Escherichia coli FimH mutants and uses thereof

Abstract

To provide designs of mutated FimH polypeptides of Escherichia coli (E. coli), which result in improved biochemical properties and immunogenicity, compositions comprising such polypeptides, and uses thereof.SOLUTION: Provided is a pharmaceutical composition comprising a mutated FimH polypeptide, which comprises at least one amino acid mutation relative to the amino acid sequence of the wild-type FimH polypeptide, and a pharmaceutically acceptable carrier.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Technical field of the invention The present disclosure relates to mutated Escherichia coli FimH polypeptides and methods of use thereof. [Background technology]

[0002] Urinary tract infections (UTIs) affect one in five women at least once in their lifetime, causing significant morbidity and mortality and placing a substantial burden on healthcare systems. While several different bacteria can cause UTIs, the most common cause (90–95% of cases) is the gram-negative bacterium Escherichia coli (E. coli). Most E. coli UTIs are caused by uropathogenic E. coli (UPEC), which colonize the gastrointestinal tract and migrate from the fecal flora to the urogenital tract, where they adhere to host urothelial cells, thereby establishing a reservoir for ascending infection of the urinary tract. Adhesion is facilitated by fimbrial adhesins, including type 1 fimbriae, that bind to mannosylated glycoproteins in the epithelial layer or are secreted into the urine. Type 1 fimbriae are highly conserved among clinical UPEC isolates and are encoded by a cluster of genes called fim, which encode accessory proteins (FimC, FimD), various structural subunits (FimE, FimF, FimG), and an adhesin called FimH. FimH is essential for all features of UTI infection in a mouse model that mimics aspects of human bladder infection (Hannan et al., PLoS Pathog. 2010 Aug 12;6(8):e1001042; doi:10.1371 / journal.ppat.1001042; Schwartz et al., Infect Immun. 2011 Oct;79(10):4250-9. doi:10.1128 / IAI.05339-11). Small molecule inhibitors that target FimH by mimicking mannosylated receptors further substantiate the role of FimH in UTI and show promise as therapeutic agents in animal models (Cusumano CK et al., Sci Transl Med. 2011;3(109):109ra115. doi:10.1126 / scitranslmed.3003021).Additionally, FimH is under positive selection in E. coli human cystitis isolates (Chen SL et al., Proc Natl Acad Sci U S A. 2009 Dec 29;106(52):22439-44. doi:10.1073 / pnas.0902179106), and positively selected residues can affect virulence in a mouse model of cystitis (Schwartz, DJ et al., Proc Natl Acad Sci U S A. 110, 15530-15537, doi:10.1073 / pnas.1315203110(2013)).

[0003] FimH contains two domains: a lectin-binding domain (FimH) responsible for binding to mannosylated glycoproteins; LD ) and a pilin domain. The pilin domain functions to link FimH to other structural subunits of the pilus, such as FimG, through a mechanism called donor strand exchange (Le Trong, I et al., J. Struct Biol. 2010 Dec;172(3):380-8. doi:10.1016 / j.jsb.2010.06.002). The FimH pilin domain forms an incomplete immunoglobulin fold that generates a groove that provides a binding site for the N-terminal β-strand of FimG, forming a strong intermolecular link between FimH and FimG. FimH LDAlthough FimH can be expressed in a soluble, stable form, full-length FimH is unstable on its own (Vetsch, M. et al., J. Mol. Biol. 322:827-840 (2002); Barnhart, M. et al., J. Mol. Biol. 322:827-840 (2002); Barnhart, M. et al., Proc. Natl. Acad. Sci. USA (2000) Jul. 5;97(14):7709-14), either in peptide form or as a fusion protein, unless complexed with the chaperone FimC or complemented with the donor strand peptide of FimG (Barnhart, M. et al., Proc. Natl. Acad. Sci. USA (2000) Jul. 5;97(14):7709-14; Sauer, M. et al., Nat. Commun. (2016) Mar. 7;7:10738; Barnhart, M. et al., J. Bacteriol. 2003 May;185(9):2723-30). The design and expression of a full-length FimH molecule by linking the FimG donor peptide to full-length FimH via a glycine-serine linker has been previously described (PCT International Publication No. WO2021 / 084429, published May 6, 2021), which was designated FimH-DSG. Summary of the Invention [Problem to be solved by the invention]

[0004] FimH LD is considered to be a poor immunogen in terms of its ability to stimulate functional immunogenicity. Several studies have shown that binding antibody titers are higher in FimH with and without adjuvants. LDStudies suggest that FimH can be elicited by IgG, but functional neutralization titers were only observed in the presence of adjuvants (PCT International Publication No. WO2021 / 084429, published May 6, 2021). Studies suggest that locking FimH in an open conformation with reduced affinity for mannoside ligands improves functional immunogenicity (Kisiela, DI et al., Proc Natl Acad Sci USA 110, 19089-19094 (2013)). Thus, there is a need in the art for novel FimH variants with reduced affinity for mannoside ligands and improved biochemical properties that result in improved functional immunogenicity compared to wild-type FimH. [Means for solving the problem]

[0005] The present disclosure relates to the design of E. coli FimH mutant polypeptides that result in improved biochemical properties and immunogenicity, compositions comprising such polypeptides, and uses thereof. For example, in one aspect, the present disclosure provides a mutated FimH polypeptide comprising at least one amino acid mutation compared to the amino acid sequence of a wild-type FimH polypeptide, wherein the mutation position is selected from the group consisting of F1, P12, G14, G15, G16, A18, P26, V27, V28, Q32, N33, L34, V35, R60, S62, Y64, G65, L68, F71, T86, L107, Y108, L109, V112, S113, A115, G116, V118, A119, A127, L129, Q133, F144, V154, V155, V156, P157, T158, V163 and V185, and the amino acid positions are numbered according to SEQ ID NO: 59.

[0006] In a further embodiment, the mutated FimH polypeptide is selected from the group consisting of F1I; F1L; F1V; F1M; F1Y; F1W; P12C; G14C; G15A; G15P; G16A; G16P; A18C; P26C; V27A; V27C; V28C; Q32C; N33C; L34C; L34N; L34S; L34T; L34D; L34E; L34K; L34R; V35C; R60P; S62C; Y64C; G65A; L68C; F71C; T86C; L10 The FimH polypeptide may comprise at least one mutation selected from the group consisting of: 7C; Y108C; L109C; V112C; S113C; A115V; G116C; V118C; A119C; A119N; A119S; A119T; A119D; A119E; A119K; A119R; A127C; L129C; Q133K; F144C; V154C; V156C; P157C; T158C; V163I; and V185I, or any combination thereof. For example, the mutated FimH polypeptide may comprise the mutations G15A and G16A. Further, for example, the mutated FimH polypeptide may comprise the mutations P12C and A18C. Further, for example, the mutated FimH polypeptide may comprise the mutations G14C and F144C. Further, for example, the mutated FimH polypeptide comprises mutations P26C and V35C. Further, for example, the mutated FimH polypeptide comprises mutations P26C and V154C. Further, for example, the mutated FimH polypeptide comprises mutations P26C and V156C. Further, for example, the mutated FimH polypeptide comprises mutations V27C and L34C. Further, for example, the mutated FimH polypeptide comprises mutations V28C and N33C. Further, for example, the mutated FimH polypeptide comprises mutations V28C and P157C. Further, for example, the mutated FimH polypeptide comprises mutations Q32C and Y108C. Further, for example, the mutated FimH polypeptide comprises mutations N33C and L109C. Further, for example, the mutated FimH polypeptide comprises mutations N33C and P157C. Further, for example, the mutated FimH polypeptide comprises the mutations V35C and L107C.Further, for example, the mutated FimH polypeptide comprises mutations V35C and L109C. Further, for example, the mutated FimH polypeptide comprises mutations S62C and T86C. Further, for example, the mutated FimH polypeptide comprises mutations S62C and L129C. Further, for example, the mutated FimH polypeptide comprises mutations Y64C and L68C. Further, for example, the mutated FimH polypeptide comprises mutations Y64C and A127C. Further, for example, the mutated FimH polypeptide comprises mutations L68C and F71C. Further, for example, the mutated FimH polypeptide comprises mutations V112C and T158C. Further, for example, the mutated FimH polypeptide comprises mutations S113C and G116C. Further, for example, the mutated FimH polypeptide comprises mutations S113C and T158C. Further, for example, the mutated FimH polypeptide comprises mutations V118C and V156C. Further, for example, the mutated FimH polypeptide comprises mutations A119C and V155C. Further, for example, the mutated FimH polypeptide comprises mutations L34N and V27A. Further, for example, the mutated FimH polypeptide comprises mutations L34S and V27A. Further, for example, the mutated FimH polypeptide comprises mutations L34T and V27A. Further, for example, the mutated FimH polypeptide comprises mutations L34D and V27A. Further, for example, the mutated FimH polypeptide comprises mutations L34E and V27A. Further, for example, the mutated FimH polypeptide comprises mutations L34K and V27A. Further, for example, the mutated FimH polypeptide comprises mutations L34R and V27A. Further, for example, the mutated FimH polypeptide comprises mutations A119N and V27A. Further, for example, the mutated FimH polypeptide comprises the mutations A119S and V27A.Further, for example, the mutated FimH polypeptide comprises the mutations A119T and V27A.Further, for example, the mutated FimH polypeptide comprises mutations A119D and V27A. Further, for example, the mutated FimH polypeptide comprises mutations A119E and V27A. Further, for example, the mutated FimH polypeptide comprises mutations A119K and V27A. Further, for example, the mutated FimH polypeptide comprises mutations A119R and V27A. Further, for example, the mutated FimH polypeptide comprises mutations G15A and V27A. Further, for example, the mutated FimH polypeptide comprises mutations G16A and V27A. Further, for example, the mutated FimH polypeptide comprises mutations G15P and V27A. Further, for example, the mutated FimH polypeptide comprises mutations G16P and V27A. Further, for example, the mutated FimH polypeptide comprises mutations G15A, G16A, and V27A. Further, for example, the mutated FimH polypeptide comprises the mutations G65A and V27A. Further, for example, the mutated FimH polypeptide comprises the mutations V27A and Q133K. Further, for example, the mutated FimH polypeptide comprises the mutations G15A, G16A, V27A, and Q133K. Further, for example, the mutated FimH polypeptide comprises any one of SEQ ID NOs: 2-58 and 60-64. Further, for example, isolated mutated FimH polypeptides disclosed herein are provided.

[0007] In a further example, the present disclosure provides a pharmaceutical composition comprising (i) a mutated FimH polypeptide disclosed herein, and (ii) a pharmaceutically acceptable carrier.

[0008] In a further example, the present disclosure provides an immunogenic composition comprising a mutated FimH polypeptide disclosed herein. For example, the immunogenic composition further comprises at least one additional antigen, such as a polysaccharide or sugar conjugate or protein. Furthermore, for example, the immunogenic composition further comprises at least one adjuvant.

[0009] In a further example, the present disclosure provides a nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence of a mutated FimH polypeptide disclosed herein.

[0010] In a further example, the present disclosure provides a mutated FimH polypeptide as disclosed herein, wherein the polypeptide is immunogenic.

[0011] The present disclosure further provides recombinant mammalian cells comprising a polynucleotide encoding a mutated FimH polypeptide disclosed herein.

[0012] The present disclosure further provides a culture comprising a recombinant cell disclosed herein, wherein the culture is at least 5 liters, at least 10 liters, at least 20 liters, at least 50 liters, at least 100 liters, at least 200 liters, at least 500 liters, at least 1000 liters, or at least 2000 liters in size.

[0013] The present disclosure further provides methods for producing a mutated FimH polypeptide disclosed herein, comprising culturing a recombinant mammalian cell disclosed herein under suitable conditions, thereby expressing the polypeptide; and collecting the polypeptide.

[0014] The present disclosure further provides a method for (i) inducing an immune response in a subject against extraintestinal pathogenic Escherichia coli (E. coli), or (ii) inducing opsonophagocytosis and / or the production of neutralizing antibodies specific to extraintestinal pathogenic Escherichia coli (E. coli), comprising administering to the subject an effective amount of a composition disclosed herein. In one example, the subject is at risk of developing a urinary tract infection. In a further example, the subject is at risk of developing bacteremia. In a further example, the subject is at risk of developing sepsis. In another example, the subject is at risk of developing Crohn's disease.

[0015] The present disclosure further provides a method of eliciting an immune response against E. coli in a mammal, comprising administering to the mammal an effective amount of a composition disclosed herein. In one example, the immune response comprises opsonophagocytosis and / or neutralizing antibodies against E. coli. In a further example, the immune response protects the mammal from E. coli infection.

[0016] The present disclosure further provides a method of preventing, treating, or ameliorating a bacterial infection, disease, or condition in a subject, comprising administering to the subject an immunologically effective amount of a composition disclosed herein. [Brief explanation of the drawings]

[0017] [Figure 1] 1A and 1B show circular dichroism spectra of FimHLD and the FimH-DSG mutant, where Fig. 1A shows the circular dichroism spectrum in the near UV region and Fig. 1B shows the circular dichroism spectrum in the far UV region. [Figure 2] FIG. 2 shows the relative immunogenicity of FimHLD variants in the yeast mannan neutralization assay in PD3. [Figure 3] Figures 3A-3B show the immunogenicity of FimHLD and FimH-DSG mutants in yeast mannan neutralization assays at PD2 (Figure 3A) and PD3 (Figure 3B). [Figure 4] FIG. 4 is a diagram showing the major purification steps utilized in the isolation of wild-type and mutated forms of His-tagged FimH-DsG. [Figure 5] 5A and 5B show the purification profile of FimH-DSG WT. Figure 5A shows the elution profile of FimH-DSG WT on an SP-Sepharose column, and Figure 5B shows SDS-PAGE analysis of the eluted fractions. [Figure 6]6A and 6B show the purification profile of the FimH-DSG G15A G16A V27A mutant, where Fig. 6A shows the elution profile of the FimH-DSG G15A G16A V27A mutant on an SP-Sepharose column, and Fig. 6B shows SDS-PAGE analysis of the eluted fractions. [Figure 7] 7A-7B show analytical SECs of FimH-DSG proteins. The analytical SEC of FimH-DSG G15A G16A V27A is shown in FIG. 7A, and the analytical SEC of wild-type FimH-DSG WT is shown in FIG. 7B. [Figure 8] FIG. 8 shows a schematic representation of the mechanism of FimH HMW complex formation. [Figure 9] FIG. 9 shows the immunization schedule and subsequent challenges for non-human primates, as described in Example 21 herein. [Figure 10] Figure 10 shows the rise in O-antigen serotype-specific antibodies after vaccination of NHPs with the FimH-DSG G15A G16A V27A mutant + tetravalent Escherichia coli (E. coli) O-antigens (O1a, O2, O6, and O25b). Legend: Placebo (circles); FimH-DSG G15A G16A V27A (squares); FimH-DSG G15A G16A V27A + tetravalent O-antigen (triangles). [Figure 11] Figures 11A-11B show that immunization with FimH-DSG G15A G16A V27A + / - 4plex O antigen elicits a strong functional anti-FimH antibody response. Figure 11A shows the results of a direct Luminex FimH IgG assay, and Figure 11B shows the results of an E. coli binding inhibition assay. As used herein, the term "4plex" has the same meaning as and is interchangeable with the term "tetravalent." [Figure 12]Figure 12 shows that bacteriuria is reduced in vaccinated non-human primates (NHPs) after challenge as described in Example 21. Legend: placebo (circles); FimH-DSG G15A G16A V27A (squares); FimH-DSG G15A G16A V27A + tetravalent O antigen (triangles); *p≦0.007 compared to the placebo group. [Figure 13] Figures 13A-13C show that biomarkers of infection are reduced in vaccinated NHPs after challenge with three groups: placebo, FimH-DSG G15A G16A V27A alone, and FimH-DSG G15A G16A V27A + tetravalent O antigen, as described in Example 21. Figure 13A shows quantification of MPO in urine, Figure 13B shows quantification of IL-8 in urine, and Figure 13C shows the percentage of animals with increased PMNs in urine sediment. Legend: placebo (circles); FimH-DSG G15A G16A V27A (squares); FimH-DSG G15A G16A V27A + tetravalent O antigen (triangles). DETAILED DESCRIPTION OF THE INVENTION

[0018] Sequence Identifier SEQ ID NO: 1 is the wild-type E. coli FimH LD The amino acid sequence of (FimHLD_WT) is shown below. SEQ ID NO: 2 sets forth the amino acid sequence of mutant E. coli FimHLD_G65A_V27A. SEQ ID NO: 3 sets forth the amino acid sequence of mutant E. coli FimHLD_F1I. SEQ ID NO: 4 sets forth the amino acid sequence of mutant E. coli FimHLD_F1L. SEQ ID NO: 5 sets forth the amino acid sequence of mutant E. coli FimHLD_F1V. SEQ ID NO: 6 sets forth the amino acid sequence of mutant E. coli FimHLD_F1M. SEQ ID NO: 7 sets forth the amino acid sequence of mutant E. coli FimHLD_F1Y. SEQ ID NO: 8 sets forth the amino acid sequence of mutant E. coli FimHLD_F1W. SEQ ID NO: 9 sets forth the amino acid sequence of mutant E. coli FimHLD_Q133K. SEQ ID NO: 10 sets forth the amino acid sequence of mutant E. coli FimHLD_G15A. SEQ ID NO: 11 sets forth the amino acid sequence of mutant E. coli FimHLD_G15P. SEQ ID NO: 12 sets forth the amino acid sequence of mutant E. coli FimHLD_G16A. SEQ ID NO: 13 sets forth the amino acid sequence of mutant E. coli FimHLD_G16P. SEQ ID NO: 14 sets forth the amino acid sequence of mutant E. coli FimHLD_G15A_G16A. SEQ ID NO: 15 sets forth the amino acid sequence of mutant E. coli FimHLD_R60P. SEQ ID NO: 16 sets forth the amino acid sequence of mutant E. coli FimHLD_G65A. SEQ ID NO: 17 sets forth the amino acid sequence of mutant E. coli FimHLD_P12C_A18C. SEQ ID NO: 18 sets forth the amino acid sequence of mutant E. coli FimHLD_G14C_F144C. SEQ ID NO: 19 sets forth the amino acid sequence of mutant E. coli FimHLD_P26C_V35C. SEQ ID NO: 20 sets forth the amino acid sequence of mutant E. coli FimHLD_P26C_V154C. SEQ ID NO: 21 sets forth the amino acid sequence of mutant E. coli FimHLD_P26C_V156C. SEQ ID NO: 22 sets forth the amino acid sequence of mutant E. coli FimHLD_V27C_L34C. SEQ ID NO: 23 sets forth the amino acid sequence of mutant E. coli FimHLD_V28C_N33C. SEQ ID NO: 24 sets forth the amino acid sequence of mutant E. coli FimHLD_V28C_P157C. SEQ ID NO: 25 sets forth the amino acid sequence of mutant E. coli FimHLD_Q32C_Y108C. SEQ ID NO: 26 sets forth the amino acid sequence of mutant E. coli FimHLD_N33C_L109C. SEQ ID NO: 27 sets forth the amino acid sequence of mutant E. coli FimHLD_N33C_P157C. SEQ ID NO: 28 sets forth the amino acid sequence of mutant E. coli FimHLD_V35C_L107C. SEQ ID NO: 29 sets forth the amino acid sequence of mutant E. coli FimHLD_V35C_L109C. SEQ ID NO: 30 sets forth the amino acid sequence of mutant E. coli FimHLD_S62C_T86C. SEQ ID NO: 31 sets forth the amino acid sequence of mutant E. coli FimHLD_S62C_L129C. SEQ ID NO: 32 sets forth the amino acid sequence of mutant E. coli FimHLD_Y64C_L68C. SEQ ID NO: 33 sets forth the amino acid sequence of mutant E. coli FimHLD_Y64C_A127C. SEQ ID NO: 34 sets forth the amino acid sequence of mutant E. coli FimHLD_L68C_F71C. SEQ ID NO: 35 sets forth the amino acid sequence of mutant E. coli FimHLD_V112C_T158C. SEQ ID NO: 36 sets forth the amino acid sequence of mutant E. coli FimHLD_S113C_G116C. SEQ ID NO: 37 sets forth the amino acid sequence of mutant E. coli FimHLD_S113C_T158C. SEQ ID NO: 38 sets forth the amino acid sequence of mutant E. coli FimHLD_V118C_V156C. SEQ ID NO: 39 sets forth the amino acid sequence of mutant E. coli FimHLD_A119C_V155C. SEQ ID NO: 40 sets forth the amino acid sequence of mutant E. coli FimHLD_L34N_V27A. SEQ ID NO: 41 sets forth the amino acid sequence of mutant E. coli FimHLD_L34S_V27A. SEQ ID NO: 42 sets forth the amino acid sequence of mutant E. coli FimHLD_L34T_V27A. SEQ ID NO: 43 sets forth the amino acid sequence of mutant E. coli FimHLD_A119N_V27A. SEQ ID NO: 44 sets forth the amino acid sequence of mutant E. coli FimHLD_A119S_V27A. SEQ ID NO: 45 sets forth the amino acid sequence of mutant E. coli FimHLD_A119T_V27A. SEQ ID NO: 46 sets forth the amino acid sequence of mutant E. coli FimH-DSG_A115V. SEQ ID NO: 47 sets forth the amino acid sequence of mutant E. coli FimH-DSG_V163I. SEQ ID NO: 48 sets forth the amino acid sequence of mutant E. coli FimH-DSG_V185I. SEQ ID NO: 49 sets forth the amino acid sequence of mutant E. coli FimH-DSG_DSG_V3I. SEQ ID NO: 50 sets forth the amino acid sequence of mutant E. coli FimHLD_G15A_V27A. SEQ ID NO: 51 sets forth the amino acid sequence of mutant E. coli FimHLD_G16A_V27A. SEQ ID NO: 52 sets forth the amino acid sequence of mutant E. coli FimHLD_G15P_V27A. SEQ ID NO: 53 sets forth the amino acid sequence of mutant E. coli FimHLD_G16P_V27A. SEQ ID NO: 54 sets forth the amino acid sequence of mutant E. coli FimHLD_G15A_G16A_V27A. SEQ ID NO: 55 sets forth the amino acid sequence of mutant E. coli FimHLD_V27A_R60P. SEQ ID NO: 56 sets forth the amino acid sequence of mutant E. coli FimHLD_G65A_V27A. SEQ ID NO: 57 sets forth the amino acid sequence of mutant E. coli FimHLD_V27A_Q133K. SEQ ID NO: 58 sets forth the amino acid sequence of mutant E. coli FimHLD_G15A_G16A_V27A_Q133K. SEQ ID NO: 59 sets forth the amino acid sequence of wild-type E. coli full-length FimH (FimH-DSG_WT) containing the donor strand FimG peptide connected via a linker. SEQ ID NO: 60 sets forth the amino acid sequence of mutant E. coli FimH-DSG_V27A. SEQ ID NO: 61 sets forth the amino acid sequence of mutant E. coli FimH-DSG_G15A_V27A. SEQ ID NO: 62 sets forth the amino acid sequence of mutant E. coli FimH DSG_G15A_G16A_V27A. SEQ ID NO: 63 sets forth the amino acid sequence of mutant E. coli FimH DSG_V27A_Q133K. SEQ ID NO: 64 sets forth the amino acid sequence of mutant E. coli FimH DSG_G15A_G16A_V27A_Q133K. SEQ ID NO: 65 sets forth the amino acid sequence of the mouse Ig kappa signal peptide sequence. SEQ ID NOs: 66-108 set forth the amino acid and nucleic acid sequences of nanostructure-associated polypeptides or fragments thereof. SEQ ID NO: 109 - primer for PCR. SEQ ID NO: 110 - primer for PCR. SEQ ID NO: 111 - PCR probe. SEQ ID NO: 112 sets forth the O25b 2401 WzzB amino acid sequence. SEQ ID NO: 113 sets forth the O25a:K5:H1 WzzB amino acid sequence. SEQ ID NO: 114 sets forth the O25a ETEC ATCC WzzB amino acid sequence. SEQ ID NO: 115 sets forth the K12 W3110 WzzB amino acid sequence. SEQ ID NO: 116 sets forth the Salmonella LT2 WzzB amino acid sequence. SEQ ID NO: 117 sets forth the O25b 2401 FepE amino acid sequence. SEQ ID NO: 118 sets forth the O25a:K5:H1 FepE amino acid sequence. SEQ ID NO: 119 sets forth the O25a ETEC ATCC FepE amino acid sequence. SEQ ID NO: 120 sets forth the O157 FepE amino acid sequence. SEQ ID NO: 121 sets forth the Salmonella LT2 FepE amino acid sequence. SEQ ID NO: 122 sets forth the primer sequence for LT2wzzB_S. SEQ ID NO: 123 sets forth the primer sequence for LT2wzzB_AS. SEQ ID NO: 124 sets forth the primer sequence for O25bFepE_S. SEQ ID NO: 125 sets forth the primer sequence for O25bFepE_A. SEQ ID NO: 126 sets forth the primer sequence for wzzB P1_S. SEQ ID NO: 127 sets forth the primer sequence for wzzB P2_AS. SEQ ID NO: 128 sets forth the primer sequence for wzzB P3_S. SEQ ID NO: 129 sets forth the primer sequence for wzzB P4_AS. SEQ ID NO: 130 sets forth the primer sequence for O157 FepE_S. SEQ ID NO: 131 sets forth the primer sequence for O157 FepE_AS. SEQ ID NO: 132 sets forth the primer sequence for pBAD33_adaptor_S. SEQ ID NO: 133 sets forth the primer sequence for pBAD33_adaptor_AS. SEQ ID NO: 134 sets forth the primer sequence for JUMPSTART_r. SEQ ID NO: 135 sets forth the primer sequence for gnd_f. SEQ ID NO: 136 sets forth the amino acid sequence of the human IgG receptor FcRn large subunit p51 signal peptide. SEQ ID NO: 137 sets forth the amino acid sequence of the human IL10 protein signal peptide. SEQ ID NO: 138 sets forth the amino acid sequence of the human respiratory syncytial virus A (strain A2) fusion glycoprotein F0 signal peptide. SEQ ID NO: 139 sets forth the amino acid sequence of the influenza A hemagglutinin signal peptide. SEQ ID NOs: 140-147 list the SignalP 4.1 (DTU Bioinformatics) sequences from various species used for signal peptide prediction.

[0019] Detailed Description The present disclosure relates to methods of using Escherichia coli (E. coli) FimH mutant polypeptides (variants), compositions comprising FimH variants, methods for producing and purifying FimH variants, nucleic acids encoding FimH variants, host cells comprising such nucleic acids, and compositions comprising FimH variants.

[0020] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referencing each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. Any method described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further explain the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0021] Several documents are cited throughout the text of this disclosure. Each of the documents cited herein (including any patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated herein by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0022] definition As used herein, the term "about" means approximately or approximately, and in the context of numerical values ​​or ranges described herein, means, in one embodiment, ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0023] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," and "the" and similar references should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0024] A "fragment" in reference to an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A fragment truncated at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. A fragment truncated at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that functions to initiate translation. A fragment of an amino acid sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0025] As used herein, the terms "wild-type" or "WT" or "native" refer to an amino acid sequence found in nature, including allelic variants. A wild-type amino acid sequence, peptide, or protein has an amino acid sequence that has not been intentionally modified.

[0026] As used herein, reference to a "variant" or "mutant" of an amino acid sequence (peptide, protein, or polypeptide) or a "mutated" polypeptide includes amino acid insertion variants / mutants, amino acid addition variants / mutants, amino acid deletion variants / mutants, and / or amino acid substitution variants / mutants. The term "variant" or "mutant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those of natural origin. The term "variant" or "mutant" particularly includes fragments of the amino acid sequence.

[0027] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion is also possible with appropriate screening of the resulting products. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can occur at any position in the protein. Amino acid deletion variants containing deletions at the N- and / or C-terminal ends of a protein are also called N- and / or C-terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides, and / or to replacing amino acids with other amino acids with similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one member of a family of amino acids that are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; lysine, arginine; and Phenylalanine, tyrosine.

[0028] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is preferably provided over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably provided for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 consecutive amino acids in some embodiments. In some embodiments, the degree of similarity or identity is provided for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using a best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, matrix:Blosum62, gap open 10.0, gap extension 0.5.

[0029] As used herein, "sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between these sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between these sequences.

[0030] The terms "% identical", "% identity" or similar terms are intended to refer in particular to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences to be compared. The percentage is purely statistical, and the differences between the two sequences can be, but are not necessarily, randomly distributed over the entire length of the sequences to be compared. Comparison of two sequences is usually carried out by comparing these sequences after optimal alignment over a segment or "window of comparison" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, available at the National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include (i) an expectation threshold set to 10; (ii) a word size set to 28; (iii) maximum matches in the query range set to 0; (iv) match / mismatch scores set to 1, -2; (v) gap costs set to linear; and (vi) a filter for low complexity regions being used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include (i) an expectation threshold set to 10; (ii) a word size set to 3; (iii) maximum matches in the query range set to 0; (iv) a matrix set to BLOSUM62; (v) gap costs set to presence: 11, extension: 1; and (vi) a conditional composition score matrix adjustment.

[0031] Percentage identity is obtained by determining the number of corresponding identical positions in the sequences to be compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0032] In some embodiments, the degree of similarity or identity is provided for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence.For example, if the reference nucleic acid sequence is 200 nucleotides, the degree of identity is provided for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 nucleotides, in some embodiments, consecutive nucleotides.In some embodiments, the degree of similarity or identity is provided for the entire length of the reference sequence.

[0033] According to the present disclosure, a homologous amino acid sequence exhibits at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98%, or at least 99% identity of amino acid residues. The amino acid sequence variants / mutants described herein can be easily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be easily prepared with the aid of known peptide synthesis techniques, such as, for example, by solid-phase synthesis and similar methods.

[0034] In one embodiment, a fragment or variant / mutant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant / mutant" of an amino acid sequence refers to any fragment or variant / mutant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to an antigen or antigenic sequence, one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant / mutant," as used herein, particularly refers to a variant / mutant molecule or sequence that contains an amino acid sequence in which one or more amino acids have been altered compared to the amino acid sequence of the parent molecule or sequence, yet is still capable of performing one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response. In one embodiment, the alterations in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, for example, the immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments, the immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0035] As used herein, "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0036] I. E. coli FimH Polypeptide Fimbrial adhesins, including type 1 fimbriae, bind to mannosylated glycoproteins in the epithelial layer or are secreted into the urine. Type 1 fimbriae are highly conserved among clinical UPEC isolates and are encoded by a cluster of genes called fim, which encode accessory proteins (FimC, FimD), various structural subunits (FimE, FimF, FimG), and an adhesin called FimH. FimH contains two domains: a lectin-binding domain (FimH) responsible for binding to mannosylated glycoproteins; LD ), and a pilin domain. The pilin domain functions to link FimH to other structural subunits of the pilus, such as FimG, by a mechanism called donor strand exchange. The FimH pilin domain forms an incomplete immunoglobulin fold, resulting in a groove that provides a binding site for the N-terminal β-strand of FimG, forming a strong intermolecular link between FimH and FimG. FimH LD Although FimH can be expressed in a soluble, stable form, full-length FimH is unstable on its own unless complexed with the chaperone FimC, either in peptide form or as a fusion protein, or complemented with the donor chain peptide of FimG. Therefore, expression of a stable full-length FimH molecule was enabled by linking the FimG donor peptide to the C-terminus of full-length FimH via a glycine-serine linker, which is designated FimH-DSG.

[0037] In one aspect, the present disclosure provides mutated FimH polypeptides, such as those shown in Table 1. Such variants provide mutations in the amino acid sequence compared to the amino acid sequence of the corresponding wild-type (WT) FimH polypeptide. In some aspects, such variants are immunogenic to the wild-type FimH protein or to bacteria expressing the wild-type FimH polypeptide. In certain aspects, FimH variants possess certain beneficial characteristics, such as increased immunogenic properties, compared to the corresponding wild-type FimH polypeptide.

[0038] As used herein, the term "FimH polypeptide" refers to any domain of a full-length wild-type E. coli FimH polypeptide, any combination of domains of a full-length wild-type E. coli FimH polypeptide, or a full-length E. coli FimH polypeptide, or a fragment of any of these. For example, in one embodiment, the present disclosure provides a mutated FimH polypeptide. LD The present disclosure provides a novel FimH polypeptide that is a mutated FimH polypeptide or a FimH-DSG polypeptide, which has reduced affinity for mannoside ligands (verified by biochemical and biophysical analyses) that improves functional immunogenicity. LD and wild-type FimH for the FimH-DSG mutant. LD Evaluation of the neutralizing response of these mutants compared to the wild-type strains is described.

[0039] The introduced amino acid mutations in the FimH mutant polypeptide can include amino acid substitutions, deletions, or additions. In some embodiments, the only mutations in the amino acid sequence of the FimH polypeptide mutant are amino acid substitutions compared to the wild-type FimH protein.

[0040] [Table 1-1]

[0041] [Table 1-2]

[0042] [Table 1-3]

[0043] [Table 1-4]

[0044] [Table 1-5]

[0045] [Table 1-6]

[0046] The amino acid sequence of the wild-type FimH polypeptide is well known in the art. LD The amino acid sequence of the domain is provided herein as SEQ ID NO: 1. A full-length wild-type FimH polypeptide comprising the FimG donor peptide linked to the C-terminus of full-length FimH via a glycine-serine linker is provided herein as SEQ ID NO: 59. Nucleic acid sequences encoding such amino acid sequences are also well known in the art.

[0047] In one aspect of the present disclosure, certain mutated FimH polypeptides confer a reduced affinity for mannoside ligands, resulting in a locked-open conformation that results in improved functional immunogenicity. Thus, such FimH mutants may be useful as antigens in immunogenic compositions, such as vaccines against E. coli infection. Wild-type FimH LD Since FimH is considered a poor immunogen in terms of its ability to stimulate functional immunogenicity, such FimH mutants may provide improved antigens to be used in such immunogenic compositions.

[0048] In one embodiment, as described in Example 1, FimH mutants were designed in an attempt to lock the FimH lectin domain in an open conformation to reduce affinity for mannoside ligands. Such mutants can contain at least 1, 2, 3, 4, 5, or more mutations. Mutations can include naturally occurring amino acid substitutions common among urinary tract infection isolates (e.g., V27A); LDsubstitutions on the ligand-binding side of FimH (such as those at positions F1 and Q133); LD glycine switch mutations in FimH (such as those at positions G15, G16, and G65); LD Introduction of cysteine ​​pairs for disulfide bond stabilization in FimH (such as at position pairs P12-A18; G14-F144; P26-V35; P26-V154; P26-V156; V27-L34; V28-N33; V28-P157; Q32-Y108; N33-L109; N33-P157; V35-L107; V35-L109; S62-T86; S62-L129; Y64-A127; L68-F71; V112-T158; S113-T158; V118-V156; and / or A119-V155); LD and any combination of the types of mutations at the amino acid positions noted above. In another aspect, the present disclosure provides FimH mutants as provided in SEQ ID NOS: 2-58 and 60-64, or any combination of the mutants noted in any of such sequences. In another aspect, the present disclosure provides FimH mutants according to any of SEQ ID NOS: 23, 50, 51, 52, 53, 54, 60, and 62. In a further aspect, the present disclosure provides a FimH mutant according to SEQ ID NOS: 62.

[0049] In a further aspect, the present disclosure provides isolated FimH mutants of any of the FimH mutants provided in SEQ ID NOs: 2-58 and 60-64. For example, in one aspect, the present disclosure provides isolated FimH mutants according to any of SEQ ID NOs: 23, 50, 51, 52, 53, 54, 60, and 62. In a further aspect, the present disclosure provides isolated FimH mutants according to SEQ ID NO: 62.

[0050] Thus, in some specific embodiments, the present disclosure provides FimH mutants comprising a combination of introduced mutations, the combination of mutations being set forth in any of the mutants provided in Table 1 (i.e., in SEQ ID NOS: 2-58 and 60-64). Any combination of amino acid substitutions provided in each of the mutants in Table 1 can be made in the wild-type FimH polypeptide sequence to arrive at different FimH mutants. FimH mutants based on the native FimH polypeptide sequence of any other subtype or strain and comprising any of the combinations of mutations described herein are also within the scope of the present disclosure.

[0051] A further embodiment of the present disclosure is a polypeptide that is 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% identical to any of SEQ ID NOs: 1 to 64. In a preferred embodiment, the polypeptide is 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% identical to SEQ ID NO: 62. Another aspect of the present invention is a polypeptide that is 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% identical to any of SEQ ID NOs: 1-64. In preferred embodiments, the polypeptide is 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% identical to SEQ ID NO:62.

[0052] The FimH mutants provided by the present disclosure can be prepared by routine methods known in the art, such as by expression in a recombinant host system using a suitable vector. Suitable recombinant host cells include, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of suitable insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells (clonal isolates derived from the parent Trichoplusia ni BTI-TN-5B1-4 cell line (Invitrogen)). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (typically transformed with sheared adenovirus type 5 DNA, HEK293 or Expi 293 cells), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Suitable avian cells include, for example, chicken embryonic stem cells (e.g., EBx® cells), chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus vector systems, are known to those skilled in the art and are described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego, Calif. Avian cell expression systems are also known to those skilled in the art and are described, for example, in U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168; and 6,500,668. Similarly, bacterial and mammalian cell expression systems are also known in the art and are described, for example, in Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

[0053] Numerous suitable vectors for expressing recombinant proteins in insect or mammalian cells are well known and conventional in the art. Suitable vectors can contain numerous components, including, but not limited to, one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as transcriptional control elements (e.g., promoters, enhancers, terminators) and / or one or more translational signals; and a signal or leader sequence (e.g., of mammalian origin or from a heterologous mammalian or non-mammalian species) for targeting to the secretory pathway in a selected host cell. For example, for expression in insect cells, a suitable baculovirus expression vector, such as pFastBac (Invitrogen), is used to produce recombinant baculovirus particles. The baculovirus particles are amplified and used to infect insect cells to express the recombinant protein. For expression in mammalian cells, a vector that will drive expression of the construct in the desired mammalian host cell (e.g., Chinese hamster ovary cells) is used.

[0054] FimH mutant polypeptides can be isolated using any suitable method. For example, methods for purifying FimH mutant polypeptides by immunoaffinity chromatography are known in the art. Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687 (2004). Suitable methods for purifying desired proteins, including precipitation and various types of chromatography, such as hydrophobic interaction, ion exchange, affinity, chelation, and size exclusion, are well known in the art. Two or more of the above or other suitable methods can be used to create a suitable purification scheme. If desired, FimH mutant polypeptides can contain a "tag" that facilitates purification, such as an epitope tag or a histidine (His) tag. Such tagged polypeptides can be conveniently isolated from conditioned medium, for example, by chelation or affinity chromatography.

[0055] The term "antigen," as used herein, refers to a molecule that can be recognized by an antibody. Examples of antigens include polypeptides, peptides, lipids, polysaccharides, and nucleic acids that contain antigenic determinants, such as those recognized by immune cells.

[0056] II. Nucleic Acids Encoding FimH Variants In another aspect, the present disclosure provides nucleic acid molecules encoding the FimH mutants disclosed herein. Such nucleic acid molecules include DNA, cDNA, and RNA sequences. In one embodiment, the nucleic acid molecules can be incorporated into a vector, such as an expression vector.

[0057] In one aspect, a nucleic acid encoding an Escherichia coli (E. coli) FimH mutant poly(pol) peptide or any fragment thereof is disclosed. One or more nucleic acid constructs encoding a FimH mutant polypeptide or a fragment thereof can be used for genomic integration and subsequent polypeptide expression. For example, a single nucleic acid construct encoding a FimH mutant polypeptide or a fragment thereof can be introduced into a host cell. Alternatively, the coding sequence for the polypeptide can be carried by two or more nucleic acid constructs, which are then introduced into the host cell simultaneously or sequentially.

[0058] 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 aspects, genomic integration is achieved.

[0059] The nucleic acid construct may comprise genomic DNA or cDNA containing one or more introns. Some genes are expressed more efficiently when introns are present. In some embodiments, the nucleic acid sequence is suitable for expressing an exogenous polypeptide in the mammalian cell.

[0060] In some aspects, the nucleic acid encoding the polypeptide or fragment thereof is codon optimized to increase the level of expression in any particular cell.

[0061] In some embodiments, the nucleic acid construct comprises a signal sequence encoding a peptide that directs secretion of the E. coli-derived polypeptide or fragment thereof. In some embodiments, the nucleic acid comprises the native signal sequence of the E. coli-derived polypeptide FimH. In some embodiments, where the E. coli-derived polypeptide or fragment thereof comprises an endogenous signal sequence, the nucleic acid sequence encoding the signal sequence can be codon optimized to increase the level of expression of the protein in the host cell.

[0062] In some embodiments, the signal sequence is any one of the following lengths: 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.

[0063] In some embodiments, if a polypeptide or fragment thereof contains a signal sequence, the endogenous signal sequence naturally associated with the polypeptide can be replaced with a signal sequence not associated with the wild-type polypeptide to improve the level of expression of the polypeptide or fragment in cultured cells. Thus, in some embodiments, the nucleic acid does not contain the native signal sequence of a polypeptide or fragment thereof derived from E. coli. In some embodiments, the nucleic acid does not contain 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 derived from E. coli or a fragment thereof. For example, a polypeptide or fragment thereof derived from E. coli FimH can be expressed with a heterologous peptide, preferably a signal sequence (e.g., an IgK 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 at the N-terminus of the mature E. coli FimH protein occurs immediately before the first phenylalanine residue of the mature E. coli FimH protein. The heterologous sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.

[0064] In preferred embodiments, the signal sequence is an IgK signal sequence. In some embodiments, the nucleic acid encodes a polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 1-64. In some embodiments, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 23, 50, 51, 52, 53, 54, 60, 61, or 62. In some embodiments, the nucleic acid encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 62. In preferred embodiments, the signal sequence is a mouse IgK signal sequence.

[0065] Suitable mammalian expression vectors for producing FimH mutant polypeptides or fragments thereof are known in the art and are commercially available, such as the pSecTag2 expression vector from Invitrogen™. An ​​exemplary mouse Ig kappa signal peptide sequence comprises the sequence ETDTLLLWVLLLWVPGSTG (SEQ ID NO: 65). In some embodiments, the vector comprises the pBudCE4.1 mammalian expression vector from Thermo Fisher. Additional exemplary suitable vectors include the pcDNA™ 3.1 mammalian expression vector (Thermo Fisher).

[0066] In some aspects, the signal sequence does not include a hemagglutinin signal sequence.

[0067] In some embodiments, the nucleic acid comprises the native signal sequence of the FimH polypeptide or a fragment thereof. In some embodiments, the signal sequence is not an IgK signal sequence. In some embodiments, the signal sequence comprises a hemagglutinin signal sequence.

[0068] In one aspect, a vector comprising the coding sequence of a FimH mutant polypeptide or a fragment thereof is disclosed herein. Exemplary vectors include plasmids capable of autonomous replication or replication in mammalian cells. Typical expression vectors include suitable promoters, enhancers, and terminators that are useful for regulating the expression of the coding sequence in the expression construct. The vector may also include a selection marker for obtaining a phenotypic trait (such as resistance to antibiotics such as ampicillin or neomycin) for selecting transformed host cells.

[0069] Suitable promoters are known in the art. Exemplary promoters include, for example, the CMV promoter, adenovirus, EF1a, GAPDH metallothionine promoter, SV40 early promoter, SV40 late promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, and the like. The promoter may be a constitutive or inducible promoter. One or more vectors may be used (e.g., one vector encoding all subunits or domains or fragments thereof, or multiple vectors collectively encoding the subunits or domains or fragments thereof).

[0070] Internal ribosome entry sites (IRES) and 2A peptide sequences can also be used. IRES and 2A peptides offer alternative approaches for co-expression of multiple sequences. IRES is a nucleotide sequence that allows translation initiation within a messenger RNA (mRNA) sequence as part of the larger process of protein synthesis. Normally, in eukaryotes, translation can only be initiated at the 5' end of an mRNA molecule. IRES elements allow the expression of multiple genes from a single transcript. IRES-based polycistronic vectors that express multiple proteins from a single transcript can reduce the risk of non-expressing clones leaking from selection. 2A peptides allow multiple proteins in a single open reading frame to be translated into a polyprotein, which is subsequently cleaved into individual proteins via a ribosome-skipping mechanism. 2A peptides can provide more balanced expression of multiple protein products. Exemplary IRES sequences include, for example, the EV71 IRES, EMCV IRES, and HCV IRES. With respect to genome 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. Sometimes, protein expression levels can vary depending on the integration site. Therefore, it may be desirable to select multiple clones according to the expression level of the recombinant protein to identify clones that achieve the desired expression level.

[0071] Exemplary nucleic acid constructs are further described in figures, for example, Figures 2A-2T of PCT International Publication No. WO2021 / 084429, published May 6, 2021, which is incorporated herein by reference.

[0072] In one embodiment, the nucleic acid sequence encodes an amino acid sequence having 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% identity to any one of SEQ ID NOs: 1-64. In a preferred embodiment, the nucleic acid sequence encodes an amino acid sequence having 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% identity to SEQ ID NO: 62. In another aspect of the invention, a 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% or 99.9% identity to any of SEQ ID NOs: 1-64. In preferred embodiments, 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% or 99.9% identity to SEQ ID NO:62.

[0073] In certain aspects of the present disclosure, the RNA is messenger RNA (mRNA), which refers to an RNA transcript that encodes a peptide or protein. As established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide-coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides. In one aspect, the RNA described herein can have modified nucleosides. In some aspects, the RNA includes modified nucleosides in place of at least one (e.g., all) uridines.

[0074] In some embodiments, the compositions or medical preparations described herein comprise RNA encoding an amino acid sequence comprising a FimH mutant polypeptide. Similarly, the methods described herein include administering such RNA. One possible platform for use herein is based on an antigen-encoding RNA vaccine to induce robust neutralizing antibodies and concomitant / concomitant T cell responses, preferably to achieve protective immunization with a minimal vaccine dose. The administered RNA is preferably in-vitro transcribed RNA. Three different RNA platforms are particularly preferred: unmodified uridine-containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), and self-amplifying RNA (saRNA). In a particularly preferred aspect, the RNA is in-vitro transcribed RNA.

[0075] III.Host cells In one aspect, the present disclosure relates to a cell in which a sequence encoding a FimH mutant polypeptide or a fragment thereof is expressed in a mammalian host cell. In one embodiment, the polypeptide is transiently expressed in the host cell. In another embodiment, the polypeptide is stably integrated into the genome of the host cell, which expresses the polypeptide or a fragment thereof when cultured under suitable conditions. In a preferred embodiment, the polynucleotide sequence is expressed with high efficiency and genome stability.

[0076] Suitable mammalian host cells are known in the art. Preferably, the host cells are suitable for producing proteins on an industrial manufacturing scale. Exemplary mammalian host cells include any one of the following and their derivatives: Chinese hamster ovary (CHO) cells, COS cells (a cell line derived from monkey kidney (African green monkey)), Vero cells, Hela cells, baby hamster kidney (BHK) cells, human embryonic kidney (HEK) cells, NSO cells (a mouse myeloma cell line), and C127 cells (a non-tumorigenic mouse cell line). Further exemplary mammalian host cells include mouse Sertoli (TM4), buffalo rat liver (BRL 3A), mouse mammary tumor (MMT), rat hepatocellular carcinoma (HTC), mouse myeloma (NSO), mouse hybridoma (Sp2 / 0), mouse thymoma (EL4), Chinese hamster ovary (CHO) and CHO cell derivatives, mouse embryonic (NIH / 3T3, 3T3 Li), rat cardiac muscle (H9c2), mouse myoblasts (C2C12), and mouse kidney (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.

[0077] Any cell capable of cell culture can be used in accordance with the present invention. In some embodiments, the cell is a mammalian cell. Non-limiting examples of mammalian cells that can be used in accordance with the present invention include the BALB / c mouse myeloma line (NSO / 1, ECACC No: 85110503); human retinoblastoma cells (PER.C6, Cru cells, Leiden, The Netherlands); SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney 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 28 yophil 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 tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and human hepatocellular carcinoma line (Hep G2). In some preferred embodiments, the cells are CHO cells. In some preferred aspects, the cells are GS cells.

[0078] In addition, any number of commercially available and non-commercially available hybridoma cell lines can be used in accordance with the present invention. The term "hybridoma" as used herein refers to a cell or progeny resulting from the fusion of an immortalized cell and an antibody-producing cell. The hybridoma thus obtained is an immortalized cell that produces an antibody. The individual cells used to produce a hybridoma can be derived from any mammalian source, including, but not limited to, rats, pigs, rabbits, sheep, pigs, goats, and humans. In some embodiments, a hybridoma is a trioma cell line that is generated when the progeny of a heterohybrid myeloma fusion, which is the product of the fusion between a human cell and a mouse myeloma cell line, is subsequently fused with a plasma cell. In some embodiments, a hybridoma is any immortalized hybrid cell line that produces an antibody, such as a quadroma (see, for example, Milstein et al., Nature, 537:3053 (1983)). Those skilled in the art will recognize that hybridoma cell lines may have different nutritional requirements and / or require different culture conditions for optimal growth, and will be able to modify conditions as needed.

[0079] In some embodiments, the cell comprises a first gene of interest, wherein the first gene of interest is chromosomally integrated. 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), a dependent gene, or a combination thereof. In some embodiments, the gene of therapeutic interest comprises a gene encoding a difficult-to-express (DtE) protein.

[0080] In some embodiments, a first gene of interest is located between two distinct recombination target sites (RTSs) in a site-specific integration (SSI) mammalian cell, wherein the two RTSs are chromosomally integrated within the NL1 locus or the NL2 locus. See, e.g., U.S. Patent Application Publication No. 20200002727 for a description of the NL1, NL2, NL3, NL4, NL5, and NL6 loci. In some embodiments, the first gene of interest is located within the NL1 locus. In some embodiments, the cell comprises a second gene of interest, wherein the second gene of interest is chromosomally integrated. In some embodiments, the second gene of interest comprises a reporter gene, a selection gene, a therapeutic gene (such as a FimH mutant polypeptide or a fragment thereof), a dependent gene, or a combination thereof. In some embodiments, the therapeutic gene 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 within the NL1 locus or the NL2 locus. In some embodiments, the first gene of interest is located within the NL1 locus and the second gene of interest is located within the NL2 locus. In some embodiments, the cell comprises a third gene of interest, wherein the third gene of interest is chromosomally integrated. In some embodiments, the third gene of interest comprises a reporter gene, a selection gene, a therapeutic gene (such as a polypeptide or fragment thereof derived from E. coli), a dependent gene, or a combination thereof. In some embodiments, the therapeutic gene 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 within the NL1 locus or the NL2 locus. In some embodiments, the third gene of interest is located within a locus distinct 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 in 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 located within 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 located within the NL2 locus. In some embodiments, the cell comprises a site-specific recombinase gene. In some embodiments, the site-specific recombinase gene is chromosomally integrated.

[0081] In another aspect, the present disclosure provides a mammalian cell comprising at least four distinct RTSs, wherein the cell comprises: (a) at least two distinct RTSs chromosomally integrated within the NL1 locus or the NL2 locus; (b) a first gene of interest integrated between the at least two RTSs of (a), the first gene of interest comprising a reporter gene, a gene encoding a DtE protein, a dependent gene, or a combination thereof; and (c) a second gene of interest integrated within a second chromosomal locus distinct from the locus of (a), the second gene of interest comprising a reporter gene, a gene encoding a DtE protein (such as a polypeptide or fragment thereof derived from Escherichia coli (E. coli)), a dependent gene, or a combination thereof. In some aspects, the present disclosure provides a mammalian cell comprising at least four distinct RTSs, wherein the cell comprises: (a) at least two distinct RTSs chromosomally integrated within the Fer1L4 locus; (b) at least two distinct RTSs chromosomally integrated within the NL1 or NL2 locus; (c) a first gene of interest chromosomally integrated within the Fer1L4 locus, the first gene of interest comprising a reporter gene, a gene encoding a DtE protein, a dependent gene, or a combination thereof; and (d) a second gene of interest chromosomally integrated within the NL1 or NL2 locus of (b), the second gene of interest comprising a reporter gene, a gene encoding a DtE protein (such as a polypeptide or fragment thereof derived from E. coli), a dependent gene, or a combination thereof.

[0082] In some aspects, the present disclosure provides mammalian cells comprising at least six distinct RTSs, wherein the cells comprise: (a) at least two distinct RTSs and a first gene of interest that are chromosomally integrated within the Fer1L4 locus; (b) at least two distinct RTSs and a second gene of interest that are chromosomally integrated within the NL1 locus; and (c) at least two distinct RTSs and a third gene of interest that are chromosomally integrated within the NL2 locus.

[0083] 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 terms also refer 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, wherein the gene of interest is chromosomally integrated into the host cell. In some embodiments, a gene of interest is operably linked to a heterologous promoter, wherein the gene of interest is chromosomally integrated into the host cell. In some embodiments, a dependent gene is operably linked to a promoter, wherein the dependent gene is chromosomally integrated into the host cell genome. In some embodiments, a dependent gene is operably linked to a heterologous promoter, wherein the dependent gene is chromosomally integrated into the host cell genome. In some embodiments, a gene encoding a DtE protein is operably linked to a promoter, wherein the gene encoding a DtE protein is chromosomally integrated into the host cell genome. In some embodiments, the gene encoding the DtE protein is operably linked to a heterologous promoter, wherein the gene encoding the DtE protein is chromosomally integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a promoter, wherein the recombinase gene is chromosomally integrated into the host cell. In some embodiments, the recombinase gene is operably linked to a promoter, wherein the recombinase gene is chromosomally integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a heterologous promoter, wherein the recombinase gene is not chromosomally integrated into the host cell genome. In some embodiments, the recombinase gene is operably linked to a heterologous promoter, wherein the recombinase gene is not chromosomally integrated into the host cell genome.

[0084] As used herein, the term "chromosomally integrated" or "chromosomal integration" refers to the stable integration of a nucleic acid sequence into the chromosome of a host cell, for example, a mammalian cell, i.e., a nucleic acid sequence that is chromosomally integrated into the genomic DNA (gDNA) of a host cell, for example, 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 clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) gene editing system (CRISPR / CAS).

[0085] IV. Compositions and Formulations In one aspect, the present disclosure includes compositions comprising at least one FimH mutant polypeptide or fragment thereof described herein. In some aspects, the compositions elicit an immune response comprising antibodies that can confer immunity against pathogenic species of E. coli.

[0086] In some embodiments, the composition comprises a FimH mutant polypeptide as the only antigen. In some embodiments, the composition does not comprise a conjugate.

[0087] In some embodiments, a composition comprises a FimH mutant polypeptide and at least one additional antigen. In some embodiments, a composition comprises a FimH mutant polypeptide and an additional E. coli antigen. In some embodiments, a composition comprises a FimH mutant polypeptide and an E. coli-derived glycoconjugate.

[0088] In some embodiments, the composition comprises a FimH mutant polypeptide and a polypeptide derived from E. coli FimC or a fragment thereof.

[0089] In one embodiment, the disclosure includes a composition comprising a FimH mutant polypeptide and a sugar comprising a structure selected from any one of the sugar structures disclosed in PCT International Publication No. WO2021 / 084429 published on May 6, 2021 and WO2020 / 039359 published on February 27, 2020, and U.S. Patent Application Publication No. US2020 / 0061177 published on February 27, 2020, each of which is incorporated by reference herein in its entirety. In one aspect, the disclosure includes a composition comprising a FimH mutant polypeptide; 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., Formulas 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 O62D1, Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (Strain 7) 3-1)), 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 O 92, 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 O1 21, 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 O1 43, Formula O144, Formula O145, Formula O146, Formula O147, Formula O148, Formula O149, Formula O150, Formula O151, Formula O152, Formula O153, Formula 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, preferably 31 to 90).

[0090] In some embodiments, the composition comprises any one of the sugars disclosed herein. In preferred embodiments, the composition comprises any one of the conjugates disclosed herein.

[0091] In some embodiments, the composition comprises at least one glycoconjugate from E. coli serotype O25, preferably serotype O25b. In one embodiment, the composition comprises at least one glycoconjugate from E. coli serotype O1, preferably serotype O1a. In one embodiment, the composition comprises at least one glycoconjugate from E. coli serotype O2. In one embodiment, the composition comprises at least one glycoconjugate from E. coli serotype O6.

[0092] In one embodiment, the composition comprises at least one glycoconjugate selected from any one of the following 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 any one of the following E. coli serotypes: O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In another embodiment, the composition comprises at least three glycoconjugates selected from any one of the following E. coli serotypes: O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6. In a further embodiment, the composition comprises glycoconjugates derived from each of the following E. coli serotypes: O25, O1, O2, and O6, preferably O25b, O1a, O2, and O6.

[0093] In a preferred embodiment, the glycoconjugates of any of the above compositions individually comprise a CRM 197 In another preferred embodiment, the glycoconjugates of any of the above compositions are individually conjugated to an SCP.

[0094] Thus, in some embodiments, a composition comprises a FimH mutant polypeptide and an O antigen from at least one E. coli serotype. In a preferred embodiment, a composition comprises a FimH mutant polypeptide and an O antigen from more than one E. coli serotype. For example, the composition may comprise O antigens from two different E. coli serotypes (or "v", valency) to 12 different serotypes (12v). In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from three different serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from four different E. coli serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from five different E. coli serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from six different E. coli serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from seven different E. coli serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from eight different E. coli serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from nine different E. coli serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from ten different E. coli serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from eleven different E. coli serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from twelve different serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from thirteen different serotypes. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from fourteen different serotypes. In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 15 different serotypes.In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 16 different serotypes. In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 17 different serotypes. In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 18 different serotypes. In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 19 different serotypes. In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 20 different serotypes.

[0095] Preferably, the number of E. coli saccharides may range from 1 serotype (or "v", 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 one 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.

[0096] In one aspect, a composition comprises a glycoconjugate comprising a FimH mutant polypeptide and an O antigen from at least one E. coli serogroup, wherein the O antigen is conjugated to a carrier protein. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from more than one E. coli serotype, each O antigen conjugated to a carrier protein. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from two different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from three different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from four different E. coli serotypes, each O antigen conjugated to a carrier protein. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from five different E. coli serotypes, each conjugated to a carrier protein. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from six different E. coli serotypes, each conjugated to a carrier protein. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from seven different E. coli serotypes, each conjugated to a carrier protein. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from eight different E. coli serotypes, each conjugated to a carrier protein. In one embodiment, a composition comprises a FimH mutant polypeptide and O antigens from nine different E. coli serotypes, each conjugated to a carrier protein. In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 10 different E. coli serotypes, each O antigen conjugated to a carrier protein.In one embodiment, the composition comprises an O antigen from a FimH mutant polypeptide and an O antigen from 11 different E. coli serotypes, each conjugated to a carrier protein. In one embodiment, the composition comprises a FimH mutant polypeptide and an O antigen from 12 different serotypes, each conjugated to a carrier protein. In one embodiment, the composition comprises a FimH mutant polypeptide and an O antigen from 13 different serotypes, each conjugated to a carrier protein. In one embodiment, the composition comprises a FimH mutant polypeptide and an O antigen from 14 different serotypes, each conjugated to a carrier protein. In one embodiment, the composition comprises a FimH mutant polypeptide and an O antigen from 15 different serotypes, each conjugated to a carrier protein. In one embodiment, the composition comprises a FimH mutant polypeptide and an O antigen from 16 different serotypes, each conjugated to a carrier protein. In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 17 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 18 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 19 different serotypes, each O antigen conjugated to a carrier protein. In one embodiment, the composition comprises a FimH mutant polypeptide and O antigens from 20 different serotypes, each O antigen conjugated to a carrier protein.

[0097] In another aspect, the composition comprises O polysaccharides derived from at least one E. coli serotype. In a preferred embodiment, the composition comprises O polysaccharides derived from more than one E. coli serotype. For example, the composition may comprise O polysaccharides derived from two different E. coli serotypes to 12 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides derived from three different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides derived from four different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides derived from five different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides derived from six different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides derived from seven different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides derived from eight different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 9 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 10 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 11 different E. coli serotypes. In one embodiment, the composition comprises O polysaccharides from 12 different serotypes. In one embodiment, the composition comprises O polysaccharides from 13 different serotypes. In one embodiment, the composition comprises O polysaccharides from 14 different serotypes. In one embodiment, the composition comprises O polysaccharides from 15 different serotypes. In one embodiment, the composition comprises O polysaccharides from 16 different serotypes. In one embodiment, the composition comprises O polysaccharides from 17 different serotypes. In one embodiment, the composition comprises O polysaccharides from 18 different serotypes. In one embodiment, the composition comprises O polysaccharides from 19 different serotypes. In one embodiment, the composition comprises O polysaccharides from 20 different serotypes.

[0098] In a preferred embodiment, the composition comprises O polysaccharides from at least one E. coli serotype, each O polysaccharide conjugated to a carrier protein. In a preferred embodiment, the composition comprises O polysaccharides from more than one E. coli serotype, each O polysaccharide conjugated to a carrier protein. For example, the composition may comprise O polysaccharides from two different E. coli serotypes to 12 different E. coli serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from three different E. coli serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from four different E. coli serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from five different E. coli serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from six different E. coli serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from seven different E. coli serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from eight different E. coli serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from nine different E. coli serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from ten different E. coli serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from eleven different E. coli serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 12 different serotypes, each O polysaccharide conjugated to a carrier protein.In one embodiment, the composition comprises O polysaccharides from 13 different serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 14 different serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 15 different serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 16 different serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 17 different serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 18 different serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 19 different serotypes, each O polysaccharide conjugated to a carrier protein. In one embodiment, the composition comprises O polysaccharides from 20 different serotypes, each O polysaccharide conjugated to a carrier protein.

[0099] In a most preferred embodiment, the composition comprises O polysaccharides from at least one E. coli serotype, wherein the O polysaccharide is conjugated to a carrier protein and comprises an O antigen and a core saccharide. In a preferred embodiment, the composition comprises O polysaccharides from more than one E. coli serotype, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. For example, the composition may comprise O polysaccharides from 12 different E. coli serotypes from two different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from three different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide. In one embodiment, the composition comprises O polysaccharides from four 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 from 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 from 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 from 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 from 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 from nine different E. coli serotypes, each O polysaccharide being conjugated to a carrier protein and comprising an O antigen and a core saccharide.In one embodiment, the composition comprises O polysaccharides from 10 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 from 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 from 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 from 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 from 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 from 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 from 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 from 17 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 from 18 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 from 19 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 from 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.

[0100] In another preferred embodiment, the composition comprises a FimH mutant polypeptide and a CRM197 and an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises a CRM of formula O25a, where n is at least 30, and a core sugar. 197 In 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 sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises the formula O1a, where n is at least 30, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises the formula O2, where n is at least 30, and a core sugar. 197 wherein the O polysaccharide comprises formula O6, where n is at least 30, and a core sugar.

[0101] 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 O17, where n is at least 30, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, wherein the O polysaccharide comprises the formula O15, where n is at least 30, and a core sugar. 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 30, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, the O polysaccharide comprising the formula O75, where n is at least 30, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, the O polysaccharide comprising the formula O4, where n is at least 30, and a core sugar. 197In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, the O polysaccharide comprising formula O16, where n is at least 30, and a core sugar. 197 In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM, the O polysaccharide comprising formula O13, where n is at least 30, and a core sugar. 197 wherein the O polysaccharide comprises a structure of formula O7, where n is at least 30, and a core sugar.

[0102] In another embodiment, the composition comprises a CRM 197 and a core sugar. In another embodiment, the O polysaccharide comprises formula O8, where n is 1-20, preferably 2-5, more preferably 3. In another embodiment, the composition further comprises an O polysaccharide conjugated to a CRM 197 and a core sugar. In another embodiment, the O polysaccharide comprises formula O9, where n is 1-20, preferably 4-8, and more preferably 5. In another embodiment, the O polysaccharide comprises formula O9a, where n is 1-20, preferably 4-8, and more preferably 5.

[0103] In some embodiments, the O polysaccharide is selected from any one of formula O20ab, formula O20ac, formula O52, formula O97, and formula O101, where n is 1-20, preferably 4-8, and more preferably 5.

[0104] As noted above, the composition may comprise any combination of FimH mutant polypeptides and conjugated O polysaccharides (antigens). 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 comprising the formula O25b (wherein n is at least 30) and a core sugar conjugated to a CRM; 197 an O polysaccharide comprising formula O1a, where n is at least 30, and a core sugar conjugated to a CRM; 197 an O polysaccharide conjugated to a CRM, the O polysaccharide comprising the formula O2, where n is at least 30, and a core sugar; and (iv) a CRM 197 wherein the O polysaccharide comprises a core sugar and a formula O6, wherein n is at least 30.

[0105] In one embodiment, a composition comprises a FimH mutant polypeptide and at least one O polysaccharide derived from any E. coli serotype that is not 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.

[0106] In one embodiment, the composition comprises a FimH mutant polypeptide and a respective O polysaccharide. 197 In one embodiment, the composition comprises an O polysaccharide from two different E. coli serotypes, each O polysaccharide comprising an O antigen and a core sugar conjugated to a CRMH mutant polypeptide. 197 In one embodiment, the composition comprises O polysaccharides from three different E. coli serotypes, each O polysaccharide comprising an O antigen and a core sugar, conjugated to a CRMH mutant polypeptide. 197 In one embodiment, the composition comprises a FimH mutant polypeptide and O polysaccharides from five different E. coli serotypes, each O polysaccharide comprising a CRMP. 197In one embodiment, the composition comprises a FimH mutant polypeptide and O polysaccharides from six different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a FimH mutant polypeptide and O polysaccharides from seven different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a FimH mutant polypeptide and O polysaccharides from eight different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a FimH mutant polypeptide and O polysaccharides from nine different E. coli serotypes, each O polysaccharide comprising a CRMP. 197 In one embodiment, the composition comprises a FimH mutant polypeptide and a CRMP, wherein the O polysaccharide comprises an O antigen and a core saccharide. 197 In one embodiment, the composition comprises an O polysaccharide from 10 different E. coli serotypes, each O polysaccharide comprising an O antigen and a core sugar conjugated to a CRMH mutant polypeptide. 197 In one embodiment, the composition comprises O polysaccharides from 11 different E. coli serotypes, wherein the O polysaccharides comprise an O antigen and a core sugar. In one embodiment, the composition comprises a FimH mutant polypeptide and O polysaccharides from 12 different serotypes, wherein each O polysaccharide comprises a CRM 197 In one embodiment, the composition comprises a FimH mutant polypeptide and a CRMP, wherein the O polysaccharide comprises an O antigen and a core saccharide. 197 In one embodiment, the composition comprises an O polysaccharide from 13 different serotypes, each O polysaccharide conjugated to a CRMP. 197In one embodiment, the composition comprises an O polysaccharide from 14 different serotypes, each O polysaccharide comprising an O antigen and a core sugar conjugated to a CRMH mutant polypeptide. 197 In one embodiment, the composition comprises an O polysaccharide from 15 different serotypes, each O polysaccharide comprising an O antigen and a core sugar conjugated to a CRMH mutant polypeptide. 197 In one embodiment, the composition comprises an O polysaccharide from 16 different serotypes, each O polysaccharide conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from 17 different serotypes, each O polysaccharide conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from 18 different serotypes, each O polysaccharide conjugated to a CRMP. 197 In one embodiment, the composition comprises an O polysaccharide from 19 different serotypes, each O polysaccharide conjugated to a CRMP. 197 The O polysaccharides are conjugated to the O antigen and the core sugar, and contain O polysaccharides from 20 different serotypes.

[0107] In one aspect, the invention provides a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O25b, where n is 15±2. In one aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O25b, where n is 17±2. In one aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O25b, where n is 55±2. In another aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O25b, where n is 51±2. In another aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises the formula O25b, where n is an integer greater than 30, preferably n is an integer between 31 and 100. 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 CRMP. 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 sugar 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.

[0108] In one aspect, 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. Thus, a comparison of OPA activity in pre- and post-immunization sera using the immunogenic composition of the invention can be performed to compare their responses to serotype O25B and assess the potential for increased responders. 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 provide a method for the production of a immunized population of E. coli serotype O25B responders (i.e., in vitro In one embodiment, the immunogenic composition elicits 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 composition of the invention elicits 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 composition of the invention increases the proportion of responders (i.e., individuals with sera having a titer of at least 1:8 as determined by an in vitro opsonophagocytic killing assay) to E. coli serotype O25B compared to a pre-immune population. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of a human subject against E. coli serotype O25B compared to a pre-immune population.

[0109] In one aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O1a, where n is an integer greater than 30, preferably n is an integer between 31 and 100. In another aspect, the invention relates to a composition comprising a FimH mutant polypeptide 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 FimH polypeptide 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 embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In one 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 sugar 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.

[0110] 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. Thus, a comparison of OPA activity in pre- and post-immunization sera using the immunogenic composition of the present invention can be performed to compare their responses to serotype O1A and assess the potential for increased responders. 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 provide a method for the production of a immunized population of E. coli serotype O1A responders (i.e., in vitro In one embodiment, the immunogenic composition elicits 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 composition of the invention elicits 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 composition of the invention increases the proportion of responders (i.e., individuals with sera having a titer of at least 1:8 as determined by an in vitro opsonophagocytic killing assay) to E. coli serotype O1A compared to a pre-immune population. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of a human subject against E. coli serotype O1A compared to a pre-immune population.

[0111] In one aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a sugar covalently attached to a carrier protein, wherein the sugar comprises a group of formula O2, where n is an integer greater than 30, preferably n is an integer between 31 and 100. In another aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a sugar covalently attached to a carrier protein, wherein the sugar comprises a group of formula O2, where n is 43±2. In another aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a sugar covalently attached to a carrier protein, wherein the sugar comprises a group of formula O2, where n is 47±2. In another aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a sugar covalently attached to a carrier protein, wherein the sugar comprises a group of formula O2, where n is 17±2. In another aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises the formula O2, where 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 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 sugar 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.

[0112] 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. Thus, a comparison of the OPA activity of pre- and post-immunization sera using the immunogenic composition of the present invention can be performed to compare their responses to serotype O2 and assess the potential for enhanced responders. 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 provide a method for the production of a immunized population of E. coli serotype O2 responders (i.e., in vitro In one embodiment, the immunogenic composition elicits 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 composition of the invention elicits 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 composition of the invention increases the proportion of responders (i.e., individuals with sera having a titer of at least 1:8 as determined by an in vitro opsonophagocytic killing assay) to E. coli serotype O2 compared to a pre-immune population. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of a human subject against E. coli serotype O2 compared to a pre-immune population.

[0113] In one aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises a formula O6, where n is an integer greater than 30, preferably n is an integer between 31 and 100. In one aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises a formula O6, where n is 42±2. In another aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises a formula O6, where n is 50±2. In another aspect, the invention relates to a composition comprising a FimH mutant polypeptide and a conjugate comprising a saccharide covalently attached to a carrier protein, wherein the saccharide comprises a formula O6, where n is 17±2. In another aspect, the invention relates to a composition comprising a conjugate comprising a FimH mutant polypeptide and a saccharide covalently attached to a carrier protein, wherein the saccharide comprises formula O6, where n is 18±2. In one embodiment, the saccharide further comprises an E. coli R1 core saccharide moiety. In one 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 sugar 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.

[0114] 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. Thus, the OPA activity of pre- and post-immunization sera using the immunogenic composition of the present invention can be compared to assess their response to serotype O6 to assess the potential for increased responders. 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 provide a method for the production of a immunized population of E. coli serotype O6 responders (i.e., in vitro In one embodiment, the immunogenic composition elicits 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 composition of the invention elicits 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 composition of the invention increases the proportion of responders (i.e., individuals with sera having a titer of at least 1:8 as determined by an in vitro opsonophagocytic killing assay) to E. coli serotype O6 compared to a pre-immune population. In one embodiment, the immunogenic composition of the invention significantly increases the OPA titer of a human subject against E. coli serotype O6 compared to a pre-immune population.

[0115] In one embodiment, a composition comprises a conjugate comprising a FimH mutant polypeptide and a saccharide covalently attached to a carrier protein, 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., For example, 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 O7 5, 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 O 101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O11 1, 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 O1 33, 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 O and n is an integer between 1 and 100, preferably between 31 and 90. In one embodiment, the saccharide further comprises an E. coli R1 core sugar moiety. In one embodiment, the saccharide further comprises an E. coli R2 core sugar moiety. In one embodiment, the saccharide further comprises an E. coli R3 core sugar moiety. In another embodiment, the saccharide further comprises an E. coli R4 core sugar 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, 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 sugar 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, or 29 additional conjugates, up to a maximum of 30 additional conjugates, each conjugate comprising a sugar covalently attached to a carrier protein, wherein the sugar comprises a structure selected from any one of the formulas above.

[0116] A. Sugar 1. Sugars and O-polysaccharides In one embodiment, the sugars are produced by expression (not necessarily overexpression) of different Wzz proteins (eg, WzzB) to control the size of the sugar.

[0117] As used herein, the term "sugar" refers to a single sugar moiety or monosaccharide unit and to combinations of two or more single sugar moieties or monosaccharide units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. Sugars may be linear or branched.

[0118] In one embodiment, the sugar is produced in a recombinant Gram-negative bacterium. In one embodiment, the sugar is produced in a recombinant E. coli cell. In one embodiment, the sugar is produced in a recombinant Salmonella cell. 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 Examples of suitable bacterial strains include E. coli GAR2401, Salmonella enterica serovar Typhimurium strain LT2, E. coli GAR2401, Salmonella enterica serovar Enteritidis CVD1943, Salmonella enterica serovar Typhimurium strain CVD1925, Salmonella enterica serovar Paratyphi A CVD1902, and Shigella flexneri CVD1208S. In one embodiment, the bacterium is not E. coli GAR2401. This genetic approach to sugar production allows for efficient production of O polysaccharides and O antigen molecules as vaccine components.

[0119] As used herein, the term "wzz protein" refers to, for example, wzzB, wzz, wzz SF , wzz ST , fepE, wzz fepE, wzz1, and wzz2. GenBank accession numbers for exemplary wzz gene sequences are AF011910 for E4991 / 76, AF011911 for F186, AF011912 for M70 / 1-1, AF011913 for 79 / 311, AF011914 for Bi7509-41, AF011915 for C664-1992, AF011916 for C258-94, AF011917 for C722-89, and AF011919 for EDL933. GenBank accession numbers for G7 and Bi316-41 wzz gene sequences are U39305 and U39306, respectively. Additional GenBank accession numbers for exemplary wzz gene sequences are: NP_459581 for Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 FepE; AIG66859 for E. coli O157:H7 strain EDL933 FepE; NP_461024 for Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 WzzB; NP_416531 for E. coli K-12 substrain MG1655 WzzB; and NP_415119 for E. coli K-12 substrain MG1655 FepE. In preferred embodiments, the wzz family proteins are selected from the group consisting of wzzB, wzz, and wzz. SF , wzz ST , fepE, wzz fepE , wzz1 and wzz2, most preferably wzzB, and more preferably fepE.

[0120] Exemplary wzzB sequences include those set forth in SEQ ID NOs: 112 to 116. Exemplary FepE sequences include those set forth in SEQ ID NOs: 117 to 121.

[0121] In some aspects, modified sugars (modified relative to the corresponding wild-type sugar) can be produced in Gram-negative bacteria by expressing (not necessarily overexpressing) a wzz family protein (e.g., fepE) from a Gram-negative bacterium and / or by switching off (i.e., suppressing, deleting, or removing) a second wzz gene (e.g., wzzB) to produce higher molecular weight sugars, such as lipopolysaccharides containing medium or long O antigen chains with an increased number of repeat units compared to the corresponding wild-type O polysaccharide. For example, modified sugars can be produced by expressing (not necessarily overexpressing) wzz2 and switching off wzz1. Alternatively, modified sugars can be produced by expressing (not necessarily overexpressing) wzz / fepE and switching off wzzB. In another embodiment, modified sugars can be produced by expressing (not necessarily overexpressing) wzzB but switching off wzz / fepE. In another embodiment, modified sugars can be produced by expressing fepE. Preferably, the wzz family protein is derived from a strain heterologous to the host cell. Methods for determining the length of sugars are known in the art. Such methods include, but are not limited to, nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography. Methods for producing high molecular weight sugars described herein, such as lipopolysaccharides containing medium or long O-antigen chains, are described in PCT International Publication No. WO2020 / 039359 and corresponding U.S. Patent Application Publication No. US2020 / 0061177, each of which is incorporated herein by reference in its entirety.

[0122] In some embodiments, the sugar is produced by expressing a wzz-family protein having an amino acid sequence having at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, and SEQ ID NO: 121. In one embodiment, the wzz-family protein comprises a sequence selected from any one of SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, and SEQ ID NO: 121. Preferably, the wzz family protein has at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, and SEQ ID NO: 116. In some embodiments, the sugar is produced by expressing a protein having an amino acid sequence with at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a fepE protein.

[0123] 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 containing medium or long O-antigen chains with an increased number of repeating units of at least 1, 2, 3, 4, or 5 compared to the corresponding wild-type O polysaccharide. In one aspect, the invention relates to saccharides produced by Gram-negative bacteria that express (but do not necessarily overexpress) a wzz-family protein (e.g., wzzB) in culture to produce high molecular weight saccharides containing medium or long O-antigen chains with an increased number of repeating units of at least 1, 2, 3, 4, or 5 compared to the corresponding wild-type O antigen. See the description of O polysaccharides and O antigens below for additional exemplary saccharides with an increased number of repeating units compared to the corresponding wild-type saccharide. Desired chain lengths are those that result in improved or maximal immunogenicity in the context of a given vaccine construct.

[0124] In another embodiment, the saccharide comprises any one of the formulas selected from Table A, wherein the number of repeating 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, , 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, and more than 100 repeat units. Preferably, the saccharide comprises an increase of 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 repeating units compared to the corresponding wild-type O polysaccharide. Methods for determining saccharide length are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography.

[0125] In a preferred embodiment, the present invention relates to saccharides produced in recombinant E. coli host cells in which the endogenous wzz O-antigen length regulator gene (e.g., wzzB) has been deleted and replaced with a (second) wzz gene (e.g., Salmonella fepE) from a Gram-negative bacterium heterologous to the recombinant E. coli host cell, producing high molecular weight saccharides, such as lipopolysaccharides, containing medium or long O-antigen chains. In some embodiments, the recombinant E. coli host cell contains a wzz gene from Salmonella, preferably Salmonella enterica.

[0126] 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 an integrated gene in the chromosomal DNA of the host cell. Methods for stable expression of 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 a heterologous gene for an O antigen as a stably maintained plasmid vector. In another embodiment, the host cell comprises a heterologous gene for an O antigen as an integrated gene in the chromosomal DNA of the host cell. Methods for stable expression of plasmid vectors in E. coli host cells and Salmonella host cells are known in the art. Methods for integrating heterologous genes into the chromosome of E. coli host cells and Salmonella host cells are known in the art.

[0127] 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, but are not limited to, sugar alcohols, polyols, aldol sugars, or keto sugars, such as 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 carbon sources may be added to the medium before the start of cultivation, or may be added in portions or continuously during cultivation.

[0128] 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.

[0129] 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.

[0130] 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 additional carbon sources. Suitable inorganic salts, micronutrients, growth factors, and supplemental carbon sources for culturing E. coli are known in the art.

[0131] 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 sources, and various vitamins. In some embodiments, the medium does not optionally contain such additional components such as peptone, NZ amine, soybean enzymatic hydrolysate, additional yeast extract, malt extract, supplemental carbon sources, and various vitamins.

[0132] Examples of suitable supplemental carbon sources include, but are not limited to, other carbohydrates such as glucose, fructose, mannitol, starch or starch hydrolysates, cellulose hydrolysates, and molasses; organic acids such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, and fumaric acid; and alcohols such as glycerol, inositol, mannitol, and sorbitol.

[0133] 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; nitrate or nitrite, and other nitrogen-containing materials, including amino acids, meat extract, peptone, fish meal, fish hydrolysate, corn infusion, casein hydrolysate, soybean meal hydrolysate, yeast extract, dry yeast, ethanol-yeast distillate, soybean flour, cottonseed flour, and the like, in pure or crude preparations.

[0134] In some embodiments, the medium comprises 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.

[0135] In some embodiments, the medium comprises suitable growth factors. Examples of suitable micronutrients, growth factors, etc. include, but are not limited to, 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 compounds or present in natural sources. 12, 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, nitriloacetate, etc. Those skilled in the art can empirically determine the amount according to methods and techniques known in the art.

[0136] In another embodiment, the modified sugars described herein (as compared to the corresponding wild-type sugars) are synthetically produced, e.g., in vitro. Synthetic production or synthesis of sugars can facilitate avoiding costly and time-intensive production processes. In one embodiment, the sugars are synthesized, e.g., by using a sequential glycosylation strategy or a combination of a sequential glycosylation strategy and a [3+2] block synthesis strategy from a suitably protected monosaccharide intermediate. For example, thiol glycosides and glycosyl trichloroacetimidate derivatives can be used as glycosyl donors in glycosylation. In one embodiment, the sugars synthesized in vitro have the same structure as sugars produced by recombinant means, such as engineering the wzz family proteins described above.

[0137] The sugars produced (by recombinant or synthetic means) can be, for example, from the following Escherichia coli (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 (180 / C3 strain)), 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, 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, O62D1, O63, O64, O65, O66, O68, O69, O70, O71, O73 (e.g., O73 (73-1 strain)), O74, O75, O76, O77, O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O98, O99, O100, O1 01, O102, O103, O104, O105, O106, O107, O108, O109, O110, 0111, O112, O 113, O114, O115, O116, O117, O118, O119, O120, O121, O123, O124, O125, O126, O127, O128, O129, O130, O131, O132, O133, O134, O135, O136, O137 , O138, O139, O140, O141, O142, O143, O144, O145, O146, O147, O148, O14 9, comprising a structure derived from any E. coli serotype, including any one of O150, O151, O152, O153, O154, O155, O156, O157, O158, O159, O160, O161, O162, 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;

[0138] Individual polysaccharides are typically purified (enriched with respect to the amount of polysaccharide-protein conjugate) by methods known in the art, such as, for example, dialysis, concentration procedures, diafiltration procedures, tangential flow filtration, sedimentation, elution, centrifugation, sedimentation, ultrafiltration, depth filtration, and / or column chromatography (ion exchange chromatography, multimode ion exchange chromatography, DEAE, and hydrophobic interaction chromatography). Preferably, the polysaccharides are purified by a method comprising tangential flow filtration.

[0139] The purified polysaccharides can be activated (e.g., chemically activated) to render them capable of reacting (e.g., directly to a carrier protein or via a linker such as an eTEC spacer) and then incorporated into the glycoconjugates of the invention, as further described herein.

[0140] In one preferred embodiment, the saccharides of the invention are derived from the E. coli serotype 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.

[0141] As used herein, reference to any of the above serotypes refers to a serotype that includes a repeating unit structure (O unit, as described below) that is known in the art and 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 A. As another example, the term "O25b" serotype refers to a serotype that includes formula O25b, as shown in Table A.

[0142] As used herein, serotypes are generally referred to herein unless otherwise specified, such as, for example, the term formula "O18" is generally referred to as including formula O18A, formula O18ac, formula O18A1, formula O18B, and formula O18B1.

[0143] As used herein, the term "O1" generally refers to encompassing species of formulas that include the generic designation "O1" in their formula names set forth in Table A, such as any one of formula O1A, formula O1A1, formula O1B, and formula O1C, respectively, as set forth in Table A. Thus, "O1 serotype" generally refers to serotypes that include any one of formula O1A, formula O1A1, formula O1B, and formula O1C.

[0144] As used herein, the term "O6" generally refers to species of formulae that include the generic designation "O6" in their formula names set forth in Table A, such as any one of the formulae O6:K2;K13;K15; and O6:K54, respectively, set forth in Table A. Thus, "O6 serotype" generally refers to serotypes that encompass any one of the formulae O6:K2;K13;K15; and O6:K54.

[0145] Other examples of terms generally referring to species of formulas that include a generic term in the formula name listed in Table A include "O4," "O5," "O18," and "O45."

[0146] As used herein, the term "O2" refers to the formula O2 shown in Table A. The term "O2 O antigen" refers to a saccharide that encompasses the formula O2 shown in Table A.

[0147] As used herein, references to O antigens from the above serotypes refer to the saccharide comprising the formula labeled with the corresponding serotype name. For example, the term "O25B O antigen" refers to the saccharide comprising the formula O25B shown in Table A.

[0148] As another example, the term "O1 O antigen" refers generally to saccharides that include formulas containing the term "O1," such as formula O1A, formula O1A1, formula O1B, and formula O1C, each shown in Table A.

[0149] As another example, the term "O6 O antigen" refers generally to saccharides that include formulas containing the term "O6," such as formula O6:K2; formula O6:K13; formula O6:K15, and formula O6:K54, each shown in Table A.

[0150] 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 includes ultracentrifugation.

[0151] 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 the repeating units of an O antigen without the 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.

[0152] Methods for purifying O polysaccharides, including the core oligosaccharide, from LPS are known in the art. For example, after purification of LPS, the purified LPS can 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 lyophilized and stored at 4°C. In certain embodiments, deletion of capsule synthesis genes is described to allow for simple purification of O polysaccharide.

[0153] O polysaccharide can 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 for O polysaccharide preparation. Preparation of LPS can be accomplished by methods known in the art.

[0154] In certain embodiments, O polysaccharides purified 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 a vaccine antigen in a conjugate or complexed vaccine.

[0155] 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, In a preferred embodiment, the O polysaccharide has a molecular weight that is increased by at least 1-fold and at most 5-fold compared to the corresponding wild-type O polysaccharide. In another embodiment, the O polysaccharide has a molecular weight that is at least two-fold and at most four-fold increased compared to the corresponding wild-type O polysaccharide. The 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 O antigen repeating units. In one embodiment, the increase in molecular weight of the O polysaccharide is due to a wzz family protein.

[0156] 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 increased by 0, 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 kDa or more. In one embodiment, the O polysaccharides of the present invention have a molecular weight increased by at least 1 kDa and at most 200 kDa compared to the corresponding wild-type O polysaccharide. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 15 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 18 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 21 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 22 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 30 kDa and at most 200 kDa. In one embodiment, the molecular weight is increased by at least 1 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 100 kDa, hi one embodiment, the molecular weight is increased by at least 15 kDa and at most 100 kDa.In one embodiment, the molecular weight is increased by at least 20 kDa and at most 100 kDa. In one embodiment, the molecular weight is increased by at least 1 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 5 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 15 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 18 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 30 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 90 kDa. In one embodiment, the molecular weight is increased by at least 12 kDa and at most 85 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 75 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 70 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 60 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 50 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 49 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 48 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 47 kDa. In one embodiment, the molecular weight is increased by at least 10 kDa and at most 46 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 45 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 44 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 43 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 42 kDa. In one embodiment, the molecular weight is increased by at least 20 kDa and at most 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 O antigen repeating units. In one embodiment, the increase in molecular weight of the O polysaccharide is due to a wzz family protein.

[0157] In another embodiment, the O polysaccharide comprises any one of the formulas selected from Table A, wherein the number of repeating units, n, in the O polysaccharide 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 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, and many more than 100 repeat units. Preferably, the saccharide comprises an increase of 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 repeating units compared to the corresponding wild-type O polysaccharide.

[0158] 2.O antigen O antigens are part of the lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria. O antigens are located 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 Escherichia coli (E. coli) serotyping scheme includes O polysaccharides 1 to 181.

[0159] O antigens comprise oligosaccharide repeating units (O units), the wild-type structure of which typically contains 2-8 residues derived from a wide range of sugars. Exemplary E. coli O antigen O units are described in Table A and in PCT International Publication No. WO2021 / 084429, published May 6, 2021, which is incorporated by reference in its entirety. In some embodiments, the present disclosure includes compositions comprising at least one FimH mutant polypeptide and at least one of the O antigens described in Table A and in PCT International Publication No. WO2021 / 084429, published May 6, 2021, which is incorporated by reference in its entirety.

[0160] In one embodiment, a saccharide of the invention may be a single oligosaccharide unit. In one embodiment, a saccharide of the invention is one repeating oligosaccharide unit of the relevant serotype. In such an embodiment, the saccharide may comprise a structure selected from any one of formula O1a, formula O2, formula O6, formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O25b, formula O52, formula O97, and formula O101. In a further embodiment, the saccharide may comprise a structure selected from any one of formula O1a, formula O2, formula O6, and formula O25b.

[0161] In one embodiment, the sugar of the present invention may be an oligosaccharide. Oligosaccharides have a small number of repeating units (typically 5 to 15 repeating units) and are typically derived synthetically or by hydrolysis of a polysaccharide. In such an embodiment, the sugar may comprise a structure selected from any one of formulas O1a, O2, O6, O8, O9a, O9, O20ab, O20ac, O25b, O52, O97, and O101. In a further embodiment, the sugar may comprise a structure selected from any one of formulas O1a, O2, O6, and O25b.

[0162] Preferably, all saccharides of the present invention and in the immunogenic compositions of the present invention are polysaccharides. High molecular weight polysaccharides can induce a specific antibody immune response due to epitopes present on their antigenic surface. Isolation and purification of high molecular weight polysaccharides are preferably contemplated for use in the conjugates, compositions and methods of the present invention.

[0163] In some embodiments, the number of repeating O units in each individual O-antigen polymer (and thus the length and molecular weight of the polymer chain) 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 that result in two different O Ag modal chain lengths, one long and one short. Expression (not necessarily overexpression) of a wzz family protein (e.g., wzzB) in Gram-negative bacteria can allow for manipulation of O-antigen length to shift or bias bacterial production of a particular length range of O-antigens and enhance the production of high yields of high molecular weight lipopolysaccharides. In one embodiment, "short" modal length as used herein refers to a small number of repeating O units, e.g., 1 to 20 repeating O units. In one embodiment, "long" modal length as used herein refers to a number of repeating O units greater than 20, up to a maximum of 40. In one embodiment, a "very long" mode length as used herein refers to greater than 40 repeating O units.

[0164] In one embodiment, the sugar produced has an increase of at least 10 repeating units, 15 repeating units, 20 repeating units, 25 repeating units, 30 repeating units, 35 repeating units, 40 repeating units, 45 repeating units, 50 repeating units, 55 repeating units, 60 repeating units, 65 repeating units, 70 repeating units, 75 repeating units, 80 repeating units, 85 repeating units, 90 repeating units, 95 repeating units, or 100 repeating units compared to the corresponding wild-type O polysaccharide.

[0165] In another embodiment, the saccharide of the invention 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, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 12 , 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 unit increases. Preferably, the saccharide comprises an increase of 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 repeating units compared to the corresponding wild-type O polysaccharide. See, e.g., Table 21. Methods for determining saccharide length are known in the art. Such methods include nuclear magnetic resonance, mass spectrometry, and size exclusion chromatography, as described in Example 13.

[0166] 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 sugar or KDO residue) by the molecular weight of the repeating unit (i.e., the molecular weight of the structure in the corresponding formula shown in Table 1, which can theoretically be calculated as the sum of the molecular weights of each monosaccharide in the formula). The molecular weight of each monosaccharide in a 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 taken into consideration. As defined herein, "n" refers to the number of repeating units in a polysaccharide molecule (shown in parentheses in Table 1). As is known in the art, in biopolymers, repeat structures may incorporate regions of imperfect repeats, e.g., missing branches. Furthermore, it is known in the art that polysaccharides isolated and purified from natural sources, such as bacteria, may be heterogeneous in size and branching. In such cases, n may represent the mean or median value for n of the molecules in the population.

[0167] In one embodiment, the O polysaccharide has an increase of at least one repeating unit of the O antigen compared to the corresponding wild-type O polysaccharide. The repeating units of the O antigen are shown in Table 1. In one embodiment, the O polysaccharide has an increase of at least one repeating unit of the O antigen compared to the corresponding wild-type O polysaccharide. The repeating units of the O antigen are shown in Table 1. In one embodiment, the O polysaccharide has an increase of at least one repeating unit of the O antigen compared to the corresponding wild-type O polysaccharide. 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more total repeating units. Preferably, the saccharide has a total of at least 3 and at most 80 repeating 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, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23 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 unit increases.

[0168] In one embodiment, the saccharide is selected from the group consisting of O antigens, wherein 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 at least 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, and The O antigens include those O antigens whose n is an integer of at most 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 value can be combined with any maximum value to define a range. Exemplary ranges include, for example, at least 1 to at most 1000; at least 10 to at most 500; and at least 20 to at most 80, preferably at most 90. In one preferred embodiment, n is at least 31 to at most 90. In a preferred embodiment, n is 40 to 90, more preferably 60 to 85.

[0169] In one embodiment, the saccharide comprises an O antigen wherein n in any one of the O antigen formulas is at least 1 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 5 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 10 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 25 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 50 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 75 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 100 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 125 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 150 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 175 and at most 200. In one embodiment, n in any one of the O antigen formulas is at least 1 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 5 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 10 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 25 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 50 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 75 and at most 100. In one embodiment, n in any one of the O antigen formulas is at least 1 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 5 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 10 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 20 and at most 75.In one embodiment, n in any one of the O antigen formulas is at least 25 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 30 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 40 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 50 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 30 and at most 90. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 85. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 75. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 70. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 60. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 50. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 49. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 48. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 47. In one embodiment, n in any one of the O antigen formulas is at least 35 and at most 46. In one embodiment, n in any one of the O antigen formulas is at least 36 and at most 45. In one embodiment, n in any one of the O antigen formulas is at least 37 and at most 44. In one embodiment, n in any one of the O antigen formulas is at least 38 and at most 43. In one embodiment, n in any one of the O antigen formulas is at least 39 and at most 42. In one embodiment, n in any one of the O antigen formulas is at least 39 and at most 41.

[0170] For example, in one embodiment, n in the sugar 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 at least 35 and at most 60. 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 at least 55 and at most 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.

[0171] Sugar structures can be determined by methods and means known in the art, such as, for example, NMR, including 1D, 1H, and / or 13C, 2D TOCSY, DQF-COSY, NOESY, and / or HMQC.

[0172] 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. 00kDa; 15kDa to 150kDa; 12kDa to 120kDa; 12kDa to 75kDa; 12kDa to 50kDa; 12kDa to 60kDa; 35kDa to 75kDa; 40kDa to 60kDa; 35kDa to 60kDa; 20kDa to 60kDa; 12kDa to 20kDa; or 20kDa to 50kDa. 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 kDa; 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 1,000 kDa; 30 kDa to 60 kDa; 30 kDa to 50 kDa, or 5 kDa to 60 kDa. Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0173] As used herein, the term "molecular weight" of a polysaccharide or carrier protein-polysaccharide conjugate refers to the molecular weight calculated by size exclusion chromatography (SEC) coupled with a multi-angle laser light scattering detector (MALLS).

[0174] Polysaccharides may become slightly reduced in size during normal purification procedures. Additionally, as described herein, polysaccharides can be subjected to sizing techniques prior to conjugation. Mechanical or chemical sizing can be used. Chemical hydrolysis can be performed using acetic acid. Mechanical sizing can be performed using high-pressure homogenization shear. The molecular weight ranges listed above refer to purified polysaccharides prior to conjugation (e.g., prior to activation).

[0175] [Table 2-1]

[0176] [Table 2-2]

[0177] [Table 2-3]

[0178] [Table 2-4]

[0179] [Table 2-5]

[0180] [Table 2-6]

[0181] [Table 2-7]

[0182] [Table 2-8]

[0183] [Table 2-9]

[0184] 3. Core oligosaccharides The core oligosaccharide is located between lipid A and the outer region of the O antigen 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 involves a ketosidic bond between the hemiketal function of the innermost 3-deoxy-d-manno-octa-2-ulosonic acid (KDO) residue and the hydroxyl group of the GlcNAc residue of lipid A. The core oligosaccharide region shows high 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.

[0185] 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 residue has the following formula: KDO:

[0186] [ka] Includes:

[0187] The outer region of the core oligosaccharide shows greater variation than the inner core region, and differences in this region distinguish the five chemotypes in Escherichia coli (E. coli): R1, R2, R3, R4, and K-12. The generalized structures of the carbohydrate backbones of the outer core oligosaccharides of the five known chemotypes are well known in the art. Hep II is the last residue of the inner core oligosaccharide. All outer core oligosaccharides share a structural theme, with a (hexose)3 carbohydrate backbone and two side chain residues, although the order of the hexoses in the backbone and the nature, position, and linkage of the side chain residues can all vary. The structures of the R1 and R4 outer core oligosaccharides are very similar, differing only in a single β-linked residue.

[0188] The core oligosaccharide of wild-type E. coli is classified in the art into five different chemotypes based on the structure of the distal oligosaccharide: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12.

[0189] In a preferred embodiment, the compositions described herein comprise a glycoconjugate comprising a core oligosaccharide in which the O polysaccharide is bound to an O antigen. In one embodiment, the composition induces 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. In another embodiment, the composition induces an immune response against at least two core E. coli chemotypes. In another embodiment, the composition induces an immune response against at least three core E. coli chemotypes. In another embodiment, the composition induces an immune response against at least four core E. coli chemotypes. In another embodiment, the composition induces an immune response against all five core E. coli chemotypes.

[0190] In another preferred embodiment, the compositions described herein comprise a saccharide conjugate in which the O polysaccharide does not comprise a core oligosaccharide bound to an O antigen. In one embodiment, such a composition induces 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, even though the saccharide conjugate having the O polysaccharide does not comprise a core oligosaccharide.

[0191] E. coli serotypes can be characterized according to one of five chemotypes. Table B lists exemplary serotypes characterized according to chemotype. Bolded serotypes represent serotypes most commonly associated with the indicated core chemotype. Thus, in preferred embodiments, the composition induces 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, including an immune response against any one of the corresponding E. coli serotypes.

[0192] [Table 3]

[0193] In some embodiments, a 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.

[0194] 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, O6, O2, O1, O75, O4, O16, O18, O13, O20, O21, O91, and O163, where n is 1-100, preferably 31-100, preferably 31-90, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R1 core moiety in the saccharide.

[0195] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R2 chemotype, e.g., selected from saccharides having formula O21, O44, O11, O89, O162, and O9, where n is 1-100, preferably 31-100, preferably 31-90, 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.

[0196] 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, O15, O153, O21, O17, O11, O159, O22, O86, and O93, where n is 1-100, preferably 31-100, preferably 31-90, 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.

[0197] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having an R4 chemotype, e.g., selected from saccharides having formula O2, formula O1, formula O86, formula O7, formula O102, formula O160, and formula O166, where n is 1-100, preferably 31-100, preferably 31-90, more preferably 35-90, and most preferably 35-65. In some embodiments, the saccharide in the composition further comprises an E. coli R4 core moiety.

[0198] In some embodiments, the composition comprises a saccharide comprising a structure derived from a serotype having a K-12 chemotype (e.g., selected from a saccharide having the formula O25b and a saccharide having the formula O16, where n is 1-100, preferably 31-100, preferably 31-90, more preferably 35-90, and most preferably 35-65). In some embodiments, the saccharide in the composition further comprises an E. coli K-12 core moiety.

[0199] 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.

[0200] 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.

[0201] 4. Conjugated O Antigen 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 poses a practical challenge for production. For commercial use, saccharide size can affect compatibility with various conjugation synthesis strategies, product uniformity, and conjugate immunogenicity. 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 various Gram-negative bacterial strains, including E. coli.

[0202] In one embodiment, the purified sugars are chemically activated to produce activated sugars that can react with the carrier protein. Once activated, each sugar is separately conjugated to a carrier protein to form a conjugate, i.e., a glycoconjugate. As used herein, the term "glycoconjugate" refers to a sugar covalently bound to a carrier protein. In one embodiment, the sugar is directly bound to the carrier protein. In another embodiment, the sugar is bound to the carrier protein via a spacer / linker.

[0203] Conjugates can be prepared by a scheme in which the 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.

[0204] In one embodiment, each saccharide is conjugated to the same carrier protein.

[0205] When the protein carrier is the same for two or more sugars in the composition, the sugars can be conjugated to the same molecule of carrier protein (e.g., a carrier molecule having two or more different sugars conjugated thereto).

[0206] In a preferred embodiment, the sugars are each individually conjugated to different molecules of the protein carrier (each molecule of the protein carrier has only one type of sugar conjugated to it), in which embodiment the sugars are said to be individually conjugated to the carrier protein.

[0207] 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 saccharide conjugate is purified (enriched in terms of the amount of polysaccharide-protein conjugate) by various techniques. These techniques include concentration / diafiltration, precipitation / elution, column chromatography, and depth filtration. After the individual saccharide conjugates are purified, they are mixed to formulate the immunogenic composition of the present invention.

[0208] a. Activation. The present invention further relates to activated polysaccharides produced from any of the embodiments described herein, in which the polysaccharide is activated with a chemical reagent that generates 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 substantially reduce the molecular weight of the polysaccharide. For example, in some embodiments, the degree of activation does not cleave the polysaccharide backbone. 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 in the carrier protein (as determined by amino acid analysis), such as those of a reference polysaccharide. For example, in some embodiments, the degree of activation does not significantly increase the number of modified lysine residues in the carrier protein (as determined by amino acid analysis) by more than three times compared to the number of modified lysine residues in the carrier protein of a conjugate with a reference polysaccharide at the same degree of activation. In some embodiments, the degree of activation does not increase the level of unconjugated free sugar. In some embodiments, the degree of activation does not decrease the optimal sugar / protein ratio.

[0209] In some embodiments, the activated saccharide has a percentage of activation of 1-100%, e.g., 2-80%, 2-50%, 3-30%, and 4-25%, for example, where the number of moles of thiol per saccharide repeat unit of the activated saccharide is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, or about 100%. Preferably, the activation level is at most 50%, more preferably at most 25%. In one embodiment, the activation level is at most 20%. Any minimum value can be combined with any maximum value to define a range.

[0210] In one embodiment, the polysaccharide is activated with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide is then coupled to a carrier protein (preferably a CRM). 197 or tetanus toxoid) directly or via a spacer (linker) group.

[0211] For example, the spacer can be cystamine or cysteamine to yield a thiolated polysaccharide that can be coupled to a carrier by 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 76yophilized76e, 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]propionate (SBAP)). In one embodiment, a cyanate ester (which may be generated by CDAP chemistry) is coupled with hexanediamine or adipic acid dihydrazide (ADH), and the amino-derivatized sugar is attached to a carrier protein (e.g., CRM) using carbodiimide (e.g., EDAC or EDC) chemistry via a carboxyl group on the protein carrier. 197 ) to be conjugated.

[0212] Other suitable conjugation techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU. Conjugation may involve 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 may involve reduction of the anomeric terminus to a primary hydroxyl group, optional protection / deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a CDI carbamate intermediate, and coupling of the CDI carbamate intermediate with an amino group on the protein (CDI chemistry).

[0213] b. Molecular Weight. In some embodiments, the saccharide conjugate 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 further embodiments, the sugars are from 100 kDa to 1000 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 8 00kDa;150kDa~700kDa;150kDa~600kDa;150kDa~500kDa;150kDa~400kDa;150kDa~300kDa;200kDa~ 1,000kDa;200kDa~900kDa;200kDa~800kDa;200kDa~700kDa;200kDa~600kDa;200kDa~500kDa;200k Da~400kDa;200kDa~300kDa;250kDa~1,000kDa;250kDa~900kDa;250kDa~800kDa;250kDa~700kDa;2 50kDa~600kDa;250kDa~500kDa;250kDa~400kDa;250kDa~350kDa;300kDa~1,000kDa;300kDa~900kDa The sugar conjugates 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, and 500 kDa to 600 kDa. In one embodiment, sugar conjugates having such molecular weights are produced by single-end conjugation. In another embodiment, sugar conjugates having such molecular weights are produced by reductive amination chemistry (RAC) prepared in an aqueous buffer.Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0214] In some embodiments, the 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, the glycoconjugates have a molecular weight of 500 kDa to 10,000 kDa. In other embodiments, the glycoconjugates have a molecular weight of 1,000 kDa to 8,000 kDa. In still other embodiments, the 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 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; 200 kDa to 3,000 kDa; 200 kDa to 1,000 kDa; 500 kDa to 20,000 kDa; 500 kDa to 15,000 kDa; 500 kDa to 12,500 kDa; 500 kDa to 10,000 kDa. Da;500kDa~7,500kDa;500kDa~6,000kDa;500kDa~5,000kDa;500kDa~4,000kDa;500kDa~3,000kDa;500kDa~2,000kDa;500 kDa~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~4,000kDa;750kDa~3,000kDa;750kDa~2,000kDa;750kDa~1,500 kDa;1,000kDa~15,000kDa;1,000kDa~12,500kDa;1,000kDa~10,000kDa;1,000kDa~7,500kDa;1,000kDa~6,000kDa;1,000k The glycoconjugates may have a molecular weight of between 5,000 kDa, 1,000 kDa and 4,000 kDa, 1,000 kDa and 2,500 kDa, 2,000 kDa and 15,000 kDa, 2,000 kDa and 12,500 kDa, 2,000 kDa and 10,000 kDa, 2,000 kDa and 7,500 kDa, 2,000 kDa and 6,000 kDa, 2,000 kDa and 5,000 kDa, 2,000 kDa and 4,000 kDa, or 2,000 kDa and 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.

[0215] In further embodiments, the glycoconjugates of the invention have a molecular weight of between 1,000 kDa and 20,000 kDa; between 1,000 kDa and 15,000 kDa; between 2,000 kDa and 10,000 kDa; between 2,000 kDa and 7,500 kDa; between 2,000 kDa and 5,000 kDa; between 3,000 kDa and 20,000 kDa; between 3,000 kDa and 15,000 kDa; between 3,000 kDa and 12,500 kDa; between 4,000 kDa and 10,000 kDa; between 4,000 kDa and 7,500 kDa; between 4,000 kDa and 6,000 kDa; or between 5,000 kDa and 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.

[0216] 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.

[0217] The molecular weight of the glycoconjugate can be measured by SEC-MALLS. Any whole 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 sugar to carrier protein ratio (w / w). In some embodiments, the polysaccharide to carrier protein ratio (w / w) in the glycoconjugate is 0.5 to 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 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0). In other embodiments, the sugar to carrier protein ratio (w / w) is 0.5-2.0, 0.5-1.5, 0.8-1.2, 0.5-1.0, 1.0-1.5, or 1.0-2.0. In further embodiments, the sugar to carrier protein ratio (w / w) is 0.8-1.2. In preferred embodiments, the polysaccharide to carrier protein ratio in the conjugate is 0.9-1.1. In some such embodiments, the carrier protein is a CRM. 197 is.

[0218] The glycoconjugates were also analyzed based on their molecular size distribution (K d ) can also be characterized. Size exclusion chromatography media (CL-4B) can be used to determine the relative molecular size distribution of the conjugates. Size exclusion chromatography (SEC) is used in a gravity-fed column to profile the molecular size distribution of the conjugates. Large molecules, which are excluded from small pores in the media, elute more quickly than small molecules. A fraction collector is used to collect the column eluate. Fractions are tested colorimetrically with a sugar assay. K d For the determination of the fraction at which molecules are completely excluded (V0), (K d = 0), and the fraction showing maximum retention (V i ), (K d The column is calibrated to establish the fraction at which a particular sample characteristic is reached (V e ) into the formula K d=(V e -V0) / (V i -V0) by K d Associate with.

[0219] c. Free Sugars. The glycoconjugates and immunogenic compositions of the present invention may contain free saccharides that are not covalently conjugated to a carrier protein but are nevertheless present in the glycoconjugate composition. The free saccharides may be non-covalently associated with the glycoconjugate (i.e., non-covalently bound, adsorbed, or entrapped within or with it). In preferred embodiments, the glycoconjugate comprises at most 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises less than about 25% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises at most about 20% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises at most about 15% free polysaccharide relative to the total amount of polysaccharide. In another preferred embodiment, the saccharide conjugate comprises at most about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the saccharide conjugate comprises less than about 8% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the saccharide conjugate comprises at most about 6% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the saccharide conjugate comprises at most about 5% free polysaccharide relative to the total amount of polysaccharide.

[0220] d. Covalent Bonds. 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 frequent embodiments, the carrier protein is a CRM. 197 In another embodiment, at least one bond between the carrier protein and the saccharide is present every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 saccharide repeat units of the polysaccharide. In one embodiment, the carrier protein is a CRM 197 Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure.

[0221] e. Lysine Residues. Another method of characterizing the glycoconjugates of the present invention is to characterize the extent of conjugated lysine (degree of conjugation) of the carrier protein (e.g., CRMP) that becomes conjugated to the sugar. 197The degree of conjugation depends on the number of lysine residues in the carrier protein. Evidence for 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 skilled in the art. Conjugation results in a reduction in 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 present invention is between 4 and 7. In some such embodiments, the carrier protein is a CRM 197 is.

[0222] The frequency of attachment of glycans to lysines on the carrier protein is another parameter for characterizing the glycoconjugates of the present invention. For example, in some embodiments, at least one covalent bond between the carrier protein and the polysaccharide occurs for every 4 saccharide repeating units of the polysaccharide. In other embodiments, at least one covalent bond between the carrier protein and the polysaccharide occurs for every 10 saccharide repeating units of the polysaccharide. In other embodiments, at least one covalent bond between the carrier protein and the polysaccharide occurs for every 15 saccharide repeating units of the polysaccharide. In other embodiments, at least one covalent bond between the carrier protein and the polysaccharide occurs for every 25 saccharide repeating units of the polysaccharide.

[0223] f O-acetylation. In some embodiments, sugars of the present invention are O-acetylated. In some embodiments, sugar conjugates comprise sugars 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% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more, or about 100%. % O-acetylation refers to the percentage of a given sugar relative to 100% (each repeating unit is fully acetylated relative to its acetylated structure).

[0224] In some embodiments, the sugar conjugate is prepared by reductive amination. In some embodiments, the sugar conjugate is a single-end-linked conjugated sugar in which the sugar is directly covalently bound to the carrier protein. In some embodiments, the sugar conjugate is covalently bound to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

[0225] g. Reductive Amination In one embodiment, the sugar is conjugated to the carrier protein by reductive amination (such as described in U.S. Patent Application Publication Nos. 2006 / 0228380, 2007 / 0231340, 2007 / 0184071 and 2007 / 0184072, WO2006 / 110381, WO2008 / 079653, and WO2008 / 143709).

[0226] Reductive amination involves (1) oxidation of a sugar, and (2) reduction of the activated sugar and carrier protein to form a conjugate. Prior to oxidation, the sugar may be hydrolyzed. Mechanical or chemical hydrolysis can be used. Chemical hydrolysis can be carried out using acetic acid.

[0227] The oxidation step may involve reaction with periodate. As used herein, the term "periodate" refers to both periodate and periodic acid. The term also refers to metaperiodate (IO4 - ) and orthoperiodate (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 metaperiodate, preferably in the presence of sodium periodate (NaIO). In another embodiment, the polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of periodic acid.

[0228] 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 oxidizing agent to selectively oxidize the primary hydroxyl. In the reaction, the actual oxidizing agent is an N-oxoammonium salt in the catalytic cycle. In some aspects, the stable nitroxyl or nitroxide radical compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. In some aspects, the stable nitroxyl or nitroxide radical compound bears a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In some aspects, the stable nitroxyl radical compound is TEMPO or a derivative thereof. In some aspects, the oxidizing agent is a molecule bearing an N-halo moiety. In one embodiment, the oxidizing agent 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 oxidizing agent is N-chlorosuccinimide.

[0229] After the sugar oxidation step, the sugar is said to be activated, and is hereinafter referred to as "activated." The activated sugar and carrier protein can be lyophilized (freeze-dried) independently (separate lyophilization) or together (co-lyophilization). In one embodiment, the activated sugar and carrier protein are co-lyophilized. In another embodiment, the activated polysaccharide and carrier protein are lyophilized independently.

[0230] In one embodiment, lyophilization is carried out in the presence of a non-reducing sugar, which may include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinit.

[0231] The next step in the conjugation process is the reduction of the activated sugar and carrier protein to form a conjugate using a reducing agent (so-called reductive amination). Suitable reducing agents include cyanoborohydrides such as sodium cyanoborohydride, sodium triacetoxyborohydride, or sodium or zinc borohydride in the presence of a Bronsted or Lewis acid, amine boranes such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe'PrN-BH3, benzylamine-BH3, or 5-ethyl-2-methylpyridine borane (PEMB), borane-pyridine, or borohydride exchange resin. In one embodiment, the reducing agent is sodium cyanoborohydride.

[0232] 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 pH 6.0-8.5, 7.0-8.0, or 7.0-7.5). 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 lyophilized activated sugars and carrier proteins.

[0233] 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 (NaBH4). After conjugation (reduction reaction and, if necessary, capping), the glycoconjugate can be purified (enriched in terms of the amount of polysaccharide-protein conjugate) by various techniques known to those skilled in the art. These techniques include dialysis, concentration / diafiltration operations, tangential flow filtration sedimentation / elution, column chromatography (DEAE or hydrophobic interaction chromatography), and depth filtration. The glycoconjugate can be purified by diafiltration and / or ion exchange chromatography and / or size exclusion chromatography. In one embodiment, the glycoconjugate is purified by diafiltration or ion exchange chromatography or size exclusion chromatography. In one embodiment, the glycoconjugate is sterile filtered.

[0234] In a preferred embodiment, glycoconjugates derived from E. coli serotypes selected from any one of O25B, O1a, O2, and O6 are prepared by reductive amination. In a preferred embodiment, glycoconjugates derived from E. coli serotypes O25B, O1a, O2, and O6 are prepared by reductive amination.

[0235] In one aspect, the present invention provides a carrier protein, e.g.,

[0236] [ka] [wherein n is an integer of 1 or more] A CRM linked to a sugar of formula O25B, represented by 197 In preferred embodiments, n is an integer of at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, and at most 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 value can be combined with any maximum value to define a range. Exemplary ranges include, for example, at least 1 and at most 1000; at least 10 and at most 500; and at least 20 and at most 80. In one preferred embodiment, n is at least 31 and at most 90, more preferably 40-90, and most preferably 60-85.

[0237] In another aspect, the present invention provides a carrier protein, e.g., CRM, conjugated to a saccharide having any one of the following structures shown in Table A, where n is an integer greater than or equal to 1: 197 The present invention relates to a conjugate comprising:

[0238] Without being bound by theory or mechanism, it is believed that in some embodiments, stable conjugates require a level of antigen modification that balances preservation of the structural integrity of important immunogenic epitopes of the antigen.

[0239] h. Activation and Formation of Aldehydes. In some embodiments, the sugars of the present invention are activated, resulting in the formation of aldehydes. In those embodiments in which sugars are activated, the percentage (%) of activation (or degree of oxidation (DO)) refers to the moles of sugar repeating units per mole of aldehyde of the activated polysaccharide. For example, in some embodiments, sugars are activated by periodate oxidation of vicinal diols on the repeating units of the polysaccharide, resulting in the formation of aldehydes. Varying the molar equivalents (meq) of sodium periodate relative to the sugar repeating unit and the temperature during oxidation results in varying levels of degree of oxidation (DO).

[0240] Sugar and aldehyde concentrations are typically determined by colorimetric assays. An alternative reagent is TEMPO (2,2,6,6-tetramethylpiperidine 1-oxyl radical)-N-chlorosuccinimide (NCS) mixture, which leads to the formation of aldehydes from primary alcohol groups.

[0241] In some embodiments, the activated saccharide has a degree of oxidation of 1 to 100, e.g., 2 to 80, 2 to 50, 3 to 30, and 4 to 25 moles of saccharide repeating units per mole of aldehyde of the activated saccharide. 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 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, or 90 or more, or about 100. Preferably, the degree of oxidation (DO) is at least 5 and at most 50, more preferably at least 10 and at most 25. In one embodiment, the DO is at least 10 and at most 25. Any minimum value and any maximum value can be combined to define a range. The DO value can be expressed as a percentage (%) of activation. For example, in one embodiment, a DO value of 10 refers to 1 activated saccharide repeat unit out of a total of 10 saccharide repeat units in the activated saccharide, in which case a DO value of 10 can be expressed as 10% activation.

[0242] In some embodiments, the conjugates prepared by reductive amination chemistry comprise a carrier protein and a sugar, wherein the sugar 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 O5 4, 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 O 102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O11 2, 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 O1 34, 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 O15 6, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O In some embodiments, the sugar in the conjugate comprises a structure selected from any one of 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 sugar in the conjugate comprises a formula where n is an integer between 1 and 1000, 5 and 1000, preferably between 31 and 100 or 31 and 90, more preferably between 35 and 90, and most preferably between 35 and 65.

[0243] i. Single-Ended Conjugates. In some embodiments, the conjugate is a single-end-linked conjugated saccharide in which the saccharide is covalently attached to the carrier protein at one end of the saccharide. In some embodiments, the single-end-linked conjugated polysaccharide has a terminal saccharide. For example, a conjugate is single-end-linked when one end of the polysaccharide (the terminal sugar residue) is covalently attached to the carrier protein. In some embodiments, a conjugate is single-end-linked when the terminal sugar residue of the polysaccharide is covalently attached to the carrier protein via a linker. Such linkers may 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).

[0244] In some embodiments, the sugar is conjugated to the carrier protein via a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue to form a single-end linked conjugate.

[0245] In some embodiments, the conjugate is preferably not a bioconjugate. The term "bioconjugate" refers to a conjugate of a protein (e.g., a carrier protein) and an antigen, such as an O antigen (e.g., O25B), prepared in a host cell background, where the host cell machinery binds the antigen to the protein (e.g., N-linkage). Glycoconjugates include bioconjugates prepared by means that do not require preparation of the conjugate in a host cell, such as conjugation by chemical bonding of the protein and the sugar, as well as saccharide antigen (e.g., oligosaccharide and polysaccharide)-protein conjugates.

[0246] j. Thiol-Activated Sugars. In some embodiments, the sugars of the present invention are thiol-activated. In such embodiments in which the sugar is thiol-activated, the percentage (%) of activation refers to the moles of thiol per saccharide repeat unit of the activated polysaccharide. Sugar and thiol concentrations are typically determined by Ellman's assay for sulfhydryl quantification. For example, in some embodiments, the sugar comprises activation of 2-keto-3-deoxyoctanoic acid (KDO) using a disulfide amine linker. In some embodiments, the sugar is covalently attached to the carrier protein via a bivalent heterobifunctional linker (also referred to herein as a "spacer"). The linker preferably provides a thioether bond between the sugar and the carrier protein, resulting in a sugar conjugate referred to herein as a "thioether sugar conjugate." In some embodiments, the linker further provides a carbamate and amide bond, such as, for example, (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC).

[0247] In some embodiments, the single-end linked conjugate comprises a carrier protein and a sugar, wherein the sugar 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 O18A), 1, 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 O90, formula O9 1, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O 104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O11 4, 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 O1 36, 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 O 169, 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 sugar in the conjugate comprises a formula where 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.

[0248] For example, in one embodiment, a single-end linked conjugate comprises a carrier protein and a sugar having a structure selected from formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97, and formula O101, where n is an integer from 1 to 10.

[0249] 5. eTEC conjugates In one aspect, the present invention generally relates to 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 Application Publication No. WO2014027302, the entire contents of which are incorporated herein by reference), immunogenic compositions comprising such glycoconjugates, and methods for the preparation and use of 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 by a carbamate bond and the carrier protein is covalently conjugated to the eTEC spacer by an amide bond. The eTEC spacer contains seven linear atoms (ie, -C(O)NH(CH2)2SCH2C(O)-) and provides stable thioether and amide bonds between the sugar and the carrier protein.

[0250] The eTEC-binding glycoconjugates of the present invention have the general formula (I):

[0251] [ka] [wherein the atoms that make up the eTEC spacer are contained within the central box] It can be represented by:

[0252] In the glycoconjugate of the present invention, the sugar may be a polysaccharide or an oligosaccharide.

[0253] The carrier protein incorporated into the glycoconjugates of the present invention is selected from the group of carrier proteins generally suitable for such purposes, as further described herein or as known to those skilled in the art. In certain embodiments, the carrier protein is a CRM 197 is.

[0254] In another aspect, the present invention provides a method of making a sugar conjugate comprising a sugar described herein conjugated to a carrier protein via an eTEC spacer, the method comprising: a) reacting the sugar with a carbonic acid derivative in an organic solvent to produce an activated sugar; b) reacting the activated sugar with cystamine or cysteamine, or a salt thereof, to produce a thiolated sugar; c) reacting the thiolated sugar with a reducing agent to produce an activated thiolated sugar comprising one or more free sulfhydryl residues; d) reacting the activated thiolated sugar with one or more α-haloacetates. and e) reacting the thiolated sugar-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 sugar, thereby producing an eTEC-binding sugar conjugate.

[0255] In a common embodiment, 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 dimethyl sulfoxide (DMSO). In a preferred embodiment, the thiolated sugar is produced by reacting an activated sugar with a bifunctional symmetric thioalkylamine reagent, cystamine, or a salt thereof. Alternatively, the thiolated sugar can be formed by reacting an activated sugar with cysteamine, or a salt thereof. The eTEC-binding sugar conjugate produced by the methods of the present invention can be represented by the general formula (I):

[0256] In a common embodiment, the first capping reagent is N-acetyl-L-cysteine, which reacts with unconjugated α-haloacetamide groups on lysine residues of the carrier protein to form S-carboxymethylcysteine ​​(CMC) residues that are covalently attached to the activated lysine residues via a thioether bond.

[0257] In other embodiments, the second capping reagent is iodoacetamide (IAA), which reacts with the unconjugated free sulfhydryl groups of the activated thiolated sugar to provide a capped thioacetamide. Step e) often involves capping with both a first capping reagent and a second capping reagent. In certain embodiments, step e) involves capping with N-acetyl-L-cysteine ​​as the first capping reagent and IAA as the second capping reagent.

[0258] In some embodiments, the capping step e) further comprises, after reaction with the first and / or second capping reagent, reaction with a reducing agent, such as DTT, TCEP, or mercaptoethanol.

[0259] The eTEC-linked sugar conjugates and immunogenic compositions of the present invention may contain free sulfhydryl residues. In some examples, the activated thiolated sugars formed by the methods provided herein contain multiple free sulfhydryl residues, some of which may not undergo covalent conjugation to a carrier protein during the conjugation step. Such remaining free sulfhydryl residues are capped by reaction with a thiol-reactive capping reagent, such as iodoacetamide (IAA), to cap potentially reactive functional groups. Other thiol-reactive capping reagents, such as maleimide-containing reagents, are also contemplated.

[0260] Additionally, the eTEC-binding glycoconjugates and immunogenic compositions of the present invention may contain residual unconjugated carrier protein or may contain activated carrier protein that has undergone modification during the capping process step.

[0261] In some embodiments, step d) further comprises providing an activated carrier protein comprising one or more α-haloacetamide groups prior to reacting the activated thiolated sugar with the activated carrier protein. In frequent embodiments, the activated carrier protein comprises one or more α-bromoacetamide groups.

[0262] In another aspect, the invention provides an eTEC-linked glycoconjugate comprising a sugar described herein conjugated to a carrier protein via an eTEC spacer produced according to any of the methods disclosed herein.

[0263] In some embodiments, the carrier protein is a CRM 197 and CRM 197 The covalent bond between the eTEC spacer and the polysaccharide occurs at least once per 4, 10, 15, or 25 saccharide repeat units of the polysaccharide.

[0264] For each of the aspects of the invention, and particularly for certain embodiments of the methods and compositions described herein, the eTEC-binding sugar conjugate comprises a sugar described herein, such as a sugar derived from Escherichia coli (E. coli).

[0265] In another aspect, the present invention provides a method of preventing, treating, or ameliorating a bacterial infection, disease, or condition in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition of the present invention, wherein the immunogenic composition comprises an eTEC-binding glycoconjugate comprising a saccharide described herein. In some embodiments, the saccharide is derived from Escherichia coli (E. coli).

[0266] In some embodiments, the eTEC-binding glycoconjugate comprises a carrier protein and a glycoconjugate 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 O5 8, 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 O8 0, 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 O1 05, 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 O1 37, 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 and a sugar comprising a structure selected from any one of 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 sugar in the conjugate comprises a formula where 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.

[0267] The number of lysine residues in the carrier protein that become conjugated to a saccharide can be characterized as the conjugated lysine range. For example, in some embodiments of the immunogenic composition, the CRM 197 may contain 4 to 16 of the 39 lysine residues covalently attached to the sugar. Another way to express this parameter is the CRM 197 In another embodiment, the CRM has between about 10% and about 41% of the lysines covalently bound to sugars. 197 may contain 2-20 of the 39 lysine residues covalently attached to the sugar. Another way to express this parameter is the CRM 197 Approximately 5% to 50% of the lysines are covalently bonded to sugars.

[0268] In a common embodiment, the carrier protein is a CRM 197 and CRM 197 The covalent bond between the eTEC spacer and the polysaccharide occurs at least once per 4, 10, 15, or 25 saccharide repeat units of the polysaccharide.

[0269] In other embodiments, the conjugate comprises at least one covalent bond between the carrier protein and the saccharide for every 5-10 saccharide repeating units; every 2-7 saccharide repeating units; every 3-8 saccharide repeating units; every 4-9 saccharide repeating units; every 6-11 saccharide repeating units; every 7-12 saccharide repeating units; every 8-13 saccharide repeating units; every 9-14 saccharide repeating units; every 10-15 saccharide repeating units; every 2-6 saccharide repeating units; every 3-7 saccharide repeating units; every 4-8 saccharide repeating units; every 6-10 saccharide repeating units; every 7-11 saccharide repeating units; every 8-12 saccharide repeating units; every 9-13 saccharide repeating units; every 10-14 saccharide repeating units; every 10-20 saccharide repeating units; or every 4-25 saccharide repeating units.

[0270] In another embodiment, at least one bond between the carrier protein and the saccharide occurs every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 saccharide repeat units of the polysaccharide.

[0271] 6. Carrier Proteins A component of the glycoconjugates of the present invention is a carrier protein to which a sugar is conjugated. The terms "protein carrier" or "carrier protein" or "carrier" can be used interchangeably herein. The carrier protein should be suitable for standard conjugation procedures.

[0272] One component of the conjugate is a carrier protein to which the O polysaccharide is conjugated. In one embodiment, the conjugate comprises a carrier protein conjugated to the core oligosaccharide of the O polysaccharide. In one embodiment, the conjugate comprises a carrier protein conjugated to the O antigen of the O polysaccharide.

[0273] The terms "protein carrier" or "carrier protein" or "carrier" can be used interchangeably herein. The carrier protein should be suitable for standard conjugation procedures.

[0274] In a preferred embodiment, the carrier protein of the conjugate is TT, DT, DT variant (CRM 197 and the like), H. influenzae protein D, PhtX, PhtD, PhtDE fusions (particularly those described in WO01 / 98334 and WO03 / 54007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, C. difficile toxin A or B, and PsaA. In one embodiment, the carrier protein of the conjugate of the invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of the conjugate of the invention is TT (tetanus toxoid). In another embodiment, the carrier protein of the conjugate of the invention is PD (Haemophilus influenzae protein D—see, e.g., EP0594610B). In some embodiments, the carrier protein comprises poly(L-lysine) (PLL).

[0275] In a preferred embodiment, the sugar is a CRM 197 Conjugated to proteins. CRM 197 The protein is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. 197 The nontoxigenic phage β197tox was generated by nitrosoguanidine mutagenesis of the toxigenic corynephage beta. - CRM is produced by Clostridium difficile (C. difficile) infected by 197 The protein has the same molecular weight as diphtheria toxin but differs from it by a single base change in the structural gene (guanine to adenine). This single base change causes an amino acid substitution in the mature protein (glutamic acid to glycine), eliminating the toxicity of diphtheria toxin. 197 Proteins are safe and effective T cell-dependent carriers for sugars.

[0276] Thus, in some embodiments, the conjugates of the invention may be modified such that the sugar is a CRM 197 CRM as a carrier protein covalently bound to 197 Includes.

[0277] In a preferred embodiment, the carrier protein of the glycoconjugate is DT (diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM197 (a non-toxic but antigenically identical variant of diphtheria toxin), other DT mutants (CRM176, CRM228, CRM45 (Uchida et al., J. Biol. Chem. 218:3838-3844, 1973), CRM9, CRM45, CRM102, CRM103 or CRM107; and Nicholls and Youle in Genetically Engineered Toxins, Eds.: Frankel, Maecel Dekker and other mutations described by Inc. 1992; deletions or mutations of Glu-148 to Asp, Gln, or Ser and / or Ala158 to Gly and other mutations disclosed in U.S. Pat. Nos. 4,709,017 and 4,950,740; mutations of at least one or more residues of Lys516, Lys526, Phe530, and / or Lys534 and other mutations disclosed in U.S. Pat. Nos. 5,917,017 or 6,455,673; or fragments disclosed in U.S. Pat. No. 5,843,711), which are detoxified in some manner. pneumococcal pneumolysin (Kuo et al. (1995) Infect. ... lmmun 63:2706~13), OMPC (meningococcal outer membrane protein usually extracted from N. meningitidis serogroup B - EP0372501), PorB (meningococcus (N.meningitidis), PD (Haemophilus influenzae protein D - see e.g., EP 0594610B), or immunologically functional equivalents thereof, synthetic peptides (EP 0378881, EP 0427347), heat shock proteins (WO 93 / 17712, WO 94 / 03208), pertussis proteins (WO 98 / 58668, EP 0471177), cytokines, lymphokines, growth factors or hormones (WO 91 / 01146), artificial proteins containing multiple human CD4+ T cell epitopes derived from antigens from various pathogens (Falugi et al. (2001) Eur J Immunol 31:3816-3824), e.g., N19 protein (Baraldoi et al. (2004) Infect Immunol 72:4884-7), pneumococcal surface protein PspA (WO02 / 091998), iron uptake protein (WO01 / 72337), Clostridium difficile toxin A or B (WO00 / 61761), transferrin-binding protein, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa exotoxin A (especially its non-toxic mutants, e.g., exotoxin A carrying a substitution at glutamic acid 553 (Uchida, Cameron DM, RJ Collier, 1987; J. Bacteriol. 169:4967-4971). Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or purified protein derivative of tuberculin (PPD), can also be used as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins, such as cholera toxoid (e.g., as described in International Patent Application WO 2004 / 083251), E. coli LT, E. coli ST, and exotoxin A derived from Pseudomonas aeruginosa.

[0278] In some embodiments, the carrier protein is, for example, a CRM 197, diphtheria toxin fragment B (DTFB), DTFB C8, diphtheria toxoid (DT), tetanus toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid, or exotoxin A from Pseudomonas aeruginosa; detoxified exotoxin A (EPA) from Pseudomonas aeruginosa; maltose-binding protein (MBP), flagellin, detoxified hemolysin A from Staphylococcus aureus, clumping factor A, clumping factor B, cholera toxin B subunit (CTB), Streptococcus pneumoniae The carrier protein is selected from any one of C. pneumoniae pneumolysin and detoxified variants thereof, C. jejuni AcrA, C. jejuni native glycoprotein, and Streptococcal C5a peptidase (SCP). In one embodiment, the carrier protein is detoxified Pseudomonas exotoxin (EPA). In another embodiment, the carrier protein is not detoxified Pseudomonas exotoxin (EPA). In one embodiment, the carrier protein is flagellin. In another embodiment, the carrier protein is not flagellin.

[0279] In a preferred embodiment, the carrier protein of the glycoconjugate is selected from the group consisting of TT, DT, DT variants (CRM 197and the like), H. influenzae protein D, PhtX, PhtD, PhtDE fusions (particularly those described in WO01 / 98334 and WO03 / 54007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, C. difficile toxin A or B, and PsaA. In one embodiment, the carrier protein of a glycoconjugate of the present invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of a glycoconjugate of the present invention is TT (tetanus toxoid). In another embodiment, the carrier protein of a glycoconjugate of the present invention is PD (Haemophilus influenzae protein D - see, e.g., EP0594610B).

[0280] In a preferred embodiment, the capsular saccharide of the invention is a CRM 197 Conjugated to proteins. CRM 197 The protein is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. 197 CRM is produced by Corynebacterium diphtheriae (C. diphtheriae) infected with the nontoxigenic phage β197tox-, which was created by nitrosoguanidine mutagenesis of the toxigenic corynebacterium phage beta (Uchida, T. et al., 1971, Nature New Biology 233:8-11). 197 The protein has the same molecular weight as diphtheria toxin but differs from it by a single base change in the structural gene (guanine to adenine). This single base change causes an amino acid substitution in the mature protein (glutamic acid to glycine), eliminating the toxicity of diphtheria toxin. 197 Proteins are safe and effective T cell-dependent carriers for sugars. 197 and further details regarding its production can be found, for example, in US Pat. No. 5,614,382.

[0281] Thus, in a frequent embodiment, the glycoconjugates of the invention are those in which the capsular polysaccharide is a CRM 197 CRM as a carrier protein covalently bound to 197 Includes:

[0282] In a further embodiment, the carrier protein of the glycoconjugate is SCP (Streptococcal C5a peptidase). All human isolates of β-hemolytic streptococci produce a highly conserved cell wall protein, SCP (Streptococcal C5a peptidase), which specifically inactivates C5a. The scp gene encodes a polypeptide containing 1,134 to 1,181 amino acids (Brown et al., PNAS, 2005, Vol. 102, No. 51, pp. 18391-18396). The first 31 residues are an export signal presequence, which is removed during passage through the cell membrane. The next 68 residues function as a prosequence and must be removed to produce active SCP. The next 10 residues can be removed without loss of protease activity. At the other end, starting from Lys-1034, there are four consecutive 17-residue motifs followed by a cell sorting and cell wall attachment signal. This combined signal consists of a 20 residue hydrophilic sequence containing the LPTTND sequence, a 17 residue hydrophobic sequence and a short basic carboxyl terminus.

[0283] SCPs can be divided into domains (see Figure 1B in Brown et al., PNAS, 2005, Vol. 102, No. 51, pp. 18391-18396). These domains include the pre / pro domain (which contains the export signal pre-sequence (typically the first 31 residues) and the pro-sequence (typically the next 68 residues)), the protease domain (divided into two parts: protease part 1 typically occupies residues 89-333 / 334, and protease domain part 2 typically occupies residues 467 / 468-583 / 584), and the protease-associated domain (PA domain). (generally, residues 333 / 334 to 467 / 468), three fibronectin type III (Fn) domains (Fn1, generally, residues 583 / 584 to 712 / 713; Fn2, generally, residues 712 / 713 to 928 / 929 / 930; Fn3, generally, residues 929 / 930 to 1029 / 1030 / 1031), and a cell wall anchor domain (generally, residues 1029 / 1030 / 1031 to the C-terminus).

[0284] In one embodiment, the carrier protein of the glycoconjugate of the present invention is SCP from GBS (SCPB). An example of SCPB is provided in SEQ ID NO: 3 of WO 97 / 26008. See also SEQ ID NO: 3 of WO 00 / 34487.

[0285] In another embodiment, the carrier protein of the glycoconjugate of the present invention is a GAS-derived SCP (SCPA). Examples of SCPA can be found in SEQ ID NO: 1 and SEQ ID NO: 2 of WO 97 / 26008. See also SEQ ID NO: 1, 2 and 23 of WO 00 / 34487.

[0286] In a further embodiment, the carrier protein of the glycoconjugate of the invention is an SCP as set forth in SEQ ID NO: 150 or 151 of WO2014 / 136064.

[0287] B. Adjuvants In some embodiments, the immunogenic compositions disclosed herein may further comprise at least one, two, or three adjuvants. The term "adjuvant" refers to a compound or mixture that enhances the immune response to an antigen. The antigen may act primarily as a delivery system, primarily as an immunomodulator, or have both strong properties. Suitable adjuvants include those suitable for use in mammals, including humans.

[0288] Examples of known suitable delivery system-type adjuvants that can be used in humans include, but are not limited to, alum (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide), calcium phosphate, liposomes, oil-in-water emulsions such as MF59 (4.3% w / v squalene, 0.5% w / v polysorbate 80 (Tween 80), 0.5% w / v sorbitan trioleate (Span 85)), water-in-oil emulsions such as Montanide, and poly(D,L-lactide-co-glycolide) (PLG) microparticles or nanoparticles.

[0289] In one aspect, the immunogenic compositions disclosed herein comprise an aluminum salt (alum) (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide) as an adjuvant. In a further aspect, the immunogenic compositions disclosed herein comprise aluminum phosphate or aluminum hydroxide as an adjuvant. In one aspect, the immunogenic compositions disclosed herein comprise 0.1 mg / mL to 1 mg / mL or 0.2 mg / mL to 0.3 mg / mL of elemental aluminum in the form of aluminum phosphate. In one aspect, the immunogenic compositions disclosed herein comprise about 0.25 mg / mL of elemental aluminum in the form of aluminum phosphate.

[0290] Examples of known suitable immunomodulatory adjuvants that can be used in humans include, but are not limited to, saponin extract from Aquilla bark (QS21, QuilA), TLR4 agonists such as MPL (monophosphoryl lipid A), 3DMPL (3-O-deacylated MPL) or GLA-AQ, LT / CT mutants, cytokines such as various interleukins (e.g., IL-2, IL-12) or GM-CSF, AS01, etc.

[0291] Examples of known suitable immunomodulatory adjuvants that exhibit both delivery and immunomodulatory properties that can be used in humans include, but are not limited to, ISCOMS (see, e.g., Sjolander et al. (1998) J. Leukocyte Biol. 64:713; WO90 / 03184, WO96 / 11711, WO00 / 48630, WO98 / 36772, WO00 / 41720, WO2006 / 134423 and WO2007 / 026190) or GLA-EM, which is a combination of a TLR4 agonist and an oil-in-water emulsion.

[0292] For veterinary applications, such as, but not limited to, animal testing, one skilled in the art would know of various adjuvants, including Freund's complete adjuvant (CFA), Freund's incomplete adjuvant (IFA), Emulsigen, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP), and other adjuvants. 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL+TDM+CWS), in a 2% squalene / Tween 80 emulsion, can be used.

[0293] Additional exemplary adjuvants for enhancing the effectiveness of the immunogenic compositions disclosed herein include, but are not limited to, (1) SAF containing, for example, (a) 10% squalane, 0.4% Tween 80, 5% Pluronic® blocking polymer L121, and thr-MDP microfluidized to a submicron emulsion or vortexed to generate a larger particle size emulsion, and (b) RIBI™ Adjuvant System (RAS) (Ribi™) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™). (1) saponin adjuvants such as QS21, STIMULON™ (Cambridge Bioscience, Worcester, Mass.), ABISCO® (Isconova, Sweden), or particles generated therefrom, such as ISCOMATRIX® (Commonwealth Serum Laboratories, Australia), or ISCOMs (immunostimulating complexes) that may contain no additional detergents (e.g., WO 00 / 07621); (2) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (3) interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-29, IL-21, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29 ...9, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, (5) cytokines such as monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see, e.g., GB 2220211, EP 0689454) (see, e.g., WO 00 / 56358);(6) 3dMPL in combination with, for example, QS21 and / or oil-in-water emulsions (see, for example, EP 0835318, EP 0735898, EP 0761231); (7) polyoxyethylene ethers or polyoxyethylene esters (see, for example, WO 99 / 52549); (8) polyoxyethylene sorbitan ester surfactants in combination with octoxynol (see, for example, WO 01 / 21207) or polyoxyethylene alkyl ethers or esters in combination with at least one further non-ionic surfactant such as octoxynol. (9) saponin and immunostimulatory oligonucleotides (e.g., CpG oligonucleotides) (e.g., WO00 / 62800); (10) particles of immunostimulants and metal salts (see, e.g., WO00 / 23105); (11) saponin and oil-in-water emulsions (e.g., WO99 / 11241); (12) saponin (e.g., QS21) + 3dMPL + IM2 (optionally + sterol) (e.g., WO98 / 57659); (13) other substances that act as immunostimulants to enhance the efficacy of the composition. Examples of muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE).

[0294] In another embodiment, the adjuvant is a liposomal Quillaja Saponaria-21 (QS21) formulation containing 5.1 mg / mL QS-21, 5 mM succinate, 60 mM NaCl, 0.1% PS80, pH 5.6. In a further embodiment, the adjuvant is a liposomal monophosphoryl lipid A (MPLA, Synthetic, PHAD®, Avanti) formulation containing 15 mM phosphate buffer, pH 6.1, 4 mg / mL 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 mg / mL cholesterol, 0.2 mg / mL MPLA (Lot 00714551-0018-2XLipoMPL) with a liposome particle size of 71 nm as determined by dynamic light scattering. In yet a further embodiment, the adjuvant is a liposomal MPLA / QS21 formulation comprising 15 mM phosphate buffer, pH 6.1, 4 mg / mL DOPC, 1 mg / mL cholesterol, 0.2 mg / mL MPLA and 0.2 mg / mL QS-21 (Lot 00714551-0018-2XlipoMQ) with a particle size of 75 nm for the MPLA-QS21 liposomes as determined by dynamic light scattering.

[0295] In a further aspect of the present disclosure, the immunogenic composition disclosed herein comprises a CpG oligonucleotide as an adjuvant. As used herein, CpG oligonucleotide refers to an immunostimulatory CpG oligodeoxynucleotide (CpG ODN), and therefore, these terms are used interchangeably unless otherwise indicated. An immunostimulatory CpG oligodeoxynucleotide optionally contains one or more immunostimulatory CpG motifs, which are unmethylated cytosine-guanine dinucleotides, within the context of certain preferred bases. The methylation state of a CpG immunostimulatory motif generally refers to the cytosine residue in the dinucleotide. An immunostimulatory oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide that contains a 5' unmethylated cytosine linked to a 3' guanine by a phosphate bond and activates the immune system by binding to Toll-like receptor 9 (TLR-9). In another embodiment, immunostimulatory oligonucleotide can contain one or more methylated CpG dinucleotides, and activate immune system through TLR9, but not as potent as if CpG motif is unmethylated.CpG immunostimulatory oligonucleotide can contain one or more palindrome, and then contain CpG dinucleotide.CpG oligonucleotide is described in several published patents, published patent applications and other publications, including United States Patent No. 6,194,388;6,207,646;6,214,806;6,218,371;6,239,116;and 6,339,068.

[0296] Various classes of CpG immunostimulatory oligonucleotides have been identified. These are referred to as the A, B, C, and P classes and are described in more detail in WO2010 / 125480, page 3, line 22 to page 12, line 36. The methods of the present disclosure include the use of these different classes of CpG immunostimulatory oligonucleotides.

[0297] V. Purification and Production Methods In one aspect, the present disclosure relates to a method for producing a FimH mutant polypeptide. Such a method can include, for example, culturing mammalian cells under suitable conditions to express the FimH mutant polypeptide. The method can further include collecting the polypeptide from the culture. The process can further include purifying the polypeptide.

[0298] In some embodiments, the method produces FimH mutant polypeptide at a yield of about 0.1 g / L to 0.5 g / L. In some embodiments, the yield of FimH mutant polypeptide 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 21 mg / L, at least about 22 mg / L, at least about 23 mg / L, at least about 24 mg / L, at least about 25 mg / L, at least about 26 mg / L, at least about 27 mg / L, at least about 28 mg / L, at least about 29 mg / L, at least about 30 mg / L, at least about 31 mg / L, at least about 32 mg / L, at least about 33 mg / L, at least about 34 mg / L, at least about 35 mg / L, at least about 36 mg / L, at least about 37 mg / L, at least about 38 mg / L, at least about 39 mg / L, at least about 40 mg / L, at least about 41 mg / L, at least about 42 mg / L, at least about 43 mg / L, at least about 44 mg / L, at least about 45 mg / L, at least about 46 mg / L, at least about 47 mg / L, at least about 8 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.

[0299] In some embodiments, the cell culture suitable for the present disclosure is a fed-batch culture. The term "fed-batch culture," as used herein, refers to a method of culturing cells in which additional components are supplied to the culture one or more times after the start of the culture process. Such supplied components typically include nutrients for the cells that are depleted during the culture process. The fed-batch culture is typically stopped at some point, and the cells and / or components in the medium are collected and optionally isolated. In some embodiments, the fed-batch culture comprises a basal medium supplemented with a feed medium.

[0300] In some aspects, cells can be grown in batch or fed-batch culture, where the culture is terminated after sufficient expression of the polypeptide, after which the expressed polypeptide is harvested and optionally isolated. In some aspects, cells can be grown in perfusion culture, where the culture is not terminated, but rather fresh nutrients and other components are added to the culture periodically or continuously, during which the expressed polypeptide is harvested periodically or continuously.

[0301] In some aspects, the expression level or activity of the FimH mutant polypeptide 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 FimH mutant polypeptide in a bacterial cell, such as, for example, an Escherichia coli (E. coli) host cell.

[0302] In some embodiments, cells can be grown in small-scale reactors to form cell cultures ranging in volume from a few milliliters to several liters, hi some embodiments, cells can be grown in large-scale commercial bioreactors to form cell cultures, which can be in volumes ranging from approximately at least 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 therebetween. In some embodiments, the cell culture size ranges from 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, 400 L to 50,000 L, 500 L to 50,000 L, 600 L to 60,000 L, 700 L to 70,000 L, 800 L to 80,000 L, 900 L to 90,000 L, 1000 L to 50,000 L, 1000 L to 50,000 L, 2000 L to 50,000 L, 3000 L to 50,000 L, 400 L to 50,000 L, 500 L to 50,000 L, 600 L to 70,000 L, 700 L to 80,000 L, 800 L to 90,000 L, 900 L to 100,000 L, 10 ... It can range from 0L to 50,000L, 4500L to 50,000L, 1000L to 10,000L, 1000L to 20,000L, 1000L to 25,000L, 1000L to 30,000L, 15L to 2000L, 40L to 1000L, 100L to 500L, 200L to 400L, or any integer range therebetween.

[0303] The temperature of the cell culture will be selected primarily based on the temperature range at which the cell culture remains viable and produces high levels of polypeptide, the temperature at which the production or accumulation of metabolic waste products is minimized, and / or any combination of the above or other factors deemed important by the artisan. As a non-limiting example, CHO cells grow well and produce high levels of protein or polypeptide at approximately 37°C. Generally, most mammalian cells can grow well and / or produce high levels of protein or polypeptide 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 protein or polypeptide 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 at one or more points during the cell culture process.

[0304] The terms "culture" and "cell culture," as used herein, 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.

[0305] In some embodiments, cells can be grown in one of a variety of chemically defined media, where the components of the media are known and controlled. In some embodiments, cells can be grown in complex media, where not all of the components of the media are known and / or controlled. Chemically defined growth media for culturing mammalian cells have been widely developed and published over the last several decades. All components of the defined media are well characterized, and such defined media do not contain complex additives such as serum or hydrolysates. Early media formulations were developed to enable cell growth and viability with little or no concern for protein production. More recently, media formulations have been developed for the express purpose of supporting the culture of highly proliferative recombinant protein-producing cells. Such media are preferred for use in the methods of the present invention. Such media generally contain large amounts of nutrients, particularly amino acids, that support the growth and / or maintenance of cells at high densities. If necessary, those skilled in the art can modify these media for use in the methods of the present invention. For example, one skilled in the art can reduce the amount of phenylalanine, tyrosine, tryptophan and / or methionine in these media for use as basal or feed media in the methods as disclosed herein.

[0306] Not all components of complex media are well characterized; therefore, complex media can 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 the defined media described herein. In some embodiments, defined media typically contain approximately 50 chemical entities at known concentrations in water. Many of them also contain one or more well-characterized proteins, such as insulin, IGF-1, transferrin, or BSA, while others do not require a protein component and are therefore referred to as protein-free defined media. Typical chemical components of media fall into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and a miscellaneous category that cannot be succinctly classified.

[0307] The cell culture medium may optionally be supplemented with supplementary components. As used herein, the term "supplementary components" refers to components that enhance growth and / or survival beyond a minimal proportion, including, but not limited to, hormones and / or other growth factors, specific ions (such as 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, supplementary components can be added to the initial cell culture. In some embodiments, supplementary components can be added after the initiation of cell culture. Typically, trace elements refer to various inorganic salts present at micromolar or lower levels. For example, commonly included trace elements include zinc, selenium, copper, etc. In some embodiments, iron (ferrous or ferric salts) can be included as a trace element initially in the cell culture medium at micromolar concentrations. Among trace elements, manganese is also often included as a divalent cation (MnCl2 or MnSO4) in the nanomolar to micromolar range. Many less common trace elements are typically added at nanomolar concentrations.

[0308] In some embodiments, the medium used in the methods of the present invention is suitable for cell culture, e.g., 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 It is a medium suitable for supporting high cell densities, such as 1000 cells / mL. In some embodiments, the cell culture is a mammalian cell fed-batch culture, preferably a CHO cell fed-batch culture.

[0309] In some embodiments, the cell culture medium comprises phenylalanine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tryptophan at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises methionine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises leucine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises serine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises threonine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises glycine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises two of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine and tyrosine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM.In some embodiments, the cell culture medium comprises phenylalanine and tryptophan at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine and tryptophan at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tryptophan and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises three of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, and tryptophan at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM.

[0310] In some embodiments, the cell culture medium comprises phenylalanine, tryptophan, and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises tyrosine, tryptophan, and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM.

[0311] In some embodiments, the cell culture medium comprises four of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM.

[0312] In some embodiments, the cell culture medium includes phenylalanine, tyrosine, tryptophan, and methionine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium includes five of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium includes six of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium includes seven of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 1 mM. In some embodiments, the cell culture medium comprises phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at concentrations of less than 2 mM, less than 1 mM, between 0.1 and 2 mM, between 0.1 and 1 mM, between 0.5 and 1.5 mM, or between 0.5 and 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, aspartate, glutamate, and asparagine at concentrations of 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, or 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, aspartate, glutamate, 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 glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, 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 one, two, three, four, five, six, seven, eight, or nine of valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate, 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, aspartate, glutamate, 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 valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate, 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 comprises isoleucine at a concentration of 3 mM, 5 mM, 7 mM, 10 mM, 15 mM, or 20 mM, preferably greater than 10 mM.

[0313] In some embodiments, the cell culture medium comprises leucine 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 described above is for use in the methods disclosed herein. In some embodiments, the cell culture medium described above is used as a basal medium in the methods disclosed herein. In some embodiments, the cell culture medium described above is used as a feed medium in the methods disclosed herein.

[0314] The methods of the present disclosure can be used with any cell culture method that is suitable for the desired process (e.g., production of a recombinant protein). As a non-limiting example, cells can be grown in batch or fed-batch culture, where the culture is terminated after sufficient expression of the recombinant protein (e.g., an antibody), and the expressed protein is then harvested. Alternatively, as another non-limiting example, cells can be grown in batch-refeed culture, where 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 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.

[0315] The cells can be grown in any convenient volume selected by the practitioner. For example, the cells can be grown in small-scale reaction vessels ranging in volume from a few milliliters to several liters. Alternatively, the cells can be grown in large-scale commercial bioreactors ranging in volume from approximately at least 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 therebetween.

[0316] The temperature of the cell culture will be 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, but 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 aspects, the cell culture is grown at one or more points during the cell culture process 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 of skill in the art would be able to select a suitable temperature or temperatures to grow the cells at depending on the specific needs of the cells and the particular production requirements of the practitioner. The cells can be grown for any amount of time depending on the needs of the practitioner and the requirements of the cells themselves. In some embodiments, the cells are grown at 37°C. In some aspects, the cells are grown at 36.5°C.

[0317] In some embodiments, cells can be grown in the initial growth phase (or growth phase) for a longer or shorter amount of time depending on the needs of the technician and the cells themselves.In some embodiments, cells are grown for a sufficient period of time to achieve a predetermined cell density.In some embodiments, cells are grown for a sufficient period of time to achieve a cell density that is a given percentage of the maximum cell density that cells would eventually reach if grown without disturbance.For example, cells can be grown for a sufficient period of time to achieve a desired viable cell density of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99 percent of the maximum cell density. In some embodiments, the cells are grown until 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 until the cell density does not increase by more than 5% per day of culture.

[0318] In some embodiments, cells are grown for a defined 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, the 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. The practitioner 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.

[0319] The cell culture can be agitated or shaken during the initial cultivation phase to increase oxygenation and distribution of nutrients to the cells. In accordance with the present invention, one skilled in the art will appreciate that it may be advantageous to control or regulate certain internal conditions of the bioreactor during the initial growth phase, including, but not limited to, pH, temperature, oxygenation, etc.

[0320] At the end of the initial growth phase, a second set of culture conditions can be applied to shift at least one of the culture conditions to cause a metabolic shift in the culture. The metabolic shift can be achieved, for example, by changing the temperature, pH, osmolality, or chemical induction level of the cell culture. In one non-limiting embodiment, the culture conditions are shifted by shifting the temperature of the culture. However, as is known in the art, temperature shifting is not the only mechanism by which a suitable metabolic shift can be achieved. For example, such a metabolic shift can also be achieved by shifting other culture conditions, including, but not limited to, pH, osmolality, and sodium butyrate level. The timing of the culture shift will be determined by the practitioner of the present invention based on protein production requirements or the needs of the cells themselves.

[0321] When the temperature of a culture is shifted, the temperature shift can be relatively gradual. For example, it can take several hours or days to complete the temperature change. Alternatively, the temperature shift can be relatively sudden. For example, the temperature change can be completed in less than several hours. Given suitable production and control equipment, such as those standard in commercial large-scale production of polypeptides or proteins, the temperature change can be completed in less than an hour.

[0322] In some embodiments, once the cell culture conditions have been shifted as described above, the cell culture is maintained through a subsequent production phase under a second set of culture conditions that promote cell culture survival and viability and are suitable for expression of the desired polypeptide or protein at commercially relevant levels.

[0323] As described above, the culture can be shifted by shifting one or more of a number of culture conditions, including, but not limited to, temperature, pH, osmolality, and sodium butyrate level. In some aspects, the temperature of the culture is shifted. In this embodiment, the culture is maintained during the subsequent production phase at a temperature or temperature range lower than the temperature or temperature range of the initial growth phase. As discussed above, multiple separate temperature shifts can be used to increase cell density or viability, or to increase recombinant protein expression.

[0324] The term "titer," as used herein, refers to, for example, the total amount of recombinantly expressed protein produced by a cell culture in a given amount of medium volume. Titer is typically expressed in grams of protein per liter of medium.

[0325] In some embodiments, cell proliferation is increased by at least 5%, 10%, 15%, 20%, or 25% compared to control cultures. In some embodiments, cell proliferation is increased by at least 10% compared to control cultures. In some embodiments, cell proliferation is increased by at least 20% compared to control cultures.

[0326] In some embodiments, productivity is determined by titer and / or volumetric productivity. 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.

[0327] purification In some aspects, methods for producing a FimH mutant polypeptide include isolating and / or purifying the polypeptide. In some aspects, the expressed polypeptide is secreted into the culture medium, so that cells and other solids can be removed by centrifugation and / or filtration. In preferred embodiments, the polypeptide or fragment thereof is soluble.

[0328] FimH mutant polypeptides produced according to the methods described herein can be harvested from host cells and isolated using any suitable method, generally known in the art (e.g., Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687 (2004)). Suitable methods for purifying polypeptides 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 may include two or more of these or other suitable methods. In some embodiments, one or more of the polypeptides may include a "tag" to facilitate purification or detection. Examples include, for example, His tags (which bind to metal ions, e.g., hexahistidine), antibodies, maltose-binding protein (MBP) (which binds to amylose), glutathione-S-transferase (GST) (which binds to glutathione), FLAG tags (which bind to anti-flag antibodies), Strep tags (which bind to streptavidin or its derivatives). Polypeptides with such tags can be easily isolated, for example, from conditioned medium by chelation or affinity chromatography. Optionally, the tag sequence can be cleaved after purification. In one embodiment, the FimH mutant polypeptide does not comprise a purification tag.

[0329] In one embodiment, the FimH mutant polypeptide can be isolated by first obtaining a cell culture supernatant and then subjecting the supernatant to both ultrafiltration and diafiltration methods. Such filtration methods are known to those skilled in the art. After ultrafiltration and diafiltration, the resulting cell-free solution is then subjected to a chromatography step, such as Ni-NTA chromatography using a nickel affinity resin. This step can then be followed by dialysis, which can then be followed by cation exchange chromatography using an SP column or the like. The use of an acidic pH (e.g., less than about 6.0, less than about 5.5, less than about 5.0, less than about 4.5, about 4.4, about 4.3, about 4.2, about 4.1, or about 4.0 or less) during purification on SP-Sepharose may be desirable under certain conditions.

[0330] Although specific strains of E. coli may be mentioned herein, it should be understood that polypeptides or fragments thereof derived from E. coli are not limited to specific strains unless specified.

[0331] VI. Use of the Composition In one aspect, the present disclosure provides the use of a FimH mutant polypeptide, a nucleic acid encoding such a mutant, a vector for expressing such a mutant, or a composition comprising such a mutant or nucleic acid as a medicament or in the manufacture of a medicament for eliciting an immune response against or preventing E. coli infection in a subject.

[0332] In other aspects, the present disclosure provides methods for eliciting an immune response against Escherichia coli (E. coli) in a subject, such as a human, comprising administering to the subject an effective amount of a FimH mutant polypeptide, a nucleic acid molecule encoding a FimH mutant polypeptide, or a composition comprising a FimH mutant polypeptide or nucleic acid molecule. The present disclosure also provides methods for preventing Escherichia coli (E. coli) infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition, such as a vaccine, comprising a FimH mutant polypeptide, a nucleic acid encoding a FimH mutant polypeptide, or a vector expressing a FimH mutant polypeptide. In certain aspects, the pharmaceutical composition comprises a FimH mutant polypeptide disclosed herein. In some aspects of the methods provided herein above, the subject is a human.

[0333] In another aspect, the disclosure provides methods for inducing an immune response in a subject against extraintestinal pathogenic E. coli or for inducing opsonophagocytosis and / or the production of neutralizing antibodies in a subject specific for extraintestinal pathogenic E. coli, comprising administering to the subject an effective amount of any of the compositions described herein, such as a composition comprising a FimH mutant polypeptide described herein. In further aspects of such methods, the subject is at risk of developing a urinary tract infection, and / or at risk of developing bacteremia, and / or at risk of developing sepsis.

[0334] In a further aspect, the present disclosure provides a method of eliciting an immune response against E. coli in a mammal, comprising administering to the mammal an effective amount of any of the compositions described herein. For example, in one aspect, the immune response comprises opsonophagocytosis and / or neutralizing antibodies against E. coli. In a further aspect, the immune response protects the mammal from E. coli infection.

[0335] In a further aspect, the present disclosure provides a method of preventing, treating, or ameliorating a bacterial infection, disease, or condition in a subject, comprising administering to the subject an immunologically effective amount of any of the compositions described herein.

[0336] In the method of the present disclosure, the composition can be administered to a subject with or without the administration of an adjuvant.The effective amount administered to a subject is an amount sufficient to induce an immune response to an E. coli antigen, such as FimH protein, in the subject.Subjects that can be selected for treatment include subjects at risk of developing E. coli infection, such as subjects at risk of developing urinary tract infection, and / or at risk of developing bacteremia, and / or at risk of developing sepsis due to exposure or potential exposure to E. coli.

[0337] As used herein, "subject" refers to a mammal, preferably a human.In one embodiment, the subject is at risk of any one of the following conditions selected from the group consisting of urinary tract infection, cholecystitis, cholangitis, diarrhea, hemolytic uremic syndrome, neonatal meningitis, urosepsis, intraperitoneal infection, meningitis, complicated pneumonia, wound infection, post-prostate biopsy related infection, neonatal / infant sepsis, neutropenic fever and other bloodstream infections; pneumonia, bacteremia, and sepsis.

[0338] Administration of compositions provided by the present disclosure, such as pharmaceutical compositions, can be carried out using standard routes of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration.

[0339] The total dose of the composition provided to a subject in a single administration can be varied, as known to those of skill in the art.

[0340] It is also possible to provide one or more booster administrations of one or more of the immunogenic compositions. If a boosting vaccination is performed, typically, such a boosting vaccination will be administered to the same subject between one week and ten years, preferably between two weeks and six months, after the subject's initial administration of the composition (in such a case referred to as a "priming vaccination"). In an alternative boosting regimen, after the priming vaccination, the subject may be administered a different vector, for example, one or more adenovirus or other vectors, such as modified Ankara vaccinia virus (MVA), or DNA, or protein. The immunogenic compositions provided by the present disclosure can be used together with one or more other immunogenic compositions.

[0341] Dosage of the composition Dosage regimens can be adjusted to produce the optimal desired response. For example, a single dose of mutated FimH polypeptide can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the situation. It should be noted that dosage values ​​can vary with the type and severity of the condition to be alleviated and can include single or multiple doses. Furthermore, it should be understood that for any particular subject, specific dosing regimens should be adjusted over time according to the individual's needs and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0342] In some embodiments, the amount of FimH mutant polypeptide in the composition can range from about 10 μg to about 300 μg of each protein antigen, hi some embodiments, the amount of FimH mutant polypeptide in the composition can range from about 20 μg to about 200 μg of each protein antigen.

[0343] The amount of glycoconjugate in each dose is selected to induce an immunoprotective response without significant adverse side effects in a typical vaccine. Such amounts will vary depending on the specific immunogen used and how it is delivered. The amount of a particular glycoconjugate in an immunogenic composition can be calculated based on the total polysaccharide content of that conjugate (conjugated and unconjugated). For example, a glycoconjugate containing 20% ​​free polysaccharide will have approximately 80 g of conjugated polysaccharide and approximately 20 g of unconjugated polysaccharide in a 100 g polysaccharide dose. The amount of glycoconjugate may vary depending on the E. coli serotype. The sugar concentration can be determined by uronic acid assay.

[0344] The "immunogenic amounts" of the different polysaccharide components in the immunogenic composition may vary, and each may contain about 1.0 g, about 2.0 g, about 3.0 g, about 4.0 g, about 5.0 g, about 6.0 g, about 7.0 g, about 8.0 g, about 9.0 g, about 10.0 g, about 15.0 g, about 20.0 g, about 30.0 g, about 40.0 pg, about 50.0 pg, about 60.0 pg, about 70.0 pg, about 80.0 pg, about 90.0 pg or about 100.0 g of any particular polysaccharide antigen. Generally, each dose will contain between 0.1 g and 100 g, particularly between 0.5 g and 20 g, more particularly between 1 g and 10 g, and even more particularly between 2 g and 5 g of polysaccharide for a given serotype. Any whole integer within any of the above ranges is contemplated as an embodiment of the present disclosure. In one embodiment, each dose will contain 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g, 8 g, 9 g, 10 g, 15 g, or 20 g of polysaccharide for a given serotype.

[0345] VII. Combinations with saccharides and / or polypeptides derived from Klebsiella pneumoniae or fragments thereof Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative pathogen known to cause urinary tract infections, bacteremia, and sepsis. Multidrug-resistant K. pneumoniae infections are an increasing cause of mortality in at-risk and vulnerable populations. O-antigen serotypes are highly prevalent among strains causing invasive disease worldwide, making derived O-antigen glycoconjugates attractive as vaccine antigens.

[0346] In one aspect, any of the compositions disclosed herein may further comprise at least one saccharide that is, or is derived from, at least one K. pneumoniae serotype selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12. In a preferred embodiment, any of the compositions disclosed herein may further comprise a polypeptide derived from K. pneumoniae selected from a polypeptide or immunogenic fragment thereof derived from a K. pneumoniae type I pilus protein; or a polypeptide or immunogenic fragment thereof derived from a K. pneumoniae type III pilus protein; or a combination thereof.

[0347] As is known in the art, K. pneumoniae O1 and O2 O antigens and their corresponding v1 and v2 subtypes are polymeric galactans that differ in the structure of their repeating units. K. pneumoniae O1 and O2 antigens contain homopolymeric galactose units (or galactans). K. pneumoniae O1 and O2 antigens each contain a D-galactan I unit (sometimes referred to as an O2a repeating unit), while the O1 antigen differs in that it has a D-galactan II cap structure. D-galactan III (d-Gal-III) is a variant of D-galactan I. The structures of the basic galactans I and III that define two distinct serotype O2 subtypes, O2v1 and O2v2; and the structures of the derived chimeras resulting from capping with galactan II to give rise to subtypes O1v1 and O1v2, are shown in Kelly SD et al., J Biol Chem 2019;294:10863-76; and Clarke BR et al., J Biol Chem 2018;293:4666-79.

[0348] In some embodiments, the saccharide derived from K. pneumoniae O1 comprises a repeating unit of [→3)-β-D-Galf-(1→3)-α-D-Galp-(1→]. In some embodiments, the saccharide derived from K. pneumoniae O1 comprises a repeating unit of [→3)-α-D-Galp-(1→3)-β-D-Galp-(1→]. In some embodiments, the saccharide derived from K. pneumoniae O1 comprises a repeating unit of [→3)-β-D-Galf-(1→3)-α-D-Galp-(1→] and [→3)-α-D- In some embodiments, the saccharide from Klebsiella pneumoniae O1 comprises a repeating unit of Galp-(1→3)-β-D-Galp-(1→]. In some embodiments, the saccharide from Klebsiella pneumoniae O1 comprises a repeating unit of β-D-Galp-(1→3)-[α-D-Galp-(1→4)]-α-D-Galp-(1→] (referred to as a D-Gal-III repeating unit) (Kol O. et al. (1992) Carbohydr. Res. 236, 339-344; Whitfield C. et al. (1991) J. Bacteriol. 173, 1420-1431).

[0349] In some embodiments, the saccharide derived from K. pneumoniae O2 is [→3)-α- D -Galp-(1→3)-β- D In some embodiments, the saccharide from K. pneumoniae O2 comprises a repeating unit of -Galf-(1→], which may be a component of the K. pneumoniae serovar O2a antigen. In some embodiments, the saccharide from K. pneumoniae O2 comprises a repeating unit of [→3)-β- D -GlcpNAc-(1→5)-β- D-Galf-(1→] (which may be a component of the K. pneumoniae serovar O2c antigen). In some embodiments, the saccharide derived from K. pneumoniae O2 comprises a modification of the O2a repeating unit by the side chain addition of a (1→4)-linked Galp residue (which may be a component of the K. pneumoniae O2afg antigen). In some embodiments, the saccharide derived from K. pneumoniae O2 comprises a modification of the O2a repeating unit by the side chain addition of a (1→2)-linked Galp residue (which may be a component of the K. pneumoniae O2aeh antigen). (Whitfield C. et al. (1992) J. Bacteriol. 174, 4913-4919).

[0350] Without being bound by mechanism or theory, it has been disclosed in the art that the O-antigen polysaccharide structures of Klebsiella pneumoniae serotypes O3 and O5 are identical to those of Escherichia coli (E. coli) serotypes O9a (formula O9a) and O8 (formula O8), respectively.

[0351] In some embodiments, the saccharide derived from K. pneumoniae O4 comprises a repeating unit of [→4)-α-D-Galp-(1→2)-β-D-Ribf-(1→)]. In some embodiments, the saccharide derived from K. pneumoniae O7 comprises repeating units of [→2-αL-Rhap-(1→2)-β-D-Ribf-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap-(1→]. In some embodiments, the saccharide derived from K. pneumoniae O8 serovar comprises the same repeating unit structure as K. pneumoniae O2a, but is non-stoichiometrically O-acetylated. In some embodiments, the saccharide derived from K. pneumoniae O12 serovar comprises repeating units of [α-Rhap-(1→3)-β-GlcpNAc] disaccharide repeating units.

[0352] In one aspect, the invention includes a composition comprising a polypeptide or fragment thereof derived from E. coli FimH; and at least one saccharide that is or is derived from at least one K. pneumoniae serotype selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12. In some embodiments, the composition comprises saccharides from or derived from one or more of serotypes O1, O2, O3, and O5, or a combination thereof. In some embodiments, the composition comprises saccharides from or derived from each of serotypes O1, O2, O3, and O5.

[0353] In another aspect, the present invention provides at least one saccharide that is or is derived from at least one K. pneumoniae serotype selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8, and O12; and at least one saccharide that is or is derived from at least one K. pneumoniae serotype selected from O1 (e.g., formula O1A, formula O1B, and formula O1C), O2, O3, O4 (e.g., formula O4:K52 and formula O4:K6), O5 (e.g., formula O5ab and formula O5ac (strain 180 / C3)), O6 (e.g., Formulas 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 O 38, 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 O8 2, 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 0111, Formula O112, Formula O113, Formula O114, Formula O115, Formula O116, Formula O1 17, 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 O14 6, 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 O and O187, wherein n is an integer between 1 and 100. In some embodiments, the composition comprises a saccharide derived from an E. coli O antigen having a structure selected from any one of: O164, O165, O166, O167, O...

Claims

1. A nucleic acid comprising nucleotides encoding a mutated FimH polypeptide comprising an amino acid substitution compared to the amino acid sequence of a wild-type FimH polypeptide, G15A and G16A and V27A; G15A and V27A; or G16A and V27A and wherein the mutated FimH polypeptide induces the production of neutralizing antibodies specific for E. coli.

2. An immunogenic composition comprising a nucleic acid comprising nucleotides encoding a mutated FimH polypeptide, wherein the mutated FimH polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 50, 51, 54, 61 and 62.

3. The immunogenic composition of claim 1 or 2, wherein the nucleic acid is RNA.

4. The immunogenic composition of claim 3, wherein the nucleic acid is a nucleoside-modified mRNA (modRNA).

5. The immunogenic composition according to any one of claims 1 to 4, for inducing an immune response in a subject against E. coli.

6. The immunogenic composition of claim 5 further comprising at least one additional antigen.

7. 7. The immunogenic composition of claim 6, wherein the additional antigen is an E. coli saccharide comprising a structure selected from the group consisting of O25b, O1A, O2 and O6.

8. 8. The immunogenic composition of claim 7, wherein the composition comprises E. coli saccharides comprising each of the structures O25b, O1A, O2 and O6.

9. The immunogenic composition of claim 1 , further comprising an adjuvant.

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