. In some embodiments, cell proliferation is increased by at least 10% compared to a control culture. In some embodiments, cell proliferation is increased by at least 20% compared to a control culture.

[0166] In some embodiments, productivity is determined by titer and / or volumetric productivity. As used herein, the term "titer" refers to the total amount of recombinantly expressed protein produced by, for example, a cell culture in a given volume of medium. Titer is usually expressed in grams of protein per liter of medium.

[0167] In some embodiments, productivity is determined by titer. In some embodiments, productivity is increased by at least 5%, 10%, 15%, 20%, or 25% compared to a control culture. In some embodiments, productivity is increased by at least 10% compared to a control culture. In some embodiments, productivity is increased by at least 20% compared to a control culture.

[0168] In some embodiments, the maximum cell density of the cell culture is 1 x 10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×10 7 cells / mL, 1×10 8 cells / mL, or 5 x 10 8 In some embodiments, the maximum cell density of the cell culture is greater than 5 x 10 cells / mL. 6 In some embodiments, the highest concentration of cell culture Large cell density is 1 x 10 8 > cells / mL.

[0169] V. Purification In some embodiments, the method of producing an E. coli derived polypeptide or fragment thereof comprises isolating and / or purifying the E. coli derived polypeptide or fragment thereof. In some embodiments, the expressed E. coli derived polypeptide or fragment thereof is secreted into the culture medium, so that cells and other solids can be removed by centrifugation and / or filtration.

[0170] Polypeptides or fragments thereof derived from E. coli produced according to the methods described herein can be recovered and purified from host cells using any suitable method. Suitable methods for purifying polypeptides or fragments thereof include precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelation, and size exclusion, all of which are known in the art. A suitable purification scheme can include two or more of these or other suitable methods. In some embodiments, one or more of the E. coli derived polypeptides or fragments thereof can contain a "tag" that facilitates purification, such as an epitope tag, HIS tag, or Strep tag. Such tagged polypeptides can be conveniently purified, for example, from conditioned medium by chelation or affinity chromatography. Optionally, the tag sequence can be cleaved after purification.

[0171] In some embodiments, the polypeptide or fragment thereof derived from E. coli may contain a tag for affinity purification. Affinity purification tags are known in the art. Examples include His tags (which bind to metal ions), antibodies, maltose-binding protein (MBP) (which bind to amylose), glutathione S-transferase (GST) (which bind to glutathione), FLAG tags, and Strep tags (which bind to streptavidin or its derivatives).

[0172] In a preferred embodiment, the E. coli derived polypeptide or fragment thereof does not contain a purification tag. In some embodiments, the yield of the polypeptide or fragment thereof from E. coli is at least about 1 mg / L, at least about 2 mg / L, at least about 3 mg / L, at least about 4 mg / L, at least about 5 mg / L, at least about 6 mg / L, at least about 7 mg / L, at least about 8 mg / L, at least about 9 mg / L, at least about 10 mg / L, at least about 11 mg / L, at least about 12 mg / L, at least about 13 mg / L, at least about 14 mg / L, at least about 15 mg / L, at least about 16 mg / L, at least about 17 mg / L, at least about 18 mg / L, at least about 19 mg / L, at least about 20 mg / L, at least about 25 mg / L, at least about 30 mg / L, at least about 35 mg / L, at least about 40 mg / L, at least about 45 mg / L, at least about 50 mg / L, at least about 55 mg / L, at least about 60 mg / L, at least about 65 mg / L, at least about 70 mg / L, at least about 75 mg / L, at least about 80 mg / L, at least about 85 mg / L, at least about 90 mg / L, at least about 95 mg / L, or at least about 100 mg / L.

[0173] In some embodiments, the culture is at least about 10 liters in size, e.g., at least about 10 L, at least about 20 L, at least about 30 L, at least about 40 L, at least about 50 L, at least about 60 L, at least about 70 L, at least about 80 L, at least about 90 L, at least about 100 L, at least about 150 L, at least about 200 L, at least about 250 L, at least about 300 L, at least about 400 L, at least about 500 L, at least about 600 L, at least about 700 L, at least about 800 L, at least about 900 L, at least about 1000 L, at least about 2000 L, at least about 3000 L, at least about 4000 L, at least about 5000 L, at least about 6000 L, at least about 10,000 L, at least about 15,000 L, at least about 20,000 L, at least about 25,000 L, at least about 30,000 L, at least about 35,000 L, at least about 40,000 L L, at least about 45,000 L, at least about 50,000 L, at least about 55,000 L, at least about 60,000 L, at least about 65,000 L, at least about 70,000 L, at least about 75,000 L, at least about 80,000 L, at least about 85,000 L, at least about 90,000 L, at least about 95,000 L, at least about 100,000 L, etc.

[0174] VI. Compositions and Formulations In one aspect, the invention includes a composition comprising a polypeptide or fragment thereof derived from E. coli. In some embodiments, the composition generates an immune response comprising antibodies that can confer immunity against pathogenic species of E. coli.

[0175] In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli as the only antigen. In some embodiments, the composition does not comprise a conjugate.

[0176] In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and an additional antigen. In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and an additional E. coli antigen. In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and an additional E. coli antigen. In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and a glycoconjugate derived from E. coli.

[0177] In some embodiments, the polypeptide or fragment thereof is derived from E. coli FimH. In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli FimC.

[0178] In some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli FimH and a polypeptide or fragment thereof derived from E. coli FimC.

[0179] In one aspect, the present invention provides a method for the preparation of a polypeptide or fragment thereof from E. coli FimH, comprising combining a polypeptide or fragment thereof with a polypeptide of formula O1 (e.g., formula O1A, formula O1B, and formula O1C), formula O2, formula O3, formula O4 (e.g., formula O4:K52 and formula O4:K6), formula O5 (e.g., formula O5ab and formula O5ac (strain 180 / C3)), formula O6 (e.g., formula O6:K2;K13;K15 and formula O6:K54), formula O7, or a polypeptide of formula O8 (e.g., formula O8:K1A, formula O1B, and formula O1C), a polypeptide of formula O9 (e.g., formula O9:K2;K13;K15, and formula O9:K54), a polypeptide of formula O10 (e.g., formula O11; formula O12; formula O13; formula O14), a polypeptide of formula O15 (e.g., formula O16; formula O17; formula O18; formula O19), a polypeptide of formula O20 (e.g., formula O21; formula O22; formula O23), a polypeptide of formula O31 (e.g., formula O4:K52 and formula O4:K6), a polypeptide of formula O52 (e.g., formula O5ab and formula O5ac (strain 180 / C3)), a polypeptide of formula O63 (e.g., formula O6:K2;K13;K15, and formula O6:K54), a polypeptide of formula O74 (e.g., formula O75; formula O76; formula O77), a polypeptide of formula O78 (e.g., formula O79; formula O89; formula O91), a polypeptide of formula O116 (e.g., formula O117; formula O125), a polypeptide of formula O13 , Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O5 3, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula 62D1, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O 99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O 113, Formula O114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141 , formula O142, formula O143, formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O1 55, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O and a saccharide comprising a structure selected from any one of formulas O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187 (wherein n is an integer of 1 to 100).

[0180] In some embodiments, the composition comprises any one of the saccharides disclosed herein. In preferred embodiments, the composition comprises any one of the conjugates disclosed herein.

[0181] In some embodiments, the composition comprises at least one glycoconjugate of E. coli serotype O25, preferably serotype O25b. In one embodiment, the composition comprises at least one glycoconjugate of E. coli serotype O1, preferably serotype O1a. In one embodiment, the composition comprises at least one glycoconjugate of E. coli serotype O2. In one embodiment, the composition comprises at least one glycoconjugate of E. coli serotype O6.

[0182] In one embodiment, the composition comprises at least one glycoconjugate selected from E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises at least two glycoconjugates selected from E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises at least three glycoconjugates selected from E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In one embodiment, the composition comprises glycoconjugates of each of the E. coli serotypes O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6.

[0183] In a preferred embodiment, the glycoconjugate of any of the above compositions is, respectively, a CRM 197 It is conjugated to Thus, in some embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of at least one E. coli serotype. In preferred embodiments, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of two or more E. coli serotypes. For example, the composition can include O antigens of 12 different serotypes (12v) from two different E. coli serotypes (or "v"s for valences). In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of three different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of four different E. coli serotypes. In one embodiment, the composition comprises an O antigen of five different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of six different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of seven different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of eight different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of nine different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of ten different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of eleven different E. coli serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of twelve different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 13 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 14 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 15 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 16 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 17 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 18 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 19 different serotypes. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 20 different serotypes.

[0184] Preferably, the number of E. coli saccharides may range from 1 serotype (or "v" for valency) to 26 different serotypes (26v). In one embodiment, there is 1 serotype. In one embodiment, there are 2 different serotypes. In one embodiment, there are 3 different serotypes. In one embodiment, there are 4 different serotypes. In one embodiment, there are 5 different serotypes. In one embodiment, there are 6 different serotypes. In one embodiment, there are 7 different serotypes. In one embodiment, there are 8 different serotypes. In one embodiment, there are 9 different serotypes. In one embodiment, there are 10 different serotypes. In one embodiment, there are 11 different serotypes. In one embodiment, there are 12 different serotypes. In one embodiment, there are 13 different serotypes. In one embodiment, there are 14 different serotypes. In one embodiment, there are 15 different serotypes. In one embodiment, there are 16 different serotypes. In one embodiment, there are 17 different serotypes. In one embodiment, there are 18 different serotypes. In one embodiment, there are 19 different serotypes. In one embodiment, there are 20 different serotypes. In one embodiment, there are 21 different serotypes. In one embodiment, there are 22 different serotypes. In one embodiment, there are 23 different serotypes. In one embodiment, there are 24 different serotypes. In an embodiment, there are 25 different serotypes. In one embodiment, there are 26 different serotypes. The saccharide is conjugated to the carrier protein to form the glycoconjugate described herein.

[0185] In one aspect, the composition comprises a polypeptide or fragment thereof derived from E. coli and a glycoconjugate comprising an O antigen of at least one E. coli serogroup, wherein the O antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O antigens of two or more E. coli serotypes, wherein each O antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and and O antigens of two different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O antigens of three different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O antigens of four different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O antigens of five different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O antigens of six different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O antigens of seven different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of eight different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of nine different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of ten different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of eleven different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of twelve different serotypes, each O antigen conjugated to a carrier protein.In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 13 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 14 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 15 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 16 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 17 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and an O antigen of 18 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O antigens of 19 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O antigens of 20 different serotypes, each O antigen conjugated to a carrier protein.

[0186] In another aspect, the composition comprises O polysaccharides of at least one E. coli serotype. In a preferred embodiment, the composition comprises O polysaccharides of two or more E. coli serotypes. For example, the composition can comprise O polysaccharides of two different E. coli serotypes to 12 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides of three different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides of four different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides of five different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides of six different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides of seven different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides of eight different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides of nine different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides of ten different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides of eleven different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides of twelve different serotypes. In one embodiment, the composition comprises O polysaccharides of thirteen different serotypes. In one embodiment, the composition comprises O polysaccharides of fourteen different serotypes. In one embodiment, the composition comprises O polysaccharides of fifteen different serotypes. In one embodiment, the composition comprises O polysaccharides of sixteen different serotypes. In one embodiment, the composition comprises O polysaccharides of seventeen different serotypes. In one embodiment, the composition comprises O polysaccharides of eighteen different serotypes. In one embodiment, the composition comprises O polysaccharides of nineteen different serotypes. In one embodiment, the composition comprises O polysaccharides of 20 different serotypes.

[0187] In a preferred embodiment, the composition comprises O polysaccharides of at least one E. coli serotype, wherein the O polysaccharide is conjugated to a carrier protein. In a preferred embodiment, the composition comprises O polysaccharides of two or more E. coli serotypes, wherein each O polysaccharide is conjugated to a carrier protein. For example, the composition may comprise O polysaccharides of 12 different E. coli serotypes from two different E. coli serotypes, wherein each O polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of three different E. coli serotypes, wherein each O polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of four different E. coli serotypes, wherein each O polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of five different E. coli serotypes, wherein each O polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of six different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of seven different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of eight different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of nine different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of ten different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of eleven different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of twelve different serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of 13 different serotypes, each O polysaccharide being conjugated to a carrier protein, hi one embodiment, the composition comprises O polysaccharides of 14 different serotypes, each O polysaccharide being conjugated to a carrier protein.In one embodiment, the composition comprises O polysaccharides of 15 different serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of 16 different serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of 17 different serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of 18 different serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of 19 different serotypes, each O polysaccharide being conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides of 20 different serotypes, each O polysaccharide being conjugated to a carrier protein.

[0188] In a most preferred embodiment, the composition comprises an O polysaccharide of at least one E. coli serotype, the O polysaccharide being conjugated to a carrier protein, the O polysaccharide comprising an O antigen and and a core saccharide. In a preferred embodiment, the composition comprises O polysaccharides of two or more E. coli serotypes, each O polysaccharide conjugated to a carrier protein, and the O polysaccharide comprises an O antigen and a core saccharide. For example, the composition may comprise O polysaccharides of 12 different E. coli serotypes from two different E. coli serotypes, each O polysaccharide conjugated to a carrier protein, and the O polysaccharide comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of three different E. coli serotypes, each O polysaccharide conjugated to a carrier protein, and the O polysaccharide comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of four different E. coli serotypes, each O polysaccharide conjugated to a carrier protein, and the O polysaccharide comprises an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of five different E. coli serotypes, each O polysaccharide conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of six different E. coli serotypes, each O polysaccharide conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of seven different E. coli serotypes, each O polysaccharide conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of eight different E. coli serotypes, each O polysaccharide conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of nine different E. coli serotypes, each O polysaccharide conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of 10 different E. coli serotypes, each O polysaccharide conjugated to a carrier protein and comprising an O antigen and a core saccharide, hi one embodiment, the composition comprises O polysaccharides of 11 different E. coli serotypes, each O polysaccharide conjugated to a carrier protein and comprising an O antigen and a core saccharide.In one embodiment, the composition comprises O polysaccharides of 12 different serotypes, each O polysaccharide conjugated to a carrier protein, and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of 13 different serotypes, each O polysaccharide conjugated to a carrier protein, and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of 14 different serotypes, each O polysaccharide conjugated to a carrier protein, and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of 15 different serotypes, each O polysaccharide conjugated to a carrier protein, and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of 16 different serotypes, each O polysaccharide conjugated to a carrier protein, and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of 17 different serotypes, each O polysaccharide conjugated to a carrier protein, the O polysaccharide comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of 18 different serotypes, each O polysaccharide conjugated to a carrier protein, the O polysaccharide comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of 19 different serotypes, each O polysaccharide conjugated to a carrier protein, the O polysaccharide comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides of 20 different serotypes, each O polysaccharide conjugated to a carrier protein, the O polysaccharide comprising an O antigen and a core saccharide. In a preferred embodiment, the carrier protein is a CRM. 197 is.

[0189] In another preferred embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and a CRMP. 197 and an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises formula O25a, where n is at least 40, and a core saccharide. 197In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises the formula O25b, where n is at least 40, and a core saccharide. 197 wherein the O polysaccharide comprises a saccharide of formula O1a, where n is at least 40, and a core saccharide. In embodiments, the composition comprises a CRM 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, the O polysaccharide comprising the formula O2, where n is at least 40, and a core saccharide. 197 wherein the O polysaccharide comprises formula O6, where n is at least 40, and a core saccharide.

[0190] In another embodiment, the composition comprises a CRM 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, the O polysaccharide comprising formula O17, where n is at least 40, and a core saccharide. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises a CRM of formula O15, where n is at least 40, and a core saccharide. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises the formula O18A, where n is at least 40, and a core saccharide. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises a CRM of formula O75, where n is at least 40, and a core saccharide. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, the O polysaccharide comprising formula O4, where n is at least 40, and a core saccharide. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises a CRM of formula O16, where n is at least 40, and a core saccharide. 197In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, the O polysaccharide comprising formula O13, where n is at least 40, and a core saccharide. 197 wherein the O polysaccharide comprises formula O7, where n is at least 40, and a core saccharide.

[0191] In another embodiment, the composition comprises a CRM 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises formula O8, where n is at least 40, and a core saccharide. In another embodiment, the O polysaccharide comprises formula O8, where n is 1-20, preferably 2-5, and more preferably 3. Formula O8 is shown, for example, in Figure 10B. ... at least 1 197 and a core saccharide conjugated to the O polysaccharide, wherein the O polysaccharide comprises formula O9, where n is at least 40, and a core saccharide. In another embodiment, the O polysaccharide comprises formula O9, where n is 1-20, preferably 4-8, and more preferably 5. Formula O9 is shown, for example, in Figure 10B. In another embodiment, the O polysaccharide comprises formula O9a, where n is 1-20, preferably 4-8, and more preferably 5. Formula O9a is shown, for example, in Figure 10B.

[0192] In some embodiments, the O polysaccharide comprises a structure selected from any one of formula O20ab, formula O20ac, formula O52, formula O97, and formula O101, where n is 1 to 20, preferably 4 to 8, and more preferably 5. See, e.g., Figure 10B.

[0193] As mentioned above, the composition may comprise any combination of a polypeptide or fragment thereof derived from E. coli and a conjugated O polysaccharide (antigen). In one exemplary embodiment, the composition comprises a polysaccharide comprising formula O25b, a polysaccharide comprising formula O1A, a polysaccharide comprising formula O2, and a polysaccharide comprising formula O6. More specifically, such a composition comprises: (i) a CRM 197an O polysaccharide conjugated to a CRMP, the O polysaccharide comprising the formula O25b, where n is at least 40, and a core saccharide; and (ii) a CRMP 197 an O polysaccharide comprising the formula O1a, where n is at least 40, and a core saccharide conjugated to a CRM; 197 an O polysaccharide conjugated to a CRM, the O polysaccharide comprising the formula O2, wherein n is at least 40, and a core saccharide; and (iv) a CRM 197 an O polysaccharide conjugated to the O polysaccharide, the O polysaccharide comprising formula O6, where n is at least 40, and a core saccharide.

[0194] In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and at least one O polysaccharide derived from any E. coli serotype other than O25a. For example, in one embodiment, the composition does not include a saccharide comprising the formula O25a. Such a composition may include, for example, an O polysaccharide comprising the formula O25b, an O polysaccharide comprising the formula O1A, an O polysaccharide comprising the formula O2, and an O polysaccharide comprising the formula O6.

[0195] In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of two different E. coli serotypes, each O polysaccharide comprising a CRM 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of three different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of four different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of five different E. coli serotypes, each O polysaccharide comprising a CRMP. 197In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of six different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of seven different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of eight different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of nine different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of ten different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of eleven different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of 12 different serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of 13 different serotypes, each O polysaccharide comprising a CRMP. 197In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of 14 different serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of 15 different serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of 16 different serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of 17 different serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and a polypeptide conjugated to a polypeptide of one of the 18 species of E. coli. Each O polysaccharide contains a CRM 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of 19 different serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a polypeptide or fragment thereof derived from E. coli and O polysaccharides of 20 different serotypes, each O polysaccharide comprising a CRMP. 197 The O polysaccharide comprises an O antigen and a core saccharide.

[0196] In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises the formula O25b (where n is 15±2). In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises the formula O25b (where n is 17±2). In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises the formula O25b (where n is 55±2). In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises the formula O25b (where n is 51±2). In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In another embodiment, the saccharide further comprises an E. coli K12 core saccharide moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is a CRM 197 In one embodiment, the conjugate is prepared by single-end linkage conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

[0197] In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of binding to E. coli serotype O25B polysaccharide at a concentration of at least 0.2 pg / ml, 0.3 pg / ml, 0.35 pg / ml, 0.4 pg / ml, or 0.5 pg / ml, as determined by an ELISA assay. Therefore, the potential for responder expansion can be assessed by comparing OPA activity between serum before and after immunization with the immunogenic composition of the present invention and comparing the response to serotype O25B. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of killing E. coli serotype O25B, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, which are capable of killing E. coli serotype O25B, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic compositions of the invention increase the proportion of responders (i.e., individuals with sera having a titer of at least 1:8 as determined by in vitro OPA) to E. coli serotype O25B compared to the pre-immunization population. In one embodiment, the immunogenic compositions produce a titer of at least 1:8 against E. coli serotype O25B in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the invention produce a titer of at least 1:8 against E. coli serotype O25B in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the invention increase the proportion of responders (i.e., individuals with sera having a titer of at least 1:8 as determined by in vitro OPA) to E. coli serotype O25B compared to the pre-immunization population. In one embodiment, the immunogenic compositions of the invention significantly increase the OPA titer against E. coli serotype O25B in human subjects compared to the pre-immunization population.

[0198] In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O1a, where n is 39±2. In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O1a, where n is 13±2. In one aspect, the saccharide further comprises an E. coli R1 core saccharide moiety. In one aspect, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is a CRM 197 In one embodiment, the conjugate is prepared by single-end linkage conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

[0199] In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of binding to E. coli serotype O1A polysaccharide at a concentration of at least 0.2 pg / ml, 0.3 pg / ml, 0.35 pg / ml, 0.4 pg / ml, or 0.5 pg / ml, as determined by an ELISA assay. Therefore, the potential for responder expansion can be assessed by comparing OPA activity between serum samples taken before and after immunization with the immunogenic composition of the present invention and comparing the response to serotype O1A. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of killing E. coli serotype O1A, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, which are capable of killing E. coli serotype O1A, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic compositions of the invention increase the proportion of responders to E. coli serotype O1A (i.e., individuals with sera having a titer of at least 1:8 as determined by in vitro OPA) compared to a pre-immunization population. In one embodiment, the immunogenic compositions produce a titer of at least 1:8 against E. coli serotype O1A in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the invention produce a titer of at least 1:8 against E. coli serotype O1A in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the invention significantly increase the proportion of responders to E. coli serotype O1A (i.e., individuals with sera having a titer of at least 1:8 as determined by in vitro OPA) compared to a pre-immunization population. In one embodiment, the immunogenic compositions of the invention significantly increase OPA titers against E. coli serotype O1A in human subjects compared to a pre-immunization population.

[0200] In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises the formula O2, where n is 43±2. In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises the formula O2, where n is 47±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises the formula O2, where n is 47±2. wherein n is 17±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises the formula O2, wherein n is 18±2. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In another embodiment, the saccharide further comprises an E. coli R4 core saccharide moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is a CRM 197 In one embodiment, the conjugate is prepared by single-end linkage conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

[0201] In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of binding to E. coli serotype O2 polysaccharide at a concentration of at least 0.2 pg / ml, 0.3 pg / ml, 0.35 pg / ml, 0.4 pg / ml, or 0.5 pg / ml, as determined by an ELISA assay. Therefore, the potential for responder expansion can be assessed by comparing OPA activity between serum before and after immunization with the immunogenic composition of the present invention and comparing the response to serotype O2. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of killing E. coli serotype O2, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, which are capable of killing E. coli serotype O2, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic compositions of the invention increase the proportion of responders to E. coli serotype O2 (i.e., individuals with sera having a titer of at least 1:8 as determined by in vitro OPA) compared to a pre-immunization population. In one embodiment, the immunogenic compositions produce a titer of at least 1:8 against E. coli serotype O2 in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the invention produce a titer of at least 1:8 against E. coli serotype O2 in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the invention significantly increase the proportion of responders to E. coli serotype O2 (i.e., individuals with sera having a titer of at least 1:8 as determined by in vitro OPA) compared to a pre-immunization population. In one embodiment, the immunogenic compositions of the invention significantly increase OPA titers against E. coli serotype O2 in human subjects compared to a pre-immunization population.

[0202] In one aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O6, where n is 42±2. In another aspect, the invention relates to a composition comprising a polypeptide or fragment thereof derived from E. coli and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O6, where n is 50±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O6, where n is 17±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O6, where n is 18±2. In one aspect, the saccharide further comprises an E. coli R1 core saccharide moiety. In one aspect, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is a CRM 197 In one embodiment, the conjugate is prepared by single-end bond conjugation. The conjugates are prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

[0203] In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of binding to E. coli serotype O6 polysaccharide at a concentration of at least 0.2 pg / ml, 0.3 pg / ml, 0.35 pg / ml, 0.4 pg / ml, or 0.5 pg / ml, as determined by an ELISA assay. Therefore, the potential for responder expansion can be assessed by comparing OPA activity between serum before and after immunization with the immunogenic composition of the present invention and comparing the response to serotype O6. In one embodiment, the immunogenic composition elicits IgG antibodies in humans, which are capable of killing E. coli serotype O6, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic composition elicits functional antibodies in humans, which are capable of killing E. coli serotype O6, as determined by an in vitro opsonophagocytosis assay. In one embodiment, the immunogenic compositions of the invention increase the proportion of responders to E. coli serotype O6 (i.e., individuals with sera having a titer of at least 1:8 as determined by in vitro OPA) compared to a pre-immunization population. In one embodiment, the immunogenic compositions produce a titer of at least 1:8 against E. coli serotype O6 in at least 50% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the invention produce a titer of at least 1:8 against E. coli serotype O6 in at least 60%, 70%, 80%, or at least 90% of subjects as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic compositions of the invention significantly increase the proportion of responders to E. coli serotype O6 (i.e., individuals with sera having a titer of at least 1:8 as determined by in vitro OPA) compared to a pre-immunization population. In one embodiment, the immunogenic compositions of the invention significantly increase OPA titers against E. coli serotype O6 in human subjects compared to a pre-immunization population.

[0204] In one aspect, the composition comprises a conjugate comprising a polypeptide or fragment thereof derived from E. coli and a saccharide covalently attached to a carrier protein, the saccharide being selected from the group consisting of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180 / C3)), Formula O6 (e.g., Formula O6:K2;K13;K15, and Formula O6:K8;K9;K10;K11), and Formula O6:K12;K13;K14. 6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula O62D1, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69 , Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (Strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, Formula O117 , formula O118, formula O119, formula O120, formula O121, formula O123, formula O124, formula O125, formula O126, formula O127, formula O128, formula O12 9, Formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O1 40, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145, Formula O146, Formula O147, Formula O148, Formula O149, Formula O150, Formula O Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187 (wherein n is an integer of 1 to 100). In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In one embodiment, the saccharide further comprises an E. coli R2 core saccharide moiety. In one embodiment, the saccharide further comprises an E. coli R3 core saccharide moiety. In another embodiment, the saccharide further comprises an E. coli R4 core saccharide moiety. In one embodiment, the saccharide further comprises an E. coli K12 core saccharide moiety. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is a CRM 197In one embodiment, the conjugate is prepared by single-end linkage conjugation. In one embodiment, the conjugate is prepared by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent. In one embodiment, the composition further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 additional conjugates, up to a maximum of 30 additional conjugates, each conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises a structure selected from any one of the formulas above.

[0205] A. Sugars In one embodiment, the saccharides are produced by expression (not necessarily overexpression) of different Wzz proteins (eg, WzzB) to control the size of the saccharide.

[0206] As used herein, the term "saccharide" refers to a monosaccharide moiety or unit, and to combinations of two or more monosaccharide moieties or units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. Sugars may be linear or branched.

[0207] In one embodiment the saccharides are produced in recombinant Gram-negative bacteria. In one embodiment the saccharides are produced in recombinant E. coli cells. In one embodiment the saccharides are produced in recombinant Salmonella cells. Exemplary bacteria include E.coli O25K5H1, E.coli BD559, E.coli GAR2831, E.coli GAR865, E.coli GAR868, E.coli GAR869, E.coli GAR872, E.coli GAR878, E.coli GAR896, E.coli GAR1902, E.coli O25a ETC NR-5, E.coli O157:H7:K-, Salmonella enterica serotype Typhimurium strain LT2, E.coli GAR2401, Salmonella enterica serotype Enteritidis CVD 1943, Salmonella enterica serotype Typhimurium CVD 1925, Salmonella enterica serotype Paratyphi A CVD 1902, and and Shigella flexneri CVD 1208S. In one embodiment, the bacterium is not E. coli GAR2401. Genetic approaches for the production of these saccharides allow for the efficient production of O polysaccharides and O antigen molecules as vaccine components.

[0208] As used herein, the term "wzz protein" refers to, for example, wzzB, wzz, wzz SF , wzz ST , fepE, wzz fepE" refers to polypeptides that determine the chain length, such as wzz1, wzz2, and wzz3. Exemplary wzz gene sequences have GenBank accession numbers AF011910 (E4991 / 76), AF011911 (F186), AF011912 (M70 / 1-1), AF011913 (79 / 311), AF011914 (Bi7509-41), AF011915 (C664-1992), AF011916 (C258-94), AF011917 (C722-89), and AF011919 (EDL933). The GenBank accession numbers for the G7 and Bi316-41 wzz gene sequences are U39305 and U39306, respectively. Additional GenBank accession numbers for exemplary wzz gene sequences are NP_459581 (Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 FepE), AIG66859 (E. coli O157:H7 strain EDL933 FepE), NP_461024 (Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 WzzB), NP_416531 (E. coli K-12 substrain MG1655 WzzB), and NP_415119 (E. coli K-12 substrain MG1655 FepE). In preferred embodiments, the wzz family proteins are wzzB, wzz, wzz SF , wzz ST , fepE, wzz fepE , wzz1, and wzz2, most preferably wzzB, and more preferably fepE.

[0209] Exemplary wzzB sequences include those shown in SEQ ID NOs: 30 to 34. Exemplary FepE sequences include those shown in SEQ ID NOs: 35 to 39. In some embodiments, to generate high molecular weight saccharides, such as lipopolysaccharides comprising medium or long chain O-antigen chains, modified saccharides (modified relative to the corresponding wild-type saccharide) can be produced in Gram-negative bacteria by expressing (not necessarily overexpressing) a wzz family protein from Gram-negative bacteria (e.g., fepE) and / or switching off (i.e., suppressing, deleting, removing) a second wzz gene (e.g., wzzB). For example, modified saccharides can be produced by expressing (not necessarily overexpressing) wzz2 and switching off wzz1. Or, alternatively, modified saccharides can be produced by expressing (not necessarily overexpressing) wzzfepE and switching off wzzB. In another embodiment, modified saccharides can be produced by expressing (not necessarily overexpressing) wzzB but switching off wzzfepE. In another embodiment, modified saccharides can be produced by expressing fepE. Preferably, the wzz family protein is derived from a strain that is heterologous to the host cell.

[0210] In some embodiments, the saccharides are produced by expressing a wzz-family protein having an amino acid sequence that is at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. In one embodiment, the wzz-family protein comprises a sequence selected from any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. Preferably, the wzz-family protein comprises a sequence selected from any one of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39. 33, SEQ ID NO: 34. In some embodiments, the saccharide is produced by expressing a protein having an amino acid sequence that is at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the fepE protein.

[0211] In one aspect, the invention relates to saccharides produced by expressing a wzz family protein, preferably fepE, in Gram-negative bacteria to produce high molecular weight saccharides comprising medium or long chain O antigen chains, the saccharides having at least 1, 2, 3, 4, or 5 more repeat units than the corresponding wild-type O polysaccharide. In one aspect, the invention relates to saccharides produced by Gram-negative bacteria in culture that express (but do not necessarily overexpress) a wzz family protein (e.g., wzzB) from Gram-negative bacteria to produce high molecular weight saccharides comprising medium or long chain O antigen chains, the saccharides having at least 1, 2, 3, 4, or 5 more repeat units than the corresponding wild-type O antigen. For further examples of saccharides having an increased number of repeat units compared to the corresponding wild-type saccharide, see the discussion of O polysaccharides and O antigens below. The desired chain length is one that provides improved or maximal immunogenicity in the context of a given vaccine construct.

[0212] In another embodiment, the saccharide comprises any one of the formulas selected from Table 1, wherein the number of repeat units in the saccharide, n, is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 2, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 100 or more repeat units larger. Preferably, the saccharide contains at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 more repeating units than the corresponding wild-type O polysaccharide. See, e.g., Table 24. Methods for determining the length of saccharides are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography, as described in Example 13.

[0213] In preferred embodiments, the present invention relates to saccharides produced in recombinant E. coli host cells for the production of high molecular weight saccharides such as lipopolysaccharides comprising medium or long chain O-antigen chains, wherein the gene for the endogenous wzz O-antigen length regulator (e.g., wzzB) has been deleted and replaced with a (second) wzz gene of a Gram-negative bacterium (e.g., Salmonella fepE) that is heterologous to the recombinant E. coli host cell. In some embodiments, the recombinant E. coli host cell comprises a Salmonella, preferably Salmonella enterica, wzz gene.

[0214] In one embodiment, the host cell comprises a heterologous gene for a wzz family protein as a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene for a wzz family protein as a gene integrated into the chromosomal DNA of the host cell. Methods for stably expressing plasmid vectors in E. coli host cells and for integrating heterologous genes into the chromosome of E. coli host cells are known in the art. In one embodiment, the host cell comprises the heterologous O antigen gene as a stably maintained plasmid vector. In another embodiment, the host cell comprises the heterologous O antigen gene as a gene integrated into the chromosomal DNA of the host cell. Methods for stably expressing plasmid vectors in E. coli and Salmonella host cells are known in the art. Methods for integrating heterologous genes into the chromosomes of E. coli and Salmonella host cells are known in the art.

[0215] In one aspect, the recombinant host cells are cultured in a medium containing a carbon source. Carbon sources for culturing E. coli are known in the art. Exemplary carbon sources include sugar alcohols, polyols, aldol sugars, or keto sugars, including, but not limited to, arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, rhamnose, raffinose, sorbitol, sorbose, sucrose, trehalose, pyruvate, succinate, and methylamine. In a preferred embodiment, the medium contains glucose. In some embodiments, the medium contains a polyol or aldol sugar, such as mannitol, inositol, sorbose, glycerol, sorbitol, lactose, and arabinose, as a carbon source. All of the carbon sources may be added to the medium before the start of the culture, or may be added stepwise or continuously during the culture.

[0216] An exemplary culture medium for recombinant host cells comprises elements selected from any one of KH2PO4, K2HPO4, (NH4)2SO4, sodium citrate, Na2SO4, aspartic acid, glucose, MgSO4, FeSO4-7H2O, Na2MoO4-2H2O, H3BO3, CoCl2-6H2O, CuCl2-2H2O, MnCl2-4H2O, ZnCl2, and CaCl2-2H2O. Preferably, the medium comprises KH2PO4, K2HPO4, (NH4)2SO4, sodium citrate, Na2SO4, aspartic acid, glucose, MgSO4, FeSO4-7H2O, Na2MoO4-2H2O, H3BO3, CoCl2-6H2O, CuCl2-2H2O, MnCl2-4H2O, ZnCl2, and CaCl2-2H2O.

[0217] As used herein, a medium may be solid or liquid, synthetic (i.e., artificial) or natural, and may contain sufficient nutrients for the cultivation of recombinant host cells. Preferably, the medium is a liquid medium.

[0218] In some embodiments, the medium may further comprise suitable inorganic salts. In some embodiments, the medium may further comprise micronutrients. In some embodiments, the medium may further comprise growth factors. In some embodiments, the medium may further comprise an additional carbon source. In some embodiments, the medium may further comprise suitable inorganic salts, micronutrients, growth factors, and supplemental carbon sources. Suitable inorganic salts, micronutrients, growth factors, and supplemental carbon sources for culturing E. coli are known in the art.

[0219] In some embodiments, the medium may optionally contain additional components such as peptone, NZ amine, soybean enzymatic hydrolysate, additional yeast extract, malt extract, supplemental carbon source, and various vitamins. In some embodiments, the medium does not contain additional components such as peptone, NZ amine, soybean enzymatic hydrolysate, additional yeast extract, malt extract, supplemental carbon source, and various vitamins.

[0220] Examples of suitable supplemental carbon sources include other carbohydrates such as glucose, fructose, mannitol, starch or starch hydrolysates, cellulose hydrolysates and molasses, acetate, propionate, lactate, formate, malate, citrate, and fumarate. and alcohols such as glycerol, inositol, mannitol and sorbitol.

[0221] In some embodiments, the medium further comprises a nitrogen source. Suitable nitrogen sources for culturing E. coli are known in the art. Examples of suitable nitrogen sources include, but are not limited to, ammonia, including ammonia gas and aqueous ammonia, ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, and ammonium acetate, urea, nitrates, or nitrites, and other nitrogen-containing substances, such as amino acids in pure or crude preparations, meat extract, peptone, fish meal, fish hydrolysates, corn steep liquor, casein hydrolysates, soybean meal hydrolysates, yeast extract, dry yeast, ethanol yeast distillate, soybean flour, cottonseed meal, and the like.

[0222] In some embodiments, the medium contains inorganic salts. Examples of suitable inorganic salts include, but are not limited to, salts of potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper, molybdenum, tungsten, and other trace elements, and phosphate.

[0223] In some embodiments, the medium contains suitable growth factors. Examples of suitable micronutrients, growth factors, etc. include coenzyme A, pantothenic acid, pyridoxine-HCl, biotin, thiamine, riboflavin, flavin mononucleotide, flavin adenine dinucleotide, DL-6,8-thioctic acid, folic acid, vitamin B, as pure or partially purified chemical compounds or as present in natural substances.12 These include, but are not limited to, other vitamins, amino acids such as cysteine ​​and hydroxyproline, bases such as adenine, uracil, guanine, thymine, and cytosine, sodium thiosulfate, p- or r-aminobenzoic acid, niacinamide, nitriloacetic acid, etc. These amounts can be determined empirically by one skilled in the art according to methods and techniques known in the art.

[0224] In another embodiment, the modified sugars described herein (relative to the corresponding wild-type sugars) are synthetically produced, e.g., in vitro. Synthetic production or synthesis of sugars can facilitate avoiding cost- and time-intensive production processes. In one embodiment, sugars are synthetically synthesized from suitably protected monosaccharide intermediates, e.g., by using a sequential glycosylation strategy or a combination of sequential glycosylation and a [3+2] block synthesis strategy. For example, thioglycosides and glycosyl trichloroacetimidate derivatives can be used as glycosyl donors in glycosylation. In one embodiment, sugars synthetically synthesized in vitro have the same structure as sugars produced by recombinant techniques, such as by engineering the wzz family proteins described above.

[0225] The saccharides produced (by recombinant or synthetic means) can be, for example, those of the following E. coli serotypes: O1 (e.g., O1A, O1B, and O1C), O2, O3, O4 (e.g., O4:K52 and O4:K6), O5 (e.g., O5ab and O5ac (strain 180 / C3)), O6 (e.g., O6:K2;K13;K15 and O6:K54), O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18 (e.g., O18A, O18ac, O18A1, O18B1, O18C1, O18D1, O18E1, O18F1, O18F2, O18F3, O18F4, O18F5, O18F6, O18F7, O18F8, O18F9, O18F1, O18F9, O18F1, O18F1, O18F2, O18F3, O18F4, O18F5, O18F6, O18F7, O18F8, O18F9 ...1, O18F1, O18F1, O18F2, O18F O18B, and O18B1), O19, O20, O21, O22, O23 (e.g., O23A), O24, O25 (e.g., O25a and O25b), O26, O27, O28, O29, O30, O32, O33, O34, O35, O36, O37, O38, O39, O40, O41, O42, O43, O44, O45 (e.g., O45 and O45rel), O46, O48, O49, O50, O51, O52, O53, O54, O55, O56, O57, O58, O59, O60, O61, O62, 62D1, O63, O64, O65, O66, O68, O69, O70, O71, O73 (e.g., O73 (strain 73-1)), O74, O75, O76, O77, O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O9 8, O99, O100, O101, O102, O103, O104, O105, O106, O107, O108, O109, O110, O111, O112, O113, O11 4, O115, O116, O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O13 1, O132, O133, O134, O135, O136, O137, O138, O139, O140, O141, O142, O143, O144, O145, O146, O1 47, O148, O149, O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, O1 and a construct derived from any E. coli serotype, including any one of O163, O164, O165, O166, O167, O168, O169, O170, O171, O172, O173, O174, O175, O176, O177, O178, O179, O180, O181, O182, O183, O184, O185, O186, and O187.

[0226] Individual polysaccharides are typically purified (concentrated relative to the amount of polysaccharide-protein conjugate) by methods known in the art, such as dialysis, concentration, diafiltration, tangential flow filtration, precipitation, elution, centrifugation, precipitation, ultrafiltration, depth filtration, and / or column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE, and hydrophobic interaction chromatography). Preferably, the polysaccharides are purified by methods including tangential flow filtration.

[0227] The purified polysaccharide may be activated (e.g., chemically activated) to allow it to react (e.g., directly to a carrier protein or via a linker such as an eTEC spacer), as further described herein, before being incorporated into the glycoconjugates of the invention.

[0228] In a preferred embodiment, the saccharides of the invention are derived from an E. coli serotype, and the serotype is O25a. In another preferred embodiment, the serotype is O25b. In another preferred embodiment, the serotype is O1A. In another preferred embodiment, the serotype is O2. In another preferred embodiment, the serotype is O6. In another preferred embodiment, the serotype is O17. In another preferred embodiment, the serotype is O15. In another preferred embodiment, the serotype is O18A. In another preferred embodiment, the serotype is O75. In another preferred embodiment, the serotype is O4. In another preferred embodiment, the serotype is O16. In another preferred embodiment, the serotype is O13. In another preferred embodiment, the serotype is O7. In another preferred embodiment, the serotype is O8. In another preferred embodiment, the serotype is O9.

[0229] As used herein, reference to any of the above serotypes refers to a serotype that includes a repeating unit structure (O unit, described below) that is known in the art and is unique to the corresponding serotype. For example, the term "O25a" serotype (also known in the art as serotype "O25") refers to a serotype that includes formula O25, as shown in Table 1. As another example, the term "O25b" serotype refers to a serotype that includes formula O25b, as shown in Table 1.

[0230] As used herein, serotypes are referred to collectively herein unless otherwise specified, e.g., the term formula "O18" collectively means formula O18A, formula O18ac, formula O18A1, formula O18B, and formula O18B1.

[0231] As used herein, the term "O1" collectively refers to a species of formula that includes the generic designation "O1" in its formula name according to Table 1, such as any one of formula O1A, formula O1A1, formula O1B, and formula O1C, respectively, as shown in Table 1. Thus, "O1 serotype" collectively refers to a serotype that includes any one of formula O1A, formula O1A1, formula O1B, and formula O1C.

[0232] As used herein, the term "O6" collectively refers to species of formulae that include the generic designation "O6" in their formula names according to Table 1, such as any one of the formulae O6:K2;K13;K15; and O6:K54, respectively, as shown in Table 1. Thus, "O6 serotype" collectively refers to serotypes that encompass any one of the formulae O6:K2;K13;K15; and O6:K54.

[0233] Other examples of terms that refer generically to a class of formulas that include a generic term in the formula name according to Table 1 include "O4," "O5," "O18," and "O45." As used herein, the term "O2" refers to formula O2 as shown in Table 1. The term "O2 O antigen" refers to a saccharide that encompasses formula O2 as shown in Table 1.

[0234] As used herein, reference to O antigens of the above serotypes refers to saccharides that encompass the formula with the corresponding serotype name. For example, the term "O25B O antigen" refers to saccharides that encompass the formula O25B as shown in Table 1.

[0235] As another example, the term "O1 O antigen" refers collectively to saccharides that include formulas that include the term "O1," such as formula O1A, formula O1A1, formula O1B, and formula O1C, each shown in Table 1.

[0236] As another example, the term "O6 O antigen" refers collectively to saccharides that include formulas that include the term "O6," such as formula O6:K2, formula O6:K13, formula O6:K15, and formula O6:K54, each shown in Table 1.

[0237] B. O polysaccharide As used herein, the term "O polysaccharide" refers to any structure containing an O antigen, provided that the structure does not contain whole cells or lipid A. For example, in one embodiment, the O polysaccharide comprises lipopolysaccharide without attached lipid A. Steps for removing lipid A are known in the art and include, for example, heat treatment with the addition of acid. An exemplary process involves treatment with 1% acetic acid at 100°C for 90 minutes. This process is combined with a process for isolating the removed lipid A. An exemplary process for isolating lipid A involves ultracentrifugation.

[0238] In one embodiment, O polysaccharide refers to a structure consisting of an O antigen, in which case O polysaccharide is synonymous with the term O antigen. In a preferred embodiment, O polysaccharide refers to a structure comprising repeating units of an O antigen but not a core saccharide. Thus, in one embodiment, the O polysaccharide does not comprise an E. coli R1 core portion. In another embodiment, the O polysaccharide does not comprise an E. coli R2 core portion. In another embodiment, the O polysaccharide does not comprise an E. coli R3 core portion. In another embodiment, the O polysaccharide does not comprise an E. coli R4 core portion. In another embodiment, the O polysaccharide does not comprise an E. coli K12 core portion. In another preferred embodiment, O polysaccharide refers to a structure comprising an O antigen and a core saccharide. In another embodiment, O polysaccharide refers to a structure comprising an O antigen, a core saccharide, and a KDO portion.

[0239] Methods for purifying O polysaccharides, including the core oligosaccharide, from LPS are known in the art. For example, after LPS purification, the purified LPS may be hydrolyzed by heating in 1% (v / v) acetic acid at 100°C for 90 minutes, followed by ultracentrifugation at 142,000 x g for 5 hours at 4°C. The supernatant containing the O polysaccharide is freeze-dried and stored at 4°C. In certain embodiments, deletions of capsule synthesis genes are described that allow for easy purification of the O polysaccharide.

[0240] The O polysaccharide may be isolated by methods including, but not limited to, mild acid hydrolysis to remove lipid A from LPS. Other embodiments may include the use of hydrazine as an agent to prepare the O polysaccharide. Preparation of LPS may be accomplished by methods known in the art.

[0241] In certain embodiments, purified O polysaccharides from wild-type, modified, or attenuated Gram-negative bacterial strains that express (but do not necessarily overexpress) a Wzz protein (e.g., wzzB) are provided for use in conjugate vaccines. In preferred embodiments, O polysaccharide chains are purified from Gram-negative bacterial strains that express (but do not necessarily overexpress) a wzz protein for use as vaccine antigens, either as conjugates or combined vaccines.

[0242] In one embodiment, the O polysaccharide is about 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 25 fold, 26 fold, 27 fold, 28 fold, 29 fold, 30 fold, 31 fold, 32 fold, 33 fold, 34 fold, 35 fold, 36 fold, 37 fold, 38 fold, 39 fold, 40 fold, 41 fold, 42 fold, 43 fold, 44 fold, 45 fold, 46 fold, 47 fold, 48 fold, 49 fold, 50 fold , 51-fold, 52-fold, 53-fold, 54-fold, 55-fold, 56-fold, 57-fold, 58-fold, 59-fold, 60-fold, 61-fold, 62-fold, 63-fold, 64-fold, 65-fold, 66-fold, 67-fold, 68-fold, 69-fold, 70-fold, 71-fold, 72-fold, 73-fold, 74-fold, 75-fold, 76-fold, 77-fold, 78-fold, 79-fold, 80-fold, 81-fold, 82-fold, 83-fold, 84-fold, 85-fold, 86-fold, 87-fold, 88-fold, 89-fold, 90-fold, 91-fold, 92-fold, 93-fold, 94-fold, 95-fold, 96-fold, 97-fold, 98-fold, 99-fold, 100-fold, or more. In a preferred embodiment, the O polysaccharide has a molecular weight that is increased by more than 1-fold and not more than 5-fold compared to the corresponding wild-type O polysaccharide. In another embodiment, the O polysaccharide has a molecular weight that is increased by at least two-fold and not more than four-fold compared to the corresponding wild-type O polysaccharide. The increased molecular weight of the O polysaccharide compared to the corresponding wild-type O polysaccharide is preferably associated with an increased number of repeating units in the O antigen. In one embodiment, the increased molecular weight of the O polysaccharide is due to a wzz family protein.

[0243] In one embodiment, the O polysaccharide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, The O polysaccharides of the present invention have a molecular weight that is increased by 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 100 kDa, or more. In one embodiment, the O polysaccharides of the present invention have a molecular weight that is increased by at least 1 kDa but not more than 200 kDa compared to the corresponding wild-type O polysaccharide. In one embodiment, the molecular weight is increased by at least 5 kDa but not more than 200 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa but not more than 200 kDa. In one embodiment, the molecular weight increases by at least 12 kDa and not more than 200 kDa. In one embodiment, the molecular weight increases by at least 15 kDa and not more than 200 kDa. In one embodiment, the molecular weight increases by at least 18 kDa and not more than 200 kDa. In one embodiment, the molecular weight increases by at least 20 kDa and not more than 200 kDa. In one embodiment, the molecular weight increases by 21 kDa to 200 kDa. In one embodiment, the molecular weight increases by 22 kDa to 200 kDa. In one embodiment, the molecular weight increases by 30 kDa to 200 kDa. In one embodiment, the molecular weight increases by 1 kDa to 100 kDa. In one embodiment, the molecular weight increases by 5 kDa to 100 kDa. In one embodiment, the molecular weight increases by 10 kDa to 100 kDa. In one embodiment, the molecular weight increases by 12 kDa to 100 kDa. In one embodiment, the molecular weight increases by 15 kDa to 100 kDa. In one embodiment, the molecular weight increases by 20 kDa to 100 kDa. In one embodiment, the molecular weight increases by 1 kDa to 75 kDa. In one embodiment, the molecular weight increases by 5 kDa to 75 kDa. In one embodiment, the molecular weight increases by 10 kDa to 75 kDa. In one embodiment, the molecular weight increases by 12 kDa to 75 kDa. In one embodiment, the molecular weight increases by 15 kDa to 75 kDa. In one embodiment, the molecular weight increases by 18 kDa to 75 kDa. In one embodiment, the molecular weight increases by 20 kDa to 75 kDa. In one embodiment, the molecular weight increases by 30 kDa to 75 kDa. In one embodiment, the molecular weight increases by 10 kDa to 90 kDa. In one embodiment, the molecular weight increases by 12 kDa to 85 kDa. In one embodiment, the molecular weight increases by 10 kDa to 75 kDa. In one embodiment, the molecular weight increases by 10 kDa to 70 kDa. In one embodiment, the molecular weight increases by 10 kDa to 60 kDa. In one embodiment, the molecular weight increases by at least 10 kDa and not more than 50 kDa. In one embodiment, the molecular weight increases by at least 10 kDa and not more than 49 kDa. In one embodiment, the molecular weight increases by at least 10 kDa and not more than 48 kDa. In one embodiment, the molecular weight increases by at least 10 kDa and not more than 47 kDa. In one embodiment, the molecular weight increases by at least 10 kDa and not more than 46 kDa. In one embodiment, the molecular weight increases by at least 20 kDa and not more than 45 kDa. In one embodiment, the molecular weight increases by at least 20 kDa and not more than 44 kDa. In one embodiment, the molecular weight increases by at least 20 kDa and not more than 43 kDa. In one embodiment, the molecular weight increases by at least 20 kDa and not more than 42 kDa.In one embodiment, the molecular weight is increased by at least 20 kDa and not more than 41 kDa. Such an increase in molecular weight of the O polysaccharide compared to the corresponding wild-type O polysaccharide is preferably associated with an increase in the number of repeating units of the O antigen. In one embodiment, the increase in molecular weight of the O polysaccharide is due to a wzz family protein. See, e.g., Table 21.

[0244] In another embodiment, the O polysaccharide comprises any one of the formulas selected from Table 1, wherein the number of repeat units in the O polysaccharide, n, is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 11 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 100 or more repeat units larger. Preferably, the saccharide comprises at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 more repeat units than the corresponding wild-type O polysaccharide. See, e.g., Table 21.

[0245] C. O antigen O antigens are part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. O antigens are present on the cell surface and are variable cellular components. The variability of O antigens provides the basis for serotyping of Gram-negative bacteria. The current E. coli serotyping scheme includes O polysaccharides 1 to 181.

[0246] O antigens contain repeating oligosaccharide units (O units), whose wild-type structures typically contain two to eight residues of a wide range of sugars. Exemplary E. coli O antigen O units are shown in Table 1. See also Figures 9A-9C and 10A-10B.

[0247] In one embodiment, a saccharide of the invention may be a single oligosaccharide unit. In one embodiment, a saccharide of the invention is a single repeating unit of an oligosaccharide of the relevant serotype. In such an embodiment, the saccharide may comprise a structure selected from any one of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101.

[0248] In one embodiment, the saccharide of the present invention can be an oligosaccharide. Oligosaccharides have a low number of repeating units (usually 5-15 repeating units) and are typically obtained synthetically or by hydrolysis of a polysaccharide. In such an embodiment, the saccharide can comprise a structure selected from any one of formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101.

[0249] Preferably, all of the saccharides in the saccharides of the invention and immunogenic compositions of the invention are polysaccharides. High molecular weight polysaccharides can induce a specific antibody immune response due to epitopes present on the surface of antigens. Isolation and purification of high molecular weight polysaccharides is preferably contemplated for use in the conjugates, compositions, and methods of the invention.

[0250] In some embodiments, the number of repeating O units (and thus the length and molecular weight of the polymer chain) in each individual O-antigen polymer depends on the wzz chain length regulator, an inner membrane protein. Different wzz proteins result in different ranges of modal lengths (from 4 to over 100 repeating units). The term "modal length" refers to the number of repeating O units. Gram-negative bacteria often have two different Wzz proteins, resulting in two different OAg modal chain lengths: long and short. Expression (not necessarily overexpression) of a wzz family protein (e.g., wzzB) in Gram-negative bacteria can manipulate O-antigen length to alter or bias bacterial production of O-antigens with a particular length range, enhancing the production of high-yield, high-molecular-weight lipopolysaccharides. In one embodiment, "short" modal length, as used herein, refers to a low number of repeating O units, e.g., 1 to 20. In one embodiment, "long" modal length, as used herein, refers to a number of repeating O units greater than 20, and up to 40. In one aspect, "very long chain" modal length as used herein means more than 40 repeating O units.

[0251] In one embodiment, the saccharide produced has at least 10 more repeat units, 15 more repeat units, 20 more repeat units, 25 more repeat units, 30 more repeat units, 35 more repeat units, 40 more repeat units, 45 more repeat units, 50 more repeat units, 55 more repeat units, 60 more repeat units, 65 more repeat units, 70 more repeat units, 75 more repeat units, 80 more repeat units, 85 more repeat units, 90 more repeat units, 95 more repeat units, or 100 more repeat units compared to the corresponding wild-type O polysaccharide.

[0252] In another aspect, the saccharides of the invention have a nucleotide sequence similar to that of the corresponding wild-type O polysaccharide: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 100, or more repeating units. Preferably, the saccharide contains at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 more repeating units compared to the corresponding wild-type O polysaccharide. See, e.g., Table 21. Methods for determining the length of saccharides are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography, as described in Example 13.

[0253] Methods for determining the number of repeating units in a saccharide are also known in the art. For example, the number of repeating units (or "n" in a formula) can be calculated by dividing the molecular weight of the polysaccharide (not including the molecular weight of the core saccharide or KDO residue) by the molecular weight of the repeating unit (i.e., the molecular weight of the structure that can theoretically be calculated as the sum of the molecular weights of each monosaccharide in the corresponding formula, for example, as shown in Table 1). The molecular weight of each monosaccharide in the formula is known in the art. For example, the molecular weight of the repeating unit of formula O25b is about 862 Da. For example, the molecular weight of the repeating unit of formula O1a is about 845 Da. For example, the molecular weight of the repeating unit of formula O2 is about 829 Da. For example, the molecular weight of the repeating unit of formula O6 is about 893 Da. When determining the number of repeating units in a conjugate, the molecular weight of the carrier protein and the protein:polysaccharide ratio are factored into the calculation. As defined herein, "n" is the number of repeating units in a polysaccharide molecule (represented in parentheses in Table 1). As is known in the art, the repeating structures in biopolymers may be interspersed with regions of imperfect repeating, e.g., missing branches. Furthermore, it is known in the art that polysaccharides isolated or purified from natural sources, such as bacteria, may be heterogeneous in size and branching. In such cases, n may represent the average or median n of the molecules in the population.

[0254] In one embodiment, the O polysaccharide has at least one more O antigen repeat unit compared to the corresponding wild-type O polysaccharide. The O antigen repeat units are shown in Table 1. In one embodiment, the O polysaccharide has at least one more O antigen repeat unit than the corresponding wild-type O polysaccharide. The O antigen repeat units are shown in Table 1. In one embodiment, the O polysaccharide has at least one more O antigen repeat unit than the corresponding wild-type O polysaccharide. , 54, 55, 56, 57, 58, 59, 60, 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, 100 or more total repeat units. Preferably, the saccharide has a total of 3 to 80 repeat units. In another embodiment, the O polysaccharide has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 2 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, 100 or more repeating units.

[0255] In one aspect, the saccharide is selected from the group consisting of saccharides in which n in any of the O antigen formulas (such as those shown in Table 1 (see also Figures 9A-9C and Figures 10A-10B)) is 1, 2, 3, 4, 5, 10, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40 or more, and up to 200, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50. Any minimum number may be combined with any maximum number to define a range. Exemplary ranges include, for example, from 1 to 1000, from 10 to 500, and from 20 to 80, preferably 90 or less. In a preferred embodiment, n is from 31 to 90. In a preferred embodiment, n is 40-90, more preferably 60-85.

[0256] In one embodiment, the saccharide comprises an O antigen wherein n in any one of the O antigen formulas is between 1 and 200, inclusive. In one embodiment, n in any one of the O antigen formulas is between 5 and 200, inclusive. In one embodiment, n in any one of the O antigen formulas is between 10 and 200, inclusive. In one embodiment, n in any one of the O antigen formulas is between 25 and 200, inclusive. In one embodiment, n in any one of the O antigen formulas is between 50 and 200, inclusive. In one embodiment, n in any one of the O antigen formulas is between 75 and 200, inclusive. In one embodiment, n in any one of the O antigen formulas is between 100 and 200, inclusive. In one embodiment, n in any one of the O antigen formulas is between 125 and 200, inclusive. In one embodiment, n in any one of the O antigen formulas is between 150 and 200, inclusive. In one embodiment, n in any one of the O antigen formulas is between 175 and 200, inclusive. In one embodiment, n in any one of the O antigen formulas is between 1 and 100, inclusive. In one embodiment, n in any one of the O antigen formulas is between 5 and 100, inclusive. In one embodiment, n in any one of the O antigen formulas is between 10 and 100, inclusive. In one embodiment, n in any one of the O antigen formulas is between 25 and 100, inclusive. In one embodiment, n in any one of the O antigen formulas is between 50 and 100, inclusive. In one embodiment, n in any one of the O antigen formulas is between 75 and 100, inclusive. In one embodiment, n in any one of the O antigen formulas is between 1 and 75, inclusive. In one embodiment, n in any one of the O antigen formulas is between 5 and 75, inclusive. In one embodiment, n in any one of the O antigen formulas is between 10 and 75, inclusive. In one embodiment, n in any one of the O antigen formulas is between 20 and 75, inclusive. In one embodiment, n in any one of the O antigen formulas is between 25 and 75, inclusive. In one embodiment, n in any one of the O antigen formulas is equal to or greater than 30 and equal to or less than 75. In one embodiment, n in any one of the O antigen formulas is equal to or greater than 40 and equal to or less than 75. In one embodiment, n in any one of the O antigen formulas is equal to or greater than 50 and equal to or less than 75. In one embodiment, n in any one of the O antigen formulas is equal to or greater than 30 and equal to or less than 90.In one embodiment, n in any one of the O antigen formulas is between 35 and 85, inclusive. In one embodiment, n in any one of the O antigen formulas is between 35 and 75, inclusive. In one embodiment, n in any one of the O antigen formulas is between 35 and 70, inclusive. In one embodiment, n in any one of the O antigen formulas is between 35 and 60, inclusive. In one embodiment, n in any one of the O antigen formulas is between 35 and 50, inclusive. In one embodiment, n in any one of the O antigen formulas is between 35 and 49, inclusive. In one embodiment, n in any one of the O antigen formulas is between 35 and 48, inclusive. In one embodiment, n in any one of the O antigen formulas is between 35 and 47, inclusive. In one embodiment, n in any one of the O antigen formulas is between 35 and 46, inclusive. In one embodiment, n in any one of the O antigen formulas is between 36 and 45, inclusive. In one embodiment, n in any one of the O antigen formulas is between 37 and 44, inclusive. In one embodiment, n in any one of the O antigen formulas is equal to or greater than 38 and equal to or less than 43. In one embodiment, n in any one of the O antigen formulas is equal to or greater than 39 and equal to or less than 42. In one embodiment, n in any one of the O antigen formulas is equal to or greater than 39 and equal to or less than 41.

[0257] For example, in one embodiment, n of the saccharide is 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 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, or 90, most preferably 40. In another embodiment, n is 35 or greater and 60 or less. For example, in one embodiment, n is any one of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60, preferably 50. In another preferred embodiment, n is equal to or greater than 55 and equal to or less than 75. For example, in one embodiment, n is 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69, most preferably 60.

[0258] The structure of the saccharide may be determined by methods and tools known in the art, such as NMR, including 1D, 1H, and / or 13C, 2D TOCSY, DQF-COSY, NOESY, and / or HMQC.

[0259] In some embodiments, the purified polysaccharide prior to conjugation has a molecular weight of between 5 kDa and 400 kDa. In other such embodiments, the saccharide has a molecular weight of between 10 kDa and 400 kDa, between 5 kDa and 400 kDa, between 5 kDa and 300 kDa, between 5 kDa and 200 kDa, between 5 kDa and 150 kDa, between 10 kDa and 100 kDa, between 10 kDa and 75 kDa, between 10 kDa and 60 kDa, between 10 kDa and 40 kDa, between 10 kDa and 100 kDa, between 10 kDa and 20 kDa. and having a molecular weight of 0 kDa, 15 kDa to 150 kDa, 12 kDa to 120 kDa, 12 kDa to 75 kDa, 12 kDa to 50 kDa, 12 to 60 kDa, 35 kDa to 75 kDa, 40 kDa to 60 kDa, 35 kDa to 60 kDa, 20 kDa to 60 kDa, 12 kDa to 20 kDa, or 20 kDa to 50 kDa. In further embodiments, the polysaccharide has a molecular weight of 7 kDa to 15 kDa, 8 kDa to 16 kDa, 9 kDa to 25 kDa, 10 kDa to 100, 10 kDa to 60 kDa, 10 kDa to 70 kDa, 10 kDa to 160 kDa, 15 kDa to 600 kDa, 20 kDa to 1000 kDa, 20 kDa to 600 kDa, 20 kDa to 400 kDa, 30 kDa to 1000 kDa, 30 kDa to 60 kDa, 30 kDa to 50 kDa, or 5 kDa to 60 kDa. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0260] As used herein, the term "molecular weight" of a polysaccharide or carrier protein-polysaccharide conjugate means the molecular weight calculated by size exclusion chromatography (SEC) in combination with a multi-angle laser light scattering detector (MALLS).

[0261] Polysaccharides may undergo slight size reduction during normal purification processes. Additionally, as described herein, polysaccharides may be subjected to sizing techniques prior to conjugation. Mechanical or chemical sizing may be used. Chemical hydrolysis using acetic acid may be performed. Mechanical sizing may be performed using high-pressure homogenization shear. The molecular weight ranges described above refer to purified polysaccharides prior to conjugation (e.g., prior to activation).

[0262] [Table 1-1]

[0263] [Table 1-2]

[0264] [Table 1-3]

[0265] [Table 1-4]

[0266] [Table 1-5]

[0267] [Table 1-6]

[0268] [Table 1-7]

[0269] [Table 1-8]

[0270] D. Core Oligosaccharides The core oligosaccharide is located between lipid A and the O-antigen outer region in wild-type E. coli LPS. More specifically, the core oligosaccharide is the portion of the polysaccharide that contains the linkage between the O-antigen and lipid A in wild-type E. coli. This linkage is formed by the hemiketal functional group of the innermost 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue and the lipid A. The core oligosaccharide region contains a ketosidic bond between the hydroxyl group of the GlcNAc residue of the nucleotide. The core oligosaccharide region shows a high degree of similarity among wild-type E. coli strains. It usually contains a limited number of sugars. The core oligosaccharide comprises an inner core region and an outer core region.

[0271] More specifically, the inner core is composed primarily of L-glycero-D-manno-heptose (heptose) and KDO residues. The inner core is highly conserved. The KDO residues include KDO of the formula:

[0272] [ka]

[0273] The outer region of the core oligosaccharide shows greater diversity than the inner core region, and differences in this region distinguish the five chemotypes of E. coli: R1, R2, R3, R4, and K-12. See Figure 24, which shows the generalized structures of the carbohydrate backbones of the outer core oligosaccharides of the five known chemotypes. Hep II is the last residue on the inner core oligosaccharide. All outer core oligosaccharides share a structural theme of a (hexose) trisaccharide backbone and two side chain residues, although the order of the hexoses within the backbone, as well as the nature, position, and linkage of the side chain residues, can all vary. The structures of the R1 and R4 outer core oligosaccharides are highly similar, differing only by a single β-linked residue.

[0274] In the art, the core oligosaccharide of wild-type E. coli is classified into five different chemotypes based on the structure of the terminal oligosaccharides: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12.

[0275] In a preferred embodiment, the compositions described herein comprise a glycoconjugate in which the O polysaccharide comprises a core oligosaccharide bound to an O antigen. In another embodiment, the composition elicits an immune response against at least one of the following core E. coli chemotypes: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12. In another embodiment, the composition elicits an immune response against at least two core E. coli chemotypes. In another embodiment, the composition elicits an immune response against at least three core E. coli chemotypes. In another embodiment, the composition elicits an immune response against at least four core E. coli chemotypes. In another embodiment, the composition elicits an immune response against all five core E. coli chemotypes.

[0276] In another preferred embodiment, the compositions described herein comprise glycoconjugates in which the core oligosaccharide attached to the O antigen is not included in the O polysaccharide. In one embodiment, such compositions elicit an immune response against at least one of the core E. coli chemotypes E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, despite the glycoconjugate having an O polysaccharide that does not include the core oligosaccharide.

[0277] E. coli serotypes may be characterized by one of five chemotypes. Table 2 lists exemplary serotypes characterized by chemotype. Serotypes in bold represent the serotypes most commonly associated with the core chemotype indicated. Thus, in preferred embodiments, the present compositions are directed against E. coli R1, E. coli R2, E. coli R3, E. coli R4, E. coli R5, E. coli R6, E. coli R7, E. coli R8, E. coli R9, E. coli R10, E. coli R11, E. coli R12, E. coli R13, E. coli R14, E. coli R15, E. coli R16, E. coli R17, E. coli R18, E. coli R19, E. coli R119, E. coli R119, E. coli R120, E. coli R130, E. coli R140, E. coli R150, E. coli R160, E. coli R171, E. coli R182, E. coli R191, E. coli R192, E. coli R193, E. coli R194, E. coli R195, E. coli R196, E. coli R197, E. coli R198, E. coli R199, E. coli R200, E. coli R210, E. coli R211, E. coli R212, E. coli R213, E. coli R214, E. coli R220, E. coli R230, E. coli R240, E. coli R250, E. coli R260, E. coli R270, E. coli R280, E. coli R290, E. coli R215, E. coli R216, E. coli R217, E. coli R218, E. coli R221, E. coli R230, E. coli R241, The present invention elicits an immune response against at least one of the core E. coli chemotypes R3, E. coli R4, and E. coli K12, and this immune response includes an immune response against any one of the corresponding E. coli serotypes.

[0278] [Table 2]

[0279] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, e.g., selected from saccharides having formula O25a, formula O6, formula O2, formula O1, formula O75, formula O4, formula O16, formula O8, formula O18, formula O9, formula O13, formula O20, formula O21, formula O91, and formula O163, where n is 1 to 100. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety, e.g., as shown in Figure 24.

[0280] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R1 chemotype, e.g., selected from saccharides having formula O25a, formula O6, formula O2, formula O1, formula O75, formula O4, formula O16, formula O18, formula O13, formula O20, formula O21, formula O91, and formula O163, where n is 1 to 100, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety in the saccharide.

[0281] In some embodiments, the composition comprises saccharides comprising a structure derived from a serotype having an R2 chemotype, e.g., selected from saccharides having formula O21, formula O44, formula O11, formula O89, formula O162, and formula O9, where n is 1-100, preferably 31-100, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R2 core moiety, e.g., as shown in Figure 24.

[0282] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R3 chemotype, e.g., selected from saccharides having formula O25b, formula O15, formula O153, formula O21, formula O17, formula O11, formula O159, formula O22, formula O86, and formula O93, where n is 1-100, preferably 31-100, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R3 core moiety, e.g., as shown in Figure 24.

[0283] In some embodiments, the compositions include saccharides having, for example, formula O2, formula O1, formula O86, formula O7, formula O102, formula O160, and formula O166 (wherein n is 1 to 100, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65). In some embodiments, the saccharide in the composition further comprises an E. coli R4 core moiety, for example, as shown in Figure 24.

[0284] In some embodiments, the composition comprises saccharides comprising a structure derived from a serovar having a K-12 chemotype (e.g., selected from saccharides having the formula O25b and saccharides having the formula O16), where n is between 1 and 1000, preferably between 31 and 100, more preferably between 35 and 90, and most preferably between 35 and 65. In some embodiments, the saccharides in the composition further comprise an E. coli K-12 core moiety, e.g., as shown in Figure 24.

[0285] In some embodiments, the saccharide comprises a core saccharide. Thus, in one embodiment, the O polysaccharide further comprises an E. coli R1 core portion. In another embodiment, the O polysaccharide further comprises an E. coli R2 core portion. In another embodiment, the O polysaccharide further comprises an E. coli R3 core portion. In another embodiment, the O polysaccharide further comprises an E. coli R4 core portion. In another embodiment, the O polysaccharide further comprises an E. coli K12 core portion.

[0286] In some embodiments, the saccharide does not comprise a core saccharide. Thus, in one embodiment, the O polysaccharide does not comprise an E. coli R1 core portion. In another embodiment, the O polysaccharide does not comprise an E. coli R2 core portion. In another embodiment, the O polysaccharide does not comprise an E. coli R3 core portion. In another embodiment, the O polysaccharide does not comprise an E. coli R4 core portion. In another embodiment, the O polysaccharide does not comprise an E. coli K12 core portion.

[0287] E. Conjugated O Antigens Chemical conjugation of O antigens, or preferably O polysaccharides, to protein carriers can improve the immunogenicity of O antigens or O polysaccharides. However, variability in polymer size presents practical challenges in production. In commercial applications, the size of the saccharides can affect compatibility with various conjugation synthesis strategies, product uniformity, and the immunogenicity of the conjugates. Controlling the expression of Wzz family protein chain length regulators by manipulating the O antigen synthesis pathway allows for the production of O antigen chains of desired lengths in a variety of Gram-negative bacterial strains, including E. coli.

[0288] In one embodiment, purified saccharides are chemically activated to generate activated saccharides that can react with a carrier protein. Once activated, each saccharide is separately conjugated to a carrier protein to form a conjugate, i.e., a glycoconjugate. As used herein, the term "glycoconjugate" refers to a saccharide covalently attached to a carrier protein. In one embodiment, the saccharide is directly linked to the carrier protein. In another embodiment, the saccharide is linked to the protein via a spacer / linker. The conjugates can be prepared by a scheme in which a carrier is attached to the O antigen at one or more sites along the O antigen or by a scheme in which at least one residue of the core oligosaccharide is activated.

[0289] In one embodiment, each saccharide is conjugated to the same carrier protein. If the protein carrier is the same for two or more saccharides in the composition, these saccharides may be conjugated to the same molecule of carrier protein (e.g., two or more different saccharides are conjugated to the carrier molecule).

[0290] In a preferred embodiment, each saccharide is individually conjugated to a different molecule of the protein carrier (only one type of saccharide is conjugated to each molecule of the protein carrier), in which embodiment the saccharides are said to be individually conjugated to the carrier protein.

[0291] Chemical activation of the saccharide and subsequent conjugation to the carrier protein can be achieved by the activation and conjugation methods disclosed herein. After conjugation of the polysaccharide to the carrier protein, the glycoconjugate is purified (concentrated relative to the amount of polysaccharide-protein conjugate) by a variety of techniques. These techniques include concentration / diafiltration, precipitation / elution, column chromatography, and depth filtration. After the individual glycoconjugates are purified, they are combined to formulate the immunogenic compositions of the invention.

[0292] Activation. The present invention further relates to activated polysaccharides produced by any of the embodiments described herein, wherein the polysaccharides are activated with a chemical reagent to provide reactive groups for conjugation to a linker or carrier protein. In some embodiments, the saccharides of the present invention are activated prior to conjugation to a carrier protein. In some embodiments, the degree of activation does not significantly reduce the molecular weight of the polysaccharide. For example, in some embodiments, the degree of activation does not cleave the backbone of the polysaccharide. In some embodiments, the degree of activation does not significantly reduce the molecular weight of the polysaccharide. 197 The degree of activation does not significantly affect the degree of conjugation as measured by the number of modified lysine residues (determined by amino acid analysis) in the carrier protein, such as . For example, in some embodiments, the degree of activation does not significantly increase the number of modified lysine residues in the carrier protein (determined by amino acid analysis) by three times compared to the number of modified lysine residues in the carrier protein of the conjugate with the reference polysaccharide at the same degree of activation. In some embodiments, the degree of activation does not increase the level of unconjugated free sugars. In some embodiments, the degree of activation does not decrease the optimal sugar / protein ratio.

[0293] In some embodiments, the activated saccharide has an activation ratio of 1-100%, e.g., 2-80%, 2-50%, 3-30%, and 4-25%, where the moles of thiol per saccharide repeat unit of the activated saccharide are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, ≥20%, ≥30%, ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, or ≥90%, or about 100%. Preferably, the activation ratio is at most 50%, more preferably at most 25%. In one embodiment, the activation ratio is at most 20%. Any minimum number may be combined with any maximum number to define a range.

[0294] In one embodiment, the polysaccharide is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to generate a cyanate ester. The activated polysaccharide is then coupled to a carrier protein (preferably CRMP). 197 or tetanus toxoid) directly or via a spacer (linker) group.

[0295] For example, the spacer may be cystamine or cysteamine, resulting in a thiolated polysaccharide, which can be coupled to the carrier via a thioether bond obtained after reaction with a maleimide-activated carrier protein (e.g., using N-[γ-maleimidobutyryloxy]succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using iodoacetimide, N-succinimidyl bromoacetate (SBA; SIB), N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), sulfosuccinimidyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), N-succinimidyl iodoacetate (SIA), or succinimidyl 3-[bromoacetamido]proprionate (SBAP)). In one embodiment, a cyanate ester (optionally made by CDAP chemistry) is coupled to hexanediamine or adipic acid dihydrazide (ADH), and an amino-derivatized saccharide is coupled to the cyanate ester using carbodiimide (e.g., EDAC or EDC) chemistry. and then attaching the carrier protein (e.g., CRM) via the carboxyl group of the protein carrier. 197 ) is conjugated to

[0296] Other suitable conjugation techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Conjugation may require a carbonyl linker, which can be formed by reacting the free hydroxyl group of the sugar with CDI, followed by reaction with the protein to form a carbamate bond. This can involve reducing the anomeric terminus to a primary hydroxyl group, optionally protecting / deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling the CDI carbamate intermediate with the amino group of the protein (CDI chemistry).

[0297] Molecular Weight. In some embodiments, the glycoconjugate comprises a saccharide having a molecular weight of 10 kDa to 2,000 kDa. In other embodiments, the saccharide has a molecular weight of 50 kDa to 1,000 kDa. In other embodiments, the saccharide has a molecular weight of 70 kDa to 900 kDa. In other embodiments, the saccharide has a molecular weight of 100 kDa to 800 kDa. In other embodiments, the saccharide has a molecular weight of 200 kDa to 600 kDa. In a further embodiment, the saccharide is selected from the group consisting of 100 kDa to 1,000 kDa, 100 kDa to 900 kDa, 100 kDa to 800 kDa, 100 kDa to 700 kDa, 100 kDa to 600 kDa, 100 kDa to 500 kDa, 100 kDa to 400 kDa, 100 kDa to 300 kDa, 150 kDa to 1,000 kDa, 150 kDa to 900 kDa, 150 kDa to 800kDa, 150kDa~700kDa, 150kDa~600kDa, 150kDa~500kDa, 150kDa~400kDa, 150kDa~300kDa, 200kDa a~1,000kDa, 200kDa~900kDa, 200kDa~800kDa, 200kDa~700kDa, 200kDa~600kDa, 200kDa~500kDa, 20 0kDa~400kDa, 200kDa~300, 250kDa~1,000kDa, 250kDa~900kDa, 250kDa~800kDa, 250kDa~700kDa, 2 50kDa~600kDa, 250kDa~500kDa, 250kDa~400kDa, 250kDa~350kDa, 300kDa~1,000kDa, 300kDa~900kDa The glycoconjugates may have molecular weights of 300 kDa to 800 kDa, 300 kDa to 700 kDa, 300 kDa to 600 kDa, 300 kDa to 500 kDa, 300 kDa to 400 kDa, 400 kDa to 1,000 kDa, 400 kDa to 900 kDa, 400 kDa to 800 kDa, 400 kDa to 700 kDa, 400 kDa to 600 kDa, or 500 kDa to 600 kDa. In one embodiment, glycoconjugates having such molecular weights are produced by single-end conjugation. In another embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC) prepared in an aqueous buffer.Any integer within any of the above ranges is contemplated as an aspect of this disclosure.

[0298] In some embodiments, glycoconjugates of the present invention have a molecular weight of 400 kDa to 15,000 kDa, 500 kDa to 10,000 kDa, 2,000 kDa to 10,000 kDa, 3,000 kDa to 8,000 kDa, or 3,000 kDa to 5,000 kDa. In other embodiments, glycoconjugates have a molecular weight of 500 kDa to 10,000 kDa. In other embodiments, glycoconjugates have a molecular weight of 1,000 kDa to 8,000 kDa. In still other embodiments, glycoconjugates have a molecular weight of 2,000 kDa to 8,000 kDa or 3,000 kDa to 7,000 kDa. In further embodiments, the glycoconjugates of the present invention have a molecular weight of 200 kDa to 20,000 kDa, 200 kDa to 15,000 kDa, 200 kDa to 10,000 kDa, 200 kDa to 7,500 kDa, 200 kDa to 5,000 kDa, 20 0kDa~3,000kDa, 200kDa~1,000kDa, 500kDa~20,000kDa, 500kDa~15,000kDa, 500kDa~12,500kDa, 500kDa~ 10,000kDa, 500kDa~7,500kDa, 500kDa~6,000kDa, 500kDa~5,000kDa, 500kDa~4,000kDa, 500kDa~3,000kDa , 500kDa~2,000kDa, 500kDa~1,500kDa, 500kDa~1,000kDa, 750kDa~20,000kDa, 750kDa~15,000kDa, 750kDa ~12,500kDa, 750kDa~10,000kDa, 750kDa~7,500kDa, 750kDa~6,000kDa, 750kDa~5,000kDa, 750kDa~4,000k Da, 750kDa~3,000kDa, 750kDa~2,000kDa, 750kDa~1,500kDa, 1,000kDa~15,000kDa, 1,000kDa~12,500kDa, 1,000kDa~10,000kDa, 1,000kDa~7,500kDa, 1,000kDa~6,000kDa, 1,000kDa~5,000kDa, 1,000kDa~4,000kDa The glycoconjugates have molecular weights of 1,000 kDa to 2,500 kDa, 2,000 kDa to 15,000 kDa, 2,000 kDa to 12,500 kDa, 2,000 kDa to 10,000 kDa, 2,000 kDa to 7,500 kDa, 2,000 kDa to 6,000 kDa, 2,000 kDa to 5,000 kDa, 2,000 kDa to 4,000 kDa, or 2,000 kDa to 3,000 kDa. In one embodiment, glycoconjugates having such molecular weights are produced by eTEC conjugation as described herein. In another embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC). In another embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC) prepared in DMSO.

[0299] In further embodiments, the glycoconjugates of the invention have a molecular weight of 1,000 kDa to 20,000 kDa, 1,000 kDa to 15,000 kDa, 2,000 kDa to 10,000 kDa, 2,000 kDa to 7,500 kDa, 2,000 kDa to 5,000 kDa, 3,000 kDa to 20,000 kDa, 3,000 kDa to 15,000 kDa, 3,000 kDa to 12,500 kDa, 4,000 kDa to 10,000 kDa, 4,000 kDa to 7,500 kDa, 4,000 kDa to 6,000 kDa, or 5,000 kDa to 7,000 kDa. In one embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC). In another embodiment, glycoconjugates having such molecular weights are produced by reductive amination chemistry (RAC) prepared in DMSO. In another embodiment, glycoconjugates having such molecular weights are produced by eTEC conjugation as described herein.

[0300] In further embodiments, the glycoconjugates of the invention have a molecular weight of 5,000 kDa to 20,000 kDa, 5,000 kDa to 15,000 kDa, 5,000 kDa to 10,000 kDa, 5,000 kDa to 7,500 kDa, 6,000 kDa to 20,000 kDa, 6,000 kDa to 15,000 kDa, 6,000 kDa to 12,500 kDa, 6,000 kDa to 10,000 kDa, or 6,000 kDa to 7,500 kDa.

[0301] The molecular weight of the glycoconjugate can be measured by SEC-MALLS. Any integer within any of the above ranges is contemplated as an embodiment of the present disclosure. The glycoconjugates of the present invention can also be characterized by the saccharide to carrier protein ratio (w / w). In some embodiments, the polysaccharide to carrier protein ratio (w / w) in the glycoconjugate is between 0.5 and 3 (e.g., about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about In other embodiments, the saccharide to carrier protein ratio (w / w) is 0.5 to 2.0, 0.5 to 1.5, 0.8 to 1.2, 0.5 to 1.0, 1.0 to 1.5, or 1.0 to 2.0. In further embodiments, the saccharide to carrier protein ratio (w / w) is 0.8 to 1.2. In preferred embodiments, the polysaccharide to carrier protein ratio in the conjugate is 0.9 to 1.1. In some such embodiments, the carrier protein is a CRM. 197 is.

[0302] The glycoconjugates were characterized by a molecular size distribution (K d ) can also be characterized. To determine the relative molecular size distribution of the conjugates, a size exclusion chromatography medium (CL-4B) can be used. Size exclusion chromatography (SEC) is used to profile the molecular size distribution of the conjugates in a gravity-fed column. Large molecules that are excluded from the pores of the medium elute faster than small molecules. A fraction collector is used to collect the column effluent. Fractions are tested colorimetrically by sugar assay. K d To determine the fraction of molecules completely excluded (V0), (K d = 0), and the fraction representing maximum retention (V i ), (K d Calibrate the column to establish the fraction (V) at which a specified sample attribute is reached. e ) is the formula K d =(V e -V o ) / (V i -V0) is related to Kd.

[0303] Free saccharides. The glycoconjugates and immunogenic compositions of the present invention may contain free saccharides that are present in the glycoconjugate composition despite not being covalently conjugated to a carrier protein. The free saccharides may be non-covalently associated with the glycoconjugate (i.e., non-covalently bound, adsorbed, or entrapped in or by the glycoconjugate). In preferred embodiments, the glycoconjugate contains up to 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharides relative to the total amount of polysaccharides. In preferred embodiments, the glycoconjugate contains less than about 25% free polysaccharides relative to the total amount of polysaccharides. In preferred embodiments, the glycoconjugate contains up to about 20% free polysaccharides relative to the total amount of polysaccharides. In preferred embodiments, the glycoconjugate contains up to about 15% free polysaccharides relative to the total amount of polysaccharides. In another preferred embodiment, the glycoconjugate contains up to about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% free polysaccharides relative to the total amount of polysaccharides. In a preferred embodiment, the glycoconjugate contains less than about 8% free polysaccharides relative to the total amount of polysaccharides. In a preferred embodiment, the glycoconjugate contains up to about 6% free polysaccharides relative to the total amount of polysaccharides. In a preferred embodiment, the glycoconjugate contains up to about 5% free polysaccharides relative to the total amount of polysaccharides. See, for example, Table 19, Table 20, Table 21, Table 22, Table 23, Table 24, and Table 25.

[0304] In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide every 5-10 saccharide repeat units, every 2-7 saccharide repeat units, every 3-8 saccharide repeat units, every 4-9 saccharide repeat units, every 6-11 saccharide repeat units, every 7-12 saccharide repeat units, every 8-13 saccharide repeat units, every 9-14 saccharide repeat units, every 10-15 saccharide repeat units, every 2-6 saccharide repeat units, every 3-7 saccharide repeat units, every 4-8 saccharide repeat units, every 6-10 saccharide repeat units, every 7-11 saccharide repeat units, every 8-12 saccharide repeat units, every 9-13 saccharide repeat units, every 10-14 saccharide repeat units, every 10-20 saccharide repeat units, every 4-25 saccharide repeat units, or every 2-25 saccharide repeat units. In many embodiments, the carrier protein is a CRM 197 In another embodiment, at least one bond between the carrier protein and the saccharide is a bond between two of the polysaccharides, In one embodiment, the carrier protein is a CRM. 197 Any integer within any of the above ranges is contemplated as an aspect of this disclosure.

[0305] Lysine residues. Another way to characterize the glycoconjugates of the invention is to measure the number of carrier proteins (e.g., CRMs) conjugated to the saccharide, which can be characterized as the extent of lysine conjugated (degree of conjugation). 197The degree of conjugation is determined by the number of lysine residues in the carrier protein. Evidence of lysine modification of the carrier protein due to covalent attachment to the polysaccharide can be obtained by amino acid analysis using routine methods known to those of skill in the art. Conjugation reduces the number of lysine residues recovered compared to the carrier protein starting material used to generate the conjugate material. In preferred embodiments, the degree of conjugation of glycoconjugates of the invention is 2-15, 2-13, 2-10, 2-8, 2-6, 2-5, 2-4, 3-15, 3-13, 3-10, 3-8, 3-6, 3-5, 3-4, 5-15, 5-10, 8-15, 8-12, 10-15, or 10-12. In one embodiment, the degree of conjugation of glycoconjugates of the invention is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In preferred embodiments, the degree of conjugation of the glycoconjugates of the invention is between 4 and 7. In some such embodiments, the carrier protein is a CRM 197 is.

[0306] The frequency of attachment of saccharide chains to lysines of a carrier protein is another parameter that characterizes the glycoconjugates of the present invention. For example, in some embodiments, there is at least one covalent bond between the carrier protein and the polysaccharide for every four saccharide repeat units of the polysaccharide. In other embodiments, the covalent bond between the carrier protein and the polysaccharide occurs at least once for every 10 saccharide repeat units of the polysaccharide. In other embodiments, the covalent bond between the carrier protein and the polysaccharide occurs at least once for every 15 saccharide repeat units of the polysaccharide. In further embodiments, the covalent bond between the carrier protein and the polysaccharide occurs at least once for every 25 saccharide repeat units of the polysaccharide.

[0307] O-Acetylation. In some embodiments, the saccharides of the present invention are O-acetylated. In some embodiments, glycoconjugates comprise saccharides having a degree of O-acetylation of 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 75-100%, 80-100%, 90-100%, 50-90%, 60-90%, 70-90%, or 80-90%. In other embodiments, the degree of O-acetylation is ≧10%, ≧20%, ≧30%, ≧40%, ≧50%, ≧60%, ≧70%, ≧80%, or ≧90%, or about 100%. The degree of O-acetylation (%) refers to the percentage of a given sugar relative to 100% (where each repeat unit is fully acetylated relative to the acetylated structure).

[0308] In some embodiments, the glycoconjugate is prepared by reductive amination. In some embodiments, the glycoconjugate is a single-end-linked conjugated saccharide, which is directly covalently attached to the carrier protein. In some embodiments, the glycoconjugate is covalently attached to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

[0309] Reductive Amination. In one embodiment, saccharides are converted to saccharides by reductive amination (e.g., as described in U.S. Patent Application Publication Nos. 2006 / 0228380, 2007 / 0231340, 2007 / 0184071, and 2007 / 0184072, WO2006 / 110381, WO2008 / 079653, and and as described in WO2008 / 143709), are conjugated to a carrier protein.

[0310] Reductive amination involves (1) oxidation of a saccharide, and (2) reduction of the activated saccharide and a carrier protein to form a conjugate. The saccharide is optionally hydrolyzed prior to oxidation. Mechanical or chemical hydrolysis can be used. Chemical hydrolysis can be performed using acetic acid.

[0311] The oxidation step may involve reaction with periodic acid. As used herein, the term "periodic acid" refers to both periodate and periodic acid. The term also refers to metaperiodic acid (IO4 - ) and orthoperiodic acid (IO6 5- ) and various salts of periodic acid (e.g., sodium periodate and potassium periodate). In one embodiment, the polysaccharide is oxidized in the presence of metaperiodic acid, preferably in the presence of sodium periodate (NalO). In another embodiment, the polysaccharide is oxidized in the presence of orthoperiodic acid, preferably in the presence of periodic acid.

[0312] In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxide radical compound, such as a piperidine-N-oxy or pyrrolidine-N-oxy compound, in the presence of an oxidant that selectively oxidizes primary hydroxyls. In the reaction, the actual oxidant is an N-oxoammonium salt in the catalytic cycle. In one aspect, the stable nitroxyl or nitroxide radical compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. In one aspect, the stable nitroxyl or nitroxide radical compound has a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, the stable nitroxyl radical compound is TEMPO or a derivative thereof. In one aspect, the oxidant is a molecule having an N-halo moiety. In one aspect, the oxidant is selected from any one of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione, dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinane-2,4,6-trione, diiodoisocyanuric acid, and 1,3,5-triiodo-1,3,5-triazinane-2,4,6-trione. Preferably, the oxidant is N-chlorosuccinimide.

[0313] After the saccharide oxidation step, the saccharide is said to be activated, and is hereinafter referred to as "activated." The activated saccharide and carrier protein can be lyophilized (freeze-dried) independently (by separate lyophilization) or together (by co-lyophilization). In one embodiment, the activated saccharide and carrier protein are co-lyophilized. In another embodiment, the activated saccharide and carrier protein are lyophilized independently.

[0314] In one embodiment, lyophilization is carried out in the presence of a non-reducing sugar, contemplated non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit.

[0315] The next step in the conjugation process is the reduction of the activated saccharide and the carrier protein with a reducing agent to form the conjugate (so-called reductive amination). Suitable reducing agents are cyanoborohydrides such as sodium cyanoborohydride, sodium triacetoxyborohydride, or sodium or zinc borohydride in the presence of a Bronsted or Lewis acid, pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuM e'PrN-BH3, benzylamine-BH3, or an amine borane such as 5-ethyl-2-methylpyridine borane (PEMB), borane-pyridine, or a borohydride exchange resin. In one embodiment, the reducing agent is sodium cyanoborohydride.

[0316] In some embodiments, the reduction reaction is carried out in an aqueous solvent (e.g., selected from PBS, MES, HEPES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, bicine, or HEPB, at a pH of 6.0-8.5, 7.0-8.0, or 7.0-7.5), while in other embodiments, the reaction is carried out in an aprotic solvent. In some embodiments, the reduction reaction is carried out in DMSO (dimethyl sulfoxide) or DMF (dimethylformamide) solvent. DMSO or DMF solvent can be used to reconstitute the lyophilized activated polysaccharide and carrier protein.

[0317] At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugate, which can be capped using a suitable capping agent. In one embodiment, the capping agent is sodium borohydride (NaBH). After conjugation (reduction reaction and optional capping), the glycoconjugate may be purified (concentrated relative to the amount of polysaccharide-protein conjugate) by a variety of techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration, tangential flow filtration precipitation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. The glycoconjugate may be purified by diafiltration and / or ion exchange chromatography and / or size exclusion chromatography. In some embodiments, the glycoconjugate is purified by diafiltration or ion exchange chromatography or size exclusion chromatography. In one embodiment, the glycoconjugate is sterile filtered.

[0318] In a preferred embodiment, glycoconjugates of E. coli serotypes selected from any one of O25B, O1, O2, and O6 are prepared by reductive amination. In a preferred embodiment, glycoconjugates of E. coli serotypes O25B, O1, O2, and O6 are prepared by reductive amination.

[0319] In one aspect, the present invention provides a method for producing a medicament for a medicament comprising:

[0320] [ka]

[0321] wherein n is any integer equal to or greater than 1; 197In a preferred embodiment, n is an integer greater than or equal to 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and less than or equal to 200, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50. Any minimum number may be combined with any maximum number to define a range. Exemplary ranges include, for example, from 1 to 1000, from 10 to 1200, and from 12 to 1400. This includes from 31 to 90, more preferably from 40 to 90, and most preferably from 60 to 85.

[0322] In another aspect, the invention provides a carrier protein, e.g., CRM, conjugated to a saccharide having any one of the following structures shown in Table 1 (see also Figures 9A-9C and 10A-10B), where n is an integer equal to or greater than 1: 197 The present invention relates to a conjugate comprising:

[0323] Without wishing to be bound by theory or mechanism, it is believed that in some embodiments, stable conjugates require a level of saccharide antigen modification that is balanced with preserving the structural integrity of important immunogenic epitopes of the antigen.

[0324] Activation and Aldehyde Formation. In some embodiments, saccharides of the present invention are activated, resulting in the formation of aldehydes. In such embodiments in which saccharides are activated, percent activation (or degree of oxidation (DO)) (see, e.g., Example 32) refers to the moles of saccharide repeat unit per mole of aldehyde in the activated polysaccharide. For example, in some embodiments, saccharides are activated by periodate oxidation of vicinal diols in the polysaccharide repeat unit, resulting in the formation of aldehydes. Varying the molar equivalents (meq) of sodium periodate relative to the saccharide repeat unit and the temperature during oxidation results in different levels of degree of oxidation (DO).

[0325] The concentrations of sugars and aldehydes are usually determined by colorimetric assays. An alternative reagent is the combination of TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl radical)-N-chlorosuccinimide (NCS), which leads to the formation of aldehydes from primary alcohol groups.

[0326] In some embodiments, the activated saccharide has a degree of oxidation of 1 to 100 moles of saccharide repeat unit per mole of aldehyde in the activated saccharide, e.g., 2 to 80, 2 to 50, 3 to 30, and 4 to 25 moles. The degree of activation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, ≥ 20, ≥ 30, ≥ 40, ≥ 50, ≥ 60, ≥ 70, ≥ 80, or ≥ 90, or about 100. Preferably, the degree of oxidation (DO) is 5 to 50, more preferably 10 to 25. In one embodiment, the DO is 10 to 25. Any minimum number and any maximum number may be combined to define a range. The value of the degree of oxidation may be expressed as a percent activation. For example, in one embodiment, a DO value of 10 means that out of a total of 10 saccharide repeat units in the activated saccharide, one is an activated saccharide repeat unit, in which case a DO value of 10 can be expressed as 10% activation.

[0327] In some embodiments, the conjugates prepared by reductive amination chemistry comprise a carrier protein and a saccharide, wherein the saccharide is selected from the group consisting of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180 / C3)), Formula O6 (e.g., Formula O6:K2;K13;K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18B, Formula O18C, Formula O18D, Formula O18E, Formula O18F, Formula O18H, Formula O18H, Formula O18I ... ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O3 4, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O5 7, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula 62D1, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O129, Formula O130, Formula O131, Formula O132, Formula O133, Formula O134, Formula O135, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143 , formula O144, formula O145, formula O146, formula O147, formula O148, formula O149, formula O150, formula O151, formula O152, formula O153, formula O154, formula O155, formula O156, formula O157, formula O158, formula O159 , Formula O160, Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187. In some embodiments, the saccharide in the conjugate comprises a formula wherein n is an integer between 1 and 1000, between 5 and 1000, preferably between 31 and 100, more preferably between 35 and 90, and most preferably between 35 and 65.

[0328] Single-end-linked conjugates. In some embodiments, the conjugate is a single-end-linked conjugated saccharide, where the saccharide is covalently attached to the carrier protein at one end of the saccharide. In some embodiments, the single-end-linked conjugated saccharide has a terminal saccharide. For example, if one of the ends of the polysaccharide (the terminal saccharide residue) is covalently attached to the carrier protein, the conjugate is single-end-linked. In some embodiments, if the terminal saccharide residue of the polysaccharide is covalently attached to the carrier protein via a linker, the conjugate is single-end-linked. Such linkers can include, for example, a cystamine linker (A1), a 3,3'-dithiobis(propanoic acid dihydrazide) linker (A4), and a 2,2'-dithio-N,N'-bis(ethane-2,1-diyl)bis(2-(aminooxy)acetamide) linker (A6).

[0329] In some embodiments, the saccharide is conjugated to the carrier protein via a 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue to form a single-end-linked conjugate. See, e.g., Examples 26, 27, 28, and Figure 17.

[0330] In some embodiments, the conjugate is preferably not a bioconjugate. The term "bioconjugate" refers to a conjugate of an antigen, such as an O antigen (e.g., O25B), with a protein (e.g., a carrier protein) that is prepared in the background of a host cell, where the host cell machinery attaches the antigen to the protein (e.g., N-linkage). Glycoconjugates include not only bioconjugates but also conjugates of proteins with saccharide antigens (e.g., oligosaccharides and polysaccharides) that are prepared by techniques that do not require preparation of the conjugate in a host cell, such as conjugation by chemical bonding of the saccharide to the protein.

[0331] Thiol-activated saccharides. In some embodiments, the saccharides of the present invention are thiol-activated. In such embodiments in which the saccharide is thiol-activated, the percent activation is: This refers to the moles of thiol per saccharide repeat unit of the activated polysaccharide. The saccharide and thiol concentrations are typically determined by Ellman's assay for sulfhydryl quantification. For example, in some embodiments, the saccharide comprises 2-keto-3-deoxyoctanoic acid (KDO) activation with a disulfide amine linker. See, e.g., Example 10 and Figure 31. In some embodiments, the saccharide is covalently attached to the carrier protein via a divalent heterobifunctional linker (also referred to herein as a "spacer"). The linker preferably provides a thioether bond between the saccharide and the carrier protein, resulting in a glycoconjugate referred to herein as a "thioether glycoconjugate." In some embodiments, the linker further provides a carbamate bond and an amide bond, such as (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC). See, e.g., Example 21.

[0332] In some embodiments, the single-end linked conjugate comprises a carrier protein and a saccharide, wherein the saccharide is selected from the group consisting of Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180 / C3)), Formula O6 (e.g., Formula O6:K2;K13;K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, , Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55 , Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula 62D1, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O 90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O 103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O11 3, Formula O114, Formula O115, Formula O116, Formula O117, Formula O118, Formula O119, Formula O120, Formula O121, Formula O123, Formula O124, Formula O125, formula O126, formula O127, formula O128, formula O129, formula O130, formula O131, formula O132, formula O133, formula O134, formula O1 35, Formula O136, Formula O137, Formula O138, Formula O139, Formula O140, Formula O141, Formula O142, Formula O143, Formula O144, Formula O145,Formula O146, Formula O147, Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O 157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168 , Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187. In some embodiments, the saccharide in the conjugate comprises a formula where n is an integer of 1 to 1000, 5 to 1000, preferably 31 to 100, more preferably 35 to 90, and most preferably 35 to 65.

[0333] For example, in one embodiment, the single-end linked conjugate comprises a carrier protein and a nucleotide of Formula O8, Formula O9a, Formula O9, Formula O20ab, Formula O20ac, Formula O52, Formula O97, and Formula O1 01 (wherein n is an integer of 1 to 10).

[0334] F. eTEC conjugates In one aspect, the present invention broadly relates to glycoconjugates (including immunogenic compositions comprising such glycoconjugates) comprising the above-described E. coli-derived saccharides covalently conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer (e.g., as described in U.S. Pat. No. 9,517,274 and International Patent Publication No. 2014027302, the entire contents of which are incorporated by reference herein), as well as methods of preparing and using such glycoconjugates and immunogenic compositions. The glycoconjugates comprise saccharides covalently conjugated to a carrier protein via one or more eTEC spacers, wherein the saccharide is covalently conjugated to the eTEC spacer via a carbamate bond and the carrier protein is covalently conjugated to the eTEC spacer via an amide bond. The eTEC spacer contains seven linear atoms (ie, -C(O)NH(CH2)2SCH2C(O)-), providing stable thioether and amide bonds between the saccharide and the carrier protein.

[0335] The eTEC-binding glycoconjugates of the present invention have the general formula (I):

[0336] [ka]

[0337] where the atoms that make up the eTEC spacer are contained in the central box. In the glycoconjugate of the present invention, the saccharide may be a polysaccharide or an oligosaccharide.

[0338] The carrier protein incorporated into the glycoconjugates of the present invention is selected from the group of carrier proteins widely suitable for such purposes as further described herein or known to those of skill in the art. In certain embodiments, the carrier protein is a CRM 197 is.

[0339] In another aspect, the invention provides a method for making a glycoconjugate comprising a saccharide described herein conjugated to a carrier protein via an eTEC spacer, the method comprising the steps of: a) reacting the saccharide with a carbonic acid derivative in an organic solvent to form an activated saccharide; b) reacting the activated saccharide with cystamine or cysteamine or a salt thereof to form a thiolated saccharide; c) reacting the thiolated saccharide with a reducing agent to form an activated thiolated saccharide comprising one or more free sulfhydryl residues; and d) reacting the activated thiolated saccharide with one or more free sulfhydryl residues. and e) reacting the thiolated saccharide-carrier protein conjugate with an activated carrier protein containing a plurality of α-haloacetamide groups to form a thiolated saccharide-carrier protein conjugate; and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α-haloacetamide groups of the activated carrier protein, and / or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues of the activated thiolated saccharide to form an eTEC-linked glycoconjugate. and generating a

[0340] In many embodiments, the carbonate derivative is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT) or 1,1'-carbonyldiimidazole (CDI). Preferably, the carbonate derivative is CDT and the organic solvent is a polar aprotic solvent such as dim...

Claims

1. A recombinant mammalian cell comprising a polynucleotide encoding a polypeptide or fragment thereof derived from E. coli.

2. 2. The recombinant cell of claim 1, wherein the polypeptide is derived from E. coli fimbrial H (FimH).

3. The recombinant cell of claim 2 , wherein the polypeptide comprises a phenylalanine residue at the N-terminus of the polypeptide.

4. 3. The recombinant cell of claim 2, wherein the polypeptide comprises a phenylalanine residue within the first 20 residues of the N-terminus.

5. 3. The recombinant cell of claim 2, wherein the polypeptide comprises a phenylalanine residue at position 1 of the polypeptide.

6. 6. The recombinant cell of claim 5, wherein the polypeptide does not contain a glycine residue immediately preceding the phenylalanine residue at position 1 of the polypeptide.

7. 3. The recombinant cell of claim 2, wherein the polypeptide does not contain an N-glycosylation site at position 7 of the polypeptide.

8. 7. The recombinant cell of claim 6, wherein the polypeptide does not contain an Asn residue at position 7 of the polypeptide.

9. 9. The recombinant cell of claim 8, wherein the polypeptide comprises a residue at position 7 selected from the group consisting of Ser, Asp, Thr, and Gln.

10. 6. The recombinant cell of claim 5, wherein the polypeptide does not contain an N-glycosylation site at position 70 of the polypeptide.

11. 11. The recombinant cell of claim 10, wherein the polypeptide does not contain an Asn residue at position 70 of the polypeptide.

12. The recombinant cell of claim 10, wherein the polypeptide does not contain a Ser residue at position 70 of the polypeptide.

13. 2. The recombinant cell of claim 1, wherein said polypeptide comprises a residue substitution at an N-glycosylation site of said polypeptide selected from the group consisting of Ser, Asp, Thr, and Gln.

14. The recombinant cell of claim 13, wherein the N-glycosylation site comprises position N235 of the polypeptide.

15. 14. The recombinant cell of claim 13, wherein the N-glycosylation site comprises position N228 of the polypeptide.

16. 14. The recombinant cell of claim 13, wherein the N-glycosylation sites comprise positions N235 and N228 of the polypeptide.

17. The recombinant cell of claim 2 , wherein the polypeptide comprises SEQ ID NO:

3.

18. The recombinant cell of claim 2 , wherein the polypeptide comprises SEQ ID NO:

2.

19. 2. The recombinant cell of claim 1, wherein the polypeptide comprises an aliphatic hydrophobic amino acid residue at position 1 of the polypeptide.

20. 20. The recombinant cell of claim 19, wherein the aliphatic hydrophobic amino acid residue is selected from the group consisting of Ile, Leu, and Val.

21. The recombinant cell of claim 1 , wherein the polypeptide comprises a fragment of FimH.

22. 22. The recombinant cell of claim 21, wherein the polypeptide comprises the lectin domain of FimH.

23. 23. The recombinant cell of claim 22, wherein the lectin domain has a mass of approximately 17,022 daltons.

24. The recombinant cell of claim 1 , wherein the polypeptide is complexed with a FimC polypeptide or a fragment thereof.

25. 25. The recombinant cell of claim 24, wherein the FimC polypeptide or fragment thereof comprises a glycine residue at position 37 of the FimC polypeptide or fragment thereof.

26. The recombinant cell of claim 2 , wherein the polypeptide is in a low affinity conformation.

27. The recombinant cell of claim 2 , wherein the polypeptide is stabilized by FimG.

28. The recombinant cell of claim 2 , wherein the polypeptide is stabilized by a donor strand peptide of FimG (DsG).

29. 29. The recombinant cell of claim 28, wherein the polynucleotide sequence further encodes a linker sequence.

30. 30. The recombinant cell of claim 29, wherein the linker comprises between 4 and 15 amino acid residues.

31. 30. The recombinant cell of claim 29, wherein the linker comprises between 5 and 10 amino acid residues.

32. 30. The recombinant cell of claim 29, wherein the linker comprises seven amino acid residues.

33. 2. The recombinant cell of claim 1, wherein the polypeptide does not contain a signal peptide selected from the group consisting of a native FimH leader peptide, an influenza hemagglutinin signal peptide, and a human respiratory syncytial virus A (A2 strain) fusion glycoprotein F0 signal peptide.

34. 10. The method of claim 1, wherein the polypeptide comprises a mouse IgK signal peptide sequence. Recombinant cells.

35. The recombinant cell according to claim 1, wherein the polypeptide comprises any one signal peptide sequence selected from the signal peptide of the human IgG receptor FcRn large subunit p51 and the signal peptide of the human IL10 protein.

36. 3. The recombinant cell of claim 2, wherein the polypeptide comprises an arginine to proline mutation at amino acid position 60 (R60P) according to the numbering of SEQ ID NO:

3.

37. 2. The recombinant cell of claim 1, wherein the expression level of said polypeptide is higher than the expression level of a corresponding wild-type polypeptide expressed in the periplasm of a wild-type E. coli cell.

38. 2. The recombinant cell of claim 1, wherein the expression level of the polypeptide is greater than 10 mg / L.

39. The recombinant cell of claim 1 , wherein the polynucleotide sequence is integrated into the genomic DNA of the mammalian cell.

40. The recombinant cell of claim 1 , wherein the polynucleotide sequence is codon-optimized for expression in the cell.

41. The recombinant cell of claim 1 , wherein the cell is a human embryonic kidney cell.

42. 41. The recombinant cell of claim 40, wherein the human embryonic kidney cells comprise HEK293 cells.

43. 43. The recombinant cell of claim 42, wherein the HEK293 cell is selected from any one of a HEK293T cell, a HEK293TS cell, and a HEK293E cell.

44. The recombinant cell of claim 1 , wherein the cell is a CHO cell.

45. 45. The recombinant cell of claim 44, wherein the CHO cell is a CHO-K1 cell, a CHO-DUXB11 cell, a CHO-DG44 cell, or a CHO-S cell.

46. The recombinant cell of claim 1 , wherein the polypeptide is soluble.

47. The recombinant cell of claim 1 , wherein the polypeptide is secreted from the cell.

48. 3. The recombinant cell of claim 2, wherein the polypeptide comprises an N28Q substitution according to the numbering of SEQ ID NO:

1.

49. 3. The recombinant cell of claim 2, wherein the polypeptide comprises an N28D substitution according to the numbering of SEQ ID NO:

1.

50. 3. The recombinant cell of claim 2, wherein the polypeptide comprises an N28S substitution according to the numbering of SEQ ID NO:

1.

51. 3. The recombinant cell of claim 2, wherein the polypeptide comprises a substitution selected from any one of N28Q, V48C, and L55C according to the numbering of SEQ ID NO:

1.

52. 3. The recombinant cell of claim 2, wherein the polypeptide comprises the substitution N92S according to the numbering of SEQ ID NO:

1.

53. 2. The recombinant cell of claim 1, wherein the polypeptide or fragment thereof derived from FimH comprises a substitution selected from any one of V48C and L55C according to the numbering of SEQ ID NO:

1.

54. 10. A culture comprising the recombinant cell of claim 1, said culture being at least 5 liters in size.

55. 50. The culture of claim 49, wherein the yield of the polypeptide or fragment thereof is at least 0.05 g / L.

56. 56. The culture of claim 55, wherein the yield of the polypeptide or fragment thereof is at least 0.10 g / L.

57. 10. A method for producing a polypeptide or fragment thereof from E. coli, comprising the steps of expressing said polypeptide or fragment thereof by culturing the recombinant mammalian cell of claim 1 under suitable conditions, and recovering said polypeptide or fragment thereof.

58. 58. The method of claim 57, further comprising purifying the polypeptide or fragment thereof.

59. 58. The method of claim 57, wherein the cell comprises a nucleic acid encoding any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:

27.

60. 58. The method of claim 57, wherein the yield of the polypeptide or fragment thereof is at least 0.05 g / L.

61. 58. The method of claim 57, wherein the yield of the polypeptide or fragment thereof is at least 0.10 g / L.

62. A composition comprising a polypeptide having at least 70% identity to any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:

29.

63. 63. The composition of claim 62, further comprising a saccharide comprising a structure selected from the formula of any one of Table 1.

64. 64. The composition of claim 63, wherein the saccharide is covalently attached to a carrier protein.

65. The carrier protein is poly(L-lysine), CRM １９７ , diphtheria toxin fragment B (DTFB), DTFB C8, diphtheria toxoid (DT), tetanus toxoid (TT), fragment C of TT, pertussis toxoid, cholera toxoid, or any one of Pseudomonas aeruginosa exotoxin A, detoxified P. aeruginosa exotoxin A (EPA), maltose binding protein (MBP), detoxified S. aureus hemolysin A, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, and native glycoproteins of C. jejuni.

66. The carrier protein is a CRM １９７ 65. The composition of claim 64, wherein:

67. 65. The composition of claim 64, wherein the carrier protein is tetanus toxoid (TT).

68. 65. The composition of claim 64, wherein the carrier protein is poly(L-lysine).

69. 65. The composition of claim 64, wherein the saccharide is covalently attached to the carrier protein by reductive amination.

70. 65. The composition of claim 64, wherein the saccharide is covalently attached to the carrier protein by CDAP chemistry.

71. 65. The composition of claim 64, wherein the saccharide is covalently attached to the carrier protein by a single end-linked conjugation.

72. 65. The composition of claim 64, wherein the saccharide is covalently attached to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

73. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:27.

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