Methods for producing bioconjugates of Escherichia coli O-antigen polysaccharides, compositions thereof, and methods of use thereof

Abstract

Provided are methods for producing bioconjugates of O-antigen polysaccharides covalently linked to a carrier protein using recombinant host cells. The recombinant host cells used in the methods described herein encode specific oligosaccharyl transferases, depending on the O-antigen polysaccharide bioconjugate to be produced. The oligosaccharyl transferase can be a PglB oligosaccharyl transferase or a variant thereof. Also provided are compositions containing bioconjugates, and methods for vaccinating subjects against extraintestinal pathogenic Escherichia coli (ExPEC) using the bioconjugates and compositions described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 819,762, filed on March 18, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Reference to sequence listings submitted electronically

[0004] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an ASCII formatted Sequence Listing with the file name "004852_11612 Sequence_Listing," a creation date of March 11, 2020, and a size of 199 KB. The Sequence Listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background Art

[0005] Extraintestinal pathogenic Escherichia coli ( Escherichia coli ) (ExPEC) strains, along with commensal E. coli strains, are normally harmless residents of the human gastrointestinal tract. ExPEC isolates cannot be easily distinguished from commensal isolates by serotype, although many clonal lineages are ExPEC-dominant, as determined by the O-antigen, capsule, and flagellar antigen serotype (abbreviated O:K:H, e.g., O25:K1:H4). In contrast to commensal E. coli, ExPEC strains express a wide variety of virulence factors that enable them to colonize the gastrointestinal tract and cause a wide range of extraintestinal infections that are associated with a significant healthcare cost burden due to hospitalizations and mortality. Neonates, the elderly, and immunocompromised patients are particularly susceptible to ExPEC infections, including invasive ExPEC disease (IED).

[0006] ExPEC strains are the most common cause of urinary tract infections (UTIs) and are important contributing factors for surgical site infections and neonatal meningitis. These strains are also associated with abdominal and pelvic infections and nosocomial pneumonia, and occasionally involve other extraintestinal infections, such as osteomyelitis, cellulitis, and wound infections. All of these major infection sites can lead to ExPEC bacteremia. ExPEC is the most common cause of community-onset bacteremia and the main etiological pathogen of nosocomial bacteremia, and is found in approximately 17% to 37% of clinically significant blood isolates. Patients with ExPEC-positive blood cultures typically develop sepsis syndrome, severe sepsis, or septic shock. Increased resistance of ExPEC to first-line antibiotics has been observed, including cephalosporins, fluoroquinolones, and trimethoprim / sulfamethoxazole. The emergence and rapid global spread of ExPEC sequence type 131 (ST131) are considered to be the main drivers of increased drug resistance, including multidrug resistance. This clone was found in 12.5% ​​to 30% of all ExPEC clinical isolates, predominantly displayed serotype O25b:H4, and showed high-level resistance to fluoroquinolones, often accompanied by trimethoprim / sulfamethoxazole resistance and extended-spectrum β-lactamases that confer resistance to cephalosporins.

[0007] O-antigens comprise the immunodominant components of the cell wall lipopolysaccharide (LPS) in Gram-negative bacteria, including E. coli. There are currently >180 serologically distinct E. coli O-antigens identified, with the vast majority of ExPEC isolates classified within fewer than 20 O-antigen serotypes. Full-length E. coli O-antigens typically consist of approximately 10 to 25 repeating sugar units attached to a highly conserved LPS core structure, with each component consisting of a rfb and rfa The enzymes predominantly encoded in the gene cluster are synthesized. After polymerization of the O-antigen, the O-antigen polysaccharide backbone may be modified, usually by the addition of acetyl or glucose residues. These modifications effectively increase serotype diversity by generating antigenically distinct serotypes that share a common polysaccharide backbone but differ in side branches. Genes encoding O-antigen modification enzymes are usually located on the chromosome rfb Clusters, and in some cases, these genes are found within lysogenic bacterial phages.

[0008] ExPEC isolates belonging to the O4 serogroup have been commonly identified in contemporary surveillance studies of blood isolates in the United States and the European Union. The structure of the O4 polysaccharide has been determined to be: -->2) α-L-Rha (1->6) α-D-Glc (1->3) α-L-FucNAc (1->3) β-D-GlcNAc (1->, from E. coli O4:K52 strain (Jann et al., Carbohydr. Res. (1993) Vol. 248, pp. 241-250). A different form of the O4 polysaccharide structure has been determined for O4:K3, O4:K6, and O4:K12 strains, in which the above structure is modified by the addition of α-D-Glc (1->3) attached to a rhamnose residue of the polysaccharide (Jann et al., 1993, supra); this form of the polysaccharide is referred to herein as 'glucosylated O4'. The enzyme responsible for the modification of the O-antigen in E. coli O4 strains has not yet been identified.

[0009] Efforts to develop vaccines to prevent ExPEC infections have focused on O-antigen polysaccharide conjugates. Pseudomonas aeruginosa ) exotoxin A, a 12-valent O-antigen conjugate vaccine was synthesized and tested for safety and immunogenicity in a phase 1 clinical study (Cross et al., J. Infect. Dis (1994) Vol. 170, pp. 834-40). This candidate vaccine has never been approved for clinical use. Recently, a bioconjugation system in E. coli has been developed in which both the polysaccharide antigen and the carrier protein are synthesized in vivo and subsequently expressed in E. coli by the oligosaccharyltransferase PglB (Campylobacter jejuni ( Campylobacter jejuni ) enzyme) activity in vivo conjugation (Wacker et al., Proc. Nat. Acad. Sci (2006) Vol. 103, pp. 7088-93). This N-linked protein glycosylation system enables the transfer of different polysaccharides to carrier proteins, allowing a straightforward method for purifying the conjugate.

[0010] Bioconjugation has been successfully used to produce conjugate polysaccharides for tetravalent O-antigen vaccine candidates against E. coli (Poolman and Wacker, J. Infect. Dis (2016) Vol. 213(1), pp. 6-13). However, the development of a successful ExPEC vaccine requires coverage of the dominant serotypes, and the presence of further O-antigen modifications in subsets of ExPEC isolates presents further challenges in terms of coverage of isolates displaying both unmodified and modified LPS. Furthermore, the production efficiency of the multiple components of more complex vaccine compositions covering multiple serotypes is becoming increasingly important, and therefore there remains a need to improve the production of individual bioconjugates of specific O-antigens. Summary of the Invention

[0011] Given the increasing antibiotic resistance among ExPEC isolates and the presence of further O-antigen modifications in predominant O-serotypes, there is a need for improved prophylactic and therapeutic treatments for these infections. The present invention addresses this need by defining the genetic composition of contemporary clinical isolates, including identifying genes encoding O-antigen modification enzymes, thereby allowing the engineering of recombinant host cells capable of synthesizing bioconjugates of O-antigens, including bioconjugates containing selected O-antigen modifications. Additionally, in one aspect of the present invention, host cells and methods for the improved production of bioconjugates of specific O-antigens using variants of oligosaccharyltransferases (OSTs) are provided, based on the advantages of using certain OST variants for bioconjugates of certain E. coli O-antigens in an unpredictable serotype-dependent manner. The use of such OST variants may also, in certain cases, affect the glycosylation pattern of the bioconjugates, for example by increasing the relative number of glycans coupled to the carrier protein compared to bioconjugates produced using wild-type or other OST variants, and thus novel bioconjugates produced by such methods are also provided as an aspect of the present invention.

[0012] In one aspect, provided is a method for preparing E. coli O covalently linked to a carrier protein. x A method for bioconjugating an antigen-polysaccharide, the method comprising:

[0013] (i) providing a recombinant host cell comprising:

[0014] a. About O x - Antigenic polysaccharide rfb nucleotide sequence of the gene cluster;

[0015] b. A nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably SEQ ID NO: 2; and

[0016] c. Encoding oligosaccharyltransferase PglB y the nucleotide sequence of; and

[0017] (ii) culturing the recombinant host cell under conditions for bioconjugate production,

[0018] in:

[0019] When O x -When the antigen is O1A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0020] When O x -When the antigen is glucosylated O4 antigen polysaccharide, PglB y comprising an amino acid mutation N311V or amino acid mutations Y77H and N311V, and the recombinant host cell further comprising a sequence encoding a glucosyltransferase GtrS having at least 80% identity to SEQ ID NO: 4 and capable of modifying E. coli O4 antigen polysaccharide by adding glucose to produce a glucosylated E. coli O4 antigen polysaccharide, and nucleotide sequences encoding a translocase GtrA and a glycosyltransferase GtrB having at least 80% sequence identity to SEQ ID NOs: 7 and 8, respectively, wherein the translocase is capable of translocating glucose linked to a bacterial terpene alcohol, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol;

[0021] When O x -When the antigen is O6A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0022] When O x -When the antigen is O8 antigen polysaccharide, PglB y does not contain amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483, and 669;

[0023] When O x -When the antigen is O15 antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0024] When O x -When the antigen is O16 antigen polysaccharide, PglB y amino acid mutations including Y77H, S80R, Q287P, K289R, and N311V;

[0025] When O x -When the antigen is O18A antigen polysaccharide, PglB y does not comprise amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483, and 669; and

[0026] When O x -When the antigen is O75 antigen polysaccharide, PglB y Contains the amino acid mutation N311V,

[0027] wherein in each case the amino acid mutation is relative to wild-type PglB having the amino acid sequence of SEQ ID NO: 6,

[0028] wherein the O1A, glucosylated O4, O6A, O8, O15, O16, O18A and O75 antigen polysaccharides have the structures of formula (O1A), (O4-Glc+), (O6A), (O8), (O15), (O16), (O18A) and (O75) as shown in Table 1, respectively, and each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0029] In one embodiment, O x - The antigen is O1A antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0030] In one embodiment, O x - The antigen is a glucosylated 04 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Comprising the amino acid mutation N311V. In one embodiment, x - The antigen is a glucosylated 04 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations Y77H and N311V. x -In an embodiment where the antigen is a glucosylated O4 antigen polysaccharide, the recombinant host cell preferably further comprises a sequence encoding GtrS having at least 80% identity to SEQ ID NO: 4, and nucleotide sequences encoding GtrA and GtrB having at least 80% sequence identity to SEQ ID NOs: 7 and 8, respectively.

[0031] In one embodiment, O x - The antigen is O6A antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0032] In one embodiment, O x - The antigen is O8 antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669 are not included.

[0033] In one embodiment, O x - The antigen is O15 antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0034] In one embodiment, O x - The antigen is O16 antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations Y77H, S80R, Q287P, K289R and N311V.

[0035] In one embodiment, O x - The antigen is O18A antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y It does not contain amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669, and preferably contains the amino acid sequence of SEQ ID NO: 6.

[0036] In one embodiment, O x - The antigen is O75 antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains the amino acid mutation N311V.

[0037] In one specific aspect, provided is a method for preparing E. coli O covalently linked to a carrier protein. x - a method for preparing a bioconjugate of an antigen-polysaccharide, the method comprising:

[0038] (i) providing a recombinant host cell comprising:

[0039] a. About O x - Antigenic polysaccharide rfb nucleotide sequence of the gene cluster;

[0040] b. A nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably SEQ ID NO: 2; and

[0041] c. Encoding oligosaccharyltransferase PglB y the nucleotide sequence of; and

[0042] (ii) culturing the recombinant host cell under conditions for bioconjugate production,

[0043] Wherein relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains the amino acid mutation N311V,

[0044] Among them O x - The antigen is an O1A antigen polysaccharide, a glucosylated O4 antigen polysaccharide, an O6A antigen polysaccharide, an O15 antigen polysaccharide, an O16 antigen polysaccharide or an O75 antigen polysaccharide, and when O x When the antigen is a glucosylated O4 antigen polysaccharide, the recombinant host cell further comprises a sequence encoding a glucosyltransferase GtrS having at least 80% identity to SEQ ID NO: 4 and capable of modifying E. coli O4 antigen polysaccharide by adding glucose to produce a glucosylated E. coli O4 antigen polysaccharide, and nucleotide sequences encoding a translocase GtrA and a glycosyltransferase GtrB having at least 80% sequence identity to SEQ ID NOs: 7 and 8, respectively, wherein the translocase is capable of translocating glucose linked to a bacterial terpene alcohol and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol, and

[0045] wherein the O1A, glucosylated O4, O6A, O15, O16 and O75 antigen polysaccharides have the structures of formula (O1A), (O4-Glc+), (O6A), (O15), (O16) and (O75) as shown in Table 1, respectively, and each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0046] In certain embodiments, the method further comprises isolating the bioconjugate from the recombinant host cell.

[0047] In certain embodiments, the carrier protein is selected from the group consisting of detoxified Pseudomonas aeruginosa exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus ( S. aureus ) hemolysin A, clumping factor A, clumping factor B, Escherichia coli heat-labile enterotoxin, detoxified variant of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae ( Streptococcus pneumoniae ) pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, Neisseria meningitidis ( Neisseria meningitidis) and the outer membrane protein (OMPC) from non-typeable Haemophilus influenzae ( Haemophilus influenzae ) of protein D.

[0048] In certain embodiments, the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA). Preferably, the EPA carrier protein comprises 1-10, preferably 2-4, more preferably 4 glycosylation sites. In certain embodiments, each glycosylation site comprises a glycosylation consensus sequence having SEQ ID NO: 2. In a specific embodiment, the EPA carrier protein comprises SEQ ID NO: 3.

[0049] In certain embodiments, the recombinant host cell is an E. coli cell, such as an E. coli K-12 strain, such as strain W3110.

[0050] In another aspect, provided is a method of preparing an O-protein covalently linked to a carrier protein as described herein. x Methods of bioconjugating antigen-polysaccharide bioconjugates produce the bioconjugates.

[0051] In another aspect, provided are compositions comprising such bioconjugates. In some embodiments, the composition comprises at least 2, preferably at least 3, more preferably at least 5, and even more preferably at least 7 such bioconjugates.

[0052] In certain embodiments, the composition according to the present invention comprises a bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to a carrier protein, wherein the glucosylated O4 antigen polysaccharide has a structure of formula (O4-Glc+) as shown in Table 1, and n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. In certain embodiments, the composition according to the present invention further comprises at least a bioconjugate of a O25B antigen polysaccharide of E. coli covalently linked to a carrier protein, wherein the O25B antigen polysaccharide has a structure of formula (O25B) as shown in Table 1, and n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. In certain embodiments, the composition according to the present invention further comprises at least a bioconjugate of an Escherichia coli O2 antigen polysaccharide covalently linked to a carrier protein, wherein the O2 antigen polysaccharide has a structure of formula (O2) as shown in Table 1, and n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0053] In certain embodiments, the compositions of the present invention comprise: (i) a bioconjugate of an E. coli O1A antigen polysaccharide covalently coupled to a carrier protein, (ii) a bioconjugate of an E. coli O2 antigen polysaccharide covalently coupled to a carrier protein, (iii) a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide covalently coupled to a carrier protein, (iv) a bioconjugate of an E. coli O6A antigen polysaccharide covalently coupled to a carrier protein, (v) a bioconjugate of an E. coli O8 antigen polysaccharide covalently coupled to a carrier protein, (vi) a bioconjugate of an E. coli O15 antigen polysaccharide covalently coupled to a carrier protein, (vii) a bioconjugate of an E. coli O16 antigen polysaccharide covalently coupled to a carrier protein. coli O25B antigen polysaccharide bioconjugates, (viii) a bioconjugate of an Escherichia coli O25B antigen polysaccharide covalently coupled to a carrier protein, and (ix) a bioconjugate of an Escherichia coli O75 antigen polysaccharide covalently coupled to a carrier protein, wherein the O1A, O2, glucosylated O4, O6A, O8, O15, O16, O25B and O75 antigen polysaccharides have the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O8), (O15), (O16), (O25B) and (O75) as shown in Table 1, respectively, and each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. In certain embodiments, such compositions further comprise: (x) a bioconjugate of an E. coli O18A antigen polysaccharide covalently coupled to a carrier protein, wherein the O18A antigen polysaccharide has a structure of formula (O18A) as shown in Table 1, and n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. In certain embodiments, the composition of the present invention is an immunogenic composition.

[0054] In other aspects, provided are methods of vaccinating a subject against extraintestinal pathogenic E. coli (ExPEC), comprising administering to the subject such a bioconjugate or composition as described herein. In still other aspects, provided are such a bioconjugate or composition as described herein for use in vaccinating against extraintestinal pathogenic E. coli (ExPEC).

[0055] In other aspects, provided are methods for preparing E. coli O covalently linked to a carrier protein. x A recombinant host cell for a bioconjugate of an antigen-polysaccharide, the recombinant host cell comprising:

[0056] (a) About O x - Antigenic polysaccharide rfb nucleotide sequence of the gene cluster;

[0057] (b) a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2; and

[0058] (c) Encoding oligosaccharyltransferase PglB y The nucleotide sequence of

[0059] in:

[0060] When O x -When the antigen is O1A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0061] When O x -When the antigen is glucosylated O4 antigen polysaccharide, PglB y comprising an amino acid mutation N311V or amino acid mutations Y77H and N311V, and the recombinant host cell further comprising a sequence encoding a glucosyltransferase GtrS having at least 80% identity to SEQ ID NO: 4 and capable of modifying E. coli O4 antigen polysaccharide by adding glucose to produce a glucosylated E. coli O4 antigen polysaccharide, and nucleotide sequences encoding a translocase GtrA and a glycosyltransferase GtrB having at least 80% sequence identity to SEQ ID NOs: 7 and 8, respectively, wherein the translocase is capable of translocating glucose linked to a bacterial terpene alcohol, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol;

[0062] When O x -When the antigen is O6A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0063] When O x -When the antigen is O6A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0064] When O x -When the antigen is O15 antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0065] When O x -When the antigen is O16 antigen polysaccharide, PglB yamino acid mutations including Y77H, S80R, Q287P, K289R, and N311V;

[0066] When O x -When the antigen is O18A antigen polysaccharide, PglB y does not comprise amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483, and 669; and

[0067] When O x -When the antigen is O75 antigen polysaccharide, PglB y Contains the amino acid mutation N311V,

[0068] wherein in each case the amino acid mutation is relative to wild-type PglB having the amino acid sequence of SEQ ID NO: 6, and

[0069] wherein the O1A, glucosylated O4, O6A, O8, O15, O16, O18A and O75 antigen polysaccharides have the structures of formula (O1A), (O4-Glc+), (O6A), (O8), (O15), (O16), (O18A) and (O75) as shown in Table 1, respectively, and each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0070] In certain embodiments, provided are host cells wherein the x - the antigen is O1A antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0071] In certain embodiments, provided are recombinant host cells of the present invention, wherein the x - The antigen is a glucosylated 04 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y In certain embodiments, the recombinant host cell of the present invention is provided, wherein the O x - The antigen is a glucosylated 04 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations Y77H and N311V. xIn certain embodiments where the antigen is a glucosylated O4 antigen polysaccharide, the recombinant host cell further comprises a sequence encoding GtrS having the amino acid sequence of SEQ ID NO: 4, and nucleotide sequences encoding GtrA and GtrB having the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

[0072] In certain embodiments, provided are recombinant host cells of the present invention, wherein the x - the antigen is O6A antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0073] In certain embodiments, provided are recombinant host cells of the present invention, wherein the x - The antigen is O8 antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669 are not included.

[0074] In certain embodiments, provided are recombinant host cells of the present invention, wherein the x - The antigen is O15 antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0075] In certain embodiments, provided are recombinant host cells of the present invention, wherein the x - The antigen is O16 antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations Y77H, S80R, Q287P, K289R and N311V.

[0076] In certain embodiments, provided are recombinant host cells of the present invention, wherein the x - The antigen is O18A antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669 are not included.

[0077] In certain embodiments, provided are recombinant host cells of the present invention, wherein thex - The antigen is O75 antigen polysaccharide, relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains the amino acid mutation N311V.

[0078] In certain embodiments, provided are recombinant host cells of the present invention, wherein the carrier protein is selected from the group consisting of detoxified Pseudomonas aeruginosa exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, clumping factor A, clumping factor B, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.

[0079] In certain embodiments, provided are recombinant host cells of the present invention, wherein the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA). In certain embodiments thereof, the EPA carrier protein comprises 1-10, preferably 2-4, more preferably 4 glycosylation sites. In certain embodiments, each glycosylation site comprises a glycosylation consensus sequence having SEQ ID NO: 2. In certain embodiments, the EPA carrier protein comprises SEQ ID NO: 3.

[0080] In certain embodiments, provided are recombinant host cells of the invention, wherein the recombinant host cell is an E. coli cell, such as an E. coli K-12 strain, such as strain W3110.

[0081] In certain embodiments of the host cells and methods for preparing bioconjugates of E. coli glucosylated O4 antigen polysaccharide covalently linked to a carrier protein according to the present invention, rfb The gene cluster comprises a sequence encoding an enzyme that produces an E. coli O4 antigen polysaccharide (formula (O4-Glc-) in Table 1) and is at least 80%, such as at least 90%, such as at least 95%, such as at least 98% identical to SEQ ID NO: 9. In certain embodiments, rfb The gene cluster comprises SEQ ID NO: 9.

[0082] In certain embodiments of the host cells and methods for preparing a bioconjugate of a glucosylated E. coli O4 antigen polysaccharide covalently linked to a carrier protein according to the present invention, the glucosyltransferase capable of modifying the E. coli O4 antigen polysaccharide to produce the glucosylated E. coli O4 antigen polysaccharide has an amino acid sequence that has at least 90%, preferably at least 95%, and preferably at least 98% sequence identity to SEQ ID NO: 4. In certain embodiments, the glucosyltransferase comprises SEQ ID NO: 4.

[0083] In certain embodiments of the host cells and methods for preparing bioconjugates of E. coli glucosylated O4 antigen polysaccharide covalently linked to a carrier protein according to the present invention, the translocase is capable of translocating bacterial terpene alcohol-linked glucose and has at least 90%, preferably at least 95%, and preferably at least 98% sequence identity to SEQ ID NO: 7. In certain embodiments, the translocase comprises SEQ ID NO: 7.

[0084] In certain embodiments of the host cells and methods for preparing bioconjugates of glucosylated E. coli O4 antigen polysaccharide covalently linked to a carrier protein according to the present invention, the glycosyltransferase is capable of glucosylating a bacterial terpene alcohol and has at least 90%, preferably at least 95%, and preferably at least 98% sequence identity to SEQ ID NO: 8. In certain embodiments, the glycosyltransferase comprises SEQ ID NO: 8. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings.It is to be understood that the invention is not limited to the precise embodiments shown in the drawings.

[0086] In the attached figure:

[0087] Figure 1 Shown are ELISA IgG titers against unmodified (GLC-) or glucose-modified ((GLC+) O4 LPS in sera from two rabbits immunized with Glc-modified O4 polysaccharide bioconjugates as described in Example 4; ELISA titers were determined in quadruplicate;

[0088] Figure 2 As described in Example 4, it is shown that gtrS coli O4 isolates in the state of IgG titers in whole cell ELISA using pooled sera from rabbits immunized with Glc-modified O4 bioconjugates; the following isolates are gtrSNegative: A2625, stGVXN4988, OC24784, OC24787, and OC24788; the following isolates were gtrS positive: Y1382, E551, OC24334, stGVXN4983, stGVXN4994, and OC24794; negative control strain OC9487 (ATCC 35383; serotype O75) was also included;

[0089] Figure 3 Shown are pooled sera from rabbits immunized with free modified O4 polysaccharide. gtrS Western blot of LPS extracted from positive and negative O4 isolates;

[0090] Figure 4A and 4B Antibody responses induced by glucosylated O4 (O4-Glc+)-EPA bioconjugate are shown; Figure 4A Shown are serum antibody levels measured by ELISA at days 0, 14, and 42 after immunization; individual titers (log10EC50 titers) and GMT ± 95% CI are shown; the gray dashed line indicates the threshold above which the dilution curve of the sample has a 4PL fit; Figure 4B Shown are the results of an opsonophagocytic (OPK) assay to determine the functionality of antibodies in serum samples obtained on day 42 after immunization with glucosylated O4 (O4-Glc+)-EPA bioconjugate (4.0 μg); Wilcoxon rank sum test with Bonferroni correction; *P≤0.05, ***P≤0.0001;

[0091] Figure 5 Figure 4 shows the boosting effect of the glucosylated O4 (O4Glc+)-EPA bioconjugate in Sprague Dawley rats immunized at three different doses as described in Example 4; serum antibody levels were measured by ELISA on days 0, 14, and 42 after immunization; individual titers (log10 EC50 titers) are shown for each animal; the line between the data points connects the IgG titers for each animal over time; the gray dashed line indicates the threshold above which the dilution curve for the sample has a 4PL fit; statistical analysis was performed with the Wilcoxon signed-rank test and the Bonferroni correction for multiple comparisons (P = 0.012 for 4.0 μg / dose on day 14 vs. day 0; P = 0.006 for all doses on day 42 vs. day 0; P = 0.006 for all doses on day 42 vs. day 14);

[0092] Figure 6 Functionality of antibodies induced by the O4-Glc+-EPA bioconjugate is shown; Sprague Dawley rats were immunized intramuscularly three times with formulation buffer or 4.00 µg / dose of the O4(Glc+)-EPA bioconjugate; antibody functionality was determined by opsonophagocytic killing assay (OPKA) using O4(Glc+) and O4(Glc-) E. coli strains; individual opsonization titers (OI) and GMT ± 95% CI are shown;

[0093] Figure 7 Capillary electrophoresis readout of a PglB screening for O4-Glc+ bioconjugate production for each test strain is shown using a monoclonal antibody to detect O4-Glc+ bioconjugates in the periplasmic fraction. The monoglycosylated product is approximately 180 kDa, the diglycosylated product is approximately 320 kDa, and the triglycosylated product is approximately 450 kDa. A) First round of screening. Wt PglB is in lane 3, N311V-PglB is in lanes 2 and 4, the empty control strain is in lane 1, and the other PglB variants are in lanes 5 and 6. B) Second round of screening. N311V PglB is in lane 3, N311V+Y77H PglB is in lane 9, the empty control strain is in lanes 1 and 2, and the other PglB variants are in the remaining lanes.

[0094] Figure 8 Shown are antibody responses induced by the ExPEC10V vaccine in New Zealand white rabbits. Animals received three intramuscular immunizations with either ExPEC10V or saline administered at intervals of 2 weeks. The ExPEC10V vaccine was administered at three different concentrations (Group 1: high dose, Group 2: medium dose, and Group 3: low dose, Table 11), and the control group received only saline (Group 4, 0.9% (w / v) sodium chloride solution). Antibody levels were measured by ELISA on day 0 (before vaccination) and days 14, 27, and 42 (after vaccination). Individual titers (EC50 titers) and geometric mean titers (GMT) ± 95% CI are shown. The Wilcoxon rank sum test was performed with a Bonferroni correction for multiple comparisons. Comparison of ExPEC10V vaccinated animals (Groups 1, 2, and 3) relative to saline controls (Group 4). **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. LOD: limit of detection.

[0095] Figure 9Figure 2 shows the antibody response induced by ExPEC10V. New Zealand white rabbits received three intramuscular immunizations with ExPEC10V (105.6 μg total polysaccharide) or 0.9% w / v sodium chloride solution (control). IgG titers were determined by ELISA on day 1 (before immunization, n=20 / group), day 31 (after immunization, n=20 / group), and day 50 (after immunization, n=10 / group). The figure shows the individual titers and geometric means ± 95% confidence intervals for each group. Differences in IgG titers between ExPEC10V and control groups were analyzed using the Tobit model with a likelihood ratio test. P values ​​≤ 0.05 were considered significant. *P ≤ 0.05, ****P ≤ 0.0001.

[0096] FIG10 shows the overall study design for the Phase 1 / 2a clinical trial with the ExPEC10V vaccine in humans. Figure 10A The overall study design for Cohort 1 is shown, and Figure 10B The overall study design is shown for Cohort 2. See Example 11 for details. DETAILED DESCRIPTION

[0097] Throughout the background and specification, various publications, articles, and patents are cited or described; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, and the like included in this specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these matters constitute part of the prior art with respect to any invention disclosed or claimed.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In other respects, certain terms used herein have the meaning as set forth in the specification.

[0099] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0100] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series.

[0101] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0102] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprises" may be replaced by the term "contains" or "includes," or sometimes when used herein, by the term "having."

[0103] As used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. As used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of an aspect or embodiment of the present invention, any of the above-mentioned terms "comprising," "containing," "including," and "having" may be replaced with the term "consisting of" or "consisting essentially of" to alter the scope of the present disclosure.

[0104] As used herein, the term "and / or" connecting multiple recited elements should be understood to include both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first element and the second element together. Any one of these options should be understood to fall within this meaning and, therefore, satisfy the requirements of the term "and / or" as used herein. The simultaneous applicability of more than one option should also be understood to fall within this meaning and, therefore, satisfy the requirements of the term "and / or."

[0105] The identification of a structural modification of the O-antigen within the Escherichia coli O4 serotype, namely the glucose branch (Jann et al., 1993), has posed a challenge to the discovery and development of glycoconjugate vaccines targeting bacterial isolates within this serotype. The proportion of clinical contemporary O4 isolates expressing unmodified (without glucose side branches) and modified (with glucose side branches) forms of the O4 O-antigen is unknown. Obtaining information about this property is crucial for the selection of relevant antigenic structures. In addition, it has not been determined to what extent vaccine-induced antibodies raised against one form of the O4 polysaccharide will cross-react with another form. The purification of O-antigen without lipid A and the subsequent chemical conjugation to a carrier protein are lengthy and laborious processes. In addition, the purification, lipid A detoxification and chemical conjugation processes can result in loss of epitopes, antigenic heterogeneity and reduced immunogenicity of the conjugated polysaccharide. The synthesis of glycoconjugates by bioconjugation can overcome these limitations of classical purification and chemical conjugation, but the in vivo synthesis of glucose-branched O4 O-antigen requires the activity of a polysaccharide branching enzyme, which is located in rfb To date, the O-antigen modification enzyme responsible for glucose branching in O4 E. coli strains has not been identified. rfb The gene cluster cloned into a bioconjugating E. coli strain expressing PglB is insufficient to synthesize glucose-branched O4 glycoconjugates, but instead only produces non-glucose-branched O4 bioconjugates (the structures of the glycans of which are shown in Formula (O4) in Table 1). As used herein, the terms "glucosylated O4," "glucose-branched O4," "O4 Glc+," and "Glc+ O4" O-antigen refer to an O4 O-antigen with a glucose side branch, and its structure is shown in Formula (O4-Glc+) in Table 1.

[0106] Disclosed herein are genes encoding O-antigen modification enzymes responsible for the glucose branching of E. coli O4 antigen polysaccharides. Also disclosed herein are host cells, such as recombinantly engineered host cells, comprising nucleic acids encoding enzymes capable of producing in vivo bioconjugates comprising glucosylated O4 antigen polysaccharides covalently bound to a carrier protein. Such host cells can be used to produce bioconjugates comprising glucosylated O4 antigen linked to a carrier protein, which can be used, for example, in the formulation of therapeutic and / or prophylactic compositions (e.g., vaccines). Further provided herein are compositions comprising bioconjugates of glucosylated O4 antigen polysaccharides, alone or in combination with other E. coli antigens (e.g., O1, O2, O6, O8, O15, O16, O18, O25, and / or O75 antigen polysaccharides and serosubtypes thereof). Such compositions can be used in prophylactic and / or therapeutic methods, such as vaccination of hosts infected with E. coli, and can be used to generate antibodies, which can be used, for example, in therapeutic methods, such as immunization of a subject.

[0107] As used herein, the terms "O-antigen," "O-antigen polysaccharide," "O-antigen saccharide," and "OPS" refer to the O-antigen of Gram-negative bacteria. Typically, the O-antigen is a polymer of immunogenic repeating polysaccharide units. In a specific embodiment, the terms "O-antigen," "O-antigen polysaccharide," and "OPS" refer to the O-antigen of Escherichia coli. Different serotypes of E. coli express different O-antigens. In E. coli, the gene products involved in the biogenesis of O-antigens are expressed by rfb Gene cluster encoding.

[0108] As used herein, “ rfb Cluster and rfb The term "gene cluster" refers to the gene cluster that encodes the enzymatic machinery that can synthesize the O-antigen backbone structure. rfb The cluster may apply to any O-antigen biosynthetic cluster and preferably refers to an O-antigen biosynthetic cluster from Escherichia Escherichia ), specifically the gene cluster of Escherichia coli.

[0109] As used herein, the term "O1A" refers to the O1A antigen of Escherichia coli (a serosubtype of Escherichia coli serotype O1). The term "O2" refers to the O2 antigen of Escherichia coli (a serosubtype of Escherichia coli serotype O2). The term "O6A" refers to the O6A antigen of Escherichia coli (a serosubtype of Escherichia coli serotype O6). The term "O8" refers to the O8 antigen of Escherichia coli (a serosubtype of Escherichia coli serotype O8). The term "O15" refers to the O15 antigen of Escherichia coli (a serotype of Escherichia coli serotype O15). The term "O16" refers to the O16 antigen of Escherichia coli (a serotype of Escherichia coli serotype O16). The term "O18A" refers to the O18A antigen of Escherichia coli (a serosubtype of Escherichia coli serotype O18). The term "O25B" refers to the O25B antigen from Escherichia coli (a serosubtype of Escherichia coli serotype O25). The term "O75" refers to the O75 antigen of E. coli (E. coli serotype O75).

[0110] The structures of the E. coli O-antigen polysaccharides referred to throughout this application are shown below in Table 1. A single repeating unit is shown for each E. coli O-antigen polysaccharide.

[0111] Table 1: Structure of Escherichia coli O-antigen polysaccharide

[0112]

[0113]

[0114] .

[0115] All monosaccharides described herein have their ordinary meanings as known in the art. Monosaccharides can have either a D or L configuration. If D or L is not specified, the sugar is understood to have a D configuration. Monosaccharides are often referred to by abbreviations commonly known and used in the art. For example, Glc refers to glucose; D-Glc refers to D-glucose; and L-Glc refers to L-glucose. Other common abbreviations for monosaccharides include: Rha, rhamnose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Fuc, fucose; Man, mannose; Man3Me, 3-O-methyl-mannose; Gal, galactose; FucNAc, N-acetylfucosamine; and Rib, ribose. The suffix "f" refers to furanose, and the suffix "p" refers to pyranose.

[0116] As used with respect to O-antigen, the terms "RU," "repeat unit," and "repeating unit" refer to the biological repeat unit (BRU) of an O-antigen when it is synthesized in vivo by cellular machinery (e.g., glycosyltransferases). The number of RUs of an O-antigen may vary depending on the serotype, and in embodiments of the present invention, typically ranges from about 1-100 RUs, preferably from about 1 to 50 RUs, such as 1-50 RUs, 1-40 RUs, 1-30 RUs, 1-20 RUs, and 1-10 RUs, and more preferably at least 3 RUs, at least 4 RUs, at least 5 RUs, such as 3-50 RUs, preferably 5-40 RUs, such as 7-25 RUs, such as 10-20 RUs. However, in some cases, the number of RUs of an O-antigen may be 1-2. The structure of each O-antigen specifically described herein is shown as containing one RU, where the variable "n" specifies the number of RUs. In each O-antigen polysaccharide in the bioconjugate of the present invention, n is independently an integer from 1 to 100, such as 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, preferably at least 3, more preferably at least 5, such as 3 to 50, preferably 5 to 40 (e.g., 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 or 40), but in some cases, can be 1 to 2. In some embodiments, n is independently an integer from about 7 to 25, such as about 10 to 20. The values ​​may vary between individual O-antigen polysaccharides in the composition and are provided here as average values, i.e., if a bioconjugate is described herein as having n independently being an integer between 5 and 40, the composition contains a majority of O-antigen polysaccharides having 5 to 40 repeating units, but may also contain some O-antigen polysaccharides having fewer than 5 or more than 40 repeating units.

[0117] The term "glycoconjugate" refers to a sugar or saccharide antigen (e.g., oligosaccharides and polysaccharides)-protein conjugate linked to another chemical species, including but not limited to proteins, peptides, lipids, etc. Glycoconjugates can be prepared by chemical methods, for example, by chemical (synthetic) bonding of proteins and sugar or saccharide antigens. The term glycoconjugate also includes bioconjugates.

[0118] The term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and a sugar or saccharide antigen (e.g., oligosaccharides and polysaccharides) produced in a host cell setting, preferably a bacterial host cell, such as an E. coli host cell, wherein the host cell machinery links the antigen to the protein (e.g., N-linked). Preferably, the term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and an O-antigen, preferably an E. coli O-antigen (e.g., O1A, O2, glycosylated O4, O6A, O8, O15, O16, O18A, O25B, O75, etc.) produced in a host cell setting, wherein the host cell machinery links the antigen to the protein (e.g., N-linked). Because bioconjugates are produced in host cells by host cell machinery, the antigen and protein are covalently linked via glycosidic bonds or linkages in the bioconjugate. Bioconjugates can be prepared in recombinant host cells that have been engineered to express the cellular machinery required to synthesize O-antigens and / or to link O-antigens to target proteins. As described herein, bioconjugates have advantageous properties over chemically prepared glycoconjugates (in which polysaccharides are purified from bacterial cell walls and subsequently chemically coupled to a carrier protein), for example, bioconjugates require fewer chemicals in their manufacture and are more consistent in the resulting final product and contain fewer or no free (i.e., not bound to a carrier protein) polysaccharides. Therefore, in typical embodiments, bioconjugates are preferred over chemically produced glycoconjugates.

[0119] The term "about" when used in conjunction with a number refers to any number that is within ±1, ±5, or ±10% of the referenced number.

[0120] The term "percentage (%) sequence identity" or "% identity" describes the number of matches ("hits") of the identical amino acids of two or more compared amino acid sequences compared to the number of amino acid residues constituting the overall length of the amino acid sequence. In other words, when sequences are compared and aligned for maximum correspondence as measured using a sequence comparison algorithm as known in the art, or when manually aligned and visually inspected, alignment is used, for two or more sequences, the percentage (e.g., 90%, 95%, 97% or 98% identity) of identical amino acid residues can be determined. The sequences compared to determine sequence identity may therefore differ by amino acid substitution, addition or deletion. Suitable programs for aligning protein sequences are known to those skilled in the art. The percentage sequence identity of protein sequences can be determined, for example, using programs such as CLUSTALW, ClustalOmega, FASTA or BLAST, for example, using NCBI BLAST algorithm (Altschul SF et al. (1997), Nucleic Acids Res. 25: 3389-3402).

[0121] For example, for amino acid sequences, sequence identity and / or similarity can be determined by using standard techniques known in the art, or by inspection, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using default settings. In certain embodiments, percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc.

[0122] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, which is obtained from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to default values.

[0123] Another useful algorithm is gapped BLAST as reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402.

[0124] The term "invasive extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED)" is defined herein as an acute illness consistent with a systemic bacterial infection, confirmed microbiologically by isolation and identification of E. coli from the blood or other normally sterile body sites, or by isolation and identification of E. coli from the urine of patients who have signs and symptoms of invasive disease (systemic inflammatory response syndrome (SIRS), sepsis, or septic shock) and no other identifiable source of infection.

[0125] Bioconjugates of Glucosylated O4 Antigen Polysaccharide from Escherichia coli

[0126] In one aspect, provided herein are bioconjugates of glucosylated O4 antigen polysaccharides of E. coli covalently linked to a carrier protein. As used herein, the term "O4" refers to an O4 antigen from E. coli (E. coli serotype O4). It is known that O-antigen structural modifications exist within E. coli O4 serotypes. In particular, some O4 serotypes express modified O-antigens with branched glucose units. As used herein, "glucosylated O4 antigen," "glucosylated O4 antigen polysaccharide," "O4-Glc+ antigen polysaccharide," and "O4-Glc+ antigen" refer to O4 antigens (e.g., E. coli O4 antigens) with glucose branches, wherein D-glucose is linked to L-rhamnose in the repeating unit L-Rha→D-Glc→L-FucNAc→D-GlcNAc. In a specific embodiment, the E. coli glucosylated O4 antigen polysaccharide comprises a structure of formula (O4-Glc+) as shown in Table 1, wherein n is an integer from 1 to 100. In a preferred embodiment, n is an integer from 3 to 50, such as 5 to 40, such as 7 to 25, such as 10 to 20.

[0127] Independent of glucose branching state, E. coli O4 strains carry essentially the same rfb The gene cluster encodes genes responsible for the production of the O4 antigen polysaccharide. However, the in vivo synthesis of the modified O4 antigen with glucose branches requires the activity of a polysaccharide branching enzyme located in rfbGene cluster. To the best of the inventors' knowledge, the identity of the polysaccharide branching enzyme responsible for glucose modification of the O4 antigen has hitherto remained unknown. Here, the inventors have discovered the sequence of the polysaccharide branching enzyme responsible for glucose modification of the O4 antigen. Identification of this enzyme allows for the production of bioconjugates with modified O4 antigen polysaccharides having glucose branches. Glucose-modified forms of the O4 antigen polysaccharide are present in the predominant serotype and can therefore be used to provide an improved immune response, for example for prophylactic or therapeutic applications.

[0128] In particular, provided herein is a gene encoding a glucosyltransferase specific for E. coli serotype O4. gtrS The sequence of the gene, the glucosyltransferase that glucosylates the O4 antigen. gtrA 、 gtrB and gtrS The gene encodes the enzyme responsible for glucosylation of O-antigens. gtrA and gtrB Genes are highly homologous and interchangeable, but gtrS Gene encoding serotype-specific O-antigen glucosyltransferase. gtrS The gene encodes the GtrS enzyme, which modifies the O4 antigen by introducing a glucose branch. Characterization of contemporary clinical E. coli isolates of serotype O4 revealed the presence of gtrS , indicating that the E. coli O4 antigenic polysaccharide modified by the addition of glucose residues is predominant in the current infectious isolates.

[0129] In one embodiment, provided herein is a strain from E. coli serotype O4. gtrS A nucleic acid of a gene encoding a GtrS glucosyltransferase comprising the amino acid sequence of SEQ ID NO: 4. In another embodiment, gtrSThe nucleic acid encodes a GtrS protein from Escherichia coli serotype O4 having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 4, preferably 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 4. A GtrS protein having at least 80% identity to the amino acid sequence of SEQ ID NO: 4 is capable of specifically glucosylating E. coli O4 antigen polysaccharide to obtain a glucosylated O4 antigen having the formula (O4-Glc+) as shown in Table 1. In view of the present disclosure, one of ordinary skill in the art will be able to prepare mutant forms of the GtrS protein of SEQ ID NO: 4 having at least 80% sequence identity to SEQ ID NO: 4 and test such sequences for glucosylation activity against E. coli O4 antigens. The following describes in more detail a GtrS protein encoding an E. coli serotype O4 glucosyltransferase. gtrS The invention relates to a recombinant host cell containing the nucleic acid sequence of the gene, and the use of the recombinant host cell in producing glucose-modified O4 antigen polysaccharide and its bioconjugate.

[0130] about gtrA and gtrB The sequence of the encoded protein may comprise an amino acid sequence having at least about 80% identity to SEQ ID NOs: 7 and 8, respectively, gtrA and gtrB The encoded proteins act as bacterial terpene alcohol-linked glucose translocase (GtrA, flips bacterial terpene alcohol-linked glucose on the inner membrane to the periplasm) and bacterial terpene alcohol glucosyltransferase (GtrB, links glucose to bacterial terpene alcohols), respectively. In certain embodiments, nucleic acid sequences encoding GtrA and GtrB proteins are also present in the host cells of the present invention, and the GtrA and GtrB proteins have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 7 and 8, respectively, and have bacterial terpene alcohol-linked glucose translocase and bacterial terpene alcohol glucosyltransferase activity, respectively, and the host cells further comprise an O4-specific rfb coli O4-specific GtrS coding sequence as described above, an oligosaccharyltransferase as described herein, and a sequence encoding a carrier protein having one or more glycosylation consensus sequences as described herein to produce a bioconjugate of E. coli glucosylated O4 serotype (comprising a glycan structure of the formula (O4-Glc+) in Table 1).

[0131] The bioconjugates of the glucosylated O4 antigen polysaccharide of E. coli provided herein are preferably covalently linked to a carrier protein via a glycosidic bond. In view of the present disclosure, any carrier protein known to those skilled in the art can be used. Suitable carrier proteins include, but are not limited to, detoxified Pseudomonas aeruginosa exotoxin A (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, aggregation factor A, aggregation factor B, E. coli heat-labile enterotoxin, detoxified variants of E. coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli Sat protein, the passenger domain of E. coli Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein (OMPC) of Neisseria meningitidis, and protein D from non-typeable Haemophilus influenzae. Bioconjugation to a variety of different carrier proteins containing the desired consensus glycosylation sequence has been described, showing that a wide range of proteins can be glycosylated using this technology (see, for example, WO 06 / 119987, WO 2015 / 124769, WO 2015 / 158403, WO 2015 / 82571, WO 2017 / 216286 and WO 2017 / 67964, which collectively show a wide variety of carrier proteins that can be successfully used in bioconjugation).

[0132] In certain embodiments, the carrier protein is modified, for example, in a manner that renders the protein less toxic and / or more susceptible to glycosylation. In a specific embodiment, the carrier protein used herein is modified so as to maximize the number of glycosylation sites in the carrier protein in a manner that allows for lower concentrations of the protein to be administered, for example, in immunogenic compositions, particularly in bioconjugate form thereof.

[0133] Thus, in certain embodiments, the carrier proteins described herein are modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycosylation sites typically associated with carrier proteins (e.g., relative to the number of glycosylation sites associated with the carrier protein in its natural / native, i.e., "wild-type" state). The introduction of glycosylation sites into the carrier protein can be achieved by inserting a glycosylation consensus sequence into any position in the primary structure of the protein, for example, by adding new amino acids to the primary structure of the protein so that the glycosylation site is fully or partially added, or by mutating existing amino acids in the protein to generate a glycosylation site. One of ordinary skill in the art will recognize that the amino acid sequence of a protein can be easily modified using methods known in the art, such as recombinant methods involving modification of the nucleic acid sequence encoding the protein. In specific embodiments, the glycosylation consensus sequence is introduced into a specific region of the carrier protein, such as a surface structure of the protein, at the N or C terminus of the protein, and / or in a loop stabilized by a disulfide bridge at the bottom of the protein. In some embodiments, the glycosylation consensus sequence can be extended by adding lysine residues for more efficient glycosylation.

[0134] Illustrative examples of glycosylation consensus sequences that can be inserted into or generated in a carrier protein include Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 1); and Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro (SEQ ID NO: 2).

[0135] In some embodiments, the E. coli glucosylated O4 antigen polysaccharide is covalently linked (e.g., N-linked) to an asparagine (Asn) residue in a carrier protein, wherein the Asn residue is present in a glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, more preferably having SEQ ID NO: 2. Typically, the carrier protein comprises 1-10 glycosylation sites, preferably 2 to 4 glycosylation sites, and most preferably 4 glycosylation sites, e.g., 1-10, preferably 2-4, and more preferably 4 glycosylation sites, each of which comprises a glycosylation consensus sequence having an amino acid sequence of SEQ ID NO: 1, and more preferably an amino acid sequence of SEQ ID NO: 2.

[0136] In a specific embodiment, the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A. For EPA, various detoxified protein variants have been described in the literature and can be used as carrier proteins. For example, according to Lukac et al., 1988, Infect Immun, 56: 3095-3098, and Ho et al., 2006, Hum Vaccin , 2: 89-98, by mutating and deleting the catalytically essential residues L552V and ΔE553, detoxification can be achieved. As used herein, "EPA" refers to detoxified Pseudomonas aeruginosa exotoxin A. In those embodiments where the carrier protein is EPA, the E. coli glucosylated O4 antigen polysaccharide can be covalently linked to an Asn residue in a glycosylation site (which comprises a glycosylation consensus sequence with SEQ ID NO: 1), and preferably to an Asn residue in a glycosylation site (which comprises a glycosylation consensus sequence with SEQ ID NO: 2). Preferably, the EPA carrier protein comprises 1-10 glycosylation sites, preferably 2 to 4 glycosylation sites, most preferably 4 glycosylation sites, e.g., 1-10, preferably 2-4, and more preferably 4 glycosylation sites, each of which comprises a glycosylation consensus sequence with an amino acid sequence of SEQ ID NO: 1, and more preferably SEQ ID NO: 2.

[0137] In some embodiments, the EPA carrier protein comprises four glycosylation sites, each comprising a glycosylation consensus sequence, for example, a glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 2. As used herein, "EPA-4 carrier protein" and "EPA-4" refer to a detoxified Pseudomonas aeruginosa exotoxin A carrier protein comprising four glycosylation sites, each comprising a glycosylation consensus sequence having SEQ ID NO: 2. An exemplary preferred example of an EPA-4 carrier protein is an EPA carrier protein comprising the amino acid sequence of SEQ ID NO: 3.

[0138] Composition

[0139] In another aspect, provided herein are compositions comprising a bioconjugate of a glucosylated 04 antigen polysaccharide of E. coli covalently linked to a carrier protein. The compositions provided herein can include any bioconjugate of a glucosylated 04 antigen polysaccharide of E. coli covalently linked to a carrier protein (e.g., EPA) as described herein.

[0140] In some embodiments, the composition is an immunogenic composition. As used herein, an "immunogenic composition" refers to a composition that can elicit an immune response in a host or subject to which the composition is applied. The immunogenic composition may further comprise a pharmaceutically acceptable carrier. In some embodiments, the composition is a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle that is administered with the composition and is non-toxic and should not interfere with the efficacy of the active ingredient. For example, saline solutions and dextrose and glycerol aqueous solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. Other examples of suitable pharmaceutically acceptable carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0141] In one embodiment, the composition of the invention comprises a bioconjugate of the invention in Tris-buffered saline (TBS) pH 7.4 (e.g., containing Tris, NaCl, and KCl, e.g., 25 mM, 137 mM, and 2.7 mM, respectively). In other embodiments, the composition of the invention comprises a bioconjugate of the invention in about 10 mM KH2PO4 / Na2HPO4 buffer at a pH of about 7.0, the buffer having about 5% (w / v) sorbitol, about 10 mM methionine, and about 0.02% (w / v) polysorbate 80. In other embodiments, the compositions of the invention comprise a bioconjugate of the invention in about 10 mM KH2PO4 / Na2HPO4 buffer at a pH of about 7.0, said buffer having about 8% (w / v) sucrose, about 1 mM EDTA, and about 0.02% (w / v) polysorbate 80 (see, e.g., WO 2018 / 077853 for suitable buffers for bioconjugates of E. coli O-antigen covalently bound to an EPA carrier protein).

[0142] In some embodiments, the compositions described herein are monovalent formulations and contain, for example, one E. coli O-antigen polysaccharide, such as an E. coli glucosylated O4 antigen polysaccharide, in isolated form or as part of a glycoconjugate or bioconjugate. Also provided herein are compositions (e.g., pharmaceutical compositions and / or immunogenic compositions) that are multivalent compositions, such as bivalent, trivalent, tetravalent, etc. compositions. For example, a multivalent composition comprises more than one antigen, such as an E. coli O-antigen, glycoconjugate, or bioconjugate thereof. In specific embodiments, a multivalent composition provided herein comprises a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide and at least one additional antigen.

[0143] In one embodiment, the composition (eg, pharmaceutical composition and / or immunogenic composition) is a monovalent composition comprising a bioconjugate of a glucosylated 04 antigen polysaccharide of E. coli covalently linked to a carrier protein as described herein.

[0144] In another embodiment, the composition (eg, pharmaceutical composition and / or immunogenic composition) is a multivalent composition comprising a glucosylated 04 antigen polysaccharide of E. coli covalently linked to a carrier protein as described herein and at least one additional antigen.

[0145] In some embodiments, the additional antigen is an antigenic saccharide or polysaccharide, more preferably an E. coli O-antigen polysaccharide, such as an E. coli O-antigen of one or more serotypes O2, O6, O8, O15, O16, O18, O25, and O75 and serosubtypes thereof. In some embodiments, each of the additional E. coli O-antigen polysaccharides is a glycoconjugate, meaning that the E. coli O-antigen polysaccharide is covalently linked, for example, by chemical or enzymatic means, to another chemical species, such as a protein, peptide, lipid, or the like, most preferably a carrier protein. In a preferred embodiment, each of the additional E. coli O-antigen polysaccharides is a bioconjugate, wherein the O-antigen polysaccharide is covalently linked, for example, to a carrier protein, enzymatically via a glycosidic bond by host cell machinery. In certain embodiments, the compositions provided herein may comprise 1-20 additional glycoconjugates, more preferably bioconjugates of E. coli O-antigen polysaccharides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 additional glycoconjugates or preferably bioconjugates of E. coli O-antigen polysaccharides. Other antigens may be included in the compositions provided herein, such as peptide, protein or lipid antigens, etc.

[0146] In some embodiments, the composition (e.g., a pharmaceutical composition and / or an immunogenic composition) comprises a bioconjugate of a glucosylated E. coli O4 antigen polysaccharide and at least one additional antigen polysaccharide selected from the group consisting of an E. coli O1A antigen polysaccharide, an E. coli O2 antigen polysaccharide, an E. coli O6A antigen polysaccharide, an E. coli O8 antigen polysaccharide, an E. coli O15 antigen polysaccharide, an E. coli O16 antigen polysaccharide, an E. coli O18A antigen polysaccharide, an E. coli O25B antigen polysaccharide, and an E. coli O75 antigen polysaccharide. Preferably, each additional O-antigen polysaccharide is covalently linked to a carrier protein, and more preferably, the bioconjugate is formed.

[0147] In one embodiment, the O1A antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with a glucosylated O4 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O1A antigen polysaccharide comprises a structure of formula (O1A) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. Preferably, the O1A antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, such as EPA.

[0148] In one embodiment, the O2 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with a glucosylated O4 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O2 antigen polysaccharide comprises a structure of formula (O2) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. Preferably, the O2 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, such as EPA.

[0149] In one embodiment, the O6A antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with a glucosylated O4 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O6A antigen polysaccharide comprises a structure of formula (O6A) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. Preferably, the O6A antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, such as EPA.

[0150] In one embodiment, the O8 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with a glucosylated O4 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O8 antigen polysaccharide comprises a structure of formula (O8) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. Preferably, the O8 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, such as EPA.

[0151] In one embodiment, the O15 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with a glucosylated O4 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O15 antigen polysaccharide comprises a structure of formula (O15) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. Preferably, the O15 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, such as EPA.

[0152] In one embodiment, the O16 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with a glucosylated O4 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O16 antigen polysaccharide comprises a structure of formula (O16) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. Preferably, the O16 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, such as EPA.

[0153] In one embodiment, the O18A antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with a glucosylated O4 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O18A antigen polysaccharide comprises a structure of formula (O18A) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. Preferably, the O18A antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, such as EPA.

[0154] In one embodiment, the O25B antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with a glucosylated O4 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the O25B antigen polysaccharide comprises a structure of formula (O25B) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. Preferably, the O25B antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, such as EPA.

[0155] In one embodiment, the 075 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate or bioconjugate) is used in the compositions provided herein (e.g., in combination with a glucosylated 04 antigen polysaccharide or a bioconjugate thereof). In a specific embodiment, the 075 antigen polysaccharide comprises a structure of formula (075) as shown in Table 1, wherein n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20. Preferably, the 075 antigen polysaccharide is part of a bioconjugate and is covalently linked to a carrier protein, such as EPA.

[0156] In another embodiment, the composition (e.g., a pharmaceutical composition and / or an immunogenic composition) comprises at least E. coli O1A, O2, glucosylated O4, O6A, and O25B antigenic polysaccharides, preferably a bioconjugate of the O1A, O2, glucosylated O4, O6A, and O25B antigenic polysaccharides covalently linked to a carrier protein such as EPA (i.e., a pentavalent composition).

[0157] In a preferred embodiment, the composition (e.g., a pharmaceutical composition and / or an immunogenic composition) comprises at least Escherichia coli O1A, O2, glucosylated O4, O6A, O8, O15, O16, O25B and O75 antigen polysaccharides, preferably a bioconjugate of O1A, O2, glucosylated O4, O6A, O8, O15, O16, O25B and O75 antigen polysaccharides covalently linked to a carrier protein such as EPA (i.e., a 9-valent composition).

[0158] In another preferred embodiment, the composition (e.g., a pharmaceutical composition and / or an immunogenic composition) comprises at least E. coli O1A, O2, glucosylated O4, O6A, O8, O15, O16, O18A, O25B and O75 antigenic polysaccharides, preferably a bioconjugate of O1A, O2, glucosylated O4, O6A, O8, O15, O16, O18A, O25B and O75 antigenic polysaccharides covalently linked to a carrier protein such as EPA (i.e., a 10-valent composition).

[0159] Also contemplated herein are compositions that optionally further comprise additional O-antigens from other E. coli serotypes (eg, in isolated form or as part of a glycoconjugate or bioconjugate).

[0160] In some embodiments, the additional E. coli O1A, O2, O6A, O8, O15, O16, O18A, O25B, and / or O75 antigen polysaccharides are each covalently linked to a carrier protein. The O-antigen polysaccharide can be linked to the carrier protein by chemical or other synthetic methods, or the O-antigen polysaccharide can be part of a bioconjugate, and preferably part of a bioconjugate. In view of the present disclosure, any carrier protein known to those skilled in the art can be used. Suitable carrier proteins include, but are not limited to, detoxified Pseudomonas aeruginosa exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, clumping factor A, clumping factor B, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae. Preferably, the carrier protein is EPA.

[0161] In some embodiments, particularly when part of a bioconjugate, the additional E. coli O1A, O2, O6A, O8, O15, O16, O18A, O25B and / or O75 antigenic polysaccharide is each covalently linked to an asparagine (Asn) residue in the carrier protein, wherein the Asn residue is present in a glycosylation site comprising the glycosylation consensus sequence Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 1), preferably, wherein the Asn residue is present in a glycosylation site comprising the glycosylation consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro (SEQ ID NO: 2). The carrier protein may comprise 1-10 glycosylation sites, preferably 2 to 4 glycosylation sites, and most preferably 4 glycosylation sites, each of which comprises a glycosylation consensus sequence. In a specific embodiment, the carrier protein is an EPA-4 carrier protein, such as an EPA-4 carrier protein comprising the amino acid sequence of SEQ ID NO: 3.

[0162] In a specific embodiment, provided herein are compositions (e.g., pharmaceutical compositions and / or immunogenic compositions) comprising: (i) a molecule comprising SEQ ID NO: 3) a bioconjugate of a glucosylated O4 antigen polysaccharide of Escherichia coli covalently linked to a detoxified Pseudomonas aeruginosa exotoxin A carrier protein (EPA-4 carrier protein), wherein the glucosylated O4 antigen polysaccharide of Escherichia coli comprises a structure of formula (O4-Glc+); (ii) a bioconjugate of an O1A antigen polysaccharide of Escherichia coli covalently linked to an EPA-4 carrier protein, wherein the O1A antigen polysaccharide of Escherichia coli comprises a structure of formula (O1A); (iii) a bioconjugate of an O2 antigen polysaccharide of Escherichia coli covalently linked to an EPA-4 carrier protein, wherein the O2 antigen polysaccharide of Escherichia coli comprises a structure of formula (O2); (iv) a bioconjugate of an O6A antigen polysaccharide of Escherichia coli covalently linked to an EPA-4 carrier protein, wherein the O6A antigen polysaccharide of Escherichia coli comprises a structure of formula (O6A); (v) a bioconjugate of an O8 antigen polysaccharide of Escherichia coli covalently linked to an EPA-4 carrier protein, wherein the O8 antigen polysaccharide of Escherichia coli comprises a structure of formula (O6A). coli O75 antigenic polysaccharide covalently linked to an EPA-4 carrier protein, wherein the E. coli O75 antigenic polysaccharide comprises a structure of formula (O75), wherein each of the formulas is provided in Table 1, and for each of the formulas, independently, n is an integer from 1 to 100, such as from 1 to 50, preferably from 3 to 50, such as from 5 to 40.

[0163] In a specific embodiment, the composition (e.g., a pharmaceutical composition and / or an immunogenic composition) further comprises: (x) a bioconjugate of an Escherichia coli O18A antigenic polysaccharide covalently linked to an EPA-4 carrier protein, wherein the Escherichia coli O18A antigenic polysaccharide comprises a structure of formula (O18A) as shown in Table 1, wherein n with respect to the structure is an integer from 1 to 100, e.g., from 1 to 50, preferably from 3 to 50, e.g., from 5 to 40.

[0164] In some embodiments, the compositions provided herein comprise a bioconjugate of a glucosylated 04 antigen polysaccharide of E. coli and a bioconjugate of at least a 025B antigen polysaccharide of E. coli, wherein the bioconjugate of the 025B antigen polysaccharide of E. coli is present in the composition at a concentration that is about 1.5 to 6 times, e.g., about 2 to 4 times, e.g., 1.5, 2, 3, 4, 5, or 6 times, greater than the concentration of any other bioconjugate present in the composition.

[0165] In certain embodiments, the composition comprises a bioconjugate of E. coli O1A, O2, glucosylated O4, O6A, O8, O15, O16, O25B, and O75 antigen polysaccharides, wherein the bioconjugate of O1A:O2:glucosylated O4:O6A:O8:O15:O16:O25B:O75 is present in a ratio of 1:1:1:1:1:1:1:2:1 or 2:1:1:2:1:1:1:1:4:1 (by weight of O-antigen polysaccharide).

[0166] In certain embodiments, the composition comprises a bioconjugate of E. coli O1A, O2, glucosylated O4, O6A, O8, O15, O16, O18A, O25B, and O75 antigen polysaccharides, wherein the bioconjugate of O1A:O2:glucosylated O4:O6A:O8:O15:O16:O18A:O25B:O75 is present in a ratio of 1:1:1:1:1:1:1:1:2:1 or 2:1:1:2:1:1:1:1:1:4:1 (by weight of O-antigen polysaccharide).

[0167] In some embodiments, the compositions provided herein comprise a bioconjugate of a glucosylated E. coli O4 antigen polysaccharide and a bioconjugate of at least an E. coli O25B antigen polysaccharide, wherein the bioconjugate of the E. coli O25B antigen polysaccharide is present in the composition at a concentration of 2 to 50 μg / mL, preferably 8 to 40 μg / mL, more preferably 16-32 μg / mL, such as 16, 18, 20, 22, 24, 26, 28, 30, or 32 μg / mL. In such embodiments, the concentration of the bioconjugate of the E. coli O25B antigen polysaccharide is preferably about 1.5 to 6 times, such as about 2 to 4 times, such as 1.5, 2, 3, 4, 5, or 6 times, greater than the concentration of any other bioconjugate present in the composition.

[0168] In certain embodiments, the compositions described herein (e.g., pharmaceutical compositions and / or immunogenic compositions) comprise an adjuvant or are administered in combination with an adjuvant. The adjuvant for administration in combination with the compositions described herein can be administered prior to (e.g., within 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, 10 minutes), concurrently with, or after (e.g., within 72 hours, 48 ​​hours, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, 10 minutes) administration of the composition. As used herein, the term "adjuvant" refers to a compound that, when combined with or administered as part of a composition described herein, increases, enhances, and / or strengthens the immune response to the E. coli O-antigen polysaccharide in the bioconjugate, but does not generate an immune response to the E. coli O-antigen polysaccharide in the bioconjugate when the adjuvant compound is administered alone. In some embodiments, the adjuvant enhances the immune response to the E. coli O-antigen polysaccharide in its bioconjugate without producing an allergic or other adverse reaction. Adjuvants can enhance the immune response through several mechanisms including, for example, lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages.

[0169] Examples of suitable adjuvants include, but are not limited to, aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, aluminum sulfate, and aluminum oxide, including nanoparticles or nanoaluminum adjuvant formulations containing aluminum adjuvants), calcium phosphate, monophosphoryl lipid A (MPL) or 3-deoxyacylated monophosphoryl lipid A (3D-MPL) (see, e.g., British Patent GB2220211, EP0971739, EP1194166, US6491919), AS01, AS02, AS03, and AS04 (all GlaxoSmithKline; see, e.g., EP1126876, US7357936 for AS04, EP0671948, EP0761231, US5750110 for AS02), MF59 (Novartis), imidazopyridine compounds (see WO2007 / 109812), imidazoquinoxaline compounds (see WO2007 / 109813), delta-insulin, STING-activating synthetic cyclic dinucleotides (e.g., US20150056224), combinations of lecithin and carbomer homopolymers (e.g., US6676958), and saponins such as QuilA and QS21 (see, e.g., Zhu D and W Tuo, 2016, Nat Prod Chem Res 3: e113 (doi:10.4172 / 2329-6836.1000e113), Matrix M, Iscoms, Iscomatrix, etc., optionally in combination with QS7 (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Patent No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (e.g., squalene or peanut oil), optionally in combination with an immunostimulant such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Another adjuvant is CpG (Bioworld Today, November 15, 1998). Further examples of adjuvants are liposomes containing immunostimulants such as MPL and QS21, as in AS01E and AS01B (e.g., US 2011 / 0206758). Other examples of adjuvants are CpG (Bioworld Today, November 15, 1998). Today, November 15, 1998) and imidazoquinolines (e.g., imiquimod and R848). See, e.g., Reed G et al., 2013, Nature Med, 19: 1597-1608. In certain embodiments, the adjuvant contains a toll-like receptor 4 (TLR4) agonist. TLR4 agonists are well known in the art, see, for example, Ireton GC and SG Reed, 2013, Expert Rev Vaccines 12: 793-807. In certain embodiments, the adjuvant includes a TLR4 agonist comprising lipid A or an analog or derivative thereof, such as MPL, 3D-MPL, RC529 (e.g., EP1385541), PET-lipid A, GLA (a pyranosyl lipid adjuvant, a synthetic disaccharide glycolipid; e.g., US20100310602, US8722064), SLA (e.g., Carter D et al., 2016, Clin Transl Immunology 5:e108 (doi: 10.1038 / cti.2016.63), which describes a structure-function approach to optimizing TLR4 ligands for human vaccines), PHAD (a phosphorylated hexaacyl disaccharide), 3D-PHAD (whose structure is identical to GLA), 3D-(6-acyl)-PHAD (3D(6A)-PHAD). (PHAD, 3D-PHAD, and 3D(6A)PHAD are synthetic lipid A variants, see, e.g., avantilipids.com / divisions / adjuvants, which also provides the structures of these molecules), E6020 (CAS No. 287180-63-6), ONO4007, OM-174, and the like.

[0170] In certain embodiments, the compositions described herein do not comprise an adjuvant and are not administered in combination with an adjuvant.

[0171] In certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein (e.g., drugs and / or immunogenicity) can be formulated for subcutaneous, parenteral, oral, sublingual, buccal, intradermal, transdermal, colorectal, intraperitoneal, rectal administration, intravenous, intranasal, intratracheal, intramuscular, topical, transdermal or intradermal administration. In a specific embodiment, the compositions provided herein (e.g., drugs and / or immunogenicity) are formulated for intramuscular injection.

[0172] How to use

[0173] The bioconjugates and compositions provided herein can be used to induce antibodies against E. coli glucosylated O4 antigens in a subject and to vaccinate a subject against E. coli, particularly extraintestinal pathogenic E. coli (ExPEC). As used herein, "subject" means any animal, preferably a mammal, to which a bioconjugate or composition provided herein will be or has been administered. As used herein, the term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, non-human primates (NHPs) such as monkeys or apes, humans, and the like. In certain embodiments, the subject is a human. The human subject can be of any age. In certain embodiments, the subject is a human between about two months and about 18 years old, for example, between 1 and 18 years old. In certain embodiments, the subject is a human who is at least 18 years old. In certain embodiments, the subject is a human between 15 and 50 years old, for example, between 18 and 45 years old, for example, between 20 and 40 years old. In certain embodiments, the subject is a man. In certain embodiments, the subject is a woman. In certain embodiments, the subject is immunocompromised. In certain embodiments, the subject is a human being who is at least 50 years old, at least 55 years old, at least 60 years old, or at least 65 years old. In certain embodiments, the subject is a human being who is no more than 100 years old, no more than 95 years old, no more than 90 years old, no more than 85 years old, no more than 80 years old, or no more than 75 years old. In certain embodiments, the subject is a human being who is at least 60 years old and no more than 85 years old. In certain embodiments, the subject is a human being in a stable health state. In certain embodiments, the subject is an adult who is at least 60 years old and no more than 85 years old in a stable health state. In certain embodiments, the subject is a human being with a history of urinary tract infection (UTI, i.e., a bacterial infection in the urethra, bladder, ureters, and / or kidneys), i.e., a human being who has had at least one episode of UTI in his or her lifetime. In certain embodiments, the subject is a human being who has had a history of UTI in the past 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year. In certain embodiments, the subject is a human being who has had a history of UTI in the past two years. In certain embodiments, the subject is a human subject with a history of recurrent UTIs, i.e., at least two UTIs within six months or at least three UTIs within a year. In certain embodiments, the subject is a human subject with a history of recurrent UTIs within the past two years. In certain embodiments, the subject is a human 60 years of age or older with a stable health condition. In certain embodiments, the subject is a human 60 years of age or older with a history of UTIs within the past two years. In certain embodiments, the subject is a human at least 60 years of age and less than 75 years of age with a history of UTIs within the past two years. In certain embodiments, the subject is a human subject 75 years of age or older with a history of UTIs within the past two years.In certain embodiments, the subject is a patient scheduled to undergo an elective genitourinary and / or abdominal procedure or surgical procedure, such as a transrectal ultrasound-guided prostate needle biopsy (TRUS-PNB).

[0174] In one aspect, provided herein is a method of inducing antibodies against E. coli glucosylated O4 antigen in a subject, comprising administering to the subject any bioconjugate of E. coli glucosylated O4 antigen covalently linked to a carrier protein described herein, alone or further in combination with other E. coli O-antigen polysaccharides or glycoconjugates or bioconjugates thereof, or a composition comprising a bioconjugate of E. coli glucosylated O4 antigen covalently linked to a protein, alone or further in combination with other E. coli O-antigen polysaccharides or glycoconjugates or bioconjugates thereof.

[0175] In certain embodiments, antibodies induced, elicited, or identified against a glucosylated O4 antigen of E. coli have opsonophagocytic activity. In specific embodiments, the antibodies induced, elicited, or identified are cross-reactive antibodies capable of mediating opsonophagocytic killing of both glucosylated and non-glucosylated O4 strains of E. coli.

[0176] In certain embodiments, antibodies induced, elicited, or identified against a glucosylated E. coli O4 antigen specifically recognize unmodified and glucose-modified O4 antigen polysaccharides. In certain embodiments, antibodies induced, elicited, or identified against a glucosylated E. coli O4 antigen specifically recognize E. coli serotype O4. In certain embodiments, antibodies induced by a bioconjugate of a glucosylated E. coli O4 antigen preferentially bind to the glucosylated O4 antigen compared to the non-glucosylated O4 antigen.

[0177] Antibodies induced by the bioconjugates and compositions described herein can include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that specifically binds to E. coli O-antigen polysaccharide, such as a glucosylated O4 antigen polysaccharide.

[0178] Antibodies induced, triggered or identified using the bioconjugates or compositions provided herein can be used to monitor the efficacy and / or disease progression of therapy. Any immunoassay system known in the art can be used for this purpose, including but not limited to competitive and non-competitive assay systems using the following techniques: such as radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), immunoassay based on electrochemiluminescence (ECL), "sandwich" immunoassay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, immunoradiometric assay, fluorescence immunoassay, protein A immunoassay, and immunoelectrophoresis assay. Several of these assays, such as immunoassays based on ECL, can be performed in multiple formats, and typically multiple assay formats are preferred.

[0179] Antibodies induced, elicited or identified using bioconjugates of E. coli glucosylated O4 antigen polysaccharides can be used, for example, to detect E. coli O4 strains, particularly glucosylated O4 strains, from a variety of E. coli strains, and / or to diagnose infection by E. coli O4 or glucosylated O4 strains.

[0180] In another aspect, provided herein are methods for vaccinating a subject against E. coli (e.g., extraintestinal pathogenic E. coli, ExPEC), comprising administering to the subject any of the bioconjugates of the glucosylated O4 antigen of E. coli covalently linked to a carrier protein, described herein, alone or in combination with other E. coli O-antigens, or glycoconjugates or bioconjugates thereof, or a composition comprising a bioconjugate of the glucosylated O4 antigen of E. coli covalently linked to a carrier protein, alone or in combination with other E. coli O-antigens, or glycoconjugates or bioconjugates thereof. It will be understood by those skilled in the art that the subject will be vaccinated against the E. coli strain whose O antigen, or glycoconjugate or bioconjugate thereof, is present in the administered composition. For example, administration of a composition comprising polysaccharides of the O1A, O2, glucosylated O4, O6A, and O25B antigens can be used to vaccinate a subject against E. coli serotypes O1A, O2, O4, O6A, and O25B.

[0181] In certain embodiments, vaccination is for the prevention of invasive ExPEC disease (IED), such as urosepsis, bacteremia, sepsis, etc. In certain embodiments, vaccination is for the prevention or reduction of the occurrence or severity of urinary tract infections. In certain embodiments, IEDs may be hospital-acquired, such as in patients undergoing genitourinary and / or abdominal surgery or surgical procedures. In certain embodiments, IEDs may be healthcare-associated, such as in patients receiving healthcare for another condition, such as via a central line, catheter, etc., such as in a hospital, outpatient surgical center, end-stage renal disease facility, long-term care facility, etc. In certain embodiments, IEDs may be community-acquired, such as in patients who have not recently been exposed to healthcare risks.

[0182] In another aspect, provided herein are methods of inducing an immune response against E. coli (e.g., ExPEC) in a subject, comprising administering to the subject any bioconjugate of an E. coli glucosylated O4 antigen covalently linked to a carrier protein, alone or in combination with other E. coli O-antigens, or glycoconjugates or bioconjugates thereof, or a composition comprising a bioconjugate of an E. coli glucosylated O4 antigen covalently linked to a carrier protein, alone or in combination with other E. coli O-antigens, or glycoconjugates or bioconjugates thereof. In one embodiment, the subject has an E. coli (e.g., ExPEC) infection at the time of administration. In a preferred embodiment, the subject does not have an E. coli (e.g., ExPEC) infection at the time of administration.

[0183] In certain embodiments, the compositions and bioconjugates described herein can be administered to a subject to induce an immune response, which includes the production of antibodies, preferably antibodies with opsonophagocytic activity. Such antibodies can be isolated using techniques known to those skilled in the art (e.g., immunoaffinity chromatography, centrifugation, precipitation, etc.).

[0184] The ability of the bioconjugates and compositions described herein to generate an immune response in a subject can be evaluated using any method known to those skilled in the art or described herein. In some embodiments, the ability of a bioconjugate to generate an immune response in a subject can be evaluated by immunizing a subject (e.g., mouse, rat, rabbit, or monkey) or a collection of subjects with a bioconjugate described herein and immunizing another subject (e.g., mouse, rat, rabbit, or monkey) or a collection of subjects with a control (PBS). The subject or collection of subjects can then be challenged with ExPEC, and the ability of the ExPEC to cause disease (e.g., UTI, bacteremia, or other disease) in the subject or collection of subjects can be determined. One skilled in the art will recognize that if the subject or collection of subjects immunized with the control develops disease after challenge with ExPEC, but the subject or collection of subjects immunized with a bioconjugate described herein or a composition thereof develops less disease or no disease, then the bioconjugate is capable of generating an immune response in the subject. The ability of the bioconjugates described herein, or compositions thereof, to induce antisera that cross-react with the O antigen from ExPEC can be tested, for example, by an immunoassay such as an ELISA (see, e.g., Van den Dobbelsteen et al., 2016, Vaccine 34: 4152-4160), or an ECL-based immunoassay.

[0185] For example, the ability of the bioconjugates described herein to generate an immune response in a subject can be evaluated using a serum bactericidal assay (SBA) or an opsonophagocytic killing assay (OPK assay, or OPKA), which represent established and recognized methods that have been used to obtain approval for vaccines based on glycoconjugates. Such assays are well known in the art and, briefly, comprise the following steps: antibodies to a target of interest (e.g., an O-antigen polysaccharide, such as a glucosylated O4 antigen polysaccharide of E. coli) are generated and isolated by administering to a subject (e.g., a mouse, rat, rabbit, or monkey) a compound that elicits such antibodies. The bactericidal ability of the antibody can then be evaluated, for example, by culturing the bacteria in question (e.g., E. coli of the relevant serotype) in the presence of the antibody and complement and, depending on the assay, neutrophils, and determining the ability of the antibody to mediate killing and / or neutralization of the bacteria, for example, using standard microbiological methods. For examples of OPK assays for E. coli bioconjugate vaccines, see, for example, Abbanat et al., 2017, Clin. Vaccine Immunol. 24:e00123-17. OPK assays can be performed in a single or multiplex format, with multiplex formats (e.g., simultaneous detection of multiple serotypes) generally being preferred. Multiplex OPK assays are sometimes referred to herein as "MOPAs."

[0186] In some embodiments, the methods described herein comprise administering an effective amount of a bioconjugate of a glucosylated E. coli O4 antigen covalently linked to a carrier protein as described herein, alone or in combination with other E. coli O-antigens, or glycoconjugates or bioconjugates thereof, or a composition comprising a bioconjugate of a glucosylated E. coli O4 antigen covalently linked to a carrier protein, alone or in combination with other E. coli O-antigens, or glycoconjugates or bioconjugates thereof. In one embodiment, an "effective amount" is an amount that vaccinates a subject against E. coli (e.g., ExPEC). In another embodiment, an "effective amount" is an amount that induces an immune response against E. coli (e.g., ExPEC) in a subject, such as an immune response comprising the production of antibodies, preferably antibodies with opsonophagocytic activity.

[0187] In certain embodiments wherein the compositions provided herein comprise a bioconjugate of a glucosylated E. coli O4 antigen polysaccharide and at least a bioconjugate of an E. coli O25B antigen polysaccharide, the effective amount of the E. coli O25B antigen polysaccharide is about 1.5 to 6 times, e.g., about 2 to 4 times, e.g., 1.5, 2, 3, 4, 5, or 6 times, the concentration of any other bioconjugate present in the composition. In such embodiments, the effective amount of the E. coli O25B antigen polysaccharide is, e.g., about 5 to 18 μg / administration, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 μg / administration.

[0188] In certain embodiments, a bioconjugate or composition according to the present invention is administered to a subject once. In certain embodiments, a bioconjugate or composition according to the present invention is administered to a subject more than once, for example, in a prime-boost regimen. In certain embodiments, the time between two administrations is at least two weeks, at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least five years, at least ten years, or at least fifteen years. In humans, the desired immune response can generally be generated by a single administration of a bioconjugate or composition according to the present invention. In certain embodiments, repeated administration, for example, after ten years, is provided.

[0189] host cells

[0190] Provided herein are host cells, e.g., prokaryotic host cells, capable of producing E. coli O antigens and bioconjugates comprising such E. coli O antigens. The host cells provided herein are preferably modified (e.g., by genetic engineering) to contain one or more nucleic acids encoding host cell machinery (e.g., glycosyltransferases) for producing E. coli O-antigen polysaccharides and / or bioconjugates thereof.

[0191] Any host cell known to those skilled in the art can be used to produce the E. coli O antigen polysaccharide described herein (e.g., E. coli glucosylated O4 antigen polysaccharide), and the bioconjugates comprising the E. coli O antigen polysaccharide described herein (e.g., bioconjugates of E. coli glucosylated O4 antigen polysaccharide), including archaea, prokaryotic host cells, and eukaryotic host cells. In a preferred embodiment, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells for producing the E. coli O antigen polysaccharide described herein, and the bioconjugates comprising the E. coli O antigen polysaccharide described herein include, but are not limited to, Escherichia species, Shigella ( Shigella ) species, Klebsiella spp. ( Klebsiella ) species, Xanthomonas spp. ( Xhantomonas ) species, Salmonella spp. ( Salmonella ) species, Yersinia spp. ( Yersinia ) species, Lactococcus spp. ( Lactococcus ) species, Lactobacillus spp. ( Lactobacillus ) species, Pseudomonas spp. ( Pseudomonas ) species, Corynebacterium spp. ( Corynebacterium ) species, Streptomyces spp. ( Streptomyces ) species, Streptococcus spp. ( Streptococcus ) species, Staphylococcus spp. ( Staphylococcus ) species, Bacillus spp. ( Bacillus ) species and Clostridium spp. ( Clostridium ) species.

[0192] In a specific embodiment, the host cell used to produce the E. coli O-antigen polysaccharide described herein, and the bioconjugates comprising the E. coli O-antigen polysaccharide described herein, is a prokaryotic host cell, and preferably is E. coli.

[0193] In certain embodiments, host cells used to produce the E. coli O-antigen polysaccharides and bioconjugates described herein are engineered to contain heterologous nucleic acid, e.g., a nucleic acid comprising a desired O-antigen serotype. rfb In one embodiment, the heterologous nucleic acid may be a nucleic acid encoding a gene cluster, a nucleic acid encoding one or more carrier proteins and / or glycosyltransferases. rfb Genes and / or heterologous nucleic acids encoding proteins involved in glycosylation pathways (e.g., prokaryotic and / or eukaryotic glycosylation pathways) are introduced into host cells as described herein. Such nucleic acids can encode proteins including, but not limited to, oligosaccharyltransferases and / or glycosyltransferases.

[0194] Described herein are sequences of various genes and gene clusters encoding glycosyltransferases that can be used to prepare recombinant host cells that can, for example, be used to prepare E. coli O-antigen polysaccharides and bioconjugates thereof. Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, a protein having a particular amino acid sequence can be encoded by a variety of different nucleic acids. Thus, those skilled in the art will appreciate that the nucleic acids provided herein can be altered in such a way that their sequence differs from the sequences provided herein without affecting the amino acid sequence of the protein encoded by the nucleic acid.

[0195] Provided herein are host cells (e.g., recombinant host cells) for producing the following bioconjugates: E. coli glucosylated O4 antigen polysaccharide, O1A antigen polysaccharide, O2 antigen polysaccharide, O6A antigen polysaccharide, O8 antigen polysaccharide, O15 antigen polysaccharide, O16 antigen polysaccharide, O18A antigen polysaccharide, O25B antigen polysaccharide, or O75 antigen polysaccharide. The host cells provided herein contain nucleic acids encoding enzymes (e.g., glycosyltransferases) capable of producing E. coli O antigen polysaccharides. The host cells provided herein can naturally express nucleic acids capable of producing the desired O antigen, or the host cells can be prepared to express such nucleic acids. In certain embodiments, the nucleic acid is heterologous to the host cell and is introduced into the host cell using genetic methods known in the art. For example, the nucleic acid can be introduced into the host cell by genetic manipulation (e.g., the gene cluster is expressed on one or more plasmids or integrated into the host cell genome (see, e.g., International Patent Application Publication Nos. WO 2014 / 037585, WO 2014 / 057109, WO 2015 / 052344).

[0196] In one embodiment, provided herein are host cells (e.g., recombinant host cells) capable of producing a bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to a carrier protein. Preferably, such host cells comprise a gene encoding gtrS The nucleic acid sequence of the gene, as known to the inventors, gtrS A gene is identified herein for the first time as encoding a polysaccharide branching enzyme capable of transferring glucose to E. coli O4 antigen (i.e., a glucosyltransferase specific for E. coli O4 antigen polysaccharide), and in particular, transferring glucose to L-Rha via an α-1,3-glycosidic bond. An example of an amino acid sequence of such a branching enzyme is provided in SEQ ID NO: 4. Other examples include amino acid sequences having at least 80% identity thereto. gtrSIllustrative examples of nucleic acid sequences of genes include, but are not limited to, SEQ ID NO: 5, or a degenerate nucleic acid sequence encoding SEQ ID NO: 4, or a nucleic acid sequence encoding a functional O4-specific GtrS enzyme that is at least 80% identical to SEQ ID NO: 4.

[0197] In a specific embodiment, a host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of a glucosylated 04 antigen polysaccharide of E. coli covalently linked to a carrier protein comprises a nucleotide sequence encoding a glucosyltransferase having at least 80% sequence identity to SEQ ID NO: 4, for example, about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4. Given the redundancy of the genetic code, for example, using codon-optimized sequences, one of ordinary skill in the art can prepare nucleic acid variants encoding the amino acid sequence of the glucosyltransferase.

[0198] In certain embodiments, a host cell (e.g., a recombinant host cell) comprising a nucleotide sequence encoding a glucosyltransferase (GtrS) having at least 80% sequence identity to SEQ ID NO: 4, capable of producing a bioconjugate of a glucosylated 04 antigen polysaccharide from E. coli covalently linked to a carrier protein, further comprises a nucleotide sequence encoding a bacterial terpene alcohol-linked glucose translocase (GtrA) having at least 80% sequence identity to SEQ ID NO: 7, and a nucleotide sequence encoding a bacterial terpene alcohol glucosyltransferase (GtrB) having at least 80% sequence identity to SEQ ID NO: 8. In certain embodiments, the nucleic acid sequences encode GtrA and GtrB proteins that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 7 and 8, respectively, and have bacterial terpene alcohol-linked glucose translocase (SEQ ID NO: 7) and bacterial terpene alcohol glucosyltransferase (SEQ ID NO: 8) activity, respectively. Given the redundancy of the genetic code, one of ordinary skill in the art can prepare nucleic acid variants encoding the amino acid sequences of bacterial terpene alcohol-linked glucose translocases and bacterial terpene alcohol glucosyltransferases, for example, using codon-optimized sequences when necessary.

[0199] The host cell (eg, recombinant host cell) provided herein is capable of producing a bioconjugate of a glucosylated O4 antigen polysaccharide of E. coli covalently linked to a carrier protein, further comprising a rfbThe nucleotide sequence of the gene cluster can be used to produce Escherichia coli O4 antigen polysaccharide rfb An example of a gene cluster is provided herein as SEQ ID NO: 9. Another example can be found in GenBank, locus AY568960. Degenerate nucleic acid sequences encoding the same enzyme as that encoded by this sequence, or sequences encoding an enzyme that is at least 80% identical, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, can also be used.

[0200] In a specific embodiment, provided herein is a host cell (e.g., a recombinant host cell, preferably a recombinant prokaryotic host cell, preferably a recombinant E. coli host cell) that produces a glucosylated O4 antigen polysaccharide, wherein the host cell comprises gtrS , About Escherichia coli O4 antigen polysaccharide rfb Gene cluster and nucleic acid encoding vector protein. Such host cells can be modified using recombinant methods to contain gtrS Gene, rfb One or more plasmids containing the gene cluster and / or nucleic acid encoding the vector protein, or containing some or all of the relevant genes integrated into the host cell genome, e.g. gtrS 、 rfb Cluster and / or nucleic acid encoding a carrier protein. In certain embodiments, the gene or gene cluster has been integrated into the genome of the host cell using homologous recombination. The advantage of gene integration into the genome of the host cell is stability in the absence of antibiotic selection.

[0201] In another embodiment, provided herein is a host cell (e.g., a recombinant host cell, preferably a recombinant prokaryotic host cell) that produces a glucosylated O4 antigen polysaccharide, wherein the host cell comprises GtrS (glucosyltransferase), and O4 rfb Cluster-encoded enzymes. In certain embodiments, some or all of the above enzymes are heterologous to the host cell.

[0202] In other specific embodiments, provided herein are host cells (e.g., recombinant host cells, preferably recombinant prokaryotic host cells) that produce a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide, preferably a glucosylated O4 antigen polysaccharide, wherein the host cell further comprises a nucleotide sequence encoding an oligosaccharyltransferase and / or a nucleotide sequence encoding a carrier protein. In one embodiment, the oligosaccharyltransferase is heterologous to the host cell. In another embodiment, the carrier protein is heterologous to the host cell. Preferably, the host cell comprises a heterologous nucleotide sequence encoding a glucosyltransferase having at least 80% sequence identity to SEQ ID NO: 4. In a preferred embodiment, the O4 cluster rfb The gene is heterologous to the host cell. Preferably, the sequence encoding the enzyme capable of introducing branched glucose side chains into the O4 antigen is gtrS A gene encoding a glucosyltransferase having at least 80% sequence identity to SEQ ID NO: 4 is heterologous to the host cell. A nucleic acid is heterologous to the host cell if the same sequence does not naturally occur in the host cell. Heterologous nucleic acid can be introduced into the parent cell, for example, by genetic engineering, for example, by transformation (e.g., chemical transformation or electroporation) and / or recombination. In certain embodiments, a heterologous nucleic acid, such as a desired rfb loci, gtrS The coding sequence, carrier protein coding sequence and / or glycosyltransferase coding sequence are integrated into the genome of the host cell, preferably a bacterial host cell, preferably an E. coli host cell. In a preferred embodiment, the endogenous rfb Loci and (if applicable) gtrS The coding sequences have been inactivated, preferably deleted from the genome of the recombinant host cell, and preferably these are replaced with the desired heterologous rfb The loci and (if applicable) the desired gtrS In certain embodiments, the host cell is K-12 of E. coli (as a non-limiting example, E. coli strain W3110 is a K-12 strain), or B strain of E. coli (as a non-limiting example, E. coli strain BL21 is a B strain), or any other well-defined strain of E. coli, such as a laboratory strain or a production strain, as opposed to the original wild-type isolate. In a preferred embodiment, by rfb Loci and gtrSA gene encoding a glucosyltransferase having at least 80% sequence identity to SEQ ID NO: 4 is introduced into such E. coli, the host cells being derived from an E. coli strain that does not express the O4 antigen or glucosylated O4 antigen. An advantage of using a well-characterized strain, such as E. coli K-12 or E. coli B, as a precursor host cell is the possibility of using similar production processes for different O-antigen bioconjugates, since the properties of the production strain are well-defined. Although bioconjugates of different O-antigens will behave differently, and the expression process can be optimized depending on the production strain, at least the basic process for producing the O-antigen bioconjugate using such a well-defined precursor strain is more predictable than when an unknown strain, such as a wild-type isolate, is used as a precursor to the production host strain. In this way, experience with the production of earlier described E. coli O-antigen bioconjugates, such as the O1A, O2, O6A and O25B bioconjugates described in, for example, WO 2015 / 124769 and WO 2017 / 035181, can be used as a basis for designing the production of other E. coli O-antigen bioconjugates. gtrS different, gtrA and gtrB The genes are not serotype specific, and in certain embodiments, these are homologous to the host cell (e.g., E. coli K12 strain W3110 includes gtrA and gtrB genes, which can be introduced together with the O4 serotype-specific recombination gtrS The gene (which encodes the glucosyltransferase of SEQ ID NO: 4 or a glucosyltransferase having at least 80% identity thereto) functions together to replace the endogenous gtrS In other embodiments, for example, when the host cell does not have an endogenous gtrA and / or gtrB In the case of genes, gtrA and gtrB One or both genes encoding GtrA and GtrB proteins having at least about 80% identity to SEQ ID NOs: 7 and 8, respectively, and having bacterial terpene alcohol-linked glucose translocase and bacterial terpene alcohol glucosyltransferase activities, respectively, are also recombinantly introduced into the host cell.

[0203] Also provided herein are host cells (e.g., recombinant host cells) capable of producing a bioconjugate of an E. coli O1A, O2, O6A, O8, O15, O16, O18A, O25B, or O75 antigen polysaccharide covalently linked to a carrier protein. Such host cells (e.g., recombinant host cells) contain a specific O-antigen polysaccharide. rfb Nucleotide sequence of the gene cluster. rfbThe gene cluster can be isolated from a wild-type E. coli strain and combined with nucleic acids encoding oligosaccharyltransferases (e.g., PglB) and carrier proteins (e.g., EPA) in a host cell to obtain a recombinant host cell that produces the desired E. coli O-antigen or a bioconjugate thereof. For example, using bioconjugation technology, such as that described in WO 2014 / 037585, WO 2009 / 104074, and WO 2009 / 089396, such host cells can be recombinantly modified to contain a gene encoding an oligosaccharyltransferase (e.g., PglB) and a carrier protein (e.g., EPA). rfb Preferably, the host cell contains one or more plasmids containing a gene cluster, an oligosaccharyl transferase (e.g., PglB), and a carrier protein (e.g., EPA). rfb In certain embodiments, the gene cluster encodes oligosaccharyltransferases, carrier proteins, and, where applicable, gtrS The nucleic acid of the gene is also integrated into the genome of the host cell. In certain embodiments, the heterologous or homologous gtrA and gtrB The gene is also integrated into the genome of the host cell.

[0204] Preparation of bioconjugates for O1A, O2, O6A, and O25B antigens has been described in detail in WO 2015 / 124769 and WO 2017 / 035181. rfb locus) has been described in: Iguchi A, et al., DNA Research, 2014, 1-7 (doi: 10.1093 / dnares / dsu043), and DebRoy C, et al., PLoS One. 2016, 11 (1): e0147434 (doi: 10.1371 / journal.pone.0147434; corrected: PLoS One. 2016, 11 (4): e0154551, doi: 10.1371 / journal.pone.0154551). rfb The nucleic acid sequences of the clusters and the amino acid sequences of the proteins encoded therein can also be found in public databases such as GenBank. rfb Exemplary sequences of clusters are also provided in SEQ ID NOs: 9 and 11-19. Thus, for each desired bioconjugate mentioned above, the corresponding rfb Clusters are introduced into host cells to obtain proteins specific for the desired O-antigen. rfbCluster, and host cells containing nucleic acids encoding oligosaccharyltransferases and carrier proteins. For the reasons indicated above, preferably the host cell is a recombinant host cell and is preferably derived from a strain with relatively well-known properties, such as an E. coli laboratory or production strain, such as E. coli K12 or E. coli BL21, etc. Preferably, rfb The cluster is heterologous to the host cell, for example, it is introduced into a precursor cell of the host cell and preferably integrated into its genome. rfb If the gene cluster is present in the precursor cell, it has been replaced in the host cell with the gene for the target O-antigen. rfb Preferably, the oligosaccharyltransferase is heterologous to the host cell, and in certain embodiments, the nucleic acid encoding such oligosaccharyltransferase is integrated into the genome of the host cell.

[0205] Any host cell provided herein (e.g., a recombinant host cell, preferably a recombinant prokaryotic host cell) comprising a protein encoded by N - Nucleic acids encoding additional enzymes active in glycosylation, for example, the host cells provided herein can further comprise a nucleic acid encoding an oligosaccharyltransferase or a nucleic acid encoding other glycosyltransferases.

[0206] The host cells provided herein contain a nucleic acid encoding an oligosaccharyl transferase. The oligosaccharyl transferase transfers lipid-linked oligosaccharides to a host cell comprising N -glycosylation consensus motif of the nascent polypeptide chain. The nucleic acid encoding the oligosaccharyl transferase can be native to the host cell or can be introduced into the host cell using genetic methods. In a preferred embodiment, the oligosaccharyl transferase is heterologous to the host cell. Escherichia coli does not naturally contain oligosaccharyl transferases, and therefore, if E. coli is used as a host cell for the production of bioconjugates, a heterologous oligosaccharyl transferase is contained in such host cells, for example, after introduction by genetic engineering. In view of the present disclosure, the oligosaccharyl transferase can be from any source known in the art.

[0207] In certain embodiments, having N - glycosyltransferase activity of oligosaccharyltransferases, e.g. O - Glycosyltransferases, such as PglL as a non-limiting example, can be used in combination with their own different glycosylation consensus sequences in a carrier protein, as described, for example, in WO 2016 / 82597. Other glycosyltransferases, such as O -glycosyltransferases, and therefore also can be used as oligosaccharyltransferases according to the invention.

[0208] In certain preferred embodiments, the oligosaccharyltransferase is from Campylobacter ( Campylobacter For example, in one embodiment, the oligosaccharyltransferase is an oligosaccharyltransferase from Campylobacter jejuni (i.e., pglB; see, e.g., Wacker et al., 2002, Science 298:1790-1793; see also, e.g., NCBI Gene ID: 3231775, UniProt Accession No. 086154). In another embodiment, the oligosaccharyltransferase is from Campylobacter larvae ( Campylobacter lari ) (see, e.g., NCBI Gene ID: 7410986).

[0209] In a specific embodiment, the oligosaccharyltransferase is a PglB oligosaccharyltransferase from Campylobacter jejuni, including the native (wild-type) protein or any variant thereof, such as those described in International Patent Application Publications WO 2016 / 107818 and WO 2016 / 107819. PglB can transfer lipid-linked oligosaccharides to asparagine residues in the consensus sequences SEQ ID NO: 1 and SEQ ID NO: 2. In a specific embodiment, the PglB oligosaccharyltransferase comprises SEQ ID NO: 6 or a variant thereof. In certain embodiments, one or more endogenous glycosylation consensus sequences in wild-type PglB have been mutated to prevent PglB from glycosylation, such as SEQ ID NO: 6 comprising the mutation N534Q. Examples of variant PglB oligosaccharyltransferases suitable for use in recombinant host cells provided herein include the PglB oligosaccharyltransferase of SEQ ID NO: 6 comprising at least one mutation selected from N311V, K482R, D483H, A669V, Y77H, S80R, Q287P, and K289R. In a specific embodiment, the variant PglB oligosaccharyltransferase has SEQ ID NO: 6 comprising the mutation N311V. In another specific embodiment, the variant PglB oligosaccharyltransferase has SEQ ID NO: 6 comprising the mutations Y77H and N311V. In another specific embodiment, the variant PglB oligosaccharyltransferase has SEQ ID NO: 6 comprising the mutations N311V, K482R, D483H, and A669V. In another specific embodiment, the variant PglB oligosaccharyltransferase has SEQ ID NO: 6 comprising mutations Y77H, S80R, Q287P, K289R, and N311V. It is discovered and described herein that certain PglB oligosaccharyltransferase variants provide surprisingly improved yields in the production of bioconjugates of E. coli O-antigens of specific serotypes. For a given E. coli O-antigen, improved or optimal PglB variants are unpredictable. Thus, in certain aspects, the present invention also provides methods for producing bioconjugates of specific E. coli O-antigens using specific PglB variants as oligosaccharyltransferases.Further variants of PglB preferably having one or more specific amino acids at the indicated positions disclosed in the combinations herein (e.g., 77Y, 80S, 287Q, 289K, 311N, 482K, 483D, 669A; or 311V; or 311V, 482R, 483H, 669V; or 77H, 80R, 287P, 289R, 311V; or 77H, 311V; etc.) which have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 6 and still have oligosaccharyltransferase activity can also be used in the production of bioconjugates.

[0210] In a specific embodiment, the host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of a glucosylated O4 antigen polysaccharide of Escherichia coli covalently linked to a carrier protein further comprises a nucleotide sequence encoding a PglB oligosaccharyltransferase from Campylobacter jejuni, wherein the PglB oligosaccharyltransferase has an amino acid sequence of SEQ ID NO: 6, or preferably SEQ ID NO: 6 comprising the mutation N311V, or more preferably SEQ ID NO: 6 comprising the mutations Y77H and N311V.

[0211] In other specific embodiments, the host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of an E. coli O1A, O6A or O15 antigen polysaccharide covalently linked to a carrier protein further comprises a nucleotide sequence encoding a PglB oligosaccharyltransferase from Campylobacter jejuni having an amino acid sequence of SEQ ID NO: 6, or preferably comprising mutations N311V, K482R, D483H and A669V.

[0212] In a specific embodiment, the host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of an Escherichia coli O16 antigen polysaccharide covalently linked to a carrier protein further comprises a nucleotide sequence encoding a PglB oligosaccharyltransferase from Campylobacter jejuni, wherein the PglB oligosaccharyltransferase has an amino acid sequence of SEQ ID NO: 6, or preferably comprises mutations Y77H, S80R, Q287P, K289R and N311V.

[0213] In a specific embodiment, the host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of an Escherichia coli O75 antigen polysaccharide covalently linked to a carrier protein further comprises a nucleotide sequence encoding a PglB oligosaccharyltransferase from Campylobacter jejuni, wherein the PglB oligosaccharyltransferase has an amino acid sequence of SEQ ID NO: 6, or preferably SEQ ID NO: 6 comprising the mutation N311V.

[0214] In a specific embodiment, the host cell (e.g., a recombinant host cell) capable of producing a bioconjugate of an E. coli O8, O18A, O25B or O2 antigen polysaccharide covalently linked to a carrier protein further comprises a nucleotide sequence encoding a PglB oligosaccharyltransferase from Campylobacter jejuni, wherein the PglB oligosaccharyltransferase has an amino acid sequence of SEQ ID NO: 6, preferably wherein SEQ ID NO: 6 does not comprise amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669.

[0215] In some embodiments, any of the host cells provided herein comprises a nucleic acid encoding a carrier protein, such as a protein to which an O-antigen polysaccharide produced by the host cell glycosylation machinery can be attached to form a bioconjugate. In view of the present disclosure, the host cell can comprise a nucleic acid encoding any carrier protein known to those skilled in the art, including but not limited to detoxified Pseudomonas aeruginosa exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, clumping factor A, clumping factor B, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.

[0216] In a preferred embodiment, the host cell further comprises a nucleic acid encoding a detoxified Pseudomonas aeruginosa exotoxin A (EPA). Preferably, the EPA carrier protein comprises 1-10 glycosylation sites, preferably 2 to 4 glycosylation sites, most preferably 4 glycosylation sites, such as 1-10, preferably 2-4, and more preferably 4 glycosylation sites, each of which comprises a glycosylation consensus sequence having an amino acid sequence of SEQ ID NO: 1, and more preferably having an amino acid sequence of SEQ ID NO: 2. In a specific embodiment, the host cell further comprises a nucleic acid encoding an EPA-4 carrier protein comprising SEQ ID NO: 3.

[0217] In certain embodiments, the carrier protein used to generate the bioconjugates by the host cells described herein comprises a "tag," i.e., an amino acid sequence that allows for the isolation and / or identification of the carrier protein. For example, adding a tag to a carrier protein can be used for the purification of the protein, and therefore, for the purification of conjugate vaccines comprising the tagged carrier protein. Exemplary tags that can be used herein include, but are not limited to, histidine (HIS) tags (e.g., hexahistidine tags or 6XHis-Tags), FLAG-TAG, and HA tags. In certain embodiments, once the tags used herein are no longer needed, e.g., after the protein has been purified, they are removable, e.g., by chemical reagents or enzymatic means. In other embodiments, the carrier protein does not comprise a tag.

[0218] In certain embodiments, the vector proteins described herein comprise a signal sequence that targets the vector protein to the periplasmic space of the host cell in which the vector protein is expressed. In a specific embodiment, the signal sequence is derived from E. coli DsbA, E. coli outer membrane porin A (OmpA), E. coli maltose binding protein (MalE), Erwinia solani ( Erwinia carotovorans ) pectate lyase (PelB), FlgI, NikA or Bacillus ( Bacillus ) species endo-xylanase (XynA), heat-labile E. coli enterotoxin LTIIb, Bacillus endoxylanase XynA, or E. coli flagellin (FlgI). In one embodiment, the signal sequence comprises SEQ ID NO: 10. After the protein is translocated to the periplasm, the signal sequence may be cleaved off and, thus, may no longer be present in the final carrier protein of the bioconjugate.

[0219] In certain embodiments, additional modifications can be introduced (e.g., using recombinant techniques) into the host cells described herein. For example, host cell nucleic acids (e.g., genes) encoding proteins that form part of a glycosylation pathway that may compete with or interfere with one or more heterologous genes involved in glycosylation introduced recombinantly into the host cell can be deleted or modified in the host cell background (genome) in a manner that renders them inactive / dysfunctional (i.e., the deleted / modified host cell nucleic acids do not encode functional proteins). In certain embodiments, when nucleic acids are deleted from the genome of the host cells provided herein, they are replaced with desired sequences, e.g., sequences that can be used to produce an O-antigen polysaccharide or a bioconjugate thereof.

[0220] Exemplary genes or gene clusters that can be deleted in the host cell (and in some cases, replaced by other desired nucleic acid sequences) include genes or gene clusters of the host cell involved in glycolipid biosynthesis, e.g. waL (See, e.g., Feldman et al., 2005, PNAS USA 102:3016-3021), lipid A core biosynthetic cluster ( waa ), galactose clusters ( gal ), arabinose clusters ( ara ), colonic acid cluster ( wc ), capsular polysaccharide cluster, undecaprenol-p biosynthesis genes (e.g. uppS 、 uppP ), und-P recycling genes, metabolic enzymes involved in nucleotide-activated sugar biosynthesis, enterobacterial common antigen cluster ( eca ) and prophage O antigen modification clusters such as gtrABS In a specific embodiment, the host cells described herein are modified such that they do not produce any O-antigen polysaccharide other than the desired O-antigen polysaccharide, such as a glucosylated O4 antigen polysaccharide.

[0221] In a specific embodiment, waL A gene is deleted or functionally inactivated from the genome of a host cell (e.g., a recombinant host cell) as provided herein. waL "and" waL The term "gene" refers to an O-antigen ligase gene encoding a membrane-bound enzyme whose active site is located in the periplasm. The encoded enzyme transfers undecaprenylphosphate (UPP)-bound O-antigen to the lipid A core to form lipopolysaccharide. waL Deletion or disruption of a gene (e.g., waLstrains) disrupt the transfer of O-antigen to lipid A and, conversely, may enhance the transfer of O-antigen to another biomolecule, such as a carrier protein.

[0222] In another specific embodiment, waL Gene, gtrA Gene, gtrB Gene, gtrS Genes and rfb One or more of the gene clusters are deleted or functionally inactivated from the original genome of the prokaryotic host cell provided herein.

[0223] In one embodiment, the host cell used herein is E. coli, which produces a bioconjugate of a glucosylated O4 antigen polysaccharide, wherein waL The gene is deleted or functionally inactivated from the host cell genome and a specific gene for the E. coli O4 antigen polysaccharide is inserted. gtrS In certain embodiments of the production strain for the bioconjugate of glucosylating O4 O-antigen, a gene encoding a glucosyltransferase having at least 80% sequence identity to SEQ ID NO: 4 is added. gtrS Gene inserted into parent strain gtrS The gene is located in order to be responsible for the glucosylation of the O4 antigen gtrS Gene replacement in the parent strain gtrS An example of such a parent strain is Escherichia coli K-12 strain W3110. gtrA and gtrB The genes may be homologous to the parent strain, or alternatively, one or both of these genes may be heterologous to the parent strain. gtrS Genes, these gtrA and gtrB The gene is not specific for the O-antigen structure.

[0224] Also provided herein is a method for preparing a recombinant host cell. The recombinant host cell produced by the method described herein can be used to produce a bioconjugate of an E. coli O antigen. The method includes introducing one or more recombinant nucleic acid molecules into the cell to produce the recombinant host cell. Generally, the recombinant nucleic acid molecule is heterologous. In view of the present disclosure, any method known in the art can be used to introduce the recombinant nucleic acid molecule into the host cell. Any method known to those of ordinary skill in the art can be used to introduce the recombinant nucleic acid into the host cell described herein, such as electroporation, chemical transformation, heat shock, natural transformation, phage transduction, and conjugation. In a specific embodiment, a plasmid is used to introduce the recombinant nucleic acid into the host cell described herein. For example, heterologous nucleic acid can be expressed in the host cell by a plasmid (e.g., an expression vector). In another specific embodiment, a method for inserting the nucleic acid into the genome as described in, for example, International Patent Application Publication WO 2014 / 037585, WO 2014 / 057109, or WO 2015 / 052344 is used to introduce the heterologous nucleic acid into the host cell described herein.

[0225] In one embodiment, a method for preparing a recombinant host cell for producing a bioconjugate of a glucosylated E. coli O4 antigen polysaccharide covalently linked to a carrier protein comprises introducing one or more recombinant nucleic acid molecules into a cell, preferably an E. coli cell, to produce a recombinant host cell. In such an embodiment, the recombinant nucleic acid molecules introduced into the cell include (i) a recombinant nucleic acid molecule for the E. coli O4 antigen polysaccharide; rfb (ii) a nucleotide sequence encoding a glucosyltransferase having at least 80% sequence identity to SEQ ID NO: 4, wherein the glucosyltransferase is capable of modifying E. coli O4 antigen polysaccharide to produce a glucosylated E. coli O4 antigen polysaccharide; (iii) a nucleotide sequence encoding a carrier protein; and (iv) a nucleotide sequence encoding an oligosaccharyltransferase capable of covalently linking the glucosylated E. coli O4 antigen polysaccharide to the carrier protein to produce a bioconjugate. In a preferred embodiment, the nucleotide sequence encoding the glucosyltransferase having at least 80% sequence identity to SEQ ID NO: 4 replaces the endogenous gtrS Gene. Deletion of endogenous gtrS This has the advantage that it will not interfere with the formation of glucosylated O4 antigen polysaccharide structures. In certain embodiments, regarding the E. coli O4 antigen polysaccharide rfb The nucleotide sequence of the gene cluster replaces the endogenous rfb If the cell has not yet encoded gtrA and / or gtrBIf the cell already encodes the translocase (gtrA) and glycosyltransferase (gtrB) nucleotide sequences encoding at least 80% identity to SEQ ID NOs: 7 and 8, respectively, can be introduced into the cell. gtrA and gtrB coli K-12 strain W3110), there is no need to introduce or alter these genes.

[0226] In a specific embodiment, the glucosyltransferase (gtrS specific for adding a glucose branch to the O4 antigen) has SEQ ID NO: 4.

[0227] In a specific embodiment, the oligosaccharyltransferase is PglB from Campylobacter jejuni. In one such embodiment, the oligosaccharyltransferase comprises the amino acid sequence of SEQ ID NO: 6. In another such embodiment, the oligosaccharyltransferase comprises the amino acid sequence of SEQ ID NO: 6, said SEQ ID NO: 6 comprising the mutation N311V. In another such embodiment, the oligosaccharyltransferase comprises the amino acid sequence of SEQ ID NO: 6, said SEQ ID NO: 6 comprising the mutations Y77H and N311V.

[0228] In another embodiment, the carrier protein comprises at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably SEQ ID NO: 2. In another embodiment, the carrier protein is EPA, preferably EPA-4, such as EPA-4 comprising SEQ ID NO: 3.

[0229] In certain embodiments according to the present invention, E. coli strains routinely used as tools and model organisms in molecular biology can, for example, be used as the parent of the host cell. Non-limiting examples include E. coli K12 strains (e.g., W1485, W2637, W3110, MG1655, DH1, DH5α, DH10, etc.), B strains (e.g., BL-21, REL606, etc.), C strains, or W strains. In a specific embodiment, the host strain is derived from the parent strain W3110. For example, such strains can be obtained from the Yale E. coli Genetic Collection ( E. coli For more information about E. coli, see, for example, Ecoliwiki.net.

[0230] Methods of producing conjugates and bioconjugates

[0231] Also provided are methods for producing glycoconjugates of the E. coli O-antigen polysaccharide described herein. Glycoconjugates, including bioconjugates, can be prepared in vitro or in vivo, for example using the recombinant host cells described herein for production.

[0232] In some embodiments, the glycoconjugates can be prepared by chemical synthesis, i.e., prepared outside the host cell (in vitro). For example, methods known to those skilled in the art can be used, including conjugating E. coli O antigen polysaccharides to carrier proteins using activated reactive groups in the polysaccharide / oligosaccharide and the carrier protein. See, for example, Pawlowski et al., 2000, Vaccine 18:1873-1885; and Robbins et al., 2009, Proc Natl Acad Sci USA 106:7974-7978), the disclosure of which is incorporated herein by reference. Such methods include extracting antigenic polysaccharides / oligosaccharides from host cells, purifying the polysaccharides / oligosaccharides, chemically activating the polysaccharides / oligosaccharides, and conjugating the polysaccharides / oligosaccharides to a carrier protein.

[0233] In some embodiments, the host cells described herein can be used to produce bioconjugates comprising an E. coli O antigen polysaccharide covalently linked to a carrier protein. Methods for producing such bioconjugates using host cells are known in the art. See, for example, WO 2003 / 074687 and WO 2006 / 119987. Such methods include culturing any of the recombinant host cells described herein under conditions in which the bioconjugate is produced. In view of the present disclosure, the bioconjugates can be isolated, separated, and / or purified from the recombinant host cells using any method known in the art. For example, the bioconjugates can be purified by any method known in the art for purifying proteins, such as chromatography (e.g., ion exchange, anion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. See, for example, the methods described in WO 2009 / 104074. Furthermore, the bioconjugates can be fused to heterologous polypeptide sequences to facilitate purification. The actual conditions used to purify a particular bioconjugate will depend in part on such factors as the net charge, hydrophobicity, and / or hydrophilicity of the bioconjugate and will be apparent to those skilled in the art. Preparation of bioconjugates of O1A, O2, O6A, and O25B, and vaccine compositions containing these, have been described, for example, in WO 2015 / 124769 and WO 2017 / 035181.

[0234] Also provided are bioconjugates produced by the methods described herein, ie, using the recombinant host cells described herein.

[0235] In some embodiments, a method for preparing a bioconjugate of an E. coli O-antigen polysaccharide covalently linked to a carrier protein comprises: (i) providing a recombinant host cell comprising (a) a vector for the O-antigen polysaccharide; rfb (b) a nucleotide sequence encoding a carrier protein, preferably EPA, comprising at least one glycosylation site (which comprises a glycosylation consensus sequence having SEQ ID NO: 1, preferably SEQ ID NO: 2), and more preferably comprising four glycosylation sites, each of which comprises a glycosylation consensus sequence having SEQ ID NO: 2; and (c) a nucleotide sequence encoding an oligosaccharyltransferase, such as PglB oligosaccharyltransferase or a variant thereof.

[0236] In certain embodiments, the E. coli O-antigen polysaccharide produced using the recombinant host cells described herein is covalently bound to a carrier protein at a specific polysaccharide / protein ratio (w / w) by weight. This ratio of the amount of O-antigen polysaccharide by weight covalently bound to the carrier protein by weight is referred to as the "polysaccharide / protein ratio" or "polysaccharide / protein ratio" or "PS / protein ratio". In some embodiments, the O-antigen polysaccharide is covalently bound to the carrier protein at a polysaccharide / protein (w / w) ratio of about 1:20 to 20:1, preferably 1:10 to 10:1, more preferably 1:3 to 3:1. In certain non-limiting embodiments of the bioconjugates described herein, the polysaccharide / protein ratio is about 0.1 to 0.5, for example, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, or 0.5. In such embodiments, the weight ratio of O-antigen polysaccharide to protein is about 1:10 to 1:2, e.g., 1:10: 1:9: 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, or 1:2, depending on the specific O-antigen serotype. In certain embodiments, the polysaccharide / protein ratio is about 0.15 to about 0.45. Generally, a higher polysaccharide / protein ratio of O-antigen polysaccharide to carrier protein is preferred because, in some cases, large amounts of carrier protein can cause immunological interference. In addition, a higher polysaccharide / protein ratio will help to obtain sufficient O-antigen polysaccharide for administration in the form of a bioconjugate while keeping the amount of carrier protein relatively low, which is particularly beneficial for multivalent compositions in which multiple serotypes are to be covered by the composition, e.g., compositions comprising bioconjugates from at least 4 different O-antigens, at least 5 different O-antigens, at least 6 different O-antigens, at least 7 different O-antigens, at least 8 different O-antigens, at least 9 different O-antigens, at least 10 different O-antigens, etc.

[0237] The glycan / protein ratio of the conjugates according to the present invention can be determined by determining the amount of protein and the amount of glycans. The amount of protein can be determined by measuring the UV absorbance at 280 nm (A280). The amount of glycans can be determined based on ion chromatography-pulsed amperometric detection (IC-PAD) of the sugars in the repeating unit (e.g., Man for O8 in Table 1, and GlcNAc for the other glycans in Table 1), after which the structural information of the repeating unit can be used to calculate the total glycan amount (e.g., the repeating unit of O1A has a molar mass of 845 Da, and 1 mole of such repeating unit contains 1 mole of GlcNAc, which allows calculation of the total glycan amount when the amount of GlcNAc has been determined by IC-PAD).

[0238] In some embodiments, the bioconjugate of the E. coli O25B antigen polysaccharide covalently linked to a carrier protein produced using recombinant host cells according to the cells and methods described herein has a degree of acetylation at position 2 of the L-Rh saccharide. The degree of O-acetylation of the O25B antigen polysaccharide in the bioconjugate is preferably at least 30%, preferably at least 50%, for example at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0239] Similarly, the degree of O-acetylation of the E. coli 016 antigen polysaccharide in the bioconjugate is preferably at least 30%, preferably at least 50%, such as at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0240] In a specific embodiment, a method for preparing a bioconjugate of an O-antigen polysaccharide comprises providing a recombinant host cell comprising a nucleic acid sequence encoding a specific oligosaccharyltransferase, particularly a PglB oligosaccharyltransferase or a variant thereof, depending on the O-antigen polysaccharide bioconjugate to be produced. The specific oligosaccharyltransferase variant may affect the yield of the bioconjugate produced by the host cell. Generally, higher yields are preferred because yields will affect the cost of producing a specific bioconjugate, which is particularly important for multivalent compositions comprising several different bioconjugates. In some embodiments, the method further comprises isolating the bioconjugate from the recombinant host cell.

[0241] In a specific embodiment, when the O-antigen is O1A, O6A or O15 antigen polysaccharide, the PglB oligosaccharyltransferase comprises amino acid mutations N311V, K482R, D483H and A669V, wherein the amino acid mutations are relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6.

[0242] In another specific embodiment, when the O-antigen is a glucosylated O4 antigen polysaccharide, the PglB oligosaccharyltransferase comprises an amino acid mutation N311V, or an amino acid mutation of Y77H and N311V, wherein the amino acid mutation is relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6.

[0243] In another specific embodiment, when the O-antigen is an O16 antigen polysaccharide, the PglB oligosaccharyltransferase comprises amino acid mutations Y77H, S80R, Q287P, K289R and N311V, wherein the amino acid mutations are relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6.

[0244] In another specific embodiment, when the O-antigen is the O75 antigen polysaccharide, the PglB oligosaccharyltransferase comprises an amino acid mutation of N311V, wherein the amino acid mutation is relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6.

[0245] In another specific embodiment, when the O-antigen is O8, O18A, O25B or O2 antigen polysaccharide, the PglB oligosaccharyltransferase comprises the amino acid sequence of SEQ ID NO: 6, wherein SEQ ID NO: 6 does not comprise the amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669. In certain embodiments thereof, the PglB oligosaccharyltransferase comprises the amino acid sequence of SEQ ID NO: 6.

[0246] In some embodiments, the carrier protein is selected from detoxified Pseudomonas aeruginosa exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, clumping factor A, clumping factor B, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.

[0247] In certain embodiments, the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA). Preferably, the EPA carrier protein comprises 1-10, preferably 2-4, more preferably 4 glycosylation sites. Preferably, each glycosylation site comprises a glycosylation consensus sequence having the amino acid sequence of SEQ ID NO: 2. In a specific embodiment, the host cell comprises a nucleic acid encoding an EPA-4 carrier protein comprising SEQ ID NO: 3.

[0248] In certain embodiments, the recombinant host cell is an E. coli cell, such as an E. coli K-12 strain, such as strain W3110.

[0249] Also provided herein are bioconjugates of O-antigen polysaccharides produced according to the O-antigen / PglB oligosaccharyltransferase pairing indicated above, using recombinant host cells encoding oligosaccharyltransferases. Also provided are compositions comprising such bioconjugates. In certain embodiments, the composition comprises at least two, preferably at least three, more preferably at least five, and even more preferably at least seven such bioconjugates.

[0250] In some embodiments, according to the O-antigen / PglB oligosaccharyltransferase pairing indicated above, the bioconjugate of the O-antigen polysaccharide produced by a recombinant host cell encoding the oligosaccharyltransferase preferably has one or more preferred properties described herein, such as the glycan / protein ratio and / or the amount or ratio of the polyglycosylated carrier protein.

[0251] Implementation Plan

[0252] Embodiment 1 is to prepare Escherichia coli O that is covalently linked to a carrier protein x A method for bioconjugating an antigen-polysaccharide, the method comprising:

[0253] (i) providing a recombinant host cell comprising:

[0254] a. About O x - Antigenic polysaccharide rfb nucleotide sequence of the gene cluster;

[0255] b. A nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably SEQ ID NO: 2; and

[0256] c. Encoding oligosaccharyltransferase PglB y the nucleotide sequence of; and

[0257] (ii) culturing the recombinant host cell under conditions for bioconjugate production,

[0258] in:

[0259] When O x -When the antigen is O1A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0260] When O x -When the antigen is glucosylated O4 antigen polysaccharide, PglB y comprising an amino acid mutation N311V or amino acid mutations Y77H and N311V, and the recombinant host cell further comprising a sequence encoding a glucosyltransferase GtrS having at least 80% identity to SEQ ID NO: 4 and capable of modifying E. coli O4 antigen polysaccharide by adding glucose to produce a glucosylated E. coli O4 antigen polysaccharide, and nucleotide sequences encoding a translocase GtrA and a glycosyltransferase GtrB having at least 80% sequence identity to SEQ ID NOs: 7 and 8, respectively, wherein the translocase is capable of translocating glucose linked to a bacterial terpene alcohol, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol;

[0261] When O x -When the antigen is O6A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0262] When O x -When the antigen is O8 antigen polysaccharide, PglB y does not contain amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483, and 669;

[0263] When O x -When the antigen is O15 antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0264] When O x -When the antigen is O16 antigen polysaccharide, PglB y amino acid mutations including Y77H, S80R, Q287P, K289R, and N311V;

[0265] When O x -When the antigen is O18A antigen polysaccharide, PglB ydoes not comprise amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483, and 669; and

[0266] When O x -When the antigen is O75 antigen polysaccharide, PglB y Contains the amino acid mutation N311V,

[0267] wherein in each case the amino acid mutation is relative to wild-type PglB having the amino acid sequence of SEQ ID NO: 6, and

[0268] wherein the O1A, glucosylated O4, O6A, O8, O15, O16, O18A and O75 antigen polysaccharides have the structures of formula (O1A), (O4-Glc+), (O6A), (O8), (O15), (O16), (O18A) and (O75) as shown in Table 1, respectively, and each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0269] Embodiment 2 is the method of embodiment 1, wherein the x - the antigen is O1A antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0270] Embodiment 3 is the method of embodiment 1, wherein the x - The antigen is a glucosylated 04 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Comprising the amino acid mutation N311V or the amino acid mutations Y77H and N311V.

[0271] Embodiment 4 is the method of embodiment 3, wherein the recombinant host cell further comprises a sequence encoding GtrS having the amino acid sequence of SEQ ID NO: 4, and nucleotide sequences encoding GtrA and GtrB having the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

[0272] Embodiment 5 is the method of embodiment 1, wherein the x - the antigen is O6A antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0273] Embodiment 6 is the method of embodiment 1, wherein the x - the antigen is the O8 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669 are not included.

[0274] Embodiment 7 is the method of embodiment 1, wherein the x - the antigen is the O15 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0275] Embodiment 8 is the method of embodiment 1, wherein the x - the antigen is O16 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations Y77H, S80R, Q287P, K289R and N311V.

[0276] Embodiment 9 is the method of embodiment 1, wherein the x - the antigen is O18A antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669 are not included.

[0277] Embodiment 10 is the method of embodiment 1, wherein the x - the antigen is the O75 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains the amino acid mutation N311V.

[0278] Embodiment 11 is to prepare Escherichia coli O covalently linked to a carrier protein x - a method for preparing a bioconjugate of an antigen-polysaccharide, the method comprising:

[0279] (i) providing a recombinant host cell comprising:

[0280] (a) About O x - Antigenic polysaccharide rfb nucleotide sequence of the gene cluster;

[0281] (b) a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2; and

[0282] (c) Encoding oligosaccharyltransferase PglB y the nucleotide sequence of; and

[0283] (ii) culturing the recombinant host cell under conditions for bioconjugate production,

[0284] Wherein relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains the amino acid mutation N311V,

[0285] Among them O x - The antigen is an O1A antigen polysaccharide, a glucosylated O4 antigen polysaccharide, an O6A antigen polysaccharide, an O15 antigen polysaccharide, an O16 antigen polysaccharide or an O75 antigen polysaccharide, and when O x When the antigen is a glucosylated O4 antigen polysaccharide, the recombinant host cell further comprises a sequence encoding a glucosyltransferase GtrS having at least 80% identity to SEQ ID NO: 4 and capable of modifying E. coli O4 antigen polysaccharide by adding glucose to produce a glucosylated E. coli O4 antigen polysaccharide, and nucleotide sequences encoding a translocase GtrA and a glycosyltransferase GtrB having at least 80% sequence identity to SEQ ID NOs: 7 and 8, respectively, wherein the translocase is capable of translocating glucose linked to a bacterial terpene alcohol and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol, and

[0286] wherein the O1A, glucosylated O4, O6A, O15, O16 and O75 antigen polysaccharides have the structures of formula (O1A), (O4-Glc+), (O6A), (O15), (O16) and (O75) as shown in Table 1, respectively, and each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0287] Embodiment 12 is the method of any one of embodiments 1 to 11, further comprising isolating the bioconjugate from the recombinant host cell.

[0288] Embodiment 13 is the method of any one of embodiments 1 to 12, wherein the carrier protein is selected from the group consisting of detoxified Pseudomonas aeruginosa exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, clumping factor A, clumping factor B, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.

[0289] Embodiment 14 is the method of embodiment 13, wherein the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA).

[0290] Embodiment 15 is the method of embodiment 14, wherein the EPA carrier protein comprises 1-10, preferably 2-4, more preferably 4 glycosylation sites.

[0291] Embodiment 16 is the method of embodiment 15, wherein each glycosylation site comprises a glycosylation consensus sequence having SEQ ID NO: 2.

[0292] Embodiment 17 is the method of embodiment 16, wherein the EPA carrier protein comprises SEQ ID NO: 3.

[0293] Embodiment 18 is the method of any one of embodiments 1-17, wherein the recombinant host cell is an E. coli cell, such as an E. coli K-12 strain, such as strain W3110.

[0294] Embodiment 19 is a bioconjugate produced by the method of any one of embodiments 1-18.

[0295] Embodiment 20 is a composition comprising the bioconjugate of embodiment 19.

[0296] Embodiment 21 is a composition comprising at least 2, preferably at least 3, more preferably at least 5, and still more preferably at least 7 bioconjugates of embodiment 19.

[0297] Embodiment 22 is a composition of embodiment 20 or 21, comprising a bioconjugate of a glucosylated O4 antigen polysaccharide of Escherichia coli covalently linked to a carrier protein, wherein the glucosylated O4 antigen polysaccharide has a structure of the formula (O4-Glc+) as shown in Table 1, and n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0298] Embodiment 23 is a composition of any one of embodiments 20 to 22, further comprising at least a bioconjugate of an Escherichia coli O25B antigen polysaccharide covalently linked to a carrier protein, wherein the O25B antigen polysaccharide has a structure of formula (O25B) as shown in Table 1, and n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0299] Embodiment 24 is a composition of any one of embodiments 20 to 23, further comprising at least a bioconjugate of an E. coli O2 antigen polysaccharide covalently linked to a carrier protein, wherein the O2 antigen polysaccharide has a structure of formula (O2) as shown in Table 1, and n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0300] Embodiment 25 is a composition of any one of embodiments 20 to 24, comprising: (i) a bioconjugate of an E. coli O1A antigen polysaccharide covalently coupled to a carrier protein, (ii) a bioconjugate of an E. coli O2 antigen polysaccharide covalently coupled to a carrier protein, (iii) a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide covalently coupled to a carrier protein, (iv) a bioconjugate of an E. coli O6A antigen polysaccharide covalently coupled to a carrier protein, (v) a bioconjugate of an E. coli O8 antigen polysaccharide covalently coupled to a carrier protein, (vi) a bioconjugate of an E. coli O15 antigen polysaccharide covalently coupled to a carrier protein, (vii) a bioconjugate of an E. coli O15 antigen polysaccharide covalently coupled to a carrier protein, coli O25B antigen polysaccharide covalently coupled to a carrier protein, and (ix) a bioconjugate of an E. coli O75 antigen polysaccharide covalently coupled to a carrier protein, wherein the O1A, O2, glucosylated O4, O6A, O8, O15, O16, O25B and O75 antigen polysaccharides have the structures of formula (O1A), (O2), (O4-Glc+), (O6A), (O8), (O15), (O16), (O25B) and (O75) as shown in Table 1, respectively, and each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0301] Embodiment 26 is the composition of embodiment 25, further comprising: (x) a bioconjugate of an Escherichia coli O18A antigen polysaccharide covalently coupled to a carrier protein, wherein the O18A antigen polysaccharide has a structure of formula (O18A) as shown in Table 1, and n is an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0302] Embodiment 27 is the composition of any one of embodiments 20 to 26, wherein the composition is an immunogenic composition.

[0303] Embodiment 28 is a method of vaccinating a subject against Escherichia coli, particularly extraintestinal pathogenic Escherichia coli (ExPEC), comprising administering to the subject the bioconjugate of embodiment 19, or the composition or immunogenic composition of any one of embodiments 20 to 27.

[0304] Embodiment 29 is the bioconjugate of embodiment 19, or the composition or immunogenic composition of any one of embodiments 20 to 27, for use in vaccination against extraintestinal pathogenic Escherichia coli (ExPEC).

[0305] Embodiment 30 is for preparing Escherichia coli O covalently linked to a carrier protein x A recombinant host cell for a bioconjugate of an antigen-polysaccharide, the recombinant host cell comprising:

[0306] (a) About O x - Antigenic polysaccharide rfb nucleotide sequence of the gene cluster;

[0307] (b) a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence having SEQ ID NO: 1, preferably having SEQ ID NO: 2; and

[0308] (c) Encoding oligosaccharyltransferase PglB y The nucleotide sequence of

[0309] in:

[0310] When O x -When the antigen is O1A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0311] When O x -When the antigen is glucosylated O4 antigen polysaccharide, PglB ycomprising an amino acid mutation N311V or amino acid mutations Y77H and N311V, and the recombinant host cell further comprising a sequence encoding a glucosyltransferase GtrS having at least 80% identity to SEQ ID NO: 4 and capable of modifying E. coli O4 antigen polysaccharide by adding glucose to produce a glucosylated E. coli O4 antigen polysaccharide, and nucleotide sequences encoding a translocase GtrA and a glycosyltransferase GtrB having at least 80% sequence identity to SEQ ID NOs: 7 and 8, respectively, wherein the translocase is capable of translocating glucose linked to a bacterial terpene alcohol, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol;

[0312] When O x -When the antigen is O6A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0313] When O x -When the antigen is O8 antigen polysaccharide, PglB y does not contain amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483, and 669;

[0314] When O x -When the antigen is O15 antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V;

[0315] When O x -When the antigen is O16 antigen polysaccharide, PglB y amino acid mutations including Y77H, S80R, Q287P, K289R, and N311V;

[0316] When O x -When the antigen is O18A antigen polysaccharide, PglB y does not comprise amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483, and 669; and

[0317] When O x -When the antigen is O75 antigen polysaccharide, PglB y Contains the amino acid mutation N311V;

[0318] wherein in each case the amino acid mutation is relative to wild-type PglB having the amino acid sequence of SEQ ID NO: 6, and

[0319] wherein the O1A, glucosylated O4, O6A, O8, O15, O16, O18A and O75 antigen polysaccharides have the structures of formula (O1A), (O4-Glc+), (O6A), (O8), (O15), (O16), (O18A) and (O75) as shown in Table 1, respectively, and each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0320] Embodiment 31 is the recombinant host cell of embodiment 30, wherein said x - the antigen is O1A antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0321] Embodiment 32 is the recombinant host cell of embodiment 30, wherein said x - The antigen is a glucosylated 04 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Comprising the amino acid mutation N311V or the amino acid mutations Y77H and N311V.

[0322] Embodiment 33 is the recombinant host cell of embodiment 32, wherein the recombinant host cell further comprises a sequence encoding GtrS having the amino acid sequence of SEQ ID NO: 4, and nucleotide sequences encoding GtrA and GtrB having the amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

[0323] Embodiment 34 is the recombinant host cell of embodiment 30, wherein said x - the antigen is O6A antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0324] Embodiment 35 is the recombinant host cell of embodiment 30, wherein said x - the antigen is the O8 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669 are not included.

[0325] Embodiment 36 is the recombinant host cell of embodiment 30, wherein saidx - the antigen is the O15 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V.

[0326] Embodiment 37 is the recombinant host cell of embodiment 30, wherein said x - the antigen is O16 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains amino acid mutations Y77H, S80R, Q287P, K289R and N311V.

[0327] Embodiment 38 is the recombinant host cell of embodiment 30, wherein said x - the antigen is O18A antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669 are not included.

[0328] Embodiment 39 is the recombinant host cell of embodiment 30, wherein said x - the antigen is the O75 antigen polysaccharide, and relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, PglB y Contains the amino acid mutation N311V.

[0329] Embodiment 40 is the recombinant host cell of any one of embodiments 30 to 39, wherein the carrier protein is selected from the group consisting of detoxified Pseudomonas aeruginosa exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, clumping factor A, clumping factor B, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.

[0330] Embodiment 41 is the recombinant host cell of any one of embodiments 30-40, wherein the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA).

[0331] Embodiment 42 is the recombinant host cell of embodiment 41, wherein the EPA carrier protein comprises 1-10, preferably 2-4, more preferably 4 glycosylation sites.

[0332] Embodiment 43 is the recombinant host cell of embodiment 42, wherein each glycosylation site comprises a glycosylation consensus sequence having SEQ ID NO: 2.

[0333] Embodiment 44 is the recombinant host cell of embodiment 43, wherein the EPA carrier protein comprises SEQ ID NO: 3.

[0334] Embodiment 45 is the recombinant host cell of any one of embodiments 30 to 44, wherein the recombinant host cell is an E. coli cell, such as an E. coli K-12 strain, such as strain W3110.

[0335] Embodiment 46 is a bioconjugate according to embodiment 19, wherein the bioconjugate is a bioconjugate of E. coli glucosylated 04 antigen polysaccharide covalently linked to a carrier protein.

[0336] Embodiment 47 is a bioconjugate according to embodiment 46, wherein the carrier protein is an EPA carrier protein comprising SEQ ID NO: 3.

[0337] Embodiment 48 is a bioconjugate according to embodiment 46 or 47, wherein the glucosylated O4 antigen polysaccharide has a structure of formula (O4-Glc+) as shown in Table 1, and n is an integer from 5 to 40.

[0338] Embodiment 49 is a composition comprising the bioconjugate according to any one of embodiments 46-48.

[0339] Embodiment 50 is a composition according to embodiment 49, further comprising one or more conjugates, each of which comprises an E. coli antigenic polysaccharide covalently coupled to a carrier protein.

[0340] Embodiment 51 is a composition according to embodiment 50, wherein the one or more conjugates comprise E. coli antigenic polysaccharides of one or more of the following E. coli serotypes: O1A, O2, O6A, O8, O15, O16, O18A, O25B and O75, wherein the O1A, O2, O6A, O8, O15, O16, O25B and O75 antigenic polysaccharides have the structures of formula (O1A), (O2), (O6A), (O8), (O15), (O16), (O18A), (O25B) and (O75) as shown in Table 1, respectively, and each n is independently an integer from 1 to 100, preferably from 3 to 50, such as from 5 to 40, such as from 7 to 25, such as from 10 to 20.

[0341] Embodiment 52 is a composition according to embodiment 51, comprising a conjugate of the following E. coli serotypes: O1A, O2, O6A, O8, O15, O16, O18A, O25B and O75.

[0342] Embodiment 53 is a composition according to embodiment 52, wherein each of the conjugates is a bioconjugate. Example

[0343] The following examples of the present invention are intended to further illustrate the nature of the present invention. It should be understood that the following examples do not limit the present invention, and the scope of the present invention is determined by the appended claims.

[0344] Example 1 : Epidemiological data of Escherichia coli infection

[0345] To determine the distribution of O-serotypes of Escherichia coli causing bacteremia, a global surveillance study was conducted. Between 2011 and 2017, more than 3200 E. coli bloodstream isolates were collected from patients aged 60 years or older hospitalized in countries across North America, Europe, Asia-Pacific, and South America. The O-antigen serotype of each strain was determined using classical agglutination techniques and sequence-based O genotyping. See Table 2.

[0346] Separated human blood samples were analyzed to determine the identity of the pathogens present and their antibiotic resistance patterns. Following analysis, E. coli isolates were obtained from the samples. E. coli identity was verified by MALDI-TOF MS. Further analysis of the E. coli isolates was performed using an antiserum-based agglutination assay to determine their O-antigen serotype (DebRoy et al. (2011) Animal health research reviews / Conference of Research Workers inAnimal Diseases 12, 169-185). Isolates that could not be typed by agglutination were sequenced by whole genome sequencing followed by O serotype-specific wzy and wzx The O genotyping of the gene sequence was further analyzed.

[0347] Table 2: Distribution of the most common bacteremia-associated Escherichia coli O-serotypes, based on O-serotyping by agglutination plus O-genotyping of isolates that could not be typed by agglutination, in a collection of 3217 blood isolates collected worldwide between 2011 and 2017. Subjects were hospitalized in the following countries: United States, Canada, Argentina, Brazil, United Kingdom, Germany, Spain, Italy, Netherlands, France, Japan, Thailand, South Korea, and Australia.

[0348] O-serotype Prevalence rate n (%) O25 737 (22.9%) O2 268 (8.3%) O6 261 (8.1%) O1 255 (7.9%) O75 145 (4.5%) O15 110 (3.4%) O8 104 (3.2%) O16 103 (3.2%) O4 96 (3.0%) O18 91 (2.8%)

[0349] Stratification of the global collection of bacteremia-associated E. coli by geographic location revealed that the prevalence of the top 10 O serotypes was independent of location, suggesting that these are the dominant O-serotypes globally associated with bacteremia-causing E. coli.

[0350] In a global collection (n=345) of bacteremia-associated multidrug-resistant E. coli isolates (i.e., those resistant to at least three classes of clinically relevant antimicrobial drugs), the prevalence of the top 10 O-serotypes was 75.4%.

[0351] Taking all information from epidemiological analyses into account, the 10 dominant O-serotypes could cover an estimated 60–80% of E. coli-associated bacteremic infections, assuming coverage of a subset of nontypeable strains.

[0352] A multivalent vaccine that covers a significant proportion of the E. coli serotypes that cause bacteremia would be very useful. Therefore, the O-serotypes of Table 2 would be good candidates for a multivalent vaccine based on O-antigens. Such vaccines can be advantageously prepared using bioconjugation technology.

[0353] One of the top 10 (Table 2) serotypes is O4. Therefore, it would be beneficial to prepare a bioconjugate vaccine comprising the O-antigen polysaccharide of E. coli serotype O4 coupled to a carrier protein.

[0354] Example 2 Characterization of genes encoding O-antigen modifying enzymes in contemporary O4 clinical isolates

[0355] Two variants of the E. coli O4 antigenic polysaccharide have been described (see, e.g., Jann B et al., 1993, Carbohydr. Res. 248: 241-250): one variant with an unbranched structure (the structure is shown as (O4-Glc-) in Table 1), and another variant with an additional glucose side branch substituted (the structure is shown as (O4-Glc+) in Table 1). The proportion of these two variants found in contemporary clinical isolates is unknown. Although both variants react with O4 antiserum, it is not known whether there are immunological differences between these variants. In addition, the enzyme responsible for attaching the glucose side branch to generate the (O4-Glc+) antigenic polysaccharide has not been identified to date, and its putative coding sequence is likely located in the O4 rfb outside the gene cluster.

[0356] A collection of 32 agglutination-confirmed E. coli O4 clinical isolates originally isolated from subjects in the United States and the European Union during 2011-2012 was subjected to whole genome sequence analysis. rfb The gene cluster sequences were aligned with those of the reference strain and compared at the nucleotide level. In addition to some naturally occurring single nucleotide polymorphisms, all characterized isolates showed the same sequence identity as the O4 reference strain. rfb cluster, indicating that independent of their Glc branching status, E. coli O4 strains carry the same rfb Therefore, in order to produce the E. coli O4-Glc+ antigen polysaccharide, a gene with an unknown sequence is likely required, which encodes an E. coli O4-specific branching enzyme and must be located in the E. coli O4 rfb The sequence of this unknown gene needs to be identified and employed if one wishes to produce bioconjugates with the E. coli O4-Glc+ antigen polysaccharide in a strain that would otherwise only produce bioconjugates with the E. coli O4-Glc- antigen polysaccharide.

[0357] Then it may encode an O-antigen modification enzyme rfb The presence of genes outside the gene cluster was analyzed by whole genome sequence data. First, Shigella flexneri ( Shigella flexneri ) gtrAB Homologs of Escherichia coli gtrAB The downstream open reading frame was putatively identified as an E. coli O4-specific gene gtrS , which may encode a putative E. coli O4-specific branching enzyme GtrS, which is responsible for adding glucose branches to the E. coli O4 antigen.

[0358] The amino acid sequence of the O4-specific GtrS enzyme is provided as SEQ ID NO: 4. An exemplary nucleic acid sequence encoding this protein is provided as SEQ ID NO: 5.

[0359] Of the E. coli O4 isolates characterized, approximately 80% were found to carry the gtrS coli O4-specific genes were also determined by PCR using sequence-specific primers in an independent collection of 20 agglutination-confirmed E. coli O4 clinical isolates isolated from subjects in the United States and the European Union between 2014 and 2016. gtrS This analysis confirmed that 17 of the 20 isolates carried O4 gtrS sequence, which corresponds to a prevalence of 85%.

[0360] Example 3 :O4 gtrS Cloning into E. coli W3110, production and structural confirmation of Glc-modified O4 bioconjugates

[0361] To test whether bioconjugates containing O4-antigen polysaccharides modified with branched glucose could be prepared, an E. coli O4-antigen EPA bioconjugate production strain with a putative branching enzyme was constructed. O4- gtrS Gene (SEQ ID NO: 5, see Example 2) replaced the endogenous O16-gtrS gene, and through homologous recombination, O16 rfb Cluster replacement with Escherichia coli strain W3110 Δ wzzE-wecG ∆ waL Δ wbbI-JK O4 rfb Alternatively, in some strains, O4 rfb The cluster is encoded on a plasmid.

[0362] Subsequently, a plasmid encoding a detoxified Pseudomonas aeruginosa exotoxin A (EPA) carrier protein (variants with 2 or 4 consensus glycosylation sites, referred to as 'EPA-2' and 'EPA-4', respectively) and the oligosaccharyl transferase PglB was introduced into the strain. The O4-EPA bioconjugate modified with Glc was produced by growing the E. coli production strain in a bioreactor culture and inducing PglB and EPA expression by IPTG and arabinose, respectively. The O4-EPA bioconjugate was extracted from the biomass periplasmic extract.

[0363] To confirm the detailed polysaccharide composition and bonding of the O4-EPA bioconjugate, multiple NMR experiments were performed on the bioconjugate with the EPA-4 carrier protein (data not shown). The assignments obtained were consistent with those published in the literature (Jansson, PE et al., 1984, Carbohydr. Res. 134 (2): 283-291; Jann B et al., 1993, Carbohydr. Res. 248: 241-250). The ID spectrum recorded at 313 K showed a large HOD signal and a small sharp signal from the O4 pentasaccharide RU, with five anomeric signals, two NAc signals, and two H6 signals (Rha and FucNAc).

[0364] 1D proton distribution was confirmed by using 2D proton-proton and proton-carbon related NMR experiments. First, 2D TOCSY (120 ms) experiments confirmed the expected cross peaks from H1 and H6 (for Rha and FucNAc) about O4 pentasaccharide RU, and the small peak from terminal RU and EPA. In the methyl region, TOCSY showed a cross peak from H6 to H1 and from H6 to H5 for α-Rha and for α-FucNAc about O4 RU. Other peaks observed were from EPA amino acids and terminal Rha (tRha). Secondly, carbon NMR spectra contained good dispersion and diagnostic singlets about O4 RU. By using HSQC experiments, carbon was indirectly profiled via the proton attached. HSQC-DEPT experiments provided the inversion peak of CH2 group. HSQC gives cross peaks for the pentasaccharide RU (five anomeric, ring, two N-acetyl and two methyl (Rha and FucNAc)) groups and the EPA amino acid in characteristic regions. Each proton / carbon pair for O4 can be assigned based on proton assignments and literature.

[0365] Therefore, the structural characterization experiments confirmed that the putative E. coli O4- gtrS Genes that can produce Glc-branched O4 bioconjugates (comprising a polysaccharide antigen structure as shown by the formula (O4-Glc+) in Table 1).

[0366] Example 4 : Immunogenicity of Glc-branched O4 bioconjugates in rabbits

[0367] Glc-modified O4 bioconjugates (i.e., polysaccharides having a structure containing the formula (O4-Glc+) as shown in Table 1) were used for rabbit immunization using the Rapid Rabbit Protocol (Eurogentec). Sera from immunized rabbits were analyzed by ELISA for anti-O4 IgG titers against purified O4 lipopolysaccharide (LPS) with (Glc+; i.e., containing glucosylated O4 polysaccharides) or without Glc branches (Glc-; i.e., containing non-glucosylated O4 polysaccharides). Immunization with the bioconjugate resulted in high IgG titers in both rabbits ( Figure 1 In both cases, antibody titers induced by the O4 bioconjugate were higher against Glc+ LPS compared to Glc-LPS.

[0368] The serum was also pooled and characterized using gtrS A test set of E. coli O4 isolates from the state of the disease was used in whole cell ELISA studies. Five strains were tested gtrS Negative (no Glc branch) and six gtrS Positive (Glc branch) E. coli O4 isolates, as well as negative control strains. Pooled sera from rabbits immunized with Glc-modified O4 bioconjugates contained high titers of IgG that specifically recognized the tested O4 isolates ( Figure 2 Consistent with the LPS ELISA, all O4 isolates tested were recognized by the immune sera. gtrS Positive isolates showed gtrS Negative isolates had overall higher binding ( Figure 2 ). In particular, the following isolates were gtrS Positive: Y1382, E551, OC24334, stGVXN4983, stGVXN4994, and OC24794, and the following isolates were gtrS Negative: A2625, stGVXN4988, OC24784, OC24787 and OC24788. The immune sera did not bind to the negative control strain of the irrelevant serotype O E. coli OC9487 (ATCC 35383).

[0369] From silver-stained polyacrylamide gel gtrS Extracted LPS profiles from a test set of positive and negative isolates did not reveal significant differences between isolates expressing the unmodified and modified forms of the O4 antigen, confirming that the observed differences were not explained by quantitative differences in LPS expression levels (data not shown).

[0370] Western blotting of extracted LPS using pooled immune sera was performed to evaluate recognition of the O4 O-antigen by IgG elicited in response to immunization with Glc-modified O4 bioconjugates. Binding of IgG from rabbits immunized with modified O4 to both modified and unmodified O4 LPS was observed, including specific recognition of LPS bands across a wide range of sizes, including high molecular weight LPS bands ( Figure 3 ).

[0371] In the further experiments below, when reference is made to an 'O4' bioconjugate or production strain or 'EcoO4', this refers to a bioconjugate or production strain of Glc-branched O4 having the glycan structure (O4-Glc+) in Table 1, unless explicitly stated otherwise (the terms 'O4' and 'O4-Glc+' are therefore used interchangeably for the bioconjugates or production strains in these experiments).

[0372] Example 5 : Immunogenicity of Glc-branched O4 bioconjugates in rats

[0373] Sprague Dawley rats were immunized intramuscularly three times with formulation buffer or (O4-Glc+)-EPA bioconjugate (i.e., a bioconjugate of glucosylated O4 antigen polysaccharide covalently coupled to an EPA carrier protein; the carrier protein was EPA-2 as described above in Example 3) at three different doses (0.04 μg, 0.40 μg, or 4.0 μg). Serum antibody levels were measured by ELISA on days 0, 14, and 42 after immunization.

[0374] Immunization with 0.04 μg, 0.40 μg, and 4.00 μg (O4-Glc+)-EPA bioconjugate induced a significant increase in IgG antibody levels on day 42 after immunization when compared to formulation buffer ( Figure 4A The antibodies induced by the (O4-Glc+)-conjugate were functional, i.e., able to mediate the killing of the (O4-Glc+) E. coli strain ( Figure 4B ).

[0375] Antibody levels induced by 0.04 μg, 0.40 μg, and 4.0 μg (O4-Glc+)-EPA bioconjugates were significantly increased on day 42 compared to those detected at baseline (day 42 vs. day 0, P = 0.006 for all doses) and day 14 post-immunization (day 42 vs. day 14, P = 0.006 for all doses). Figure 5In the group receiving 4.0 µg of the bioconjugate, titers also increased significantly on day 14 compared to day 0, indicating that a single dose of 4.0 µg of (O4-Glc+)-EPA bioconjugate induced a significant increase in IgG titers (day 14 vs. day 0, P = 0.012). The significant increase in IgG titers observed between day 14 and day 42 for all three concentrations of the bioconjugate tested suggests that a third dose of the (O4-Glc+)-EPA bioconjugate was able to boost the antibody response ( Figure 5 ).

[0376] The functionality of antibodies induced by the O4-Glc+-EPA conjugate was determined by opsonophagocytic killing assay (OPKA) using O4 (Glu+) and O4 (Glu-) E. coli strains in rats immunized three times intramuscularly with formulation buffer or the bioconjugate at 4.00 μg / dose. The antibodies induced by the (O4-Glc+)-EPA bioconjugate were functional, i.e., able to mediate killing of the (O4-Glc+) E. coli strain ( Figure 4B , Figure 6 Notably, antibodies induced by the (O4-Glc+)-EPA bioconjugate were able to mediate the killing of both (O4-Glc+) and (O4-Glc-) (i.e., polysaccharides having the structure of formula (O4-Glc-) in Table 1, i.e., O4 polysaccharides without Glc branches) E. coli strains ( Figure 6 ).

[0377] In conclusion, antibodies induced by the O4-Glc+-EPA bioconjugate were cross-reactive and able to mediate killing of E. coli O4 strains with and without glucose branches.

[0378] Example 6 :Production strains for Escherichia coli O-antigen bioconjugates and the resulting bioconjugate products

[0379] In addition to the (04-Glc+)-EPA bioconjugate prepared as described above, nine (9) other bioconjugates were produced. In particular, the additional bioconjugates produced included E. coli 01A-EPA bioconjugate, 02-EPA bioconjugate, 06A-EPA bioconjugate, 08-EPA bioconjugate, 015-EPA bioconjugate, 016-EPA bioconjugate, 018A-EPA bioconjugate, 025B-EPA bioconjugate, and 075-EPA bioconjugate. The chemical structures of the glycans of these conjugates are found in the respective formulas in Table 1. The composition comprising the 10 bioconjugates is referred to herein as 'ExPEC10V'. The composition comprising the 01A-EPA, 02-EPA, 06A-EPA, and 025B-EPA bioconjugates is referred to as 'ExPEC4V' (and previously described, for example, in WO 2015 / 124769 and WO 2017 / 035181).

[0380] Escherichia coli W3110 parent strain

[0381] The non-pathogenic Escherichia coli K12 strain W3110 is used as the parent strain for making up all ten production strains. Escherichia coli K12 strain W3110 derives from Escherichia coli genetic preservation center (Coli Genetic Stock Center) (Yale University, New Haven (CT), USA, product number CGSC#4474). Its relevant genotype has previously been described (Escherichia coli W3110, F-, λ-, IN (rrnD-rrnE) 1, rph-1), and its genomic sequence has previously been disclosed (people such as Hayashi K, 2006, Mol. Syst. Biol. 2006.0007 (doi:10.1038 / msb4100049). Escherichia coli W3110 bacterial strain is genetically modified to allow production of every kind of Escherichia coli O-antigen bioconjugate (table 3).

[0382] Bioconjugate production strains

[0383] Both "ExPEC4V" and "ExPEC10V" compositions contain the O2-EPA and O25B-EPA bioconjugates from the same production strain. The "ExPEC4V" composition contains the O1A-EPA bioconjugate from either the stGVXN4411 or stLMTB10217 production strain, while the "ExPEC10V" composition contains the O1A-EPA bioconjugate from the stLMTB10217 production strain. The "ExPEC4V" composition contains the O6A-EPA bioconjugate from the stGVXN4112 production strain, while the "ExPEC10V" composition contains the O6A-EPA bioconjugate from the stLMTB10923 production strain. In addition, the "ExPEC10V" composition contains 04-EPA (i.e., (04-Glc+)-EPA), 08-EPA, 015-EPA, 016-EPA, 018A-EPA, and 075-EPA bioconjugates from production strains that were not used in "ExPEC4V." As shown below, different production strains may differ in the plasmids used to express the EPA carrier protein and / or the oligosaccharyltransferase PglB. An overview of several production strains is given in Table 3 below.

[0384] Table 3 Overview of genetic engineering of Escherichia coli production strains for O-antigen bioconjugates of ExPEC4V and ExPEC10V vaccine compositions

[0385]

[0386] O-antigen biosynthesis ( rfb ) gene cluster

[0387] Among all E. coli O-antigen producing strains, the naturally occurring E. coli W3110 genome O16::IS5-antigen biosynthesis ( rfb ) gene clusters were replaced with selected O-antigen-specific biosynthetic clusters from selected serotypes of E. coli strains encoding serotype-specific O-antigen structures (see Table 1 for these O-antigen structures). Ten donors were selected or confirmed after whole genome analysis of E. coli blood isolates. rfb Cluster. W3110 O16::IS5 defective in O-antigen biosynthesis rfb The replacement of the gene cluster has been achieved in a single homologous recombination event. rfb In the case of gene clusters, donor DNA is flanked by gnd and rmlCA Genes are recombined, while other strains rfb Gene clusters are flanked by gnd and galF Genes are recombined. rfb The sequences of the clusters are provided in SEQ ID NOs: 9 and 11-19.

[0388] O-antigen ligase ( waL )Gene

[0389] All E. coli O-antigen producing strains carry artificially introduced waL The gene encoding the O-antigen ligase is deleted in the Escherichia coli W3110 genome. strains in which the transfer of the O-antigen to lipid A is disrupted, which instead directs the transfer of the O-antigen to the carrier protein to increase product yield.

[0390] O-antigen glucosylation ( gtrABS )Gene

[0391] The E. coli W3110 genome responsible for glucosylation of O16 O-antigen in E. coli O8, O15, O16, O18A, O25B, and O75 production strains gtrABS The gene has been deleted. Although the gtrA and gtrB Genes are highly homologous and interchangeable, but gtrS This gene encodes a serotype-specific O-antigen glycosyltransferase. In E. coli W3110, GtrS can transfer a glucose (Glc) residue to the GlcNAc sugar in the α-l-Rha-(1→3)-d-GlcNAc motif of the E. coli O16 O-antigen. This transfer does not occur in E. coli O1A, O2, and O6A producing strains. gtrABS These O-antigens lack the α-l-Rha-(1→3)-d-GlcNAc motif, which is the gtrS Among the E. coli O4 production strains, W3110 gtrS The gene has been replaced with E. coli O4 gtrS Genes for proper glucosylation of E. coli O4 O-antigen.

[0392] Oligosaccharyltransferase PglB

[0393] All E. coli O-antigen producing strains express a variant of the Campylobacter jejuni glycosyltransferase PglB, which can be N -Glycosylation transfers the O-antigen to an amino acid consensus sequence on the carrier protein. PglB has broad substrate recognition, but due to low product yields, several production strains expressing PglB variants with modified substrate specificity have been prepared, resulting in improved product yields (see, for example, WO 2016 / 107818, WO 2016 / 107819). pglBThe gene was placed behind an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible promoter on the plasmid. Table 4 below lists the PglB variants encoded by the plasmids used to produce the E. coli O-antigen production strains used for bioconjugates of the ExPEC4V and ExPEC10V compositions described above. Further plasmids with variations in the vector backbone, antibiotic resistance markers, and / or alternative PglB variants have also been successfully tested for bioconjugate production.

[0394] Table 4 :PglB and EPA plasmids used in Escherichia coli O-antigen production strains

[0395] Plasmid name Gene <![CDATA[Description 1 > pGVXN114 Campylobacter jejuni codon usage; SpR pGVXN970 Optimized E. coli codon usage; SpR pGVXN971 <![CDATA[ pglB N534Q ]]> Optimized E. coli codon usage; the native glycosylation site of PglB is inactivated; SpR pGVXN1217 <![CDATA[ pglB N311V ]]> Optimized E. coli codon usage; substrate-optimized PglB; SpR pGVXN1221 <![CDATA[ pglB N311V,K482R,D483H,A669V ]]> Optimized E. coli codon usage; substrate-optimized PglB; SpR pGVXN2381 <![CDATA[ pglB Y77H,S80R,Q287P,K289R,N311V ]]> Optimized E. coli codon usage; substrate-optimized PglB; SpR pGVXN659 EPA-4 EPA with four bioconjugation sites; AmpR pGVXN1076 EPA-4 EPA with four bioconjugation sites; KanR

[0396] 1 SpR, spectinomycin resistance; AmpR, ampicillin resistance; KanR, kanamycin resistance

[0397] Carrier protein (EPA)

[0398] All E. coli O-antigen production strains express a genetically detoxified Pseudomonas aeruginosa ADP-ribosyltransferase toxin (EPA) as a carrier protein for the O-antigen. The EPA toxin differs from the wild-type EPA toxin by two residues: Leu552 is changed to Val and Glu553 (in the catalytic domain) is deleted. The deletion of Glu553 is reported to significantly reduce toxicity. In addition to the detoxification mutations, four (EPA-4) consensus N -glycosylation site motif. epa The genes were placed behind an L-arabinose (Ara) inducible promoter on the plasmid (Table 4). Table 4 is limited to the plasmids used in the production strains of the bioconjugates used in the "ExPEC4V" and "ExPEC10V" compositions described above. Variations in vector backbone, antibiotic resistance markers, and / or EPA variants were observed, e.g., in the consensus N Plasmids differing in the number of glycosylation site motifs (eg, with two such motifs, EPA-2) have also been successfully tested for bioconjugate production.

[0399] Example 7 Optimization of oligosaccharyltransferases for the generation of bioconjugates with glucosylated O4 (O4-Glc+) antigens

[0400] Yield optimization with respect to bioconjugate production can be achieved by modification of the Campylobacter jejuni oligosaccharyltransferase PglB, which may result in more efficient or higher degree of O-antigen conversion to the EPA carrier protein. NSuch an optimization strategy was applied in an E. coli strain used to produce bioconjugates with glucosylated O4 (O4-Glc+) O-antigen polysaccharides and resulted in PglB variants optimized for (O4-Glc+) specificity, improving bioconjugate product yields.

[0401] In this method, an O4-Glc+ O-antigen polysaccharide production strain containing an EPA expression plasmid was transformed with various PglB expression plasmids, each containing a different amino acid substitution in the PglB protein that altered substrate specificity. The bioconjugate production level and profile of each strain were evaluated at the shake flask level in an osmotic shock experiment, and readout was performed on periplasmic extracts by capillary electrophoresis immunoassay using an O4-Glc+-specific monoclonal antibody.

[0402] One of the tested PglB variants containing the N311V amino acid substitution was found to significantly improve the product yield of the glucosylated O4 bioconjugate ( Figure 7 A).

[0403] In a further improvement where the N311V PglB-variant was further modified, the Y77H amino acid substitution further enhanced the O4-Glc+-specific product yield and showed an increased degree of di- and tri-glycosylated products compared to the N311V PglB variant, where the other modifications were found to be neutral or had a negative effect on product yield ( Figure 7 B) Plasmid pLMTB4008 (SpR) encodes an E. coli codon usage-optimized, (O4-Glc+)-substrate-optimized PglB variant with mutations Y77H and N311V.

[0404] A PglB variant with optimized substrate specificity for the O4-Glc+ O-antigen polysaccharide, containing N311V and Y77H amino acid substitutions relative to wild-type (wt) Campylobacter jejuni glycosyltransferase PglB, was found to double the bioconjugate yield compared to the PglB-N311V variant from the first round of optimization.

[0405] Similarly, using screening, the best-yielding PglB variants were also identified for production of E. coli O-antigen bioconjugates for the other nine serotypes in the ExPEC10V composition.

[0406] For bioconjugates with O1A, O6A or O15 antigenic polysaccharides, PglB with amino acid mutations N311V, K482R, D483H and A669V was found to give the highest yield.

[0407] For bioconjugates with 02, 08, 018A or 025B antigenic polysaccharides, wild-type PglB (ie, without amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669) was found to give the highest yield.

[0408] For the bioconjugate with the O16 antigen polysaccharide, PglB with the amino acid mutations Y77H, S80R, Q287P, K289R and N311V was found to give the highest yield.

[0409] For the bioconjugate with the O75 antigen polysaccharide, PglB with the amino acid mutation N311V was found to give the highest yield.

[0410] From these results it can be seen that the optimal PglB variant is different for different O-antigens and that the optimal PglB variant for producing a bioconjugate with a given O-antigen polysaccharide is unpredictable.

[0411] Example 8 Bioconjugates of O-antigens from 10 Escherichia coli serotypes and their quality attributes

[0412] The O-glycan residues of target O-antigens are structurally diverse and have variable repeat units. The specificity and affinity of the glycosyltransferase PglB are linked to the glycan structure. Therefore, preparing bioconjugates with desired quality attributes (e.g., purity, glycan / protein ratio, etc.) is a challenging and non-trivial task. The correct combination of PglB and EPA carrier protein determines the yield and may affect the glycosylation efficiency. By optimizing PglB and carrier protein, bioconjugates with the desired quality attributes were produced. Maintaining a low threshold of total carrier protein may also be important, particularly when one or more O-antigen bioconjugates are combined and administered in a single composition or vaccine, as very large amounts of carrier protein may cause immune interference. To avoid such phenomena, conjugates with higher glycan / protein ratios are preferred. Therefore, for the ExPEC10V vaccine, a bioconjugate with a glycosylation ratio at least comparable to that of the previously described ExPEC4V vaccine that has been clinically tested was developed.

[0413] Each bioconjugate was produced by culturing the respective host cells (Example 6, Table 3) in a bioreactor (10 L and / or 200 L volume) according to previously described methods and expressing the bioconjugate. Each drug substance was manufactured batchwise by bacterial fed-batch fermentation to generate a biomass of the expressed bioconjugate containing the corresponding polysaccharide serotype. The cells were cultured and induced with IPTG and arabinose. The bioconjugate was isolated from the periplasm of the bioreactor culture by osmotic shock followed by chromatographic purification. This process was performed for each of the 10 bioconjugates.

[0414] The E. coli O-antigen bioconjugates thus prepared, which were the drug substances (DS) for ExPEC10V and ExPEC4V, showed comparable critical quality attributes: (1) process-related purity (measured by RP-HPLC) greater than 95%, (2) polysaccharide / protein ratios ranging from approximately 0.1 to 0.5, with a majority ranging from 0.15 to 0.45, and (3) bacterial endotoxins (European Pharmacopoeia (Ph. Eur.) 2.2.3) less than 0.5 EU / μg polysaccharide. The average length of the individual polysaccharide chains was typically about 10-20 repeat units (measured using high-resolution SDS-PAGE).

[0415] For all ten bioconjugates that were DS of the ExPEC10V compositions described above, the structures of the polysaccharide repeating units were confirmed (by NMR and MS / MS of intact or trypsin-digested conjugates) to be those shown in the formulas for the corresponding serotypes in Table 1 .

[0416] Of the ten serotypes for which bioconjugates were prepared for the ExPEC10V composition, serotype O18 had the lowest bioconjugate production yield.

[0417] The ExPEC10V drug product (DP) comprises a mixture of the ten monovalent DSs described above.

[0418] Example 9 :Toxicology of ExPEC10V vaccine

[0419] A single-dose pilot toxicity and local tolerance study (non-GLP) with ExPEC10V was conducted in female NZW rabbits. One group (n=2) received an intramuscular (IM) injection of a control (saline) on day 0, while a second group (n=4) received an IM injection of ExPEC10V at 105.6 µg total polysaccharide (PS) / dose (9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 9.6: 19.2: 9.6 µg PS / dose for O-serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively) using a dosing volume of 0.6 mL (176 µg PS / mL). Necropsy was performed on day 2.

[0420] No mortality was observed. In addition, no vaccine-related effects were noted for clinical observations (including injection site effects using Draize scoring), body weight, food consumption, and body temperature. Histopathologically, no vaccine-related changes were observed at the site of administration or at the draining (iliac) lymph nodes. A minimal increase in the germinal center formation of the spleen was observed in one of the four treated animals (day 2) and was considered a normal immune response to the injected vaccine. Overall, the administration of a single IM dose of ExPEC10V to female rabbits was well tolerated.

[0421] Example 10 : Immunogenicity of ExPEC10V blend formulation in rabbits

[0422] The ExPEC4V vaccine (a bioconjugate containing E. coli serotypes O1A, O2, O6A, and O25B) has previously been shown to be immunogenic in rats, rabbits, and humans (see, e.g., WO 2015 / 124769; WO 2017 / 035181; Huttner et al., 2017, Lancet Infect Dis, http: / / dx.doi.org / 10.1016 / S1473-3099(17)30108-1; RW Frenck Jr, et al., abstract 5587, ASM Microbe 2018). The novel bioconjugates of the present invention containing glucosylated E. coli serotype O4 were shown to be immunogenic in Examples 4 and 5 above. The immunogenicity of the bioconjugates of E. coli serotypes O8, O15, O16, O18A, and O75 (all with EPA-2 as a carrier protein in this experiment) when administered separately (monovalently) to rats confirmed that each of these bioconjugates was also immunogenic, as ELISA data indicated that each of these bioconjugates could elicit high levels of E. coli O-antigen-specific antibodies (not shown).

[0423] The immunogenicity of a 10-valent vaccine containing a mixture of the 10 bioconjugates described above was also tested. New Zealand White (NZW) rabbits (female, 12-16 weeks old) received three intramuscular immunizations with ExPEC10V or saline administered 2 weeks apart (Table 5; administered on days 0, 14, and 27). The 10 polysaccharides that were part of the ExPEC10V vaccine used in these experiments were conjugated to the carrier protein EPA (EPA-4), which contains four glycosylation sites. The vaccine was formulated at three different doses: Group 1 ('high dose'): 8 ug / dose of O1A, O2, O6A, O4, O8, O15, O16, O18, and O75, and 16 ug / dose of O25B; Group 2 ('medium dose'): 4 ug / dose of O2, O4, O8, O15, O16, O18, and O75, 8 ug / dose of O1A and O6A, and 16 ug / dose of O25B; Group 3 ('low dose'): 0.4 ug / dose of O2, O4, O8, O15, O16, O18, and O75, 0.8 ug / dose of O1A and O6A, and 1.6 ug / dose of O25B. Animals from the control group (Group 4) received only saline (0.9% (w / v) sodium chloride solution) (Table 5).

[0424] Antibody responses were assessed on day 0 (before immunization) and on days 14, 27, and 42 after immunization. Serum antibody levels induced by each bioconjugate and carrier protein EPA included in the vaccine were measured by ELISA (total IgG) using type-specific LPS as a coating material. Antibody titers were reported as EC50 values, which correspond to the half-maximal effective concentration based on replicates of a 12-step titration curve drawn in a 4-parameter logistic nonlinear regression model. Functional activity was determined by OPK.

[0425] Table 5. Description of experimental groups.

[0426]

[0427] The results are shown in Figure 8 and are summarized in Table 6.

[0428] Table 6. Summary of E. coli O-antigen-specific antibody responses induced by ExPEC10V in NZW rabbits.

[0429]

[0430] Dark grey squares show serotype-specific antibody responses where the p-value was statistically significant. Light grey squares show serotype-specific antibody responses where the p-value was not statistically significant (ns). Wilcoxon rank sum test with Bonferroni correction for multiple comparisons. Comparison of ExPEC10V vaccinated animals (Groups 1, 2, and 3) versus saline controls (Group 4). *p≤0.05, **p≤0.01. # P values ​​were statistically significant after excluding outlier animals from the control group (sensitivity analysis).

[0431] For O1A, O2, O4, O6A, O16, O18A, and O25B, the high dose of ExPEC10V (Group 1) induced significantly higher IgG antibody levels at all time points investigated (days 14, 27, and 42 post-immunization) when compared to saline controls. Figure 8 , Table 6). Significantly higher antibody titers induced by the O8 and O75 conjugates when compared to saline controls were observed on days 27 and 42 after immunization ( Figure 8 , Table 6).

[0432] For O1A, O2, O4, O6A, O16, and O25B, the medium (Group 2) and low (Group 3) doses of ExPEC10V induced significantly higher antibody levels at all time points investigated (days 14, 27, and 42 post-immunization) when compared to saline controls ( Figure 8 , Table 6). Significantly higher antibody titers induced by the O8, O18A, and O75 conjugates when compared to saline controls were observed on days 27 and 42 post-immunization, suggesting that boosting doses in rabbits increased responses to these O serotypes ( Figure 8 , Table 6).

[0433] For the O15 conjugate, sensitivity analysis omitting outlier animals from the control group showed that all three doses of ExPEC10V vaccine induced significant increases in antibody responses when compared to saline controls at days 14, 27, and 42 post-immunization ( Figure 8 , Table 6).

[0434] At all time points investigated (days 14, 27, and 42), antibodies induced by the carrier protein EPA were significantly higher than EPA antibody titers in the saline-treated (control) group for the three doses of ExPEC10V tested (high, medium, and low). Figure 8 ).

[0435] Comparisons between doses (not shown) showed that at day 14 post-vaccination, for most of the tested conjugates (O1A, O2, O4, O6A, O15, O16, O18A, and O25B), high doses of ExPEC10V induced significantly higher antibody responses when compared to low doses. For O1A, O2, O4, O18A, O25B, and O75, medium doses of ExPEC10V also induced significantly higher antibody responses compared to low doses. For the O8 conjugate, all three formulations of ExPEC10V induced similar antibody levels at day 14 post-vaccination.

[0436] For the O1A, O2, O4, O16, O25B, and O75 conjugates, low doses of ExPEC10V induced a significant increase in antibody responses at day 42 post-vaccination (after priming and two booster doses) when compared to high and medium doses of ExPEC10V. These findings are consistent with other experience with conjugate vaccines, where, for example, no clear relationship between dose and magnitude of antibody response to primary vaccination was observed in infants vaccinated with pneumococcal conjugate vaccines (Poolman JT et al. Expert Rev Vaccines. 2013, 12(12):1379-94).

[0437] For the O6A, O8, and O15 conjugates, there were no significant differences between the three doses of ExPEC10V tested at day 42 post-vaccination. For the O18A conjugate, the high dose of ExPEC10V induced significantly higher antibody responses when compared to the medium dose at day 42 post-vaccination.

[0438] For the carrier protein (EPA), high and medium doses of ExPEC10V induced significantly higher antibody responses when compared to the low dose at day 14 post-vaccination. The high dose of the vaccine also induced significantly higher antibody responses when compared to the low dose at day 42 post-vaccination.

[0439] In conclusion, three formulations of ExPEC10V (high, medium, and low) were immunogenic in rabbits when administered via intramuscular injection on days 0, 14, and 27.

[0440] To date, functional antibodies induced by this vaccine in rabbits capable of killing E. coli strains have been shown for serotypes O1A, O2, O4, O6A, O15, O16 and O25B.

[0441] In further experiments, as part of a toxicology study, a GMP batch of the ExPEC10V vaccine was prepared (for production, see Example 8 above) and injected into NZW rabbits (Table 7). In this study, NZW rabbits (male and female) received three intramuscular injections (0.6 mL) of the ExPEC10V vaccine (days 1, 15, and 29), and a control group received a 0.9% (w / v) sodium chloride solution (saline). Each dose of the vaccine contained 9.6 μg polysaccharide (PS) of serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, and O75, and 19.2 μg PS of serotype O25B, corresponding to 105.6 μg total PS (176 μg total PS / mL) and 382.8 μg total EPA (638 μg EPA / mL). IgG titers against O-antigen and carrier protein (EPA) were determined from samples collected before the treatment period (day 1) and on days 31 and 50 after immunization.

[0442] When compared to the control group that received saline only, a significant increase in antibody responses against all O-antigens and the carrier protein EPA was observed at days 31 and 50 after vaccination in the group that received ExPEC10V ( Figure 9 , Table 8). For the O1A serotype, significantly higher antibody responses were also observed on day 1 (baseline) when vaccinated animals were compared to the control group. These results suggest that some animals were pre-exposed to E. coli or had antibodies that cross-reacted with O1A-LPS.

[0443] Table 7. Experimental groups and ExPEC10V doses used in NZW rabbits.

[0444]

[0445] *Each dose (0.6 mL dosing volume) contains 9.6:9.6:9.6:9.6:9.6:9.6:9.6:9.6:9.6:19.2:9.6 μg polysaccharide (PS) (176 μg total PS / mL) for serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively. Each dose contains 382.8 μg EPA protein (638 μg EPA / mL).

[0446] Table 8. Immunogenicity of ExPEC10V in NZW rabbits as part of toxicology studies.

[0447]

[0448] Antibody responses induced by ExPEC10V. Light grey squares indicate serotypes for which a significant increase in antibody responses was observed in the vaccine group compared to the control. Tobit model with likelihood ratio test. ****P ≤ 0.0001.

[0449] Example 11 : Phase 1 / 2a trial of the ExPEC10V vaccine in humans

[0450] Currently, there is no vaccine available to prevent IED. The serotypes that comprise the ExPEC10V vaccine (O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75) were selected to address invasive disease caused by the most clinically relevant ExPEC strains, which also represent the majority of ExPEC isolates that cause antimicrobial-resistant IED, including ST131. The selected serotypes represent the 10 prevalent ExPEC O-serotypes that cause bloodstream infections in the elderly population and are responsible for approximately 70% of bloodstream infections caused by ExPEC.

[0451] Because the mechanism of action of the conjugate vaccine in the prevention of invasive disease is not expected to be affected by antibiotic resistance mechanisms, the ExPEC10V vaccine is considered to provide protection against IED caused by both drug-resistant and drug-susceptible O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75 serotypes.

[0452] There is prior clinical experience with ExPEC4V, an earlier vaccine candidate, which contains a subset of the four E. coli O-antigen conjugates (O1A, O2, O6A, and O25B) also found in ExPEC10V. Based on results from four clinical studies (two completed Phase 1 studies, one completed Phase 2 study, and one ongoing Phase 2 study), ExPEC4V was well tolerated by study participants, and no vaccine-related safety signals were observed at doses up to 16 μg polysaccharide (PS) / serotype (O1A, O2, O6A, and O25B). Most adverse events (AEs) were Grade 1 and 2, with very few Grade 3 AEs reported. Late-onset solicited local AEs (AEs that started after Day 5 post-vaccination) were observed, primarily with higher doses of ExPEC4V. In each study, the ExPEC4V vaccine was shown to be immunogenic, demonstrating dose-dependent vaccine immune responses and increases in O-antigen-specific immunoglobulin G (IgG) titers as measured by enzyme-linked immunosorbent assay (ELISA). Functional activity of the antibodies was confirmed using the ExPEC4V optimized opsonophagocytic killing assay (OPKA). Co-analysis of ELISA and OPKA test results showed correlation between assay responses (Pearson correlation coefficients ≥0.61 and ≥0.48 for days 30 and 360, respectively, in the Phase 2 clinical trial [Study 4V-BAC2001]), confirming the use of ELISA as the primary measure of ExPEC4V antibody titers and predicting functional antibody activity. Analysis of immunogenicity data has confirmed the durability of immune responses up to three years after vaccination with ExPEC4V. It has also been observed that sera from humans vaccinated with ExPEC4V and having high titers of serotype-specific opsonophagocytic antibodies were able to mediate in vivo protection when passively transferred into mice that were subsequently challenged intraperitoneally with E. coli strains of serotypes O25B or O2 (not shown). Thus, ExPEC4V-specific opsonophagocytic human antibodies mediated bacterial killing in vivo, which is consistent with other conjugate vaccines where the proposed mechanism of protection is through the induction of opsonophagocytic antibodies that mediate bacterial killing.

[0453] ExPEC10V includes a total of ten serotypes and increases the coverage of bloodstream infections caused by ExPEC in adults over 60 years old from approximately 50% (ExPEC4V) to approximately 70%. Based on clinical experience with ExPEC4V, as well as preclinical data for ExPEC10V as discussed in the Examples above, it is expected that administration of ExPEC10V will also induce immune responses in humans against E. coli serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75.

[0454] A randomized, observer-blinded, first-in-human Phase 1 / 2a study was conducted in healthy individuals aged 60 to 85 years to evaluate the safety, reactogenicity, and immunogenicity of three different doses of the ExPEC10V vaccine (Study 10V-BAC1001). The study design included two cohorts: a total of 1,004 participants were enrolled in the study, including 404 participants (100 participants / ExPEC10V dose) in healthy individuals aged ≥60 to ≤85 years in Cohort 1, and an additional 600 participants in healthy individuals aged ≥60 years with a history of UTI within the past 5 years in Cohort 2.

[0455] ExPEC10V is a 10-valent vaccine candidate in development for the prevention of invasive extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED) in adults over 60 years of age. ExPEC10V consists of O-antigen polysaccharides (PS) of ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively, bioconjugated to a carrier protein, which is a genetically detoxified form of exotoxin A (EPA) derived from Pseudomonas aeruginosa, the production of which has been described above. The O4 PS is a glucosylated form with the structure (O4-Glc+) in Table 1.

[0456] Goals and End Points

[0457] Cohort 1 - Phase 1 / 2a observer-blind period, followed by open-label, long-term follow-up (N=404):

[0458]

[0459]

[0460] Cohort 2 - Double-blind period followed by double-blind long-term follow-up (N=600):

[0461]

[0462]

[0463] Overall design

[0464] This was a randomized, multicenter, interventional study involving two cohorts.

[0465] For Cohort 1, the study had an observer-blind, active-controlled design and included a total of 404 adult participants aged ≥60 to ≤85 years, in stable health, with or without a history of UTI. The study design for Cohort 1 consisted of three periods: a screening period of up to 28 days, a 181-day follow-up period with observer-blind vaccination on Day 1, and an open-label LTFU period from Day 182 after vaccination until 3 years (Day 1096) ( Figure 10A Only participants from the selected dose groups of ExPEC10V (approximately 100 participants) and those from the Prevnar 13 group progressed to LTFU. The end of Cohort 1 was the last participant's 3-year visit (Day 1096).

[0466] For Cohort 2, the study had a double-blind, placebo-controlled design and included a total of 600 adult participants aged ≥60 years, in stable health, with a history of UTI within the past 5 years. Enrollment began after completion of the Phase 1 / 2a primary analysis and ExPEC10V dose selection from Cohort 1. The study design for Cohort 2 consisted of three periods: a screening period of up to 28 days, a double-blind 181-day follow-up period with vaccination on Day 1, and a double-blind LTFU period from Day 182 after vaccination until 3 years (Day 1096). Figure 10B All participants in Cohort 2 progressed to LTFU. The end of the study was the 3-year visit (Day 1096) for the last participant in Cohort 2.

[0467] Cohort 1: 1 period

[0468] In Phase 1 of Cohort 1, a total of 84 participants were enrolled in a staggered approach following a stepwise dose escalation schedule, with appropriate safety assessments before progression from one step to the next. The study commissioned an internal Data Review Committee (DRC) to review physical examination data (baseline and targeted), baseline demographics, and safety data (including requested local and systemic AEs, unrequested AEs, SAEs, clinical laboratory data, and vital signs) for these 84 Phase 1 participants. During this period of the study, participants were enrolled and randomized in six steps:

[0469] Step 1: Four sentinel participants were enrolled and randomized; two participants in the ExPEC10V low-dose group (Table 11), and one participant each in the ExPEC4V and Prevnar 13 groups.

[0470] Step 2: 24 participants were enrolled and randomized; 18 participants in the ExPEC10V low-dose group (Table 11), and 3 participants each in the ExPEC4V and Prevnar 13 groups.

[0471] Step 3: Four sentinel participants were enrolled and randomized; two participants in the ExPEC10V medium-dose group (Table 11), and one participant each in the ExPEC4V and Prevnar 13 groups.

[0472] Step 4: 24 participants were enrolled and randomized; 18 participants in the ExPEC10V medium-dose group (Table 11), and 3 participants each in the ExPEC4V and Prevnar 13 groups.

[0473] Step 5: Four sentinel participants were enrolled and randomized; two participants in the ExPEC10V high-dose group (Table 11), and one participant each in the ExPEC4V and Prevnar 13 groups.

[0474] Step 6: 24 participants were enrolled and randomized; 18 participants in the ExPEC10V high-dose group (Table 11), and 3 participants each in the ExPEC4V and Prevnar 13 groups.

[0475] On Day 1, all participants received a single intramuscular (IM) injection of ExPEC10V (1 of 3 doses), ExPEC4V, or Prevnar 13, according to the assigned study vaccination group. Four sentinel participants in each of Steps 1, 3, and 5 were contacted by telephone 24 hours after vaccination to collect safety information. Blinded 24-hour post-vaccination safety data for each group of four sentinel participants were reviewed by the Principal Investigator (PI), Study Physician (SRP), and Sponsor Medical Manager (SML). Randomization of additional participants to the next step was halted until completion of this Day 2 sentinel safety assessment.

[0476] In the absence of any clinically significant findings, an additional 24 participants were enrolled (for steps 2, 4, and 6) and randomized to one of three study vaccination groups (Table 11) to receive a single IM injection of ExPEC10V (1 of 3 doses), ExPEC4V, or Prevnar 13 on Day 1.

[0477] Following vaccination of an additional 24 participants at each dose level (low dose in step 2, medium dose in step 4, and high dose in step 6), safety data 14 days post-vaccination were reviewed by the DRC for all 28 (4+24) participants at each dose level before progression to the next dose level or Phase 2a.

[0478] Cohort 1: Phase 2a

[0479] Based on acceptable safety and reactogenicity (in the absence of any safety concerns or any events meeting specific study hold rules), as determined by the DRC after review of safety data 14 days after vaccination for the initial 84 participants, the remaining 320 participants from Cohort 1 were randomized and dosed in Phase 2a of the study. These additional 320 participants were enrolled and randomized in parallel in a 2:2:2:1:1 ratio to one of five study vaccination groups to receive a single IM injection of ExPEC10V (1 of 3 doses), ExPEC4V, or Prevnar 13 on Day 1 (Table 11).

[0480] In addition to performing the 14-day safety review for the initial 84 participants, the DRC also evaluated the safety data for Cohort 1 over the course of the study and reviewed any events that met study-specific vaccination hold rules or any other safety concerns that may have arisen.

[0481] For Cohort 1, the primary analysis occurred when all participants had completed the Day 30 visit (Visit 4) or had discontinued prematurely. The final analysis occurred when all participants had completed the Day 181 visit or had discontinued prematurely. For participants who progressed to the open-label long-term follow-up (LTFU) period (selected dose groups of ExPEC10V and Prevnar 13), annual follow-up analyses included safety and immunogenicity data (multiplex ECL-based immunoassay and MOPA) collected through visits at Year 1 (Day 366), Year 2 (Day 731), and Year 3 (Day 1096) post-vaccination.

[0482] Queue 2

[0483] In Cohort 2, the safety, reactogenicity, and immunogenicity of ExPEC10V at the selected dose (based on the results of the primary analysis of Cohort 1) were evaluated in participants aged ≥60 years with a history of UTI within the past 5 years and in stable health. For Cohort 2, the study had a double-blind, placebo-controlled design and enrolled a total of 600 participants, who were randomized in parallel in a 2:1 ratio (400 participants in the ExPEC10V group and 200 participants in the placebo group).

[0484] On Day 1, all participants received a single IM injection of the selected dose of ExPEC10V or placebo according to the assigned study vaccination group (Table 12).

[0485] For Cohort 2, the primary analysis included safety and immunogenicity data and occurred when all participants had completed the Day 30 visit (Visit 4) or had discontinued early. The final analysis occurred when all participants had completed the Day 181 visit or had discontinued early. For all participants, annual follow-up analyses included safety and immunogenicity data (multiplex ECL-based immunoassay and MOPA) collected until the visits at Year 1 (Day 366), Year 2 (Day 731), and Year 3 (Day 1096) post-vaccination.

[0486] Fecal sample analysis was performed using metagenomics in a selected subset of participants to assess the effect of ExPEC10V on the prevalence of pathogens (e.g., Clostridium difficile) and ExPEC10V serotypes in the gut microbiota.

[0487] Number of participants

[0488] A total of 1004 participants were included in the study; 404 participants in cohort 1 and 600 participants in cohort 2.

[0489] intervention group

[0490] Description of the intervention

[0491] ExPEC10V: An E. coli bioconjugate vaccine in phosphate-buffered saline containing the O-antigens (PS) of ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively, bioconjugated to the EPA carrier protein. A single 0.5 mL IM (deltoid muscle) injection of one of three doses of ExPEC10V is administered on day 1.

[0492] ExPEC4V: An E. coli bioconjugate vaccine in saline buffer containing the O-antigen PS of ExPEC serotypes O1A, O2, O6A, and O25B, respectively, bioconjugated to the EPA carrier protein (4:4:4:8 µg PS / ExPEC serotype). A single 0.5 mL IM (deltoid muscle) injection of ExPEC4V on day 1.

[0493] Prevnar 13: A sterile suspension of capsular antigenic saccharides of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, individually linked to the nontoxic diphtheria CRM197 protein. Supplied in a single-dose prefilled syringe for a single 0.5 mL IM (deltoid muscle) injection on day 1.

[0494] Placebo: Normal saline. On Day 1, a single 0.5 mL IM (deltoid muscle) injection of placebo.

[0495] The ExPEC study intervention materials are described in Table 9.

[0496] Table 9. BAC1001MV ExPEC investigational vaccines.

[0497]

[0498] EPA = genetically detoxified form of exotoxin A derived from Pseudomonas aeruginosa; PS = polysaccharide

[0499] ExPEC4V consists of the O-antigen polysaccharides (PS) of ExPEC serotypes O1A, O2, O6A, and O25B, respectively, bioconjugated to the EPA carrier protein.

[0500] ExPEC10V consists of O-antigen polysaccharides (PS) of ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively, bioconjugated to the EPA carrier protein.

[0501] Dosage is based on PS only. EPA (µg) is the measured value.

[0502] ExPEC10V consists of 10 monovalent drug substances (DSs). For this clinical study, two different concentrations (medium and high) of drug product (DP) were produced (Table 10). A third (low) concentration was clinically achieved by diluting the high concentration 1:1 with dilution buffer, which was the same as the formulation buffer. Each DP was formulated in sodium / potassium phosphate buffer (0.02% [w / w] polysorbate 80, 5% [w / w] sorbitol, 10 mM methionine) at pH 7.0.

[0503] Table 10: Composition of the ExPEC10V vaccine used in the Phase 1 / 2a clinical study

[0504]

[0505] EPA = genetically detoxified Pseudomonas aeruginosa exotoxin A used as a carrier protein

[0506] a The active ingredient is a biologically synthesized conjugate consisting of PS antigen and carrier protein (EPA); the dosage is calculated based on the PS portion only.

[0507] b The "low concentration" is obtained clinically by diluting the "high concentration" 1:1 with dilution buffer.

[0508] Safety Assessment

[0509] Key safety evaluations included requested local and systemic AEs, unsolicited AEs, SAEs, physical examinations, vital sign measurements, and clinical laboratory tests.

[0510] Immunogenicity evaluation

[0511] Key immunogenicity evaluations of the collected sera included evaluation of vaccine-elicited ExPEC10V and ExPEC4V serotype-specific total IgG antibody levels, as measured by a multiplex ECL-based immunoassay, and ExPEC10V and ExPEC4V serotype-specific functional antibodies, as measured by a multiplex format (MOPA) opsonophagocytic killing assay (OPKA). Immunogenicity evaluation of pneumococcal antibody titers elicited by Prevnar 13 was not performed.

[0512] The levels of serum antibodies induced by ExPEC10V were measured using a multiplex electrochemiluminescence (ECL)-based immunoassay. This assay combines high-binding carbon electrodes in a multi-well 96-well microplate format coated with different E. coli O-LPS antigens or the carrier protein EPA. A secondary antibody (anti-human IgG) labeled with SULFO-TAG was used to detect the levels of antigen-specific antibodies present in serum samples. SULFO-TAG emits light in the presence of electrical stimulation, and its intensity increases proportionally to the amount of bound IgG antibodies. This assay is qualified according to the International Conference on Harmonisation (ICH) recommendations.

[0513] The levels of functional antibodies induced by ExPEC10V were measured by a multiplex opsonophagocytosis assay (MOPA). Briefly, heat-inactivated serum samples were serially diluted and incubated with different E. coli strains that are specifically resistant to different types of antibiotics. After this, human complement and phagocytes (HL60) were added to the reaction, and after a second incubation period, aliquots of the reaction mixture were transferred to different PVDF hydrophilic membrane filter plates containing culture medium supplemented with a specific antibiotic that selectively allows the growth of strains resistant to that specific antibiotic. After overnight growth, colony forming units (CFU) were counted to determine the number of surviving bacteria. This assay is qualified according to ICH recommendations.

[0514] The following measures of immunogenicity were assessed and tabulated by study vaccination group for all immunogenicity time points for ExPEC10V serotype antibodies as measured by multiplex ECL-based immunoassay and MOPA, and EPA as measured by multiplex ECL-based immunoassay only:

[0515] - Proportion of participants with a ≥2-fold and ≥4-fold increase in serum antibody titers from Day 1 (pre-vaccination)

[0516] - Geometric mean titer (GMT)

[0517] - GMR: fold change from baseline calculated from post-baseline / baseline values.

[0518] For the LTFU period, a descriptive summary of immunogenicity is provided for each serotype.

[0519] The overall evidence available at the time of the primary analysis of Cohort 1 (Day 30 results) was considered for later dose selection.

[0520] Table 11: Cohort 1: Vaccination schedule

[0521]

[0522] * ExPEC10V consists of the O-antigen polysaccharide (PS) of ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively, bioconjugated to a carrier protein, which is a genetically detoxified form of exotoxin A (EPA) derived from Pseudomonas aeruginosa.

[0523] **ExPEC4V consists of the O-antigen polysaccharide (PS) of ExPEC serotypes O1A, O2, O6A, and O25B, respectively, bioconjugated to a carrier protein, which is a genetically detoxified form of exotoxin A (EPA) derived from Pseudomonas aeruginosa.

[0524] *** Prevnar 13, Pneumococcal 13-valent Conjugate Vaccine (Diphtheria CRM197 Protein), is a sterile suspension of capsular antigen saccharides of Streptococcus pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, individually linked to the avirulent diphtheria CRM197 protein.

[0525] Table 12: Cohort 2: Vaccination schedule

[0526]

[0527] a ExPEC10V consists of the O-antigen polysaccharide (PS) of ExPEC serotypes O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B, and O75, respectively, bioconjugated to a carrier protein, which is a genetically detoxified form of exotoxin A (EPA) derived from Pseudomonas aeruginosa.

[0528] Participants enrolled in Cohort 2 of the study were randomized in a 2:1 ratio (ExPEC10V:placebo). The dose of ExPEC10V used in Cohort 2 was based on the results of the primary analysis (Day 30) of Cohort 1.

[0529] state

[0530] Enrollment and vaccination of Cohort 1 of the aforementioned study have been completed. The study is being conducted in a blinded fashion. Based on ongoing review of safety data, no major safety concerns have been identified, and the ExPEC10V vaccine has an acceptable safety profile.

[0531] Immunogenicity analysis of Cohort 1 clinical samples is ongoing in a blinded fashion. ECL data have passed 100% acceptance quality limit (AQL) checks and have been uploaded for data management. Analysis of MOPA samples is ongoing. Data unblinding and statistical analysis are being performed by a clinical research organization (CRO).

[0532] Vaccination of Cohort 2 will be initiated once the ExPEC10V dose for that cohort has been identified based on the completed primary analysis of the Day 30 results from Cohort 1.

[0533] Those skilled in the art will appreciate that changes may be made to the above-described embodiments without departing from the broad inventive concept thereof. It should therefore be understood that this invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by this description.

[0534] sequence

[0535] SEQ ID NO: 1 (glycosylation consensus sequence)

[0536] Asn-X-Ser(Thr), where X can be any amino acid except Pro

[0537] SEQ ID NO: 2 (optimized glycosylation consensus sequence)

[0538] Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any amino acid except Pro

[0539] SEQ ID NO: 3 (EPA carrier protein containing four glycosylation consensus sequences (EPA-4))

[0540]

[0541] SEQ ID NO: 4 ( O4 GtrS amino acid sequence)

[0542] MNNLIMNNWCKLSIFIIAFILLWLRRPDILTNAQFWAEDSVFWYKDAYENGFLSSLTTPRNGYFQTVSTFIVGLTALLNPDYAPFVSNFFGIMIRSVIIWFLFTERFNFLTLTTRIFLSIYFLCMPGLDEVHANITNAHWYLSLYVSMILIARNPSSKSWRFHDIFFILLSGLSGPFIIFILAASCFKFINNCKDHISVRSFINFYLRQP YALMIVCALIQGTSIILTFNGTRSSAPLGFSFDFVISSIISSNIFLFTFVPWDIAKAGWDNLLLSYFLSVSILSCAAFVFVKGTWRMKVFATLPLLIIIFSMAKPQLTDSAPQLPTLINGQGSRYFVNHIIAIFSLLCVYLLECVRGKVATLFSKIYLTILLFVMGCLNFVITPLPMNWREGATLINNAKTGDVISIQVLPPGLTLELRKK

[0543] SEQ ID NO: 5 ( Exemplary O4 gtrS nucleic acid sequence)

[0544]

[0545] SEQ ID NO: 6 ( Example of PglB sequence

[0546] MLKKEYLKNPYLLVLFAMIILAYVFSVFCRFYWVWWASEFNEYFFNNQLMIISNDGYAFAEGARDMIAGFHQPNDLSYYGSSLALTYWLYKITPFSFESIILYMSTFLSSLVIPTILLANEYKRPLMGFVAALLASIANSYYNRTMSGYYDTDMLVIVLPMFILFFMVRMILKKDFF SLIALPLFIGIYLWWYPSSYTLNVALIGLFLIYTLIFHRKEKIFYIAVILSSLTLSNIAWFYQSAIIVILFALFALEQKRLNFMIIGILGSATLIFLILSGGVDPILYQLKFYIFRSDESANLTQGFMYFNVNQTIQEVENVDLSFMRRISGSEIVFLFSLFGFVLLRKHKSMIMA LPILVLGFLALKGGLRFTIYSVPVMALGFGFLLSEFKAIMVKKYSQLTSNVCIVFATILTLAPVFIHIYNYKAPTVFSQNEASLLNQLKNIANREDYVVTWWDYGYPVRYYSDVKTLVDGGKHLGKDNFFPSFALSKDEQAAANMARLSVEYTEKSfyAPQNDILKTDILQAMMKDYN QSNVDLFLASLSKPDFKIDTPKTRDIYLYMPARMSLIFSTVASFSFINLDTGVLDKPFTFSTAYPLDVKNGEIYLSNGVVLSDDFRSFKIGDNVVSVNSIVEINSIKQGEYKITPIDDKAQFYIFYLKDSAIPYAQFILMDKTMFNSAYVQMFFLGNYDKNLFDLVINSRDAKVFKLKI

[0547] SEQ ID NO: 7 (Exemplary gtrA amino acid sequence; E. coli W3110 yfdG, GenBank: BAA16209.1)

[0548] MLKLFAKYTSIGVLNTLIHWVVFGVCIYVAHTNQALANFAGFVVAVSFSFFANAKFTFKASTTTMRYMLYVGFMGTLSATVGWAADRCALPPMITLVTFSAISLVCGFVYSKPIVFRDAK

[0549] SEQ ID NO: 8 (Exemplary gtrB amino acid sequence—E. coli W3110 yfdH, GenBank: BAA16210.1)

[0550] MKISLVVPVFNEEEAIPIFYKTVREFEELKSYEVEIVFINDGSKDATESIINALAVSDPLVVPLSFTRNFGKEPALFAGLDHATGDAIIPIDVDLQDPIEVIPHLIEKWQAGADMVLAKRSDRSTDGRLKRKTAEWFYKLHNKISNPKIEENVGDFRLMSRDVVENIKLMPERNLFMKGILSWVGGKTDIVEYVRAERIAGDTKFNGWKLWNLALEGITSFSTFPLRIWTYIGLVVASVAFIYGAWMILDTIIFGNAVRGYPSLLVSILFLGGIQMIGIGVLGEYIGRTYIETKKRPKYIIKRVKK

[0551] SEQ ID NO: 9 (Exemplary 04 rfb Locus nucleotide sequence – O4-EPA producing strain BVEC-L- 00684f)

[0552]

[0553] SEQ ID NO: 10 (Exemplary signal sequence of EPA carrier protein)

[0554] MKKIWLALAG LVLAFSASA

[0555] SEQ ID NO: 11 (Exemplary O1A rfb Locus nucleotide sequence – O1A-EPA production strain stGVXN4411 and stLMTB10217)

[0556]

[0557] SEQ ID NO: 12 (Exemplary O2 rfb Locus nucleotide sequence – O2-EPA producing strain stGVXN4906)

[0558]

[0559] SEQ ID NO: 13 (Exemplary O6A rfb Locus nucleotide sequence – O6A-EPA producing strain stGVXN4112 and stLMTB10923)

[0560]

[0561] SEQ ID NO: 14 (Exemplary 08 rfb Locus Nucleotide Sequence – O8-EPA Production Strain stLMTB11734)

[0562]

[0563] SEQ ID NO: 15 (Exemplary 015 rfb Locus Nucleotide Sequence – O15-EPA Production Strain stLMTB11738)

[0564]

[0565] SEQ ID NO: 16 (Exemplary 016 rfb Locus nucleotide sequence – O16-EPA producing strain stLMTB11739)

[0566]

[0567] SEQ ID NO: 17 (Exemplary O18A rfb Locus nucleotide sequence – O18A-EPA producing strain BVEC- L-00559)

[0568]

[0569] SEQ ID NO: 18 (Exemplary O25B rfb locus nucleotide sequence – O25B-EPA production strain stGVXN4459)

[0570]

[0571] SEQ ID NO: 19 (Exemplary 075 rfb Locus Nucleotide Sequence – O75-EPA Production Strain stLMTB11737)

[0572]

Claims

1. A method for preparing Escherichia coli O covalently linked to a carrier protein x - A method for preparing a bioconjugate of an antigen-polysaccharide, the method comprising: (i) providing a recombinant prokaryotic host cell comprising: a. About O x - Nucleotide sequence of the rfb gene cluster of the antigenic polysaccharide; b. a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence Asn-X-Ser or Asn-X-Thr, wherein X is any amino acid except Pro; and c. Encoding oligosaccharyltransferase PglB y The nucleotide sequence of (ii) culturing the recombinant prokaryotic host cell under conditions for bioconjugate production, in: When O x -When the antigen is O1A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V; When O x -When the antigen is glycosylated O4 antigen polysaccharide, PglB y The recombinant host cell further comprises: a sequence encoding a glucosyltransferase GtrS, which is SEQ ID NO: 4 and is capable of modifying the E. coli O4 antigen polysaccharide by adding glucose to produce the E. coli glucosylated O4 antigen polysaccharide; and a nucleotide sequence of a translocase GtrA and a glycosyltransferase GtrB of SEQ ID NO: 7 and 8, respectively, wherein the translocase is capable of translocating glucose linked to a bacterial terpene alcohol, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol; When O x -When the antigen is O8 antigen polysaccharide, PglB y does not contain amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483, and 669; When O x -When the antigen is O15 antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V; When O x -When the antigen is O16 antigen polysaccharide, PglB y Contains amino acid mutations Y77H, S80R, Q287P, K289R, and N311V; When O x -When the antigen is O18A antigen polysaccharide, PglB y does not comprise amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669; and When O x -When the antigen is O75 antigen polysaccharide, PglB y Contains the amino acid mutation N311V, wherein in each case the amino acid mutation is relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, and wherein the O1A, glucosylated O4, O8, O15, O16, O18A and O75 antigenic polysaccharides have the following structures, respectively: And each n is independently an integer from 1 to 100.

2. A method for preparing Escherichia coli O covalently linked to a carrier protein x - A method for preparing a bioconjugate of an antigen-polysaccharide, the method comprising: (i) providing a recombinant prokaryotic host cell comprising: a. About O x - Nucleotide sequence of the rfb gene cluster of the antigenic polysaccharide; b. a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence Asn-X-Ser or Asn-X-Thr, wherein X is any amino acid except Pro; and c. Encoding oligosaccharyltransferase PglB y The nucleotide sequence of (ii) culturing the recombinant prokaryotic host cell under conditions for bioconjugate production, wherein: When O x -When the antigen is O6A antigen polysaccharide, PglB y The invention comprises amino acid mutations N311V, K482R, D483H and A669V, wherein the amino acid mutations are relative to the wild-type PglB having an amino acid sequence of SEQ ID NO: 6; and the O6A antigen polysaccharide has the following structure: wherein n is independently an integer from 1 to 100.

3. The method of claim 1, wherein the glycosylation consensus sequence is Asp-X-Asn-Z-Ser or Asp-X-Asn-Z-Thr or Glu-X-Asn-Z-Ser or Glu-X-Asn-Z-Thr, wherein X and Z are independently selected from any amino acid except Pro.

4. The method of claim 1, wherein each n is independently an integer from 3 to 50.

5. The method of claim 1, wherein each n is independently an integer from 5 to 40.

6. The method of claim 1, wherein each n is independently an integer from 7 to 25.

7. The method of claim 1, wherein each n is independently an integer from 10 to 20.

8. The method of claim 1, wherein said O x - The antigen is a glucosylated O4 antigen polysaccharide, and relative to the wild-type PglB having an amino acid sequence of SEQ ID NO: 6, the PglB y Comprising the amino acid mutation N311V or the amino acid mutations Y77H and N311V.

9. A method for preparing Escherichia coli O covalently linked to a carrier protein x - A method for preparing a bioconjugate of an antigen-polysaccharide, the method comprising: (i) providing a recombinant prokaryotic host cell comprising: a. About O x - Nucleotide sequence of the rfb gene cluster of the antigenic polysaccharide; b. a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence Asn-X-Ser or Asn-X-Thr, wherein X is any amino acid except Pro; and c. Encoding oligosaccharyltransferase PglB y The nucleotide sequence of (ii) culturing the recombinant prokaryotic host cell under conditions for bioconjugate production, Wherein relative to the wild-type PglB of the amino acid sequence of SEQ ID NO: 6, PglB y Contains the amino acid mutation N311V, Among them, x - the antigen is O1A antigen polysaccharide, glycosylated O4 antigen polysaccharide, O6A antigen polysaccharide, O15 antigen polysaccharide, O16 antigen polysaccharide or O75 antigen polysaccharide, And when O x When the antigen is a glucosylated O4 antigen polysaccharide, the recombinant prokaryotic host cell further comprises: a sequence encoding a glucosyltransferase GtrS, the glucosyltransferase GtrS being SEQ ID NO: 4 and being capable of modifying the E. coli O4 antigen polysaccharide by adding glucose to produce the E. coli glucosylated O4 antigen polysaccharide; and nucleotide sequences of a translocase GtrA and a glycosyltransferase GtrB being SEQ ID NO: 7 and 8, respectively, wherein the translocase is capable of translocating glucose linked to a bacterial terpene alcohol, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol, and The O1A, glucosylated O4, O6A, O15, O16 and O75 antigen polysaccharides have the following structures respectively: And each n is independently an integer from 1 to 100.

10. The method of claim 9, wherein the glycosylation consensus sequence is Asp-X-Asn-Z-Ser or Asp-X-Asn-Z-Thr or Glu-X-Asn-Z-Ser or Glu-X-Asn-Z-Thr, wherein X and Z are independently selected from any amino acid except Pro.

11. The method of claim 9, wherein each n is independently an integer from 3 to 50.

12. The method of claim 9, wherein each n is independently an integer from 5 to 40.

13. The method of claim 9, wherein each n is independently an integer from 7 to 25.

14. The method of claim 9, wherein each n is independently an integer from 10 to 20.

15. The method of any one of claims 1 to 14, further comprising isolating the bioconjugate from the recombinant prokaryotic host cell.

16. The method of any one of claims 1 to 14, wherein the carrier protein is selected from the group consisting of detoxified Pseudomonas aeruginosa exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, clumping factor A, clumping factor B, Escherichia coli heat-labile enterotoxin, detoxified variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli Sat protein, passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.

17. The method of claim 16, further comprising isolating the bioconjugate from the recombinant prokaryotic host cell.

18. The method of claim 17, wherein the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA).

19. The method of claim 18, wherein the EPA carrier protein comprises 1-10 glycosylation sites.

20. The method of claim 19, wherein the EPA carrier protein comprises 2-4 glycosylation sites.

21. The method of claim 19, wherein the EPA carrier protein comprises 4 glycosylation sites.

22. The method of claim 21, wherein each glycosylation site comprises a glycosylation consensus sequence of SEQ ID NO:

2.

23. The method of claim 15, wherein the carrier protein comprises the sequence of SEQ ID NO:

3.

24. The method of claim 15, wherein the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA), wherein the EPA carrier protein comprises 2-4 glycosylation sites, wherein each glycosylation site comprises a glycosylation consensus sequence of SEQ ID NO:

2.

25. The method of claim 23, wherein the recombinant prokaryotic host cell is an E. coli cell.

26. The method of claim 23, wherein the recombinant prokaryotic host cell is an Escherichia coli K-12 strain.

27. The method of claim 23, wherein the recombinant prokaryotic host cell is strain W3110.

28. The method of claim 24, wherein the recombinant prokaryotic host cell is an E. coli cell.

29. The method of claim 24, wherein the recombinant prokaryotic host cell is an E. coli K-12 strain.

30. The method of claim 24, wherein the recombinant prokaryotic host cell is strain W3110.

31. A method for preparing Escherichia coli O covalently linked to a carrier protein x - a recombinant prokaryotic host cell of a bioconjugate of an antigenic polysaccharide, the recombinant prokaryotic host cell comprising: a. About O x - Nucleotide sequence of the rfb gene cluster of the antigenic polysaccharide; b. a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence Asn-X-Ser or Asn-X-Thr, wherein X can be any amino acid except Pro; and c. Encoding oligosaccharyltransferase PglB y The nucleotide sequence of in: When O x -When the antigen is O1A antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H, and A669V; When O x -When the antigen is glycosylated O4 antigen polysaccharide, PglB y The recombinant host cell further comprises: a sequence encoding a glucosyltransferase GtrS, which is SEQ ID NO: 4 and is capable of modifying the E. coli O4 antigen polysaccharide by adding glucose to produce the E. coli glucosylated O4 antigen polysaccharide; and a nucleotide sequence of a translocase GtrA and a glycosyltransferase GtrB of SEQ ID NO: 7 and 8, respectively, wherein the translocase is capable of translocating glucose linked to a bacterial terpene alcohol, and the glycosyltransferase is capable of glucosylating the bacterial terpene alcohol; When O x -When the antigen is O8 antigen polysaccharide, PglB y does not contain amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483, and 669; When O x -When the antigen is O15 antigen polysaccharide, PglB y Contains amino acid mutations N311V, K482R, D483H and A669V; when O x -When the antigen is O16 antigen polysaccharide, PglB y Contains amino acid mutations Y77H, S80R, Q287P, K289R, and N311V; When O x -When the antigen is O18A antigen polysaccharide, PglB y does not comprise amino acid mutations at positions 77, 80, 287, 289, 311, 482, 483 and 669; and When O x -When the antigen is O75 antigen polysaccharide, PglB y Contains the amino acid mutation N311V; wherein in each case the amino acid mutation is relative to the wild-type PglB having the amino acid sequence of SEQ ID NO: 6, and wherein the O1A, glucosylated O4, O8, O15, O16, O18A and O75 antigenic polysaccharides have the following structures, respectively: and And each n is independently an integer from 1 to 100.

32. A method for preparing Escherichia coli O covalently linked to a carrier protein x - a recombinant prokaryotic host cell of a bioconjugate of an antigenic polysaccharide, the recombinant prokaryotic host cell comprising: a. About O x - Nucleotide sequence of the rfb gene cluster of the antigenic polysaccharide; b. a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising a glycosylation consensus sequence Asn-X-Ser or Asn-X-Thr, wherein X is any amino acid except Pro; and c. Encoding oligosaccharyltransferase PglB y The nucleotide sequence of Among them, x - Antigen is O6A antigen polysaccharide, PglB y The amino acid mutations N311V, K482R, D483H and A669V are included, and the O6A antigen polysaccharide has the following structure: and n is independently an integer from 1 to 100.

33. The recombinant prokaryotic host cell of claim 31, wherein the glycosylation consensus sequence is Asp-X-Asn-Z-Ser or Asp-X-Asn-Z-Thr or Glu-X-Asn-Z-Ser or Glu-X-Asn-Z-Thr, wherein X and Z are independently selected from any amino acid except Pro.

34. The recombinant prokaryotic host cell of claim 31, wherein each n is independently an integer from 3 to 50.

35. The method of claim 31, wherein each n is independently an integer from 5 to 40.

36. The method of claim 31, wherein each n is independently an integer from 7 to 25.

37. The method of claim 31, wherein each n is independently an integer from 10 to 20.

38. The recombinant prokaryotic host cell of claim 31, wherein said x - The antigen is a glucosylated O4 antigen polysaccharide, and relative to the wild-type PglB having an amino acid sequence of SEQ ID NO: 6, the PglB y Comprising the amino acid mutation N311V or the amino acid mutations Y77H and N311V.

39. The recombinant host cell of claim 38, wherein the recombinant prokaryotic host cell further comprises a sequence encoding GtrS having an amino acid sequence of SEQ ID NO: 4, and nucleotide sequences encoding GtrA and GtrB having amino acid sequences of SEQ ID NOs: 7 and 8, respectively.

40. The recombinant prokaryotic host cell of any one of claims 31 to 39, wherein the carrier protein is selected from the group consisting of detoxified Pseudomonas aeruginosa exotoxin A (EPA), Escherichia coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified Staphylococcus aureus hemolysin A, clumping factor A, clumping factor B, Escherichia coli heat-labile enterotoxin, a detoxified variant of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, a detoxified variant of cholera toxin, Escherichia coli Sat protein, a passenger domain of Escherichia coli Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa PcrV, outer membrane protein of Neisseria meningitidis (OMPC), and protein D from non-typeable Haemophilus influenzae.

41. The recombinant prokaryotic host cell of any one of claims 31 to 39, wherein the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA).

42. The recombinant prokaryotic host cell of claim 41, wherein the EPA carrier protein comprises 1-10 glycosylation sites.

43. The recombinant prokaryotic host cell of claim 41, wherein the EPA carrier protein comprises 2-4 glycosylation sites.

44. The recombinant prokaryotic host cell of claim 41, wherein the EPA carrier protein comprises four glycosylation sites.

45. The recombinant prokaryotic host cell of claim 42, wherein each glycosylation site comprises a glycosylation consensus sequence of SEQ ID NO:

2.

46. ​​The recombinant prokaryotic host cell of any one of claims 31-39, wherein the vector comprises the amino acid sequence of SEQ ID NO:

3.

47. The recombinant prokaryotic host cell of any one of claims 31 to 39, wherein the carrier protein is detoxified Pseudomonas aeruginosa exotoxin A (EPA), wherein the EPA carrier protein comprises 2-4 glycosylation sites, wherein each glycosylation site comprises a glycosylation consensus sequence of SEQ ID NO:

2.

48. The recombinant prokaryotic host cell of any one of claims 31 to 39, wherein the recombinant prokaryotic host cell is an E. coli cell.

49. The recombinant prokaryotic host cell of any one of claims 31 to 39, wherein the recombinant prokaryotic host cell is an Escherichia coli K-12 strain.

50. The recombinant prokaryotic host cell of any one of claims 31 to 39, wherein the recombinant prokaryotic host cell is strain W3110.

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