RU2831010C1 - Fimh escherichia coli mutants and use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to mutant polypeptides FimH Escherichia coli and use thereof. Disclosed are versions of the mutant polypeptide FimH for inducing production of antibodies specific to uropathogenic E. coli in a subject. Also disclosed are a nucleic acid molecule coding said polypeptide, a pharmaceutical composition comprising said polypeptide or nucleic acid molecule, a recombinant mammalian cell for producing a mutant FimH polypeptide, a culture for producing the mutant FimH polypeptide, a method for producing the mutant FimH polypeptide, a method for inducing the production of uropathogenic E. coli-specific antibodies in a subject, as well as a method for preventing, treating or relieving uropathogenic E. coli infection in a subject.

EFFECT: invention provides obtaining mutant polypeptides FimH Escherichia coli with low affinity to mannoside ligands and improved biochemical properties, which provide higher functional immunogenicity compared to wild type FimH.

21 cl, 13 dwg, 24 tbl, 21 ex

Description

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 63/130,153, filed December 23, 2020, U.S. Provisional Patent Application No. 63/185,425, filed May 7, 2021, and U.S. Provisional Patent Application No. 63/282,244, filed November 23, 2021. The entire contents of each of these applications are hereby incorporated by reference.

LINK TO SEQUENCE LIST

This application is filed electronically via EFS-Web and includes an electronically submitted sequence listing in .txt format. The 220 KB .txt file contains a sequence listing titled "PC072713_ST25_17Nov2021.txt" generated on November 17, 2021. The sequence listing contained in this .txt file is part of the disclosure and is incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to mutant FimH polypeptides of Escherichia coli and methods of using them.

LEVEL OF TECHNOLOGY

Urinary tract infections (UTIs) affect one in five women at least once in their lifetime and are responsible for significant morbidity and mortality, placing a significant burden on healthcare systems. Although 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 faecal flora to the urogenital tract, where they adhere to host uroepithelial cells, thereby creating a reservoir for ascending urinary tract infections. Adhesion is facilitated by fimbrial adhesins, including type 1 fimbriae, which bind to mannosylated glycoproteins in the epithelial layer or are secreted into the urine. Type 1 fimbriae are highly conserved among clinical isolates of UPEC and are encoded by a cluster of genes termed fim, which encode accessory proteins (FimC, FimD), various structural subunits (FimE, FimF, FimG), and an adhesion protein termed FimH. All characteristics of UTI infection in mouse models that mimic aspects of human bladder infection are dependent on FimH (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 provide further support for the role of FimH in UTI and show promising therapeutic properties in animal models (Cusumano C.K., et al. Sci Transl Med. 2011; 3(109):109ra115. doi: 10.1126/scitranslmed.3003021). In addition, FimH is under positive selection in E. coli isolates from human cystitis (Chen SL, et al. Proc Natl Acad Sci U.S.A. 2009 Dec 29; 106(52):22439-44. doi: 10.1073/pnas.0902179106), and residues resulting from positive selection can influence virulence in mouse models of cystitis (Schwartz, D.J. et al. Proc Natl Acad Sci U.S.A. 110, 15530-15537, doi:10.1073/pnas.1315203110 (2013)).

FimH consists of two domains: a lectin-binding domain (FimH _LD ), responsible for binding to mannosylated glycoproteins, and a pilin domain. The pilin domain serves 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 pilin domain of FimH forms an incomplete fold of the immunoglobulin, resulting in a groove that provides a binding site for the N-terminal β-chain of FimG, forming a strong intermolecular linkage between FimH and FimG. Although FimH _LD can be expressed in a soluble, stable form, full-length FimH by itself is unstable (Vetsch, M., et al. J. Mol. Biol. 322:827–840 (2002); Barnhart MM, et al., Proc. Natl Acad Sci USA (2000) Jul 5; 97(14):7709–14), unless it is complexed with the chaperone FimC or supplemented with the donor chain peptide FimG in peptide form or as a fusion protein (Barnhart MM, et al., Proc Natl Acad Sci USA (2000) Jul 5; 97(14): 7709–14; Sauer MM et al. Nat Commun. (2016) Mar 7; 7:10738; Barnhart MM et al., J Bacteriol. 2003 May; 185(9):2723-30). The development and expression of the full-length FimH molecule by coupling the donor peptide FimG to full-length FimH via a glycine-serine linker has been described previously (see International PCT Publication No. WO2021/084429, published May 6, 2021), and the product is designated FimH-DSG.

FimH _LD is considered a poor immunogen in terms of its ability to stimulate functional immunogenicity. Some studies suggest that although binding antibody titers can be induced by FimH _LD with and without adjuvant, functional neutralizing antibody titers were only observed in the presence of adjuvant (see International PCT Publication No. WO2021/084429, published May 6, 2021). Studies show that fixing 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)). Accordingly, there is a need in the art for new FimH mutants with reduced affinity for mannoside ligands and improved biochemical properties that provide increased functional immunogenicity compared to wild-type FimH.

ESSENCE OF THE INVENTION

The present invention relates to the development of mutant E. coli FimH polypeptides that have improved biochemical properties and immunogenicity, compositions containing such polypeptides, and their use. For example, in one aspect, the present invention provides a mutant FimH polypeptide that comprises at least one amino acid mutation relative to the amino acid sequence of a wild-type FimH polypeptide, wherein the position of the mutation 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, wherein the amino acid positions are numbered according to SEQ ID NO:59.

In another aspect, a mutant FimH polypeptide is provided, comprising at least one mutation 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; L107C; 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, a mutant FimH polypeptide comprises the G15A and G16A mutations. In addition, the mutant FimH polypeptide comprises the P12C and A18C mutations. In addition, the mutant FimH polypeptide comprises the G14C and F144C mutations. In addition, the mutant FimH polypeptide comprises the P26C and V35C mutations. In addition, the mutant FimH polypeptide comprises the P26C and V154C mutations. In addition, the mutant FimH polypeptide contains the mutations P26C and V156C. In addition, the mutant FimH polypeptide contains the mutations V27C and L34C. In addition, the mutant FimH polypeptide contains the mutations V28C and N33C. In addition, the mutant FimH polypeptide contains the mutations V28C and P157C. In addition, the mutant FimH polypeptide contains the mutations Q32C and Y108C. In addition, the mutant FimH polypeptide contains the mutations N33C and L109C. In addition, the mutant FimH polypeptide contains the mutations N33C and P157C. In addition, the mutant FimH polypeptide contains the mutations V35C and L107C. In addition, the mutant FimH polypeptide contains the mutations V35C and L109C. In addition, the mutant FimH polypeptide contains the mutations S62C and T86C. In addition, the mutant FimH polypeptide contains the S62C and L129C mutations. In addition, the mutant FimH polypeptide contains the Y64C and L68C mutations. In addition, the mutant FimH polypeptide contains the Y64C and A127C mutations. In addition, the mutant FimH polypeptide contains the L68C and F71C mutations. In addition, the mutant FimH polypeptide contains the V112C and T158C mutations. In addition, the mutant FimH polypeptide contains the S113C and G116C mutations. In addition, the mutant FimH polypeptide contains the S113C and T158C mutations. In addition, the mutant FimH polypeptide contains the V118C and V156C mutations. In addition, the mutant FimH polypeptide contains the A119C and V155C mutations. In addition, the mutant FimH polypeptide contains the L34N and V27A mutations. In addition, the mutant FimH polypeptide contains the L34S and V27A mutations. In addition, the mutant FimH polypeptide contains the L34T and V27A mutations. In addition, the mutant FimH polypeptide contains the L34D and V27A mutations. In addition, the mutant FimH polypeptide contains the L34E and V27A mutations. In addition, the mutant FimH polypeptide contains the L34K and V27A mutations. In addition, the mutant FimH polypeptide contains the L34R and V27A mutations. In addition, the mutant FimH polypeptide contains the A119N and V27A mutations. In addition, the mutant FimH polypeptide contains the A119S and V27A mutations. In addition, the mutant FimH polypeptide contains the A119T and V27A mutations. In addition, the mutant FimH polypeptide contains the A119D and V27A mutations. In addition, the mutant FimH polypeptide contains the A119E and V27A mutations. In addition, the mutant FimH polypeptide contains the A119K and V27A mutations. In addition, the mutant FimH polypeptide contains the A119R and V27A mutations. In addition, the mutant FimH polypeptide contains the G15A and V27A mutations. In addition, the mutant FimH polypeptide contains the G16A and V27A mutations. In addition, the mutant FimH polypeptide contains the G15P and V27A mutations. In addition, the mutant FimH polypeptide contains the G16P and V27A mutations. In addition, the mutant FimH polypeptide contains the G15A, G16A, and V27A mutations. In addition, the mutant FimH polypeptide contains the G65A and V27A mutations. In addition, the mutant FimH polypeptide contains the V27A and Q133K mutations. In addition, the mutant FimH polypeptide comprises the mutations G15A, G16A, V27A and Q133K. In addition, for example, the mutant FimH polypeptide comprises the sequence of any of SEQ ID NOs: 2-58 and 60-64. In addition, for example, the mutant FimH polypeptide is disclosed herein, wherein the polypeptide is isolated.

In another example, the present invention relates to a pharmaceutical composition comprising (i) a mutant FimH polypeptide disclosed herein and (ii) a pharmaceutically acceptable carrier.

In another example, the present invention relates to an immunogenic composition comprising a mutant FimH polypeptide disclosed herein. For example, the immunogenic composition further comprises at least one additional antigen, such as a polysaccharide or glycoconjugate or protein. Furthermore, for example, the immunogenic composition further comprises at least one adjuvant.

In another example, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of a mutant FimH polypeptide disclosed herein.

In another example, the present invention relates to a mutant FimH polypeptide as disclosed herein, wherein said polypeptide is immunogenic.

The present invention also relates to a recombinant mammalian cell comprising a polynucleotide encoding a mutant FimH polypeptide disclosed herein.

The present invention also relates to a culture comprising a recombinant cell as disclosed herein, wherein the volume of said 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.

The present invention also relates to a method for producing a mutant FimH polypeptide disclosed herein, comprising culturing a recombinant mammalian cell disclosed herein under suitable conditions, thereby allowing expression of the polypeptide; and harvesting the polypeptide.

The present disclosure also provides a method of (i) inducing an immune response in a subject against extraintestinal pathogenic E. coli or (ii) inducing the production of opsonophagocytic and/or neutralizing antibodies in a subject specific for extraintestinal pathogenic E. coli, wherein the method comprises 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 another example, the subject is at risk of developing bacteremia. In another example, the subject is at risk of developing sepsis. In another example, the subject is at risk of developing Crohn's disease.

The present invention also relates to a method for inducing an immune response against E. coli in a mammal, comprising administering to the mammal an effective amount of the composition disclosed herein. In one example, the immune response comprises opsonophagocytic and/or neutralizing antibodies to E. coli. In another example, the immune response protects the mammal from E. coli infection.

The present invention also relates to a method for preventing, treating or ameliorating a bacterial infection, disease or condition in a subject, comprising administering to the subject an immunologically effective amount of the composition disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1B show the circular dichroism spectra of the FimH _LD and FimH-DSG mutants. Figure 1A shows the near-UV circular dichroism spectra, and Figure 1B shows the far-UV circular dichroism spectra.

Figure 2 shows the relative immunogenicity of FimH _LD mutants in the yeast mannan binding neutralization assay in the presence of PD3.

Figures 3A-3B show the immunogenicity of FimH _LD and FimH-DSG mutants in the yeast mannan binding neutralization assay in the presence of PD2 (Figure 3A) and PD3 (Figure 3B).

Fig. 4 is a diagram showing the main purification steps used to isolate His-tagged wild-type and mutant FimH-DsG.

Figures 5A-5B show the FimH-DSG WT purification profile.

Fig. 5A is the elution profile of FimH-DSG WT on an SP-Sepharose column, and Fig. 5B is the SDS-PAGE analysis of the eluted fractions.

Figures 6A-6B show the purification profile of the FimH-DSG mutant G15A G16A V27A.

Figure 6A shows the elution profile of the FimH-DSG G15A G16A V27A mutant on an SP-Sepharose column, and Figure 6B shows the SDS-PAGE analysis of the eluted fractions.

Figures 7A-7B show the analytical SEC of FimH-DSG proteins. The analytical SEC of FimH-DSG G15A G16A V27A is shown in Figure 7A, and that of WT wild-type FimH-DSG is shown in Figure 7B.

Fig. 8 shows a schematic representation of the mechanism of formation of the HMW FimH complex.

Figure 9 shows a schedule of immunization of non-human primates and subsequent challenge described in Example 21 of the present disclosure.

Figure 10 shows the increase in the level of serotype-specific antibodies to the O-antigen after vaccination of NHP with the FimH-DSG G15A G16A V27A mutant + 4-valent E. coli O-antigens (O1a, O2, O6, and O25b). Legend: Placebo (circles); FimH-DSG G15A G16A V27A (squares); FimH-DSG G15A G16A V27A + 4-valent O-antigen (triangles).

Figures 11A-11B show that immunization with FimH-DSG G15A G16A V27A+/-4plex O antigens elicited strong functional anti-FimH antibody responses.

Figure 11A shows the results of the direct anti-FiinH IgG assay in the Luminex system, and Figure 11B shows the results of the E. coli binding inhibition assay. As used herein, the term "4plex" has the same meaning as "4-valent," which are used interchangeably.

Figure 12 shows that bacteriuria is reduced in vaccinated nonhuman primates (NHPs) following challenge as described in Example 21. Legend: placebo (circles); FimH-DSG G15A G16A V27A (squares); FimH-DSG G15A G16A V27A+4-valent O-antigen (triangles); *p≤0.007 relative to placebo.

Figures 13A-13C show that biomarkers of infection are reduced in vaccinated NHPs following challenge with three groups: placebo, FimH-DSG G15A G16A V27A only, and FimH-DSG G15A G16A V27A+4-valent O antigens, as described in Example 21. Figure 13A shows quantification of urinary MPO, Figure 13B shows quantification of urinary IL-8, and Figure 13C shows the percentage of animals with elevated PMN in urine sediment. Legend: placebo (circles); FimH-DSG G15A G16A V27A (squares); FimH-DSG G15A G16A V27A+4-valent O antigen (triangles).

SEQUENCE IDENTIFIERS

SEQ ID NO: 1 represents the amino acid sequence of wild-type E. coli FimH-LD (FimHLD_WT).

SEQ ID NO: 2 represents the amino acid sequence of the mutant FimHLD_G65A_V27A of E. coli.

SEQ ID NO: 3 represents the amino acid sequence of the E. coli FimHLD_F1I mutant.

SEQ ID NO: 4 represents the amino acid sequence of the E. coli FimHLD_F1L mutant.

SEQ ID NO: 5 represents the amino acid sequence of the E. coli FimHLD_F1V mutant.

SEQ ID NO: 6 represents the amino acid sequence of the E. coli FimHLD_F1M mutant.

SEQ ID NO: 7 represents the amino acid sequence of the E. coli FimHLD_F1Y mutant.

SEQ ID NO: 8 represents the amino acid sequence of the E. coli FimHLD_F1W mutant.

SEQ ID NO: 9 represents the amino acid sequence of the E. coli FimHLD_Q133K mutant.

SEQ ID NO: 10 represents the amino acid sequence of the E. coli FimHLD_G15A mutant.

SEQ ID NO: 11 represents the amino acid sequence of the E. coli FimHLD_G15P mutant.

SEQ ID NO: 12 represents the amino acid sequence of the E. coli FimHLD_G16A mutant.

SEQ ID NO: 13 represents the amino acid sequence of the E. coli FimHLD_G16P mutant.

SEQ ID NO: 14 represents the amino acid sequence of the E. coli FimHLD_G15A_G16A mutant.

SEQ ID NO: 15 represents the amino acid sequence of the E. coli FimHLD_R60P mutant.

SEQ ID NO: 16 represents the amino acid sequence of the E. coli FimHLD_G65A mutant.

SEQ ID NO: 17 represents the amino acid sequence of the mutant E. coli FimHLD_P12C_A18C.

SEQ ID NO: 18 represents the amino acid sequence of the E. coli FimHLD_G14C_F144C mutant.

SEQ ID NO: 19 represents the amino acid sequence of the E. coli FimHLD_P26C_V35C mutant.

SEQ ID NO: 20 represents the amino acid sequence of the E. coli FimHLD_P26C_V154C mutant.

SEQ ID NO: 21 represents the amino acid sequence of the E. coli FimHLD_P26C_V156C mutant.

SEQ ID NO: 22 represents the amino acid sequence of the E. coli FimHLD_V27C_L34C mutant.

SEQ ID NO: 23 represents the amino acid sequence of the E. coli FimHLD_V28C_N33C mutant.

SEQ ID NO: 24 represents the amino acid sequence of the E. coli FimHLD_V28C_P157C mutant.

SEQ ID NO: 25 represents the amino acid sequence of the E. coli FimHLD_Q32C_Y108C mutant.

SEQ ID NO: 26 represents the amino acid sequence of the E. coli FimHLD_N33C_L109C mutant.

SEQ ID NO: 27 represents the amino acid sequence of the mutant E. coli FimHLD_N33C_P157C.

SEQ ID NO: 28 represents the amino acid sequence of the mutant E. coli FimHLD_V35C_L107C.

SEQ ID NO: 29 represents the amino acid sequence of the E. coli FimHLD_V35C_L109C mutant.

SEQ ID NO: 30 represents the amino acid sequence of the E. coli FimHLD_S62C_T86C mutant.

SEQ ID NO: 31 represents the amino acid sequence of the E. coli FimHLD_S62C_L129C mutant.

SEQ ID NO: 32 represents the amino acid sequence of the E. coli FimHLD_Y64C_L68C mutant.

SEQ ID NO: 33 represents the amino acid sequence of the mutant E. coli FimHLD_Y64C_A127C.

SEQ ID NO: 34 represents the amino acid sequence of the E. coli FimHLD_L68C_F71C mutant.

SEQ ID NO: 35 represents the amino acid sequence of the mutant E. coli FimHLD_V112C_T158C.

SEQ ID NO: 36 represents the amino acid sequence of the E. coli FimHLD_S113C_G116C mutant.

SEQ ID NO: 37 represents the amino acid sequence of the mutant E. coli FimHLD_S113C_T158C.

SEQ ID NO: 38 represents the amino acid sequence of the E. coli FimHLD_V118C_V156C mutant.

SEQ ID NO: 39 represents the amino acid sequence of the mutant E. coli FimHLD_A119C_V155C.

SEQ ID NO: 40 represents the amino acid sequence of the E. coli FimHLD_L34N_V27A mutant.

SEQ ID NO: 41 represents the amino acid sequence of the E. coli FimHLD_L34S_V27A mutant.

SEQ ID NO: 42 represents the amino acid sequence of the E. coli FimHLD_L34T_V27A mutant.

SEQ ID NO: 43 represents the amino acid sequence of the E. coli FimHLD_A119N_V27A mutant.

SEQ ID NO: 44 represents the amino acid sequence of the E. coli FimHLD_A119S_V27A mutant.

SEQ ID NO: 45 represents the amino acid sequence of the mutant E. coli FimHLD_A119T_V27A.

SEQ ID NO: 46 represents the amino acid sequence of the E. coli FimH-DSG_A115V mutant.

SEQ ID NO: 47 represents the amino acid sequence of the E. coli FimH-DSG_V163I mutant.

SEQ ID NO: 48 represents the amino acid sequence of the E. coli FimH-DSG_V185I mutant.

SEQ ID NO: 49 represents the amino acid sequence of the mutant E. coli FimH-DSG_DSG_V3I.

SEQ ID NO: 50 represents the amino acid sequence of the E. coli FimHLD_G15A_V27A mutant.

SEQ ID NO: 51 represents the amino acid sequence of the E. coli FimHLD_G16A_V27A mutant.

SEQ ID NO: 52 represents the amino acid sequence of the E. coli FimHLD_G15P_V27A mutant.

SEQ ID NO: 53 represents the amino acid sequence of the E. coli FimHLD_G16P_V27A mutant.

SEQ ID NO: 54 represents the amino acid sequence of the mutant E. coli FimHLD_G15A_G16A_V27A.

SEQ ID NO: 55 represents the amino acid sequence of the E. coli FimHLD_V27A_R60P mutant.

SEQ ID NO: 56 represents the amino acid sequence of the E. coli FimHLD_G65A_V27A mutant.

SEQ ID NO: 57 represents the amino acid sequence of the E. coli FimHLD_V27A_Q133K mutant.

SEQ ID NO: 58 represents the amino acid sequence of the mutant E. coli FimHLD_G15A_G16A_V27A_Q133K.

SEQ ID NO: 59 represents the amino acid sequence of full-length wild-type E. coli FimH, including the donor chain of the FimG peptide connected via a linker (FimH-DSG_WT).

SEQ ID NO: 60 represents the amino acid sequence of the E. coli FimH-DSG_V27A mutant.

SEQ ID NO: 61 represents the amino acid sequence of the E. coli FimH-DSG_G15A_V27A mutant.

SEQ ID NO: 62 represents the amino acid sequence of DSG_G15A_G16A_V27A mutant E. coli FimH.

SEQ ID NO: 63 represents the amino acid sequence of DSG_V27A_Q133K mutant E. coli FimH.

SEQ ID NO: 64 represents the amino acid sequence of DSG_G15A_G16A_V27A_Q133K mutant E. coli FimH.

SEQ ID NO: 65 represents the amino acid sequence of the signal peptide of the kappa chain of mouse Ig.

SEQ ID NO: 66-108 represent amino acid sequences and nucleic acid sequences for a nanostructure-related polypeptide or fragment thereof.

SEQ ID NO: 109 - PCR primer.

SEQ ID NO: 110 - PCR primer.

SEQ ID NO: 111 - PCR probe.

SEQ ID NO: 112 represents the amino acid sequence of O25b 2401 WzzB.

SEQ ID NO: 113 represents the amino acid sequence of 025a:K5:H1 WzzB.

SEQ ID NO: 114 represents the amino acid sequence of 025a ETEC ATCC WzzB.

SEQ ID NO: 115 represents the amino acid sequence of K12 W3110 WzzB.

SEQ ID NO: 116 represents the amino acid sequence of Salmonella LT2 WzzB.

SEQ ID NO: 117 represents the amino acid sequence of O25b 2401 FepE.

SEQ ID NO: 118 represents the amino acid sequence of O25a:K5:H1 FepE.

SEQ ID NO: 119 represents the amino acid sequence of O25a of ETEC ATCC FepE.

SEQ ID NO: 120 represents the amino acid sequence of O157 FepE.

SEQ ID NO: 121 represents the amino acid sequence of Salmonella FepE LT2.

SEQ ID NO: 122 represents the primer sequence for LT2wzzB_S.

SEQ ID NO: 123 represents the primer sequence for LT2wzzB_AS.

SEQ ID NO: 124 represents the primer sequence for 025bFepE_S.

SEQ ID NO: 125 represents the primer sequence for 025bFepE_A.

SEQ ID NO: 126 represents the primer sequence for P1_S wzzB.

SEQ ID NO: 127 represents the primer sequence for P2_AS wzzB.

SEQ ID NO: 128 represents the primer sequence for P3_S wzzB.

SEQ ID NO: 129 represents the primer sequence for P4_AS wzzB.

SEQ ID NO: 130 represents the primer sequence for FepE_S 0157.

SEQ ID NO: 131 represents the primer sequence for FepE_AS 0157.

SEQ ID NO: 132 represents the primer sequence for pBAD33_adaptor_S.

SEQ ID NO: 133 represents the primer sequence for pBAD33 adaptor AS.

SEQ ID NO: 134 represents the primer sequence for JUMPSTART_r.

SEQ ID NO: 135 represents the primer sequence for gnd_f.

SEQ ID NO: 136 represents the amino acid sequence of the signal peptide of the large subunit p51 of the human IgG receptor FcRn.

SEQ ID NO: 137 represents the amino acid sequence of the human IL-10 protein signal peptide.

SEQ ID NO: 138 represents the amino acid sequence of the signal peptide of the fusion glycoprotein F0 of human respiratory syncytial virus A (strain A2).

SEQ ID NO: 139 represents the amino acid sequence of the signal peptide of the hemagglutinin of influenza A virus.

SEQ ID NO: 140-147 represent P4.1 signal sequences (DTU Bioinformatics) obtained from different species used for signal peptide prediction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to mutant E. coli FimH polypeptides (mutants), compositions containing FimH mutants, methods for producing and purifying FimH mutants, nucleic acids that encode FimH mutants, host cells that contain such nucleic acids, and methods for using compositions containing FimH mutants.

In the context of the present specification, the recitation of ranges of values is provided only as a shorthand way of referring to each individual value falling within the recited range. Unless otherwise indicated in the specification, each individual value is included in the specification as if it were separately recited. All methods disclosed in the present specification may be performed in any suitable order unless otherwise indicated in the present specification or unless the context clearly contradicts otherwise. The use of any and all examples or illustrative language (e.g., "such as") provided in the present specification is intended only to further illustrate the invention and does not limit the scope of the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

Throughout the text of this specification, several documents are cited. Each of the documents cited in this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), both supra and infra, are incorporated herein by reference in their entirety. Nothing in this specification should be construed as an admission that such publications constitute prior art with respect to the appended claims.

Definitions

In the context of the present description, the term “about” means approximately or nearly, and in the context of a numerical value or range presented in one of the embodiments described herein, means ±20%, ±10%, ±5%, or ±3% of the stated or claimed numerical value or range.

The terms “a,” “an,” and “the” and similar references used in the context of the description of the invention (especially in the context of the claims) are to be construed as embracing both the singular and the plural unless otherwise indicated in the description or unless the context clearly indicates otherwise.

"Fragment" in relation to an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e. a sequence that is an amino acid sequence that is shortened at the N-terminus and/or the C-terminus. A fragment that is shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a shortened open reading frame that lacks the 3' end of the open reading frame. A fragment that is shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a shortened open reading frame that lacks the 5' end of the open reading frame, provided that the shortened open reading frame contains a start codon that serves to initiate translation. The fragment of the amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues of the amino acid sequence. The fragment of the amino acid sequence preferably comprises at least 6, in particular 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.

As used herein, the term "wild type" or "WT" or "native" refers to a naturally occurring amino acid sequence, including allelic variants. A wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified.

In the context of the present description, the term "variants" of an amino acid sequence (of a peptide, protein or polypeptide) or "mutants" or a reference to a "mutant" polypeptide comprises 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 homologues, in particular those that occur in nature. The term "variant" or "mutant" includes, in particular, fragments of an amino acid sequence.

Amino acid insertion variants comprise insertions of one, two or more amino acids into a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are introduced into a particular region of the amino acid sequence, although random insertion is also possible with appropriate screening of the resulting product. Amino acid addition variants comprise amino- and/or carboxy-terminal fusions of one or more amino acids, for example 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, for example the removal of 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletions can be at any position in the protein. Amino acid deletion variants that comprise a deletion at the N-terminus and/or C-terminus of the protein are also referred to as N-terminal and/or C-terminal truncation variants. Amino acid substitution variants are characterized by the deletion of at least one residue in the sequence and the insertion of another residue in its place. Preference is given to modifications located at positions of the amino acid sequence that are not conservative, between homologous proteins or peptides and/or the replacement of amino acids with others having similar properties. Preferably, the amino acid substitutions in peptide and protein variants are conservative amino acid substitutions, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid substitution involves the substitution of one amino acid from a family of amino acids related in their side chains. Naturally occurring amino acids are generally classified into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (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.

Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence which 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 given for an amino acid region whose length 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 length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, then in some embodiments of contiguous amino acids the degree of similarity or identity is preferably given 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 amino acids. In some embodiments, the degree of similarity or identity is relative to the full length of the reference amino acid sequence. Alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using the best sequence alignment method, such as Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, gap opening penalty 10.0, gap extension penalty 0.5.

In the context of the present description, the term "sequence similarity" refers to the percentage of amino acids that are either 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 the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

The terms "% identical", "% identity" or similar terms are intended to refer specifically to the percentage of nucleotides or amino acids that are identical in an optimal alignment of the sequences being compared. The percentage referred to is purely statistical and the differences between the two sequences may be distributed, optionally, randomly over the entire length of the sequences being compared. Comparison of two sequences is typically performed by comparing the sequences, after optimal alignment, against a segment or "comparison window" to identify local regions of the corresponding sequences. Optimal alignment for comparison may be performed manually or by the local homology algorithm of Smith and Waterman (1981), Ads App. Math. 2, 482, by the local homology algorithm of Needleman and Wunsch (1970), J. Mol. Biol. 48, 443, using the similarity search algorithm of Pearson and Lipman (1988), Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm available on the NCBI website (e.g., blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seg&LINK_LOC=align2seg). In some embodiments, the parameters used for the BLASTN algorithm on the NCBI website include: (i) expected threshold: 10; (ii) word size: 28; (iii) maximum number of matches in the query range: 0; (iv) hit/mismatch score: 1, -2; (v) gap penalties: Linear; and (vi) a filter is used for low complexity regions. In some embodiments, the parameters used for the BLASTP algorithm on the NCBI website include: (i) expected threshold: 10; (ii) word size: 3; (iii) maximum number of matches in the query range: 0; (iv) matrix: BLOSUM62; (v) gap extension penalty: 11, extension:11; and (vi) conditional compositional correction of the substitution matrix.

Percent identity is obtained by determining the number of identical positions at which the compared sequences match, dividing this number by the number of positions compared (e.g. the number of positions in the reference sequence) and multiplying this result by 100.

In some embodiments, the degree of similarity or identity is indicated for a region whose length 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 length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, then in some embodiments of contiguous nucleotides, the degree of identity is given 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, the degree of similarity or identity is indicated for the full length of the reference sequence.

The homologous amino acid sequences of the invention have at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identical amino acid residues. Variants/mutants of the amino acid sequence described herein can be readily obtained by a person skilled in the art, for example by manipulating recombinant DNA. Manipulations of DNA sequences to obtain peptides or proteins having substitutions, additions, insertions or deletions are described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily obtained by known peptide synthesis methods, such as, for example, solid phase synthesis and similar methods.

In one aspect, a fragment or variant/mutant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or a "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 to or similar to the properties of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant/mutant" as used herein specifically refers to a variant/mutant molecule or sequence that comprises an amino acid sequence with one or more amino acids altered compared to the amino acid sequence of the parent molecule or sequence and that is still capable of performing one or more functions of the parent molecule or sequence, such as inducing an immune response. In one aspect, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In various embodiments, the function of the functional fragment or functional variant may be reduced but still substantially 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 the parent molecule or sequence. However, in other embodiments, the immunogenicity of the functional fragment or functional variant may be increased compared to the parent molecule or sequence.

As used herein, the term "isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living organism is not "isolated," but the same nucleic acid or peptide, partially or completely separated from materials coexisting with it in its natural state, is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as, for example, a host cell.

I. E. coli FimH polypeptides

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 UPEC clinical isolates and are encoded by a cluster of genes termed fim, which encode accessory proteins (FimC, FimD), various structural subunits (FimE, FimF, FimG), and an adhesion protein termed FimH. FimH consists of two domains: a lectin-binding domain (FimH _LD ), responsible for binding to mannosylated glycoproteins, and a pilin domain. The pilin domain serves to link FimH to other pilus structural subunits, such as FimG, through a mechanism termed donor strand exchange. The pilin domain of FimH forms an incomplete fold of the immunoglobulin, resulting in a groove that provides a binding site for the N-terminal β-chain of FimG, forming a strong intermolecular linkage between FimH and FimG. Although FimH _LD can be expressed in a soluble stable form, full-length FimH by itself is unstable unless it is complexed with the chaperone FimC or supplemented with a FimG donor chain peptide in peptide form or as a fusion protein. Accordingly, expression of a stable full-length FimH molecule is possible by linking a FimG donor peptide to the C-terminus of full-length FimH via a glycine-serine linker to yield a product designated FimH-DSG.

In one aspect, the present invention provides mutant FimH polypeptides, such as those shown in Table 1. Such mutants comprise mutations in the amino acid sequence relative to the amino acid sequence of the corresponding wild-type (WT) FimH polypeptide. In some aspects, such mutants are immunogenic with respect to the wild-type FimH protein or bacteria expressing the wild-type FimH polypeptide. In some aspects, FimH mutants have certain advantageous characteristics, such as increased immunogenic properties compared to the corresponding wild-type FimH polypeptide.

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 wild-type E. coli FimH polypeptide or any fragment thereof. For example, in one embodiment, the present invention relates to a mutant FimH polypeptide that is a mutant FimH _LD polypeptide or a FimH-DSG polypeptide. The present invention relates to novel FimH _LD and FimH-DSG mutants with reduced affinity for mannoside ligands (as confirmed by biochemical and biophysical assays) that improves functional immunogenicity, and describes an assessment of the neutralizing responses of these mutants compared to wild-type FimH _LD .

The introduced amino acid mutations in the mutant FimH polypeptides may include amino acid substitutions, deletions, or additions. In some aspects, the only mutations in the amino acid sequence of the mutant FimH polypeptide are amino acid substitutions relative to the wild-type FimH protein.

The amino acid sequence of a wild-type FimH polypeptide is well known in the art. For example, the amino acid sequence of the FimH _LD domain is provided herein as SEQ ID NO:1. The full-length wild-type FimH polypeptide, including the FimG donor peptide linked to the C-terminus of the 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.

In one aspect of the invention, certain mutant FimH polypeptides allow for a fixed open conformation, resulting in decreased affinity for mannoside ligands and improved functional immunogenicity. Accordingly, such FimH mutants can be used as antigens in an immunogenic composition, such as a vaccine, against E. coli infection. Since wild-type FimH _LD is considered a poor immunogen in terms of its ability to stimulate functional immunogenicity, such FimH mutants allow for improved antigens that can be used in such immunogenic compositions.

In one aspect described in Example 1, FimH mutants are created that allow the FimH lectin domain to be locked in an open conformation to reduce affinity for mannoside ligands. Such mutants can include at least 1, 2, 3, 4, 5 or more mutations. The mutations can include: naturally occurring amino acid substitutions that are common among urinary tract infection isolates (such as V27A); substitutions in the ligand binding site of the FimH _LD (e.g., at positions F1 and Q133); glycine switch mutations in the FimH _LD (e.g., at positions G15, G16 and G65); introduction of cysteine pairs to stabilize the disulfide bond in FimH _LD (e.g., in the 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); non-polar-to-polar mutations in the FimH _LD (e.g., at positions V27, L34, A119, or any combination thereof); cavity filling mutations at the pilin-lectin interface of the FimH-DSG (e.g., at positions A115, V163, V185, or V3 in the DSG sequence); or any combination of these types of mutations and at the amino acid positions noted above. In another aspect, the present invention provides FimH mutants depicted in SEQ ID NOS: 2-58 and 60-64, or any combination of mutants depicted in any of such sequences. In another aspect, the present invention provides a FimH mutant of any of SEQ ID NOS: 23, 50, 51, 52, 53, 54, 60, and 62. In another aspect, the present invention provides a FimH mutant of SEQ ID NO: 62.

In another aspect, the present invention relates to any of the FimH mutants of SEQ ID NOs: 2-58 and 60-64, wherein said mutants are isolated. For example, in one aspect, the present invention provides a FimH mutant of any of SEQ ID NOs: 23, 50, 51, 52, 53, 54, 60 and 62, wherein said FimH mutant is isolated. In another aspect, the present invention provides a FimH mutant according to SEQ ID NO: 62, wherein said FimH mutant is isolated.

Accordingly, in some specific aspects, the present invention provides a FimH mutant comprising a combination of introduced mutations, wherein the mutant comprises a combination of mutations set forth in any of the mutants set forth in Table 1 (i.e., SEQ ID NOs: 2-58 and 60-64). Any combination of amino acid substitutions set forth in each of the mutants in Table 1 can be used in the wild-type FimH polypeptide sequence to produce various FimH mutants. FimH mutants based on the native FimH polypeptide sequence of any other subtype or strain comprising any combination of the mutations set forth herein are also within the scope of the present invention.

Another aspect of the present invention 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: 1-64. In a preferred aspect, 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. In another aspect, the present invention provides 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 a preferred aspect, 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.

The FimH mutants of the present invention can be produced by conventional methods known in the art, such as 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 (a clonal isolate derived from the parental Trichoplusia ni cell line BTI-TN-5B1-4 (Invitrogen)). Examples of suitable mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells, typically transformed with shear-fragmented adenovirus type 5 DNA), 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 embryo fibroblasts, chicken embryonic germ cells, quail fibroblasts (e.g., ELL-O), and duck cells. Suitable insect cell expression systems, such as baculovirus vector-based 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, among others, 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.

A variety of suitable vectors for expressing recombinant proteins in insect or mammalian cells are well known and commonly used in the art. Suitable vectors may contain a number of 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 a transcriptional control element (e.g., a promoter, enhancer, terminator) and/or one or more translation signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell (e.g., mammalian or heterologous mammalian or non-mammalian species). 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 is used that will direct expression of the construct in the desired mammalian host cell (e.g., Chinese hamster ovary cells).

The mutant FimH polypeptides can be isolated by any suitable methods. For example, methods for purifying mutant FimH protein polypeptides by immunoaffinity chromatography are known in the art. Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687 (2004). Suitable methods for purifying the desired proteins, including precipitation and various types of chromatography such as hydrophobic interaction chromatography, ion exchange, affinity, chelation, and size exclusion chromatography, are well known in the art. Suitable purification schemes can be designed using two or more of these or other suitable methods. If desired, the mutant FimH polypeptides can include a "tag" to facilitate purification, such as an epitope tag or a histidine (His) tag. Such tagged polypeptides are conveniently isolated, for example, from conditioned media by chelation chromatography or affinity chromatography.

As used herein, the term "antigen" 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 determinants recognized by an immune cell.

II. Nucleic acids encoding FimH mutants

In another aspect, the present invention relates to nucleic acid molecules that encode the FimH mutant disclosed herein. Such nucleic acid molecules include DNA, cDNA and RNA sequences. In one embodiment, the nucleic acid molecule can be included in a vector, such as an expression vector.

In one aspect, nucleic acids encoding mutant E. coli FimH polypeptides or any fragment thereof are provided. One or more nucleic acid constructs encoding mutant FimH polypeptides or fragments thereof can be used for genomic integration and subsequent expression of the polypeptide. For example, one nucleic acid construct encoding a mutant FimH polypeptide or fragment thereof can be introduced into a host cell. Alternatively, the coding sequences of the polypeptide can be carried by two or more nucleic acid constructs, which are then introduced into the host cell simultaneously or sequentially.

For example, in one exemplary embodiment, one nucleic acid construct encodes a lectin domain and a pilin domain of E. coli FimH. In another exemplary embodiment, one nucleic acid construct encodes a lectin domain and a second nucleic acid construct encodes a pilin domain of E. coli FimH. In some aspects, genomic integration is achieved.

The nucleic acid construct may comprise genomic DNA containing one or more introns, or cDNA. Some genes are expressed more efficiently when introns are present. In some aspects, the nucleic acid sequence is suitable for expressing exogenous polypeptides in said mammalian cell.

In some aspects, a nucleic acid encoding a polypeptide or fragment thereof is codon optimized to enhance expression levels in any particular cell.

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

In some aspects, the signal sequence is any 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 aspects, the signal sequence is 20 amino acids in length. In some aspects, the signal sequence is 21 amino acids in length.

In some aspects, where the polypeptide or fragment thereof comprises a signal sequence, in order to increase the expression level of the polypeptide or fragment thereof in cultured cells, the endogenous signal sequence naturally associated with the polypeptide can be replaced by a signal sequence not associated with the wild-type polypeptide. Accordingly, in some aspects, the nucleic acid does not comprise a native signal sequence of an E. coli-derived polypeptide or fragment thereof. In some aspects, the nucleic acid does not comprise a native signal sequence of an E. coli FimH-derived polypeptide. In some aspects, the E. coli-derived polypeptide or fragment thereof can be expressed by a heterologous peptide, which is preferably a signal sequence, or another peptide having a specific cleavage site at the N-terminus of the mature protein or the E. coli-derived polypeptide or fragment thereof. For example, a polypeptide derived from E. coli FimH or a fragment thereof can be expressed by a heterologous peptide (e.g., an IgK signal sequence), which is preferably a signal sequence, or another peptide having a specific cleavage site at the N-terminus of the mature E. coli FimH protein. In preferred aspects, the specific cleavage site at the N-terminus of the mature E. coli FimH protein is immediately before the original phenylalanine residue of the mature E. coli FimH protein. The heterologous sequence selected is preferably a sequence that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.

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

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

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

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

In one aspect, the present disclosure provides vectors that include coding sequences for a mutant FimH polypeptide or fragment thereof. Examples of vectors include plasmids that are capable of replicating autonomously or replicating in a mammalian cell. Typical expression vectors contain suitable promoters, enhancers, and terminators that can be used to regulate expression of the coding sequence(s) in the expression construct. Vectors can also include selection markers to provide a phenotypic trait for selection of transformed host cells (e.g., by conferring resistance to antibiotics such as ampicillin or neomycin).

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

Internal ribosome entry site (IRES) and peptide 2A sequences can also be used. IRES and peptide 2A provide alternative approaches to coexpressing multiple sequences. An IRES is a nucleotide sequence that allows translation to be initiated in the middle of a messenger RNA (mRNA) sequence as part of a larger protein synthesis process. Normally in eukaryotes, translation can only be initiated at the 5' end of an mRNA molecule. IRES elements allow expression of multiple genes in a single transcript. IRES-based polycistronic vectors that express multiple proteins from a single transcript can reduce the escape of non-expressing clones from selection. Peptide 2A allows translation of multiple proteins in a single open reading frame into a polyprotein that is subsequently cleaved into individual proteins by ribosome skipping. Peptide 2A may allow more balanced expression of multiple protein products. Examples of IRES sequences include, for example, EV71 IRES, EMCV IRES, HCV IRES. As for genomic integration, it can be site-specific or random. Site-specific recombination can be achieved by introducing homologous sequence(s) into the nucleic acid constructs provided herein. Such a homologous sequence substantially corresponds to an endogenous sequence at a specific target site in the host genome. Alternatively, random integration can be used. Sometimes, the expression level of a protein can vary depending on the integration site. Therefore, several clones can be selected according to the expression level of the recombinant protein to identify a clone that achieves the desired expression level.

Examples of nucleic acid constructs are further described in the figures, such as Figs. 2A-2T, of PCT International Publication No. WO2021/084429, published May 6, 2021, which is incorporated herein by reference.

In one aspect, the nucleic acid sequence encodes an amino acid sequence 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%, at least 99% identical to any of SEQ ID NOs: 1-64. In a preferred aspect, the nucleic acid sequence encodes an amino acid sequence 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 SEQ ID NO: 62. In another aspect of the present invention, the nucleic acid sequence encodes an amino acid sequence 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 a preferred aspect, the nucleic acid sequence encodes an amino acid sequence 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 SEQ ID NO: 62.

In some aspects of the present invention, the RNA is messenger RNA (mRNA), which refers to an RNA transcript encoding a peptide or protein. As recognized in the art, mRNA typically comprises a 5'-untranslated region (5'-UTR), a region encoding a peptide, 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, wherein DNA refers to a nucleic acid comprising deoxyribonucleotides. In one aspect, the RNA provided herein can comprise modified nucleosides. In some aspects, the RNA comprises a modified nucleoside in place of at least one (e.g., each) uridine.

In some embodiments, the compositions or medicinal products disclosed herein comprise an RNA encoding an amino acid sequence comprising a mutant FimH polypeptide. Likewise, the methods disclosed herein comprise administering such RNA. One platform suitable for use in the context of the invention is based on an antigen-encoding RNA vaccine to induce strong neutralizing antibodies and an accompanying/concomitant T-cell response to achieve protective immunization, preferably with minimal vaccine doses. The RNA administered is preferably in vitro transcribed RNA. Particularly preferred are three different RNA platforms, namely unmodified uridine-containing mRNA (uRNA), nucleoside-modified mRNA (modRNA) and self-amplifying RNA (saRNA). In one particularly preferred aspect, the RNA is in vitro transcribed RNA.

III. Host cells

In one aspect, the invention relates to cells in which sequences encoding a mutant FimH polypeptide or a fragment thereof are 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 host cell genome and, when cultured under suitable conditions, expresses the polypeptide or a fragment thereof. In a preferred embodiment, the polynucleotide sequence is expressed with high efficiency and is genetically stable.

Suitable mammalian host cells are known in the art. Preferably, the host cell is suitable for producing protein on an industrial scale. Examples of mammalian host cells include any of the following cells and derivatives thereof: Chinese hamster ovary (CHO) cells, COS cells (a cell line derived from monkey kidney, the 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-tumor mouse cell line). Additional typical mammalian host cells include mouse Sertoli cells (TM4), buffalo rat liver cells (BRL 3A), mouse mammary tumor cells (MMT), rat hepatoma cells (HTC), mouse myeloma cells (NSO), mouse hybridoma cells (Sp2/0), mouse thymoma cells (EL4), Chinese hamster ovary (CHO) cells and CHO cell derivatives, mouse embryonic cells (NIH/3T3, 3T3 Li), rat myocardial cells (H9c2), mouse myoblasts (C2C12), and mouse kidney cells (miMCD-3). Other 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.

Any cell capable of undergoing cell culture may be used in accordance with the present invention. In some aspects, the cell is a mammalian cell. Non-limiting examples of mammalian cells that may be used in accordance with the present invention include the BALB/c mouse myeloma cell line (NSO/1, ECACC No.: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); the SV40-transformed monkey kidney line CV1 (COS-7, ATCC CRL 1651); the human embryonic kidney line (293 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 28yophil 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-1587); 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 cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). In some preferred embodiments, the cells are CHO cells. In some preferred aspects, the cells are GS cells.

In addition, any number of commercially available and non-commercial hybridoma cell lines can be used in accordance with the present invention. The term "hybridoma" as used herein refers to a cell or progeny of a cell obtained by fusing an immortalized cell and an antibody-producing cell. Such a resulting hybridoma is an immortalized antibody-producing cell. The individual cells used to create the hybridoma can be obtained from any mammal, including, but not limited to, a rat, a pig, a rabbit, a sheep, a pig, a goat, and a human. In some aspects, the hybridoma is a trioma cell line that occurs when the progeny of heterohybrid myeloma fusions, which are the fusion product between human cells and a mouse sheloma cell line, are subsequently fused with a plasma cell. In some aspects, a hybridoma is any immortalized hybrid cell line that produces antibodies, such as, for example, quadroms (see, for example, Milstein et al., Nature 537:3053 (1983)). One skilled in the art will appreciate that hybrid cell lines may have different nutritional requirements and/or may require different culture conditions for optimal growth, which one may be able to modify as needed.

In some aspects, the cell comprises a first gene of interest, wherein the first gene of interest is chromosomally integrated. In some aspects, the first gene of interest comprises a reporter gene, a selection gene, a gene of interest (e.g., encoding a polypeptide derived from E. coli, or a fragment thereof), an accessory gene, or a combination thereof. In some aspects, the gene of therapeutic interest comprises a gene encoding a difficult to express protein (DtE).

In some aspects, a first gene of interest is located between two separate recombination target sites (RTSs) in a mammalian cell via site-specific integration (SSI), wherein the two RTSs are chromosomally integrated at the NL1 locus or the NL2 locus. See, for example, U.S. Patent Application Publication No. 20200002727 for a description of the NL1 locus, the NL2 locus, the NL3 locus, the NL4 locus, the NL5 locus, and the NL6 locus. In some aspects, the first gene of interest is located at the NL1 locus. In some aspects, the cell comprises a second gene of interest, wherein the second gene of interest is chromosomally integrated. In some aspects, the second gene of interest comprises a reporter gene, a selectable gene, a gene of therapeutic interest (such as a mutant FimH polypeptide or fragment thereof), an accessory gene, or a combination thereof. In some aspects, a gene of therapeutic interest comprises a gene encoding a DtE protein. In some aspects, a second gene of interest is located between two RTSs. In some aspects, the second gene of interest is located at the NL1 locus or the NL2 locus. In some aspects, the first gene of interest is located at the NL1 locus and the second gene of interest is located at the NL2 locus. In some aspects, the cell comprises a third gene of interest, wherein the third gene of interest is chromosomally integrated. In some aspects, the third gene of interest comprises a reporter gene, a selectable gene, a gene of therapeutic interest (such as a polypeptide derived from E. coli or a fragment thereof), an accessory gene, or a combination thereof. In some aspects, the gene of therapeutic interest comprises a gene encoding a DtE protein. In some aspects, the third gene of interest is located between two RTSs. In some aspects, the third gene of interest is located at the NL1 locus or the NL2 locus. In some aspects, the third gene of interest is located at a locus different from the NL1 locus and the NL2 locus. In some aspects, the first gene of interest, the second gene of interest, and the third gene of interest are located at three separate loci. In some aspects, at least one of the first gene of interest, the second gene of interest, and the third gene of interest is located at the NL1 locus, and at least one of the first gene of interest, the second gene of interest, and the third gene of interest is located at the NL2 locus. In some aspects, the cell comprises a site-specific recombinase gene. In some aspects, the site-specific recombinase gene is chromosomally integrated.

In another aspect, the present invention relates to a mammalian cell comprising at least four different RTSs, wherein the cell comprises (a) at least two different RTSs chromosomally integrated at the NL1 locus or the NL2 locus; (b) a first gene of interest inserted between the at least two RTSs of (a), wherein the first gene of interest comprises a reporter gene, a gene encoding a DtE protein, an accessory gene, or a combination thereof; (c) and a second gene of interest chromosomally integrated at a second locus different from locus (a), wherein the second gene of interest comprises a reporter gene, a gene encoding a DtE protein (such as a polypeptide derived from E. coli or a fragment thereof), an accessory gene, or a combination thereof. In some aspects, the present invention relates to a mammalian cell comprising at least four different RTSs, wherein the cell comprises (a) at least two different RTSs chromosomally integrated at the Fer1L4 locus; (b) at least two different RTSs chromosomally integrated at the NL1 locus or the NL2 locus; (c) a first gene of interest chromosomally integrated at the Fer1L4 locus, wherein the first gene of interest comprises a reporter gene, a gene encoding a DtE protein, an accessory gene, or a combination thereof; and (d) a second gene of interest chromosomally integrated at the NL1 locus or the NL2 locus of (b), wherein the second gene of interest comprises a reporter gene, a gene encoding a DtE protein (such as derived from a polypeptide from E. coli or a fragment thereof), an accessory gene, or a combination thereof.

In some aspects, the present invention relates to a mammalian cell comprising at least six different RTSs, wherein the cell comprises (a) at least two different RTSs and a first gene of interest chromosomally integrated at the Fer1L4 locus; (b) at least two different RTSs and a second gene of interest chromosomally integrated at the NL1 locus; and (c) at least two different RTSs and a third gene of interest chromosomally integrated at the NL2 locus.

As used herein, the terms "in functional combination," "in functional order," and "operably linked" refer to the linking of nucleic acid sequences such that a nucleic acid molecule is produced that is capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule. This term also refers to the joining of amino acid sequences such that a functional protein is formed. In some aspects, a gene of interest is operably linked to a promoter, wherein the gene of interest is chromosomally integrated in a host cell. In some aspects, a gene of interest is operably linked to a heterologous promoter; wherein the gene of interest is chromosomally integrated in a host cell. In some aspects, an accessory gene is operably linked to a promoter, wherein the accessory gene is chromosomally integrated into the genome of a host cell. In some aspects, an accessory gene is operably linked to a heterologous promoter; wherein the accessory gene is chromosomally integrated into the genome of a host cell. In some aspects, 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 aspects, a gene encoding a DtE protein is operably linked to a heterologous promoter, wherein the gene encoding a DtE protein is chromosomally integrated into the host cell genome. In some aspects, a recombinase gene is operably linked to a promoter, wherein the recombinase gene is chromosomally integrated in the host cell. In some aspects, a recombinase gene is operably linked to a promoter, wherein the recombinase gene is not integrated into the host cell genome. In some aspects, a recombinase gene is operably linked to a heterologous promoter, wherein the recombinase gene is not chromosomally integrated into the host cell genome.

As used herein, the term "chromosomally integrated" or "chromosomal integration" refers to the stable incorporation of a nucleic acid sequence into a chromosome of a host cell, such as a mammalian cell, i.e., a nucleic acid sequence that is chromosomally integrated into the genomic DNA (gDNA) of the host cell, such as a mammalian cell. In some aspects, the nucleic acid sequence integrated into the chromosome is stable. In some aspects, the nucleic acid sequence that is chromosomally integrated is not located on a plasmid or in a vector. In some aspects, the nucleic acid sequence that is chromosomally integrated is not excised. In some aspects, chromosomal integration is mediated by clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein (Cas) gene editing system (CRISPR/CAS).

IV. Compositions and formulations

In one aspect, the present invention relates to a composition comprising at least one mutant FimH polypeptide or fragment thereof as described herein. In some aspects, the composition elicits an immune response, including antibodies, that can provide immunity to pathogenic E. coli species.

In some aspects, the composition comprises a mutant FimH polypeptide as the only antigen. In some aspects, the composition does not include a conjugate.

In some aspects, the composition comprises a mutant FimH polypeptide and at least one additional antigen. In some aspects, the composition comprises a mutant FimH polypeptide and an additional E. coli antigen. In some aspects, the composition comprises a mutant FimH polypeptide and an E. coli glycoconjugate.

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

In one embodiment, the invention provides a composition comprising a mutant FimH polypeptide and a saccharide comprising any structure selected from the saccharide structures disclosed in PCT International Publication Nos. WO2021/084429, published May 6, 2021, and WO2020/039359, published February 27, 2020, and US Publication No. US2020/0061177, published February 27, 2020, each of which is incorporated herein by reference in its entirety. In one aspect, the invention provides a composition comprising a mutant FimH polypeptide; and a saccharide comprising a structure selected from: 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 05ab and Formula 05ac (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 023A), 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 62D-1, Formula O63, Formula O64, Formula O65, Formula O66, formula O68, formula O69, formula O70, formula O71, formula O73 (e.g., formula O73 (strain 73-1)), formula O74, formula O75, formula O76, formula O77, formula O78, formula O79, formula O80, formula O81, formula O82, formula O83, formula O84, formula O85, formula O86, formula O87, formula O88, formula O89, formula O90, formula O91, formula O92, formula O93, formula O95, formula O96, formula O97, formula O98, formula O99, formula O100, formula O101, formula O102, formula O103, formula O104, formula O105, formula O106, formula O107, formula O108, formula O109, formula O110, formula O111, formula O112, formula O113, Formulas O114, Formulas O115, Formulas O116, Formulas O117, Formulas O118, Formulas O119, Formulas O120, Formulas O121, Formulas O123, Formulas O124, Formulas O125, Formulas O126, Formulas O127, Formulas O128, Formulas O129, Formulas O130, Formulas O131, Formulas O132, Formulas O133, Formulas O134, Formulas O135, Formulas O136, Formulas O137, Formulas O138, Formulas O139, Formulas O140, Formulas O141, Formulas O142, Formulas O143, Formulas O144, Formulas O145, Formulas O146, Formulas O147, Formulas O148, Formulas O149, Formulas O150, Formulas O151, Formulas O152, Formulas O153, Formulas O154, Formulas 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, where n is an integer from 1 to 100, preferably from 31 to 90.

In some embodiments, the composition comprises any of the saccharides disclosed herein. In preferred embodiments, the composition comprises any of the conjugates disclosed herein.

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.

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

In a preferred embodiment, a glycoconjugate of any of the above compositions is individually conjugated to CRM _197. In another preferred embodiment, any glycoconjugate of the above compositions is individually conjugated to SCP.

Accordingly, in some embodiments, the composition comprises a mutant FimH polypeptide and an O-antigen from at least one E. coli serotype. In a preferred embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from more than 1 E. coli serotype. For example, the composition can comprise an O-antigen from two different E. coli serotypes (or "v", valence) and up to 12 different serotypes (12v). In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 3 different serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 4 different E. coli serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 5 different E. coli serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 6 different E. coli serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 7 different serotypes of E. coli. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 8 different serotypes of E. coli. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 9 different serotypes of E. coli. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 10 different serotypes of E. coli. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 11 different serotypes of E. coli. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 12 different serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 13 different serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 14 different serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 15 different serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 16 different serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 17 different serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 18 different serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 19 different serotypes. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 20 different serotypes.

Preferably, the number of E. coli saccharides may vary from 1 serotype (or "v", valency) to 26 different serotypes (26v). In one embodiment, one serotype is present. In one embodiment, 2 different serotypes are present. In one embodiment, 3 different serotypes are present. In one embodiment, 4 different serotypes are present. In one embodiment, 5 different serotypes are present. In one embodiment, 6 different serotypes are present. In one embodiment, 7 different serotypes are present. In one embodiment, 8 different serotypes are present. In one embodiment, 9 different serotypes are present. In one embodiment, 10 different serotypes are present. In one embodiment, 11 different serotypes are present. In one embodiment, 12 different serotypes are present. In one embodiment, 13 different serotypes are present. In one embodiment, 14 different serotypes are present. In one embodiment, 15 different serotypes are present. In one embodiment, 16 different serotypes are present. In one embodiment, 17 different serotypes are present. In one embodiment, 18 different serotypes are present. In one embodiment, 19 different serotypes are present. In one embodiment, 20 different serotypes are present. In one embodiment, 21 different serotypes are present. In one embodiment, 22 different serotypes are present. In one embodiment, 23 different serotypes are present. In one embodiment, 24 different serotypes are present. In one embodiment, 25 different serotypes are present. In one embodiment, 26 different serotypes are present. The saccharides are conjugated to a carrier protein to form glycoconjugates as disclosed herein.

In one aspect, the composition comprises a mutant FimH polypeptide; and a glycoconjugate that comprises an O-antigen from at least one serogroup of E. coli, wherein the O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide; and an O-antigen from more than 1 E. coli serotype, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 2 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 3 different E. coli serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 4 different serotypes of E. coli, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 5 different serotypes of E. coli, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 6 different serotypes of E. coli, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 7 different serotypes of E. coli, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 8 different serotypes of E. coli, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 9 different serotypes of E. coli, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises an O-antigen from a mutant FimH polypeptide and 10 different serotypes of E. coli, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises an O-antigen from a mutant FimH polypeptide and 11 different serotypes of E. coli, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 12 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 13 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 14 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 15 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 16 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 17 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 18 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 19 different serotypes, wherein each O-antigen is conjugated to a carrier protein. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-antigen from 20 different serotypes, wherein each O-antigen is conjugated to a carrier protein.

In another aspect, the composition comprises an O-polysaccharide from at least one serotype of E. coli. In a preferred embodiment, the composition comprises an O-polysaccharide from more than 1 serotype of E. coli. For example, the composition may comprise an O-polysaccharide from two different serotypes of E. coli and up to 12 different serotypes of E. coli. In one embodiment, the composition comprises an O-polysaccharide from 3 different serotypes of E. coli. In one embodiment, the composition comprises an O-polysaccharide from 4 different serotypes of E. coli. In one embodiment, the composition comprises an O-polysaccharide from 5 different serotypes of E. coli. In one embodiment, the composition comprises an O-polysaccharide from 6 different serotypes of E. coli. In one embodiment, the composition comprises an O-polysaccharide from 7 different serotypes of E. coli. In one embodiment, the composition comprises O-polysaccharide from 8 different serotypes of E. coli. In one embodiment, the composition comprises O-polysaccharide from 9 different serotypes of E. coli. In one embodiment, the composition comprises O-polysaccharide from 10 different serotypes of E. coli. In one embodiment, the composition comprises O-polysaccharide from 11 different serotypes of E. coli. In one embodiment, the composition comprises O-polysaccharide from 12 different serotypes. In one embodiment, the composition comprises O-polysaccharide from 13 different serotypes. In one embodiment, the composition comprises O-polysaccharide from 14 different serotypes. In one embodiment, the composition comprises O-polysaccharide from 15 different serotypes. In one embodiment, the composition comprises O-polysaccharide from 16 different serotypes. In one embodiment, the composition comprises O-polysaccharide from 17 different serotypes. In one embodiment, the composition comprises O-polysaccharide from 18 different serotypes. In one embodiment, the composition comprises O-polysaccharide from 19 different serotypes. In one embodiment, the composition comprises O-polysaccharide from 20 different serotypes.

In a preferred embodiment, the composition comprises an O-polysaccharide from at least one serotype of E. coli, wherein the O-polysaccharide is conjugated to a carrier protein. In a preferred embodiment, the composition comprises an O-polysaccharide from more than 1 serotype of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. For example, the composition may comprise an O-polysaccharide from two different serotypes of E. coli and up to 12 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 3 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 4 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 5 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 6 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 7 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 8 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 9 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 10 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 11 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 12 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises an O-polysaccharide from 13 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharide from 14 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharide from 15 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharide from 16 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharide from 17 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharide from 18 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharide from 19 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein. In one embodiment, the composition comprises O-polysaccharide from 20 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein.

In a most preferred embodiment, the composition comprises an O-polysaccharide of at least one serotype of E. coli, wherein the O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core (central) saccharide. In a preferred embodiment, the composition comprises an O-polysaccharide from more than 1 serotype of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. For example, the composition may comprise an O-polysaccharide from two different serotypes of E. coli and up to 12 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 3 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 4 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 5 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 6 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 7 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 8 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 9 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 10 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 11 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 12 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 13 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 14 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 15 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 16 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 17 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 18 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 19 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises an O-polysaccharide from 20 different serotypes, wherein each O-polysaccharide is conjugated to a carrier protein, and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In a preferred embodiment, the carrier protein is CRM ₁₉₇ .

In another preferred embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide conjugated to CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O25a, wherein n is at least 30, and a core saccharide. In a preferred embodiment, the composition further comprises an O-polysaccharide conjugated to CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O25b, wherein n is at least 40, and a core saccharide. In another embodiment, the composition further comprises an O-polysaccharide conjugated to CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O1a, wherein n is at least 30, and a core saccharide. In another embodiment, the composition further comprises an O-polysaccharide conjugated to CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O2, wherein n is at least 30, and a core saccharide. In another embodiment, the composition further comprises an O-polysaccharide conjugated to CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O6, wherein n is at least 30, and a core saccharide.

In another embodiment, the composition further comprises an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O17, wherein n is at least 30, and a core saccharide. In another embodiment, the composition further comprises an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O15, wherein n is at least 30, and a core saccharide. In another embodiment, the composition further comprises an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula 018A, wherein n is at least 30, and a core saccharide. In another embodiment, the composition further comprises an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O75, wherein n is at least 30, and a core saccharide. In another embodiment, the composition further comprises an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O4, wherein n is at least 30, and a core saccharide. In another embodiment, the composition further comprises an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O16, wherein n is at least 30, and a core saccharide. In another embodiment, the composition further comprises an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O13, wherein n is at least 30, and a core saccharide. In another embodiment, the composition further comprises an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O7, wherein n is at least 30, and a core saccharide.

In another embodiment, the composition further comprises an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O8, wherein n is at least 30, and a core saccharide. In another embodiment, the O-polysaccharide comprises the formula O8, wherein n is 1-20, preferably 2-5, more preferably 3. In another embodiment, the composition further comprises an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O9, wherein n is at least 30, and a core saccharide. In another embodiment, the O-polysaccharide comprises the formula O9, wherein n is 1-20, preferably 4-8, more preferably 5. In another embodiment, the O-polysaccharide comprises the formula O9a, wherein n is 1-20, preferably 4-8, more preferably 5.

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

As described above, the composition may include a mutant FimH polypeptide and any combination of conjugated O-polysaccharides (antigens). In one exemplary embodiment, the composition includes a polysaccharide that includes formula O25b, a polysaccharide that includes formula O1a, a polysaccharide that includes formula O2, and a polysaccharide that includes formula O6. More specifically, such a composition includes: (i) an O-polysaccharide conjugated to CRM ₁₉₇ , wherein the O-polysaccharide includes formula O25b, wherein n is at least 30, and a core saccharide; (ii) an O-polysaccharide conjugated to CRM ₁₉₇ , wherein the O-polysaccharide includes formula O1a, wherein n is at least 30, and a core saccharide; (iii) an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O2, wherein n is at least 30, and a core saccharide; and (iv) an O-polysaccharide conjugated with CRM ₁₉₇ , wherein the O-polysaccharide comprises the formula O6, wherein n is at least 30, and a core saccharide.

In one embodiment, the composition comprises a mutant FimH polypeptide and at least one O-polysaccharide derived from any serotype of E. coli, wherein the serotype is not O25a. For example, in one embodiment, the composition does not include a saccharide that includes the formula O25a. Such a composition may include, for example, an O-polysaccharide that includes the formula O25b, an O-polysaccharide that includes the formula O1a, an O-polysaccharide that includes the formula O2, and an O-polysaccharide that includes the formula O6.

In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 2 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 3 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 4 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 5 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 6 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 7 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 8 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 9 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 10 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 11 different serotypes of E. coli, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 12 different serotypes, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 13 different serotypes, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 14 different serotypes, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 15 different serotypes, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 16 different serotypes, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 17 different serotypes, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 18 different serotypes, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 19 different serotypes, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide. In one embodiment, the composition comprises a mutant FimH polypeptide and an O-polysaccharide from 20 different serotypes, wherein each O-polysaccharide is conjugated to CRM ₁₉₇ , and wherein the O-polysaccharide comprises an O-antigen and a core saccharide.

In one aspect, the invention relates to a composition that comprises a mutant FimH polypeptide and a conjugate that comprises a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O25b, wherein n is 15±2. In one aspect, the invention relates to a composition that comprises a mutant FimH polypeptide and a conjugate that comprises a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O25b, wherein n is 17±2. In one aspect, the invention relates to a composition that comprises a mutant FimH polypeptide and a conjugate that comprises a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O25b, wherein n is 55±2. In another aspect, the invention relates to a composition that comprises a mutant FimH polypeptide and a conjugate that comprises a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O25b, wherein n is 51±2. In another aspect, the invention relates to a composition that comprises a mutant FimH polypeptide and a conjugate that comprises a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O25b, wherein n is an integer greater than 30, preferably n is an integer from 31 to 100. In one embodiment, the saccharide further comprises an R1 moiety of the E. coli core saccharide. In another embodiment, the saccharide further comprises a K12 moiety of the E. coli core saccharide. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM _197. In one embodiment, the conjugate is obtained by single-end conjugation. In one embodiment, the conjugate is prepared by chemical reductive amination, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

In one aspect, the immunogenic composition induces the formation of IgG antibodies in humans, wherein said antibodies are capable of binding to the polysaccharide of E. coli serotype O25b 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 the serum before and after immunization with the immunogenic composition of the invention can be performed and the responses to serotype O25b can be compared to assess a potential increase in the number of patients with a response to treatment. In one embodiment, the immunogenic composition induces the formation of IgG antibodies in humans, wherein said antibodies are capable of killing E. coli serotype O25b, as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition induces the formation of functional antibodies in humans, wherein said antibodies are capable of killing E. coli serotype O25b, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of patients responding to treatment of E. coli serotype O25b infection (i.e., individuals with a serum having a titer of at least 1:8, as determined by an in vitro opsonophagocytic killing assay) compared to a previously immunized population. In one embodiment, the immunogenic composition induces 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 significantly increases the proportion of patients responding to treatment of E. coli serotype O25b infection (i.e., individuals with a serum having a titer of at least 1:8, as determined by an in vitro OPA) compared to a previously immunized population. In one embodiment, the immunogenic composition of the invention significantly increases OPA titers in humans against E. coli serotype O25b compared to a previously immunized population.

In one aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O1a, wherein n is an integer greater than 30, preferably n is an integer from 31 to 100. In another aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O1a, wherein n is 39±2. In another aspect, the invention relates to a composition that includes a FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O1a, wherein n is 13±2. In one embodiment, the saccharide further comprises an R1 moiety of the E. coli core saccharide. In one embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM _197. In one embodiment, the conjugate is produced by single-end conjugation. In one embodiment, the conjugate is produced by reductive amination chemistry,

preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

In one embodiment, the immunogenic composition induces the formation of IgG antibodies in humans, wherein said antibodies are capable of binding to the polysaccharide of E. coli serotype O1a at a concentration of at least 0.2 pg/ml, 0.3 pg/ml, 0.35 pg/ml, 0.4 pg/ml or 0.5 pg/ml, as determined by an ELISA assay. Therefore, a comparison of the OPA activity of the serum before and after immunization with the immunogenic composition of the invention can be performed and the responses to serotype O1a can be compared to evaluate a potential increase in the number of patients with a response to treatment. In one embodiment, the immunogenic composition induces the formation of IgG antibodies in humans, wherein said antibodies are capable of killing E. coli serotype O1a, as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition induces functional antibodies in humans, wherein said antibodies are capable of killing E. coli serotype O1a, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of patients responding to treatment of E. coli serotype O1a infection (i.e., individuals with a serum titer of at least 1:8, as determined by an in vitro opsonophagocytic killing assay) compared to a previously immunized population. In one embodiment, the immunogenic composition induces 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 significantly increases the proportion of patients responding to treatment of E. coli serotype O1a infection (i.e., individuals with a serum titer of at least 1:8 as determined by an in vitro OPA) compared to a previously immunized population. In one embodiment, the immunogenic composition of the invention significantly increases OPA titers in humans against E. coli serotype O1a compared to a previously immunized population.

In one aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O2, wherein n is an integer greater than 30, preferably n is an integer from 31 to 100. In another aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O2, wherein n is 43±2. In another aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O2, wherein n is 47±2. In another aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O2, wherein n is 17±2. In another aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O2, wherein n is 18±2. In one embodiment, the saccharide further comprises an R1 moiety of the E. coli core saccharide. In another embodiment, the saccharide further comprises an R4 moiety of the E. coli core saccharide. In another embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM _197. In one embodiment, the conjugate is obtained by single-end conjugation. In one embodiment, the conjugate is obtained by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

In one embodiment, the immunogenic composition induces the formation of IgG antibodies in humans, wherein said antibodies are capable of binding to the polysaccharide of E. coli serotype O2 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 the serum before and after immunization with the immunogenic composition of the invention can be performed and the responses to serotype O2 can be compared to evaluate a potential increase in patients with a response to treatment. In one embodiment, the immunogenic composition induces the formation of IgG antibodies in humans, wherein said antibodies are capable of killing E. coli serotype O2, as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition induces the formation of functional antibodies in humans, wherein said antibodies are capable of killing E. coli serotype O2, as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition of the invention increases the proportion of patients responding to treatment of E. coli serotype O2 infection (i.e., individuals with a serum having a titer of at least 1:8, as determined by an in vitro opsonophagocytic killing assay) compared to a previously immunized population. In one embodiment, the immunogenic composition induces 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 significantly increases the proportion of patients responding to treatment of E. coli serotype O2 infection (i.e., individuals with a serum having a titer of at least 1:8, as determined by an in vitro OPA) compared to a previously immunized population. In one embodiment, the immunogenic composition of the invention significantly increases OPA titers in humans against E. coli serotype O2 compared to a previously immunized population.

In one aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O6, wherein n is an integer greater than 30, preferably n is an integer from 31 to 100. In one aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O6, wherein n is 42±2. In another aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O6, wherein n is 50±2. In another aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O6, wherein n is 17±2. In another aspect, the invention relates to a composition that includes a mutant FimH polypeptide and a conjugate that includes a saccharide covalently linked to a carrier protein, wherein the saccharide comprises the formula O6, wherein n is 18±2. In one embodiment, the saccharide further comprises an R1 moiety of the E. coli core saccharide. In one embodiment, the saccharide further comprises a KDO moiety. Preferably, the carrier protein is CRM _197. In one embodiment, the conjugate is obtained by single-end conjugation. In one embodiment, the conjugate is obtained by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent.

In one embodiment, the immunogenic composition induces the formation of IgG antibodies in humans, wherein said antibodies are capable of binding to the polysaccharide of E. coli serotype O6 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 the serum before and after immunization with the immunogenic composition of the invention can be performed and the responses to serotype O6 can be compared to evaluate a potential increase in the number of patients with a response to treatment. In one embodiment, the immunogenic composition induces the formation of IgG antibodies in humans, wherein said antibodies are capable of killing E. coli serotype O6, as determined by an in vitro opsonophagocytic assay. In one embodiment, the immunogenic composition induces the formation of functional antibodies in humans, wherein said antibodies are capable of killing E. coli serotype O6, as determined by an in vitro opsonophagocytic killing assay. In one embodiment, the immunogenic composition of the invention increases the proportion of patients responding to treatment of E. coli serotype O6 infection (i.e., individuals with a serum having a titer of at least 1:8, as determined by an in vitro opsonophagocytic killing assay) compared to a previously immunized population. In one embodiment, the immunogenic composition induces 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 significantly increases the proportion of patients responding to treatment against E. coli serotype O6 (i.e., individuals with a serum having a titer of at least 1:8, as determined by an in vitro OPA) compared to a previously immunized population. In one embodiment, the immunogenic composition of the invention significantly increases OPA titers in humans against E. coli serotype O6 compared to a previously immunized population.

In one aspect, a composition comprises a mutant FimH polypeptide and a conjugate comprising a saccharide covalently linked to a carrier protein, wherein the saccharide comprises a structure selected from any 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 O45 rel), 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 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formulas O107, Formulas O108, Formulas O109, Formulas O110, Formulas O111, Formulas O112, Formulas O113, Formulas O114, Formulas O115, Formulas O116, Formulas O117, Formulas O118, Formulas O119, Formulas O120, Formulas O121, Formulas O123, Formulas O124, Formulas O125, Formulas O126, Formulas O127, Formulas O128, Formulas O129, Formulas O130, Formulas O131, Formulas O132, Formulas O133, Formulas O134, Formulas O135, Formulas O136, Formulas O137, Formulas O138, Formulas O139, Formulas O140, Formulas O141, Formulas O142, Formulas O143, Formulas O144, Formulas O145, Formulas O146, Formulas O147, Formulas O148, Formulas O149, Formulas O150, Formulas O151, Formulas O152, Formulas O153, Formulas O154, Formulas O155, Formulas O156, Formulas O157, Formulas O158, Formulas O159, Formulas O160, Formulas O161, Formulas O162, Formulas O163, Formulas O164, Formulas O165, Formulas O166, Formulas O167, Formulas O168, Formulas O169, Formulas O170, Formulas O171, Formulas O172, Formulas O173, Formulas O174, Formulas O175, Formulas O176, Formulas O177, Formulas O178, Formulas O179, Formulas O180, Formulas O181, Formulas O182, Formulas O183, Formulas O184, Formulas O185, Formulas O186 and Formulas O187, where n is an integer from 1 to 100, preferably from 31 to 90. In one embodiment, the saccharide further comprises an R1 moiety of an E. coli core saccharide. In one embodiment, the saccharide further comprises an R2 moiety of an E. coli core saccharide. In one embodiment, the saccharide further comprises an R3 moiety of an E. coli core saccharide. In another embodiment, the saccharide further comprises an R4 moiety of an E. coli core saccharide. In another embodiment, the saccharide further comprises a K12 moiety of an E. coli core saccharide. In another embodiment, the saccharide further comprises a KD0 moiety. Preferably, the carrier protein is CRM _197. In one embodiment, the conjugate is obtained by single-end conjugation. In one embodiment, the conjugate is obtained by reductive amination chemistry, preferably in DMSO buffer. In one embodiment, the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer. Preferably, the composition further comprises a pharmaceutically acceptable diluent. In one embodiment, the composition further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 additional conjugates, up to a maximum of 30 additional conjugates, wherein each conjugate comprises a saccharide covalently bound to a carrier protein, and wherein the saccharide comprises a structure selected from any of the stated formulas.

A. Saccharides

1. Saccharides and O-polysaccharides

In one embodiment, the saccharide is produced by expressing (not necessarily overexpressing) various Wzz proteins (e.g., WzzB) to control the size of the saccharide.

As used herein, the term "saccharide" refers to a single sugar unit or monosaccharide unit, as well as combinations of two or more individual sugar units or monosaccharide units covalently linked to form disaccharides, oligosaccharides, and polysaccharides. The saccharide may be linear or branched.

In one embodiment, the saccharide is produced in a recombinant gram-negative bacterium. In one embodiment, the saccharide is produced in a recombinant E. coli cell. In one embodiment, the saccharide is produced in a recombinant salmonella cell. Examples of 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 serovar Typhimurium strain LT2, E. coli GAR2401, Salmonella enterica serotype Enteritidis CVD 1943, Salmonella enterica serotype Typhimurium CVD 1925, Salmonella enterica serotype Paratyphi A CVD 1902, and Shigella flexneri. In one embodiment, the bacterium is not E. coli GAR24 O1. This genetic approach to producing saccharides allows for the efficient production of O-polysaccharides and O-antigen molecules as vaccine components.

The term "wzz protein" as used herein refers to a chain length determining polypeptide such as, for example, wzzB, wzz, wzzSF, wzzST, fepE, wzzfepE, wzzl, and wzz2. GenBank accession numbers for exemplary wzz gene sequences are as follows: AFO11910 for E4991/76, AF011911 for F186, AF011912 for M70/1-1, AF011913 for 79/311, AF011914 for Bi750-41, AF01915 for C664-1992, AF01916 for C258-94, AF01917 for C722-89, and AF01919 for EDL933. The GenBank accession numbers for the G7 and Bi316-41 wzz gene sequences are U3930 and U3930, respectively. Additional GenBank accession numbers for representative wzz gene sequences are: NP 459581 for Salmonella enterica subtype Enterica, serovar Typhimurium, strain LT2 FepE; AIG66859 for E. coli O157:H7 strain EDL933 FepE; NP_46104 for Salmonella enterica, subtype Enterica, serovar Typhimurium, strain LT2 WzzB; NP_416531 for E. coli K-12 substrain MG1655 WzzB; NP_415119 for E. coli K-12 substrain MG1655 FepE. In preferred aspects, the wzz family protein is any of wzzB, wzz, wzzSF, wzzST, fepE, wzzfepE, wzzl and wzz2, most preferably wzzB, more preferably fepE.

Exemplary wzzB sequences include those shown in SEQ ID Nos: 112-116. Exemplary FepE sequences include those shown in SEQ ID Nos: 117-121.

In some aspects, a modified saccharide (modified relative to a corresponding wild-type saccharide) can be produced by expressing (not necessarily overexpressing) a wzz family protein (e.g., fepE) from a gram-negative bacterium and/or by knocking down (i.e., repressing, deleting, removing) a second wzz gene (e.g., wzzB) in a gram-negative bacterium to produce high molecular weight saccharides, such as intermediate or long chain O-antigen-containing lipopolysaccharides that have an increased number of repeat units relative to a corresponding wild-type O-polysaccharide. For example, modified saccharides can be produced by expressing (not necessarily overexpressing) wzz2 and knocking down wzz1. Or, alternatively, modified saccharides can be produced by expressing (not necessarily overexpressing) wzz/fepE and knocking down wzzB. In another embodiment, the modified saccharides can be produced by expressing (not necessarily overexpressing) wzzB but knocking out wzz/fepE. In another embodiment, the modified saccharides can be produced by expressing fepE. Preferably, the wzz family protein is obtained from a strain heterologous to the host cell. Methods for determining the length of saccharides are known in the art. Such methods include, but are not limited to, nuclear magnetic resonance, mass spectroscopy, and size exclusion chromatography. Methods for producing the high molecular weight saccharides disclosed herein, such as lipopolysaccharides containing intermediate or long chain O-antigens, are described in PCT International Publication WO 2020/039359 and the corresponding US Publication US 2020/0061177, each of which is incorporated herein by reference in its entirety.

In some embodiments, the saccharide is produced by expressing a wzz family protein having an amino acid sequence that is at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the sequence of any 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 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 a sequence that is at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116. In some embodiments, the saccharide is produced by expressing a protein whose amino acid sequence is at least 30%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the fepE protein sequence.

In one aspect, the invention relates to saccharides obtained by expressing a wzz family protein, preferably fepE, in a gram-negative bacterium to form high molecular weight saccharides comprising intermediate or long O-antigen chains that are increased by at least 1, 2, 3, 4 or 5 repeating units compared to the corresponding wild-type O-polysaccharide. In one aspect, the invention relates to saccharides produced by a gram-negative bacterium in a culture that expresses (not necessarily overexpresses) a wzz family protein (e.g., wzzB) from the gram-negative bacterium to form high molecular weight saccharides comprising intermediate or long O-antigen chains that are increased by at least 1, 2, 3, 4 or 5 repeating units compared to the corresponding wild-type O-antigen. Descriptions of O-polysaccharides and O-antigens for additional illustrative saccharides having an increased number of repeat units compared to the corresponding wild-type saccharides are provided below. The desired chain length is the chain length that provides improved or maximal immunogenicity in the context of a given vaccine design.

In another embodiment, the saccharide comprises any formula selected from Table A, wherein the number of repeating units n in the saccharide is greater than the number of repeating units in the corresponding wild-type O-polysaccharide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeating units. Preferably, the saccharide is increased by 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 the length of saccharides are known in the art. Such methods include nuclear magnetic resonance, mass spectroscopy and size exclusion chromatography.

In a preferred embodiment, the invention relates to a saccharide produced in a recombinant E. coli host cell, wherein the endogenous O-antigen length regulator gene wzz (e.g. wzzB) is removed and replaced with a (second) wzz gene from a Gram-negative bacterium heterologous to the recombinant E. coli host cell (e.g. Salmonella fepE), to form high molecular weight saccharides, such as lipopolysaccharides, containing intermediate or long O-antigen chains. In some embodiments, the recombinant E. coli host cell comprises a wzz gene from Salmonella, preferably from Salmonella enterica.

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

In one aspect, the recombinant host cell is cultured in a medium containing a carbon source. Carbon sources for culturing E. coli are known in the art. Examples of carbon sources include sugar alcohols, polyols, aldol sugars, or keto sugars, including 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 includes glucose. In some embodiments, the medium includes a polyol or aldol sugar as a carbon source, such as mannitol, inositol, sorbose, glycerol, sorbitol, lactose, and arabinose. All carbon sources can be added to the medium before culturing, or they can be added stepwise or continuously during culturing.

A typical culture medium for the _recombinant host cell comprises an element 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 -7H ₂ O, Na ₂ MoO ₄ -2H ₂ O, H ₃ BO ₃ , CoCl ₂ -6H ₂ O, CuCl ₂ -2H ₂ O, MnCl ₂ -4H ₂ O, ZnCl ₂ and CaCl ₂ -2H ₂ O.

The medium used in the context of the present description may be solid or liquid, synthetic (i.e. artificial) or natural, and may include sufficient nutrients for culturing the recombinant host cell. Preferably, the medium is a liquid medium.

In some embodiments, the medium may further comprise suitable inorganic salts. In some embodiments, the medium may further comprise trace nutrients. 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, trace nutrients, growth factors, and an additional carbon source. Inorganic salts, trace nutrients, growth factors, and additional carbon sources suitable for culturing E. coli are known in the art.

In some embodiments, if necessary, the medium may include additional components such as peptone, N-Z amine, soybean enzymatic hydrosylate, additional yeast extract, malt extract, additional carbon sources, and various vitamins. In some embodiments, the medium does not include additional components such as peptone, N-Z amine, soybean enzymatic hydrosylate, additional yeast extract, malt extract, additional carbon sources, and various vitamins.

Illustrative examples of suitable additional carbon sources include, but are not limited to, other carbohydrates such as glucose, fructose, mannitol, starch or starch hydrolysate, cellulose hydrolysate, 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.

In some embodiments, the medium further comprises a nitrogen source. Nitrogen sources suitable for culturing E. coli are known in the art. Illustrative examples of suitable nitrogen sources include, but are not limited to, ammonia, including gaseous ammonia 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 salts and other nitrogen-containing materials, including amino acids in pure or crude form, meat extract, peptone, fish meal, fish hydrolysate, corn extract, casein hydrolysate, soybean meal hydrolysate, yeast extract, dry yeast, ethanol-yeast distillate, soybean flour, cottonseed meal and the like.

In some embodiments, the medium includes an inorganic salt. Illustrative 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, as well as phosphoric acid.

In some embodiments, the medium includes appropriate growth factors. Illustrative examples of suitable micronutrients, growth factors, and the like 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 B12, other vitamins, amino acids such as cysteine and hydroxyproline, bases such as adenine, uracil, guanine, thymine, and cytosine, sodium thiosulfate, n- or p-aminobenzoic acid, niacinamide, nitriloacetate, and the like, either as pure or partially purified chemicals or as natural materials. Amounts can be determined empirically by one skilled in the art according to methods and techniques known in the art.

In another embodiment, the modified saccharide (compared to the corresponding wild-type saccharide) described herein is produced synthetically, such as in vitro. Synthetic production or synthesis of saccharides may eliminate expensive and time-consuming manufacturing processes. In one embodiment, the saccharide is synthesized, for example, according to a sequential glycosylation strategy or a combination of sequential glycosylation and a [3+2] block synthesis strategy from suitably protected monosaccharide intermediates. For example, thioglycosides and glycosyl tri-chloroacetimidate derivatives can be used as glycosyl donors in glycosylation. In one embodiment, the saccharide that is synthetically synthesized in vitro has a structure identical to a saccharide produced by recombinant methods, such as manipulation of a wzz family protein described above.

The saccharide obtained (by recombinant or synthetic means) includes a structure obtained from any serotype of E. coli, including, for example, any of the following serotypes of E. coli: O1 (e.g., O1A, O1B, and O1C), O2, O3, O4 (e.g., O4:K52 and O4:K6), O5 (e.g., O5ab and O5ac (strain 180/C3), O6 (e.g., O6:K2; K13; K15 and O6:K54), O7, O8, O9, O10, O11, O12, O13, O14, O15, O16, O17, O18 (e.g., O18A, O18ac, O18A1, 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 O45 rel), O46, O48, O49, O50, O51, O52, O53, O54, O55, O56, O57, O58, O59, O60, O61, O62, 62D1, O63, O64, O65, O66, O68, O69, O70, O71, O73 (e.g. O73 (strain 73-1)), O74, O75, O76, O77, O78, O79, O80, O81, O82, O83, O84, O85, O86, O87, O88, O89, O90, O91, O92, O93, O95, O96, O97, O98, O99, O100, O101, O102, O103, O104, O105, O106, O107, O108, O109, O110, O111, O112, O113, 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, O149, 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.

The individual polysaccharides are typically purified (enriched relative to the amount of polysaccharide-protein conjugate) by methods known in the art, such as, for example, dialysis, concentration steps, diafiltration steps, tangential flow filtration, precipitation, elution, centrifugation, precipitation, ultrafiltration, depth filtration and/or column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE and hydrophobic interaction chromatography). The polysaccharides are preferably purified by a method that includes tangential flow filtration.

The purified polysaccharides may be activated (e.g., chemically activated) to render them reactive (e.g., either directly with a carrier protein or via a linker such as an eTEC spacer) and then incorporated into the glycoconjugates of the invention, as further disclosed herein.

In one preferred embodiment, the saccharide of the invention is obtained from an E. coli serotype wherein the serotype is O25a. In another preferred embodiment, the serotype is O25b. In another preferred embodiment, the serotype is O1A. In another preferred embodiment, the serotype is O2. In another preferred embodiment, the serotype is O6. In another preferred embodiment, the serotype is O17. In another preferred embodiment, the serotype is O15. In another preferred embodiment, the serotype is O18A. In another preferred embodiment, the serotype is O75. In another preferred embodiment, the serotype is O4. In another preferred embodiment, the serotype is O16. In another preferred embodiment, the serotype is O13. In another preferred embodiment, the serotype is O7. In another preferred embodiment, the serotype is O8. In another preferred embodiment, the serotype is O9.

As used herein, reference to any of the serotypes listed above refers to a serotype that includes a repeating unit structure (0-units, as described below) known in the art and is unique to the corresponding serotype. For example, the term serotype "O25a" (also known in the art as serotype "O25") refers to a serotype that encompasses the formula O25 shown in Table A. As another example, the term serotype "O25b" refers to a serotype that encompasses the formula O25b shown in Table A.

As used herein, serotypes are referred to generically unless otherwise specified, for example, the term “O18 formulas” refers generically to formula O18A, formula O18ac, formula 18A1, formula O18B, and formula O18B1.

As used herein, the term "O1" generally refers to formula varieties whose names include the general term "O1" according to Table A, such as any of: formula O1A, formula O1A1, formula O1B, and formula O1C, each of which is shown in Table A. Accordingly, "serotype O1" generally refers to a serotype that encompasses any of: formula O1A, formula O1A1, formula O1B, and formula O1C.

As used herein, the term "O6" generally refers to formula varieties whose names include the general term "O6" in accordance with Table A, such as any of the formulas: O6:K2; K13; K15; and O6:K54, each of which is shown in Table A. Accordingly, "serotype O6" generally refers to a serotype that encompasses any of the formulas: O6:K2; K13; K15; and O6:K54.

Other examples of terms that generally refer to variations of a formula whose names include the general term in accordance with Table A include: “O4”, “O5”, “O18”, and “O45”.

In the context of the present description, 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.

In the context of the present description, reference to the O-antigen of the above-mentioned serotype refers to a saccharide that encompasses the formula indicating the corresponding serotype name. For example, the term "O-antigen O25 B" refers to a saccharide that encompasses the formula O25 B shown in Table A.

As another example, the term “O-antigen O1” generally refers to a saccharide that encompasses a formula including the term “O1,” such as formula O1A, formula O1A1, formula O1B, and formula O1C, each of which is shown in Table A.

As another example, the term "O-antigen O6" generally refers to a saccharide that encompasses a formula including the term "O6", such as formula O6:K2; formula O6:K13; formula O6:K15; and formula O6:K54, each of which is shown in Table A.

In the context of the present description, the term "O-polysaccharide" refers to any structure that includes an O-antigen, provided that the structure does not include a whole cell or lipid A. For example, in one embodiment, the O-polysaccharide includes a lipopolysaccharide wherein lipid A is not associated. The step of removing lipid A is known in the art and includes, for example, heat treatment with the addition of an acid. An exemplary process includes treatment with 1% acetic acid at 100 ° C for 90 minutes. This process is combined with a process for isolating lipid A as removed. An exemplary method for isolating lipid A includes ultracentrifugation.

In one embodiment, the O-polysaccharide refers to a structure consisting of an O-antigen, in which case the O-polysaccharide is synonymous with the term O-antigen. In one preferred embodiment, the O-polysaccharide refers to a structure that includes repeating units of the O-antigen without a core saccharide. Accordingly, in one embodiment, the O-polysaccharide does not include an E. coli R1 core. In another embodiment, the O-polysaccharide does not include an E. coli R2 core. In another embodiment, the O-polysaccharide does not include an E. coli R3 core. In another embodiment, the O-polysaccharide does not include an E. coli R4 core. In another embodiment, the O-polysaccharide does not include an E. coli K12 core. In another preferred embodiment, the O-polysaccharide refers to a structure that includes an O-antigen and a core saccharide. In another embodiment, the O-polysaccharide refers to a structure that includes an O-antigen, a core saccharide, and a KD0 moiety.

Methods for purifying the O-polysaccharide, which includes the core oligosaccharide, from LPS are known in the art. For example, after purifying the LPS, the purified LPS can be hydrolyzed by heating in 1% (v/v) acetic acid for 90 minutes at 100 degrees Celsius, followed by

ultracentrifugation at 142,000 g for 5 hours at 4 degrees Celsius. The supernatant containing the O-polysaccharide is dried by lyophilization and stored at 4 degrees Celsius. In some embodiments, removal of genes required for capsule synthesis is described to allow for easy purification of the O-polysaccharide.

The 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 using hydrazine as an agent to produce the O-polysaccharide. The production of LPS can be accomplished by methods known in the art.

In some embodiments, conjugate vaccines utilize O-polysaccharides purified from modified or attenuated wild-type Gram-negative bacterial strains that express (optionally overexpress) the Wzz protein (e.g., wzzB). In preferred embodiments, the O-polysaccharide chain is purified from a Gram-negative bacterial strain that expresses (optionally overexpresses) the wzz protein for use as a vaccine antigen, either as a conjugate or as a complex vaccine.

In one embodiment, the O-polysaccharide has a molecular weight that is increased by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 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 times or more compared to the corresponding wild-type O-polysaccharide. In a preferred embodiment, the O-polysaccharide has a molecular weight increased by at least 1-fold, but not more than 5-fold compared to the corresponding wild-type O-polysaccharide. In another embodiment, the O-polysaccharide has a molecular weight increased by at least 2-fold but not more than 4-fold compared to the corresponding wild-type O-polysaccharide. The increase in the molecular weight of the O-polysaccharide compared to the corresponding wild-type O-polysaccharide is preferably associated with an increase in the number of repeating units of the O-antigen. In one embodiment, the increase in the molecular weight of the O-polysaccharide is due to a protein of the wzz family.

In one embodiment, the O-polysaccharide has a molecular weight that is increased by 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 kDa or more compared to the corresponding wild-type O-polysaccharide. In one embodiment, the O-polysaccharide of the invention has a molecular weight increased by at least 1 kDa but not more than 200 kDa compared to the corresponding wild-type O-polysaccharide. In one embodiment, the molecular weight is increased by at least 5 but not more than 200 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 200 kDa. In one embodiment, the molecular weight is increased by at least 12 but not more than 200 kDa. In one embodiment, the molecular weight is increased by at least 15 but not more than 200 kDa. In one embodiment, the molecular weight is increased by at least 18 but not more than 200 kDa. In one embodiment, the molecular weight is increased by at least 20 but not more than 200 kDa. In one embodiment, the molecular weight is increased by at least 21 but not more than 200 kDa. In one embodiment, the molecular weight is increased by at least 22 but not more than 200 kDa. In one embodiment, the molecular weight is increased by at least 30 but not more than 200 kDa. In one embodiment, the molecular weight is increased by at least 1 but not more than 100 kDa. In one embodiment, the molecular weight is increased by at least 5 but not more than 100 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 100 kDa. In one embodiment, the molecular weight is increased by at least 12 but not more than 100 kDa. In one embodiment, the molecular weight is increased by at least 15 but not more than 100 kDa. In one embodiment, the molecular weight is increased by at least 20 but not more than 100 kDa. In one embodiment, the molecular weight is increased by at least 1 but not more than 75 kDa. In one embodiment, the molecular weight is increased by at least 5 but not more than 75 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 75 kDa. In one embodiment, the molecular weight is increased by at least 12 but not more than 75 kDa. In one embodiment, the molecular weight is increased by at least 15 but not more than 75 kDa. In one embodiment, the molecular weight is increased by at least 18 but not more than 75 kDa. In one embodiment, the molecular weight is increased by at least 20 but not more than 75 kDa. In one embodiment, the molecular weight is increased by at least 30 but not more than 75 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 90 kDa. In one embodiment, the molecular weight is increased by at least 12 but not more than 85 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 75 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 70 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 60 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 50 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 49 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 48 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 47 kDa. In one embodiment, the molecular weight is increased by at least 10 but not more than 46 kDa. In one embodiment, the molecular weight is increased by at least 20 but not more than 45 kDa. In one embodiment, the molecular weight is increased by at least 20 but not more than 44 kDa. In one embodiment, the molecular weight is increased by at least 20 but not more than 43 kDa. In one embodiment, the molecular weight is increased by at least 20 but not more than 42 kDa. In one embodiment, the molecular weight is increased by at least 20 but not more than 41 kDa. Such an increase in the molecular weight of the O-polysaccharide compared to the corresponding wild-type O-polysaccharide is preferably associated with an increase in the number of repeating units of the O-antigen. In one embodiment, the increase in the molecular weight of the O-polysaccharide is due to a protein of the wzz family.

In another embodiment, the O-polysaccharide comprises any formula selected from Table A, wherein the number of repeating units n in the O-polysaccharide is greater than the number of repeating units in the corresponding wild-type O-polysaccharide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeating units. Preferably, the saccharide is increased by 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 repeat units compared to the corresponding wild-type O-polysaccharide.

2. O-antigen

The O-antigen is a component of the lipopolysaccharide (LPS) of the outer membrane of gram-negative bacteria. The O-antigen is located on the cell surface and is a variable component of the cell. The variability of the O-antigen is the basis for serotyping gram-negative bacteria. The current serotyping scheme for E. coli includes O-polysaccharides 1 through 181.

The O-antigen comprises oligosaccharide repeating units (O-units), the wild-type structure of which typically contains from two to eight broad-spectrum sugar residues. O-units of typical E. coli O-antigens are described in Table A and in International PCT Publication No. WO 2021/084429, published May 6, 201, which is hereby incorporated by reference in its entirety. In some embodiments, the present invention includes a composition comprising at least one mutant FimH polypeptide and at least one of the O-antigens described in Table A of International PCT Publication No. WO 2021/084429, published May 6, 2021, which is hereby incorporated by reference in its entirety.

In one embodiment, the saccharide of the invention may be a single oligosaccharide unit. In one embodiment, the saccharide of the invention is a single repeating oligosaccharide unit of the corresponding serotype. In such embodiments, the saccharide may comprise a structure selected from 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 another embodiment, the saccharide may comprise a structure selected from formula O1a, formula O2, formula O6, and formula O25b.

In one embodiment, the saccharide of the invention may be an oligosaccharide. Oligosaccharides have a small number of repeating units (typically 5-15 repeating units) and are typically produced synthetically or by hydrolysis of polysaccharides. In such embodiments, the saccharide may comprise a structure selected from 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 another embodiment, the saccharide may comprise a structure selected from formula O1a, formula O2, formula O6, and formula O25b.

Preferably, all saccharides of the present invention, and in the immunogenic compositions of the present invention, are polysaccharides. High molecular weight

polysaccharides can induce specific antibody immune responses based on epitopes present on the antigen surface. Use in the conjugates, compositions and methods of the present invention preferably involves the isolation and purification of high molecular weight polysaccharides.

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) is dependent on the wzz chain length regulator, an inner membrane protein. Different wzz proteins provide different ranges of modal length (from 4 to >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 provide two different modal lengths of Oag chains, one longer and one shorter. Expression (not necessarily overexpression) of wzz family proteins (e.g., wzzB) in Gram-negative bacteria allows manipulation of O-antigen length to shift or bias bacterial production of O-antigens having a certain length range and to enhance the production of high yield, high molecular weight lipopolysaccharides. In one embodiment, a "short" modal length as used herein refers to a small number of repeating O-units, such as 1-20. In one embodiment, a "long" modal length as used herein refers to a number of repeating O-units greater than 20, with a maximum of 40. In one embodiment, a "very long" modal length as used herein refers to more than 40 repeating O-units.

In one embodiment, the resulting saccharide is increased by 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.

In another embodiment, the saccharide of the invention is increased by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeating units compared to the corresponding wild-type O-polysaccharide. Preferably, the saccharide is increased by 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 repeat units compared to the corresponding wild-type O-polysaccharide. See, for example, Table 21. Methods for determining the length of saccharides are known in the art. Such methods include nuclear magnetic resonance, mass spectroscopy and size exclusion chromatography, as described in Example 13.

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 (excluding the molecular weight of the core saccharide or KDO moiety) by the molecular weight of the repeating unit (i.e., the molecular weight of the structure in the corresponding formula shown, for example, in Table 1, which theoretically can be calculated as the sum of the molecular weights of each monosaccharide in the formula). The molecular weight of each monosaccharide in the formula is known in the art. The molecular weight of the repeating unit of formula O25b, for example, is about 862 Da. The molecular weight of the repeating unit of formula O1a, for example, is about 845 Da. The molecular weight of the repeating unit of formula O2, for example, is about 829 Da. The molecular weight of the repeating unit of formula O6, for example, is about 893 Da. In determining the number of repeating units in the conjugate, the molecular weight of the carrier protein and the protein:polysaccharide ratio are taken into account in the calculation. As defined herein, "n" refers to the number of repeating units (shown in parentheses in Table 1) in a polysaccharide molecule. As is known in the art, in biological macromolecules, repeating structures may be interspersed with regions of imperfect repeats, such as, for example, missing branches. In addition, 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 a case, n may represent the average or median n value for molecules in a population.

In one embodiment, the O-polysaccharide is increased by at least one repeating unit of the O-antigen compared to the corresponding wild-type O-polysaccharide. The repeating units of the O-antigens are shown in Table 1. In one embodiment, the O-polysaccharide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeating units. Preferably, the saccharide has a total of at least 3 to no more than 80 repeating units. In another embodiment, the O-polysaccharide is increased by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more repeating units compared to the corresponding wild-type O-polysaccharide.

In one embodiment, the saccharide comprises an O-antigen, wherein n in any of the O-antigen formulas (such as, for example, the formulas shown in Table 1 (see also Figs. 9A-9C and Figs. 10A-10B)) is an integer of 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 no more than 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 and any maximum value can be combined to define a range. Exemplary ranges include, for example, from at least 1 to at most 1000; from at least 10 to at most 500; and from at least 20 to at most 80, preferably at most 90. In one preferred embodiment, n is from at least 31 to at most 90. In a preferred embodiment, n is from 40 to 90, more preferably from 60 to 85.

In one embodiment, the saccharide comprises an O-antigen, wherein n in any of the O-antigen formulas is from at least 1 to at most 200. In one embodiment, n in any of the O-antigen formulas is from at least 5 to at most 200. In one embodiment, n in any of the O-antigen formulas is from at least 10 to at most 200. In one embodiment, n in any of the O-antigen formulas is from at least 25 to at most 200. In one embodiment, n in any of the O-antigen formulas is from at least 50 to at most 200. In one embodiment, n in any of the O-antigen formulas is from at least 75 to at most 200. In one embodiment, n in any of the O-antigen formulas is from at least 100 to at most 200. In one embodiment, n in any of the O-antigen formulas is from at least 125 to at most 200. In one embodiment, n in any of the O-antigen formulas is at least 150 to at most 200. In one embodiment, n in any of the O-antigen formulas is at least 175 to at most 200. In one embodiment, n in any of the O-antigen formulas is at least 1 to at most 100. In one embodiment, n in any of the O-antigen formulas is at least 5 to at most 100. In one embodiment, n in any of the O-antigen formulas is at least 10 to at most 100. In one embodiment, n in any of the O-antigen formulas is at least 25 to at most 100. In one embodiment, n in any of the O-antigen formulas is at least 50 to at most 100. In one embodiment, n in any of the O-antigen formulas is at least of the O antigen is from at least 75 to at most 100. In one embodiment, n in any of the O antigen formulas is from at least 1 to at most 75. In one embodiment, n in any of the O antigen formulas is from at least 5 to at most 75. In one embodiment, n in any of the O antigen formulas is from at least 10 to at most 75. In one embodiment, n in any of the O antigen formulas is from at least 20 to at most 75. In one embodiment, n in any of the O antigen formulas is from at least 25 to at most 75. In one embodiment, n in any of the O antigen formulas is from at least 30 to at most 75. In one embodiment, n in any of the O antigen formulas is from at least 40 to at most 75. In one embodiment, n in any of the O antigen formulas is from at least 50 to at most 75. In one embodiment, n in any of the O-antigen formulas is at least 30 to at most 90. In one embodiment, n in any of the O-antigen formulas is at least 35 to at most 85. In one embodiment, n in any of the O-antigen formulas is at least 35 to at most 75. In one embodiment, n in any of the O-antigen formulas is at least 35 to at most 70. In one embodiment, n in any of the O-antigen formulas is at least 35 to at most 60. In one embodiment, n in any of the O-antigen formulas is at least 35 to at most 50. In one embodiment, n in any of the O-antigen formulas is at least 35 to at most 50. In one embodiment, n in any of the O-antigen formulas is at least 35 to at most 49. In one embodiment, n in any of the O-antigen formulas is at least 35 to at most 48. In one embodiment, n in any of the O-antigen formulas is at least 35 to at most 47. In one embodiment, n in any of the O-antigen formulas is at least 35 to at most 46. In one embodiment, n in any of the O-antigen formulas is at least 36 to at most 45. In one embodiment, n in any of the O-antigen formulas is at least 37 to at most 44. In one embodiment, n in any of the O-antigen formulas is at least 38 to at most 43. In one embodiment, n in any of the O-antigen formulas is at least 39 to at most 42. In one embodiment, n in any of the O-antigen formulas is at least 39 to at most 41. For example, in one embodiment, n in the saccharide is 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90, most preferably 40. In another embodiment, n is from at least 35 to at most 60. For example, in one embodiment, n is any number 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 from at least 55 to 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.

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

In some embodiments, the purified polysaccharide has a molecular weight between 5 kDa and 400 kDa before conjugation. In other such embodiments, the saccharide has a molecular weight of 10 kDa to 400 kDa; from 5 kDa to 400 kDa; from 5 kDa to 300 kDa; from 5 kDa to 200 kDa; from 5 kDa to 150 kDa; from 10 kDa to 100 kDa; from 10 kDa to 75 kDa; from 10 kDa to 60 kDa; from 10 kDa to 40 kDa; from 10 kDa to 100 kDa; 10 kDa and 200 kDa; from 15 kDa to 150 kDa; from 12 kDa to 120 kDa; from 12 kDa to 75 kDa; from 12 kDa to 50 kDa; from 12 to 60 kDa; from 35 kDa to 75 kDa; from 40 kDa to 60 kDa; from 35 kDa to 60 kDa; from 20 kDa to 60 kDa; from 12 kDa to 20 kDa; or from 20 kDa to 50 kDa. In further embodiments, the polysaccharide has a molecular weight of 7 kDa to 15 kDa; 8 kDa to 16 kDa; 9 kDa to 25 kDa; 10 kDa to 100; 10 kDa to 60 kDa; 10 kDa to 70 kDa; from 10 kDa to 160 kDa; from 15 kDa to 600 kDa; from 20 kDa to 1000 kDa; 20 kDa to 600 kDa; 20 kDa to 400 kDa; 30 kDa to 1000 kDa; 30 kDa to 60 kDa; 30 kDa to 50 kDa; or 5 kDa to 60 kDa. Any integer in any of the above ranges is considered as an embodiment of the invention.

In the context of the present description, the term "molecular weight" of a polysaccharide or a carrier protein-polysaccharide conjugate refers to the molecular weight calculated by size exclusion chromatography (SEC) in combination with a multi-angle laser light scattering (MALLS) detector.

During normal purification procedures, the polysaccharide may be slightly reduced in size. In addition, as disclosed herein, the polysaccharide may be size sorted prior to conjugation. Mechanical or chemical size sorting may be used. Chemical hydrolysis may be performed using acetic acid. Mechanical size sorting may be performed using high pressure shear homogenization. The molecular weight ranges mentioned above apply to purified polysaccharides prior to conjugation (e.g., prior to activation).

3. Core oligosaccharide

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 comprises the linkage between the O-antigen and lipid A in wild-type E. coli. This linkage involves a ketosidic bond between the hemiketal moiety of the innermost 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue and the hydroxyl group of the GlcNAc residue of lipid A. The core oligosaccharide region shows a high degree of similarity among wild-type E. coli strains. It typically comprises a limited number of sugars. The core oligosaccharide comprises an inner core region and an outer core region.

More specifically, the inner core consists primarily of L-glycero-D-manno-heptose (heptose) and KDO residues. The inner core is highly conserved. The KDO residue includes the following KDO formula:

The outer region of the core oligosaccharide shows more variation than the inner core, and this region is distinguished by five E. coli chemotypes: 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. HepII is the last residue of the inner core oligosaccharide. Although all outer core oligosaccharides share a common structural theme with a (hexose) ₃ carbohydrate backbone and two side chain residues, the order of the hexoses in the backbone and the nature, position, and linkage of the side chain residues can vary. The structures of outer core oligosaccharides R1 and R4 are very similar and differ only in one β-linked residue.

The classification of wild-type E. coli core oligosaccharides in this area is based on the structure of the distal oligosaccharide in five different chemotypes: E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12.

In a preferred embodiment, the compositions provided herein comprise glycoconjugates in which the O-polysaccharide comprises a core oligosaccharide linked to an O-antigen. In one embodiment, the composition induces an immune response against at least any of the E. coli core chemotypes E. coli R1, E. coli R2, E. coli R3, E. coli RA, and E. coli K12. In another embodiment, the composition induces an immune response against at least two E. coli core chemotypes. In another embodiment, the composition induces an immune response against at least three E. coli core chemotypes. In another embodiment, the composition induces an immune response against at least four E. coli core chemotypes. In another embodiment, the composition induces an immune response against all five E. coli core chemotypes.

In another preferred embodiment, the compositions provided herein comprise glycoconjugates in which the O-polysaccharide does not comprise a core oligosaccharide linked to an O-antigen. In one embodiment, such a composition induces an immune response against at least one of the E. coli core chemotypes E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, despite the glycoconjugate comprising an O-polysaccharide that does not comprise a core oligosaccharide.

E. coli serotypes can be characterized according to one of five chemotypes. Table B lists exemplary serotypes characterized according to chemotype. Serotypes in bold are serotypes that are most often associated with the indicated core chemotype. Accordingly, in a preferred embodiment, the composition induces an immune response against at least any of the E. coli core chemotypes E. coli R1, E. coli R2, E. coli R3, E. coli R4, and E. coli K12, which includes an immune response against any of the corresponding E. coli serotypes.

In some embodiments, the composition includes a saccharide that includes a structure derived from a serotype having an R1 chemotype, such as one selected from a saccharide 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, wherein n is from 1 to 100. In some embodiments, the saccharide in said composition further includes an E. coli R1 core fragment.

In some embodiments, the composition comprises a saccharide that comprises a structure derived from a serotype having an R1 chemotype, such as selected from a saccharide having formula O25a, formula O6, formula O2, formula O1, formula O75, formula O4, formula O16, formula O18, formula O13, formula O20, formula O21, formula O91 and formula O163, wherein n is from 1 to 100, preferably from 31 to 100, preferably from 31 to 90, more preferably from 35 to 90, most preferably from 35 to 65. In some embodiments, the saccharide in said composition further comprises a saccharide comprising an E. coli R1 core fragment.

In some embodiments, the composition comprises a saccharide that comprises a structure derived from a serotype having an R2 chemotype, such as one selected from a saccharide having formula O21, formula O44, formula O11, formula O89, formula O162, and formula O9, wherein n is from 1 to 100, preferably from 31 to 100, preferably from 31 to 90, more preferably from 35 to 90, most preferably from 35 to 65. In some embodiments, the saccharide in said composition further comprises an E. coli R2 core fragment.

In some embodiments, the composition comprises a saccharide that comprises a structure derived from a serotype having an R3 chemotype, such as one selected from a saccharide having formula O25b, formula O15, formula O153, formula O21, formula O17, formula O11, formula O159, formula O22, formula O86, and formula O93, wherein n is from 1 to 100, preferably from 31 to 100, preferably from 31 to 90, more preferably from 35 to 90, most preferably from 35 to 65. In some embodiments, the saccharide in said composition further comprises an E. coli R3 core fragment.

In some embodiments, the composition comprises a saccharide that comprises a structure derived from a serotype having an R4 chemotype, such as one selected from a saccharide having formula O2, formula O1, formula O86, formula O7, formula O102, formula O160, and formula O166, wherein n is from 1 to 100, preferably from 31 to 100, preferably from 31 to 90, more preferably from 35 to 90, most preferably from 35 to 65. In some embodiments, the saccharide in said composition further comprises an E. coli R4 core fragment.

In some embodiments, the composition comprises a saccharide that comprises a structure derived from a serotype having the K-12 chemotype (e.g., selected from a saccharide having the formula O25b and a saccharide having the formula O16), wherein n is from 1 to 1000, preferably from 31 to 100, preferably from 31 to 90, more preferably from 35 to 90, most preferably from 35 to 65. In some embodiments, the saccharide in said composition further comprises a K-12 core fragment of E. coli.

In some embodiments, the saccharide comprises a core saccharide. Accordingly, in one embodiment, the O-polysaccharide further comprises an E. coli R1 core fragment. In another embodiment, the O-polysaccharide further comprises an E. coli R2 core fragment. In another embodiment, the O-polysaccharide further comprises an E. coli R3 core fragment. In another embodiment, the O-polysaccharide further comprises an E. coli R4 core fragment. In another embodiment, the O-polysaccharide further comprises an E. coli K12 core fragment.

In some embodiments, the saccharide does not include a core saccharide. Accordingly, in one embodiment, the O-polysaccharide does not include an E. coli R1 core fragment. In another embodiment, the O-polysaccharide does not include an E. coli R2 core fragment. In another embodiment, the O-polysaccharide does not include an E. coli R3 core fragment. In another embodiment, the O-polysaccharide does not include an E. coli R4 core fragment. In another embodiment, the O-polysaccharide does not include an E. coli K12 core fragment.

4. Conjugated O-antigens

Chemical coupling of O-antigens or preferably O-polysaccharides to protein carriers can improve the immunogenicity of O-antigens or O-polysaccharides. However, the variability of polymer size is a practical manufacturing problem. In commercial use, the saccharide size can affect compatibility with different synthetic conjugation strategies, product homogeneity, and conjugate immunogenicity. Control of the expression of the Wzz family protein chain length regulator by manipulation during O-antigen synthesis allows the desired O-antigen chain length to be obtained in various strains of Gram-negative bacteria, including E. coli.

In one embodiment, the purified saccharides are chemically activated to produce activated saccharides that are reactive with a carrier protein. After activation, each saccharide is individually conjugated to the carrier protein to form a conjugate, namely a glycoconjugate. As used herein, the term "glycoconjugate" refers to a saccharide covalently linked to a carrier protein. In one embodiment, the saccharide is linked directly to the carrier protein. In another embodiment, the saccharide is linked to the protein via a spacer/linker.

Conjugates can be prepared by schemes that allow the carrier to be linked to the O-antigen at one or more sites along the O-antigen, or by schemes that allow at least one core oligosaccharide residue to be activated.

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

If the carrier protein is the same for 2 or more saccharides in the composition, the saccharides may be conjugated to the same carrier protein molecule (e.g., the carrier molecules have 2 or more different saccharides conjugated to them).

In a preferred embodiment, each of the saccharides is individually conjugated to a different carrier protein molecule (each carrier protein molecule has only one type of saccharide conjugated to it). In this embodiment, the saccharides are said to be individually conjugated to the carrier protein.

Chemical activation of the saccharides and subsequent conjugation to a carrier protein can be achieved by the activation and conjugation methods disclosed herein. After conjugation of the polysaccharide to the carrier protein, the glycoconjugates are purified (enriched relative to the amount of polysaccharide-protein conjugate) by various methods. These methods include concentration/diafiltration, precipitation/elution, column chromatography, and depth filtration operations. After purification of the individual glycoconjugates, they are compounded to produce the immunogenic composition of the present invention.

a. Activation. The present invention further relates to activated polysaccharides prepared according to any of the embodiments of the present invention described herein, wherein the polysaccharide is activated with a chemical reagent to form reactive groups for conjugation with a linker or a carrier protein. In some embodiments, the saccharide of the invention is activated prior to conjugation with the carrier protein. In some embodiments, the degree of activation does not result in a significant decrease in the molecular weight of the polysaccharide. For example, in some embodiments, the degree of activation does not result in cleavage of the polysaccharide backbone. In some embodiments, the degree of activation does not significantly affect the degree of conjugation, as measured by the amount of lysine residues modified in the carrier protein, such as CRM ₁₉₇ (determined by amino acid analysis). For example, in some embodiments, the degree of activation does not significantly increase the amount of modified lysine residues (as determined by amino acid analysis) in the carrier protein by 3 times compared to the amount of lysine residues modified in the carrier protein of the 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 saccharide. In some embodiments, the degree of activation does not reduce the optimal saccharide/protein ratio.

In some embodiments, the activated saccharide has an activation percentage in which the number of moles of thiol per saccharide repeating unit of the activated saccharide is from 1 to 100%, such as from 2 to 80%, from 2 to 50%, from 3-30% and from 4-25%. The degree of activation is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or about 100%. Preferably, the degree of activation is no more than 50%, more preferably no more than 25%. In one embodiment, the degree of activation is no more than 20%. Any minimum value and any maximum value may be combined to define a range.

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

For example, the spacer can be cystamine or cysteamine to form a thiolated polysaccharide that can be linked to the carrier via a thioether linkage obtained by reaction with a maleimide-activated carrier protein (e.g., using N-[Y-maleimidobutyryloxy]succinimide ester (GMBS)) or a haloacetylated carrier protein (e.g., using 76yophi1ized76e, 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 (optionally prepared by CDAP chemistry) is coupled to hexanediamine or adipic acid dihydrazide (ADH), and the amino-derived saccharide is conjugated to a carrier protein (e.g., CRM ₁₉₇ ) via carbodiimide (e.g., EDAC or EDC) chemistry via a carboxyl group on the carrier protein.

Other suitable conjugation methods include carbodiimides, hydrazides, active esters, norborane, para-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU. Conjugation may involve a carbonyl linker, which may be formed by reaction of a free hydroxyl group of the saccharide with CDI followed by reaction with the protein to form a carbamate linkage. This process may involve reduction of the anomeric end to a primary hydroxyl group, optional protection/deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to form a carbamate intermediate of CDI, and coupling of the carbamate intermediate of CDI to an amino group on the protein (CDI chemistry).

b. Molecular weight. In some embodiments, the glycoconjugate contains a saccharide with a molecular weight of 10 kDa to 2000 kDa. In other embodiments, the saccharide has a molecular weight from 50 kDa to 1000 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 from 100 kDa to 800 kDa. In other embodiments, the saccharide has a molecular weight from 200 kDa to 600 kDa. In additional embodiments, the saccharide has a molecular weight of from 100 kDa to 1000 kDa; from 100 kDa to 900 kDa; from 100 kDa to 800 kDa; from 100 kDa to 700 kDa; from 100 kDa to 600 kDa; from 100 kDa to 500 kDa; from 100 kDa to 400 kDa; from 100 kDa to 300 kDa; from 150 kDa to 1000 kDa; from 150 kDa to 900 kDa; from 150 kDa to 800 kDa; from 150 kDa to 700 kDa; from 150 kDa to 600 kDa; 150 kDa to 500 kDa; 150 kDa to 400 kDa; 150 kDa to 300 kDa; 200 kDa to 1000 kDa; 200 kDa to 900 kDa; 200 kDa to 800 kDa; 200 kDa to 700 kDa; from 200 kDa to 600 kDa; from 200 kDa to 500 kDa; from 200 kDa to 400 kDa; from 200 kDa to 300; from 250 kDa to 1000 kDa; from 250 kDa to 900 kDa; from 250 kDa to 800 kDa; from 250 kDa to 700 kDa; from 250 kDa to 600 kDa; from 250 kDa to 500 kDa; from 250 kDa to 400 kDa; from 250 kDa to 350 kDa; from 300 kDa to 1000 kDa; from 300 kDa to 900 kDa; from 300 kDa to 800 kDa; from 300 kDa up to 700 kDa; from 300 kDa to 600 kDa; from 300 kDa to 500 kDa; from 300 kDa to 400 kDa; from 400 kDa to 1000 kDa; from 400 kDa to 900 kDa; from 400 kDa to 800 kDa; from 400 kDa to 700 kDa; from 400 kDa to 600 kDa; from 500 kDa to 600 kDa. In one embodiment, a glycoconjugate having such a molecular weight is prepared by single-end conjugation. In another embodiment, a glycoconjugate having such a molecular weight is prepared by reductive amination chemistry (RAC) in an aqueous buffer. Any integer in any of the above ranges is considered as an embodiment of the invention.

In some embodiments, the glycoconjugate of the invention has a molecular weight of 400 kDa to 15,000 kDa; from 500 kDa to 10000 kDa; from 2000 kDa to 10000 kDa; from 3000 kDa to 8000 kDa; or from 3000 kDa to 5000 kDa. In other embodiments, the glycoconjugate has a molecular weight from 500 kDa to 10,000 kDa. In other embodiments, the glycoconjugate has a molecular weight of from 1000 kDa to 8000 kDa. In other embodiments, the glycoconjugate has a molecular weight of 2000 kDa to 8000 kDa, or 3000 kDa to 7000 kDa. In further embodiments, the glycoconjugate of the invention has a molecular weight of from 200 kDa to 20,000 kDa; from 200 kDa to 15,000 kDa; from 200 kDa to 10,000 kDa; from 200 kDa to 7,500 kDa; from 200 kDa to 5,000 kDa; from 200 kDa to 3,000 kDa; 200 kDa to 1000 kDa; 500 kDa to 20000 kDa; 500 kDa to 15000 kDa; 500 kDa to 12500 kDa; 500 kDa to 10000 kDa; 500 kDa to 7500 kDa; 500 kDa to 6000 kDa ; from 500 kDa to 5000 kDa; from 500 kDa to 4000 kDa; from 500 kDa to 3000 kDa; from 500 kDa to 2000 kDa; from 500 kDa to 1500 kDa; from 500 kDa to 1000 kDa; from 750 kDa to 20000 kDa; from 750 kDa to 15,000 kDa; 750 kDa to 12,500 kDa; 750 kDa to 10,000 kDa; 750 kDa to 7,500 kDa; 750 kDa to 6,000 kDa; 750 kDa to 5,000 kDa; 750 kDa to 4,000 kDa; 750 kDa up to 3000 kDa; from 750 kDa to 2000 kDa; from 750 kDa to 1500 kDa; from 1000 kDa to 15000 kDa; from 1000 kDa to 12500 kDa; from 1000 kDa to 10000 kDa; from 1000 kDa to 7500 kDa; from 1000 kDa to 6000 kDa; from 1000 kDa to 5000 kDa; from 1000 kDa to 4000 kDa; from 1000 kDa to 2500 kDa; from 2000 kDa to 15000 kDa; from 2000 kDa to 12500 kDa; from 2000 kDa to 10000 kDa; from 2000 kDa to 7500 kDa; from 2000 kDa to 6000 kDa; from 2000 kDa to 5000 kDa; from 2000 kDa to 4000 kDa; or between 2000 kDa and 3000 kDa. In one embodiment, a glycoconjugate having such a molecular weight is obtained by conjugation via eTEC as disclosed herein . In another embodiment, a glycoconjugate having such a molecular weight is prepared by reductive amination chemistry (RAC). In another embodiment, a glycoconjugate having such a molecular weight is prepared by reductive amination chemistry (RAC) in DMSO.

In further embodiments, the glycoconjugate of the invention has a molecular weight of from 1000 kDa to 20,000 kDa; from 1000 kDa to 15,000 kDa; from 2000 kDa to 10,000 kDa; from 2000 kDa to 7500 kDa; from 2000 kDa to 5000 kDa; from 3000 kDa to 20,000 kDa; from 3000 kDa to 15,000 kDa; from 3000 kDa to 12,500 kDa; from 4000 kDa to 10,000 kDa; from 4000 kDa to 7500 kDa; from 4000 kDa to 6000 kDa; or from 5000 kDa to 7000 kDa. In one embodiment, a glycoconjugate having such a molecular weight is prepared by reductive amination chemistry (RAC). In another embodiment, a glycoconjugate having such a molecular weight is prepared by reductive amination chemistry (RAC) in DMSO. In another embodiment, a glycoconjugate having such a molecular weight is prepared by the eTEC conjugation disclosed herein.

In further embodiments, the glycoconjugate of the invention has a molecular weight of from 5,000 kDa to 20,000 kDa; from 5,000 kDa to 15,000 kDa; from 5,000 kDa to 10,000 kDa; from 5,000 kDa to 7,500 kDa; from 6,000 kDa to 20,000 kDa; from 6,000 kDa to 15,000 kDa; from 6,000 kDa to 12,500 kDa; from 6,000 kDa to 10,000 kDa; or from 6,000 kDa to 7,500 kDa.

The molecular weight of the glycoconjugate can be measured using SEC-MALLS. Any integer in any of the above ranges is considered an embodiment of the invention. The glycoconjugates of the invention can also be characterized by the ratio (w/w) of saccharide to carrier protein. In some embodiments, the ratio of polysaccharide to carrier protein in the glycoconjugate (w/w) is from 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 ratio of saccharide to carrier protein (w/w) is from 0.5 to 2.0, from 0.5 to 1.5, from 0.8 to 1.2, from 0.5 to 1.0, from 1.0 to 1.5, or from 1.0 to 2.0. In further embodiments, the ratio of saccharide to carrier protein (w/w) is from 0.8 to 1.2. In a preferred embodiment, the ratio of polysaccharide to carrier protein in the conjugate is from 0.9 to 1.1. In some such embodiments, the carrier protein is CRM ₁₉₇ .

Glycoconjugates can also be characterized by their molecular size distribution (Kd). Size exclusion chromatography medium (CL-4B) can be used to determine the relative size distribution of the conjugate molecules. Size exclusion chromatography (SEC) is performed using gravity fed columns to obtain a size distribution profile of the conjugate molecules. Large molecules excluded from the pores of the medium elute faster than small molecules. Fraction collectors are used to collect the column eluate. The fractions are tested colorimetrically using saccharide analysis. To determine Kd, columns are calibrated to establish the fraction at which molecules are completely excluded (V0), (Kd=0), and the fraction representing maximum retention (Vi), (Kd=1). The fraction at which a specified sample attribute (Ve) is achieved is related to Kd by the following expression: Kd=(Ve - Vo)/(Vi - V0).

c. Free saccharide. The glycoconjugates and immunogenic compositions of the invention may include a free saccharide that is not covalently conjugated to a carrier protein but is nonetheless present in the glycoconjugate composition. The free saccharide may be non-covalently associated with (i.e., non-covalently bound to, adsorbed to, or entrapped in, or together with) the glycoconjugate. In a preferred embodiment, the glycoconjugate comprises no more than 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 no more than about 20% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises no more than about 15% free polysaccharide relative to the total amount of polysaccharide. In another preferred embodiment, the glycoconjugate comprises no more than 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 glycoconjugate comprises less than about 8% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises no more than about 6% free polysaccharide relative to the total amount of polysaccharide. In a preferred embodiment, the glycoconjugate comprises no more than about 5% free polysaccharide relative to the total amount of polysaccharide.

d. Covalent linkage. In other embodiments, the conjugate comprises at least one covalent linkage between the carrier protein and the saccharide for every 5-10 saccharide repeat units; for every 2-7 saccharide repeat units; for every 3-8 saccharide repeat units; for every 4-9 saccharide repeat units; for every 6-11 saccharide repeat units; for every 7-12 saccharide repeat units; for every 8-13 saccharide repeat units; for every 9-14 saccharide repeat units; for every 10-15 saccharide repeat units; for every 2-6 saccharide repeat units, for every 3-7 saccharide repeat units; for every 4-8 saccharide repeat units; for every 6-10 saccharide repeat units; for every 7-11 sugar repeat units; for every 8-12 sugar repeat units; for every 9-13 sugar repeat units; for every 10-14 sugar repeat units; for every 10-20 sugar repeat units; for every 4-25 sugar repeat units; or for every 2-25 sugar repeat units. In frequent embodiments, the carrier protein is CRM ₁₉₇ . In another embodiment, at least one bond between the carrier protein and the saccharide occurs for 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 CRM _197. Any integer in any of the above ranges is considered an embodiment of the invention.

e. Lysine residues. Another way in which the glycoconjugates of the invention can be characterized is by the amount of lysine residues in the carrier protein (e.g., CRM ₁₉₇ ) that become conjugated to the saccharide, which can be characterized as the level of conjugated lysines (degree of conjugation). Evidence of modification of the lysine of the carrier protein due to covalent bonds with polysaccharides can be obtained by amino acid analysis using conventional methods known to those skilled in the art. Conjugation results in a decrease in the amount of lysine residues recovered compared to the starting material of the carrier protein used to prepare the conjugate materials. In a preferred embodiment, the degree of conjugation of the glycoconjugate of the invention is from 2 to 15, from 2 to 13, from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 5, from 2 to 4, from 3 to 15, from 3 to 13, from 3 to 10, from 3 to 8, from 3 to 6, from 3 to 5, from 3 to 4, from 5 to 15, from 5 to 10, from 8 to 15, from 8 to 12, from 10 to 15, or from 10 to 12. In one embodiment, the degree of conjugation of the glycoconjugate 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 a preferred embodiment, the degree of conjugation of the glycoconjugate of the invention is from 4 to 7. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The frequency of attachment of the saccharide chain to the lysine on the carrier protein is another parameter by which the glycoconjugates of the invention can be characterized. 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 another embodiment, at least one covalent bond occurs between the carrier protein and the polysaccharide for every 10 saccharide repeating units of the polysaccharide. In another embodiment, at least one covalent bond occurs between the carrier protein and the polysaccharide for every 15 saccharide repeating units of the polysaccharide. In another embodiment, at least one covalent bond occurs between the carrier protein and the polysaccharide for every 25 saccharide repeating units of the polysaccharide.

f. O-acetylation. In some embodiments, the saccharides of the invention are O-acetylated. In some embodiments, the glycoconjugate comprises a saccharide with a degree of O-acetylation of from 10 to 100%, from 20 to 100%, from 30 to 100%, from 40 to 100%, from 50 to 100%, from 60 to 100%, from 70 to 100%, from 75 to 100%, from 80 to 100%, from 90 to 100%, from 50 to 90%, from 60 to 90%, from 70 to 90%, or from 80 to 90%. In other embodiments, the degree of O-acetylation is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or about 100%. By % O-acetylation is meant the percentage of a given saccharide relative to 100% (where each repeat unit is fully acetylated relative to its acetylated structure).

In some embodiments, the glycoconjugate is prepared by reductive amination. In some embodiments, the glycoconjugate is a conjugated saccharide with a single terminal linkage, wherein the saccharide is covalently linked directly to the carrier protein. In some embodiments, the glycoconjugate is covalently linked to the carrier protein through a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

g. Reductive amination. In one embodiment, the saccharide is conjugated to the carrier protein by reductive amination (e.g., as described in U.S. Patent Application Publication Nos. 2006/0228380, 2007/0231340, 2007/0184071, and 2007/0184072, WO 2006/110381, WO 2008/079653, and WO 2008/143709).

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

The oxidation step may include a reaction with periodate. As used herein, the term "periodate" refers to both periodate and periodic acid. The term also includes both metaperiodate ( ^IO4- ) and orthoperiodate ( ^IO65- ), as well as various periodate salts (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 ( _NalO4 ). In another embodiment, the polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of periodic acid.

In one embodiment, the oxidizing agent is a stable nitroxyl or nitroxyl radical compound, such as a piperidine-N-oxy or pyrrolidine-N-oxy compound, in the presence of an oxidizing agent for selectively oxidizing primary hydroxyls. In said reaction, the actual oxidizing agent is an N-oxoammonium salt in the catalytic cycle. In one aspect, said stable nitroxyl or nitroxyl radical compound is a piperidine-N-oxy or pyrrolidine-N-oxy compound. In one aspect, said stable nitroxyl or nitroxyl radical compound carries a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy) moiety. In one aspect, said stable nitroxyl radical compound is TEMPO or a derivative thereof. In one aspect, said oxidizing agent is a molecule bearing an N-halogen moiety. In one aspect, said oxidizing agent is selected from any 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, said oxidizing agent is N-chlorosuccinimide.

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

In one embodiment, lyophilization occurs in the presence of a non-reducing sugar, possible non-reducing sugars include sucrose, trehalose, raffinose, stachyose, melezitose, dextran, mannitol, lactitol, and palatinitol.

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

In one embodiment, the reduction reaction is carried out in an aqueous solvent (e.g., selected from PBS, MES, HE PES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, Bicine or HERB, at a pH of 6.0 to 8.5, 7.0 to 8.0 or 7.0 to 7.5), in another embodiment, the reaction is carried out in an aprotic solvent. In one embodiment, the reduction reaction is carried out in a DMSO (dimethyl sulfoxide) solvent or in DMF (dimethylformamide). DMSO or DMF solvent can be used to reconstitute the lyophilized activated polysaccharide and carrier protein.

At the end of the reduction reaction, the conjugates may have unreacted aldehyde groups remaining, which can be blocked with a suitable blocking agent. In one embodiment, the blocking agent is sodium borohydride ( _NaBH4 ). After conjugation (reduction reaction and, optionally, capping), the glycoconjugates can be purified (enriched with respect to the amount of polysaccharide-protein conjugate) by various methods known to those skilled in the art. These methods include dialysis, concentration/diafiltration operations, precipitation/eluting filtration in tangential flow, column chromatography (DEAE or hydrophobic interaction chromatography) and depth filtration. The glycoconjugates can be purified by diafiltration and/or ion exchange chromatography and/or size exclusion chromatography. In one embodiment, the glycoconjugates are purified by diafiltration or ion exchange chromatography or size exclusion chromatography. In one embodiment, the glycoconjugates are sterile filtered.

In a preferred embodiment, the E. coli glycoconjugate is selected from any of the serotypes O25b, O1a, O2, and O6, obtained by reductive amination. In a preferred embodiment, the E. coli glycoconjugates of serotypes O25b, O1a, O2, and O6 are obtained by reductive amination.

In one aspect, the invention relates to a conjugate that comprises a carrier protein, for example CRM ₁₉₇ , linked to a saccharide of formula O25b, represented by:

where n is any integer greater than or equal to 1. In a preferred embodiment, 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 and any maximum value may be combined to define a range. Exemplary ranges include, for example, from at least 1 to at most 1000; from at least 10 to at most 500; and from at least 20 to at most 80. In one preferred embodiment, n is from at least 31 to at most 90, more preferably from 40 to 90, most preferably from 60 to 85.

In another aspect, the invention relates to a conjugate that comprises a carrier protein, for example CRM ₁₉₇ , linked to a saccharide having any of the structures shown in Table A, where n is an integer greater than or equal to 1.

Without being limited by any theory or mechanism, in some embodiments, it is believed that a stable conjugate requires a level of modification of the saccharide antigen that does not result in disruption of the structural integrity of critical immunogenic epitopes of the antigen.

h. Activation and Aldehyde Formation. In some embodiments, a saccharide of the invention is activated to form an aldehyde. In such activated saccharide embodiments, the percentage (%) of activation (or oxidation state (DO)) refers to moles of saccharide repeating unit per mole of aldehyde of the activated polysaccharide. For example, in some embodiments, the saccharide is activated via periodate oxidation of vicinal diols on the polysaccharide repeating unit to form an aldehyde. Varying the molar equivalents (meq) of sodium periodate relative to the saccharide repeating unit and the temperature during oxidation results in different levels of oxidation state (DO).

Saccharide and aldehyde concentrations are usually determined using colorimetric assays. An alternative reagent is the combination TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl (free radical))-N-chlorosuccinimide (NCS), which results in the formation of aldehydes from primary alcohol groups.

In some embodiments, the activated saccharide has an oxidation state such that the number of moles of saccharide repeating unit per mole of aldehyde of the activated saccharide is from 1 to 100, such as from 2 to 80, from 2 to 50, from 3 to 30, and from 4 to 25. The degree of activation is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, ≥20, ≥30, ≥40, ≥50, ≥60, ≥70, ≥80, or>90, or about 100. Preferably, the oxidation state (DO) is from at least 5 to at most 50, more preferably from at least 10 to no more than 25. In one embodiment, the degree of activation is from no less than 10 to no more than 25. Any minimum value and any maximum value can be combined to define a range. The degree of oxidation can be represented as a percentage (%) of activation. For example, in one embodiment, a DO value of 10 refers to one activated saccharide repeating unit per 10 saccharide repeating units in the activated saccharide, and in this case, a DO value of 10 can be represented as 10% activation.

In some embodiments, the conjugate produced by reductive amination comprises a carrier protein and a saccharide, wherein the saccharide has a structure selected from: Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180/C3)), Formula O6 (e.g., Formula O6:K2; K13; K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O6O, Formula O61, Formula O62, Formula 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formulas O110, Formulas O111, Formulas O112, Formulas O113, Formulas O114, Formulas O115, Formulas O116, Formulas O117, Formulas O118, Formulas O119, Formulas O120, Formulas O121, Formulas O123, Formulas O124, Formulas O125, Formulas O126, Formulas O127, Formulas O128, Formulas O129, Formulas O130, Formulas O131, Formulas O132, Formulas O133, Formulas O134, Formulas O135, Formulas O136, Formulas O137, Formulas O138, Formulas O139, Formulas O140, Formulas O141, Formulas O142, Formulas O143, Formulas O144, Formulas O145, Formulas O146, Formulas O147, Formulas O148, Formulas O149, Formulas O150, Formulas O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186 and Formula O187. In some embodiments, the saccharide in the conjugate comprises the formula wherein n is an integer from 1 to 1000, from 5 to 1000, preferably from 31 to 100, or from 31 to 90, more preferably from 35 to 90, most preferably from 35 to 65.

i. Conjugates with a single terminal linkage. In some embodiments, the conjugate is a conjugated saccharide with a single terminal linkage, wherein the saccharide is covalently linked at one end of the saccharide to a carrier protein. In some embodiments, a conjugated polysaccharide with a single terminal linkage has a terminal saccharide. For example, a conjugate has a single terminal linkage if one of the ends (the terminal saccharide residue) of the polysaccharide is covalently linked to a carrier protein. In some embodiments, a conjugate has a single terminal linkage if the terminal saccharide residue of the polysaccharide is covalently linked to a carrier protein through a linker. Such linkers may include, for example, a cystamine linker (A1), a 3,3'-dithio-bis(propane dihydrazide) linker (A4), and a 2,2'-dithio-N,N'-bis(ethane-2,1-diyl)bis(2-(aminooxy)acetamide) linker (A6).

In some embodiments, the saccharide is conjugated to the carrier protein via a 3-deoxy-c1-manno-oct-2-ulosonic acid (KDO) moiety to form a single-terminal conjugate.

In some embodiments, the conjugate

preferably is not a bioconjugate. The term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and an antigen, e.g., an O-antigen (e.g., O25B), produced by a host cell, wherein the host cell machinery links the antigen to the protein (e.g., by N-linkages). Glycoconjugates include bioconjugates as well as conjugates of a sugar antigen (e.g., an oligo- or polysaccharide) to a protein produced by methods that do not require the conjugate to be produced in the host cell, such as by conjugation via chemical coupling of the protein to the saccharide.

j. Thiol-Activated Saccharides. In some embodiments, a saccharide of the invention is activated with a thiol. In such thiol-activated saccharide embodiments, the percentage (%) of activation refers to moles of thiol per saccharide repeating unit of the activated polysaccharide. Saccharide and thiol concentrations are typically determined using an Ellman assay to quantify sulfhydryls. For example, in some embodiments, the saccharide comprises 2-keto-3-deoxyoctanoic acid (KDO) activation with a disulfide-amine linker. In some embodiments, the saccharide is covalently linked to the carrier protein via a divalent heterobifunctional linker (also referred to herein as a "spacer"). The linker preferably provides a thioether bond between the saccharide and the carrier protein, resulting in the formation of a glycoconjugate, referred to herein as a "thioether glycoconjugate." In some embodiments, the linker further provides carbamate and amide linkages, such as, for example, (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC).

In some embodiments, the single-terminal conjugate comprises a carrier protein and a saccharide, wherein the saccharide comprises a structure selected from 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 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, formulas O110, formulas O111, formulas O112, formulas O113, formulas O114, formulas O115, formulas O116, formulas O117, formulas O118, formulas O119, formulas O120, formulas O121, formulas O123, formulas O124, formulas O125, formulas O126, formulas O127, formulas O128, formulas O129, formulas O130, formulas O131, formulas O132, formulas O133, formulas O134, formulas O135, formulas O136, formulas O137, formulas O138, formulas O139, formulas O140, formulas O141, formulas O142, formulas O143, formulas O144, formulas O145, formulas O146, formulas O147, formulas O148, formulas O149, formulas O150, formulas O151, Formulas O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187. In some embodiments, the saccharide in the conjugate comprises the formula wherein n is an integer from 1 to 1000, from 5 to 1000, preferably from 31 to 100, more preferably from 35 to 90, most preferably from 35 to 65.

For example, in one embodiment, a single-terminal conjugate comprises a carrier protein and a saccharide 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.

5. eTEC-linked conjugates

In one aspect, the invention generally relates to glycoconjugates comprising a saccharide derived from E. coli as described above, covalently conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer (as described, for example, in U.S. Patent 9,517,274 and International Patent Application Publication WO 2014027302, which are incorporated herein by reference in their entireties), including immunogenic compositions comprising such glycoconjugates, and methods for making and using such glycoconjugates and immunogenic compositions. The glycoconjugates comprise a saccharide covalently conjugated to an eTEC spacer via one or more eTEC spacers, wherein the saccharide is covalently conjugated to the eTEC spacer via a carbamate bond, and _{wherein the carrier protein is covalently conjugated to the eTEC spacer via an amide bond. The eTEC spacer comprises seven linear atoms (i.e., -C(O)NH(CH2)2SCH2C ( O} )-) and provides stable thioether and amide bonds between the saccharide and the carrier protein.

The eTEC-linked glycoconjugates of the invention can be represented by the general formula (I):

where the atoms that make up the eTEC spacer are contained in the central square.

In the glycoconjugates of the invention, the saccharide may be a polysaccharide or an oligosaccharide.

The carrier proteins included in the glycoconjugates of the invention are selected from the group of carrier proteins generally suitable for such purposes, as described hereinafter or known to those skilled in the art. In particular embodiments, the carrier protein is CRM ₁₉₇ .

In another aspect, the invention relates to a method for producing a glycoconjugate comprising a saccharide disclosed herein conjugated to a carrier protein via an eTEC spacer, comprising the steps of a) reacting the saccharide with a carbonic acid derivative in an organic solvent to produce an activated saccharide; b) reacting the activated saccharide with cystamine or cysteamine or their salts to produce a thiolated saccharide; c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide containing one or more free sulfhydryl residues; d) reacting the activated thiolated saccharide with an activated carrier protein containing one or more α-haloacetamide groups to produce a conjugate of the thiolated saccharide with the carrier protein; and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α-haloacetamide groups of the activated carrier protein; and/or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues of the activated thiolated saccharide; thereby forming an eTEC-linked glycoconjugate.

In frequent embodiments, the carbonic acid derivative is 1,1'-carbonyl-di-(1,2,4-triazole) (CDT) or 1,1'-carbonyldiimidazole (CDI). Preferably, the carbonic acid derivative is CDT and the organic solvent is a polar aprotic solvent such as dimethyl sulfoxide (DMSO). In preferred embodiments, the thiolated saccharide is prepared by reacting the activated saccharide with a bifunctional symmetrical thioalkylamine reagent, cystamine, or salts thereof. Alternatively, the thiolated saccharide can be formed by reacting the activated saccharide with cysteamine or a salt thereof. The eTEC-linked glycoconjugates prepared by the methods of the invention can be represented by general formula (I).

In common embodiments, 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 an S-carboxymethylcysteine (CMC) residue covalently linked to an activated lysine residue via a thioether linkage.

In other embodiments, the second copying reagent is iodoacetamide (IAA), which reacts with the unconjugated free sulfhydryl groups of the activated thiolated saccharide to produce a copied thioacetamide. Often, step e) includes copying with both the first copying reagent and the second copying reagent. In some embodiments, step e) includes copying with N-acetyl-L-cysteine as the first copying reagent and IAA as the second copying reagent.

In some embodiments, step e) of copying further comprises reacting with a reducing agent, such as DTT, TCEP or mercaptoethanol, after reacting with the first and/or second copying reagent.

The eTEC-linked glycoconjugates and immunogenic compositions of the invention may include free sulfhydryl residues. In some cases, the activated thiolated saccharides formed by the methods described herein will include multiple free sulfhydryl residues, some of which may not be covalently conjugated to the carrier protein during the conjugation step. Such residual free sulfhydryl residues are blocked by reaction with a blocking reagent other than a thiol-reactive blocking reagent, such as iodoacetamide (IAA), to block the potentially reactive functional group. The use of other thiol-reactive copying reagents, such as reagents containing maleimide and the like, is also contemplated.

In addition, the eTEC-linked glycoconjugates and immunogenic compositions of the invention may include residual unconjugated carrier protein, which may include an activated carrier protein that has undergone modification during parts of the copying process.

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 saccharide with the activated carrier protein. In frequent embodiments, the activated carrier protein comprises one or more α-bromoacetamide groups.

In another aspect, the invention relates to an eTEC-linked glycoconjugate comprising a saccharide disclosed herein conjugated to a carrier protein via an eTEC spacer, prepared according to any of the methods described herein.

In some embodiments, the carrier protein is CRM ₁₉₇ and at least one covalent bond is formed between CRM ₁₉₇ and the polysaccharide through an eTEC spacer for every 4, 10, 15, or 25 saccharide repeat units of the polysaccharide.

For each aspect of the invention, in particular embodiments of the methods and compositions provided herein, the eTEC-linked glycoconjugate comprises a saccharide provided herein, such as a saccharide derived from E. coli.

In another aspect, the invention relates to a method for 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 invention, wherein said immunogenic composition comprises an eTEC-linked glycoconjugate comprising a saccharide as disclosed herein. In some embodiments, the saccharide is derived from E. coli.

In some embodiments, the eTEC-linked glycoconjugate comprises a carrier protein and a saccharide, wherein the saccharide has a structure selected from Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180/C3)), Formula O6 (e.g., Formula O6:K2; K13; K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O6O, Formula O61, Formula O62, Formula 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formulas O110, Formulas O111, Formulas O112, Formulas O113, Formulas O114, Formulas O115, Formulas O116, Formulas O117, Formulas O118, Formulas O119, Formulas O120, Formulas O121, Formulas O123, Formulas O124, Formulas O125, Formulas O126, Formulas O127, Formulas O128, Formulas O129, Formulas O130, Formulas O131, Formulas O132, Formulas O133, Formulas O134, Formulas O135, Formulas O136, Formulas O137, Formulas O138, Formulas O139, Formulas O140, Formulas O141, Formulas O142, Formulas O143, Formulas O144, Formulas O145, Formulas O146, Formulas O147, Formulas O148, Formulas O149, Formulas O150, Formulas O151, Formula O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186 and Formula O187. In some embodiments, the saccharide in the conjugate comprises the formula wherein n is an integer from 1 to 1000, from 5 to 1000, preferably from 31 to 100, more preferably from 35 to 90, most preferably from 35 to 65.

The number of lysine residues in the carrier protein that become conjugated to the saccharide can be characterized as a range of conjugated lysines. For example, in some embodiments of the immunogenic compositions, CRM ₁₉₇ can comprise 4 to 16 lysine residues out of 39 covalently linked to the saccharide. Another way of expressing this parameter is that about 10% to about 41% of the lysine residues in CRM ₁₉₇ are covalently linked to the saccharide. In other embodiments, CRM ₁₉₇ can comprise 2 to 20 lysine residues out of 39 covalently linked to the saccharide. Another way of expressing this parameter is that about 5% to about 50% of the lysine residues in CRM ₁₉₇ are covalently linked to the saccharide.

In common embodiments, the carrier protein is CRM ₁₉₇ and at least one covalent bond is formed between the CRM ₁₉₇ and the polysaccharide through an eTEC spacer for every 4, 10, 15, or 25 saccharide repeat units of the polysaccharide.

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

In another embodiment, at least one bond is formed between the carrier protein and the saccharide for 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.

6. Carrier proteins

A component of the glycoconjugate of the invention is a carrier protein to which a saccharide is conjugated. The terms "carrier protein" or "carrier protein" or "carrier" may be used interchangeably herein. Carrier proteins should be amenable to standard conjugation procedures.

One of the components 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 a core oligosaccharide of the O-polysaccharide. In one embodiment, the conjugate comprises a carrier protein conjugated to an O-antigen of the O-polysaccharide.

The terms "carrier protein" or "carrier protein" or "carrier" may be used interchangeably herein. Carrier proteins should be amenable to standard conjugation procedures.

In a preferred embodiment, the carrier protein of the conjugates is independently selected from any one of TT, DT, DT mutants (such as CRM ₁₉₇ ), H. influenzae protein D, PhtX, PhtD, PhtDE fusion proteins (in particular those described in WO O1/98334 and WO O3/54007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, C. Difficile toxin A or B and PsaA. In one embodiment, the carrier protein of the conjugates of the invention is DT (diphtheria toxoid). In another embodiment, the carrier protein of the conjugates of the invention is TT (tetanus toxoid). In another embodiment, the carrier protein of the conjugates of the invention is PD (Haemophilus influenzae protein D - see, for example, EP O594610 B). In some embodiments, the carrier protein comprises poly(L-lysine) (PLL).

In a preferred embodiment, the saccharides are conjugated to the CRM ₁₉₇ protein. The CRM ₁₉₇ protein is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. CRM ₁₉₇ is produced by C. diphtheriae infected with the non-toxigenic phage β197tox, created by nitrosoguanidine mutagenesis of the toxigenic corynephage beta. The CRM ₁₉₇ protein has the same molecular weight as diphtheria toxin, but differs from it by a single base substitution (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution of glycine to glutamic acid in the mature protein and eliminates the toxic properties of diphtheria toxin. CRM ₁₉₇ protein is a T-cell-dependent, safe and effective saccharide carrier.

Accordingly, in some embodiments, the conjugates of the invention comprise CRM ₁₉₇ as a carrier protein, wherein the saccharide is covalently linked to CRM ₁₉₇ .

In a preferred embodiment, the glycoconjugate carrier protein is selected from the group consisting of DT (diphtheria toxin), TT (tetanus toxoid) or fragment C of TT, CRM ₁₉₇ (a non-toxic but antigenically identical variant of diphtheria toxin), other mutants of DT (such as CRM176, CRM228, CRM45 (Uchida et al. J. Biol. Chem. 218; 3838-3844, 1973), CRM9, CRM45, CRM102, CRM103 or CRM107; and other mutations described by Nicholls and Youle in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; deletions or mutations of Glu-148 to Asp, Gin or Ser and/or Ala 158 to Gly and other mutations disclosed in US 4,709,017 or US 4,950,740; mutations of at least one or more residues from: Lys 516, Lys 526, Phe 530 and/or Lys 534, and other mutations disclosed in US 5,917,017 or US 6,455,673; or a fragment disclosed in US 5,843,711), pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13), including Ply detoxified in some way, such as dPLY-GMBS (WO O4081515, PCT/EP2005/010258) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE (the sequences PhtA, PhtB, PhtD or PhtE are disclosed in WO O0/37105 or WO O0/39299) and Pht fusion proteins, such as PhtDE fusion proteins, PhtBE fusion proteins, Pht A-E (WO O1/98334, WO O3/54007, WO 2009/000826), OMPC (meningococcal outer membrane protein, typically extracted from JV. meningitidis serogroup B EP O372501), PorB (from N. meningitidis), PD (protein D of Haemophilus influenzae - see e.g. EP O594610 B) or their immunologically functional equivalents, synthetic peptides (EP O378881, EP O427347), heat shock proteins (WO 93/17712, WO 94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91/01146), artificial proteins containing multiple human CD4+ T-cell epitopes from antigens derived from different pathogens (Falugi et al (2001) Eur J Immunol 31; 3816-3824), e.g. protein N19 (Baraldoi et al. (2004) Infect Immun 72; 4884-7), pneumococcal surface protein PspA (WO O2/091998), iron-binding proteins (WO O1/72337), C. difficile toxin A or B (WO O0/61761), transferrin-binding proteins, pneumococcal adhesion protein (PsaA), recombinant Pseudomonas aeruginosa oxotoxin A (in particular, their non-toxic mutants (such as oxotoxin A with glutamic acid substitution 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 tuberculin protein derivative (PPD) may 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 No. WO 2004/083251), E. coli LT, E. coli ST, and oxotoxin A from Pseudomonas aeruginosa.

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

In a preferred embodiment, the glycoconjugate carrier protein is independently selected from the group consisting of mutants of TT, DT, DT (such as CRM ₁₉₇ ), I. influenzae protein D, PhtX, PhtD, PhtDE hybrids (in particular those described in WO O1/98334 and WO O3/54007), detoxified pneumolysin, PorB, N19 protein, PspA, OMPC, C. Difficile toxin A or B and PsaA. In one embodiment, the glycoconjugate carrier protein of the invention is DT (diphtheria toxoid). In another embodiment, the glycoconjugate carrier protein of the invention is TT (tetanus toxoid). In another embodiment, the carrier protein of the glycoconjugates of the invention is PD (Haemophilus influenzae protein D - see, for example, EP O594610 B).

In a preferred embodiment, the capsular saccharides of the invention are conjugated to the CRM ₁₉₇ protein. The CRM ₁₉₇ protein is a non-toxic form of diphtheria toxin, but is immunologically indistinguishable from diphtheria toxin. CRM ₁₉₇ is produced by C. diphtheriae infected with the non-toxigenic phage β197tox, created by nitrosoguanidine mutagenesis of the toxigenic corynephage beta (Uchida, T. et al. 1971, Nature New Biology 233:8-11). The CRM ₁₉₇ protein has the same molecular weight as diphtheria toxin, but differs from it by one base substitution (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution of glycine for glutamic acid in the mature protein and eliminates the toxic properties of diphtheria toxin. The CRM ₁₉₇ protein is a T-cell-dependent, safe and effective saccharide carrier. More information on CRM ₁₉₇ and its production can be found, for example, in US 5,614,382.

Accordingly, in frequent embodiments, the glycoconjugates of the invention comprise CRM ₁₉₇ as a carrier protein, wherein the capsular polysaccharide is covalently linked to CRM ₁₉₇ .

In another embodiment, the glycoconjugate carrier protein is SCP (streptococcal C5a peptidase). All isolates of human β-hemolytic streptococci produce the highly conserved cell wall protein SCP (streptococcal C5a peptidase), which specifically inactivates C5a. The scp genes encode a polypeptide containing 1134 to 1181 amino acids (Brown et al., PNAS, 2005, vol. 102, no. 51 pp. 18391-18396). The first 31 residues constitute the export signal pre-sequence and are removed upon passage through the cytoplasmic membrane. The next 68 residues serve as the pro-sequence 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 at Lys-1034, 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.

SCP can be divided into domains (see Fig. 1B in Brown et al., PNAS, 2005, vol. 102, no. 51 pp.18391-18396). These domains are the Pre/Pro domain (which includes the export signal pre-sequence (usually the first 31 residues) and the pro-sequence (usually the next 68 residues)), the protease domain (which is divided into two parts (protease domain part 1, usually residues 89-333/334, and protease domain part 2, usually residues 467/468-583/584), the protease-associated domain (PA domain) (usually residues 333/334-467/468), three fibronectin type III (Fn) domains (Fn1, usually residues 583/584-712/713; Fn2, usually residues 712/713-928/929/930; Fn3, usually residues 929/930-1029/1030/1031), and the cell wall anchor domain (usually residues 1029/1030/1031 to the C-terminus).

In one embodiment, the carrier protein of the glycoconjugates of the invention is SCP from GBS (SCPB). An example of SCPB is provided in SEQ ID NO: 3 in WO 97/26008. See also SEQ ID NO: 3 in WO O0/34487.

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

In another embodiment, the carrier protein of the glycoconjugate of the invention is an SCP as set forth in SEQ ID NO: 150 or 151 of WO 2014/136064.

B. Adjuvants

In some aspects, 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. Antigens may act primarily as a delivery system, primarily as an immunomodulator, or may have strong attributes of both. Suitable adjuvants include adjuvants suitable for use in mammals, including humans.

Examples of known suitable adjuvants of the delivery system type 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) in microparticle or nanoparticle form.

In one aspect, the immunogenic compositions disclosed herein comprise aluminum salts (alum) as an adjuvant (e.g., aluminum phosphate, aluminum sulfate, or aluminum hydroxide). In another 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.

Examples of known suitable immunomodulatory type adjuvants that can be used in humans include, but are not limited to, saponin extracts from the bark of the Aquilla tree (QS21, Quil A), 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.

Examples of known suitable immunomodulatory type adjuvants with both delivery properties and immunomodulatory characteristics 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); WO 90/03184, WO 96/11711, WO O0/48630, WO 98/36772, WO O0/41720, WO 2006/134423, and WO 2007/026190) or GLA-EM, which is a combination of a TLR4 agonist and an oil-in-water emulsion.

For veterinary applications, it is possible to use, for example, but not limited to animal experiments, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), emulsigen, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, designated as nor-MBP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, designated 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 2% squalene/Tween 80 omulsion.

Additional illustrative adjuvants for enhancing the potency of the immunogenic compositions disclosed herein include, but are not limited to, (1) oil-in-water omulsion formulations (with or without other specific immunostimulatory agents such as muramyl peptides (see below) or bacterial cell wall components), such as, for example, (a) SAF containing 10% squalane, 0.4% Tween 80, 5% Pluronic-blocked polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to form a larger omulsion, and (b) RIBI™ Adjuvant System (RAS) (Ribi Immunochem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorus lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (2) saponin adjuvants such as QS21, STIMULON™ (Cambridge Bioscience, WO rcester, Mass.), ABISCO® (Isconova, Sweden) or ISCOMATRIX® (Commonwealth Serum Laboratories, Australia), or particles derived therefrom such as ISCOM (immunostimulatory complexes), wherein ISCOM may be devoid of additional detergent (e.g. WO 00/07621); (3) complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA); (4) cytokines such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (e.g. WO 99/44636)), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) (see e.g. GB2220211, EP0689454) (see e.g. WO O0/56358); (6) a combination of 3dMPL, for example with QS21 and/or oil-in-water omulsions (see, for example, EP0835318, EP0735898, EP0761231); (7) a polyoxyethylene ether or polyoxyethylene ester (see, for example, WO 99/52549); (8) a polyoxyethylene sorbitan ester surfactant in combination with octoxynol (for example WO 01/21207) or a polyoxyethylene alkyl ester or ester surfactant in combination with at least one further non-ionic surfactant such as octoxynol (for example WO 01/21152); (9) a saponin and an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) (e.g. WO O0/62800); (10) an immunostimulant and a metal salt particle (see e.g. WO O0/23105); (11) a saponin and an oil-in-water emulsion (e.g. WO 99/11241); (12) a saponin (e.g. QS21)+3dMPL+IM2 (optionally+sterol) (e.g. WO 98/57659); (13) other substances that act as immunostimulatory agents to enhance the effectiveness of the composition. Muramyl peptides include N-acetylmuramyl-L-threonyl-L-isoglutamine (tr-MDP), N-25-acetyl-normuramil-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-L-isoglutarninyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

In another embodiment, the adjuvant is a liposomal formulation of Quillaja Saponaria-21 (QS21) comprising 5.1 mg/mL QS-21, 5 mM succinate, 60 mM NaCl, 0.1% PS80, pH 5.6. In another embodiment, the adjuvant is a liposomal monophosphoryl lipid A (MPLA, Synthetic, PHAD®, Avanti) composition comprising 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 O0714551-0018-2XLipoMPL), with a liposomal particle size of 71 nm as determined by dynamic light scattering. In another 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 O0714551-0018-2XlipoMQ), with an MPLA-QS21 liposomal particle size of 75 nm, as determined by dynamic light scattering.

In another aspect of the present invention, the immunogenic compositions disclosed herein comprise a CpG oligonucleotide as an adjuvant. A CpG oligonucleotide as used herein refers to a CpG immunostimulatory oligodeoxynucleotide (CpG ODN), and accordingly, these terms are used interchangeably unless otherwise indicated. Immunostimulatory CpG oligodeoxynucleotides comprise one or more CpG immunostimulatory motifs that are unmethylated cytosine-guanine dinucleotides, optionally in certain preferred base contexts. The methylation status of a CpG immunostimulatory motif typically refers to the cytosine residue in the dinucleotide. An immunostimulatory oligonucleotide comprising at least one unmethylated CpG dinucleotide is an oligonucleotide that comprises a 5'-unmethylated cytosine linked by a phosphate bond to a 3'-guanine and that activates the immune system by binding to Toll-like receptor 9 (TLR-9). In another embodiment, the immunostimulatory oligonucleotide may comprise one or more methylated CpG dinucleotides that will activate the immune system via TLR9, but not as strongly as if the CpG motif(s) were/were unmethylated.

Immunostimulatory CpG oligonucleotides may comprise one or more palindromes, which in turn may encompass a CpG dinucleotide. CpG oligonucleotides are described in a number of issued patents, published patent applications, and other publications, including U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068.

Various classes of CpG immunostimulatory oligonucleotides have been identified. They are designated as classes A, B, C and P and are described in more detail in WO 2010/125480 (page 3, line 22; page 12, line 36). The methods of the present invention involve the use of these various classes of CpG immunostimulatory oligonucleotides.

V. Methods of purification and production

In one aspect, the present invention relates to methods for producing a mutant FimH polypeptide. Such methods may include, for example, culturing a mammalian cell under suitable conditions, thereby allowing for expression of the mutant FimH polypeptide. The method may further include collecting the polypeptide from the culture. The method may further include purifying the polypeptide.

In some aspects, the method provides a mutant FimH polypeptide in a yield of about 0.1 g/L to about 0.5 g/L. In some aspects, the yield of the mutant FimH 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 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.

In some aspects, a cell culture suitable for the present invention is a fed-batch culture. As used herein, the term "fed-batch culture" refers to a method of culturing cells in which additional components are added to the culture during or after the start of the culturing process. Such provided components typically comprise nutrient components for the cells that are depleted during the culturing process. The culturing of a fed-batch culture is typically stopped at a certain point in time, and the cells and/or media components are harvested and optionally isolated. In some aspects, a fed-batch culture comprises a basal medium with the addition of a feeder medium.

In some aspects, cells can be grown in batch cultures or fed-batch cultures, wherein the culture is stopped after a sufficient level of expression of the polypeptide is achieved, after which the expressed polypeptide is collected and optionally isolated. In some aspects, cells can be grown in perfusion cultures, wherein the culture is not stopped and new nutrients and other components are periodically or continuously added to the culture, and the expressed polypeptide is periodically, during fed-batch, or continuously collected.

In some aspects, the expression level or activity of the mutant FimH 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, at least 100-fold compared to expression of the mutant FimH polypeptide in a bacterial cell, such as, for example, an E. coli host cell.

In some aspects, cells can be grown in small reaction vessels to produce cell culture volumes ranging from a few milliliters to several liters. In some aspects, cells can be grown in large-scale commercial bioreactors to produce cell culture volumes that can range from about at least 1 liter to 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10000, 12000 liters and more, or any volume in between. In some embodiments, the cell culture size may be from 10 L to 5,000 L, from 10 L to 10,000 L, from 10 L to 20,000 L, from 10 L to 50,000 L, from 40 L to 50,000 L, from 100 L to 50,000 L, from 500 L to 50,000 L, from 1,000 L to 50,000 L, from 2,000 L to 50,000 L, from 3,000 L to 50,000 L, from 4,000 L to 50,000 L, from 4,500 L to 50,000 L, from 1,000 L to 10,000 L, from 1,000 L to 20,000 L, from 1,000 L to 25,000 L, from 1,000 l to 30,000 l, from 15 l to 2,000 l, from 40 l to 1,000 l, from 100 l to 500 l, from 200 l to 400 l, or may be equal to any whole number between them.

The cell culture temperature is selected primarily based on a temperature range at which the cell culture remains viable, a temperature range at which high levels of polypeptide are produced, a temperature range at which the formation or accumulation of metabolic products is minimized, and/or any combination of these or other factors deemed important by the practitioner. As one non-limiting example, CHO cells grow well and produce high levels of protein or polypeptide at about 37°C. Typically, most mammalian cells grow well and/or can produce high levels of protein or polypeptide in the range of about 25°C to 42°C, although the methods described in the present invention are not limited to these temperatures. Some mammalian cells grow well and/or can produce high levels of protein or polypeptide in the range of about 35°C to 40°C. In some aspects, during the cell culturing process, the cell culture is grown one or more times 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.

The terms "culture" and "cell culture" as used herein refer to a cell population that is suspended in a medium under conditions suitable for the survival and/or growth of the cell population. As will be understood by those skilled in the art, in some aspects, these terms as used herein refer to a combination comprising a cell population and a medium in which the population is suspended. In some aspects, the cells of a cell culture include mammalian cells.

In some aspects, the cells can be grown in one of a plurality of chemically defined media wherein the components of the medium are known and controlled. In some aspects, the cells can be grown in a complex medium wherein not all components of the medium are known and/or controlled. Over the past several decades, developments of a plurality of chemically defined media for growing mammalian cell cultures have been published. All components of certain media are well characterized, so certain media do not contain complex additives such as serum or hydrolysates. Media formulations developed to support cell growth and viability in earlier years were created with little or no regard for protein production. More recently, media formulations have been developed specifically to support high-yield cell cultures producing recombinant protein. Such media are preferred for use in the method of the invention. Such media typically contain a high amount of nutrients and, in particular, amino acids to support cell growth and/or high cell density. These media can be modified, if necessary, by one of skill in the art for use in the method of the 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 a basal medium or growth medium in the method described herein.

Not all components of complex media are well characterized, so complex media may contain, among other things, additives such as simple and/or complex carbon sources, simple and/or complex nitrogen sources, and serum. In some aspects, a complex medium suitable for the present invention contains additives such as hydrolysates in addition to the other components of a particular medium disclosed herein. In some aspects, a medium of known chemical composition typically includes about fifty chemical compounds in known concentrations in water. Most of them also contain one or more well-characterized proteins such as insulin, IGF-1, transferrin, or BSA, but others do not require protein components and are therefore referred to as media of known chemical composition that do not contain proteins. Typical chemical components of the medium are divided into five broad categories: amino acids, vitamins, inorganic salts, trace elements, and other categories that do not lend themselves to clear categorization.

The cell culture medium may optionally be supplemented with additional components. As used herein, the term "additional components" refers to components that enhance growth and/or survival above a minimal level, including, but not limited to, hormones and/or other growth factors, certain ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds typically present in very low final concentrations), amino acids, lipids, and/or glucose or another energy source. In some aspects, additional components may be added to the initial cell culture. In some aspects, additional components may be added after the cell culture has begun. Typically, trace elements refer to various inorganic salts present in micromolar or lower amounts. For example, frequently included trace elements include zinc, selenium, copper, and others. In some aspects, iron (ferrous iron or ferric salts) may be included as a micronutrient in the initial cell culture medium for cell culture at micromolar concentrations. Manganese as a divalent cation ( _MnCl2 or _MnSO4 ) is also often included among the micronutrients in the nanomolar to micromolar concentration range. Numerous less common micronutrients are typically added at nanomolar concentrations.

In some aspects, the medium used in the method of the invention is a medium suitable for maintaining a high cell density in culture, such as, for example, 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 5×10 ⁸ cells/mL. In some aspects, the cell culture is a batch culture of mammalian cells, preferably a batch culture of CHO cells.

In some aspects, the cell culture medium comprises phenylalanine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises tyrosine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises tryptophan at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises methionine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises leucine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises series at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises threonine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises glycine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises two components of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine, at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises phenylalanine and tyrosine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises phenylalanine and tryptophan at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises phenylalanine and methionine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises tyrosine and tryptophan at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises tyrosine and methionine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises tryptophan and methionine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises three components of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine, at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises phenylalanine, tyrosine, and tryptophan at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises phenylalanine, tyrosine, and methionine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM.

In some aspects, the cell culture medium comprises phenylalanine, tryptophan, and methionine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises tyrosine, tryptophan, and methionine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM.

In some aspects, the cell culture medium comprises four components of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine, at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM.

In some aspects, the cell culture medium comprises phenylalanine, tyrosine, tryptophan, and methionine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises five components of phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises six components of: phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine, at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises seven components of: phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine, at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium comprises phenylalanine, tyrosine, tryptophan, methionine, leucine, serine, threonine, and glycine at a concentration of less than 2 mM, less than 1 mM, from 0.1 to 2 mM, from 0.1 to 1 mM, from 0.5 to 1.5 mM, or from 0.5 to 1 mM. In some aspects, the cell culture medium further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 components of: glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine, at a concentration greater than 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2 mM. In some aspects, the cell culture medium further comprises at least 5 components of: glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine, at a concentration greater than 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2 mM. In some aspects, the cell culture medium further comprises glycine, valine, leucine, isoleucine, proline, serine, threonine, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration greater than 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2 mM. In some aspects, the cell culture medium further comprises at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 components of: valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate and asparagine, at a concentration greater than 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2 mM. In some aspects, the cell culture medium further comprises at least 5 components of: valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate and asparagine, at a concentration greater than 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2 mM. In some aspects, the cell culture medium further comprises valine, isoleucine, proline, lysine, arginine, histidine, aspartate, glutamate and asparagine at a concentration greater than 2 mM, 3 mM, 4 mM, 5 mM, 10 mM, 15 mM, preferably 2 mM. In some aspects, the cell culture medium comprises series at a concentration greater than 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably 10 mM. In some aspects, the cell culture medium comprises valine at a concentration greater than 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably 10 mM. In some aspects, the cell culture medium comprises cysteine at a concentration greater than 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably 10 mM. In some aspects, the cell culture medium comprises isoleucine at a concentration greater than 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably 10 mM.

In some aspects, the cell culture medium comprises leucine at a concentration greater than 3 mM, 5 mM, 7 mM, 10 mM, 15 mM or 20 mM, preferably 10 mM. In some aspects, the above cell culture medium is for use in the method disclosed herein. In some aspects, the above cell culture medium is used in the method disclosed herein as a basal medium. In some aspects, the above cell culture medium is used in the method disclosed herein as a feed medium.

The methods of the present invention can be used with any cell culture method that is suitable for the desired process (e.g., producing a recombinant protein). As a non-limiting example, cells can be grown in batch or fed-batch cultures, where the culture is stopped after a sufficient level of expression of the recombinant protein (e.g., an antibody) is achieved, after which the expressed protein is harvested. Alternatively, as another non-limiting example, cells can be grown in fed-batch conditions, where the culture is not stopped, but new nutrients and other components are periodically or continuously added to the culture, and during the culture, the expressed recombinant protein is periodically or continuously harvested. Other suitable methods (e.g., culture in centrifuge tubes) are known in the art and can be used to practice the present invention.

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

The cell culture temperature is selected primarily based on the temperature range at which the cell culture remains viable and the temperature range at which a high level of the desired product (e.g., recombinant protein) is produced. Typically, most mammalian cells grow well and can produce desired products (e.g., recombinant proteins) in the temperature range of about 25°C to 42°C, although the methods described herein are not limited to these temperatures. Some mammalian cells grow well and can produce desired products (e.g., recombinant proteins or antibodies) in the temperature range of about 35°C to 40°C. In some aspects, during the cell culturing process, the cell culture is grown one or more times 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. Those skilled in the art will be able to select an appropriate temperature or temperatures for growing the cells depending on the specific needs of the cells and the specific production requirements of the practitioner. The cells can be grown for any period 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.

In some aspects, the cells can be grown during the initial growth phase (or growth phase) for a longer or shorter period of time, depending on the needs of the practitioner and the requirements of the cells themselves. In some aspects, the cells are grown for a period of time sufficient to achieve a predetermined cell density. In some aspects, the cells are grown for a period of time sufficient to achieve a cell density that is a predetermined percentage of the maximum cell density that would ultimately be achieved by the cells if their growth were not impaired. For example, the cells can be grown for a period of time sufficient 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 aspects, the cells are grown until the cell density increases by more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in 1 day of culturing. In some aspects, the cells are grown until the cell density increases by more than 5% in 1 day of culturing.

In some aspects, the cells are allowed to grow for a certain period of time. For example, depending on the initial concentration of the cell culture, the temperature at which the cells are grown, and the intrinsic growth rate of the cells, the cells can 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 from 4 to 10 days. In some cases, the cells are allowed to grow for a month or more. A person skilled in the art will be able to select the duration of the initial growth phase depending on the requirements for protein production and the needs of the cells themselves.

During the initial phase of culturing, the cell culture may be stirred or shaken to increase oxygenation and improve the availability of nutrients to the cells. According to the present invention, it will be clear to a person skilled in the art that during the initial growth stage, it is worth monitoring or adjusting certain internal conditions of the bioreactor, including, but not limited to, pH, temperature, oxygenation, etc.

At the end of the initial growth phase, at least one of the culture conditions can be changed to obtain a second set of culture conditions, in which a metabolic shift occurs in the culture. The metabolic shift can be achieved, for example, by changing the temperature, pH, osmolality, or the level of a chemical inductive substance in the cell culture. In one of the non-limiting embodiments, the culture conditions are changed by changing the culture temperature. However, as is known in the art, changing the temperature is not the only mechanism by which the corresponding metabolic shift can be achieved. For example, such a metabolic shift can also be achieved by changing other culture conditions, including, but not limited to, pH, osmolality, and sodium butyrate levels. The timing of the metabolic shift in the culture will be determined by a person skilled in the art, based on the requirements for protein production or the needs of the cells themselves.

When changing the temperature of a culture, the change can be made relatively gradually. For example, it may take several hours or days for the temperature to change completely. Alternatively, the temperature change may be relatively abrupt. For example, the temperature change may be completed in less than a few hours. With appropriate production and control equipment, such as standard equipment for industrial large-scale production of polypeptides or proteins, the temperature change may be completed in less than an hour.

In some aspects, after changing the cell culture conditions as discussed above, the cell culture is maintained in a subsequent production phase under a second set of culture conditions that promote the survival and viability of the cell culture and are suitable for expression of the desired polypeptide or protein at a commercially acceptable level.

As discussed above, a shift can be achieved in a culture by changing one or more culture conditions, including, but not limited to, temperature, pH, osmolality, and sodium butyrate levels. In some aspects, a temperature change is used to shift the culture. According to this embodiment, during a subsequent production phase, the culture is maintained at a temperature or temperature range that is lower than the temperature or temperature range during the initial growth phase. As discussed above, multiple discrete temperature changes can be used to increase cell density or viability or to increase recombinant protein expression.

In the context of the present description, the term "titer" refers, for example, to the total amount of recombinantly expressed protein produced by a cell culture in a given volume of medium. The titer is usually expressed in grams of protein per liter of medium.

In some aspects, cell growth is increased by at least 5%, 10%, 15%, 20%, or 25% compared to a control culture. In some aspects, cell growth is increased by at least 10% compared to a control culture. In some aspects, cell growth is increased by at least 20% compared to a control culture.

In some aspects, productivity is defined in titers and/or as volumetric productivity.

In some aspects, productivity is determined in titers. In some aspects, productivity is increased by at least 5%, 10%, 15%, 20%, or 25% compared to a control culture. In some aspects, productivity is increased by at least 10% compared to a control culture. In some aspects, productivity is increased by at least 20% compared to a control culture.

Cleaning

In some aspects, a method for producing a mutant FimH polypeptide includes isolating and/or purifying the polypeptide. In some aspects, the expressed polypeptide is secreted into the medium, and thus cells and other solids can be removed by centrifugation and/or filtration. In a preferred embodiment, the polypeptide or fragment thereof is soluble.

A mutant FimH polypeptide produced according to the methods described herein can be collected from the host cells and isolated by any suitable method well known in the art (e.g., Ruiz-Arguello et al., J. Gen. Virol., 85:3677-3687 (2004)). Suitable methods for purifying the polypeptide include precipitation and various types of chromatography, such as hydrophobic interaction chromatography, ion exchange, affinity, chelation, and size exclusion chromatography, all of which are known in the art. Suitable purification schemes can include two or more of these or other suitable methods. In some aspects, one or more polypeptides can include a "tag" that facilitates purification or detection. Examples include, for example, a His tag (binds to a metal ion, such as hexahistidine), an antibody, maltose binding protein (MBP) (binds to amylose), glutathione-E-transferase (GST) (binds to glutathione), a FLAG tag (binds to an anti-FLAG antibody), a streptococcal tag (binds to streptavidin or a derivative thereof). Such tagged polypeptides are conveniently isolated, for example, from conditioned media using chelation chromatography or affinity chromatography. Optionally, the tag sequence can be cleaved after purification. In one aspect, the mutant FimH polypeptide does not include a purification tag.

In one aspect, the mutant FimH polypeptides can be isolated by first obtaining a cell culture supernatant, and then subjecting the supernatant to either ultrafiltration or diafiltration. Such filtration methods are known to those skilled in the art. Following ultrafiltration and diafiltration, the resulting cell-free solution is then subjected to a chromatography step, such as Ni-NTA chromatography using, for example, a nickel affinity resin. This step can then be followed by dialysis, which can be followed by cation exchange chromatography, such as on an SP column. Under certain conditions, it may be desirable to use 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.

Although particular strains of E. coli may be mentioned in the present description, it is to be understood that a polypeptide derived from E. coli or fragment thereof is not limited to particular strains unless otherwise stated.

VI. Application of compositions

In one aspect, the present invention relates to the use of a mutant FimH polypeptide, nucleic acids encoding such a mutant, vectors for expressing such a mutant, compositions containing such a mutant or nucleic acids, as a medicament or in the manufacture of a medicament for inducing an immune response against an E. coli infection or for preventing an E. coli infection in a subject.

In other aspects, the present invention relates to a method of inducing an immune response against E. coli in a subject, such as a human, comprising administering to the subject an effective amount of a mutant FimH polypeptide, a nucleic acid molecule encoding a mutant FimH polypeptide, or a composition comprising a mutant FimH polypeptide or nucleic acid molecule. The present invention also provides a method of preventing an E. coli infection in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition, such as a vaccine, comprising a mutant FimH polypeptide, a nucleic acid encoding a mutant FimH polypeptide, or a vector expressing a mutant FimH polypeptide. In some specific aspects, the pharmaceutical composition comprises a mutant FimH polypeptide as disclosed herein. In some aspects of the methods provided herein above, the subject is a human.

In other aspects, the present invention relates to a method for inducing an immune response in a subject against extraintestinal pathogenic E. coli or inducing the formation of opsonophagocytic and/or neutralizing antibodies in a subject specific for extraintestinal pathogenic E. coli, wherein the method comprises administering to the subject an effective amount of any of the compositions disclosed herein, such as compositions comprising a mutant FimH polypeptide as described herein. In another aspect 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.

In another aspect, the present invention relates to a method for inducing an immune response against E. coli in a mammal, comprising administering to the mammal an effective amount of any of the compositions disclosed herein. For example, in one aspect, the immune response comprises the formation of opsonophagocytic and/or neutralizing antibodies to E. coli. In another aspect, the immune response protects the mammal from E. coli infection.

In another aspect, the present invention provides a method for 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.

In the methods of the present invention, the composition can be administered to a subject with or without an adjuvant. An effective amount administered to a subject is an amount sufficient to induce an immune response in the subject to an E. coli antigen, such as the FimH protein. Subjects that can be selected for treatment include individuals at risk of developing an E. coli infection, such as at risk of developing a 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.

In the context of the present description, the term "subject" means a mammal, preferably a human. In one embodiment, the subject is at risk of developing any of the conditions selected from the group consisting of urinary tract infection, cholecystitis, cholangitis, diarrhea, hemolytic uremic syndrome, neonatal meningitis, urosepsis, intra-abdominal infection, meningitis, complicated pneumonia, wound infection, prostate biopsy associated infection, neonatal/infant sepsis, neutropenic fever and other bloodstream infections; pneumonia, bacteremia and sepsis.

Administration of the compositions provided herein, such as pharmaceutical compositions, can be accomplished by standard routes of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, transmucosal, or oral administration.

The total dose of the composition administered to a subject during a single administration may vary, as is known to practitioners.

One or more repeated administrations of one or more immunogenic compositions are also possible. In the case of a booster vaccination, typically such a booster vaccination is administered to the same subject at a time interval of from one week to 10 years, preferably from two weeks to six months, after the first administration of the composition to the subject (which in such cases is called a "prime vaccination"). In alternative booster vaccination schedules, different vectors can also be administered to the subject after the prime vaccination, for example, one or more adenovirus or other vectors, such as a modified vaccinia virus Ankara (MVA) vector, or DNA or protein. The immunogenic compositions provided herein can be used together with one or more other immunogenic compositions.

Dosages of compositions

Dosage regimens may be adjusted to provide the optimal desired response. For example, a single dose of the mutant FimH polypeptide may be administered, several divided doses may be administered over time, or the dose may be proportionally decreased or increased depending on the severity of the situation. It should be noted that the dosage amount may vary depending on the type and severity of the condition to be alleviated, and the dosage may include single or multiple doses. In addition, it should be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual needs and professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are illustrative only and are not intended to limit the scope or use of the claimed composition.

In some aspects, the amount of mutant FimH polypeptide in the composition can be from about 10 μg to about 300 μg of each protein antigen. In some aspects, the amount of mutant FimH polypeptide in the composition can be from about 20 μg to about 200 μg of each protein antigen.

The amount of glycoconjugate(s) in each dose is selected to induce an immunoprotective response without significant adverse side effects in typical vaccines. This amount will vary depending on the specific immunogen used and how it is presented.

The amount of a particular glycoconjugate in an immunogenic composition can be calculated based on the total amount of polysaccharide for that conjugate (conjugated and unconjugated). For example, a glycoconjugate with 20% free polysaccharide would contain approximately 80 g of conjugated polysaccharide and approximately 20 g of unconjugated polysaccharide in a dose of 100 g of polysaccharide. The amount of glycoconjugate may vary depending on the serotype of E. coli. The concentration of saccharide can be determined using a uronic acid assay.

The "immunogenic amount" of the different polysaccharide components in the immunogenic composition may vary, and each may be 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. Typically, each dose will contain from 0.1 g to 100 g of polysaccharide for a given serotype, in particular from 0.5 g to 20 g, more particularly from 1 g to 10 g, and even more particularly from 2 g to 5 g. Any integer in any of the above ranges is considered an embodiment of the invention. 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.

VII. COMBINATION WITH A SACCHARIDE AND/OR POLYPEPTIDE OR FRAGMENTS THEREOF ORIGINATED FROM KLEBSIELLA PNEUMONIAE

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 among vulnerable at-risk populations. O-antigen serotypes are widely distributed among strains causing invasive disease worldwide, and the resulting O-antigen glycoconjugates are attractive as vaccine antigens.

In one aspect, any of the compositions disclosed herein may further comprise at least one saccharide that 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 described herein may further comprise a polypeptide derived from K. pneumoniae, selected from a polypeptide derived from K. pneumoniae type I fimbrial protein, or an immunogenic fragment thereof; or a polypeptide derived from K. pneumoniae type III fimbrial protein, or an immunogenic fragment thereof; or a combination thereof.

As is known in the art, the 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. The K. pneumoniae O1 and O2 antigens contain homopolymeric galactose units (or galactans). The K. pneumoniae O1 and O2 antigens each contain D-galactan I units (sometimes referred to as the O2a repeating unit), but the O1 antigens differ in that they have a D-galactan II cap structure. D-galactan III (d-Gal-III) is a variant of D-galactan I. The structures of the major galactans I and III define two different subtypes of serotype O2, O2v1 and O2v2; and the structures of chimera derivatives resulting from galactan II replication that yield the O1v1 and O1v2 subtypes 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.

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

In some embodiments, the K. pneumoniae O2-derived saccharide comprises a [ _→ 3)-α-D-Galp-(1 _→ 3)-β-D-Galf-(1 _→ ] repeat unit (which may be an element of the K. pneumoniae serotype O2a antigen). In some embodiments, the K. pneumoniae O2-derived saccharide comprises a [ _→ 3)-β-D-GlcpNAc-(1 _→ 5)-β-D-Galf-((which may be an element of the K. pneumoniae serotype O2 antigen). In some embodiments, the K. pneumoniae O2-derived saccharide comprises a modification of the O2a repeat unit by the addition of (1 _→ 4)-linked Galp residues to the side chain (which may be an element of the K. pneumoniae O2afg antigen). pneumoniae). In some embodiments, the saccharide derived from K. pneumoniae O2 comprises a modification of the O2a repeat unit by adding (1-2)-linked Galp residues (which may be a member of the K. pneumoniae O2aeh antigen) to the side chain. (Whitfield C, et al. (1992) J. Bacterid. 174, 4913-4919).

Without being bound by any mechanism or theory, the structure of the O-antigen polysaccharide of K. pneumoniae serotypes O3 and O5 disclosed in this art is identical to that of E. coli serotypes O9a (formula O9a) and O8 (formula O8), respectively.

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

disaccharide repeating unit.

In one aspect, the invention provides a composition comprising a polypeptide derived from E. coli FimH, or a fragment thereof; 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.

In another aspect, the invention includes a composition comprising at least one saccharide from, or that is obtained 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 a saccharide derived from an E. coli O-antigen having a structure selected from 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 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formulas O112, Formulas O113, Formulas O114, Formulas O115, Formulas O116, Formulas O117, Formulas O118, Formulas O119, Formulas O120, Formulas O121, Formulas O123, Formulas O124, Formulas O125, Formulas O126, Formulas O127, Formulas O128, Formulas O129, Formulas O130, Formulas O131, Formulas O132, Formulas O133, Formulas O134, Formulas O135, Formulas O136, Formulas O137, Formulas O138, Formulas O139, Formulas O140, Formulas O141, Formulas O142, Formulas O143, Formulas O144, Formulas O145, Formulas O146, Formulas O147, Formulas O148, Formulas O149, Formulas O150, Formulas O151, Formulas O152, Formulas O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186, and Formula O187, wherein n is an integer from 1 to 100. In some embodiments, the composition comprises a saccharide of, or derived from, one or more serotypes O1, O2, O3, and O5 of K. pneumoniae, or a combination thereof. In some embodiments, the composition includes a saccharide from, or derived from, each of serotypes O1, O2, O3, and O5 of K. pneumoniae. In some embodiments, the composition includes a saccharide derived from an E. coli O-antigen having the formula O9, and does not include a saccharide derived from serotype O3 of K. pneumoniae. In some embodiments, the composition includes a saccharide derived from an E. coli O-antigen having the formula O8, and does not include a saccharide derived from serotype O5 of K. pneumoniae.

In another aspect, the invention relates to a composition comprising a polypeptide derived from E. coli FimH, or a fragment thereof; at least one saccharide from, or derived from, at least one serotype of K. pneumoniae selected from O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8 and O12; and a saccharide having a structure selected from Formula O1 (e.g., Formula O1A, Formula O1B, and Formula O1C), Formula O2, Formula O3, Formula O4 (e.g., Formula O4:K52 and Formula O4:K6), Formula O5 (e.g., Formula O5ab and Formula O5ac (strain 180/C3)), Formula O6 (e.g., Formula O6:K2; K13; K15 and Formula O6:K54), Formula O7, Formula O8, Formula O9, Formula O10, Formula O11, Formula O12, Formula O13, Formula O14, Formula O15, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O19, Formula O20, Formula O21, Formula O22, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formula O112, Formula O113, Formula O114, Formulas O115, Formulas O116, Formulas O117, Formulas O118, Formulas O119, Formulas O120, Formulas O121, Formulas O123, Formulas O124, Formulas O125, Formulas O126, Formulas O127, Formulas O128, Formulas O129, Formulas O130, Formulas O131, Formulas O132, Formulas O133, Formulas O134, Formulas O135, Formulas O136, Formulas O137, Formulas O138, Formulas O139, Formulas O140, Formulas O141, Formulas O142, Formulas O143, Formulas O144, Formulas O145, Formulas O146, Formulas O147, Formulas O148, Formulas O149, Formulas O150, Formulas O151, Formulas O152, Formulas O153, Formulas O154, Formulas O155, Formulas 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 from 1 to 100, preferably from 31 to 90. In some embodiments, the composition comprises a saccharide derived from an E. coli O-antigen having the formula O9, and not includes a saccharide derived from serotype O3 of K. pneumoniae. In some embodiments, the composition includes a saccharide derived from an E. coli O-antigen having the formula O8 and does not include a saccharide derived from serotype O5 of K. pneumoniae.

In some embodiments, the composition comprises at least one saccharide derived from any type of K. pneumoniae selected from the group consisting of O1, O2, O3, and O5.

In some embodiments, the composition includes at least one saccharide derived from K. pneumoniae type O1. In one aspect of this embodiment, the K. pneumoniae O antigen is selected from subtype v1 (O1v1) or subtype v2 (O1v2). In one aspect of this embodiment, the K. pneumoniae O antigen is selected from subtype v1 (O1v1) and subtype v2 (O1v2). In some embodiments, the composition includes at least one saccharide derived from K. pneumoniae type O2. In one aspect of this embodiment, the K. pneumoniae O antigen is selected from subtype v1 (O2v1) or subtype v2 (O2v2). In one aspect of this embodiment, the K. pneumoniae O antigen is selected from subtype v1 (O2v1) and subtype v2 (O2v2). In another aspect, the K. pneumoniae O antigen is selected from the group consisting of: a) serotype O1 subtype v1 (O1v1), b) serotype O1 subtype v2 (O1v2), c) serotype O2 subtype v1 (O2v1), and d) serotype O2 subtype v2 (O2v2). In one aspect of this embodiment, the K. pneumoniae O antigen is subtype v1 (O1v1). In one aspect of this embodiment, the K. pneumoniae O antigen is subtype v2 (O1v2). In one aspect of this embodiment, the K. pneumoniae O antigen is subtype v1 (O2v1). In one aspect of this embodiment, the K. pneumoniae O antigen is subtype v2 (O2v2). In another aspect of this embodiment, the composition comprises one, two, three or four K. pneumoniae O-antigens selected from the group consisting of: a) serotype O1 subtype v1 (O1v1), b) serotype O1 subtype v2 (O1v2), c) serotype O2 subtype v1 (O2v1), and d) serotype O2 subtype v2 (O2v2). In some embodiments, the composition comprises a combination of saccharides derived from K. pneumoniae, wherein the first saccharide is derived from any one type of K. pneumoniae selected from the group consisting of O1, O2, O3 and O5; and the second saccharide is derived from a saccharide derived from any one type of K. pneumoniae selected from the group consisting of O1 (and d-Gal-III variants), O2 (and d-Gal-III variants), O2ac, O3, O4, O5, O7, O8 and O12. For example, in some embodiments, the composition includes at least one saccharide derived from type O1 K. pneumoniae and at least one saccharide derived from type O2 K. pneumoniae. In a preferred embodiment, the saccharide derived from K. pneumoniae is conjugated to a carrier protein; and the saccharide derived from E. coli is conjugated to a carrier protein.

In another aspect, the invention includes a composition comprising a polypeptide derived from E. coli FimH, or a fragment thereof; and at least one saccharide derived from any type of K. pneumoniae selected from the group consisting of O1, O2, O3 and O5.

In another aspect, the invention comprises at least one saccharide obtained from any type of K. pneumoniae selected from the group consisting of O1, O2, O3 and O5; and at least one saccharide derived from E. coli having a structure selected from formula O1 (e.g., formula O1A, formula O1B, and formula O1C), formula O2, formula O3, formula O4 (e.g., formula O4:K52 and formula O4:K6), formula O5 (e.g., formula O5ab and formula O5ac (strain 180/C3)), formula O6 (e.g., formula O6:K2; K13; K15 and formula O6:K54), formula O7, formula O8, formula O9, formula O10, formula O11, formula O12, formula O13, formula O14, formula O15, formula O16, formula O17, formula O18 (e.g., formula O18A, formula O18ac, formula O18A1, formula O18B, and formula O18B1), formula O19, formula O20, formula O21, formula O22, formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O27, Formula O28, Formula O29, Formula O30, Formula O32, Formula O33, Formula O34, Formula O35, Formula O36, Formula O37, Formula O38, Formula O39, Formula O40, Formula O41, Formula O42, Formula O43, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45rel), Formula O46, Formula O48, Formula O49, Formula O50, Formula O51, Formula O52, Formula O53, Formula O54, Formula O55, Formula O56, Formula O57, Formula O58, Formula O59, Formula O60, Formula O61, Formula O62, Formula 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formula O111, Formulas O112, Formulas O113, Formulas O114, Formulas O115, Formulas O116, Formulas O117, Formulas O118, Formulas O119, Formulas O120, Formulas O121, Formulas O123, Formulas O124, Formulas O125, Formulas O126, Formulas O127, Formulas O128, Formulas O129, Formulas O130, Formulas O131, Formulas O132, Formulas O133, Formulas O134, Formulas O135, Formulas O136, Formulas O137, Formulas O138, Formulas O139, Formulas O140, Formulas O141, Formulas O142, Formulas O143, Formulas O144, Formulas O145, Formulas O146, Formulas O147, Formulas O148, Formulas O149, Formulas O150, Formulas O151, Formulas O152, Formula O153, Formula O154, Formula O155, Formula O156, Formula O157, Formula O158, Formula O159, Formula O160, Formula O161, Formula O162, Formula O163, Formula O164, Formula O165, Formula O166, Formula O167, Formula O168, Formula O169, Formula O170, Formula O171, Formula O172, Formula O173, Formula O174, Formula O175, Formula O176, Formula O177, Formula O178, Formula O179, Formula O180, Formula O181, Formula O182, Formula O183, Formula O184, Formula O185, Formula O186 and Formula O187. In some embodiments, the composition includes a saccharide derived from an E. coli O antigen having the formula O9 and does not include a saccharide derived from K. pneumoniae serotype O3. In some embodiments, the composition includes a saccharide derived from an E. coli O antigen having the formula O8 and does not include a saccharide derived from K. pneumoniae serotype O5.

In some embodiments, the composition comprises at least one saccharide derived from K. pneumoniae type O1; and at least one saccharide derived from E. coli having a structure selected from the group consisting of formula O8 and formula O9. In another embodiment, the composition comprises at least one saccharide derived from K. pneumoniae type O2; and at least one saccharide derived from E. coli having a structure selected from the group consisting of formula O8 and formula O9. In another embodiment, the composition comprises at least one saccharide derived from K. pneumoniae type O1; at least one saccharide derived from K. pneumoniae type O2; and at least one saccharide derived from E. coli having a structure selected from the group consisting of formula O8 and formula O9.

In one embodiment, the invention relates to a method for inducing an immune response to K. pneumoniae in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising at least one glycoconjugate from E. coli serotype O8 or O9, wherein said immunogenic composition does not comprise glycoconjugates from K. pneumoniae serotype 05 or O3. In one aspect, the composition comprises a saccharide derived from an E. coli O-antigen having the formula O8 and does not include a saccharide derived from K. pneumoniae serotype O5. In another aspect, the composition comprises a saccharide derived from an E. coli O-antigen having the formula O9 and does not include a saccharide derived from K. pneumoniae serotype O3.

In another embodiment, the invention relates to a method for inducing an immune response to E. coli in a subject, comprising administering to the subject an immunologically effective amount of an immunogenic composition comprising at least one glycoconjugate from K. pneumoniae serotype O5 or O3 or a variant thereof, wherein said immunogenic composition does not comprise glycoconjugates from E. coli serotype O8 or O9. In one aspect, the composition comprises a saccharide derived from K. pneumoniae serotype O5 and does not comprise a saccharide derived from an E. coli O-antigen having the formula O8. In another aspect, the composition comprises a saccharide derived from K. pneumoniae serotype O3 and does not comprise a saccharide derived from an E. coli O-antigen having the formula O9.

In some embodiments, the composition comprises at least one saccharide from, or 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; at least one saccharide derived from E. coli having a structure selected from the group consisting of formula O8 and formula O9. In some embodiments, the composition comprises at least one saccharide from, or 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; at least one saccharide derived from E. coli having a structure selected from the group consisting of formula O1A, formula O1B, formula O2, formula O6 and formula O25B.

In some embodiments, the composition further comprises a polypeptide derived from K. pneumoniae selected from a polypeptide derived from a K. pneumoniae type I fimbrial protein, or an immunogenic fragment thereof; or a polypeptide derived from a K. pneumoniae type III fimbrial protein, or an immunogenic fragment thereof, or a combination thereof. The sequences of these polypeptides are known in the art.

VIII. Nanoparticles

In another aspect, the present description discloses an immunogenic complex comprising 1) a nanostructure; and 2) at least one fimbrial polypeptide antigen or fragment thereof. Preferably, the fimbrial polypeptide or fragment thereof is obtained from E. coli fimbrial H (fimH). In a preferred embodiment, the fimbrial polypeptide is selected from any of the fimbrial polypeptides described above. For example, the fimbrial polypeptide may comprise any one amino acid sequence selected from SEQ ID NO: 1-65.

In some embodiments, the antigen is fused or conjugated to the outer portion of the nanostructure to stimulate the development of adaptive immune responses to exposed epitopes. In some embodiments, the immunogenic complex further comprises an adjuvant or other immunomodulatory compounds attached to the outer portion and/or encapsulated within the cage, which facilitates adaptation of the type of immune response generated for each pathogen.

In some embodiments, the nanostructure comprises a single assembly comprising a plurality of identical first nanostructure-related polypeptides.

In alternative embodiments, the nanostructure comprises a plurality of assemblies comprising a plurality of identical first nanostructure-related polypeptides and a plurality of second assemblies, wherein each second assembly comprises a plurality of identical second nanostructure-related polypeptides.

Various nanostructure platforms can be used to create the immunogenic compositions disclosed herein. In some embodiments, the nanostructures used are formed by multiple copies of a single subunit. In some embodiments, the nanostructures used are formed by multiple copies of multiple different subunits.

Nanostructures are typically spherical in shape and/or have axial symmetry (e.g., with a 3-fold and 5-fold axis), such as the icosahedral structure given here as an example.

In some embodiments, the antigen is presented on self-assembling nanoparticles, such as self-assembling nanostructures derived from ferritin (FR), E2p, QB and 13-01. E2p is an engineered version of dihydrolipoyl acyltransferase from Bacillus stearothermophilus. 13-01 is an engineered protein having the ability to self-assemble into hyperstable nanoparticles. The subunit sequences of these proteins are known in the art. In a first aspect, the present disclosure provides a nanostructure-related polypeptide comprising an amino acid sequence that is at least 75% identical in length to an amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NO: 66-105, and at least at one identified position of the contact surface. The nanostructure-related polypeptides can be used, for example, to produce nanostructures. Nanostructured polypeptides are designed with the ability to self-assemble into pairs to form nanostructures such as icosahedral nanostructures.

In some embodiments, the nanostructure comprises (a) a plurality of first assemblies, wherein each first assembly comprises a plurality of identical first nanostructure-related polypeptides, wherein the first nanostructure-related polypeptides comprise an amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NO: 66-105; and (b) a plurality of second assemblies, wherein each second assembly comprises a plurality of identical second nanostructure-related polypeptides, wherein the second nanostructure-related polypeptides comprise an amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NO: 66-105, and wherein the second nanostructure-related polypeptide differs from the first nanostructure-related polypeptide; wherein the plurality of first assemblies interact without forming covalent bonds with the plurality of second assemblies to form the nanostructure.

The nanostructures include symmetrically repeating, non-natural, non-covalently bonded polypeptide-polypeptide interfaces that orient the first assembly and the second assembly in the interior of the nanostructure, such as a structure with icosahedral symmetry.

SEQ ID NOs: 66-105 provide an amino acid sequence of exemplary nanostructure-related polypeptides. The number of residues at the interface of exemplary nanostructure-related polypeptides of SEQ ID NOs: 66-105 ranges from 4 to 13 residues. In various embodiments, the nanostructure-related polypeptides comprise an amino acid sequence that is identical to an amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NO: 66-105, along at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of its length, and also in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 identified positions of the interface (depending on the number of residues on the interface of the given nanostructure-related polypeptide). In other embodiments, the nanostructure-related polypeptides comprise an amino acid sequence that is identical to the amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NO: 66-105, along at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the length, as well as in at least 20%, 25%, 33%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 100% of the identified contact surface positions. In additional embodiments, the nanostructure-related polypeptides comprise a nanostructure-related polypeptide with an amino acid sequence of a nanostructure-related polypeptide selected from the group consisting of SEQ ID NO: 66-105.

In one non-limiting embodiment, the nanostructure-related polypeptides can be modified to facilitate covalent bonding with a "cargo" of interest. In one non-limiting example, the nanostructure-related polypeptides can be modified, for example, by introducing different cysteine residues at specific positions to bind to one or more antigens of interest to produce a nanostructure of the nanostructure-related polypeptides that provides a framework for delivering a large number of antigens as a vaccine to achieve an improved immune response.

In some embodiments, some or all of the native cysteine residues that are present in the nanostructured polypeptides but are not intended for conjugation may be replaced with other amino acids to facilitate conjugation at certain positions. In another non-limiting embodiment, the nanostructured polypeptides may be modified by linking (covalently or non-covalently) to a moiety to facilitate "endosome escape". For applications that involve the delivery of molecules of interest to a target cell, such as targeted delivery, a critical step may be escape from an endosome, a membrane-bound organelle that is the entry point of the delivery vehicle into the cell. Endosomes become lysosomes, which degrade their contents. Therefore, if the delivery vehicle does not somehow "escape" the endosome before it becomes a lysosome, it will be destroyed and will not be able to perform its function. There are many lipids or organic polymers that disrupt endosomes and allow penetration into the cytosol. Thus, in this embodiment, the polypeptides related to the nanostructure can be modified, for example, by introducing cysteine residues that will allow chemical conjugation of such a lipid or organic polymer to the monomer or the resulting assembly surface. In another non-limiting example, the polypeptides related to the nanostructure can be modified, for example, by introducing cysteine residues that will allow chemical conjugation of fluorophores or other imaging agents that allow visualization of the nanostructures in vitro or in vivo.

Surface amino acid residues on nanostructure-related polypeptides can be mutated to improve the stability or solubility of protein subunits or assembled nanostructures. As is known to those skilled in the art, if a nanostructure-related polypeptide has high sequence homology to an existing protein family, then multiple sequence alignments of other proteins from that family can be used to select amino acid mutations at non-conserved positions that can improve the stability and/or solubility of the protein, a process called consensus protein design (9).

The amino acid residues on the surface of the polypeptides belonging to the nanostructure can be mutated to positively charged (Arg, Lys) or negatively charged (Asp, Glu) amino acids, imparting a net positive or net negative charge to the protein surface. In one non-limiting embodiment, the surface amino acid residues on the polypeptides belonging to the nanostructure can be mutated to provide a high net charge on the inner surface of the self-assembling nanostructure. Such a nanostructure can then be used to package or encapsulate a cargo molecule with an opposite net charge due to the electrostatic interaction between the inner surface of the nanostructure and the cargo molecule. In one non-limiting embodiment, the surface amino acid residues on the polypeptides belonging to the nanostructure can be mutated primarily to arginine or lysine residues, imparting a net positive charge to the inner surface of the self-assembling nanostructure. Solutions containing the nanostructure-related polypeptides can then be mixed in the presence of a nucleic acid carrier molecule, such as double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, cDNA, microRNA, siRNA, shRNA, piRNA, or another nucleic acid, to encapsulate the nucleic acid within the self-assembling nanostructure. Such a nanostructure can be used, for example, to protect, deliver, or concentrate nucleic acids.

In one embodiment, the nanostructure has icosahedral symmetry. In this embodiment, the nanostructure may comprise 60 copies of a first nanostructure-related polypeptide and 60 copies of a second nanostructure-related polypeptide. In one such embodiment, the number of identical first nanostructure-related polypeptides in each first assembly is different from the number of identical second nanostructure-related polypeptides in each second assembly. For example, in one embodiment, the nanostructure includes twelve first assemblies and twenty second assemblies; in this embodiment, each first assembly may, for example, contain five copies of an identical first nanostructure-related polypeptide, and each second assembly may, for example, contain three copies of an identical second nanostructure-related polypeptide. In another embodiment, the nanostructure comprises twelve first assemblies and thirty second assemblies; in this embodiment, each first assembly may, for example, contain five copies of an identical first nanostructure-related polypeptide, and each second assembly may, for example, contain two copies of an identical second nanostructure-related polypeptide.

In another embodiment, the nanostructure comprises twenty first assemblies and thirty second assemblies; in this embodiment, each first assembly may, for example, contain three copies of an identical first polypeptide belonging to the nanostructure, and each second assembly may, for example, contain two copies of an identical second polypeptide belonging to the nanostructure. All of these embodiments are capable of forming synthetic nanomaterials with regular icosahedral symmetry.

EXAMPLES

For a better understanding of the invention, examples are provided below. These examples are intended for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

EXAMPLE 1: Creating an antigen

In order to lock the FimH lectin domain in an open conformation to improve functional immunogenicity, mutations were engineered in FimHLD or FimH-DSG. The mutations belonged to different classes, described in Tables 2-9 below. The amino acid sequences for the various mutant FimH polypeptides are shown in Table 1.

Example 2: Antigen expression and purification

DNA encoding the FimHLD and FimH-DSG mutants was cloned into pcDNA3.1 containing the mouse IgK signal peptide and expressed in Expi293™ cells as previously described (PCT International Publication No. WO 2021/084429, published May 6, 2021). For protein characterization and immunogenicity studies, proteins were isolated using nickel affinity resin and size exclusion chromatography as described in PCT International Publication No. WO 2021/084429, published May 6, 2021.

Example 3: Fluorescence Polarization Analysis

To determine the dissociation constants of FimH mutants for mannoside ligands, a fluorescence polarization assay was developed based on the methods described by Rabbani et al. (J. Biol. Chem. 293:1835–1849 (2018)) using fluorescein conjugated to mannoside ligands with high affinity for FimH. FimH proteins were diluted in 20 mM HE PES pH 7.4, 150 mM NaCl, 0.05 mg/mL plus BSA 0.05% in triplicate 11-point titrations in a black flat-bottomed 96-well polypropylene plate (Greiner) with a final volume of 50 μL. To each well, 50 μl of fluorescein octylbiphenylmannopyranoside ligand at a concentration of 0.7 nM in the same buffer were added. The plates were incubated overnight at room temperature with shaking at 100 rpm. After 20-24 h, the plates were read using a ClarioStar Plus plate reader with fluorescein excitation at 488 nm and emission at 530 nm.

Example 4: Thermal stability analysis (ThermoFluor analysis)

To determine the melting temperature of isolated proteins in the APO (unbound) form and in the presence of ligand, a 384-well thermostability assay was developed using SYPRO orange. Mannoside compounds (methyl α-D-mannopyranoside (Sigma M6882)) mimicking the natural FimH ligand (mannose) were used for protein association analysis. FimH protein stocks were prepared by diluting proteins in 40 mM Tris, pH 8, 400 mM NaCl (assay buffer) to 4 μM; SYPRO orange dye (Invitrogen S6650) was diluted 1:10 in assay buffer. FimH mutants at a concentration of 4 μM (5 μl) were mixed with SYPRO orange dye 1:10 (0.1 μl) and assay buffer or ligand diluted in assay buffer (5 μl) to a final reaction volume of 10 μl in a 384-well MicroAmp EnduraPlate Optical plate (Applied Biosystems 4483285). The plate was analyzed by melting curve in a QuantStudio 5 Real-Time PCR System (ThermoFisher) according to the dissociation protocol from 20°C to 98°C at a rate of 0.05°C/sec. TAMRA was specified as a target and reporter, ROX as a passive reference (however, it was not used in the assay). The data were plotted as a Maxwell-Boltzmann distribution with temperature (20°C to 98°C) on the x-axis and fluorescence from the TAMRA channel on the y-axis (each temperature reading during the melt curve measurement was assigned a specific fluorescence excitation value for the TAMRA reporter). A normalization algorithm was developed to equalize the fluorescence intensity between wells and samples, and to allow comparison of y-axis fluorescence values between plates on a scale from 0 (no fluorescence) to 1 (highest fluorescence detected). This equation is shown below. Using the lookup function in Microsoft Excel (also below), a relative fluorescence value (after normalization) of 0.5 (indicating that approximately half of the protein had dissociated) was reported to correlate with a particular temperature. This temperature, the measured melting temperature (Tm) of the protein, was calculated in this manner.

The change in melting temperature (ΔTm) was determined by subtracting the Tm of the protein+ligand from the APO state. Pivot tables in Microsoft Excel were used to organize the Tin from the plate layout.

Equation for normalizing the TAMRA fluorescence signal:

Normalization value (0 to 1) = (raw fluorescence value - minimum fluorescence value for the whole well (20°C to 98°C)) / (maximum fluorescence value for the whole well - minimum fluorescence value for the whole well)

Excel lookup function to determine Tm (0.5 normalized fluorescence or 50% protein melting):

=LOOKUP(0.5, start of normalized fluorescence values: end of values, $ start of temperature values: $ end of values)

Example 5: Confirmation of the conformational state of FimH mutants using FimH-specific neutralizing monoclonal antibodies

Neutralizing monoclonal antibodies 299-3, 304-1 and 440-2 (in-house developed) were used to confirm the conformational state of the FimH mutants; 229-3 and 304-1 bind to the same epitopes as MAbs 475 and 926 (Kisiela, DI et al. Proc Natl Acad Sci USA 110, 19089–19094 (2013)), while 440-2 recognizes a different epitope and appears to preferentially bind to the FimH _LD in the open conformation. Variants retaining similar structural integrity to the wild type are expected to bind all antibodies. Octet HTX from ForteBio was used to measure antibody reactivity to each mutant in all real-time kinetic biomolecular interaction experiments. Experiments were performed at 30°C with shaking at 1000 rpm in 96-well black plates containing 240 μl per well. Ni-NTA biosensors were equilibrated in a buffer containing 1x PBS supplemented with 0.5% BSA and 0.05% Tween 20 (PBT) prior to loading of the mutant His-tagged FimH proteins at a concentration of 5 μg/ml for 5 min. FimH-loaded biosensors were allowed to recover to baseline in PBT for 3 min prior to association with antibodies from different groups at 5 μg/ml for 5 min. Kinetic analysis of the association step and obtaining the shift response in nm (Table 1) were performed using Octet data analysis software.

Example 6: Circular Dichroism Spectroscopy

For the FimH _LD and FimH-DSG mutants, far-UV (320–250 nm) and near-UV (260–200 nm) circular dichroism spectra were recorded using a JASCO J-810 spectropolarometer (Jasco) equipped with a JASCO PTC-424S/15 (Jasco) and an Isotemp water bath (Fisher Scientific). A 1 mm cuvette was used for the far-UV region, and a 10 mm cuvette was used for the near-UV region. Proteins were diluted to 0.3 mg/ml in PBS, and spectra were recorded at 20°C in cuvettes with an optical path length of 1 mm (far-UV) or 10 mm (near-UV). Scanning was performed at 100 nm/min, DIT was set to 1 s, bandwidth to 3 s, and data step to 0.1 nm. Sensitivity was set to standard. Ten spectra were accumulated and averaged for the near-UV and five for the far-UV measurements, respectively. Spectra were manually background corrected using CD spectra obtained from PBS blank runs and converted to mean residue ellipticity using equation 1, where θMRE is the calculated mean residue ellipticity, θEXP is the experimentally measured CD signal, MW is the molecular weight of the protein, N is the number of amino acid residues, C is the protein concentration in mg/mL, and 1 is the optical path length in cm.

Equation 1:

θMRE =(θEXP ⋅ MW) / (10⋅N⋅C⋅1)

Example 7: EC-1678 animal immunogenicity study

CD-1 mice aged 6–8 weeks were obtained from Charles River Laboratories. Twenty animals of each group were immunized subcutaneously at 0, 4, and 8 weeks with 10 μg of FimH protein mixed with 20 μg of Quillaja Saponaria-21 (QS-21) from a 5.1 mg/ml QS-21 stock solution containing 5 mM succinate, 60 mM NaCl, 0.1% PS80, pH 5.6.

Example 8: Whole-cell FimH neutralization assays To assess the ability of sera from vaccinated animals to inhibit the binding of fimbriated E. coli to mannosylated substrates, a whole-cell neutralization assay using yeast mannan was used, as described in International PCT Publication No. WO 2021/084429, published May 6, 2021.

Example 9: Purification of FimH-DSG WT and FimH-DSG G15A G16A V27A mutant from CHO cells

Proteins were expressed in CHO cells as secreted proteins with C-terminal His-tags. Cell culture supernatant was collected and supplemented with 1 M Tris, pH 7.4, and 5 M NaCl to final concentrations of 20 mM and 150 mM, respectively. The 5 kDa TFF cassettes were washed with buffer and equilibrated in 20 mM Tris, pH 7.5, with 500 mM NaCl and 40 mM imidazole. The supernatant was concentrated 2-fold and diafiltered against 6 volumes of 20 mM Tris, pH 7.5, 500 mM NaCl, 40 mM imidazole. The retentate was collected and washed with 50-100 ml of 20 mM Tris, pH 7.5, 500 mM NaCl, 40 mM imidazole. The retentate was filtered and washed with a 0.2 μm bottle filter. An XK26/20 column was packed with Ni-Sepharose 6 Fast Flow resin (Cytiva Life Sciences) and equilibrated with 5 column volumes of 20 mM Tris, pH 7.5, 500 mM NaCl, 40 mM imidazole. The retentate was loaded at half flow rate and washed until a stable baseline was reached (approximately 55 column volumes). Bound protein was eluted with 20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.5. Fractions containing the protein of interest were pooled and dialyzed in a 2 kDa dialysis cassette against 20 mM sodium acetate, pH 4.3, at 4°C with two buffer changes. The protein was loaded onto an SP-Sepharose cation exchange column (Cytiva Life Sciences) equilibrated with the same buffer. The material bound to the cation exchange resin was eluted with a linear NaCl gradient using 20 mM sodium acetate, pH 4.3, 1 M NaCl buffer. Fractions were pooled and dialyzed against TBS, pH 7.4.

Example 10: Analytical size exclusion chromatography (SEC) for the determination of FimH-DSG WT and FimH-DSG mutant G15A G16A V27A

Analytical SEC was performed using a Waters X bridge Protein BEH SEC 125 column. 2.5 μm, 4.6×300 mm in TBS buffer, pH 7.4, containing 10 mM EDTA, at 25°C. The injection volume was 10 μl, the flow rate was 0.5 ml/min.

Example 11: Analysis of monosaccharides by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

Aqueous samples of wild-type and triple mutant FimH-DSG (G15A, G16A, V27A) were digested for 2 h at 120°C in 2N trifluoroacetic acid. After this time, the samples were evaporated to dryness under vacuum at 45°C for 6 h. The samples were reconstituted in Milli-Q _H2O and analyzed by HPAEC-PAD in a DIONEX ICS 3000 ion chromatography system. A Dionex CarboPac PA1 column (4×250 mm) was used, eluting isocratically with a mixture of _H2O and 200 mM NaOH. The monosaccharide composition was confirmed by comparing the retention times of the peaks detected in the FimH samples with solutions of known monosaccharide standards.

Example 12: Detection of binding of the sugar moiety of the O-antigen to FimH-DSG WT and mutant FimH-DSG G15A G16A V27A

To measure potential interactions of O-antigen with FimH-DSG WT and FimH-DSG G15A G16A V27A mutant, the HTX Octet from ForteBio was used in all real-time kinetic biomolecular interaction experiments. Experiments were performed at 30°C with shaking at 1000 rpm in 96-well black plates containing 240 μl per well. Ni-NTA biosensors were equilibrated in a buffer containing 1x PBS buffer with 0.5% BSA and 0.05% Tween 20 (PBT) prior to loading His-tagged FimH-DSG WT or FimH-DSG G15A G16A V27A mutant into the biosensor at a concentration of 5 μg/ml for 5 min. Biosensors loaded with FimH-DSG WT or FimH-DSG G15A G16A V27A were allowed to recover baseline in RBT for 3 min and then loaded with double titration (200-3.125 μg/mL) of CRM conjugates with O-antigen polysaccharide O9 or O25b or O1a or O2. Antigen-loaded biosensor without any polysaccharide was used as a reference. Biosensors loaded with FimH and O-antigen titration were immersed in RBT for 3 min to establish a new baseline. Detection of O-antigen binding to the mutant was tested in the association step with 5 μg/ml O-antigen-specific mAbs for 5 min (MAb 601 for O9, MAb ECO-80-11 for O25b, MAb ECO-48-2 for O1a and MAb ECO-172-13 for O2). Octet data analysis software was used for kinetic analysis of the association step with subtraction of the reference value and obtaining the shift response in nm (in the table).

Example 13: Protein expression and purification

FimH _LD and FimH-DSG mutants were expressed in Expi293 cells and isolated from supernatants by nickel affinity capture followed by size exclusion chromatography. It should be noted that some mutants had low expression levels and did not pass biochemical or biophysical evaluation (e.g., FimH _LD P26C V35C, N33C P157C, N33C L109C, V35C L107C, V35C L109C, S113C T158C). Mutants for which sufficient yields could be obtained were evaluated in thermostability and ligand binding assays.

Example 14: Identification of FimH _LD and FimH-DSG mutants with improved thermal stability and reduced melting temperature shift in the presence of mannoside ligand

Melting temperatures of the FimH mutant proteins were determined by differential scanning fluorimetry based on the thermal shift of SYPRO orange dye, where Tm is the temperature at which 50% of the protein unfolds. Non-covalent ligands often stabilize protein targets upon specific binding, resulting in an increase in the melting temperature of the protein. Therefore, melting temperatures were determined in the presence of methyl alpha-D-mannopyranoside, which is an alpha-D-mannose derivative with micromolar affinity for FimH (Bouckaert, J. et al. Mol Microbiol. 55, 441-455 (2005)), and the melting temperature difference (ΔTm) of the protein in the presence of ligand relative to the APO form was calculated.

Wild-type (WT) FimH-BSG proteins exhibited significantly higher melting temperatures compared to FimH _LD WT and had lower ΔTm in the presence of ligand (Table 10, Table 11). FimH-BSG WT had a melting temperature of 71.66 °C, while the melting temperature of FimH _LD WT was significantly lower (61.54 °C). In the presence of methyl alpha-D-mannopyranoside, the melting temperature of FimH _LD WT shifted by 10.99 °C, while the melting temperature of FimH-BSG in the presence of ligand shifted only by 2.13 °C. This suggests that FimH _LD is more efficiently stabilized by ligand compared to FimH-DSG, which may reflect a decrease in ligand binding to FimH-DSG.

Mutations affected the melting temperature of FimH proteins in the APO state and in the presence of ligand. The fixed mutant FimH _LD V27C L34C described previously (Kisiela, DI et al. Proc Natl Acad Sci USA 110, 19089-19094 (2013); Rodriguez, VB et al. J Biol Chem 288, 24128-24139 (2013)) showed a lower melting temperature (51.42°C) compared to the wild-type FimH _LD (61.54°C), which is consistent with published data (Kisiela, DI et al. Proc Natl Acad Sci USA 110, 19089-19094 (2013); Rodriguez, VB et al. J. Biol Chem 288, 24128-24139 (2013)). Incubation of FimH _LD V27C L34C with methyl alpha-D-mannopyranoside increased the melting temperature by 7.27 °C compared to the APO form, suggesting that this mutant is partially stabilized by the ligand and may have residual ligand binding efficiency. As for the FimH-DSG V27C L34C mutant, it was less thermally stable compared to the WT (Tm = 63.29 °C), and the temperature shift of the V27C L34C mutant in the presence of ligand was slightly reduced compared to the WT (ΔTm = 1.29 °C). Five of the six Phel FimH _LD mutants had reduced melting temperature compared to the wild-type FimH _LD , except F1L, which had a similar melting temperature. The ΔTm value of four of the six FimH _LD Phel mutants in the presence of ligand was small, indicating poor stabilization by the ligand. In contrast, the most conservative amino acid substitutions F1W and F1Y showed intermediate and comparable ΔTm values, respectively, compared to the wild-type Phel FimH _LD . Regarding the overall thermal stability, the reference mutation R60P (previously described - Rabbani et al. J. Biol. Chem. 293:1835–1849 (2018); Rodriguez, VB et al. J Biol Chem 288, 24128–24139 (2013)) and several new mutations developed in-house significantly increased the melting temperature compared to V27C L34C. Interestingly, FimH _LD V28C N33C (both sites are shifted by only one residue from the reference FimH _LD V27C L34C) had the highest melting temperature of all FimH _LD mutants (Tm=65.77°C) and had a ΔTm of 2.81°C in the presence of methyl alpha-D-mannopyranoside, indicating reduced affinity for the ligand. FimH _LD mutations in the glycine loop region (G15A, G16A) significantly increased thermal stability compared to V27C L34C, and very little shift in melting temperature was observed in the presence of ligand. Glycine loop mutations also slightly increased the thermal stability of FimH-DSG, but no temperature shift was observed in the presence of ligand, collectively suggesting that FimH _LD and FimH-DSG mutants are not stabilized by ligand and may therefore have reduced binding efficiency compared to wild type.

The FimH _LD WT sequence was obtained from the UTI-associated E. coli isolate J96 (Hull, RA et al., Infect Immun 33, 933–938 (1981)). V27A is a natural variant associated with UTI virulent isolates and Crohn's disease-associated isolates (Schwartz, DJ et al., Proc Natl Acad Sci USA 110, 15530–15537 (2013); Cespedes et al., Front Microbiol 8:639 (2017)). Incorporation of V27A into FimH _LD slightly decreased the melting temperature of FimH _LD WT, and a weaker shift was observed for V27A in the presence of methyl alpha-D-mannopyranoside compared to WT. On the other hand, V27A appeared to have a stabilizing effect in the context of FimH _LD mutants G15A, G16A, G15P, G16P at the glycine loop, all of which had higher melting temperature with V27A compared to the absence of this mutation, and had ΔTm<2°C in the presence of V27A compared to 6.05°C (G16P) without this mutation. Furthermore, FimH-DSG mutants containing V27A had slightly improved thermal stability, and did not show a temperature shift in the presence of ligand. Together, this suggests that V27A has a stabilizing effect on the melting temperature of FimH and reduces the efficiency of ligand binding.

Several FimH _LD disulfide and non-polar to polar mutants were expressed at low levels and either had poor thermal stability or showed significant temperature shifts in the presence of the mannoside compound, suggesting that they retained ligand binding efficiency (Table 12). These mutants were tested in single replicates and excluded from further analysis. The thermal stability of several other FimH-DSG mutants was similarly analyzed, two of which showed improved thermal stability and a reduced shift in the presence of ligand (FimH-DSG V27A Q133K and FimH-DSG G15A G16A V27A Q133K) (Table 13). The Q133K mutation is a mutation in the FimH binding pocket that abolishes ligand binding, as described previously (Schwartz et al., Proc Natl Acad Sci USA 110:15530–15537 (2013)). These mutants were excluded from further analysis.

Example 15: Identification of FimH mutants with reduced affinity for mannoside ligand

The dissociation constants (Kd) of the FimH mutants for the mannoside ligand were determined using fluorescence polarization assays upon direct binding to fluorescein-conjugated octylbiphenylmannopyranoside (BPMP) ligand. The Kd values of the FimH _LD mutants relative to WT are shown in Table 14. FimH _LD WT and V27A showed similar high affinity for BPMP. The reference fixed mutant FimH _LD V27C L34C (Kisiela, DI et al., Proc Natl Acad Sci USA 110, 19089–19094 (2013); Rodriguez, VV et al., J Biol Chem 288:24128–24139 (2013)) had 91-fold lower ligand affinity compared to FimH _LD WT, while the affinity of FimH _LD R60P V27A (Rabbani et al., J Biol Chem 293:1835–1849 (2018)) was 179-fold lower. The mutants described here were compared with the reference fixed mutants. The FimH _LD glycine loop mutants G15A V27A, G15P V27A, G16P V27A and G16A G16A V27A showed no binding, while the Kd of G16A V27A was 156-fold higher than that of WT. All glycine loop mutations in combination with V27A showed significantly higher Kd than the glycine loop mutants alone, suggesting that V27A has a statistically indeterminate stabilizing effect, albeit with little effect on the Kd of FimH _LD WT. Inclusion of V27A also further reduced the binding affinity of FimH _LD R60P.

In this assay, three new mutants with fixing disulfide bonds were tested, all of which had a moderate decrease in ligand binding affinity compared to FimH _LD WT (33-43-fold lower affinity). FimH _LD mutants containing non-polar-to-polar mutations (A119T V27A, A119N V27A, L34T V27A, L34N V27A) had high affinity for BPMP similar to that of FimH _LD WT. FimH _LD F1 mutants showed poor binding, with the exception of F1Y with a binding affinity similar to that of FimH _LD WT.

The ligand binding affinities of the FimH-DSG constructs are shown in Table 15. The Kd value of FimH-DSG WT is more than 100-fold higher than that of FimH _LD WT, likely reflecting the different conformational states of the two FimH forms. FimH-DSG V27A also had a lower affinity compared to FimH-DSG WT. This is consistent with previous data showing that full-length FimH with A27 in complex with FimC and FimG has reduced binding affinity for mannoside compared to FimH V27 (Schwartz et al., Proc Natl Acad Sci USA 110:15530–15537 (2013)). Introduction of the V27C L34C fixation mutation into FimH-DSG reduced the affinity for BPMP by 2.5-fold, while the glycine loop mutant of FimH-DSG G15A G16A V27A had a 28-fold lower affinity compared to FimH-DSG WT. No Kd could be calculated for FimH-DSG G15A V27A and FimH-DSG G16A V27A, indicating that these mutants cannot bind BPMP. Mutations designed to stabilize FimH-DSG in the open conformation by altering the interface of the pilin-lectin domain (A115I, V185I) improved the binding affinity compared to FimH-DSG WT, as judged by the thermostability data (Example 14).

Thus, mutations in the glycine loop of the FimH _LD or FimH-DSG protein were identified that had very low binding affinity for BPMP. Glycine mutants were selected for evaluation in functional immunogenicity studies in mice based on these data and thermostability data.

Example 16: Confirmation of the conformational state of FimH mutants using circular dichroism spectroscopy

The secondary and tertiary structures of FimH _LD and FimH-DSG mutants showing increased thermal stability and reduced binding affinity for the mannoside ligand (examples 14 and 15) were analyzed by circular dichroism (CD). Wild-type FimH _LD and conformationally fixed FimH _LD mutants have different tertiary CD profiles (Rabbani et al., J Biol Chem 293:1835–1849 (2018)). The secondary and tertiary structures of selected FimH _LD and FimH-DSG mutants and the wild type were investigated using far-UV CD (secondary structure) and near-UV CD (tertiary structure) (see Fig. 1). The far-UV CD spectrum of FimH _LD is consistent with previously published data (Rabbani et al. J Biol Chem 293:1835–1849 (2018)), and the far-UV spectrum of FimH _LD and FimH-DSG is characteristic of a protein with a high beta-sheet content. The far-UV spectrum of FimH _LD V27C L34C was slightly different from that of wild-type FimH _LD , as observed by others (Rabbani et al. J Biol Chem 293:1835–1849 (2018)), reflecting the open conformational state. The secondary structure profile of the natural mutant FimH _LD V27A was also slightly different. Overall, the secondary structures of the FimH _LD or FimH-DSG mutants were very similar to those of the wild-type proteins (Fig. 1), suggesting that the overall secondary structure was not altered in these mutants. The tertiary structure profiles of the FimH _LD mutants obtained by us had very similar spectra, which also matched the published CD spectra for FimH _LD V27 L34C and V27A R60P stabilized in the open conformational state (Rabbani et al. J Biol Chem 293:1835–1849 (2018)). The profiles of the mutants described here were also significantly different from those of the wild-type FimH _LD or FimH _LD V27A. Taken together, these data suggest that the introduced mutations shift the conformation of FimH _LD towards an open conformation, while the tertiary structure of FimH-DSG remains largely unchanged upon introduction of the conformation-stabilizing mutations analyzed here.

Example 17: Characterization of FimH mutants using neutralizing monoclonal antibodies

The conformations of several selected FimH antigens were characterized by biolayer interferometry using monoclonal antibodies specific for the lectin domains 299-3, 304-1, and 440-2. Competition assay experiments (not shown) demonstrated that antibodies 229-3 and 304-1 bind to the same epitopes of the ligand-binding site as MAbs 475 and 926 (Kisiela et al., Proc Natl Acad Sci USA 110:19089–19094 (2013)). Monoclonal antibody 440-2 binds to a different epitope and appears to preferentially bind to the FimH _LD in the open conformation. Antibodies 229-3 and 304-1 were able to recognize all FimH _LD (Table 16) and FimH-DSG (Table 17) variants, although the level of binding of FimH _LD V27A was lower. In contrast, responses to antibody 440-2 were higher in all FimH _LD or FimH-DSG mutants compared to WT or V27A. This is consistent with the CD spectroscopic profiles shown in Fig. 1, suggesting that the FimH _LD mutants are in an open conformation. The response to 440-2 was also increased in FimH-DSG WT, which, in combination with the overlapping CD spectroscopic profiles of FimH-DSG and WT mutants (Example 16), suggests that this protein is in an open conformation regardless of the presence or absence of stabilizing mutations.

Example 18: Neutralization data for the FimH mutant

To assess the relative immunogenicity of the selected mutants, mice were vaccinated with the FimH mutants. The ability of the FimH mutants to elicit functional antibody titers was quantified using the whole yeast mannan neutralization assay described above and previously published (International PCT Publication No. WO 2021/084429, published May 6, 2021). Briefly, fimbriated E. coli were incubated with serum and allowed to bind to a yeast mannan-coated microtiter plate. The plate was washed and the number of viable E. coli bound to the plate was determined using a fluorescent probe. Serum neutralization titers, which inhibit the binding of fimbriated bacteria to yeast mannan, were determined from an eight-point, two-fold dilution series of sera from vaccinated mice. Titers are the reciprocal of the serum dilution at which 50% of bacteria remain bound to the plate. A summary of mean titers and responses is shown in Table 18. Plots of individual IC ₅₀ responses of mice after doses 2 and 3 are shown in Fig. 2 and Fig. 3.

In a previous work (International PCT Publication No. WO 2021/084429, published May 6, 2021), it was shown that the previously described FimH _LD V27C L34C mutant with locking disulfide bonds (Kisiela et al., Proc Natl Acad Sci USA 110:19089–19094 (2013)) did not improve functional immunogenicity compared to FimH _LD WT. A direct comparison of the functional immunogenicity of the new FimH _LD mutants with that of another previously described conformationally restricted FimH _LD V27A R60P mutant (Rabbani et al., J Biol Chem 293:1835–1849 (2018)) is shown in Fig. 2. FimH _LD G16A V27A, FimH _LD G15A G16A V27A, and FimH _LD V27A R60P mutants resulted in higher numbers of responding mice and higher titers (p-value<0.05) than FimH _LD WT. The other mutations (G15A V27A, G16P V27A, V28C N33C) did not significantly increase functional immunogenicity, and although high titers were observed in mice that did respond, the numbers of responding mice in these groups were similar to those in the FimH _LD WT group. Thus, several mutants designed to enhance the functional immunogenicity of FimH _LD by locking FimH _LD in an open conformation improved functional immunogenicity compared to FimH _LD WT. Following 2 doses of FimH _LD and FimH-DSG mutants, neutralizing titers were observed in significantly higher numbers of animals in the FimH-DSG vaccinated groups compared to the FimH _LD group (Fig. 3). This trend continued after the 3rd dose, where 95-100% of mice responded in the FimH-DSG V27A, FimH-DSG G15A V27A and FimH-DSG G15A G16A V27A vaccinated groups. IC ₅₀ geometric mean titers (GMT) were also significantly higher in all groups vaccinated with FimH-DSG mutants after dose 3. Similar FimH-DSG mutants (V27A, G15A, V27A, G15A, G16A, V27A) generated higher GMT values compared to FimH _LD mutants (p-value<0.05).

Example 19: FimH-DSG G15A G16A V27A does not bind to host glycans and can be isolated in a homogeneous state

The mutant proteins FimH-DSG WT and FimH-DSG G15A G16A V27A were expressed in CHO cells as secreted proteins containing C-terminal His-tags. Purification of recombinant His-tagged forms of FimH-DSG was performed as shown in Fig. 4.

After ultrafiltration and diafiltration, the cell-free culture medium containing FimH-DSG WT was isolated on nickel affinity resin and subjected to cation exchange chromatography. The eluted peak was quite broad and had several distinct shoulders, indicating possible heterogeneity of the FimH-DSG WT species (Fig. 5). Interestingly, when the eluted fractions were analyzed by SDS-PAGE, only single bands corresponding to FimH-DSG WT were detected in each fraction.

During the purification process, FimH-DSG exhibited properties that indicated its poor solubility. In particular, Ni-Sepharose eluates of FimH-DSG WT always appeared turbid upon visual inspection. A shift to the acidic pH side (4.3) for subsequent purification on SP-Sepharose led to clarification of the protein solution. However, the poor solubility of FimH-DSG WT preparations and the tendency to aggregation were again observed after transfer (dialysis) of the isolated protein in TBS, pH 7.4. This led to a gradual loss of protein due to aggregation and precipitation. Removal of the precipitate by centrifugation did not stop or slow down the aggregation process, although the protein concentration at this point usually decreased to 0.2-0.4 mg/mL. Protein loss due to aggregation can be monitored in the presence of 10% glycerol included in the storage buffer (TBS, pH 7.4). However, the presence of glycerol did not prevent the formation of soluble HMW aggregates, which were detected spectrophotometrically by light scattering at 350 nm.

When the same process was used to isolate the FimH-DSG G15A G16A V27A mutant, several differences were observed. First, these included the absence of any signs of turbidity when eluting the protein on the Ni-Sepharose column. Second, the peak profile eluted on the SP-Sepharose columns was not as broad as that observed for FimH-DSG WT in Fig. 6. Finally, the FimH-DSG G15A G16A V27A mutant remained completely soluble after transfer to physiological pH buffer (TBS pH 7.4). At concentrations up to 5-6 mg/mL, the FimH-DSG G15A G16A V27A mutant showed no signs of aggregation or precipitation.

Analysis of isolated WT FimH-DSG and the G15A G16A V27A FimH-DSG mutant also revealed additional differences between the two FimH-DSG variants. Analytical size exclusion chromatography (SEC) demonstrated that the G15A G16A V27A FimH-DSG mutant eluted as a single peak with a retention time consistent with its molecular weight. In contrast, the elution profile of wild-type FimH-DSG consisted of multiple peaks where the retention time of the major peak was shorter than that of the mutant shown in Fig. 7. These data clearly indicate that WT FimH-DSG forms high molecular weight complexes detectable by SEC. The presence of HMW complexes formed by WT FimH-DSG and the propensity to aggregate may be related to the functional activity of its N-terminal lectin-binding domain. We hypothesized that during CHO fermentation and secretion into the culture medium, WT FimH-DSG binds to glycan molecule(s) released from the host CHO cell surface. Due to the branched nature of glycans, each glycan may contain more than one copy of a FimH-DSG molecule. Continuous decoration of the glycan with increasing amounts of FimH-DSG could lead to the formation of different HMW complexes and, ultimately, to loss of solubility and precipitation (see Fig. 8). To test this hypothesis, isolated wild-type and mutant FimH-DSG (and FimH _LD ) species were analyzed by high pH anion exchange chromatography with pulsed amperometric (electrochemical) detection (HPAEC-PAD). This method allows the identification of oligosaccharides or glycans in a protein sample and provides information on the composition of these oligosaccharides. Briefly, acid hydrolysis is performed to release monosaccharides followed by peak analysis against a monosaccharide standard. The results of HPAEC-PAD analysis showed that the isolated preparations of FimH-DSG WT (glycosylated) and FimH _LD (non-glycosylated, data not shown) contained significant amounts of monosaccharides. The summary information of the identified monosaccharides in FimH-DSG WT and the FimH-DSG G15A G16A V27A mutant is shown in Table 19. The monosaccharide content of the FimH-DSG G15A G16A V27A mutant was significantly lower than that of FimH-DSG WT. Furthermore, it is possible that the low monosaccharide content detected represents the sugar moieties of the N-glycan that are predicted to modify N235.

Example 20: FimH _LD and FimH-DSG G15A G16A V27A mutants do not bind to E. coli O-antigens

The repeat units of the E. coli O8 and O9 O-antigens are composed of polymannose residues. This raises the question of whether FimH can bind to the O-antigen conjugates in a combined FimH-O-antigen conjugate vaccine. Biolayer interferometry experiments were designed to test the ability of FimH _LD WT to bind either free O9 polysaccharide or CRM-conjugated O9 polysaccharide and the ability of the FimH _LD G15A G16A V27A mutant, which showed undetectable affinity for the mannoside ligand in binding assays. The O-antigen binding data are shown in Table 20. At high concentrations of free polysaccharide, a response was observed for FimH _LD WT. Higher responses were observed with the CRM-conjugated polysaccharide. However, no detectable binding was observed for the FimH _LD G15A G16A V27A mutant protein. Similar experiments were performed for FimH-DSG, where the ability of the FimH-DSG G15A G16A V27A mutant to bind to free or CRM-conjugated O-antigens of different serotypes (O9, O25b, O1a and O2) was tested (see Table 21). As expected from the mannoside ligand binding properties, the overall binding affinity of FimH-DSG WT was significantly lower than that of the corresponding FimH _LD variant. At the highest concentration of CRM-conjugate, some titratable binding was observed for FimH-DSG WT, with binding levels only slightly above the background levels observed for the free polysaccharide. As with the FimH _LD G15A G16A V27A mutant, the FimH-DSG G15A G16A V27A protein did not bind to any of the free or CRM-conjugated polysaccharides. In conclusion, unlike the parental FimH _LD WT or FimH-DSG WT antigens, the derived G15A G16A V27A mutants cannot bind 0-antigens, providing opportunities for the development of a FimH and O-antigen combination vaccine.

Example 21. Non-human primates vaccinated with mutant FimH-DSG G15A G16A V27A with and without O-antigens A.

Methods

1. FimH IgG dLIA

The mutant E. coli fimbrial antigen FimH-DSG G15A Gl6A V27A bound to spectrally distinct MagPlex-C microbeads (Luminex) was diluted in blocking buffer to a concentration of 50,000 beads/mL for 1-2 h at room temperature with shaking immediately prior to the primary incubation prior to the assay. The diluted bead mixture was added to assay plates containing appropriately diluted non-human primate serum samples, controls, and a reference standard, an in-house humanized monoclonal antibody (FimH Y202) that binds to the pilin domain of FimH-DSG, for overnight incubation at 2-8°C with shaking. After washing away unbound components, purified secondary R-phycoerythrin-conjugated goat anti-human IgG Fcγ antibody (Jackson ImmunoResearch Laboratories, 109-116-170) was added to the bead mixture and incubated for 90 min at room temperature with shaking. The magnitude of the PE fluorescent signal, measured by a Luminex FLEXMAP 3D reader, was directly proportional to the amount of anti-FimH-DSG IgG bound to the protein-coupled beads. Data were analyzed using a custom SAS application that used a log-linear regression model of a standard curve to interpolate antigen-specific antibody concentrations (μg/mL) from the mean fluorescence intensity. The lower limit of quantification (LLOQ) of 0.763 μg/mL was calculated based on the shift of the standard curve.

2. 4-valent O-Ag IgG dLIA

E. coli long O-antigen polysaccharides of serotypes O25b, O1a, O2, and O6 were covalently conjugated to poly-L-lysine, and the resulting conjugates were coupled to spectrally distinct MagPlex-C microbeads (Luminex) using a standard EDC/NHS-mediated coupling reaction protocol.

The beads were incubated overnight at 2-8°C with serially diluted nonhuman primate serum samples, controls, and polyclonal standard with shaking. After washing, bound serotype-specific IgG was detected with a PE-conjugated goat anti-human IgG Fcγ secondary antibody (Jackson ImmunoResearch Laboratories, 109-115-098) after incubation for 90 min at room temperature with shaking. Fluorescence was measured with a Luminex 200 plate reader for each of four spectrally distinct regions and expressed as mean fluorescence intensity. The plot of the standard titration curve of the polyclonal standard yielded a linear graph expressed in arbitrary units, from which signals could be interpolated as serotype-specific antibody levels (U/mL).

3. Non-human primates (NHP)

Female Cynomolgus Macagues (Macaca fasicularis) were initially obtained from Charles River Laboratories (Houston, TX) and subsequently transferred to Pfizer, Pearl River, NY (age range: 4-5 years, weight range: 3.1-5.9 kg). NHPs were housed in standard four-bed cages with free access to food and water. Animals were microchipped subcutaneously to monitor core temperature. Only NHPs without E. coli infection based on negative urine qPCR results were included in experimental groups (see Method Section 10 below).

4. Vaccination and blood sampling

Cynomolgus macaques were immunized intramuscularly (0.55 ml) at weeks 0, 4, and 14 with either vehicle control (PBS, pH 6.2), monomeric fimbrial antigen FimH-DSG G15A G16A V27A (50 μg/dose), or a mixture of tetravalent O25b, O1a, O2, and O6 O-antigen polysaccharide conjugates (1 μg/dose) in combination with monomeric fimbrial antigen FimH-DSG G15A G16A V27A (50 μg/dose). Vaccine antigens were adjuvanted with AS01b (50 μg MPL and 50 μg QS-21 per dose).

At weeks 0, 6, and 16, 10 mL of blood was collected via the femoral vein into 1 serum separator tube (BD Vacutainer) using a 21g safety needle/vacutainer. Collection tubes were left at room temperature for 30 min and centrifuged at 3000 g for 10 min. Serum in the supernatant was collected, aliquoted, and stored at 80°C.

5. Clinical isolate of E. coli

One representative clinical isolate of ST131 O25b was selected based on the patient age and the origin of the specimen collection (PFEEC0578, male, age 38, bladder) from UPEC strains collected from the Pfizer-sponsored Antimicrobial Testing Leadership and Surveillance (ATLAS) database maintained by the International Health Management Associates (IHMA) clinical laboratory. The strain carries genes encoding the production of capsular polysaccharides of unknown type.

6. Preparation of UPEC strain

A stock solution of E. coli was prepared by inoculating 12 ml of LB broth (Teknova, #L8198) followed by overnight incubation at 37°C with shaking at 275 rpm. After 18 h, 12 ml of the culture was diluted in 113 ml of LB broth in a 250 ml flask (Corning, #431407). The culture was incubated for 2-3 h at 37°C at ~275 rpm until OD ₆₀₀ values were between 2.1 and 2.7. The culture was supplemented with 25 ml of glycerol (80%, MP, #3055-044). Aliquots of 5 ml were frozen at -80°C for long-term storage. The concentration of viable bacteria in the vial was confirmed by plating serial dilutions of the stock solution onto TSA plates (BD, BBL trypticase soy agar (BBL casein soy agar), catalog no. B21283X) and analyzed after 18 hours of incubation at 30°C.

7. Model of cystitis in non-human primates.

NHPs were anesthetized with a ketamine/dexdominant mixture administered intramuscularly. To prevent bladder contamination during urethral catheterization, the anogenital area was swabbed with sterile gauze soaked in sterile saline and/or antiseptic wipes containing benzalkonium chloride. A sterile 5 French red rubber catheter, pre-coated with Surgi Lube to prevent tissue irritation, was carefully inserted through the urethra into the bladder. The bladder was then emptied of any urine either by natural flow or by syringe aspiration. A 1 mL solution containing 108 ^CFU of UPEC strain PFEEC0578 was infused directly into the bladder through the catheter.

8. Monitoring animals after infection

Following challenge, animals were observed twice daily for 1 week and once daily thereafter. NHPs were observed for 30 days post-challenge. Monitoring included observation of urine output, changes in behavior or appetite, signs of pain/discomfort, and body temperature.

9. Collection of urine through the installation of a catheter.

To collect clear urine samples, the bladders of anesthetized NHPs were catheterized as described above. After catheter placement, the bladders were emptied either by natural flow or by syringe aspiration. When the bladders were void of urine, 10 mL of saline was infused and catheter aspiration was repeated. All collected samples were immediately placed on ice for storage.

10. DNA extraction

Extraction of E. coli DNA from triplicate NHP urine samples was performed using Qiagen Minelute DNA extraction kits (Qiagen, cat. no. 51306, sometimes limited by sample volume collected). The manufacturer's Blood and Body Fluid Spin protocol was used with the following modifications: the initial sample volume was increased to 500 μl (if sample volume allowed), the volume of Buffer AL was increased to 500 μl, the volume of Proteinase K was increased to 50 μl, 50 μl of molecular grade water (Corning Inc., Ref# 46-000-CM) was warmed to 37°C and used in place of Elution Buffer EB. Finally, after addition of molecular grade water at 37°C, the spin columns were incubated for 5 min at room temperature before the final spin.

11. Quantitative real-time PCR (qPCR)

Quantitative real-time PCR (qPCR) was used to assess bacterial load in NHP urine samples. E. coli serotype O25b-specific DNA was amplified by qPCR using the following primers: forward, TTGAAAGTGATGGTTTGGTAAGAAAT (SEQ ID NO: 109); reverse, T GCAGCACGTATGATAACTTCAAAG (SEQ ID NO: 110), and a Fain fluorescent reporter probe with the sequence AGGATATTTTACCCAGCAGTGCCCCGT (SEQ ID NO: 111) was used to quantify replication.

O25b serotype-specific amplicons correspond to portions of the orf10 region of serotype O25b. Custom-designed primers and probe were purchased lyophilized from Integrated DNA Technologies and reconstituted in TE buffer (Corning Inc., cat. no. 46-009-CM) to a concentration of 100 nmol/mL.

DNA samples from the DNA extraction procedure described above were analyzed in Applied Biosystems MicroAmp Optical 96-well reaction plates (Applied Biosystems, catalog # N8010560). qPCR reactions were performed in a total volume of 25 μl using 12.5 μl of Applied Biosystems 2X Taqman Fast Advanced Master Mix (Applied Biosystems, catalog # 4444554), 0.125 μl of each recovered primer, 0.5 μl of probe, 1.75 μl of molecular biology grade water (Corning Inc., catalog # 46-000-CM), and 10 μl of sample per well.

The reaction conditions for DNA amplification were 50°C for 2 min, then 95°C for 2 min and 40 cycles of 95°C for 3 sec and 60°C for 30 sec, which were performed on either an Applied Biosystems 7500 Real-Time PCR System or an Applied Biosystems QuantStudio 6 Real-Time PCR System (Applied Biosystems).

For quantification, a linear standard curve was prepared. Aliquots of the same frozen E. coli stock solution used in each challenge experiment (preparation described above) were diluted to 1 × 10 ⁹ CFU/mL in sterile PBS (Corning, 21-040-CM). Subsequent serial dilutions were performed to obtain solutions containing E. coli at concentrations of 1 × 10 ⁸ , 1 × 10 ⁷ , 1 × 10 ⁶ , 1 × 10 ⁵ , 1 × 10 ⁴ , 1000, 100, and 10 CFU/mL. Serial dilutions were prepared in sterile PBS (Corning, 21-040-CM) or pooled double-filtered NHP urine collected from subjects before inoculation. Dilutions in PBS and pooled, double-filtered NHP urine were later found to be equivalent. The number of viable bacteria present in each dilution was confirmed by plating on TSA plates (BD, BBL Trypticase Soy Agar (Soybean Casein Digest Agar), catalog number B21283X). DNA extraction was performed at each serial dilution using the same methods used to isolate DNA from the samples as described above. These qPCR standards were run on each qPCR assay plate in duplicate.

Linear regression analysis was performed using Applied Biosystems QuantStudio software. Statistical analysis showed that the standard curve constructed behaved linearly between 100 and 1× ¹⁰⁸ bacteria/mL. Therefore, the lower limit of quantification (LLOQ) was defined as 100 bacteria/mL. In some cases, samples reached the fluorescence threshold but in the run corresponding to the amount below the LLOQ, in other cases the fluorescence threshold was not reached at all (value not determined). When any of these conditions occurred, such values are reported herein as the LLOQ value (100 bacteria/mL).

12. ELISA for the determination of myeloperoxidase (MPO)

Invitrogen Myeloperoxidase Instant ELISA kits (Invitrogen, catalog #BMS2038INST) were used to quantify myeloperoxidase (MPO) in NHP urine. PIPES buffer was added to blank urine specimens to a final concentration of 5% with 0.5 M PIPES buffer, pH 6.8 (Alfa Aesar, catalog #J61786-AK). Specimens were vortexed for 15 seconds and then diluted 1:1 with the manufacturer's supplied sample diluent. Specimens were run in duplicate and the remainder of the assay followed the manufacturer's instructions. The endpoint color intensity at 450 nm was measured on a Spectramax Plus instrument (Molecular Devices). The standards included in the assay kit were used to generate a standard curve. For the assay, the low-concentration standard (156.25 pg/mL) of the assay kit was considered the lower limit of detection (LLOD). The accompanying software for the Spectramax instrument (Softmax, Molecular Devices) allows results to be extrapolated beyond the low-concentration standard. The value corresponding to half the LLOD (78.125 pg/mL), or if the assay result was completely below the detection limit, was replaced by any values extrapolated below that value.

13. IL-8 Luminex Assay

Interleukin-8 (IL-8) was measured using the Bio-Rad IL-8 Human Cytokine Screening Panel Luminex Assay custom kit (BioRad Laboratories Inc., catalog #17005177). PIPES buffer was added to neat urine samples to a final concentration of 5% with 0.5 M PIPES buffer, pH 6.8 (Alfa Aesar, catalog #J61786-AK). Samples were vortexed for 15 seconds and then diluted 1:1 with 50% modified LXA-4 buffer (PBS IX, 0.5% BSA, 0.025% sodium azide). Samples were assayed in duplicate and the manufacturer’s instructions were followed for the remainder of the assay. The assay plate was read on a BioPlex 200 Luminex instrument (BioRad Laboratories Inc.). The Bio-Plex 200 Luminex instrument's companion software, BioPlex Manager, and the standards included in the assay kit were used to construct a standard curve and extrapolate sample concentration from fluorescence intensity. The lower limit of quantification (LLOQ) was determined using BioPlex Manager software.

14. Total nuclear cell count and urine sediment analysis under a light microscope.

Within 1 hour of collection, urine samples (500 µL to 1 mL) were fixed with formalin to a final concentration of 1% and shipped overnight on ice to Pfizer Groton, CT. Upon receipt, total nucleated cells (epithelial cells and polymorphonuclear cells) were counted on a hemocytometer. A total of approximately 300 µL was loaded into a Thermo Scientific Shandon EZ Double Cytofunnels: 100 µL in one funnel and 200 µL in the other funnel. Samples were centrifuged for 5 min at 750 rpm on Shandon Double Cytoslide Microscope Slides in a Thermo Scientific CytoSpin 4 Cytocentrifuge. For samples with high cell counts, urine was first diluted 1:10 with 0.9% saline prior to cytocentrifugation. The cytoprepared slides were then briefly fixed with methanol and stained with Giemsa and May-Grünwald stains using the Sysmex SP-10. For each urine sample, one slide per urine sample was prepared with the two sample volumes specified above.

Cytospin slides containing concentrated and stained urine sediment were assessed by light microscopy for the presence or absence of increased polymorphonuclear cells (PMNs, i.e., granulocytes with segmented or kidney-shaped nuclei, usually composed of neutrophils but also including eosinophils and basophils). Cases of no observed PMNs or rarely observed PMNs were defined as the absence of increased PMN cells. Cases of more than rare PMNs observed compared with the background epithelial cell population were defined as the presence of increased PMNs.

B. Results

1. Vaccination with mutant FimH-DSG G15A G16A V27A with and without O-antigens induces the formation of effective total and neutralizing antibodies in non-human primates.

Nonhuman primates were vaccinated with the FimH-DSG G15A G16A V27A mutant with or without the quadrivalent (O25b, 06, O1a, O2) O-antigen conjugate and AS01b adjuvant (Fig. 9). In the group vaccinated with FimH-DSG G15A, G16A, V27A and the quadrivalent O-antigen, O-antigen serotype-specific antibody titers increased approximately 400-fold at 6 weeks post-vaccination (Fig. 10 and Table 22). Total anti-FimH antibody titers were quantified by the Luminex direct immunoassay (dLIA) (Fig. 11A and Table 23). Animals in the placebo group had titers below the limit of quantification. In both vaccinated groups, titers increased after two doses and could be boosted by a third dose. Overall, titers were slightly lower in animals vaccinated with FimH-DSG G15A G16A V27A with O-antigens compared to FimH-DSG G15A G16A V27A administered alone.

The sera were evaluated in an E. coli binding inhibition assay to assess the ability of anti-FimH antibodies to block E. coli binding to yeast mannan (Fig. 11B and Table 24). The mean _IC50 value of sera from animals vaccinated with FimH-DSG G15A G16A V27A alone increased to 293.65 after the 2nd dose and to 1698.39 after the 3rd dose, while the mean _IC50 value of animals vaccinated with FimH-DSG G15A G16A V27A in combination with O-antigens was 480.12 after the 2nd dose and 756.45 after the 3rd dose.

Taken together, these data show that FimH-DSG G15A G16A V27A elicits a strong antibody response in non-human primates, and the combination with O-antigens results in high titers, although somewhat lower than with FimH alone.

2. Vaccination with mutant FimH-DSG G15A G16A V27A with and without O-antigens reduces bacteriuria and infection biomarkers in a non-human primate model.

Five weeks after the final booster vaccination, vaccinated and placebo-treated NHPs were administered ¹⁰⁸ CFU of UPEC isolate PFEEC0578 via intravesical catheterization. Bacteriuria was monitored in urine collected via the catheter for 28 days. In all placebo-treated animals, instillation of live bacteria resulted in high levels of bacteriuria on days 2 and 7 post-challenge (geometric mean approximately ¹⁰⁶ bacteria/mL urine). Compared with the placebo group, animals vaccinated with FimH-DSG G15A G16A V27A or FimH-DSG G15A G16A V27A+4-valent O-antigen demonstrated a 300-fold or 1000-fold reduction in geometric mean bacteriuria on days 2 and 7 post-challenge, respectively.

At day 14, approximately 50% of placebo-vaccinated animals still had bacteriuria, >10 ⁵ bacteria/mL urine. Finally, the majority of placebo-vaccinated NHPs cleared their infection at days 21 and 28. In contrast, the majority of animals vaccinated with FimH-DSG G15A G16A V27A or FimH-DSG G15A G16A V27A+4-valent 0 antigen cleared their infection by day 14 (Fig. 12).

Various inflammatory biomarkers were then monitored in the urine of challenged NHPs. At day 7 post-challenge, all placebo-treated animals demonstrated elevated levels of polymorphonuclear (PMN) cells in the urine sediment, which were confirmed by cytological analysis. In contrast, less than 25% of NHPs vaccinated with FimH-DSG G15A G16A V27A and none of the animals immunized with FimH-DSG G15A G16A V27A+4-valent O-antigen had elevated levels of PMN cells in the urine sediment (Fig. 13C). In parallel, myeloperoxidase (MPO) and interleukin 8 (IL-8) levels were measured in urine samples for 7 days. On day 2 after challenge, a 2-fold decrease in MPO levels (geometric mean ~200 pg/mL) was observed in both vaccinated groups compared to the placebo group (geometric mean 470 pg/mL) (Fig. 13A).

In addition, on days 2 and 7 post-challenge, urinary IL-8 concentrations in FimH-DSG G15A G16A V27A-vaccinated animals were reduced by approximately 10- and 5-fold, respectively (geometric mean 5.9 pg/mL and 9.8 pg/mL), compared to levels measured in the urine of placebo-treated NHPs (geometric mean 54.2 pg/mL and 32.7 pg/mL). In a similar trend, urinary IL-8 levels on days 2 and 7 in the NHP group immunized with FimH-DSG G15A G16A V27A in combination with O-antigens decreased by approximately 5- and 3-fold, respectively (geometric mean 11.3 pg/mL), compared with IL-8 concentrations observed in placebo-treated animals (Fig. 13B).

C. Conclusions

The FimH-DSG G15A G16A V27A mutant induces high anti-FimH IgG titers in NHPs, which can be increased by administering a 3rd dose. Animals vaccinated with a combination of the FimH-DSG G15A G16A V27A mutant with quadrivalent O-antigens showed high IgG titers to the O-antigen.

FimH-DSG G15A G16A V27A induces potent neutralizing antibodies in non-human primates. Combination with tetravalent O-antigens is also immunogenic.

The FimH-DSG G15A G16A V27A mutant with or without quadrivalent O-antigens reduces bacteriuria and infection biomarkers in a cynomolgus macaque urinary tract infection model.

The following paragraphs describe additional aspects of the invention:

C1. A mutant FimH polypeptide comprising at least one amino acid mutation relative to the amino acid sequence of a wild-type FimH polypeptide, wherein the position of the mutation 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, wherein the amino acid positions are numbered according to SEQ ID NO: 59.

C2. A mutant FimH polypeptide according to item C1, comprising at least one mutation 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; L107C; 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.

C3. A mutant FimH polypeptide according to item C2, containing mutations G15A and G16A.

C4. A mutant FimH polypeptide according to item C2, containing the mutations P12C and A18C.

C5. A mutant FimH polypeptide according to item C2, containing the mutations G14C and F144C.

C6. A mutant FimH polypeptide according to item C2, containing the mutations P26C and V35C.

C7. A mutant FimH polypeptide according to item C2, containing the mutations P26C and V154C.

C8. A mutant FimH polypeptide according to item C2, containing the mutations P26C and V156C.

C9. A mutant FimH polypeptide according to item C2, containing the mutations V27C and L34C.

C10. A mutant FimH polypeptide according to item C2, containing the mutations V28C and N33C.

C11. A mutant FimH polypeptide according to item C2, containing the mutations V28C and P157C.

C12. A mutant FimH polypeptide according to item C2, containing the mutations Q32C and Y108C.

C13. A mutant FimH polypeptide according to item C2, containing the mutations N33C and L109C.

C14. A mutant FimH polypeptide according to item C2, containing the mutations N33C and P157C.

C15. A mutant FimH polypeptide according to item C2, containing the mutations V35C and L107C.

C16. A mutant FimH polypeptide according to item C2, containing the mutations V35C and L109C.

C17. A mutant FimH polypeptide according to item C2, containing the mutations S62C and T86C.

C18. A mutant FimH polypeptide according to item C2, containing the mutations S62C and L129C.

C19. A mutant FimH polypeptide according to item C2, containing the mutations Y64C and L68C.

C20. A mutant FimH polypeptide according to item C2, containing the mutations Y64C and A127C.

C21. A mutant FimH polypeptide according to item C2, containing the mutations L68C and F71C.

C22. A mutant FimH polypeptide according to item C2, containing the mutations V112C and T158C.

C23. A mutant FimH polypeptide according to item C2, containing the mutations S113C and G116C.

C24. A mutant FimH polypeptide according to item C2, containing the mutations S113C and T158C.

C25. A mutant FimH polypeptide according to item C2, containing the mutations V118C and V156C.

C26. A mutant FimH polypeptide according to item C2, containing the mutations A119C and V155C.

C27. A mutant FimH polypeptide according to item C2, containing the mutations L34N and V27A.

C28. A mutant FimH polypeptide according to item C2, containing the mutations L34S and V27A.

C29. A mutant FimH polypeptide according to item C2, containing the mutations L34T and V27A.

C30. A mutant FimH polypeptide according to item C2, containing the mutations L34D and V27A.

C31. A mutant FimH polypeptide according to item C2, containing the mutations L34E and V27A.

C32. A mutant FimH polypeptide according to item C2, containing the mutations L34K and V27A.

C33. A mutant FimH polypeptide according to item C2, containing the mutations L34R and V27A.

C34. A mutant FimH polypeptide according to item C2, containing the mutations A119N and V27A.

C35. A mutant FimH polypeptide according to item C2, containing the mutations A119S and V27A.

C36. A mutant FimH polypeptide according to item C2, containing the mutations A119T and V27A.

C37. A mutant FimH polypeptide according to item C2, containing the mutations A119D and V27A.

C38. A mutant FimH polypeptide according to item C2, containing the mutations A119E and V27A.

C39. A mutant FimH polypeptide according to item C2, containing the mutations A119K and V27A.

C40. A mutant FimH polypeptide according to item C2, containing the mutations A119R and V27A.

C41. A mutant FimH polypeptide according to item C2, containing the mutations G15A and V27A.

C42. A mutant FimH polypeptide according to item C2, containing the mutations G16A and V27A.

C43. A mutant FimH polypeptide according to item C2, containing the mutations G15P and V27A.

C44. A mutant FimH polypeptide according to item C2, containing the mutations G16P and V27A.

C45. A mutant FimH polypeptide according to item C2, containing the mutations G15A, G16A and V27A.

C46. A mutant FimH polypeptide according to item C2, containing the mutations G65A and V27A.

C47. A mutant FimH polypeptide according to item C2, containing the mutations V27A and Q133K.

C4. A mutant FimH polypeptide according to item C2, containing the mutations G15A, G16A, V27A and Q133K.

C49. A mutant FimH polypeptide according to item C2, comprising the sequence of any of SEQ ID NOs: 2-58 and 60-64.

C50. A mutant FimH polypeptide according to any one of paragraphs C1-C49, wherein said polypeptide is isolated.

C51. A pharmaceutical composition comprising (i) a mutant FimH polypeptide according to any one of paragraphs C1-C50 and (ii) a pharmaceutically acceptable carrier.

C52. An immunogenic composition comprising a mutant FimH polypeptide according to any one of paragraphs C1-C50.

C53. An immunogenic composition according to item C52, further comprising at least one additional antigen.

C54. An immunogenic composition according to item C53, wherein at least one additional antigen is a saccharide or polysaccharide, or a glycoconjugate, or a protein.

C55. An immunogenic composition according to item C52, further comprising at least one adjuvant.

C56. A nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of a mutant FimH polypeptide according to any one of paragraphs C1-C49.

C57. A mutant FimH polypeptide according to any one of paragraphs C1-C50, wherein said polypeptide is immunogenic.

C58. A recombinant mammalian cell comprising a polynucleotide encoding a mutant FimH polypeptide according to any one of paragraphs C1-C50.

C59. A culture containing a recombinant cell according to item C58, wherein the size of said culture is at least 5 liters.

C60. A method for producing a mutant FimH polypeptide according to any one of paragraphs C1-C50, comprising culturing a recombinant mammalian cell according to paragraph C58 under suitable conditions, thereby providing expression of the polypeptide; and collecting the polypeptide.

C61. A method for (i) inducing an immune response in a subject to extraintestinal pathogenic E. coli or (ii) inducing the formation of opsonophagocytic and/or neutralizing antibodies in a subject specific to extraintestinal pathogenic E. coli, wherein the method comprises administering to the subject an effective amount of a composition according to any one of paragraphs C51-C55.

C62. The method according to item C61, wherein the subject is at risk of developing a urinary tract infection.

C63. The method of C61, wherein the subject is at risk of developing bacteremia.

C64. The method according to paragraph C61, wherein the subject is at risk of developing sepsis.

C65. A method for inducing an immune response against E. coli in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of paragraphs C51-C55.

C66. The method according to item C65, wherein the immune response includes opsonophagocytic and/or neutralizing antibodies to E. coli.

C67. The method according to item C65, wherein the immune response provides protection to the mammal from E. coli infection.

C68. A method for preventing, treating or alleviating a bacterial infection, disease or condition in a subject, comprising administering to the subject an immunologically effective amount of a composition according to any one of paragraphs C51-C55.

C69. The immunogenic composition of claim C54, wherein the additional antigen is a saccharide comprising a structure selected from 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:K 2; 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 O45 rel), 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 62D ₁ , Formula O63, Formula O64, Formula O65, Formula O66, Formula O68, Formula O69, Formula O70, Formula O71, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O74, Formula O75, Formula O76, Formula O77, Formula O78, Formula O79, Formula O80, Formula O81, Formula O82, Formula O83, Formula O84, Formula O85, Formula O86, Formula O87, Formula O88, Formula O89, Formula O90, Formula O91, Formula O92, Formula O93, Formula O95, Formula O96, Formula O97, Formula O98, Formula O99, Formula O100, Formula O101, Formula O102, Formula O103, Formula O104, Formula O105, Formula O106, Formula O107, Formula O108, Formula O109, Formula O110, Formulas O111, Formulas O112, Formulas O113, Formulas O114, Formulas O115, Formulas O116, Formulas O117, Formulas O118, Formulas O119, Formulas O120, Formulas O121, Formulas O123, Formulas O124, Formulas O125, Formulas O126, Formulas O127, Formulas O128, Formulas O129, Formulas O130, Formulas O131, Formulas O132, Formulas O133, Formulas O134, Formulas O135, Formulas O136, Formulas O137, Formulas O138, Formulas O139, Formulas O140, Formulas O141, Formulas O142, Formulas O143, Formulas O144, Formulas O145, Formulas O146, Formulas O147, Formulas O148, Formulas O149, Formulas O150, Formulas O151, Formulas 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 O17 O, 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, where n is an integer from 1 to 100.

C70. The immunogenic composition of claim C69, wherein the saccharide comprises a structure selected from 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 O10, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O21, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O28, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45 rel), Formula O55, Formula O56, Formula O58, Formula O64, Formula O69, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O75, Formula O77, Formula O78, Formula O86, Formula O88, Formula O90, Formula O98, Formula O104, Formula O111, Formula O113, Formula O114, Formula O119, Formula O121, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O136, Formula O138, Formula O141, Formula O142, Formula O143, Formula O147, Formula O149, Formula O152, Formula O157, Formula O158, Formula O159, Formula O164, Formula O173, Formula 62D ₁ , Formula O22, Formula O35, Formula O65, Formula O66, Formula O83, Formula O91, Formula O105, Formula O116, Formula O117, Formula O139, Formula O153, Formula O167 and Formula O172, where n is an integer from 31 to 100.

C71. The immunogenic composition of claim C70, wherein the saccharide comprises a structure selected from 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 O10, Formula O16, Formula O17, Formula O18 (e.g., Formula O18A, Formula O18ac, Formula O18A1, Formula O18B, and Formula O18B1), Formula O21, Formula O23 (e.g., Formula O23A), Formula O24, Formula O25 (e.g., Formula O25a and Formula O25b), Formula O26, Formula O28, Formula O44, Formula O45 (e.g., Formula O45 and Formula O45 rel), Formula O55, Formula O56, Formula O58, Formula O64, Formula O69, Formula O73 (e.g., Formula O73 (strain 73-1)), Formula O75, Formula O77, Formula O78, Formula O86, Formula O88, Formula O90, Formula O98, Formula O104, Formula O111, Formula O113, Formula O114, Formula O119, Formula O121, Formula O124, Formula O125, Formula O126, Formula O127, Formula O128, Formula O136, Formula O138, Formula O141, Formula O142, Formula O143, Formula O147, Formula O149, Formula O152, Formula O157, Formula O158, Formula O159, Formula O164, Formula O173 and formulas 62D ₁ , where n is an integer from 31 to 100.

C72. An immunogenic composition according to claim C70, comprising a structure selected from formula O1 (e.g., formula O1A, formula O1B, and formula O1C), formula O2, formula O6 (e.g., formula O6:K2; K13; K15, and formula O6:K54), formula O15, formula O16, formula O21, formula O25 (e.g., formula O25a, and formula O25b), and formula O75.

C73. An immunogenic composition according to item C70, comprising a structure selected from formula O4, formula O11, formula O21 and formula O75.

C74. The immunogenic composition of claim C69, wherein the saccharide does not comprise a structure selected from formula O8, formula O9a, formula O9, formula O20ab, formula O2Oac, formula O52, formula 097, and formula O101.

C75. The immunogenic composition of claim C69, wherein the saccharide does not comprise a structure selected from formula O12.

C76. An immunogenic composition according to item C72, wherein the saccharide is obtained by expressing a protein of the wzz family in a gram-negative bacterium to form said saccharide.

C77. An immunogenic composition according to item C7b, wherein the protein of the wzz family is selected from the group consisting of wzzB, wzz, wzzSF, wzzST, fepE, wzzfepE, wzz1 and wzz2.

C78. The immunogenic composition of claim C76, wherein the wzz family protein is wzzB.

C79. The immunogenic composition of claim C76, wherein the wzz family protein is fepE.

C80. The immunogenic composition of claim C76, wherein the wzz family protein is wzzB and fepE.

C81. An immunogenic composition according to claim C76, wherein the wzz family protein is obtained from Salmonella enterica.

C82. The immunogenic composition of claim C76, wherein the wzz family protein comprises a sequence selected from any 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.

C83. An immunogenic composition according to item C76, wherein the wzz family protein comprises a sequence that is at least 90% identical to any of the sequences of SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116.

C84. The immunogenic composition of claim C76, wherein the wzz family protein comprises a sequence selected from any of SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120 and SEQ ID NO: 121.

C85. An immunogenic composition according to item C69, wherein the saccharide is synthesized synthetically.

C86. An immunogenic composition according to any one of paragraphs C69-C85, wherein the saccharide additionally comprises an R1 fragment of E. coli.

C87. An immunogenic composition according to any one of paragraphs C69-C85, wherein the saccharide additionally comprises an R2 fragment of E. coli.

C88. An immunogenic composition according to any one of paragraphs C69-C85, wherein the saccharide further comprises an R3 fragment of E. coli.

C89. An immunogenic composition according to any one of paragraphs C69-C85, wherein the saccharide additionally comprises an R4 fragment of E. coli.

C90. An immunogenic composition according to any one of paragraphs C69-C85, wherein the saccharide further comprises a K-12 fragment of E. coli.

C91. An immunogenic composition according to any one of paragraphs C69-C90, wherein the saccharide additionally comprises a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) fragment.

C92. An immunogenic composition according to any one of paragraphs C69-C85, wherein the saccharide does not additionally contain an R1 fragment of E. coli.

C93. An immunogenic composition according to any one of paragraphs C69-C85, wherein the saccharide does not additionally contain an R2 fragment of E. coli.

C94. An immunogenic composition according to any one of paragraphs C69-C85, wherein the saccharide does not additionally contain an R3 fragment of E. coli.

C95. An immunogenic composition according to any one of paragraphs C69-C85, wherein the saccharide does not additionally contain an R4 fragment of E. coli.

C96. An immunogenic composition according to any one of paragraphs C69-C85, wherein the saccharide does not additionally contain a K-12 fragment of E. coli.

C97. An immunogenic composition according to any one of paragraphs C69-C90, wherein the saccharide does not additionally contain a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) moiety.

C98. An immunogenic composition according to any one of paragraphs C69-C91, wherein the saccharide does not contain lipid A.

C99. An immunogenic composition according to any one of paragraphs C69-C98, wherein the polysaccharide has a molecular weight of from 10 kDa to 2000 kDa or from 50 kDa to 2000 kDa.

C100. An immunogenic composition according to any one of paragraphs C69-C99, wherein the saccharide has an average molecular weight of 20-40 kDa.

C101. An immunogenic composition according to any one of paragraphs C69-C100, wherein the saccharide has an average molecular weight of from 40,000 to 60,000 kDa.

C102. An immunogenic composition according to any one of paragraphs C69-C101, wherein n is an integer from 31 to 90.

C103. An immunogenic composition comprising a mutant FimH polypeptide according to any one of paragraphs C69-C50 and a conjugate comprising a saccharide covalently linked to a carrier protein, wherein the saccharide is obtained from E. coli.

C104. An immunogenic composition comprising a mutant FimH polypeptide according to any one of claims C69-C50 and a conjugate comprising a saccharide according to any one of claims C69-C102 covalently linked to a carrier protein.

C105. An immunogenic composition comprising a mutant FimH polypeptide according to any one of claims C69 to C50 or a fragment thereof; and a conjugate according to any one of claims C69 to C102, wherein the carrier protein is selected from any of: poly(L-lysine), CRM ₁₉₇ , diphtheria toxin fragment B (DTFB), C8 DTFB, diphtheria toxoid (DT), tetanus toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid, or Pseudomonas aeruginosa exotoxin A. detoxified P. aeruginosa exotoxin A (EPA), maltose binding protein (MBP), detoxified S. aureus hemolysin A, clumping factor A, clumping factor B, cholera toxin subunit B (CTB), Streptococcus pneumoniae pneumolysin and its detoxified variants, C. jejuni AcA, natural C. jejuni glycoproteins and streptococcal C5a peptidase (SCP).

C106. An immunogenic composition according to any one of paragraphs C103-C105, wherein the carrier protein is CRM ₁₉₇ .

C107. An immunogenic composition according to any one of paragraphs C103-C105, wherein the carrier protein is tetanus toxoid (TT).

C108. An immunogenic composition according to any one of paragraphs C103-C105, wherein the carrier protein is poly(L-lysine).

C109. An immunogenic composition according to any one of paragraphs C103-C107, wherein the conjugate is obtained by reductive amination.

C110. An immunogenic composition according to any one of paragraphs C103-C107, wherein the conjugate is prepared using CDAP chemistry.

C111. An immunogenic composition according to any one of paragraphs C103-C107, wherein the conjugate is a conjugated saccharide with one terminal bond.

C112. An immunogenic composition according to any one of paragraphs C103-C107, wherein the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer.

C113. An immunogenic composition according to claim C112, wherein the saccharide is conjugated to the carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the saccharide is covalently linked to the eTEC spacer by a carbamate bond, and wherein the carrier protein is covalently linked to the eTEC spacer by an amide bond.

C114. An immunogenic composition according to any one of claims C112-C113, wherein CRM ₁₉₇ comprises from 2 to 20 or from 4 to 16 lysine residues covalently linked to the polysaccharide via an eTEC spacer.

C115. An immunogenic composition according to any one of paragraphs C103-C114, wherein the saccharide:carrier protein ratio (weight/weight) is from 0.2 to 4.

C116. An immunogenic composition according to any one of paragraphs C103-C114, wherein the ratio of saccharide to protein is not less than 0.5 and not more than 2.

C117. An immunogenic composition according to any one of paragraphs C103-C114, wherein the ratio of saccharide to protein is from 0.4 to 1.7.

C118. An immunogenic composition according to any one of claims C111 to C117, wherein the saccharide is conjugated to the carrier protein via a 3-deoxy-d-manno-oct-2-ulosonic acid (KDO) residue.

C119. An immunogenic composition according to claim C69, wherein the conjugate comprises a saccharide covalently linked to a carrier protein, wherein the saccharide comprises a structure selected from formula O8, formula O9a, formula O9, formula O20ab, formula O20ac, formula O52, formula O97 and formula O101, wherein n is an integer from 1 to 10.

C120. An immunogenic composition comprising a mutant FimH polypeptide and a saccharide according to any one of paragraphs C69-C102, and a pharmaceutically acceptable diluent.

C121. An immunogenic composition comprising a mutant FimH polypeptide and a glycoconjugate according to any one of paragraphs C103-C119, and a pharmaceutically acceptable diluent.

C122. An immunogenic composition according to item C121, containing no more than about 25% free saccharide relative to the total amount of saccharide in the composition.

C123. An immunogenic composition according to any one of paragraphs C120-C121, additionally containing an adjuvant.

C124. An immunogenic composition according to any one of paragraphs C120-C121, additionally containing aluminum.

C125. An immunogenic composition according to any one of paragraphs C120-C121, further comprising QS-21.

C126. An immunogenic composition according to any one of paragraphs C120-C121, further comprising a CpG oligonucleotide.

C127. An immunogenic composition according to any one of paragraphs C120-C121, wherein the composition does not include an adjuvant.

C128. An immunogenic composition comprising a mutant FimH polypeptide according to any one of claims C69-C118 and a saccharide obtained from E. coli conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the polysaccharide is covalently linked to the eTEC spacer by a carbamate bond, and wherein the carrier protein is covalently linked to the eTEC spacer by an amide bond.

C129. An immunogenic composition according to item C128, wherein the saccharide is an O-antigen obtained from E. coli.

C130. An immunogenic composition according to item C128, further comprising a pharmaceutically acceptable filler, carrier or diluent.

C131. An immunogenic composition according to item C128, wherein the saccharide is an O-antigen obtained from E. coli.

C132. An immunogenic composition comprising a mutant FimH polypeptide and a saccharide according to any one of claims C69-C85, conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, wherein the polysaccharide is covalently linked to the eTEC spacer by a carbamate bond, and wherein the carrier protein is covalently linked to the eTEC spacer by an amide bond.

C133. An immunogenic composition comprising a mutant FimH polypeptide and (i) an E. coli O25B antigen conjugate covalently linked to a carrier protein, (ii) an E. coli 01A antigen conjugate covalently linked to a carrier protein, (iii) an E. coli O2 antigen conjugate covalently linked to a carrier protein, and (iv) an 06 antigen conjugate covalently linked to a carrier protein, wherein the E. coli O25B antigen comprises a structure of formula O25B, wherein n is an integer greater than 30.

C134. The immunogenic composition of claim C133, wherein the carrier protein is selected from any of: poly(L-lysine), CRM ₁₉₇ , diphtheria toxin fragment B (DTFB), C8 DTFB, diphtheria toxoid (DT), tetanus toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid, or Pseudomonas aeruginosa exotoxin A; detoxified P. aeruginosa exotoxin A (EPA), maltose binding protein (MBP), detoxified S. aureus hemolysin A, clumping factor A, clumping factor B, cholera toxin subunit B (CTB), Streptococcus pneumoniae pneumolysin and its detoxified variants, C. jejuni AcrA, natural C. jejuni glycoproteins and streptococcal C5a peptidase (SCP).

C135. An immunogenic composition comprising a mutant FimH polypeptide according to any one of claims C69 to C118 and (i) an E. coli O25B antigen conjugate covalently linked to a carrier protein, (ii) an E. coli O4 antigen conjugate covalently linked to a carrier protein, (iii) an E. coli O11 antigen conjugate covalently linked to a carrier protein, and (iv) an O21 antigen conjugate covalently linked to a carrier protein, wherein the E. coli O25B antigen comprises a structure of formula O75, wherein n is an integer greater than 30.

C136. The immunogenic composition of claim C135, wherein the carrier protein is selected from any of: poly(L-lysine), CRM ₁₉₇ , diphtheria toxin fragment B (DTFB), C8 DTFB, diphtheria toxoid (DT), tetanus toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid, or Pseudomonas aeruginosa exotoxin A; detoxified P. aeruginosa exotoxin A (EPA), maltose binding protein (MBP), detoxified S. aureus hemolysin A, clumping factor A, clumping factor B, cholera toxin subunit B (CTB), Streptococcus pneumoniae pneumolysin and its detoxified variants, C. jejuni AcA, natural C. jejuni glycoproteins and streptococcal C5a peptidase (SCP).

C137. A method for producing an immunogenic composition comprising a mutant FimH polypeptide and a conjugate comprising a saccharide conjugated to a carrier protein via a (2-((2-oxoethyl)thio)ethyl)carbamate (eTEC) spacer, comprising the steps of: a) reacting the saccharide with 1,1'-carbonyl-di-(1,2,4-triazole) (CDT) or 1,1'-carbonyldiimidazole (CDI) in an organic solvent to produce an activated saccharide; b) reacting the activated saccharide with cystamine or cysteamine or their salts to produce a thiolated saccharide; c) reacting the thiolated saccharide with a reducing agent to produce an activated thiolated saccharide containing one or more free sulfhydryl residues; o!) reacting an activated thiolated saccharide with an activated carrier protein comprising one or more α-haloacetamide groups to produce a thiolated saccharide-carrier protein conjugate; and e) reacting the thiolated saccharide-carrier protein conjugate with (i) a first capping reagent capable of capping unconjugated α-haloacetamide groups of the activated carrier protein; and/or (ii) a second capping reagent capable of capping unconjugated free sulfhydryl residues; thereby producing a glycoconjugate linked to eTEC, wherein the saccharide is derived from E. coli; further comprising expressing a polynucleotide encoding a FimH-derived polypeptide or a fragment thereof in a recombinant mammalian cell, and isolating said polypeptide or fragment thereof.

C138. The method according to paragraph C137, including obtaining an immunogenic composition according to any one of paragraphs C69-C102.

C139. The method according to any one of paragraphs C137-C138, wherein the capping step e) comprises reacting the thiolated saccharide-carrier protein conjugate with (i) N-acetyl-L-cysteine as a first copying reagent, and/or (ii) iodoacetamide as a second copying reagent.

C140. The method according to any one of paragraphs C137-C139, further comprising the step of compounding the saccharide by reacting it with a triazole or imidazole to obtain a compounded saccharide, wherein the compounded saccharide is frozen, lyophilized and reconstituted in an organic solvent prior to step a).

C141. The method according to any one of paragraphs C137-C140, further comprising purifying the thiolated polysaccharide obtained in step c), wherein the purification step comprises diafiltration.

C142. The method according to any one of paragraphs C137-C141, wherein the method further comprises purifying the glycoconjugate bound to eTEC by diafiltration.

C143. The method according to any one of paragraphs C137-C142, wherein the organic solvent in step a) is a polar aprotic solvent selected from any of the following: dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP), acetonitrile, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU) and hexamethylphosphoramide (HMPA) or a mixture thereof.

C144. The method according to any one of paragraphs. _C137 -C142, wherein the medium comprises an element 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 .}

C145. The medium according to item C144, wherein said medium is used for culturing E. coli.

C146. A method for producing a saccharide according to any one of paragraphs C69-C102, comprising culturing recombinant E. coli in a medium; producing said saccharide by culturing said cell in said medium; wherein said cell produces said saccharide.

_C147 . The method according to item C146, wherein the medium comprises an element _selected from any 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 .}

C148. The method according to item C146, wherein the medium contains soy hydrolysate.

C149. The method according to item C146, wherein the medium comprises yeast extract.

C150. The method according to item C146, wherein the medium does not additionally contain soy hydrolysate and yeast extract.

C151. The method according to item C146, wherein the E. coli cell comprises a heterologous protein of the wzz family selected from any of: wzzB, wzz, wzzSF, wzzST, fepE, wzzfepE, wzzl and wzz2.

C152. The method according to item C146, wherein the E. coli cell comprises a protein of the Salmonella enterica wzz family selected from any of: wzzB, wzz, wzzSF, wzzST, fepE, wzzfepE, wzzl and wzz2.

C153. The method according to item C152, wherein the wzz family protein comprises a sequence selected from any of SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116.

C154. The method according to item C146, wherein the cultivation yields >120 _OD600 /ml.

C155. The method according to paragraph C146, further comprising purifying the saccharide.

C156. The method of claim C146, wherein the purification step comprises any of the following: dialysis, a concentration step, a diafiltration step, tangential flow filtration, precipitation, elution, centrifugation, sedimentation, ultrafiltration, depth filtration, and column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE, and hydrophobic interaction chromatography).

C157. A method for inducing an immune response in a subject, comprising administering to the subject a composition according to any one of paragraphs C69-C136.

C158. The method according to item C157, wherein the immune response includes inducing the formation of antibodies specific to serum O-polysaccharides of E. coli.

C159. The method according to item C157, wherein the immune response comprises inducing the formation of an anti-E. coli IgG antibody.

C160. The method according to item C157, wherein the immune response comprises induction of bactericidal activity against E. coli.

C161. The method according to item C157, wherein the immune response includes inducing the formation of opsonophagocytic antibodies to E. coli.

C162. The method of claim C157, wherein the immune response comprises a geometric mean titer (GMT) level of at least 1,000 to 200,000 after the initial dosing.

C163. The method of claim C157, wherein the composition comprises a saccharide comprising formula 025, wherein n is an integer from 40 to 100, wherein the immune response comprises a geometric mean titer (GMT) level of at least 1,000 to 200,000 after administration of the initial dose.

C164. The method of claim C157, wherein the subject is at risk of developing any of the conditions selected from: urinary tract infection, cholecystitis, cholangitis, diarrhea, hemolytic uremic syndrome, neonatal meningitis, urosepsis, intra-abdominal infection, meningitis, complicated pneumonia, wound infection, prostate biopsy-associated infection, neonatal/infant sepsis, neutropenic fever and other bloodstream infections; pneumonia, bacteremia and sepsis.

C165. The method according to paragraph C157, wherein the subject is a mammal.

C166. A method for (i) inducing an immune response in a subject against extraintestinal pathogenic E. coli or (ii) inducing the formation of opsonophagocytic antibodies in a subject specific for extraintestinal pathogenic E. coli, wherein the method comprises administering to the subject an effective amount of a composition according to any one of paragraphs C69-C136.

C167. The method of C166, wherein the subject is at risk of developing a urinary tract infection.

C168. The method of C166, wherein the subject is at risk of developing bacteremia.

C169. The method according to paragraph C166, wherein the subject is at risk of developing sepsis.

C170. A method for inducing an immune response in a subject, comprising administering to the subject a composition according to any one of paragraphs C69-C136.

C171. The method according to item C170, wherein the immune response comprises inducing the formation of an antibody specific to E. coli serum polysaccharides.

C172. The method according to item C170, wherein the antibody specific to E. coli serum polysaccharides is an IgG antibody.

C173. The method according to item C170, wherein the antibody specific to serum polysaccharides of E. coli is an IgG antibody that has bactericidal activity against E. coli.

C174. An immunogenic composition according to claim C69, wherein n is greater than the number of repeating units in the corresponding polysaccharide of wild-type E. coli.

C175. The composition according to item C174, where n is an integer from 31 to 100.

C176. The composition of claim C174, wherein the saccharide comprises a structure according to any one of: formula O1A, formula O1B and formula O1C, formula O2, formula O6 and formula O25B.

C177. The composition of claim C174, wherein the saccharide is produced in a recombinant host cell that expresses a wzz family protein that is at least 90% identical to the sequence of any 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.

C178. The composition according to item C177, wherein the protein comprises any of SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115 and SEQ ID NO: 116.

C179. A saccharide according to item C174, wherein the saccharide is synthesized synthetically.

C180. An immunogenic composition comprising a mutant FimH polypeptide according to any one of claims C1 to C55 and (a) a conjugate comprising a carrier protein covalently linked to a saccharide of formula O25b, wherein n is an integer from 31 to 90, (b) a conjugate comprising a carrier protein covalently linked to a saccharide of formula O1A, wherein n is an integer from 31 to 90, (c) a conjugate comprising a carrier protein covalently linked to a saccharide of formula O2, wherein n is an integer from 31 to 90, and (d) a conjugate comprising a carrier protein covalently linked to a saccharide comprising formula O6, wherein n is an integer from 31 to 90.

C181. An immunogenic composition according to item C180, further comprising a conjugate comprising a carrier protein covalently linked to a saccharide having a structure selected from any of: formula O15, formula O16, formula O17, formula O18, and formula O75, where n is an integer from 31 to 90.

C182. An immunogenic composition according to item C180, containing no more than 25% free saccharide relative to the total amount of saccharide in the composition.

C183. A method for inducing an immune response against E. coli in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of paragraphs C180-C182.

C184. The method according to item C183, wherein the immune response includes opsonophagocytic antibodies to E. coli.

C185. The method according to item C183, wherein the immune response protects the mammal from E. coli infection.

C186. A recombinant mammalian cell comprising (a) a first gene of interest encoding a mutant FimH polypeptide according to any one of claims C1 to C55, wherein said gene is integrated between at least two recombination target sites (RTS).

C187. An embodiment according to claims C186, wherein the two RTS are chromosomally integrated into the NL1 locus or the NL2 locus.

C188. An embodiment according to item C186, wherein the first gene of interest further comprises a reporter gene, a gene encoding a difficult to express protein, an accessory gene, or a combination thereof.

C189. An embodiment according to claim C186, further comprising a second gene of interest chromosomally integrated into a second locus different from locus (a), wherein the second gene of interest comprises a reporter gene, a gene encoding a difficult to express protein, an accessory gene, or a combination thereof.

C190. A recombinant cell according to item C186, wherein the polynucleotide sequence is integrated into the genomic DNA of said mammalian cell.

C191. A recombinant cell according to claim C186, wherein the polynucleotide sequence is codon optimized for expression in the cell.

C192. A recombinant cell according to item C186, wherein the cell is a human embryonic kidney cell.

C193. A recombinant cell according to claim C192, wherein the human embryonic kidney cell comprises a HEK293 cell.

C194. The recombinant cell of item C193, wherein the HEK293 cell is selected from any of HEK293T cells, HEK293TS cells, and HEK293E cells.

C195. A recombinant cell according to item C186, wherein the cell is a CHO cell.

C196. A recombinant cell according to item C195, wherein said CHO cell is a CHO-K1 cell, a CHO-DUXB11 cell, a CHO-DG44 cell, or a CHO-S cell.

C197. A recombinant cell according to item C186, wherein the polypeptide is soluble.

C198. A recombinant cell according to claim C186, wherein the polypeptide is secreted from the cell.

C199. A culture comprising a recombinant cell according to item C186, wherein said culture has a size of at least 5 liters.

C200. The culture according to item C199, wherein the yield of the polypeptide or its fragment is at least 0.05 g/l.

C201. The culture according to item C199, wherein the yield of the polypeptide or its fragment is at least 0.10 g/l.

C202. A method for producing a polypeptide derived from E. coli or a fragment thereof, comprising culturing a recombinant mammalian cell according to paragraph C186 under suitable conditions, thereby providing expression of the polypeptide or fragment thereof; and collecting the polypeptide or fragment thereof.

C203. The method according to item C202, further comprising purifying the polypeptide or a fragment thereof.

C204. The immunogenic composition of claim C54, further comprising at least one saccharide obtained from any type of K. pneumoniae selected from the group consisting of O1, O2, O3 and O5.

C205. An immunogenic composition according to item C204, further comprising a saccharide obtained from KK pneumoniae type O1.

C206. An immunogenic composition according to item C204, further comprising a saccharide obtained from K. pneumoniae type O2.

C207. The composition according to item C204, further comprising a saccharide obtained from K. pneumoniae type 03.

C208. An immunogenic composition according to item C204, further comprising a saccharide obtained from K. pneumoniae type O5.

C209. The immunogenic composition according to item C204, further comprising a saccharide obtained from K. pneumoniae type O1 and a saccharide obtained from K. pneumoniae type O2.

C210. An immunogenic composition according to any one of paragraphs. C204-C2O9, further comprising at least one saccharide comprising a structure selected from formula O1, formula O1A, formula O1B, formula O1C, formula O2, formula O3, formula O4, formula O4:K52, formula O4:K6, formula O5, formula O5ab, formula O5ac, formula O6, formula O6:K2; K13; K15, Formulas O6:K54, Formulas O7, Formulas O8, Formulas O9, Formulas O10, Formulas O11, Formulas O12, Formulas O13, Formulas O14, Formulas O15, Formulas O16, Formulas O17, Formulas O18, Formulas O18A, Formulas O18ac, Formulas O18A1, Formulas O18B, Formulas O18B1, Formulas O19, Formulas O20, Formulas O21, Formulas O22, Formulas O23, Formulas O23A, Formulas O24, Formulas O25, Formulas O25a, Formulas O25b, Formulas O26, Formulas O27, Formulas O28, Formulas O29, Formulas O30, Formulas O32, Formulas O33, Formulas O34, Formulas O35, Formulas O36, Formulas O37, Formulas O38, Formulas O39, Formulas O40, Formulas O41, Formulas O42, Formulas O43, Formulas O44, formulas O45, formulas O45, formulas O45 rel, formulas O46, formulas O48, formulas O49, formulas O50, formulas O51, formulas O52, formulas O53, formulas O54, formulas O55, formulas O56, formulas O57, formulas O58, formulas O59, formulas O60, formulas O61, formulas O62, formulas 62D1, formulas O63, formulas O64, formulas O65, formulas O66, formulas O68, formulas O69, formulas O70, formulas O71, formulas O73, formulas O73, formulas O 4, formulas O75, formulas O76, formulas O77, formulas O78, formulas O79, formulas O80, formulas O81, formulas O82, formulas O83, formulas O84, formulas O85, formulas O86, formulas O87, formulas O88, formulas O89, formulas O90, formulas O91, Formulas O92, Formulas O93, Formulas O95, Formulas O96, Formulas O97, Formulas O98, Formulas O99, Formulas O100, Formulas O101, Formulas O102, Formulas O103, Formulas O104, Formulas O105, Formulas O106, Formulas O107, Formulas O108, Formulas O1O9, Formulas O110, Formulas O111, Formulas O112, Formulas O113, Formulas O114, Formulas O115, Formulas O116, Formulas O117, Formulas O118, Formulas O119, Formulas O120, Formulas O121, Formulas O123, Formulas O124, Formulas O125, Formulas O126, Formulas O127, Formulas O128, Formulas O129, Formulas O130, Formulas O131, Formulas O132, Formulas O133, Formulas O134, Formulas O135, Formulas O136, Formulas O137, Formulas O138, Formulas O139, Formulas O140, Formulas O141, Formulas O142, Formulas O143, Formulas O144, Formulas O145, Formulas O146, Formulas O147, Formulas O148, Formulas O149, Formulas O150, Formulas O151, Formulas O152, Formulas O153, Formulas O154, Formulas O155, Formulas O156, Formulas O157, Formulas O158, Formulas O159, Formulas O160, Formulas O161, Formulas O162, Formulas O163, Formulas O164, Formulas O165, Formulas O166, Formulas O167, Formulas O168, Formulas O169, Formulas O170, Formulas O171, Formulas O172, Formulas O173, Formulas O174, Formulas O175, Formulas O176, formula O177, formula O178, formula O179, formula O180, formula O181, formula O182, formula O183, formula O184, formula O185, formula O186, formula O187, where n is an integer from 1 to 100, more preferably from 31 to 90.

C211. An immunogenic composition according to claim C210, wherein the saccharide obtained from K. pneumoniae is conjugated to a carrier protein; and the saccharide obtained from E. coli is conjugated to a carrier protein.

C212. A method for inducing an immune response against E. coli in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of paragraphs C204-C211.

C213. The method according to item C212, wherein the immune response includes opsonophagocytic antibodies to E. coli.

C214. The method according to item C212, wherein the immune response protects the mammal from E. coli infection.

C215. A method for inducing an immune response against K. pneumoniae in a mammal, comprising administering to the mammal an effective amount of a composition according to any one of paragraphs C204-C211.

C216. The method according to item C215, wherein the immune response includes opsonophagocytic antibodies to K. pneumoniae.

C217. The method according to item C215, wherein the immune response protects the mammal from K. pneumoniae infection.

C218. Compositions and methods according to any one of paragraphs. C204-C217, wherein the serotype O1 of K. pneumoniae comprises the variant O1V1 or O1V2.

C219. Compositions and methods according to any one of paragraphs. C204-C217, wherein the serotype O2 of K. pneumoniae comprises the variant O2V1 or O2V2.

C220. Use of compositions according to any one of paragraphs C1-C219 presented in the present description.

C221. The composition according to item C211, wherein the O-antigen of K. pneumoniae is selected from the group consisting of a) serotype O1 subtype v1 (O1v1), b) serotype O1 subtype v2 (O1v2), c) serotype O2 subtype v1 (O2v1), and d) serotype O2 subtype v2 (O2v2).

C222. Nucleic acid containing nucleotides encoding polypeptides according to any one of paragraphs C1-C221.

C223. Nucleic acid according to item C222, wherein the nucleic acid is RNA.

C224. A nanoparticle containing a nucleic acid according to item C222 or item C223.

C225. The immunogenic composition of the present invention, further comprising one or more conjugates comprising a saccharide selected from the group consisting of: formula O4, formula O11, formula O13, formula O21 and formula O86, wherein n is an integer from 1 to 100, preferably from 31 to 90.

C226. The immunogenic composition of the present invention, further comprising one or more conjugates comprising a saccharide selected from the group consisting of formula O1a, formula O2, formula O6 and formula O25b, wherein n is an integer from 1 to 100, preferably from 31 to 90.

--->

SEQUENCE LISTS

<110> Pfizer Inc.

Che, Ye

Chorro, Laurent Oliver

Donald, Robert George Konrad

Griffor, Matthew Curtis

Silmon de Monerri, Natalie Clare

<120> FimH mutants of E. coli and their applications

<130> PC072713

<160> 147

<170> PatentIn version 3.5

<210> 1

<211> 160

<212> Protein

<213> Escherichia coli

<400> 1

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 2

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 2

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Ala Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 3

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 3

Ile Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 4

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 4

Leu Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 5

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 5

Val Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 6

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 6

Met Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 7

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 7

Tyr Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 8

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 8

Trp Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 9

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 9

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Lys Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 10

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 10

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Ala Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 11

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 11

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Pro Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 12

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 12

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Ala

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 13

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 13

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Pro

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 14

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 14

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Ala Ala

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 15

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 15

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Pro Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 16

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 16

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Ala Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 17

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 17

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Cys Ile Gly Gly Gly

1 5 10 15

Ser Cys Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 18

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 18

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Cys Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Cys

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 19

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 19

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Cys Val Val Asn Val Gly Gln

20 25 30

Asn Leu Cys Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 20

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 20

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Cys Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Cys Val Val Pro Thr Gly Gly

145 150 155 160

<210> 21

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 21

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Cys Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Cys Pro Thr Gly Gly

145 150 155 160

<210> 22

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 22

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Cys Val Asn Val Gly Gln

20 25 30

Asn Cys Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 23

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 23

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Cys Asn Val Gly Gln

20 25 30

Cys Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 24

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 24

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Cys Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Cys Thr Gly Gly

145 150 155 160

<210> 25

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 25

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Cys

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Cys Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 26

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 26

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Cys Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Cys Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 27

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 27

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Cys Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Cys Thr Gly Gly

145 150 155 160

<210> 28

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 28

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Cys Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Cys Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 29

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 29

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Cys Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Cys Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 30

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 30

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Cys Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Cys Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 31

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 31

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Cys Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Cys Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 32

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 32

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Cys

50 55 60

Gly Gly Val Cys Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 33

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 33

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Cys

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Cys Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 34

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 34

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Cys Ser Ser Cys Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 35

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 35

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Cys

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Cys Gly Gly

145 150 155 160

<210> 36

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 36

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Cys Ser Ala Cys Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 37

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 37

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Cys Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Cys Gly Gly

145 150 155 160

<210> 38

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 38

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Cys Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Cys Pro Thr Gly Gly

145 150 155 160

<210> 39

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 39

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Cys Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Cys Val Pro Thr Gly Gly

145 150 155 160

<210> 40

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 40

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Asn Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 41

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 41

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Ser Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 42

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 42

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Thr Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 43

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 43

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Asn Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 44

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 44

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ser Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 45

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 45

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Thr Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 46

<211> 300

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 46

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Val Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro

165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn

180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile

195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu

225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr

245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp

275 280 285

Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys

290 295 300

<210> 47

<211> 300

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 47

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

Cys Asp Ile Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro

165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn

180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile

195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu

225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr

245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp

275 280 285

Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys

290 295 300

<210> 48

<211> 300

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 48

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro

165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Ile Tyr Cys Ala Lys Ser Gln Asn

180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile

195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu

225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr

245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp

275 280 285

Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys

290 295 300

<210> 49

<211> 300

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 49

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro

165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn

180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile

195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu

225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr

245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp

275 280 285

Ile Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys

290 295 300

<210> 50

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 50

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Ala Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 51

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 51

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Ala

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 52

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 52

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Pro Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 53

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 53

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Pro

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 54

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 54

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Ala Ala

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 55

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 55

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Pro Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 56

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 56

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Ala Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 57

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 57

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Lys Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 58

<211> 160

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 58

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Ala Ala

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Lys Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

<210> 59

<211> 300

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 59

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro

165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn

180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile

195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu

225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr

245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp

275 280 285

Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys

290 295 300

<210> 60

<211> 300

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 60

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro

165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn

180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile

195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu

225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr

245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp

275 280 285

Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys

290 295 300

<210> 61

<211> 300

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 61

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Ala Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro

165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn

180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile

195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu

225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr

245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp

275 280 285

Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys

290 295 300

<210> 62

<211> 300

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 62

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Ala Ala

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Gln Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro

165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn

180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile

195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu

225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr

245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp

275 280 285

Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys

290 295 300

<210> 63

<211> 300

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 63

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Gly Gly

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Lys Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro

165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn

180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile

195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu

225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr

245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp

275 280 285

Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys

290 295 300

<210> 64

<211> 300

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 64

Phe Ala Cys Lys Thr Ala Ser Gly Thr Ala Ile Pro Ile Gly Ala Ala

1 5 10 15

Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Ala Val Asn Val Gly Gln

20 25 30

Asn Leu Val Val Asp Leu Ser Thr Gln Ile Phe Cys His Asn Asp Tyr

35 40 45

Pro Glu Thr Ile Thr Asp Tyr Val Thr Leu Gln Arg Gly Ser Ala Tyr

50 55 60

Gly Gly Val Leu Ser Ser Phe Ser Gly Thr Val Lys Tyr Ser Gly Ser

65 70 75 80

Ser Tyr Pro Phe Pro Thr Thr Ser Glu Thr Pro Arg Val Val Tyr Asn

85 90 95

Ser Arg Thr Asp Lys Pro Trp Pro Val Ala Leu Tyr Leu Thr Pro Val

100 105 110

Ser Ser Ala Gly Gly Val Ala Ile Lys Ala Gly Ser Leu Ile Ala Val

115 120 125

Leu Ile Leu Arg Lys Thr Asn Asn Tyr Asn Ser Asp Asp Phe Gln Phe

130 135 140

Val Trp Asn Ile Tyr Ala Asn Asn Asp Val Val Val Pro Thr Gly Gly

145 150 155 160

Cys Asp Val Ser Ala Arg Asp Val Thr Val Thr Leu Pro Asp Tyr Pro

165 170 175

Gly Ser Val Pro Ile Pro Leu Thr Val Tyr Cys Ala Lys Ser Gln Asn

180 185 190

Leu Gly Tyr Tyr Leu Ser Gly Thr Thr Ala Asp Ala Gly Asn Ser Ile

195 200 205

Phe Thr Asn Thr Ala Ser Phe Ser Pro Ala Gln Gly Val Gly Val Gln

210 215 220

Leu Thr Arg Gln Gly Thr Ile Ile Pro Ala Asn Asn Thr Val Ser Leu

225 230 235 240

Gly Ala Val Gly Thr Ser Ala Val Ser Leu Gly Leu Thr Ala Asn Tyr

245 250 255

Ala Arg Thr Gly Gly Gln Val Thr Ala Gly Asn Val Gln Ser Ile Ile

260 265 270

Gly Val Thr Phe Val Tyr Gln Gly Gly Ser Ser Gly Gly Gly Ala Asp

275 280 285

Val Thr Ile Thr Val Asn Gly Lys Val Val Ala Lys

290 295 300

<210> 65

<211> 19

<212> Protein

<213> Mus musculus

<400> 65

Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly

1 5 10 15

Ser Thr Gly

<210> 66

<211> 207

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 66

Met Glu Gly Met Asp Pro Leu Ala Val Leu Ala Glu Ser Arg Leu Leu

1 5 10 15

Pro Leu Leu Thr Val Arg Gly Gly Glu Asp Leu Ala Gly Leu Ala Thr

20 25 30

Val Leu Glu Leu Met Gly Val Gly Ala Leu Glu Ile Thr Leu Arg Thr

35 40 45

Glu Lys Gly Leu Glu Ala Leu Lys Ala Leu Arg Lys Ser Gly Leu Leu

50 55 60

Leu Gly Ala Gly Thr Val Arg Ser Pro Lys Glu Ala Glu Ala Ala Leu

65 70 75 80

Glu Ala Gly Ala Ala Phe Leu Val Ser Pro Gly Leu Leu Glu Glu Val

85 90 95

Ala Ala Leu Ala Gln Ala Arg Gly Val Pro Tyr Leu Pro Gly Val Leu

100 105 110

Thr Pro Thr Glu Val Glu Arg Ala Leu Ala Leu Gly Leu Ser Ala Leu

115 120 125

Lys Phe Phe Pro Ala Glu Pro Phe Gln Gly Val Arg Val Leu Arg Ala

130 135 140

Tyr Ala Glu Val Phe Pro Glu Val Arg Phe Leu Pro Thr Gly Gly Ile

145 150 155 160

Lys Glu Glu His Leu Pro His Tyr Ala Ala Leu Pro Asn Leu Leu Ala

165 170 175

Val Gly Gly Ser Trp Leu Leu Gln Gly Asp Leu Ala Ala Val Met Lys

180 185 190

Lys Val Lys Ala Ala Lys Ala Leu Leu Ser Pro Gln Ala Pro Gly

195 200 205

<210> 67

<211> 156

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 67

Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp

1 5 10 15

Met Ala Glu Ala Ala Ile Arg Thr Leu Lys Ala Leu Ser Pro Asn Ile

20 25 30

Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala

35 40 45

Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu

50 55 60

Gly Met Pro Gly Lys Ala Glu Lys Asp Lys Val Cys Ala His Glu Ala

65 70 75 80

Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile

85 90 95

Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Asp Glu Leu Asp

100 105 110

Ile Leu Ala Leu Val Arg Ala Ile Glu His Ala Ala Asn Val Tyr Tyr

115 120 125

Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu

130 135 140

Arg Gln Gly Arg Glu Asp Ala Gly Pro Ala Arg Glu

145 150 155

<210> 68

<211> 156

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 68

Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp

1 5 10 15

Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile

20 25 30

Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala

35 40 45

Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu

50 55 60

Gly Met Pro Gly Lys Ala Glu Lys Asp Lys Val Cys Ala His Glu Ala

65 70 75 80

Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile

85 90 95

Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys

100 105 110

Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr

115 120 125

Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu

130 135 140

Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu

145 150 155

<210> 69

<211> 209

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 69

Met Ser Thr Ile Asn Asn Gln Leu Lys Ala Leu Lys Val Ile Pro Val

1 5 10 15

Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu

20 25 30

Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala

35 40 45

Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu

50 55 60

Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys

65 70 75 80

Glu Ala Gly Ala Thr Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr

85 90 95

Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn

100 105 110

Asn Pro Ser Thr Val Glu Ala Ala Leu Glu Met Gly Leu Thr Thr Leu

115 120 125

Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser

130 135 140

Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile

145 150 155 160

Thr Pro Ser Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala

165 170 175

Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Thr Asn Gly Glu

180 185 190

Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn

195 200 205

Pro

<210> 70

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 70

Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Thr Asp Val Pro

1 5 10 15

Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser

20 25 30

Lys Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu

35 40 45

Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser

50 55 60

Ile Gly Gly Ile Glu Pro Ser Lys Asn Arg Asp His Ser Ala Val Leu

65 70 75 80

Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr

85 90 95

Ile His Phe Val Asn Leu Asn Gly Asp Asp Val Gly Trp Asn Gly Thr

100 105 110

Thr Phe

<210> 71

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 71

Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val

1 5 10 15

Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala

20 25 30

Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp

35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr

50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val

65 70 75 80

Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile

85 90 95

Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser

100 105 110

Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Ala Glu His His Arg

115 120 125

Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala

130 135 140

Cys Ile Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala

145 150 155

<210> 72

<211> 205

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 72

Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu

1 5 10 15

Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe

20 25 30

Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala

35 40 45

Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile

50 55 60

Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val

65 70 75 80

Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile

85 90 95

Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met

100 105 110

Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Thr Ile Leu

115 120 125

Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met

130 135 140

Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn

145 150 155 160

Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly

165 170 175

Val Gly Ser Ala Leu Val Lys Gly Thr Pro Asp Glu Val Arg Glu Lys

180 185 190

Ala Lys Ala Phe Val Glu Lys Ile Arg Gly Cys Thr Glu

195 200 205

<210> 73

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 73

Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val

1 5 10 15

Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala

20 25 30

Phe Glu Ala Ala Met Ala Asp Ile Gly Gly Asp Arg Phe Ala Val Asp

35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr

50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val

65 70 75 80

Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile

85 90 95

Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser

100 105 110

Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu

115 120 125

Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala

130 135 140

Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala

145 150 155

<210> 74

<211> 177

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 74

Met Phe Thr Lys Ser Gly Asp Asp Gly Asn Thr Asn Val Ile Asn Lys

1 5 10 15

Arg Val Gly Lys Asp Ser Pro Leu Val Asn Phe Leu Gly Asp Leu Asp

20 25 30

Glu Leu Asn Ser Phe Ile Gly Phe Ala Ile Ser Lys Ile Pro Trp Glu

35 40 45

Asp Met Lys Lys Asp Leu Glu Arg Val Gln Val Glu Leu Phe Glu Ile

50 55 60

Gly Glu Asp Leu Ser Thr Gln Ser Ser Lys Lys Lys Ile Asp Glu Ser

65 70 75 80

Tyr Val Leu Trp Leu Leu Ala Ala Thr Ala Ile Tyr Arg Ile Glu Ser

85 90 95

Gly Pro Val Lys Leu Phe Val Ile Pro Gly Gly Ser Glu Glu Ala Ser

100 105 110

Val Leu His Val Thr Arg Ser Val Ala Arg Arg Val Glu Arg Asn Ala

115 120 125

Val Lys Tyr Thr Lys Glu Leu Pro Glu Ile Asn Arg Met Ile Ile Val

130 135 140

Tyr Leu Asn Arg Leu Ser Ser Leu Leu Phe Ala Met Ala Leu Val Ala

145 150 155 160

Asn Lys Arg Arg Asn Gln Ser Glu Lys Ile Tyr Glu Ile Gly Lys Ser

165 170 175

Trp

<210> 75

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 75

Met Asn Gln His Ser His Lys Asp Tyr Glu Thr Val Arg Ile Ala Val

1 5 10 15

Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Gln Cys Val Arg Ala

20 25 30

Phe Glu Glu Ala Met Ala Asp Ala Gly Gly Asp Arg Phe Ala Val Asp

35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr

50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val

65 70 75 80

Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile

85 90 95

Asp Gly Met Met Asn Val Gln Leu Ser Thr Gly Val Pro Val Leu Ser

100 105 110

Ala Val Leu Thr Pro His Arg Tyr Arg Ser Ser Arg Glu His His Glu

115 120 125

Phe Phe Arg Glu His Phe Met Val Lys Gly Val Glu Ala Ala Ala Ala

130 135 140

Cys Ile Thr Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala

145 150 155

<210> 76

<211> 201

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 76

Met Gly His Thr Lys Gly Pro Thr Pro Gln Gln His Asp Gly Ser Ala

1 5 10 15

Leu Arg Ile Gly Ile Val His Ala Arg Trp Asn Lys Thr Ile Ile Met

20 25 30

Pro Leu Leu Ile Gly Thr Ile Ala Lys Leu Leu Glu Cys Gly Val Lys

35 40 45

Ala Ser Asn Ile Val Val Gln Ser Val Pro Gly Ser Trp Glu Leu Pro

50 55 60

Ile Ala Val Gln Arg Leu Tyr Ser Ala Ser Gln Leu Gln Thr Pro Ser

65 70 75 80

Ser Gly Pro Ser Leu Ser Ala Gly Asp Leu Leu Gly Ser Ser Thr Thr

85 90 95

Asp Leu Thr Ala Leu Pro Thr Thr Thr Ala Ser Ser Thr Gly Pro Phe

100 105 110

Asp Ala Leu Ile Ala Ile Gly Val Leu Ile Lys Gly Glu Thr Met His

115 120 125

Phe Glu Tyr Ile Ala Asp Ser Val Ser His Gly Leu Met Arg Val Gln

130 135 140

Leu Asp Thr Gly Val Pro Val Ile Phe Gly Val Leu Thr Val Leu Thr

145 150 155 160

Asp Asp Gln Ala Lys Ala Arg Ala Gly Val Ile Glu Gly Ser His Asn

165 170 175

His Gly Glu Asp Trp Gly Leu Ala Ala Val Glu Met Gly Val Arg Arg

180 185 190

Arg Asp Trp Ala Ala Gly Lys Thr Glu

195 200

<210> 77

<211> 237

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 77

Met Tyr Glu Val Asp His Ala Asp Val Tyr Asp Leu Phe Tyr Leu Gly

1 5 10 15

Arg Gly Lys Asp Tyr Ala Ala Glu Ala Ser Asp Ile Ala Asp Leu Val

20 25 30

Arg Ser Arg Thr Pro Glu Ala Ser Ser Leu Leu Asp Val Ala Cys Gly

35 40 45

Thr Gly Thr His Leu Glu His Phe Thr Lys Glu Phe Gly Asp Thr Ala

50 55 60

Gly Leu Glu Leu Ser Glu Asp Met Leu Thr His Ala Arg Lys Arg Leu

65 70 75 80

Pro Asp Ala Thr Leu His Gln Gly Asp Met Arg Asp Phe Gln Leu Gly

85 90 95

Arg Lys Phe Ser Ala Val Val Ser Met Phe Ser Ser Val Gly Tyr Leu

100 105 110

Lys Thr Val Ala Glu Leu Gly Ala Ala Val Ala Ser Phe Ala Glu His

115 120 125

Leu Glu Pro Gly Gly Val Val Val Val Glu Pro Trp Trp Phe Pro Glu

130 135 140

Thr Phe Ala Asp Gly Trp Val Ser Ala Asp Val Val Arg Arg Asp Gly

145 150 155 160

Arg Thr Val Ala Arg Val Ser His Ser Val Arg Glu Gly Asn Ala Thr

165 170 175

Arg Met Glu Val His Phe Thr Val Ala Asp Pro Gly Lys Gly Val Arg

180 185 190

His Phe Ser Asp Val His Leu Ile Thr Leu Phe His Gln Arg Glu Tyr

195 200 205

Glu Ala Ala Phe Met Ala Ala Gly Leu Arg Val Glu Tyr Leu Glu Gly

210 215 220

Gly Pro Ser Gly Arg Gly Leu Phe Val Gly Val Pro Ala

225 230 235

<210> 78

<211> 138

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 78

Met Gly Met Lys Glu Lys Phe Val Leu Ile Ile Thr His Gly Asp Phe

1 5 10 15

Gly Lys Gly Leu Leu Ser Gly Ala Glu Val Ile Ile Gly Lys Gln Glu

20 25 30

Asn Val His Thr Val Gly Leu Asn Leu Gly Asp Asn Ile Glu Lys Val

35 40 45

Ala Lys Glu Val Met Arg Ile Ile Ile Ala Lys Leu Ala Glu Asp Lys

50 55 60

Glu Ile Ile Ile Val Val Asp Leu Phe Gly Gly Ser Pro Phe Asn Ile

65 70 75 80

Ala Leu Glu Met Met Lys Thr Phe Asp Val Lys Val Ile Thr Gly Ile

85 90 95

Asn Met Pro Met Leu Val Glu Leu Leu Thr Ser Ile Asn Val Tyr Asp

100 105 110

Thr Thr Glu Leu Leu Glu Asn Ile Ser Lys Ile Gly Lys Asp Gly Ile

115 120 125

Lys Val Ile Glu Lys Ser Ser Leu Lys Met

130 135

<210> 79

<211> 154

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 79

Met Lys Tyr Asp Gly Ser Lys Leu Arg Ile Gly Ile Leu His Ala Arg

1 5 10 15

Trp Asn Leu Glu Ile Ile Ala Ala Leu Val Ala Gly Ala Ile Lys Arg

20 25 30

Leu Gln Glu Phe Gly Val Lys Ala Glu Asn Ile Ile Ile Glu Thr Val

35 40 45

Pro Gly Ser Phe Glu Leu Pro Tyr Gly Ser Lys Leu Phe Val Glu Lys

50 55 60

Gln Lys Arg Leu Gly Lys Pro Leu Asp Ala Ile Ile Pro Ile Gly Val

65 70 75 80

Leu Ile Lys Gly Ser Thr Met His Phe Glu Tyr Ile Cys Asp Ser Thr

85 90 95

Thr His Gln Leu Met Lys Leu Asn Phe Glu Leu Gly Ile Pro Val Ile

100 105 110

Phe Gly Val Leu Thr Cys Leu Thr Asp Glu Gln Ala Glu Ala Arg Ala

115 120 125

Gly Leu Ile Glu Gly Lys Met His Asn His Gly Glu Asp Trp Gly Ala

130 135 140

Ala Ala Val Glu Met Ala Thr Lys Phe Asn

145 150

<210> 80

<211> 164

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 80

Met Ala Val Lys Gly Leu Gly Glu Val Asp Gln Lys Tyr Asp Gly Ser

1 5 10 15

Lys Leu Arg Ile Gly Ile Leu His Ala Arg Trp Asn Arg Lys Ile Ile

20 25 30

Leu Ala Leu Val Ala Gly Ala Val Leu Arg Leu Leu Glu Phe Gly Val

35 40 45

Lys Ala Glu Asn Ile Ile Ile Glu Thr Val Pro Gly Ser Phe Glu Leu

50 55 60

Pro Tyr Gly Ser Lys Leu Phe Val Glu Lys Gln Lys Arg Leu Gly Lys

65 70 75 80

Pro Leu Asp Ala Ile Ile Pro Ile Gly Val Leu Ile Lys Gly Ser Thr

85 90 95

Met His Phe Glu Tyr Ile Cys Asp Ser Thr Thr His Gln Leu Met Lys

100 105 110

Leu Asn Phe Glu Leu Gly Ile Pro Val Ile Phe Gly Val Leu Thr Cys

115 120 125

Leu Thr Asp Glu Gln Ala Glu Ala Arg Ala Gly Leu Ile Glu Gly Lys

130 135 140

Met His Asn His Gly Glu Asp Trp Gly Ala Ala Ala Val Glu Met Ala

145 150 155 160

Thr Lys Phe Asn

<210> 81

<211> 175

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 81

Met Gly Ala Asn Trp Tyr Leu Asp Asn Glu Ser Ser Arg Leu Ser Phe

1 5 10 15

Thr Ser Thr Lys Asn Ala Asp Ile Ala Glu Val His Arg Phe Leu Val

20 25 30

Leu His Gly Lys Val Asp Pro Lys Gly Leu Ala Glu Val Glu Val Glu

35 40 45

Thr Glu Ser Ile Ser Thr Gly Ile Pro Leu Arg Asp Met Leu Leu Arg

50 55 60

Val Leu Val Phe Gln Val Ser Lys Phe Pro Val Ala Gln Ile Asn Ala

65 70 75 80

Gln Leu Asp Met Arg Pro Ile Asn Asn Leu Ala Pro Gly Ala Gln Leu

85 90 95

Glu Leu Arg Leu Pro Leu Thr Val Ser Leu Arg Gly Lys Ser His Ser

100 105 110

Tyr Asn Ala Glu Leu Leu Ala Thr Arg Leu Asp Glu Arg Arg Phe Gln

115 120 125

Val Val Thr Leu Glu Pro Leu Val Ile His Ala Gln Asp Phe Asp Met

130 135 140

Val Arg Ala Phe Asn Ala Leu Arg Leu Val Ala Gly Leu Ser Ala Val

145 150 155 160

Ser Leu Ser Val Pro Val Gly Ala Val Leu Ile Phe Thr Ala Arg

165 170 175

<210> 82

<211> 208

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 82

Met Thr Asp Tyr Ile Arg Asp Gly Ser Ala Ile Lys Ala Leu Ser Phe

1 5 10 15

Ala Ile Ile Leu Ala Glu Ala Asp Leu Arg His Ile Pro Gln Asp Leu

20 25 30

Gln Arg Leu Ala Val Arg Val Ile His Ala Cys Gly Met Val Asp Val

35 40 45

Ala Asn Asp Leu Ala Phe Ser Glu Gly Ala Gly Lys Ala Gly Arg Asn

50 55 60

Ala Leu Leu Ala Gly Ala Pro Ile Leu Cys Asp Ala Arg Met Val Ala

65 70 75 80

Glu Gly Ile Thr Arg Ser Arg Leu Pro Ala Asp Asn Arg Val Ile Tyr

85 90 95

Thr Leu Ser Asp Pro Ser Val Pro Glu Leu Ala Lys Lys Ile Gly Asn

100 105 110

Thr Arg Ser Ala Ala Ala Leu Asp Leu Trp Leu Pro His Ile Glu Gly

115 120 125

Ser Ile Val Ala Ile Gly Asn Ala Pro Thr Ala Leu Phe Arg Leu Phe

130 135 140

Glu Leu Leu Asp Ala Gly Ala Pro Lys Pro Ala Leu Ile Ile Gly Met

145 150 155 160

Pro Val Gly Phe Val Gly Ala Ala Glu Ser Lys Asp Glu Leu Ala Ala

165 170 175

Asn Ser Arg Gly Val Pro Tyr Val Ile Val Arg Gly Arg Arg Gly Gly

180 185 190

Ser Ala Met Thr Ala Ala Ala Val Asn Ala Leu Ala Ser Glu Arg Glu

195 200 205

<210> 83

<211> 128

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 83

Met Ile Thr Val Phe Gly Leu Lys Ser Lys Leu Ala Pro Arg Arg Glu

1 5 10 15

Lys Leu Ala Glu Val Ile Tyr Ser Ser Leu His Leu Gly Leu Asp Ile

20 25 30

Pro Lys Gly Lys His Ala Ile Arg Phe Leu Cys Leu Glu Lys Glu Asp

35 40 45

Phe Tyr Tyr Pro Phe Asp Arg Ser Asp Asp Tyr Thr Val Ile Glu Ile

50 55 60

Asn Leu Met Ala Gly Arg Ser Glu Glu Thr Lys Met Leu Leu Ile Phe

65 70 75 80

Leu Leu Phe Ile Ala Leu Glu Arg Lys Leu Gly Ile Arg Ala His Asp

85 90 95

Val Glu Ile Thr Ile Lys Glu Gln Pro Ala His Cys Trp Gly Phe Arg

100 105 110

Gly Arg Thr Gly Asp Ser Ala Arg Asp Leu Asp Tyr Asp Ile Tyr Val

115 120 125

<210> 84

<211> 235

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 84

Met Gly Ser Asp Leu Gln Lys Leu Gln Arg Phe Ser Thr Cys Asp Ile

1 5 10 15

Ser Asp Gly Leu Leu Asn Val Tyr Asn Ile Pro Thr Gly Gly Tyr Phe

20 25 30

Pro Asn Leu Thr Ala Ile Ser Pro Pro Gln Asn Ser Ser Ile Val Gly

35 40 45

Thr Ala Tyr Thr Val Leu Phe Ala Pro Ile Asp Asp Pro Arg Pro Ala

50 55 60

Val Asn Tyr Ile Asp Ser Val Pro Pro Asn Ser Ile Leu Val Leu Ala

65 70 75 80

Leu Glu Pro His Leu Gln Ser Gln Phe His Pro Phe Ile Lys Ile Thr

85 90 95

Gln Ala Met Tyr Gly Gly Leu Met Ser Thr Arg Ala Gln Tyr Leu Lys

100 105 110

Ser Asn Gly Thr Val Val Phe Gly Arg Ile Arg Asp Val Asp Glu His

115 120 125

Arg Thr Leu Asn His Pro Val Phe Ala Tyr Gly Val Gly Ser Cys Ala

130 135 140

Pro Lys Ala Val Val Lys Ala Val Gly Thr Asn Val Gln Leu Lys Ile

145 150 155 160

Leu Thr Ser Asp Gly Val Thr Gln Thr Ile Cys Pro Gly Asp Tyr Ile

165 170 175

Ala Gly Asp Asn Asn Gly Ile Val Arg Ile Pro Val Gln Glu Thr Asp

180 185 190

Ile Ser Lys Leu Val Thr Tyr Ile Glu Lys Ser Ile Glu Val Asp Arg

195 200 205

Leu Val Ser Glu Ala Ile Lys Asn Gly Leu Pro Ala Lys Ala Ala Gln

210 215 220

Thr Ala Arg Arg Met Val Leu Lys Asp Tyr Ile

225 230 235

<210> 85

<211> 162

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 85

Met Ser Gly Met Arg Val Tyr Leu Gly Ala Asp His Ala Gly Tyr Glu

1 5 10 15

Leu Lys Gln Ala Ile Ile Ala Phe Leu Lys Met Thr Gly His Glu Pro

20 25 30

Ile Asp Cys Gly Ala Leu Arg Tyr Asp Ala Asp Asp Asp Tyr Pro Ala

35 40 45

Phe Cys Ile Ala Ala Ala Thr Arg Thr Val Ala Asp Pro Gly Ser Leu

50 55 60

Gly Ile Val Leu Gly Gly Ser Gly Asn Gly Glu Gln Ile Ala Ala Asn

65 70 75 80

Lys Val Pro Gly Ala Arg Cys Ala Leu Ala Trp Ser Val Gln Thr Ala

85 90 95

Ala Leu Ala Arg Glu His Asn Asn Ala Gln Leu Ile Gly Ile Gly Gly

100 105 110

Arg Met His Thr Leu Glu Glu Ala Leu Arg Ile Val Lys Ala Phe Val

115 120 125

Thr Thr Pro Trp Ser Lys Ala Gln Arg His Gln Arg Arg Ile Asp Ile

130 135 140

Leu Ala Glu Tyr Glu Arg Thr His Glu Ala Pro Pro Val Pro Gly Ala

145 150 155 160

About Ala

<210> 86

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 86

Met Gly Asp Asp Ala Arg Ile Ala Ala Ile Gly Asp Val Asp Glu Leu

1 5 10 15

Asn Ser Gln Ile Gly Val Leu Leu Ala Glu Pro Leu Pro Asp Asp Val

20 25 30

Arg Ala Ala Leu Ser Ala Ile Gln His Asp Leu Phe Asp Leu Gly Gly

35 40 45

Glu Leu Cys Ile Pro Gly His Ala Ala Ile Thr Glu Asp His Leu Leu

50 55 60

Arg Leu Ala Leu Trp Leu Val His Tyr Asn Gly Gln Leu Pro Pro Leu

65 70 75 80

Glu Glu Phe Ile Leu Pro Gly Gly Ala Arg Gly Ala Ala Leu Ala His

85 90 95

Val Cys Arg Thr Val Cys Arg Arg Ala Glu Arg Ser Ile Lys Ala Leu

100 105 110

Gly Ala Ser Glu Pro Leu Asn Ile Ala Pro Ala Ala Tyr Val Asn Leu

115 120 125

Leu Ser Asp Leu Leu Phe Val Leu Ala Arg Val Leu Asn Arg Ala Ala

130 135 140

Gly Gly Ala Asp Val Leu Trp Asp Arg Thr Arg Ala His

145 150 155

<210> 87

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 87

Met Ile Leu Ser Ala Glu Gln Ser Phe Thr Leu Arg His Pro His Gly

1 5 10 15

Gln Ala Ala Ala Leu Ala Phe Val Arg Glu Pro Ala Ala Ala Leu Ala

20 25 30

Gly Val Gln Arg Leu Arg Gly Leu Asp Ser Asp Gly Glu Gln Val Trp

35 40 45

Gly Glu Leu Leu Val Arg Val Pro Leu Leu Gly Glu Val Asp Leu Pro

50 55 60

Phe Arg Ser Glu Ile Val Arg Thr Pro Gln Gly Ala Glu Leu Arg Pro

65 70 75 80

Leu Thr Leu Thr Gly Glu Arg Ala Trp Val Ala Val Ser Gly Gln Ala

85 90 95

Thr Ala Ala Glu Gly Gly Glu Met Ala Phe Ala Phe Gln Phe Gln Ala

100 105 110

His Leu Ala Thr Pro Glu Ala Glu Gly Glu Gly Gly Ala Ala Phe Glu

115 120 125

Val Met Val Gln Ala Ala Ala Gly Val Thr Leu Leu Leu Val Ala Met

130 135 140

Ala Leu Pro Gln Gly Leu Ala Ala Gly Leu Pro Pro Ala

145 150 155

<210> 88

<211> 156

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 88

Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp

1 5 10 15

Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile

20 25 30

Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala

35 40 45

Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu

50 55 60

Gly Met Pro Gly Lys Lys Glu Lys Asp Lys Val Cys Ala His Glu Ala

65 70 75 80

Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile

85 90 95

Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys

100 105 110

Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr

115 120 125

Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu

130 135 140

Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu

145 150 155

<210> 89

<211> 209

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 89

Met Asp Asp Ile Asn Asn Gln Leu Lys Arg Leu Lys Val Ile Pro Val

1 5 10 15

Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu

20 25 30

Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala

35 40 45

Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu

50 55 60

Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys

65 70 75 80

Glu Ala Gly Ala Asp Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr

85 90 95

Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn

100 105 110

Asn Pro Ser Thr Val Glu Gln Ala Leu Glu Met Gly Leu Thr Thr Leu

115 120 125

Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser

130 135 140

Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile

145 150 155 160

Thr Pro Asp Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala

165 170 175

Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Arg Asn Gly Glu

180 185 190

Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn

195 200 205

Pro

<210> 90

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 90

Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Asp Asp Val Pro

1 5 10 15

Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser

20 25 30

Lys Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu

35 40 45

Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser

50 55 60

Ile Gly Gly Ile Glu Pro Asp Lys Asn Arg Asp His Ser Ala Val Leu

65 70 75 80

Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr

85 90 95

Ile His Phe Val Asn Leu Asn Gly Asp Asp Val Gly Trp Asn Gly Thr

100 105 110

Thr Phe

<210> 91

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 91

Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Asp Asp Val Pro

1 5 10 15

Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser

20 25 30

Glu Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu

35 40 45

Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser

50 55 60

Ile Gly Gly Ile Glu Pro Asp Lys Asn Glu Asp His Ser Ala Val Leu

65 70 75 80

Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr

85 90 95

Ile His Phe Val Asp Leu Asp Gly Asp Asp Val Gly Trp Asn Gly Thr

100 105 110

Thr Phe

<210> 92

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 92

Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val

1 5 10 15

Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala

20 25 30

Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp

35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr

50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val

65 70 75 80

Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile

85 90 95

Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser

100 105 110

Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Glu His His Arg

115 120 125

Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala

130 135 140

Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala

145 150 155

<210> 93

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 93

Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val

1 5 10 15

Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala

20 25 30

Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp

35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr

50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val

65 70 75 80

Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile

85 90 95

Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser

100 105 110

Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Glu Asp His Glu

115 120 125

Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala

130 135 140

Cys Ile Glu Ile Leu Asn Ala Arg Glu Lys Ile Ala Ala

145 150 155

<210> 94

<211> 205

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 94

Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu

1 5 10 15

Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe

20 25 30

Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala

35 40 45

Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile

50 55 60

Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val

65 70 75 80

Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile

85 90 95

Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met

100 105 110

Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu

115 120 125

Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met

130 135 140

Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn

145 150 155 160

Leu Asp Asn Val Cys Glu Trp Phe Lys Ala Gly Val Leu Ala Val Gly

165 170 175

Val Gly Asp Ala Leu Val Lys Gly Asp Pro Asp Glu Val Arg Glu Lys

180 185 190

Ala Lys Lys Phe Val Glu Lys Ile Arg Gly Cys Thr Glu

195 200 205

<210> 95

<211> 205

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 95

Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu

1 5 10 15

Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe

20 25 30

Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala

35 40 45

Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile

50 55 60

Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val

65 70 75 80

Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile

85 90 95

Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met

100 105 110

Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu

115 120 125

Lys Leu Phe Pro Gly Glu Val Val Gly Pro Glu Phe Val Glu Ala Met

130 135 140

Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asp

145 150 155 160

Leu Asp Asp Val Cys Glu Trp Phe Asp Ala Gly Val Leu Ala Val Gly

165 170 175

Val Gly Asp Ala Leu Val Glu Gly Asp Pro Asp Glu Val Arg Glu Asp

180 185 190

Ala Lys Glu Phe Val Glu Glu Ile Arg Gly Cys Thr Glu

195 200 205

<210> 96

<211> 205

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 96

Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu

1 5 10 15

Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe

20 25 30

Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala

35 40 45

Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile

50 55 60

Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val

65 70 75 80

Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile

85 90 95

Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met

100 105 110

Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Asp Ile Leu

115 120 125

Lys Leu Phe Pro Gly Glu Val Val Gly Pro Gln Phe Val Lys Ala Met

130 135 140

Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Asn

145 150 155 160

Leu Asp Asn Val Cys Lys Trp Phe Lys Ala Gly Val Leu Ala Val Gly

165 170 175

Val Gly Lys Ala Leu Val Lys Gly Lys Pro Asp Glu Val Arg Glu Lys

180 185 190

Ala Lys Lys Phe Val Lys Lys Ile Arg Gly Cys Thr Glu

195 200 205

<210> 97

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 97

Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val

1 5 10 15

Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala

20 25 30

Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp

35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr

50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val

65 70 75 80

Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile

85 90 95

Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser

100 105 110

Ala Val Leu Thr Pro His Arg Tyr Arg Asp Ser Asp Ala His Thr Leu

115 120 125

Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala

130 135 140

Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala

145 150 155

<210> 98

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 98

Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val

1 5 10 15

Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala

20 25 30

Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp

35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr

50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val

65 70 75 80

Val Asp Gly Gly Ile Tyr Asp His Glu Phe Val Ala Ser Ala Val Ile

85 90 95

Asp Gly Met Met Asn Val Gln Leu Asp Thr Gly Val Pro Val Leu Ser

100 105 110

Ala Val Leu Thr Pro His Glu Tyr Glu Asp Ser Asp Ala Asp Thr Leu

115 120 125

Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala

130 135 140

Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala

145 150 155

<210> 99

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 99

Met Asn Gln His Ser His Lys Asp His Glu Thr Val Arg Ile Ala Val

1 5 10 15

Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala

20 25 30

Phe Glu Ala Ala Met Arg Asp Ile Gly Gly Asp Arg Phe Ala Val Asp

35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr

50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val

65 70 75 80

Val Asn Gly Gly Ile Tyr Arg His Glu Phe Val Ala Ser Ala Val Ile

85 90 95

Asn Gly Met Met Asn Val Gln Leu Asn Thr Gly Val Pro Val Leu Ser

100 105 110

Ala Val Leu Thr Pro His Asn Tyr Asp Lys Ser Lys Ala His Thr Leu

115 120 125

Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala

130 135 140

Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala

145 150 155

<210> 100

<211> 156

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221>MISC_FEATURE

<222> (70)..(70)

<223> Xaa is A or K

<400> 100

Met Thr Lys Lys Val Gly Ile Val Asp Thr Thr Phe Ala Arg Val Asp

1 5 10 15

Met Ala Ser Ala Ala Ile Leu Thr Leu Lys Met Glu Ser Pro Asn Ile

20 25 30

Lys Ile Ile Arg Lys Thr Val Pro Gly Ile Lys Asp Leu Pro Val Ala

35 40 45

Cys Lys Lys Leu Leu Glu Glu Glu Gly Cys Asp Ile Val Met Ala Leu

50 55 60

Gly Met Pro Gly Lys Xaa Glu Lys Asp Lys Val Cys Ala His Glu Ala

65 70 75 80

Ser Leu Gly Leu Met Leu Ala Gln Leu Met Thr Asn Lys His Ile Ile

85 90 95

Glu Val Phe Val His Glu Asp Glu Ala Lys Asp Asp Ala Glu Leu Lys

100 105 110

Ile Leu Ala Ala Arg Arg Ala Ile Glu His Ala Leu Asn Val Tyr Tyr

115 120 125

Leu Leu Phe Lys Pro Glu Tyr Leu Thr Arg Met Ala Gly Lys Gly Leu

130 135 140

Arg Gln Gly Phe Glu Asp Ala Gly Pro Ala Arg Glu

145 150 155

<210> 101

<211> 209

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221>MISC_FEATURE

<222> (2)..(2)

<223> Xaa is S or D

<220>

<221>MISC_FEATURE

<222> (3)..(3)

<223> Xaa is T or D

<220>

<221>MISC_FEATURE

<222> (10)..(10)

<223> Xaa is A or R

<220>

<221>MISC_FEATURE

<222> (85)..(85)

<223> Xaa is T or D

<220>

<221>MISC_FEATURE

<222> (119)..(119)

<223> Xaa is either A or Q

<220>

<221>MISC_FEATURE

<222> (163)..(163)

<223> Xaa is S or D

<220>

<221>MISC_FEATURE

<222> (189)..(189)

<223> Xaa is T or R

<400> 101

Met Xaa Xaa Ile Asn Asn Gln Leu Lys Xaa Leu Lys Val Ile Pro Val

1 5 10 15

Ile Ala Ile Asp Asn Ala Glu Asp Ile Ile Pro Leu Gly Lys Val Leu

20 25 30

Ala Glu Asn Gly Leu Pro Ala Ala Glu Ile Thr Phe Arg Ser Ser Ala

35 40 45

Ala Val Lys Ala Ile Met Leu Leu Arg Ser Ala Gln Pro Glu Met Leu

50 55 60

Ile Gly Ala Gly Thr Ile Leu Asn Gly Val Gln Ala Leu Ala Ala Lys

65 70 75 80

Glu Ala Gly Ala Xaa Phe Val Val Ser Pro Gly Phe Asn Pro Asn Thr

85 90 95

Val Arg Ala Cys Gln Ile Ile Gly Ile Asp Ile Val Pro Gly Val Asn

100 105 110

Asn Pro Ser Thr Val Glu Xaa Ala Leu Glu Met Gly Leu Thr Thr Leu

115 120 125

Lys Phe Phe Pro Ala Glu Ala Ser Gly Gly Ile Ser Met Val Lys Ser

130 135 140

Leu Val Gly Pro Tyr Gly Asp Ile Arg Leu Met Pro Thr Gly Gly Ile

145 150 155 160

Thr Pro Xaa Asn Ile Asp Asn Tyr Leu Ala Ile Pro Gln Val Leu Ala

165 170 175

Cys Gly Gly Thr Trp Met Val Asp Lys Lys Leu Val Xaa Asn Gly Glu

180 185 190

Trp Asp Glu Ile Ala Arg Leu Thr Arg Glu Ile Val Glu Gln Val Asn

195 200 205

Pro

<210> 102

<211> 114

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221>MISC_FEATURE

<222> (13)..(13)

<223> Xaa is T or D

<220>

<221>MISC_FEATURE

<222> (33)..(33)

<223> Xaa is K or E

<220>

<221>MISC_FEATURE

<222> (71)..(71)

<223> Xaa is S or D

<220>

<221>MISC_FEATURE

<222> (74)..(74)

<223> Xaa is R or E

<220>

<221>MISC_FEATURE

<222> (101)..(101)

<223> Xaa is N or D

<220>

<221>MISC_FEATURE

<222> (103)..(103)

<223> Xaa is N or D

<400> 102

Met Pro Ile Phe Thr Leu Asn Thr Asn Ile Lys Ala Xaa Asp Val Pro

1 5 10 15

Ser Asp Phe Leu Ser Leu Thr Ser Arg Leu Val Gly Leu Ile Leu Ser

20 25 30

Xaa Pro Gly Ser Tyr Val Ala Val His Ile Asn Thr Asp Gln Gln Leu

35 40 45

Ser Phe Gly Gly Ser Thr Asn Pro Ala Ala Phe Gly Thr Leu Met Ser

50 55 60

Ile Gly Gly Ile Glu Pro Xaa Lys Asn Xaa Asp His Ser Ala Val Leu

65 70 75 80

Phe Asp His Leu Asn Ala Met Leu Gly Ile Pro Lys Asn Arg Met Tyr

85 90 95

Ile His Phe Val Xaa Leu Xaa Gly Asp Asp Val Gly Trp Asn Gly Thr

100 105 110

Thr Phe

<210> 103

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221>MISC_FEATURE

<222> (9)..(9)

<223> Xaa is Y or H

<220>

<221>MISC_FEATURE

<222> (82)..(82)

<223> Xaa is N or D

<220>

<221>MISC_FEATURE

<222> (87)..(87)

<223> Xaa is R or D

<220>

<221>MISC_FEATURE

<222> (105)..(105)

<223> Xaa is S or D

<220>

<221>MISC_FEATURE

<222> (119)..(119)

<223> Xaa is R or E

<220>

<221>MISC_FEATURE

<222> (121)..(121)

<223> Xaa is R or E

<220>

<221>MISC_FEATURE

<222> (124)..(124)

<223> Xaa is A or D

<220>

<221>MISC_FEATURE

<222> (126)..(126)

<223> Xaa is H or D

<220>

<221>MISC_FEATURE

<222> (128)..(128)

<223> Xaa is R or E

<220>

<221>MISC_FEATURE

<222> (150)..(150)

<223> Xaa is A or N

<400> 103

Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val

1 5 10 15

Val Arg Ala Arg Trp His Ala Asp Ile Val Asp Ala Cys Val Glu Ala

20 25 30

Phe Glu Ile Ala Met Ala Ala Ile Gly Gly Asp Arg Phe Ala Val Asp

35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr

50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val

65 70 75 80

Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile

85 90 95

Asp Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser

100 105 110

Ala Val Leu Thr Pro His Xaa Tyr Xaa Asp Ser Xaa Glu Xaa His Xaa

115 120 125

Phe Phe Ala Ala His Phe Ala Val Lys Gly Val Glu Ala Ala Arg Ala

130 135 140

Cys Ile Glu Ile Leu Xaa Ala Arg Glu Lys Ile Ala Ala

145 150 155

<210> 104

<211> 205

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221>MISC_FEATURE

<222> (126)..(126)

<223> Xaa is T or D

<220>

<221>MISC_FEATURE

<222> (139)..(139)

<223> Xaa is either Q or E

<220>

<221>MISC_FEATURE

<222> (142)..(142)

<223> Xaa is K or E

<220>

<221>MISC_FEATURE

<222> (160)..(160)

<223> Xaa is N or D

<220>

<221>MISC_FEATURE

<222> (163)..(163)

<223> Xaa is N or D

<220>

<221>MISC_FEATURE

<222> (166)..(166)

<223> Xaa is E or K

<220>

<221>MISC_FEATURE

<222> (169)..(169)

<223> Xaa is D or K

<220>

<221>MISC_FEATURE

<222> (179)..(179)

<223> Xaa is S, D or K

<220>

<221>MISC_FEATURE

<222> (183)..(183)

<223> Xaa is K or E

<220>

<221>MISC_FEATURE

<222> (185)..(185)

<223> Xaa is T, D or K

<220>

<221>MISC_FEATURE

<222> (192)..(192)

<223> Xaa is D or K

<220>

<221>MISC_FEATURE

<222> (195)..(195)

<223> Xaa is A, E or K

<220>

<221>MISC_FEATURE

<222> (198)..(198)

<223> Xaa is E or K

<220>

<221>MISC_FEATURE

<222> (199)..(199)

<223> Xaa is E or K

<400> 104

Met Lys Met Glu Glu Leu Phe Lys Lys His Lys Ile Val Ala Val Leu

1 5 10 15

Arg Ala Asn Ser Val Glu Glu Ala Ile Glu Lys Ala Val Ala Val Phe

20 25 30

Ala Gly Gly Val His Leu Ile Glu Ile Thr Phe Thr Val Pro Asp Ala

35 40 45

Asp Thr Val Ile Lys Ala Leu Ser Val Leu Lys Glu Lys Gly Ala Ile

50 55 60

Ile Gly Ala Gly Thr Val Thr Ser Val Glu Gln Cys Arg Lys Ala Val

65 70 75 80

Glu Ser Gly Ala Glu Phe Ile Val Ser Pro His Leu Asp Glu Glu Ile

85 90 95

Ser Gln Phe Cys Lys Glu Lys Gly Val Phe Tyr Met Pro Gly Val Met

100 105 110

Thr Pro Thr Glu Leu Val Lys Ala Met Lys Leu Gly His Xaa Ile Leu

115 120 125

Lys Leu Phe Pro Gly Glu Val Val Gly Pro Xaa Phe Val Xaa Ala Met

130 135 140

Lys Gly Pro Phe Pro Asn Val Lys Phe Val Pro Thr Gly Gly Val Xaa

145 150 155 160

Leu Asp Xaa Val Cys Xaa Trp Phe Xaa Ala Gly Val Leu Ala Val Gly

165 170 175

Val Gly Xaa Ala Leu Val Xaa Gly Xaa Pro Asp Glu Val Arg Glu Xaa

180 185 190

Ala Lys Xaa Phe Val Xaa Xaa Ile Arg Gly Cys Thr Glu

195 200 205

<210> 105

<211> 157

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<220>

<221>MISC_FEATURE

<222> (9)..(9)

<223> Xaa is Y or H

<220>

<221>MISC_FEATURE

<222> (38)..(38)

<223> Xaa is A or R

<220>

<221>MISC_FEATURE

<222> (82)..(82)

<223> Xaa is N or D

<220>

<221>MISC_FEATURE

<222> (87)..(87)

<223> Xaa is R or D

<220>

<221>MISC_FEATURE

<222> (97)..(97)

<223> Xaa is N or D

<220>

<221>MISC_FEATURE

<222> (105)..(105)

<223> Xaa is S, N or D

<220>

<221>MISC_FEATURE

<222> (119)..(119)

<223> Xaa is R, E or N

<220>

<221>MISC_FEATURE

<222> (121)..(121)

<223> Xaa is R, E or D

<220>

<221>MISC_FEATURE

<222> (122)..(122)

<223> Xaa is K or D

<220>

<221>MISC_FEATURE

<222> (124)..(124)

<223> Xaa is K or D

<220>

<221>MISC_FEATURE

<222> (126)..(126)

<223> Xaa is H or D

<400> 105

Met Asn Gln His Ser His Lys Asp Xaa Glu Thr Val Arg Ile Ala Val

1 5 10 15

Val Arg Ala Arg Trp His Ala Glu Ile Val Asp Ala Cys Val Ser Ala

20 25 30

Phe Glu Ala Ala Met Xaa Asp Ile Gly Gly Asp Arg Phe Ala Val Asp

35 40 45

Val Phe Asp Val Pro Gly Ala Tyr Glu Ile Pro Leu His Ala Arg Thr

50 55 60

Leu Ala Glu Thr Gly Arg Tyr Gly Ala Val Leu Gly Thr Ala Phe Val

65 70 75 80

Val Xaa Gly Gly Ile Tyr Xaa His Glu Phe Val Ala Ser Ala Val Ile

85 90 95

Xaa Gly Met Met Asn Val Gln Leu Xaa Thr Gly Val Pro Val Leu Ser

100 105 110

Ala Val Leu Thr Pro His Xaa Tyr Xaa Xaa Ser Xaa Ala Xaa Thr Leu

115 120 125

Leu Phe Leu Ala Leu Phe Ala Val Lys Gly Met Glu Ala Ala Arg Ala

130 135 140

Cys Val Glu Ile Leu Ala Ala Arg Glu Lys Ile Ala Ala

145 150 155

<210> 106

<211> 142

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 106

taatgcttaa gtcgaacaga aagtaatcgt attgtacacg gccgcataat cgaaattaat 60

acgactcact ataggggaat tgtgagcgga taacaattcc ccatcttagt atattagtta 120

agtataagaa ggagatatac tt 142

<210> 107

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 107

taaagaagga gatatcat 18

<210> 108

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 108

tgagaaggag atatcat 17

<210> 109

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 109

ttgaaagtga tggtttggta agaaat 26

<210> 110

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 110

tgcagcacgt atgataactt caaag 25

<210> 111

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 111

aggatatttt acccagcagt gccccgt 27

<210> 112

<211> 325

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 112

Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn His Asp Pro Glu

1 5 10 15

Gln Ile Asp Leu Ile Asp Leu Leu Val Gln Leu Trp Arg Gly Lys Met

20 25 30

Thr Ile Ile Ile Ser Val Ile Val Ala Ile Ala Leu Ala Ile Gly Tyr

35 40 45

Leu Ala Val Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln

50 55 60

Pro Asp Val Gly Gln Ile Ala Gly Tyr Asn Asn Ala Met Asn Val Ile

65 70 75 80

Tyr Gly Gln Ala Ala Pro Lys Val Ser Asp Leu Gln Glu Thr Leu Ile

85 90 95

Gly Arg Phe Ser Ser Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn

100 105 110

Gln Glu Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln

115 120 125

Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Gly Ala

130 135 140

Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn

145 150 155 160

Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Ile Ala Leu Gly Arg Lys

165 170 175

Asn Leu Gln Asp Ser Leu Arg Thr Gln Glu Val Val Ala Gln Glu Gln

180 185 190

Lys Asp Leu Arg Ile Arg Gln Ile Gln Glu Ala Leu Gln Tyr Ala Asn

195 200 205

Gln Glu Gln Val Thr Lys Pro Gln Val Gln Gln Thr Glu Asp Val Thr

210 215 220

Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met Ile

225 230 235 240

Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Ser Asn Tyr Tyr Gln

245 250 255

Thr Arg Gln Asn Leu Leu Asp Ile Glu Ser Leu Lys Val Asp Asp Leu

260 265 270

Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Ile Arg

275 280 285

Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu Leu

290 295 300

Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu Arg

305 310 315 320

Asn Tyr Asn Ala Lys

325

<210> 113

<211> 326

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 113

Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn Asn Asp Pro Glu

1 5 10 15

Gln Ile Asp Leu Ile Asp Leu Leu Val Gln Leu Trp Arg Gly Lys Met

20 25 30

Thr Ile Ile Ile Ser Val Ile Val Ala Ile Ala Leu Ala Ile Gly Tyr

35 40 45

Leu Ala Val Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln

50 55 60

Pro Asp Val Gly Gln Ile Ala Gly Tyr Asn Asn Ala Met Asn Val Ile

65 70 75 80

Tyr Gly Gln Ala Ala Pro Lys Val Ser Asp Leu Gln Glu Thr Leu Ile

85 90 95

Gly Arg Phe Ser Ser Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn

100 105 110

Gln Asp Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln

115 120 125

Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Gly Ala

130 135 140

Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn

145 150 155 160

Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Ile Ala Leu Gly Arg Lys

165 170 175

Asn Leu Gln Asp Ser Leu Arg Thr Gln Glu Val Val Ala Gln Glu Gln

180 185 190

Lys Asp Leu Arg Ile Arg Gln Ile Gln Glu Ala Leu Gln Tyr Ala Asn

195 200 205

Gln Ala Gln Val Thr Lys Pro Gln Ile Gln Gln Thr Gly Glu Asp Ile

210 215 220

Thr Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met

225 230 235 240

Ile Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Asn Tyr Tyr

245 250 255

Gln Thr Arg Gln Asn Leu Leu Asp Ile Glu Ser Leu Lys Val Asp Asp

260 265 270

Leu Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Ile

275 280 285

Arg Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu

290 295 300

Leu Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu

305 310 315 320

Arg Asn Tyr Asn Ala Lys

325

<210> 114

<211> 326

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 114

Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn His Asp Pro Glu

1 5 10 15

Gln Ile Asp Leu Ile Asp Leu Leu Val Gln Leu Trp Arg Gly Lys Met

20 25 30

Thr Ile Ile Ile Ser Val Val Val Ala Ile Ala Leu Ala Ile Gly Tyr

35 40 45

Leu Ala Val Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln

50 55 60

Pro Asp Val Gly Gln Ile Ala Gly Tyr Asn Asn Ala Met Asn Val Ile

65 70 75 80

Tyr Gly Gln Ala Ala Pro Lys Val Ser Asp Leu Gln Glu Thr Leu Ile

85 90 95

Gly Arg Phe Ser Phe Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn

100 105 110

Gln Lys Glu Pro Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln

115 120 125

Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Asp Ala

130 135 140

Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn

145 150 155 160

Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Leu Ala Leu Gly Arg Lys

165 170 175

Asn Leu Gln Asp Ser Leu Arg Thr Gln Glu Val Val Ala Gln Glu Gln

180 185 190

Lys Asp Leu Arg Ile Arg Gln Ile Gln Glu Ala Leu Gln Tyr Ala Asn

195 200 205

Gln Ala Gln Val Thr Lys Pro Gln Ile Gln Gln Thr Gly Glu Asp Ile

210 215 220

Thr Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met

225 230 235 240

Ile Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Asn Tyr Tyr

245 250 255

Gln Thr Arg Gln Asn Leu Leu Asp Ile Glu Asn Leu Lys Val Asp Asp

260 265 270

Leu Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Ile

275 280 285

Arg Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu

290 295 300

Leu Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu

305 310 315 320

Arg Asn Tyr Asn Ser Lys

325

<210> 115

<211> 326

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 115

Met Arg Val Glu Asn Asn Asn Val Ser Gly Gln Asn His Asp Pro Glu

1 5 10 15

Gln Ile Asp Leu Ile Asp Leu Leu Val Gln Leu Trp Arg Gly Lys Met

20 25 30

Thr Ile Ile Ile Ser Val Ile Val Ala Ile Ala Leu Ala Ile Gly Tyr

35 40 45

Leu Ala Val Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln

50 55 60

Pro Asp Val Gly Gln Ile Ala Gly Tyr Asn Asn Ala Met Asn Val Ile

65 70 75 80

Tyr Gly Gln Ala Ala Pro Lys Val Ser Asp Leu Gln Glu Thr Leu Ile

85 90 95

Gly Arg Phe Ser Ser Ala Phe Ser Ala Leu Ala Glu Thr Leu Asp Asn

100 105 110

Gln Glu Glu Arg Glu Lys Leu Thr Ile Glu Pro Ser Val Lys Asn Gln

115 120 125

Gln Leu Pro Leu Thr Val Ser Tyr Val Gly Gln Thr Ala Glu Gly Ala

130 135 140

Gln Met Lys Leu Ala Gln Tyr Ile Gln Gln Val Asp Asp Lys Val Asn

145 150 155 160

Gln Glu Leu Glu Lys Asp Leu Lys Asp Asn Ile Ala Leu Gly Arg Lys

165 170 175

Asn Leu Gln Asp Ser Leu Arg Thr Gln Glu Val Val Ala Gln Glu Gln

180 185 190

Lys Asp Leu Arg Ile Arg Gln Ile Gln Glu Ala Leu Gln Tyr Ala Asn

195 200 205

Gln Ala Gln Val Thr Lys Pro Gln Ile Gln Gln Thr Gly Glu Asp Ile

210 215 220

Thr Gln Asp Thr Leu Phe Leu Leu Gly Ser Glu Ala Leu Glu Ser Met

225 230 235 240

Ile Lys His Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Asn Tyr Tyr

245 250 255

Gln Thr Arg Gln Asn Leu Leu Asp Ile Glu Ser Leu Lys Val Asp Asp

260 265 270

Leu Asp Ile His Ala Tyr Arg Tyr Val Met Lys Pro Met Leu Pro Ile

275 280 285

Arg Arg Asp Ser Pro Lys Lys Ala Ile Thr Leu Ile Leu Ala Val Leu

290 295 300

Leu Gly Gly Met Val Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu

305 310 315 320

Arg Asn Tyr Asn Ala Lys

325

<210> 116

<211> 327

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 116

Met Thr Val Asp Ser Asn Thr Ser Ser Gly Arg Gly Asn Asp Pro Glu

1 5 10 15

Gln Ile Asp Leu Ile Glu Leu Leu Leu Gln Leu Trp Arg Gly Lys Met

20 25 30

Thr Ile Ile Val Ala Val Ile Ile Ala Ile Leu Leu Ala Val Gly Tyr

35 40 45

Leu Met Ile Ala Lys Glu Lys Trp Thr Ser Thr Ala Ile Ile Thr Gln

50 55 60

Pro Asp Ala Ala Gln Val Ala Thr Tyr Thr Asn Ala Leu Asn Val Leu

65 70 75 80

Tyr Gly Gly Asn Ala Pro Lys Ile Ser Glu Val Gln Ala Asn Phe Ile

85 90 95

Ser Arg Phe Ser Ser Ala Phe Ser Ala Leu Ser Glu Val Leu Asp Asn

100 105 110

Gln Lys Glu Arg Glu Lys Leu Thr Ile Glu Gln Ser Val Lys Gly Gln

115 120 125

Ala Leu Pro Leu Ser Val Ser Tyr Val Ser Thr Thr Ala Glu Gly Ala

130 135 140

Gln Arg Arg Leu Ala Glu Tyr Ile Gln Gln Val Asp Glu Glu Val Ala

145 150 155 160

Lys Glu Leu Glu Val Asp Leu Lys Asp Asn Ile Thr Leu Gln Thr Lys

165 170 175

Thr Leu Gln Glu Ser Leu Glu Thr Gln Glu Val Val Ala Gln Glu Gln

180 185 190

Lys Asp Leu Arg Ile Lys Gln Ile Glu Glu Ala Leu Arg Tyr Ala Asp

195 200 205

Glu Ala Lys Ile Thr Gln Pro Gln Ile Gln Gln Thr Gln Asp Val Thr

210 215 220

Gln Asp Thr Met Phe Leu Leu Gly Ser Asp Ala Leu Lys Ser Met Ile

225 230 235 240

Gln Asn Glu Ala Thr Arg Pro Leu Val Phe Ser Pro Ala Tyr Tyr Gln

245 250 255

Thr Lys Gln Thr Leu Leu Asp Ile Lys Asn Leu Lys Val Thr Ala Asp

260 265 270

Thr Val His Val Tyr Arg Tyr Val Met Lys Pro Thr Leu Pro Val Arg

275 280 285

Arg Asp Ser Pro Lys Thr Ala Ile Thr Leu Val Leu Ala Val Leu Leu

290 295 300

Gly Gly Met Ile Gly Ala Gly Ile Val Leu Gly Arg Asn Ala Leu Arg

305 310 315 320

Ser Tyr Lys Pro Lys Ala Leu

325

<210> 117

<211> 377

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 117

Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Asp Ala His Phe Pro Asp

1 5 10 15

Tyr Pro Leu Ala Ser Pro Ser Asn Asn Glu Ile Asp Leu Leu Asn Leu

20 25 30

Ile Ser Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe

35 40 45

Ala Phe Ala Cys Ala Gly Leu Leu Ile Ser Phe Ile Leu Pro Gln Lys

50 55 60

Trp Thr Ser Ala Ala Val Val Thr Pro Pro Glu Pro Val Gln Trp Gln

65 70 75 80

Glu Leu Glu Lys Ser Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile

85 90 95

Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln

100 105 110

Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met

115 120 125

Asp Gln Leu Lys Glu Ala Lys Ile Asp Glu Leu Asp Leu His Arg Ala

130 135 140

Ile Val Ala Leu Ser Glu Lys Met Lys Ala Val Asp Asp Asn Ala Ser

145 150 155 160

Lys Lys Lys Asp Glu Pro Ser Leu Tyr Thr Ser Trp Thr Leu Ser Phe

165 170 175

Thr Ala Pro Thr Ser Glu Glu Ala Gln Thr Val Leu Ser Gly Tyr Ile

180 185 190

Asp Tyr Ile Ser Thr Leu Val Val Lys Glu Ser Leu Glu Asn Val Arg

195 200 205

Asn Lys Leu Glu Ile Lys Thr Gln Phe Glu Lys Glu Lys Leu Ala Gln

210 215 220

Asp Arg Ile Lys Thr Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu

225 230 235 240

Asn Tyr Ser Leu Asp Ile Ala Asn Ala Ala Gly Ile Lys Lys Pro Val

245 250 255

Tyr Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser

260 265 270

Leu Gly Ala Asp Gly Ile Glu Arg Lys Leu Glu Ile Glu Lys Ala Val

275 280 285

Thr Asp Val Ala Glu Leu Asn Gly Glu Leu Arg Asn Arg Gln Tyr Leu

290 295 300

Val Glu Gln Leu Thr Lys Ala His Val Asn Asp Val Asn Phe Thr Pro

305 310 315 320

Phe Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro

325 330 335

Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Ile Gly Gly Met Val

340 345 350

Ala Cys Gly Gly Val Leu Leu Arg Tyr Ala Met Ala Ser Arg Lys Gln

355 360 365

Asp Ala Met Met Ala Asp His Leu Val

370 375

<210> 118

<211> 377

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 118

Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Glu Ala His Phe Pro Glu

1 5 10 15

Tyr Pro Leu Ala Ser Pro Ser Asn Asn Glu Ile Asp Leu Leu Asn Leu

20 25 30

Ile Glu Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe

35 40 45

Ala Phe Ala Cys Ala Gly Leu Leu Ile Ser Phe Ile Leu Pro Gln Lys

50 55 60

Trp Thr Ser Ala Ala Val Val Thr Pro Pro Glu Pro Val Gln Trp Gln

65 70 75 80

Glu Leu Glu Lys Thr Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile

85 90 95

Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln

100 105 110

Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met

115 120 125

Asp Gln Leu Lys Glu Ala Lys Ile Asp Pro Leu Asp Leu His Arg Ala

130 135 140

Ile Val Ala Leu Ser Glu Lys Met Lys Ala Val Asp Asp Asn Ala Ser

145 150 155 160

Lys Lys Lys Asp Glu Ser Ala Leu Tyr Thr Ser Trp Thr Leu Ser Phe

165 170 175

Thr Ala Pro Thr Ser Glu Glu Ala Gln Lys Val Leu Ala Gly Tyr Ile

180 185 190

Asp Tyr Ile Ser Ala Leu Val Val Lys Glu Ser Ile Glu Asn Val Arg

195 200 205

Asn Lys Leu Glu Ile Lys Thr Gln Phe Glu Lys Glu Lys Leu Ala Gln

210 215 220

Asp Arg Ile Lys Thr Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu

225 230 235 240

Asn Tyr Ser Leu Asp Ile Ala Asn Ala Ala Gly Ile Lys Lys Pro Val

245 250 255

Tyr Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser

260 265 270

Leu Gly Ala Asp Gly Ile Glu Arg Lys Leu Glu Ile Glu Lys Ala Val

275 280 285

Thr Asp Val Ala Glu Leu Asn Gly Glu Leu Arg Asn Arg Gln Tyr Leu

290 295 300

Val Glu Gln Leu Thr Lys Thr Asn Ile Asn Asp Val Asn Phe Thr Pro

305 310 315 320

Phe Lys Tyr Gln Leu Arg Pro Ser Leu Pro Val Lys Lys Asp Gly Gln

325 330 335

Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Val Gly Gly Met Val

340 345 350

Ala Cys Gly Gly Val Leu Leu Arg His Ala Met Ala Ser Arg Lys Gln

355 360 365

Asp Ala Met Met Ala Asp His Leu Val

370 375

<210> 119

<211> 377

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 119

Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Asp Ala His Phe Pro Asp

1 5 10 15

Tyr Pro Leu Ala Ser Pro Ser Asn Asn Glu Ile Asp Leu Leu Asn Leu

20 25 30

Ile Ser Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe

35 40 45

Ala Phe Ala Cys Ala Gly Leu Leu Ile Ser Phe Ile Leu Pro Gln Lys

50 55 60

Trp Thr Ser Ala Ala Val Val Thr Pro Pro Glu Pro Val Gln Trp Gln

65 70 75 80

Glu Leu Glu Lys Ser Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile

85 90 95

Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln

100 105 110

Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met

115 120 125

Asp Gln Leu Lys Glu Ala Lys Ile Asp Glu Leu Asp Leu His Arg Ala

130 135 140

Ile Val Ala Leu Ser Glu Lys Met Lys Ala Val Asp Asp Asn Ala Ser

145 150 155 160

Lys Lys Lys Asp Glu Pro Ser Leu Tyr Thr Ser Trp Thr Leu Ser Phe

165 170 175

Thr Ala Pro Thr Ser Glu Glu Ala Gln Thr Val Leu Ser Gly Tyr Ile

180 185 190

Asp Tyr Ile Ser Thr Leu Val Val Lys Glu Ser Leu Glu Asn Val Arg

195 200 205

Asn Lys Leu Glu Ile Lys Thr Gln Phe Glu Lys Glu Lys Leu Ala Gln

210 215 220

Asp Arg Ile Lys Thr Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu

225 230 235 240

Asn Tyr Ser Leu Asp Ile Ala Asn Ala Ala Gly Ile Lys Lys Pro Val

245 250 255

Tyr Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser

260 265 270

Leu Gly Ala Asp Gly Ile Glu Arg Lys Leu Glu Ile Glu Lys Ala Val

275 280 285

Thr Asp Val Ala Glu Leu Asn Gly Glu Leu Arg Asn Arg Gln Tyr Leu

290 295 300

Val Glu Gln Leu Thr Lys Ala His Val Asn Asp Val Asn Phe Thr Pro

305 310 315 320

Phe Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro

325 330 335

Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Ile Gly Gly Met Val

340 345 350

Ala Cys Gly Gly Val Leu Leu Arg Tyr Ala Met Ala Ser Arg Lys Gln

355 360 365

Asp Ala Met Met Ala Asp His Leu Val

370 375

<210> 120

<211> 377

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 120

Met Ser Ser Leu Asn Ile Lys Gln Gly Ser Asp Ala His Phe Pro Asp

1 5 10 15

Tyr Pro Leu Ala Ser Pro Ser Asn Asn Glu Ile Asp Leu Leu Asn Leu

20 25 30

Ile Ser Val Leu Trp Arg Ala Lys Lys Thr Val Met Ala Val Val Phe

35 40 45

Ala Phe Ala Cys Ala Gly Leu Leu Ile Ser Phe Ile Leu Pro Gln Lys

50 55 60

Trp Thr Ser Ala Ala Val Val Thr Pro Pro Glu Pro Val Gln Trp Gln

65 70 75 80

Glu Leu Glu Lys Thr Phe Thr Lys Leu Arg Val Leu Asp Leu Asp Ile

85 90 95

Lys Ile Asp Arg Thr Glu Ala Phe Asn Leu Phe Ile Lys Lys Phe Gln

100 105 110

Ser Val Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met

115 120 125

Asp Gln Leu Lys Glu Ala Lys Ile Asp Glu Leu Asp Leu His Arg Ala

130 135 140

Ile Val Ala Leu Ser Glu Lys Met Lys Ala Val Asp Asp Asn Ala Ser

145 150 155 160

Lys Lys Lys Asp Glu Pro Ser Leu Tyr Thr Ser Trp Thr Leu Ser Phe

165 170 175

Thr Ala Pro Thr Ser Glu Glu Ala Gln Thr Val Leu Ser Gly Tyr Ile

180 185 190

Asp Tyr Ile Ser Ala Leu Val Val Lys Glu Ser Ile Glu Asn Val Arg

195 200 205

Asn Lys Leu Glu Ile Lys Thr Gln Phe Glu Lys Glu Lys Leu Ala Gln

210 215 220

Asp Arg Ile Lys Met Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu

225 230 235 240

Asn Tyr Ser Leu Asp Ile Ala Asn Ala Ala Gly Ile Lys Lys Pro Val

245 250 255

Tyr Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser

260 265 270

Leu Gly Ala Asp Gly Ile Glu Arg Lys Leu Glu Ile Glu Lys Ala Val

275 280 285

Thr Asp Val Ala Glu Leu Asn Gly Glu Leu Arg Asn Arg Gln Tyr Leu

290 295 300

Val Glu Gln Leu Thr Lys Ala Asn Ile Asn Asp Val Asn Phe Thr Pro

305 310 315 320

Phe Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro

325 330 335

Gly Lys Ala Ile Ile Val Ile Leu Ser Ala Leu Ile Gly Gly Met Val

340 345 350

Ala Cys Gly Ser Val Leu Leu Arg Tyr Ala Met Ala Ser Arg Lys Gln

355 360 365

Asp Ala Met Met Ala Asp His Leu Val

370 375

<210> 121

<211> 378

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic

<400> 121

Met Pro Ser Leu Asn Val Lys Gln Glu Lys Asn Gln Ser Phe Ala Gly

1 5 10 15

Tyr Ser Leu Pro Pro Ala Asn Ser His Glu Ile Asp Leu Phe Ser Leu

20 25 30

Ile Glu Val Leu Trp Gln Ala Lys Arg Arg Ile Leu Ala Thr Val Phe

35 40 45

Ala Phe Ala Cys Val Gly Leu Leu Leu Ser Phe Leu Leu Pro Gln Lys

50 55 60

Trp Thr Ser Gln Ala Ile Val Thr Pro Ala Glu Ser Val Gln Trp Gln

65 70 75 80

Gly Leu Glu Arg Thr Leu Thr Ala Leu Arg Val Leu Asp Met Glu Val

85 90 95

Ser Val Asp Arg Gly Ser Val Phe Asn Leu Phe Ile Lys Lys Phe Ser

100 105 110

Ser Pro Ser Leu Leu Glu Glu Tyr Leu Arg Ser Ser Pro Tyr Val Met

115 120 125

Asp Gln Leu Lys Gly Ala Gln Ile Asp Glu Gln Asp Leu His Arg Ala

130 135 140

Ile Val Leu Leu Ser Glu Lys Met Lys Ala Val Asp Ser Asn Val Gly

145 150 155 160

Lys Lys Asn Glu Thr Ser Leu Phe Thr Ser Trp Thr Leu Ser Phe Thr

165 170 175

Ala Pro Thr Arg Glu Glu Ala Gln Lys Val Leu Ala Gly Tyr Ile Gln

180 185 190

Tyr Ile Ser Asp Ile Val Val Lys Glu Thr Leu Glu Asn Ile Arg Asn

195 200 205

Gln Leu Glu Ile Lys Thr Arg Tyr Glu Gln Glu Lys Leu Ala Met Asp

210 215 220

Arg Val Arg Leu Lys Asn Gln Leu Asp Ala Asn Ile Gln Arg Leu His

225 230 235 240

Tyr Ser Leu Glu Ile Ala Asn Ala Ala Gly Ile Lys Arg Pro Val Tyr

245 250 255

Ser Asn Gly Gln Ala Val Lys Asp Asp Pro Asp Phe Ser Ile Ser Leu

260 265 270

Gly Ala Asp Gly Ile Ser Arg Lys Leu Glu Ile Glu Lys Gly Val Thr

275 280 285

Asp Val Ala Glu Ile Asp Gly Asp Leu Arg Asn Arg Gln Tyr His Val

290 295 300

Glu Gln Leu Ala Ala Met Asn Val Ser Asp Val Lys Phe Thr Pro Phe

305 310 315 320

Lys Tyr Gln Leu Ser Pro Ser Leu Pro Val Lys Lys Asp Gly Pro Gly

325 330 335

Lys Ala Ile Ile Ile Ile Leu Ala Ala Leu Ile Gly Gly Met Met Ala

340 345 350

Cys Gly Gly Val Leu Leu Arg His Ala Met Val Ser Arg Lys Met Glu

355 360 365

Asn Ala Leu Ala Ile Asp Glu Arg Leu Val

370 375

<210> 122

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 122

gaagcaaacc gtacgcgtaa ag 22

<210> 123

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 123

cgaccagctc ttacacggcg 20

<210> 124

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 124

gaaataggac cactaataaa tacacaaatt aataac 36

<210> 125

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 125

ataattgacg atccggttgc from 21

<210> 126

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 126

gctatttacg ccctgattgt cttttgt 27

<210> 127

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 127

attgagaacc tgcgtaaacg gc 22

<210> 128

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 128

tgaagagcgg ttcagataac ttcc 24

<210> 129

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 129

cgatccggaa acctcctaca from 21

<210> 130

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 130

gattattcgc gcaacgctaa acagat 26

<210> 131

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 131

tgatcattga cgatccggta gcc 23

<210> 132

<211> 70

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 132

cggtagctgt aaagccaggg gcggtagcgt ggtttaaacc caagcaacag atcggcgtcg 60

tcggtatgga 70

<210> 133

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 133

agcttccata ccgacgacgc cgatctgttg cttgggttta aaccacgcta ccgcccctgg 60

ctttacagct accgagct 78

<210> 134

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 134

ggtagctgta aagccagggg cggtagcgtg 30

<210> 135

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 135

ccataccgac gacgccgatc tgttgcttgg 30

<210> 136

<211> 23

<212> Protein

<213> Homo sapiens

<400> 136

Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe

1 5 10 15

Leu Leu Pro Gly Ser Leu Gly

20

<210> 137

<211> 18

<212> Protein

<213> Homo sapiens

<400> 137

Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val

1 5 10 15

Arg Ala

<210> 138

<211> 25

<212> Protein

<213> Human respiratory syncytial virus A (strain A2)

<400> 138

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly

20 25

<210> 139

<211> 15

<212> Protein

<213> Human respiratory syncytial virus A (strain A2)

<400> 139

Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly

1 5 10 15

<210> 140

<211> 365

<212> Protein

<213> Homo sapiens

<400> 140

Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe

1 5 10 15

Leu Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr

20 25 30

His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp

35 40 45

Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu

50 55 60

Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val

65 70 75 80

Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys Glu Lys

85 90 95

Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys Gly Pro Tyr Thr

100 105 110

Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly Pro Asp Asn Thr Ser Val

115 120 125

Pro Thr Ala Lys Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp

130 135 140

Leu Lys Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile

145 150 155 160

Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr

165 170 175

Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg

180 185 190

Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys

195 200 205

Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser Ala Phe

210 215 220

Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu Arg Asn Gly Leu

225 230 235 240

Ala Ala Gly Thr Gly Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser

245 250 255

Phe His Ala Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His

260 265 270

Tyr Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val

275 280 285

Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val

290 295 300

Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu

305 310 315 320

Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu Arg

325 330 335

Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu Ala Gln Asp

340 345 350

Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala Thr Ala

355 360 365

<210> 141

<211> 62

<212> Protein

<213> Homo sapiens

<400> 141

Met Gly Val Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe

1 5 10 15

Leu Leu Pro Gly Ser Leu Gly Phe Ala Cys Lys Thr Ala Asn Gly Thr

20 25 30

Ala Ile Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala

35 40 45

Pro Val Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser

50 55 60

<210> 142

<211> 574

<212> Protein

<213> Human respiratory syncytial virus A (strain A2)

<400> 142

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe

20 25 30

Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu

35 40 45

Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn Ile

50 55 60

Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu Ile Lys

65 70 75 80

Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu Leu Gln Leu Leu

85 90 95

Met Gln Ser Thr Pro Pro Thr Asn Asn Arg Ala Arg Arg Glu Leu Pro

100 105 110

Arg Phe Met Asn Tyr Thr Leu Asn Asn Ala Lys Lys Thr Asn Val Thr

115 120 125

Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Val

130 135 140

Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu

145 150 155 160

Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys

165 170 175

Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val

180 185 190

Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile Val Asn

195 200 205

Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln

210 215 220

Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn

225 230 235 240

Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu

245 250 255

Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys

260 265 270

Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile

275 280 285

Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro

290 295 300

Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro

305 310 315 320

Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg

325 330 335

Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe

340 345 350

Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp

355 360 365

Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Ile Asn Leu Cys Asn Val

370 375 380

Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr

385 390 395 400

Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys

405 410 415

Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile

420 425 430

Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Met Asp

435 440 445

Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly

450 455 460

Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro

465 470 475 480

Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn

485 490 495

Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu

500 505 510

Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn Ile Met Ile Thr

515 520 525

Thr Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val

530 535 540

Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu Ser

545 550 555 560

Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe Ser Asn

565 570

<210> 143

<211> 64

<212> Protein

<213> Human respiratory syncytial virus A (strain A2)

<400> 143

Met Glu Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr

1 5 10 15

Ala Val Thr Phe Cys Phe Ala Ser Gly Phe Ala Cys Lys Thr Ala Asn

20 25 30

Gly Thr Ala Ile Pro Ile Gly Gly Gly Ser Ala Asn Val Tyr Val Asn

35 40 45

Leu Ala Pro Val Val Asn Val Gly Gln Asn Leu Val Val Asp Leu Ser

50 55 60

<210> 144

<211> 178

<212> Protein

<213> Homo sapiens

<400> 144

Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val

1 5 10 15

Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His

20 25 30

Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe

35 40 45

Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu

50 55 60

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys

65 70 75 80

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro

85 90 95

Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu

100 105 110

Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg

115 120 125

Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn

130 135 140

Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu

145 150 155 160

Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile

165 170 175

Arg Asn

<210> 145

<211> 57

<212> Protein

<213> Homo sapiens

<400> 145

Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val

1 5 10 15

Arg Ala Phe Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly

20 25 30

Gly Gly Ser Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val

35 40 45

Gly Gln Asn Leu Val Val Asp Leu Ser

50 55

<210> 146

<211> 562

<212> Protein

<213> Influenza A virus (strain A/Japan/305/1957 H2N2)

<400> 146

Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp

1 5 10 15

Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp

20 25 30

Thr Asn Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu

35 40 45

Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro

50 55 60

Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro

65 70 75 80

Glu Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met Glu

85 90 95

Lys Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp

100 105 110

Tyr Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys

115 120 125

Val Lys Ile Leu Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly

130 135 140

Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn

145 150 155 160

Met Val Trp Leu Thr Lys Glu Gly Ser Asp Tyr Pro Val Ala Lys Gly

165 170 175

Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val

180 185 190

His His Pro Ile Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val

195 200 205

Gly Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Thr

210 215 220

Pro Glu Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Gly Gly Arg Met

225 230 235 240

Glu Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu

245 250 255

Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys

260 265 270

Arg Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys

275 280 285

Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro

290 295 300

Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val

305 310 315 320

Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln

325 330 335

Ile Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly

340 345 350

Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn

355 360 365

Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala

370 375 380

Phe Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn

385 390 395 400

Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Gly Asn Leu Glu Arg Arg

405 410 415

Leu Glu Asn Leu Asn Lys Arg Met Glu Asp Gly Phe Leu Asp Val Trp

420 425 430

Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu

435 440 445

Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met

450 455 460

Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe

465 470 475 480

Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly Thr

485 490 495

Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu

500 505 510

Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala

515 520 525

Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Met Ala

530 535 540

Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile

545 550 555 560

Cys Ile

<210> 147

<211> 54

<212> Protein

<213> Influenza A virus (strain A/Japan/305/1957 H2N2)

<400> 147

Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Phe

1 5 10 15

Ala Cys Lys Thr Ala Asn Gly Thr Ala Ile Pro Ile Gly Gly Gly Ser

20 25 30

Ala Asn Val Tyr Val Asn Leu Ala Pro Val Val Asn Val Gly Gln Asn

35 40 45

Leu Val Val Asp Leu Ser

50

<---

Claims (24)

1. A mutant FimH polypeptide for inducing the production of antibodies specific to uropathogenic E. coli in a subject, which comprises amino acid mutations relative to the amino acid sequence of a wild-type FimH polypeptide, wherein the mutations are selected from the group consisting of: a) mutations G15A and V27A; b) mutations G16A and V27A; and c) mutations G15A, G16A and V27A, and wherein the amino acid positions are numbered according to SEQ ID NO: 59. 2. A mutant FimH polypeptide for inducing the production of antibodies specific to uropathogenic E. coli in a subject, comprising a sequence selected from the group consisting of SEQ ID NO: 50, 51, 54, 61 and 62. 3. A mutant FimH polypeptide for inducing the production of antibodies specific to uropathogenic E. coli in a subject, which comprises amino acid mutations relative to the amino acid sequence of a wild-type FimH polypeptide, and wherein said mutations comprise G15A, G16A, and V27A. 4. A mutant FimH polypeptide according to any one of claims 1 to 3, wherein the polypeptide further comprises a FimG peptide linked via a glycine-serine linker. 5. A mutant FimH polypeptide according to any one of claims 1 to 4, wherein the polypeptide is immunogenic. 6. A mutant FimH polypeptide according to any one of claims 1 to 5, wherein the polypeptide is isolated. 7. A nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence of a mutant FimH polypeptide according to any one of claims 1 to 5. 8. A nucleic acid molecule according to claim 7, wherein the nucleic acid is RNA. 9. A pharmaceutical composition for inducing the production of antibodies specific to uropathogenic E. coli in a subject, comprising (i) an effective amount of a mutant FimH polypeptide according to any one of claims 1-6 or a nucleic acid molecule according to claim 7 or 8 and (ii) a pharmaceutically acceptable carrier. 10. The pharmaceutical composition according to claim 9, additionally containing at least one additional antigen. 11. The pharmaceutical composition according to claim 9, wherein at least one additional antigen is a polysaccharide or protein. 12. The pharmaceutical composition according to claim 9, additionally containing at least one adjuvant. 13. A recombinant mammalian cell for producing a mutant FimH polypeptide, comprising a polynucleotide encoding a mutant FimH polypeptide according to any one of claims 1 to 5. 14. A culture for producing a mutant FimH polypeptide, containing a recombinant cell according to claim 13 and a cell culture medium, wherein said culture has a size of at least 5 liters. 15. A method for producing a mutant FimH polypeptide according to any one of claims 1 to 5, comprising culturing a recombinant mammalian cell according to claim 13 under suitable conditions, thereby ensuring expression of the polypeptide; and collecting the polypeptide. 16. A method for inducing the production of antibodies specific to uropathogenic E. coli in a subject, wherein the method comprises administering to the subject an effective amount of the composition according to any one of claims 9-12. 17. The method of claim 16, wherein the subject is at risk of developing a urinary tract infection. 18. The method of claim 16, wherein the subject is at risk of developing bacteremia. 19. The method of claim 16, wherein the subject is at risk of developing sepsis. 20. The method according to claim 16, wherein the antibodies protect the mammal from infection by uropathogenic E. coli . 21. A method for preventing, treating or alleviating a uropathogenic E. coli infection in a subject, comprising administering to the subject an immunologically effective amount of a composition according to any one of claims 9-12.