Production of E. coli O18 bioconjugates

Abstract

The present invention relates to host cells for producing bioconjugates of E. coli O18 antigen polysaccharide conjugated to a carrier protein. The host cells are characterized in that they contain a modified Wzy O-antigen polymerase having a specific combination of amino acid substitutions at one or more of positions 199, 377 and 395 compared to the wild-type Wzy O-antigen polymerase of SEQ ID NO: 1, which improves the yield and glycosylation pattern of the O18 bioconjugates produced by the host cells. The present invention further relates to methods for producing bioconjugates of E. coli O18 antigen polysaccharide conjugated to a carrier protein using the host cells, and compositions comprising these bioconjugates, including multivalent compositions comprising bioconjugates of additional O-antigen polysaccharide serotypes.

Description

[Technical field]

[0001] The present invention relates to the fields of medical microbiology, immunology and vaccines. In particular, the present invention relates to a host cell comprising an improved Wzy O-antigen polymerase for producing bioconjugates of E. coli O18 antigen polysaccharides with increased yield and increased degree of glycosylation. The present invention further relates to compositions comprising the bioconjugates of the present invention and the use of such compositions to induce an immune response against E. coli for the prevention or treatment of E. coli infection, particularly infection with invasive extra-intestinal pathogenic E. coli (ExPEC) disease. [Background technology]

[0002] Extraintestinal pathogenic Escherichia coli (ExPEC) strains, alongside commensal E. coli strains, are normally harmless inhabitants of the human gastrointestinal tract. Although ExPEC isolates cannot be easily distinguished from commensal isolates by serotype, many clonal lineages are dominated by ExPEC, as defined by O-antigen, capsular and flagellar antigen serotypes (abbreviated as O:K:H, e.g., O25:K1:H4). In contrast to commensal E. coli, ExPEC strains express a wide range of virulence factors that enable them to colonize the gastrointestinal tract and cause a wide range of extraintestinal infections, which are associated with significant medical costs and mortality due to hospitalization. Neonates, the elderly, and immunocompromised patients are particularly susceptible to ExPEC infections, including invasive ExPEC disease (IED).

[0003] O-antigens comprise the immunodominant components of the cell wall lipopolysaccharide (LPS) in Gram-negative bacteria, including E. coli. Currently, over 180 serologically unique E. coli O-antigens have been identified, with the majority of ExPEC isolates classified within fewer than 20 O-antigen serotypes. Full-length E. coli O-antigens typically consist of approximately 10-25 repeating sugar units attached to a highly conserved LPS core structure, with each component synthesized separately by enzymes encoded primarily in the rfb and rfa gene clusters, respectively. After polymerization of the O-antigen, the O-antigen polysaccharide backbone can be modified, typically through the addition of acetyl or glucose residues. These modifications effectively increase serotype diversity by creating antigenically distinct serotypes that share a common polysaccharide backbone but differ in side chains. Genes encoding O-antigen modifying enzymes typically reside outside the rfb cluster on the chromosome, and in some cases, these genes are found within lysogenic bacteriophages.

[0004] Efforts to develop a vaccine to prevent ExPEC infection have focused on O-antigen polysaccharide conjugates. A 12-valent O-antigen conjugate vaccine was synthesized via extraction and purification of O-antigen polysaccharide and chemical conjugation to detoxified Pseudomonas aeruginosa exotoxin A and tested for safety and immunogenicity in a Phase I clinical trial (Cross et al., J. Infect. Dis. (1994) v. 170, pp. 834-40). This candidate vaccine was never approved for clinical use. A bioconjugation system in E. coli has recently been developed in which both the antigen polysaccharide and the carrier protein are synthesized in vivo and subsequently conjugated in vivo via the activity of the Campylobacter jejuni enzyme oligosaccharyltransferase PglB expressed in E. coli (Wacker et al., Proc. Nat. Acad. Sci. (2006) v. 103, pp. 7088-93). This N-linked protein glycosylation system allows for the transfer of a variety of polysaccharides to the carrier protein and allows for a method of purifying the bioconjugate from the bacteria in which it is expressed. Bioconjugation has been successfully used to produce conjugated polysaccharides for an E. coli tetravalent O-antigen candidate vaccine (Poolman and Wacker, J. Infect. Dis. (2016) v. 213(1), pp. 6-13; WO 2015 / 124769; WO 2017 / 035181).

[0005] A composition comprising 10 bioconjugates (O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B and O75, which composition is designated ExPEC10V) has been described and is undergoing clinical trials (e.g., WO 2020 / 191082(A1)).

[0006] It was observed that the product yield for the bioconjugate with O18A antigen polysaccharide was the lowest compared to the other nine bioconjugates prepared for ExPEC10V (WO 2020 / 191082(A1)). Moreover, the O18A bioconjugate so produced appears to contain a relatively high amount of glycans composed of a limited number of O18A antigen polysaccharide repeat units, termed "sEPA" (i.e., EPA carrier protein with polysaccharide chains with only about 1-3 repeat units, which corresponds to more than 20% of the O18A bioconjugate in the periplasmic fraction in the production process of the O18A bioconjugate drug substance), and this sEPA product is not easily separated from the preferred bioconjugate in which the O18A antigen polysaccharide mainly contains glycans composed of at least 5 repeat units. ExPEC isolates belonging to the O18 serogroup are commonly identified in modern surveillance studies of US and EU blood isolates, and therefore, O18 conjugates are considered to be a relevant component of ExPEC vaccines. It would therefore be desirable to improve the yield of conjugates of O18 antigenic polysaccharides. Summary of the Invention

[0007] It is therefore an object of the present invention to provide materials and methods for increasing the product yield of bioconjugates comprising O18 antigen polysaccharide during production and / or to generate materials and methods for obtaining O18 bioconjugates having a reduced relative amount of sEPA compared to O18 bioconjugates obtained using previously described production processes. Another object of the present invention is to provide materials and methods for producing bioconjugates of E. coli O18 A antigen polysaccharide having an increased degree of glycosylation compared to O18 bioconjugates obtained using previously described materials and methods.

[0008] In a first aspect, the present invention relates to a Gram-negative bacterial host cell comprising: (a) a carrier protein comprising at least one glycosylation consensus sequence; (b) an E. coli O18 rfb locus; and (c) an oligosaccharyltransferase, wherein the cell comprises a polypeptide having Wzy O-antigen polymerase activity, the polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1, the amino acid sequence comprising: i) an isoleucine (Ile, I) at a position corresponding to position 199 of SEQ ID NO:1, a lysine (Lys, K) at a position corresponding to position 377 of SEQ ID NO:1, and an alanine (Ala, A) at a position corresponding to position 395 of SEQ ID NO:1; ii) a threonine (Thr, T) at a position corresponding to position 199 of SEQ ID NO:1, a lysine (Lys, K) at a position corresponding to position 377 of SEQ ID NO:1, and a valine (Val, V) at a position corresponding to position 395 of SEQ ID NO:1; ii) a threonine at the position corresponding to position 199 of SEQ ID NO:1, a lysine at the position corresponding to position 377 of SEQ ID NO:1, and an alanine at the position corresponding to position 395 of SEQ ID NO:1; iv) an isoleucine at the position corresponding to position 199 of SEQ ID NO:1, a lysine at the position corresponding to position 377 of SEQ ID NO:1, and a valine at the position corresponding to position 395 of SEQ ID NO:1; or v) an isoleucine at the position corresponding to position 199 of SEQ ID NO:1, a methionine (Met,M) at the position corresponding to position 377 of SEQ ID NO:1, and an alanine at the position corresponding to position 395 of SEQ ID NO:1.

[0009] In one embodiment, in a host cell, a polypeptide having Wzy O-antigen polymerase activity comprises the amino acid sequence of SEQ ID NO:1, except that the amino acid sequence comprises i) Ile at position 199, Lys at position 377, and Ala at position 395; ii) Thr at position 199, Lys at position 377 and Val at position 395; iii) Thr at position 199, Lys at position 377 and Ala at position 395; iv) Ile at position 199, Lys at position 377 and Val at position 395; or v) Ile at position 199, Met at position 377 and Ala at position 395, where all positions correspond to positions in SEQ ID NO:1.

[0010] In one embodiment, in a host cell, a polypeptide having Wzy O-antigen polymerase activity comprises an Ile at position 199, a Lys at position 377, and an Ala at position 395, which positions correspond to those in SEQ ID NO:1.

[0011] In one embodiment, the host cell comprises a polynucleotide or vector encoding a polypeptide having Wzy O-antigen polymerase activity integrated into the genome of the host cell.

[0012] In one embodiment, the host cell is an Escherichia coli host cell. In a preferred embodiment, the host cell is an E. coli K-12 strain, more preferably E. coli K-12 strain W3110.

[0013] In one embodiment, in the host cell, at least one of: a) the oligosaccharyl transferase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:6, b) the carrier protein comprises SEQ ID NO:3, and c) the E. coli O18 rfb locus is the rfb locus of an E. coli strain having serotype O18A. Preferably, the oligosaccharyl transferase comprises the amino acid sequence of SEQ ID NO:6.

[0014] In one embodiment, the host cell comprises an E. coli O18 rfb locus having a nucleotide sequence encoding a Wzy O-antigen polymerase that encodes a polypeptide having Wzy O-antigen polymerase activity as defined herein above.

[0015] In a further aspect, the present invention relates to a method for producing a bioconjugate of an E. coli O18 antigenic polysaccharide conjugated to a carrier protein, comprising (a) culturing a host cell of the invention as defined herein above to produce the bioconjugate. Preferably, the method further comprises recovering the bioconjugate.

[0016] In one embodiment, the method further comprises formulating the recovered bioconjugate into a pharmaceutical composition.

[0017] In one embodiment, the method further comprises adding one or more additional bioconjugates of an E. coli O-antigen polysaccharide conjugated to a carrier protein to the pharmaceutical composition to obtain a multivalent bioconjugate composition. Preferably, the one or more additional bioconjugates comprise at least one O-antigen polysaccharide selected from the group consisting of E. coli serotypes O1, O2, O4, O6, O8, O15, O16, O25, and O75. In a preferred embodiment, the multivalent bioconjugate composition produced by the method comprises: (i) a bioconjugate of an E. coli O18A antigen polysaccharide conjugated to a carrier protein; (ii) a bioconjugate of an E. coli O1A antigen polysaccharide conjugated to a carrier protein; (iii) a bioconjugate of an E. coli O2 antigen polysaccharide conjugated to a carrier protein; (iv) a bioconjugate of an E. coli glucosylated O4 antigen polysaccharide conjugated to a carrier protein; (v) a bioconjugate of an E. coli O6A antigen polysaccharide conjugated to a carrier protein; (vi) a bioconjugate of an E. coli O15 antigen polysaccharide conjugated to a carrier protein; (vii) a bioconjugate of an E. coli O16 antigen polysaccharide conjugated to a carrier protein; (viii) a bioconjugate of an E. coli O25B antigen polysaccharide conjugated to a carrier protein; and (ix) a bioconjugate of an E. coli O25B antigen polysaccharide conjugated to a carrier protein. and (x) a bioconjugate of an O75 antigen polysaccharide, preferably wherein the carrier protein in each bioconjugate comprises SEQ ID NO: 3. In a more preferred embodiment, the multivalent bioconjugate composition produced by the method further comprises a bioconjugate of an E. coli O8 antigen polysaccharide conjugated to a carrier protein, preferably wherein the carrier protein comprises SEQ ID NO: 3. [Brief description of the drawings]

[0018] [Figure 1] Coomassie stained SDS-PAGE loaded with periplasmic extracts from shake flask cultures of E. coli production strain W3110 for O18A bioconjugates containing SEQ ID NO:1 (lanes 1 and 3) or Wzy variants with amino acid substitutions 199I, 377K, 395A (lanes 2 and 4) induced for O18A polysaccharide bioconjugate expression. In samples loaded in lanes 1 and 2, the oligosaccharyltransferase enzyme PglB was expressed from a size-reduced expression plasmid, whereas in lanes 3 and 4, the vector backbone of the PglB expression plasmid contained an additional 4500 bp of DNA sequence including non-coding DNA and the lac repressor gene lacI that appears to be optimal for product expression. Bands representing O18A polysaccharide bioconjugate material are indicated by arrows and approximate sizes of monoglycosylated EPA (M), diglycosylated EPA (D) and triglycosylated EPA (T) are shown. Description of the invention

[0019] 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 this invention belongs. Unless otherwise specified, certain terms cited herein shall have the meaning set forth herein. All patents, published patent applications and publications cited herein are incorporated by reference as if fully set forth herein. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0020] Throughout this specification and the claims that follow, unless the context otherwise requires, the term "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a specified 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. As used herein, the term "comprising" can be replaced with the terms "containing" or "including," or, as sometimes used herein, with the term "having."

[0021] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. 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. When used herein in connection with aspects or embodiments of the present invention, any of the above terms "comprising," "containing," "including," and "having" can be replaced with the terms "consisting of" or "consisting essentially of" to vary the scope of the disclosure.

[0022] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass 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 and second elements together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be within the meaning and thus meets the requirements of the term "and / or."

[0023] The terms "about" or "approximately," when used in connection with a numerical value, mean that the value may be within the given value ±20% of the value, preferably ±10% of the value, and more preferably ±5% of the value.

[0024] Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or between two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conservative amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods.

[0025] "Sequence identity" and "sequence similarity" can be determined by alignment of two peptides or two nucleotide sequences using a global or local alignment algorithm, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith Waterman). Sequences can then be called "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (defined below) (e.g., when optimally aligned by the programs GAP or BESTFIT using default parameters). Most preferably, sequence alignment is performed in ClustalW (1.83) sequence alignment using default settings.

[0026] Alternatively, the similarity or identity percentage may be determined by searching public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention may also be used as "query sequences" to perform searches against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215:403-10). BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the wzy nucleic acid molecules of the present invention. BLAST protein searches can be performed using the BLASTx program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the National Center for Biotechnology Information homepage at http: / / www.ncbi.nlm.nih.gov / .

[0027] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series.

[0028] The inventors of the present application have surprisingly found that the use of a Wzy O-antigen polymerase that differs from a reference sequence at one or more specific positions results in a significant improvement in the production yield of O18 antigen polysaccharide bioconjugates. Moreover, the use of such a polymerase has surprisingly been found to facilitate the production of O18 antigen polysaccharide conjugates with increased glycan-to-protein ratios and / or increased occupancy of glycosylation sites.

[0029] Thus, in a first aspect, the present invention provides a host cell for use in producing a bioconjugate of an E. coli O18 antigenic polysaccharide conjugated to a carrier protein.

[0030] In one embodiment, a host cell of the invention comprises at least: (a) a carrier protein comprising at least one glycosylation consensus sequence, (b) an E. coli O18 rfb locus; c) an oligosaccharyl transferase, and (d) a polypeptide of the invention having Wzy O-antigen polymerase activity.

[0031] A polypeptide of the invention having Wzy O-antigen polymerase activity in a host cell is a polypeptide that comprises an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 and further has O-antigen polymerase activity. Whether a polypeptide of the invention has O-antigen polymerase activity can be assayed by expressing a nucleotide sequence encoding a Wzy O-antigen polymerase in a suitable host cell and determining the amount of polymerized O-antigen produced by methods known to those of skill in the art or, for example, as described in Woodward et al. (2010, Nat Chem Biol. 2010 Jun;6(6):418-423), the entire contents of which are incorporated herein by reference.

[0032] In one embodiment, a host cell of the invention comprises a polypeptide having Wzy O-antigen polymerase activity, the polypeptide comprising an amino acid sequence having at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:1, the amino acid sequence being i) an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:1; ii) a threonine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and a valine at a position corresponding to position 395 of SEQ ID NO:1; iii) a threonine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:1; iv) an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and a valine at a position corresponding to position 395 of SEQ ID NO:1, or v) comprising an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a methionine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:1.

[0033] An amino acid position corresponding to one of amino acid positions 199, 377 or 395, respectively, of SEQ ID NO:1 is understood herein to refer to a position in an amino acid sequence other than SEQ ID NO:1 that aligns with one of positions 199, 377 or 395, respectively, of SEQ ID NO:1 in a sequence alignment of SEQ ID NO:1 with a sequence other than SEQ ID NO:1, preferably in a ClustalW(1.83) sequence alignment with SEQ ID NO:1, preferably using default settings. A person skilled in the art will know how to use the amino acid sequence alignment algorithms defined herein to identify corresponding amino acid positions in Wzy O-antigen polymerase amino acid sequences other than SEQ ID NO:1.

[0034] In a preferred embodiment, the host cell of the present invention is and a valine at position 395 of SEQ ID NO:1; iii) a threonine at position 199 of SEQ ID NO:1, a lysine at position 377 of SEQ ID NO:1, and alanine at position 395 of SEQ ID NO:1; iv) an isoleucine at position 199 of SEQ ID NO:1, a lysine at position 377 of SEQ ID NO:1, and a valine at position 395 of SEQ ID NO:1; or v) an isoleucine at position 199 of SEQ ID NO:1, a methionine at position 377 of SEQ ID NO:1, and alanine at position 395 of SEQ ID NO:1. Thus, in this embodiment, a host cell of the invention comprises a polypeptide having Wzy O-antigen polymerase activity, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:1, except that the amino acid sequence comprises i) an isoleucine at position 199 of SEQ ID NO:1, a lysine at position 377 of SEQ ID NO:1, and an alanine at position 395 of SEQ ID NO:1; ii) a lysine at position 377 of SEQ ID NO:1; iii) a lysine at position 377 of SEQ ID NO:1 and an alanine at position 395 of SEQ ID NO:1; iv) an isoleucine at position 199 of SEQ ID NO:1 and a lysine at position 377 of SEQ ID NO:1; or v) an isoleucine at position 199 of SEQ ID NO:1 and an alanine at position 395 of SEQ ID NO:1.

[0035] In a particularly preferred embodiment, a host cell of the invention comprises a polypeptide having Wzy O-antigen polymerase activity, wherein the polypeptide comprises an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:1.

[0036] As will be appreciated by those skilled in the art, in one embodiment, for the expression of a polypeptide having Wzy O-antigen polymerase activity, the host cell of the present invention preferably comprises a polynucleotide encoding a polypeptide having Wzy O-antigen polymerase activity of the present invention as defined herein above. The polynucleotide may be preceded by a promoter operably linked thereto. In certain embodiments, the promoter is endogenous to the Wzy O-antigen polymerase coding sequence. In certain preferred embodiments, the promoter is an endogenous promoter that drives the expression of Wzy O-antigen polymerase in bacteria of the family Enterobacteriaceae, preferably bacteria of the genus Escherichia, more preferably bacteria of the species E. coli. In other embodiments, the promoter is heterologous to the Wzy coding sequence, for example, a strong promoter known to those skilled in the art for use in recombinant expression systems is used. For example, the promoter is an inducible or constitutive prokaryotic promoter, such as ara, phoA, tac, tet, trc, trp, PBAD, lambda PL, T5 or T7 promoter.

[0037] In certain embodiments, the present invention provides isolated polynucleotides that can be used in constructing a host cell according to the present invention. The polynucleotides can be recombinant, synthetic, or artificial. The polynucleotides can be any form of nucleic acid, such as DNA or RNA, preferably DNA. The polynucleotides can include one or more nucleotides that are not present in a naturally occurring Wzy-encoding polynucleotide, such as the naturally occurring Wzy-encoding polynucleotide of SEQ ID NO:2. Specifically, the polynucleotides for use in the present invention differ from a naturally occurring Wzy-encoding polynucleotide, such as SEQ ID NO:2, at least at one or more of the codons corresponding to positions 199, 377, and 395 in SEQ ID NO:1. Furthermore, the polynucleotides for use in the present invention can differ from a naturally occurring Wzy-encoding polynucleotide at a codon other than at least one of positions 199, 377, and 395 in SEQ ID NO:1. The polynucleotides can have one or more nucleotides at their 5' and / or 3' ends that are not present in a naturally occurring Wzy-encoding polynucleotide. Those of skill in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by a described polynucleotide to reflect the codon usage of any particular host organism in which the polypeptide is expressed. Thus, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0038] As will be further understood by the skilled artisan, in one embodiment, the polynucleotide encoding the polypeptide having Wzy O-antigen polymerase activity of the present invention as defined herein above may be included in an expression construct or vector. Preferably, in the expression construct, the polynucleotide encoding the polypeptide having Wzy O-antigen polymerase activity is operably linked to an expression control sequence for expression of the polypeptide in the host cell of the present invention, for example in an expression cassette. Such expression control sequence may include the promoter identified above and may further include a Shine-Dalgarno sequence, a transcription termination sequence, etc. In one embodiment, the expression construct is included in a vector. In one embodiment, the vector is an episomal vector, such as a plasmid or a viral vector, preferably a plasmid. In another embodiment, the polynucleotide, vector, or expression construct is integrated into the genome of the host cell. In certain embodiments, the vector or expression construct is preferably in the form of DNA. In certain embodiments, the vector comprises the polynucleotide of the present invention operably linked to a promoter, meaning that the polynucleotide is under the control of the promoter. In certain embodiments, a polynucleotide encoding a polypeptide having the Wzy O-antigen polymerase activity of the present invention as defined herein is integrated into its natural location at the rfb locus in the genome of a host cell, e.g., generated by site-directed mutagenesis using genetic modification techniques based on homologous recombination known to those skilled in the art of molecular biology.

[0039] The host cell of the invention for use in producing bioconjugates of E. coli O18 antigen polysaccharide conjugated to a carrier protein is a bacterial host cell. In one embodiment, the host cell is a Gram-negative bacterial host cell. Bacterial or prokaryotic host cells suitable for use in producing bioconjugates comprising E. coli O18 antigen include, but are not limited to, Escherichia species, Shigella species, Klebsiella species, Xhantomonas species, Salmonella species, Yersinia species, Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species. In a preferred embodiment, the host cell of the invention is an Escherichia coli host cell, more preferably an E. coli K-12 strain such as the W3110 strain, the latter being particularly preferred.

[0040] In one embodiment, the host cell is a non-naturally occurring host cell. Thus, the host cell may be derived from a naturally occurring isolate, such as a clinical isolate, that has been modified to express a polypeptide having Wzy O-antigen polymerase activity as described herein, and preferably further modified to include one or more or all of: i) an O18 rfb locus as defined herein, ii) a carrier protein comprising at least one glycosylation consensus sequence as defined herein (a nucleic acid construct for expression), and iii) an oligosaccharyl transferase as defined herein (a nucleic acid construct for expression).

[0041] Preferably, the host cell of the invention for use in producing bioconjugates of E. coli O18 antigen polysaccharide conjugated to a carrier protein further comprises an oligosaccharyl transferase (OST) for the transfer of oligosaccharides to N-glycosylation sites on the carrier protein. Thus, preferably, the host cell provided herein comprises a nucleotide sequence encoding an oligosaccharyl transferase (OST). As used herein, an oligosaccharyl transferase is an enzyme that transfers lipid-linked oligosaccharides to a residue of a nascent polypeptide chain that comprises a glycosylation consensus motif, for example, an asparagine (Asn, N) residue of a nascent polypeptide chain that comprises an N-glycosylation consensus motif, an example of such an N-glycosylation consensus motif is SEQ ID NO: 4 or Asn-X-Ser(Thr). It should be understood that throughout this application, for the N-glycosylation motif Asn-X-Ser(Thr), X can be any amino acid other than proline. Preferably, such oligosaccharyltransferase transfers an oligosaccharide to the asparagine residue of SEQ ID NO: 4 in the polypeptide chain of the carrier protein described herein. The nucleic acid encoding the oligosaccharyltransferase can be native to the host cell or can be introduced into the host cell using a genetic approach. In a preferred embodiment, the oligosaccharyltransferase is heterologous to the host cell. E. coli does not naturally contain oligosaccharyltransferase, therefore, when E. coli is used as a host cell for the production of bioconjugates, a heterologous oligosaccharyltransferase is included in such a host cell, for example, upon introduction by genetic engineering. The oligosaccharyltransferase can be derived from any source known in the art in view of the present disclosure.

[0042] In certain preferred embodiments, the oligosaccharyltransferase is from Campylobacter. For example, in one embodiment, the oligosaccharyltransferase is from Campylobacter jejuni (i.e., PglB; see, e.g., Wacker et al., 2002, Science 298:1790-1793; also see, e.g., NCBI Gene ID: 3231775, UniProt Accession No. O86154). In another embodiment, the oligosaccharyltransferase is from Campylobacter lari (see, e.g., NCBI Gene ID: 7410986).

[0043] In a specific embodiment, the oligosaccharyltransferase is PglB 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 sequence SEQ ID NO:4 and Asn-X-Ser (Thr). In a particular embodiment, the PglB oligosaccharyltransferase is a polypeptide having an oligosaccharyltransferase as defined herein, the polypeptide comprising an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:6. In a preferred embodiment, the PglB oligosaccharyltransferase comprises an amino acid sequence identical to SEQ ID NO:6. In certain embodiments, one or more endogenous glycosylation consensus sequences in wild-type PglB are mutated to avoid PglB autoglycosylation, such as SEQ ID NO: 6, which contains the mutation N534Q. An example of a variant PglB suitable for use in the host cells provided herein includes PglB of SEQ ID NO: 6, which contains the mutation N311V.

[0044] A host cell of the invention for use in producing a bioconjugate of E. coli O18 antigen polysaccharide conjugated to a carrier protein further preferably comprises a nucleotide sequence encoding a carrier protein comprising at least one glycosylation site comprising the glycosylation consensus sequence Asn-X-Ser(Thr), more preferably at least one glycosylation site having SEQ ID NO:4.

[0045] In some embodiments, the carrier protein is selected from the group consisting of P. aeruginosa (EPA), E. coli flagellin (FliC), CRM197, maltose binding protein (MBP), diphtheria toxoid, tetanus toxoid, detoxified hemolysin of S. aureus, clumping factor A, clumping 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, passenger domain of E. coli Sat protein, Streptococcus pneumoniae pneumolysin, keyhole limpet hemocyanin (KLH), P. aeruginosa PcrV, outer membrane protein (OMPC) of Neisseria meningitidis, and Haemophilus The protein is selected from the group consisting of protein D from H. influenzae.

[0046] In certain embodiments, the carrier protein is detoxified exotoxin A (EPA) of Pseudomonas aeruginosa or CRM197, preferably the carrier protein is EPA. For EPA, various detoxified protein variants have been described in the literature and can be used as carrier proteins. In certain embodiments, the EPA carrier protein used in the conjugates of the present invention is modified in such a manner that the protein is less toxic and / or more susceptible to glycosylation. For example, detoxification can be achieved by mutating and deleting catalytically essential residues L552V and ΔE553 according to Lukac et al., 1988, Infect Immun, 56:3095-3098, and Ho et al., 2006, Hum Vaccin, 2:89-98. In certain embodiments, the carrier protein used in the production of the conjugates of the invention is modified to optimize the number of glycosylation sites in the carrier protein in a manner that allows a lower concentration of the protein to be administered in its bioconjugate form, for example in an immunogenic composition. In certain embodiments, the host cell encodes an EPA comprising 1-10, preferably 2-4, preferably 4 glycosylation sites comprising a glycosylation consensus sequence having SEQ ID NO: 4. In one embodiment, the carrier protein comprises an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO: 3 and comprises 1-10, preferably 2-4, preferably 4 glycosylation sites, more preferably having SEQ ID NO: 4, comprising the glycosylation consensus sequence Asn-X-Ser(Thr). In a preferred embodiment, the carrier protein comprises an EPA comprising the amino acid sequence of SEQ ID NO: 3.

[0047] In certain preferred embodiments, a host cell according to the invention comprises a nucleotide sequence encoding an oligosaccharyltransferase comprising SEQ ID NO:6 and a nucleotide sequence encoding a carrier protein comprising SEQ ID NO:3.

[0048] Host cells of the invention for use in producing bioconjugates of E. coli O18 antigenic polysaccharide conjugated to a carrier protein further preferably contain the E. coli O18 rfb locus for the synthesis of the O18 antigenic polysaccharide structure.

[0049] In E. coli, gene products involved in O-antigen biosynthesis are encoded by the rfb locus. Thus, the host cells provided herein preferably further comprise the nucleotide sequence of the E. coli O18 rfb locus, preferably the rfb locus of an E. coli strain having serotype O18A. As used herein, "O18 rfb locus" and "O18 rfb gene cluster" refer to a locus in a Gram-negative bacterial genome that comprises a cluster of genes that together encode the enzymatic machinery capable of synthesizing the O18 antigen polysaccharide structure. The term rfb locus preferably refers to a genomic locus from the genus Escherichia, particularly E. coli.

[0050] In certain embodiments, the O18rfb locus is heterologous to the host cell, e.g., introduced into a progenitor cell of the host cell and preferably integrated into its genome.Preferably, the original rfb gene cluster, if present in the progenitor cell, is replaced by the O18rfb gene cluster in the host cell, allowing the production of O18 bioconjugates.

[0051] In a particular embodiment, the rfb gene cluster of E. coli O18 antigenic polysaccharide is the rfb gene cluster of E. coli O18A antigenic polysaccharide. Thus, preferably, the rfb gene cluster of E. coli O18A antigenic polysaccharide comprises a nucleotide sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 5, which nucleotide sequence encodes an enzyme that produces E. coli O18A antigenic polysaccharide, and preferably, the nucleotide sequence encoding Wzy O-antigen polymerase is a nucleotide sequence encoding a polypeptide having the Wzy O-antigen polymerase activity of the present invention as defined herein above. In a particular embodiment, the rfb gene cluster comprises the nucleotide sequence of SEQ ID NO: 5, except for the sequence encoding Wzy O-antigen polymerase, which is a nucleotide sequence encoding a polypeptide having the Wzy O-antigen polymerase activity of the present invention as defined herein above. The structure of the repeating unit of E. coli O18A antigenic polysaccharide is shown in Table 1 as entry (O18A).

[0052] Thus, in certain preferred embodiments, the host cell of the present invention comprises an E. coli rfb gene cluster, and the nucleotide sequence encoding the Wzy O-antigen polymerase is a nucleotide sequence encoding a polypeptide having the Wzy O-antigen polymerase activity of the present invention as defined hereinabove. Thus, in certain preferred embodiments, the endogenous Wzy O-antigen polymerase coding sequence is replaced by the nucleotide sequence encoding a polypeptide having the Wzy O-antigen polymerase activity of the present invention, the Wzy polymerase of the present invention, as defined hereinabove. Removal of the endogenous Wzy coding sequence and replacement with the sequence encoding the Wzy polymerase of the present invention can be achieved by using gene editing techniques commonly known in the art.

[0053] In one embodiment, the waaL gene is deleted from or functionally inactivated in the genome of the host cell of the invention. The terms "waaL" and "waaL gene" refer to an O-antigen ligase gene that encodes a membrane-bound enzyme with an active site located in the periplasm. The enzyme encoded by the waaL gene transfers undecaprenylphosphate (UPP)-linked O-antigens to the lipid A core to form lipopolysaccharide. Deletion or disruption of the endogenous waaL gene (e.g., ΔwaaL strain) disrupts the transfer of O-antigens to lipid A and can instead enhance the transfer of O-antigens to another available biomolecule, such as a carrier protein, expressed in the host cell of the invention.

[0054] In one embodiment of the host cell of the present invention, the E. coli gtrABS gene involved in O16 O-antigen glucosylation is deleted from or functionally inactivated in the host cell genome. In a preferred embodiment, the E. coli gtrABS gene is deleted from or functionally inactivated in the genome of an E. coli W3110 host cell. The gtrA and gtrB genes in different serotypes are highly homologous and interchangeable, while the gtrS gene encodes a serotype-specific O-antigen glycosyltransferase. In E. coli W3110, GtrS can transfer a glucose (Glc) residue to the GlcNAc sugar of the aL-Rha-(1→3)-D-G1cNAc motif of E. coli O16 O-antigen.

[0055] As will be understood by those skilled in the art, the polypeptides of the invention, such as carrier proteins, oligosaccharyl transferase, polypeptides having Wzy O-antigen polymerase activity and other enzymes of the rfb gene cluster contained in the host cells of the invention, are conveniently provided to the host cells in the form of nucleic acids encoding the polypeptides of the invention. The nucleic acids encoding the polypeptides of the invention are preferably nucleic acid constructs, in particular expression constructs comprising one or more expression cassettes for the expression of the polypeptides of the invention, and the nucleotide sequences encoding the polypeptides of the invention are operably linked to expression control sequences for the expression of the polypeptides in the host cells of the invention. Suitable expression control sequences have been mentioned above and usually comprise at least a promoter. The promoters may be constitutive promoters or promoters whose activity can be regulated, for example repressed or induced under certain conditions, such as temperature change or the presence of certain chemicals or proteins in the cell, all of which are well known in the art. The nucleic acid constructs may be extrachromosomal (e.g., plasmids or other vectors) or the nucleic acid constructs may be integrated into the genome of the host cells of the invention. Molecular biological methods for the construction and / or synthesis of nucleic acid constructs for the expression of polypeptides in the host cells of the invention are generally well known in the art.

[0056] Optionally, the chain length of the O-antigen can be manipulated by manipulating the native Wzz O-antigen chain length regulatory mechanism, for example by overexpressing or supplementing with a Wzz O-antigen chain length regulator, for example by replacing the E. coli wzzB gene with one of its counterparts from species within Salmonella and Shigella, or P. aeruginosa, such as the Salmonella enterica counterpart, fepE or other wzz homologues (see, for example, US Patent Publication No. 2018 / 0099038, WO 2020 / 039359), thereby increasing, for example, the number of repeat units of the O-antigen, with or without further overexpression of the Wzy protein. The effect of wzz-like genes has been described in the literature for these species. However, none of this is required to obtain a bioconjugate with good immunogenicity, and in a preferred embodiment, the bioconjugate according to the invention is prepared without manipulation of the Wzz chain length regulatory mechanism.

[0057] In one embodiment, the yield of E. coli O18 antigen bioconjugate produced using a host cell of the invention comprising a polypeptide having Wzy O-antigen polymerase activity and specific amino acids at the indicated positions defined herein above is at least 105%, 110%, 120%, 150% or 200% of the yield produced using the same conditions using an otherwise identical host cell expressing a reference Wzy O-antigen polymerase having the amino acid sequence of SEQ ID NO:1.

[0058] In one embodiment, the glycosylation site occupancy of an E. coli O18 antigen bioconjugate produced using a host cell of the invention comprising a polypeptide having Wzy O-antigen polymerase activity and the specific amino acids at the indicated positions defined herein above is improved over the glycosylation site occupancy of a bioconjugate produced using the same conditions with an otherwise identical host cell expressing a reference Wzy O-antigen polymerase having the amino acid sequence of SEQ ID NO: 1. Thus, in one embodiment, in a composition comprising an E. coli O18 antigen bioconjugate produced using a host cell of the invention and in which the carrier protein comprises four glycosylation sites, at least 50, 55, 60, 65, or 70% of the bioconjugate molecules are di-, tri-, or tetra-glycosylated, i.e., have between 2 and 4 of the glycosylation sites occupied. Preferably, in this embodiment, the carrier protein has at least 80, 85, 90, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:3, contains four glycosylation sites, contains the glycosylation consensus sequence Asn-X-Ser(Thr), and more preferably comprises an amino acid sequence having SEQ ID NO:4. Most preferably in this embodiment, the carrier protein comprises EPA comprising the amino acid sequence of SEQ ID NO:3. In certain embodiments, such compositions are periplasmic fractions of host cells producing O18 bioconjugates. Preferably, such compositions comprise no more than 20%, 15%, 10% or more bioconjugates having an average number of about 1-3 repeating units of O18 antigen ("sEPA").

[0059] In a further aspect, the present invention further relates to a method for producing a bioconjugate of E. coli O18 antigen polysaccharide conjugated to a carrier protein using the host cell of the present invention. Thus, one embodiment relates to a method for producing a bioconjugate of E. coli O18 antigen polysaccharide conjugated to a carrier protein, preferably the method comprises at least the step of a) culturing the host cell of the present invention to produce the bioconjugate. In one embodiment, in step a), the host cell of the present invention is cultured under conditions that result in the production of the bioconjugate. In a particular embodiment, the method relates to the production of O18A bioconjugates. The host cell of the present invention is cultured under conditions suitable for the expression of a bioconjugate comprising a carrier protein covalently linked to E. coli O18 antigen polysaccharide, such conditions being known to the skilled person, for example using methods as previously described in WO 2015 / 124769 or WO 2020 / 191082.

[0060] In one embodiment, the method for producing an O18 bioconjugate further comprises the step of b) recovering the bioconjugate. The O18 bioconjugate can be isolated, separated, and / or purified from the recombinant host cell or culture medium using any method known in the art in view of the present disclosure. For example, the O18 bioconjugate can be purified by any method known in the art for the purification of proteins, such as chromatography (e.g., ion exchange, anion exchange, affinity and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for the purification of proteins. See, for example, Saraswat et al., 2013, Biomed. Res. Int., (pp. 1-18), and also see the methods described in WO 2009 / 104074. Additionally, the bioconjugate can be fused to a heterologous polypeptide sequence to facilitate purification. Methods for purifying and characterizing bioconjugates are described, for example, in Ihssen et al., 2010, supra, and in WO 2006 / 119987, WO 2009 / 104074, especially WO 2015 / 124769 and WO 2017 / 035181, and WO 2020 / 191082(A1), the disclosures of each of which are incorporated herein by reference.

[0061] In one embodiment, the method for producing an O18 bioconjugate further comprises the step of c) formulating the recovered bioconjugate into a pharmaceutical composition. Formulation of a bioconjugate into a pharmaceutical composition typically involves preparing a composition comprising a bioconjugate of the invention and at least one pharma- ceutically acceptable excipient, as described in more detail herein below. In one embodiment, the method for producing an O18 bioconjugate further comprises the addition of an adjuvant to the pharmaceutical composition comprising the bioconjugate. Adjuvants suitable for incorporation into pharmaceutical compositions are described in more detail herein below.

[0062] In one embodiment, the method for producing an O18 bioconjugate further comprises adding one or more additional conjugates or bioconjugates of an E. coli O-antigen polysaccharide conjugated to a carrier protein to the pharmaceutical composition to obtain a multivalent composition, preferably a multivalent bioconjugate composition. Thus, the one or more additional conjugates of an E. coli O-antigen polysaccharide conjugated to a carrier protein are preferably bioconjugates of an E. coli O-antigen polysaccharide of a serotype other than O18, or more preferably other than O18A. In a preferred embodiment, the one or more further conjugates or bioconjugates comprise at least one, two, three, four, five, six, seven, eight or all nine O-antigen polysaccharides selected from the group consisting of E. coli serotypes O1, O2, O4, O6, O8, O15, O16, O25 and O75. A detailed description of the E. coli O-antigen polysaccharide serotypes is provided herein below (see also Table 1). Such additional conjugates or bioconjugates can be obtained as previously described, for example as described in WO 2020 / 191082(A1).

[0063] Thus, in a preferred embodiment, the method for producing an O18 bioconjugate further comprises adding additional bioconjugates of E. coli O-antigen polysaccharide conjugated to a carrier protein to the pharmaceutical composition to obtain a multivalent composition, the multivalent bioconjugate composition being selected from the group consisting of: (i) a bioconjugate of E. coli O18A antigen polysaccharide conjugated to a carrier protein; (ii) a bioconjugate of E. coli O1A antigen polysaccharide conjugated to a carrier protein; (iii) a bioconjugate of E. coli O2 antigen polysaccharide conjugated to a carrier protein; (iv) a bioconjugate of E. coli glucosylated O4 antigen polysaccharide conjugated to a carrier protein; (v) a bioconjugate of E. coli O6A antigen polysaccharide conjugated to a carrier protein; (vi) a bioconjugate of E. coli O15 antigen polysaccharide conjugated to a carrier protein; (vii) a bioconjugate of E. coli O2 antigen polysaccharide conjugated to a carrier protein; (viii) a bioconjugate of an O16 antigen polysaccharide conjugated to a carrier protein, and (ix) a bioconjugate of an E. coli O25B antigen polysaccharide conjugated to a carrier protein. In one embodiment, the multivalent bioconjugate composition further comprises (x) a bioconjugate of an E. coli O8 antigen polysaccharide conjugated to a carrier protein. Preferably, in these embodiments, the carrier protein in each bioconjugate (i)-(ix) comprises the amino acid sequence of SEQ ID NO:3.

[0064] In another aspect, the present invention relates to the use of a host cell of the present invention comprising a polypeptide having Wzy O-antigen polymerase activity and specific amino acids at the indicated positions as defined herein above (i.e. (i) I199, K377 and A395, or (ii) T199, K377 and V395, or (iii) T199, K377 and A395, or (iv) I199, K377 and V395, or (v) I199, M377 and A395, in each case corresponding to the positions in SEQ ID NO: 1) for improving occupancy of glycosylation sites of an E. coli O18 antigen bioconjugate. Improved glycosylation site occupancy of E. coli O18 antigen bioconjugates can be observed, for example, by a reduced amount of bioconjugates with short repeating units (where n in the O18 structures in Table 1 is only about 1-3) and / or an increased amount of bioconjugates comprised of a carrier protein comprising an O18 antigen polysaccharide conjugated to at least two or more N-glycosylation consensus sequences in the carrier protein (where n in the O18 structures in Table 1 is preferably at least 5) compared to the glycosylation site occupancy of bioconjugates produced using the same conditions with an otherwise identical host cell expressing a reference Wzy O-antigen polymerase having the amino acid sequence of SEQ ID NO:1.

[0065] In another aspect, the present invention relates to a bioconjugate of E. coli O18 antigenic polysaccharide conjugated to a carrier protein obtained or obtainable in the method of the invention for producing a bioconjugate using the host cell of the invention as described herein above.

[0066] As used herein, the terms "conjugate," "glycoconjugate," and "bioconjugate" refer to a conjugated product comprising an E. coli O-antigen covalently linked to a carrier protein. The conjugates of the invention are preferably bioconjugates that are conjugated products produced in a host cell, where the O-antigen and carrier protein are biosynthetically produced by the host cell machinery, and the O-antigen is also covalently linked to the carrier protein enzymatically (e.g., via an N-linkage by a host cell enzyme).

[0067] In yet another aspect, the present invention relates to a composition comprising a bioconjugate of an E. coli O18 antigenic polysaccharide conjugated to a carrier protein obtained or obtainable in a method of the invention for producing a bioconjugate using a host cell of the invention as described herein above.

[0068] In one embodiment, a composition comprising a bioconjugate of an E. coli O18 antigen conjugated to a carrier protein is a composition comprising a bioconjugate, wherein the carrier protein comprises four glycosylation sites, and at least 50, 55, 60, 65, or 70% of the carrier protein molecules in the composition are glycosylated with E. coli O18 antigen repeats in at least two of the N-glycosylation sequences, i.e., are di-, tri-, or tetra-glycosylated, and thus have between two and four of the glycosylation sites occupied. Preferably, in this embodiment, the carrier protein comprises an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:3, comprises four glycosylation sites, comprises the glycosylation consensus sequence Asn-X-Ser(Thr), and more preferably has SEQ ID NO:4. Most preferably in this embodiment, the carrier protein comprises EPA comprising the amino acid sequence of SEQ ID NO:3. In certain embodiments, such compositions are periplasmic fractions of host cells producing O18 bioconjugates.Preferably, such compositions contain no more than 20%, 15%, 10% or more bioconjugates having an average number of about 1-3 repeating units of E. coli O18 antigenic polysaccharide ("sEPA").

[0069] In one embodiment, the composition comprising a bioconjugate of E. coli O18 antigen conjugated to a carrier protein is a pharmaceutical composition comprising, in addition to the bioconjugate, a pharma- ceutical composition comprising a pharma- ceutical acceptable excipient. The (pharmaceutical) composition of the present invention may comprise any pharma- ceutical acceptable excipient, including carriers, fillers, preservatives, solubilizers, and / or diluents. Saline and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Further examples of suitable pharmaceutical carriers are well known to those skilled in the art and are described in textbooks.

[0070] As used herein, the term "pharmaceutical acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy of the composition according to the present invention or the biological activity of the composition according to the present invention. A "pharmaceutical acceptable carrier" may include any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulation, or other material known in the art for use in pharmaceutical formulations. It will be understood that the characteristics of a pharmaceutical acceptable carrier will depend on the route of administration of a particular application. According to certain embodiments, any pharmaceutical acceptable carrier suitable for use in vaccines can be used in the present invention in light of the present disclosure. Suitable excipients include, but are not limited to, sterile water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof, as well as stabilizers, such as Human Serum Albumin (HSA) or other suitable proteins and reducing sugars.

[0071] Suitable buffer solutions include Tris-buffered saline, phosphate buffer, and sucrose phosphate glutamate buffer. For example, Tris-buffered saline (TBS) pH 7.4 (e.g., containing Tris, NaCl, and KCl, e.g., at 25 mM, 137 mM, and 2.7 mM, respectively); phosphate buffer solution containing about 10 mM KH2PO4 / Na2HPO4 buffer solution at pH about 7.0, about 5% (w / v) sorbitol, about 10 mM methionine, and about 0.02% (w / v) polysorbate 80; or phosphate buffer solution containing about 10 mM KH2PO4 / Na2HPO4 buffer solution at pH about 7.0, about 8% (w / v) sucrose, about 1 mM EDTA, and about 0.02% (w / v) polysorbate 80.

[0072] Suitable salts include, for example, one or more of Tris-hydrochloride, sodium chloride, calcium chloride, potassium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, or mixtures of such aluminum salts).

[0073] Suitable preservatives include the preservatives 0.001% to 0.01% (w / v) of phenol, benzethonium chloride, 2-phenoxyethanol, or thimerosal. In other embodiments, no preservatives are included.

[0074] The composition may be formulated to suit the intended route of administration to a subject. For example, the composition may be formulated to suit subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, intravaginal, rectal, intravenous, buccal, intranasal, intratracheal, intramuscular, topical, transdermal, or pulmonary administration, preferably intramuscular administration.

[0075] Compositions of the invention can be included in a container, pack, or dispenser together with instructions for administration.

[0076] In certain embodiments, the compositions of the invention may be stored prior to use, for example, the compositions may be stored frozen (e.g., at about -20°C or about -70°C), may be stored under refrigerated conditions (e.g., at about 2-8°C, e.g., at about 4°C), or may be stored at room temperature.

[0077] The pharmaceutical composition may optionally include an adjuvant or may be administered in combination with an adjuvant. The adjuvant for administration in combination with the composition of the present invention may be administered before, simultaneously with, or after administration of the immunogenic composition. In another preferred embodiment, the pharmaceutical composition of the present invention does not include an adjuvant and / or is not administered in combination with an adjuvant.

[0078] As used herein, the term "adjuvant" refers to a compound that, when administered with or as part of a composition of the invention, increases, enhances and / or boosts the immune response to a conjugate comprising an E. coli O18 antigen bound to a carrier protein, but does not generate an immune response to the conjugate when the adjuvant compound is administered alone. Adjuvants can enhance the immune response by several mechanisms including, for example, lymphocyte recruitment, stimulation of B cells and / or T cells, and / or stimulation of antigen presenting cells.

[0079] Specific examples of adjuvants include aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and aluminum oxide, including nanoparticles containing alum or nanoalum formulations), calcium phosphate, monophosphoryl lipid A (MPL), or 3-de-O-acylated monophosphoryl lipid A (3D-MPL) (see, e.g., GB 2220211, EP 0971739, EP 1194166, U.S. Pat. No. 6,491,919), AS01, AS02, AS03, and AS04 (all from GlaxoSmithKline; see, e.g., European Patent No. 6,491,919 for AS04), and / or other adjuvants, such as glycerol, terpenes, and / or glycerols. No. 1,126,876, U.S. Patent No. 7,357,936, for AS02, see EP Patent No. 0,671,948, EP Patent No. 0,761,231, U.S. Patent No. 5,750,110), imidazopyridine compounds (see WO 2007 / 109812), imidazoquinoxaline compounds (see WO 2007 / 109813), delta-inulin, STING-activating synthetic cyclic di-nucleotides (e.g., U.S. Patent Application Publication No. 2015 / 0056224), combinations of lecithin and carbomer homopolymers (e.g., U.S. Patent No. 6,676,958), and saponins such as Quil A and QS21, optionally in combination with QS7 (e.g., U.S. Patent No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). In certain embodiments, the adjuvant comprises Quil-A, such as those commercially available from Brenntag (now Croda) or Invivogen. QuilA contains a water-extractable fraction of saponins derived from the Quillaja saponaria Molina tree. These saponins belong to a group of triterpenoid saponins that share a common triterpenoid backbone structure. Saponins are known to induce strong adjuvant responses to T-dependent as well as T-independent antigens, as well as strong cytotoxic CD8+ lymphocyte responses, and enhance responses to mucosal antigens.They may also be combined with cholesterol and phospholipids to form immunostimulatory complexes (ISCOMs), and QuilA adjuvants may activate both antibody- and cell-mediated immune responses to a wide range of antigens from different sources. In certain embodiments, the adjuvant is AS01, e.g., AS01B. AS01 is an adjuvant system that includes MPL (3-O-desacyl-4'-monophosphoryl lipid A), QS21 (Quillaja saponaria Molina, fraction 21), and liposomes. In certain embodiments, AS01 is commercially available (GSK) or can be made as described in WO 96 / 33739, which is incorporated herein by reference. Certain adjuvants include emulsions, which are mixtures of two immiscible fluids, e.g., oil and water, one of which is suspended as droplets inside the other and stabilized by a surfactant. An oil-in-water emulsion has water forming a continuous phase surrounding the oil droplets, whereas a water-in-oil emulsion has oil forming a continuous phase. Certain emulsions include squalene (a metabolizable oil). Certain adjuvants include block copolymers, which are copolymers formed when two monomers cluster together to form a block of repeating units. An example of a water-in-oil emulsion that includes a block copolymer, squalene, and a particulate stabilizer is TiterMax®, which is commercially available from Sigma-Aldrich. Optionally, the emulsion can be combined with or include additional immune stimulating components, such as TLR4 agonists. Certain adjuvants are oil-in-water emulsions (such as squalene or peanut oil), which are also used in MF59 (see, e.g., EP 0399843, U.S. Pat. No. 6,299,884, U.S. Pat. No. 6,451,325) and AS03, optionally combined with immune stimulants such as monophosphoryl lipid A and / or QS21 in AS02 (see, Stoute et al., 1997, N. Engl. J. Med. 336, 86-91).Further examples of adjuvants are liposomes containing immune stimulants, such as MPL and QS21, for example, in AS01E and AS01B (e.g., US Patent Application Publication No. 2011 / 0206758). Other examples of adjuvants are CpG, and imidazoquinolines (e.g., imiquimod and R848). See, for example, Reed G, et al., 2013, Nature Med, 19:1597-1608. In certain embodiments, the adjuvant is a Th1 adjuvant.

[0080] In certain embodiments, the adjuvant comprises a water-extractable fraction of a saponin, preferably a saponin obtained from Quillaja saponaria. In certain embodiments, the adjuvant comprises QS-21.

[0081] In certain embodiments, the adjuvant comprises 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 is a TLR4 agonist, comprising lipid A, or its analog or derivative.

[0082] For example, adjuvants containing TLR4 agonists can be formulated in different ways, e.g., emulsions such as water-in-oil (w / o) emulsions or oil-in-water (o / w) emulsions (examples are MF59, AS03), stable (nano-) emulsions (SE), lipid suspensions, liposomes, (polymeric) nanoparticles, virosomes, alum adsorbed, aqueous formulations (AF), etc., representing different delivery systems for the immunomodulatory molecules and / or immunogens in the adjuvant.

[0083] Immunostimulatory TLR4 agonists can optionally be combined with other immunomodulatory components, such as saponins (e.g., QuilA, QS7, QS21, Matrix M, Iscoms, Iscomatrix, etc.), aluminum salts, activators for other TLRs (e.g., imidazoquinolines, flagellins, dsRNA analogs, TLR9 agonists such as CpG, etc.) (see, e.g., Reed et al., 2013, supra).

[0084] As used herein, the term "lipid A" refers to the hydrophobic lipid portion of the LPS molecule, which contains glucosamine and is linked by a ketosidic bond to a keto-deoxyoctulosonate in the inner core of the LPS molecule, anchoring the LPS molecule to the outer leaflet of the outer membrane of gram-negative bacteria. For a review of the synthesis of LPS and lipid A structures, see, for example, Raetz, 1993, J. Bacteriology 175:5745-5753, Raetz CR and C Whitfield, 2002, Annu Rev Biochem 71:635-700, U.S. Patent Nos. 5,593,969 and 5,191,072. Lipid A, as used herein, includes naturally occurring lipid A, mixtures thereof, analogs, derivatives, and precursors. The term includes monosaccharides, e.g., the precursor of lipid A, termed lipid X, disaccharide lipid A, heptaacyl lipid A, hexaacyl lipid A, pentaacyl lipid A, tetraacyl lipid A, e.g., the tetraacyl precursor of lipid A, termed lipid IVA, dephosphorylated lipid A, monophosphoryl lipid A, diphosphoryl lipid A, e.g., lipid A from Escherichia coli and Rhodobacter sphaeroides. Some immunostimulatory lipid A structures contain six acyl chains. The four primary acyl chains directly attached to the glucosamine sugar are usually 3-hydroxyacyl chains 10-16 carbons long. Two additional acyl chains are often attached to the 3-hydroxy groups of the primary acyl chains. E. coli lipid A, for example, typically has four C14 3-hydroxyacyl chains attached to the sugar, and one C12 and one C14 attached to the 3-hydroxy group of the primary acyl chain at the 2' and 3' positions, respectively.

[0085] As used herein, the term "lipid A analog or derivative" refers to a molecule similar in structure and immunological activity to lipid A, but not necessarily occurring in nature. Lipid A analogs or derivatives may be modified, for example, to be truncated or condensed, and / or to have another amine sugar residue, e.g., a glucosamine residue substituted with a galactosamine residue, to contain 2-deoxy-2-aminogluconic acid instead of glucosamine-1-phosphate at the reducing end, to have a galacturonic acid moiety instead of a phosphate at the 4' position. Lipid A analogs or derivatives may be prepared from lipid A isolated from bacteria, for example, by chemical derivatization, or may be chemically synthesized, for example, by first determining the structure of a preferred lipid A and synthesizing the analog or derivative. Lipid A analogs or derivatives are also useful as TLR4 agonist adjuvants (see, e.g., Gregg KA et al, 2017, MBio 8, eDD492-17, doi:10.1128 / mBio.00492-17).

[0086] For example, lipid A analogs or derivatives can be obtained by deacylation of wild-type lipid A molecules, for example by alkaline treatment. Lipid A analogs or derivatives can be prepared, for example, from lipid A isolated from bacteria. Such molecules can also be chemically synthesized. Another example of a lipid A analog or derivative is a lipid A molecule isolated from a bacterial cell that has a mutation, or a deletion or insertion, of an enzyme involved in lipid A biosynthesis and / or lipid A modification.

[0087] MPL and 3D-MPL are lipid A analogs or derivatives modified to reduce the toxicity of lipid A. Lipid A, MPL, and 3D-MPL have a sugar backbone with long chain fatty acids attached, which backbone contains two glycosidically linked 6-carbon sugars and a phosphoryl moiety at the 4-position. Typically, 5-8 long chain fatty acids (usually 12-14 carbon atoms) are attached to the sugar backbone. Due to derivation from natural sources, MPL or 3D-MPL can exist as a complex or mixture of several fatty acid substitution patterns with various fatty acid lengths, e.g., heptaacyls, hexacyls, pentaacyls, etc. This is also true for some of the other lipid A analogs or derivatives described herein, but synthetic lipid A variants can also be defined and can be homogeneous. MPL and its preparation are described, for example, in U.S. Pat. No. 4,436,727. 3D-MPL is described, for example, in US Pat. No. 4,912,094 (B1) and differs from MPL by the selective removal of the 3-hydroxymyristate acyl residue ester-linked to the reducing end glucosamine at position 3 (e.g., compare the structure of MPL in column 1 of US Pat. No. 4,912,094 (B1) with 3D-MPL in column 6). In the art, 3D-MPL is often used, but it is sometimes called MPL (e.g., the first structure in Table 1 of Ireton GC and SG Reed, 2013 (above) refers to this structure as MPL®, but in fact shows the structure of 3D-MPL).

[0088] Examples of lipid A (analogs, derivatives) include MPL, 3D-MPL, RC529 (e.g., European Patent No. 1385541), PET-lipid A, GLA (glycopyranosyl lipid adjuvant, synthetic disaccharide lipid; e.g., U.S. Patent Application Publication No. 2010 / 0310602, U.S. Patent No. 8722064), 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 optimize TLR4 ligands for human vaccines), PHAD (phosphorylated hexaacyl disaccharide; its structure is the same as that of GLA), 3D-PHAD, 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 number 287180-63-6), ONO4007, OM-174, and the like. For exemplary chemical structures of 3D-MPL, RC529, PET-Lipid A, GLA / PHAD, E6020, ONO4007, and OM-174, see, e.g., Table 1 in Ireton GC and SG Reed, 2013 (supra). For the structure of SLA, see, e.g., Figure 1 in Reed SG et al, 2016, Curr Opin Immunol 41:85-90. In certain preferred embodiments, the TLR4 agonist adjuvant comprises a lipid A analog or derivative selected from 3D-MPL, GLA, or SLA. In certain embodiments, the lipid A analog or derivative is formulated in a liposome.

[0089] Exemplary adjuvants comprising lipid A analogs or derivatives include GLA-LSQ (synthetic MPL [GLA], QS21, lipid formulated as liposomes), SLA-LSQ (synthetic MPL [SLA], QS21, lipid formulated as liposomes), GLA-SE (synthetic MPL [GLA], squalene oil / water emulsion), SLA-SE (synthetic MPL [SLA], squalene oil / water emulsion), SLA-nanoalum (synthetic MPL [SLA], aluminum salt), GLA-nanoalum (synthetic MPL [GLA], aluminum salt), SLA-AF (synthetic MPL [SLA], aqueous suspension), GLA-AF (synthetic MPL [GLA], aqueous suspension), SLA-alum (synthetic MPL [SLA], aluminum salt), GLA-alum (synthetic MPL [GLA], aluminum salt), and some of the GSK ASxx series of adjuvants including AS01 (MPL, QS21, liposomes), AS02 (MPL, QS21, oil / water emulsion), AS25 (MPL, oil / water emulsion), AS04 (MPL, aluminum salt), and AS15 (MPL, QS21, CpG, liposomes). See, for example, WO 2013 / 119856, WO 2006 / 116423, U.S. Pat. No. 4,987,237, U.S. Pat. No. 4,436,727, U.S. Pat. No. 4,877,611, U.S. Pat. No. 4,866,034, U.S. Pat. No. 4,912,094, U.S. Pat. No. 4,987,237, U.S. Pat. No. 5,191,072, U.S. Pat. No. 5,593,969, U.S. Pat. No. 6,759,241, U.S. Pat. No. 9,017,698, U.S. Pat. No. 9,149,521, U.S. Pat. No. 9,149,522, U.S. Pat. No. 9,415,097, U.S. Pat. No. 9,415,101, U.S. Pat. No. 9,504,743, Reed G, et al., 2013, supra.

[0090] Non-glycolipid molecules, for example synthetic molecules such as neoseptin-3 or natural molecules such as LeIF, can also be used as TLR4 agonist adjuvants, see for example Reed SG et al, 2016 (supra).

[0091] In one embodiment, the composition of the invention comprising a bioconjugate of an E. coli O18 antigen conjugated to a carrier protein further comprises at least one additional conjugate comprising an E. coli O-antigen polysaccharide covalently bound to a carrier protein. The one or more additional bioconjugates of E. coli O-antigen polysaccharide conjugated to a carrier protein are preferably bioconjugates of E. coli O-antigen polysaccharides of a serotype other than O18, or more preferably other than O18A. In a preferred embodiment, the one or more additional conjugates or bioconjugates comprise at least one, two, three, four, five, six, seven, eight or all nine O-antigen polysaccharides selected from the group consisting of E. coli serotypes O1, O2, O4, O6, O8, O15, O16, O25 and O75.

[0092] In a preferred embodiment, the one or more additional E. coli O-antigens in the additional conjugates are selected from the group consisting of E. coli O1 antigen polysaccharide (e.g., O1A antigen polysaccharide), E. coli O2 antigen polysaccharide, E. coli O4 antigen polysaccharide (e.g., E. coli glucosylated O4 antigen polysaccharide), E. coli O6 antigen polysaccharide (e.g., O6A antigen polysaccharide), E. coli O8 antigen polysaccharide, E. coli O15 antigen polysaccharide, E. coli O16 antigen polysaccharide, E. coli O25 antigen polysaccharide (e.g., O25A or O25B antigen polysaccharide, preferably O25B antigen polysaccharide), and E. coli O75 antigen polysaccharide. Preferably, each of the additional O-antigen polysaccharides is covalently bound to a carrier protein. In certain embodiments, each of the carrier proteins, i.e., the carrier protein of each conjugate, is an EPA carrier protein. Preferably, one or more, preferably all, of the additional conjugates are bioconjugates.

[0093] As used herein, the term "O18" refers to the O18 antigen of E. coli (E. coli serotype O18) and may be either the O18A, O18B, O18A1, or O18B1 antigen (each of the four subserotypes of E. coli serotype O18), and in a preferred embodiment, O18 refers to O18A. The O18A antigen polysaccharide may comprise a structure of formula (O18A) as shown in Table 1, where n is an integer between 1 and 100, such as between 3 and 50, preferably between 5 and 40, such as between 7 and 25, for example between 10 and 20. The term "O1A" refers to the O1A antigen of E. coli (a subserotype of E. coli serotype O1). The term "O2" refers to the O2 antigen of E. coli (E. coli serotype O2). The term "O4" refers to the O4 antigen of E. coli (E. coli serotype O4), which may or may not contain glucose side chains (referred to as glucosylated O4 antigen polysaccharide (O4-Glc+) and non-glucosylated O4 antigen polysaccharide (O4-Glc-), respectively; in a preferred embodiment, O4 is O4-Glc+). The term "O6A" refers to the O6A antigen of E. coli (a subserotype of E. coli serotype O6). The term "O8" refers to the O8 antigen of E. coli (E. coli serotype O8). The term "O15" refers to the O15 antigen of E. coli (E. coli serotype O15). The term "O16" refers to the O16 antigen of E. coli (E. coli serotype O16). The term "O25B" refers to the O25B antigen from E. coli (a subserotype of E. coli serotype O25). The term "O75" refers to the O75 antigen of E. coli (E. coli serotype O75).

[0094] The structures of the E. coli O-antigen polysaccharides referred to throughout this application are shown below in Table 1. A single repeating unit for each E. coli O-antigen polysaccharide is shown.

[0095] [Table 1-1]

[0096] [Table 1-2] 1 Each n is independently an integer from 1 to 100, such as 1 to 50, 1 to 40, 1 to 30, 1 to 20 and 1 to 10, 3 to 50, 3 to 40, for example at least 5, such as 5 to 40, for example 7 to 30, for example 7 to 25, for example 10 to 20.

[0097] All monosaccharides described herein have their common meanings known in the art. Monosaccharides may have the D or L configuration. If D or L is not specified, the sugar is understood to have the D configuration. Monosaccharides are typically 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-acetylfucosylamine; and Rib, ribose. The suffix "f" refers to furanose and the suffix "p" refers to pyranose.

[0098] A composition comprising a bioconjugate of a carrier protein covalently bound to an E. coli O18 antigen polysaccharide according to the invention may further comprise at least one additional conjugate of a carrier protein covalently bound to an E. coli O-antigen polysaccharide, in certain embodiments the at least one additional conjugate comprises an E. coli O-antigen polysaccharide selected from the group consisting of E. coli O1A antigen polysaccharide, E. coli O2 antigen polysaccharide, E. coli O4 antigen polysaccharide, E. coli O6A antigen polysaccharide, E. coli O8 antigen polysaccharide, E. coli O15 antigen polysaccharide, E. coli O16 antigen polysaccharide, E. coli O25B antigen polysaccharide, and E. coli O75 antigen polysaccharide. Such a composition may be prepared by adding the conjugates together to obtain a multivalent conjugate composition. Preferably, one or more, preferably all, of the additional conjugates are also bioconjugates.

[0099] In certain embodiments, the O1A antigen polysaccharide (e.g. in isolated form or as part of a glycoconjugate, preferably a bioconjugate) is present in a (pharmaceutical) composition provided herein (e.g. a composition comprising an E. coli O18 bioconjugate obtained by a method described herein). The O1A antigen polysaccharide may comprise a structure of formula (O1A) as shown in Table 1, where n is an integer between 1 and 100, such as between 3 and 50, preferably between 5 and 40, such as between 7 and 25, for example between 10 and 20. Preferably, the O1A antigen polysaccharide is part of a conjugate, more preferably a bioconjugate, and is covalently attached to a carrier protein, such as EPA.

[0100] In certain embodiments, the O2 antigen polysaccharide (e.g. in isolated form or as part of a glycoconjugate, preferably a bioconjugate) is present in a (pharmaceutical) composition provided herein (e.g. a composition comprising an E. coli O18 bioconjugate obtained by a method described herein). The O2 antigen polysaccharide may comprise a structure of formula (O2) as shown in Table 1, wherein n is an integer between 1 and 100, such as between 3 and 50, preferably between 5 and 40, such as between 7 and 25, for example between 10 and 20. Preferably, the O2 antigen polysaccharide is part of a conjugate, more preferably a bioconjugate, and is covalently attached to a carrier protein, e.g. EPA.

[0101] In certain embodiments, the O4 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate, preferably a bioconjugate) is present in a (pharmaceutical) composition provided herein (e.g., a composition comprising an E. coli O18 bioconjugate obtained by the methods described herein). Structural modifications of O-antigens are known to exist within the E. coli O4 serotype. In particular, some O4 serotypes express modified O-antigens with branched glucose units. As used herein, "glucosylated O4 antigen", "glucosylated O4 antigen polysaccharide", "glucose-branched O4", "O4-Glc+ antigen polysaccharide", "Glc+O4", and "O4-Glc+ antigen" refer to O4 antigens with glucose branches. Some O4 serotypes have O4 antigens that do not have such branched glucose units, which are referred to herein as "non-glucosylated O4 antigen", "non-glucosylated O4 antigen polysaccharide", "O4-Glc- antigen", or "O4-Glc-". When referring to "O4 antigen" herein, this can be either O4-Glc+ antigen or O4-Glc- antigen. In a preferred embodiment, O4 refers to O4-Glc+ antigen. The structures of E. coli non-glucosylated O4 antigen and E. coli glucosylated O4 antigen are shown in Table 1 in formula (O4-Glc-) and (O4-Glc+), respectively, where n is an integer between 1 and 100, such as between 3 and 50, preferably between 5 and 40, such as between 7 and 25, such as between 10 and 20. Methods for specifically producing O4-Glc+ bioconjugates are described in WO 2020 / 191082(A1), which is incorporated herein in its entirety. Preferably, the O4 antigen polysaccharide is part of a conjugate, more preferably a bioconjugate, and is covalently attached to a carrier protein, such as EPA.

[0102] In certain embodiments, the O6A antigen polysaccharide (e.g. in isolated form or as part of a glycoconjugate, preferably a bioconjugate) is present in a (pharmaceutical) composition provided herein (e.g. a composition comprising an E. coli O18 bioconjugate obtained by a method described herein). The O6A antigen polysaccharide may comprise a structure of formula (O6A) as shown in Table 1, where n is an integer between 1 and 100, such as between 3 and 50, preferably between 5 and 40, such as between 7 and 25, for example between 10 and 20. Preferably, the O6A antigen polysaccharide is part of a conjugate, more preferably a bioconjugate, and is covalently attached to a carrier protein, such as EPA.

[0103] In certain embodiments, the O8 antigenic polysaccharide (e.g. in isolated form or as part of a glycoconjugate, preferably a bioconjugate) is present in a (pharmaceutical) composition provided herein (e.g. a composition comprising an E. coli O18 bioconjugate obtained by a method described herein). The O8 antigenic polysaccharide may comprise a structure of formula (O8) as shown in Table 1, where n is an integer between 1 and 100, such as between 3 and 50, preferably between 5 and 40, such as between 7 and 25, for example between 10 and 20. Preferably, the O8 antigenic polysaccharide is part of a conjugate, more preferably a bioconjugate, and is covalently attached to a carrier protein, e.g. EPA.

[0104] In certain embodiments, the O15 antigenic polysaccharide (e.g. in isolated form or as part of a glycoconjugate, preferably a bioconjugate) is present in a (pharmaceutical) composition provided herein (e.g. a composition comprising an E. coli O18 bioconjugate obtained by a method described herein). The O15 antigenic polysaccharide may comprise a structure of formula (O15) as shown in Table 1, wherein n is an integer between 1 and 100, such as between 3 and 50, preferably between 5 and 40, such as between 7 and 25, for example between 10 and 20. Preferably, the O15 antigenic polysaccharide is part of a conjugate, more preferably a bioconjugate, and is covalently attached to a carrier protein, such as EPA.

[0105] In certain embodiments, the O16 antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate, preferably a bioconjugate) is present in a (pharmaceutical) composition provided herein (e.g., a composition comprising an E. coli O18 bioconjugate obtained by a method described herein). The O16 antigen polysaccharide may comprise a structure of formula (O16) as shown in Table 1, where n is an integer between 1 and 100, such as between 3 and 50, preferably between 5 and 40, such as between 7 and 25, such as between 10 and 20. Preferably, the O16 antigen polysaccharide is part of a conjugate, more preferably a bioconjugate, and is covalently attached to a carrier protein, such as EPA. Conjugates of E. coli O16 antigen polysaccharide covalently attached to a carrier protein may have some acetylation at the 2-position of the L-Rha sugar. This degree of O-acetylation of the O16 antigen polysaccharide in the conjugate 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%.

[0106] In certain embodiments, the O25B antigen polysaccharide (e.g., in isolated form or as part of a glycoconjugate, preferably a bioconjugate) is present in a (pharmaceutical) composition provided herein (e.g., a composition comprising an E. coli O18 bioconjugate obtained by a method described herein). The O25B antigen polysaccharide may comprise a structure of formula (O25B) as shown in Table 1, where n is an integer between 1 and 100, such as between 3 and 50, preferably between 5 and 40, such as between 7 and 25, such as between 10 and 20. Preferably, the O25B antigen polysaccharide is part of a conjugate, more preferably a bioconjugate, and is covalently attached to a carrier protein, such as EPA. Similar to the O16 antigen polysaccharide described above, conjugates of E. coli O25B antigen polysaccharide covalently attached to a carrier protein may have some degree of acetylation at the 2-position of the L-Rha sugar. This degree of O-acetylation of the E. coli O25B antigen polysaccharide in the conjugate 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%.

[0107] In certain embodiments, the O75 antigenic polysaccharide (e.g. in isolated form or as part of a glycoconjugate, preferably a bioconjugate) is present in a (pharmaceutical) composition provided herein (e.g. a composition comprising an E. coli O18 bioconjugate obtained by a method described herein). The O75 antigenic polysaccharide may comprise a structure of formula (O75) as shown in Table 1, where n is an integer between 1 and 100, such as between 3 and 50, preferably between 5 and 40, such as between 7 and 25, for example between 10 and 20. Preferably, the O75 antigenic polysaccharide is part of a conjugate, more preferably a bioconjugate, and is covalently attached to a carrier protein, such as EPA.

[0108] In certain embodiments, a composition according to the invention comprises a bioconjugate comprising a carrier protein covalently linked to an E. coli O18 antigen polysaccharide according to the invention, and further comprises one or more additional conjugates comprising a carrier protein covalently linked to an E. coli O-antigen polysaccharide, preferably between 4 and 25 such additional conjugates. Preferably, the additional conjugates comprise one or more of the E. coli O-antigen polysaccharides as described above, most preferably at least O25B, O1A, O2, and O6. Particularly preferred are compositions comprising at least 7, preferably at least 8 additional conjugates, preferably some, most preferably all, of these additional conjugates being bioconjugates.

[0109] In a preferred embodiment, a composition according to the invention comprises at least E. coli O1A, O2, O4 (preferably glucosylated O4), O6A, O15, O16, O18 (preferably O18A), O25B and O75 antigenic polysaccharides, preferably conjugates of O1A, O2, glucosylated O4, O6A, O15, O16, O18A, O25B and O75 antigenic polysaccharides, each independently covalently linked to a carrier protein, e.g. EPA (i.e. at least a 9-valent composition). In certain embodiments, such compositions further comprise E. coli O8 antigenic polysaccharide, preferably in the form of a conjugate, preferably a bioconjugate. In certain embodiments, such compositions (with or without O8 antigenic polysaccharide) comprise additional E. coli O-antigen polysaccharides from other serotypes, preferably conjugated to a carrier protein, preferably a bioconjugate, e.g. from 2 to 10 additional serotypes. In certain embodiments, a pharmaceutical composition is provided comprising at least nine bioconjugates, each bioconjugate comprising a carrier protein covalently linked to an E. coli O-antigen polysaccharide from a different serotype, including O18A (and an O18A bioconjugate is a bioconjugate according to the invention prepared using a method according to the invention), O1A, O2, O4-Glc+, O6A, O15, O16, O25B, and O75. In certain embodiments, the carrier protein is EPA, e.g., EPA having the amino acid sequence of SEQ ID NO:3.

[0110] Compositions according to the invention may optionally comprise, be combined with, or be administered together with further proteins, carbohydrates and lipopolysaccharides to which an immune response is desired.

[0111] In another aspect, the present invention relates to a pharmaceutical composition of the present invention for use as a medicament.

[0112] In a further aspect, the present invention relates to the use of the pharmaceutical composition described herein as a medicament for inducing an immune response against E. coli.

[0113] As used herein, the terms "immunogen" or "immunogenic" or "antigen" are used interchangeably to describe a molecule that is capable of generating an immunological response against itself upon administration to a recipient, either alone, in combination with an adjuvant, or presented on a display vehicle.

[0114] As used herein, an "immunological response" or "immune response" to an antigen or composition refers to the development in a subject of a humoral and / or cellular immune response to the antigen or antigens present in the composition.

[0115] In certain embodiments, the pharmaceutical compositions described herein are for use in the prevention or treatment of a disease caused by E. coli infection. In a preferred embodiment, the disease caused by E. coli infection is invasive extraintestinal pathogenic E. coli (ExPEC) disease (IED).

[0116] As used herein, the term "extraintestinal pathogenic E. coli" or "ExPEC" generally refers to genetically related pathogenic E. coli strains that invade, colonize, and induce disease in body sites outside the gastrointestinal tract. ExPEC bacteria include uropathogenic (UPEC) E. coli, neoplastic meningococcal (NMEC) E. coli, sepsis-associated (SePEC) E. coli, adherent invasive (AIEC) E. coli, and avian pathogenic (APEC) E. coli. Diseases associated with ExPEC or ExPEC infection include, but are not limited to, urinary tract infection (UTI), surgical site infection, bacteremia, abdominal or pelvic infections such as intra-abdominal infection (IAI), pneumonia, hospital-acquired pneumonia, osteomyelitis, cellulitis, pyelonephritis, wound infections, meningitis, neonatal meningitis, peritonitis, cholangitis, soft tissue infections, pyomyositis, septic arthritis and sepsis.

[0117] In a further aspect, the present invention relates to a method for preventing or treating a disease caused by E. coli infection, in particular invasive extraintestinal pathogenic E. coli (ExPEC) disease (IED), in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition as described herein.

[0118] As used herein, the term "effective amount" refers to an amount of an active ingredient or component that induces a desired biological or pharmaceutical response in a subject. Effective amounts can be determined empirically and in a routine manner for the stated purpose. For example, in vitro assays can be optionally used to help identify optimal dosage ranges.

[0119] As used herein, a "subject" or "patient" refers to any animal, preferably a mammal, most preferably a human, that is to be vaccinated or has been vaccinated by a method or composition according to an embodiment of the present invention. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like, most preferably humans. In certain embodiments, the subject is a human adult. As used herein, the term "human adult" refers to a human aged 18 years or older. In certain embodiments, the subject is (i) a human female between about 16 and about 50 years of age, e.g., between about 16 and about 35 years of age; (ii) A human adult who is over 50 years of age, or over 55 years of age, or over 60 years of age, or over 65 years of age; (iii) a human subject suffering from a recurrent UTI; (iv) a human subject having or at risk of developing E. coli bacteremia or sepsis; (v) a human subject having a condition requiring the use of a catheter; (vi) a human subject undergoing a pre-scheduled surgery; (vii) a postmenopausal human female, or (viii) a human subject having diabetes.

[0120] In yet another aspect, the present invention also relates to the use of a pharmaceutical composition as described herein for the manufacture of a vaccine or medicament for preventing or treating a disease caused by E. coli infection, in particular invasive extraintestinal pathogenic E. coli (ExPEC) disease (IED).

[0121] Various publications, articles and patents are cited or described in the "Background" and throughout the specification, and each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a context for the invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0122] Array description:

[0123] [Table 2-1]

[0124] [Table 2-2]

[0125] [Table 2-3]

[0126] [Table 2-4]

[0127] [Table 2-5]

[0128] [Table 2-6] EXAMPLES

[0129] The following examples of the present invention are presented to further illustrate the nature of the present invention, but it should be understood that the following examples are not intended to limit the present invention, the scope of which is defined by the appended claims.

[0130] A bioconjugation system in E. coli has recently been developed in which both the antigen polysaccharide and the carrier protein are synthesized in vivo and subsequently conjugated in vivo via the activity of the Campylobacter jejuni enzyme oligosaccharyltransferase PglB expressed in E. coli (Wacker et al., Proc. Nat. Acad. Sci. (2006) v. 103, pp. 7088-93). This N-linked protein glycosylation system allows for the transfer of a variety of polysaccharides to the carrier protein and allows for a method of purifying the bioconjugate from the bacteria in which it is expressed. Bioconjugation has been successfully used to produce conjugated polysaccharides for an E. coli tetravalent O-antigen candidate vaccine (Poolman and Wacker, J. Infect. Dis. (2016) v. 213(1), pp. 6-13; WO 2015 / 124769; WO 2017 / 035181). A composition comprising 10 bioconjugates (O1A, O2, O4, O6A, O8, O15, O16, O18A, O25B and O75, a composition designated ExPEC10V) has been described and is in clinical trials (e.g., WO 2020 / 191082(A1)). Each of the bioconjugates is produced by a separate production strain that exhibits O serotype-dependent yield variation. For O-antigen producing strains of some O-serotypes, improved yields could be obtained by expressing variants of the C. jejuni glycosyltransferase PglB with altered substrate specificity (see, e.g., WO 2016 / 107818, WO 2016 / 107819). However, for the O18 O-serotype, which exhibited the lowest bioconjugate production yield among the 10 serotypes for which bioconjugates were made for the ExPEC10V composition, the use of variants of the C. jejuni PglB protein and the use of alternative PglB homologs did not result in improved yields. Furthermore, further platform modifications aimed at increasing O18 bioconjugation yields were not successful in achieving this goal.Among these modifications was overexpression of WecA, a protein that initiates O-unit biosynthesis by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate to undecaprenyl phosphate (Und-P) to form Und-PP-GlcNAc. In addition, replacing the native Wzz O-antigen chain length regulator present in the E. coli K-12 bioconjugation platform strain with O-antigen chain length regulators from other Gram-negative bacterial species resulted in an increase in O18A O-antigen chain length but did not result in a significant increase in product yield.

[0131] Example 1: Yield-improved O18 bioconjugate producing strains However, the inventors obtained strains with increased O18 bioconjugate product yields and found that the Wzy O-antigen polymerase in these strains had amino acid mutations compared to Wzy in the existing O18 bioconjugate producing strain (which had a Wzy O-antigen polymerase with SEQ ID NO: 1). Based on this, the inventors created a novel producer of O18 bioconjugate that is otherwise identical except for three amino acid mutations in its Wzy O-antigen polymerase compared to the O18 bioconjugate producing strain previously described (see WO 2020 / 191082). In particular, the Wzy O-antigen polymerase in the previously described strain had the amino acids threonine (T), methionine (M), and valine (V) at positions 199, 377, and 395, respectively (i.e., the Wzy O-antigen polymerase of the previously described strain is set forth in SEQ ID NO:1), whereas the new production strain had the amino acids isoleucine (I), lysine (K), and alanine (A) at these respective positions.

[0132] The yield of O18 bioconjugate in the filtered periplasmic fraction of the 200 L bioreactor performed after osmotic shock (i.e., at an early point in the process where reasonable quantification of yield is possible) was approximately 2.4-fold higher for the new strain versus the previously described strain.

[0133] The occupancy of glycosylation sites of EPA carrier proteins with four N-glycosylation consensus sequences was increased in the new strains relative to previously described strains, i.e., the new strains derived more di-, tri-, and tetra-glycosylated EPA carrier proteins (e.g., Figure 1).

[0134] Example 2: Construction of modified Wzy O-antigen polymerase Next, the inventors performed mutagenesis studies of the Wzy O-antigen polymerase amino acid sequence to identify specific combinations of amino acid substitutions at these three positions in the Wzy O-antigen polymerase sequence of SEQ ID NO:1 that affect the yield and glycosylation pattern of the O18 bioconjugate.

[0135] Various combinations of amino acid substitutions at one or more of positions 199, 377, and 395 in the amino acid sequence of SEQ ID NO:1 of Wzy O-antigen polymerase were generated using site-directed mutagenesis using standard procedures. The various combinations generated are shown in Table 3.

[0136] Example 3: Production of O-antigen polysaccharides using modified Wzy O-antigen polymerase The wzy gene present in the rfb cluster from E. coli serotype O18A (example provided as SEQ ID NO: 5) inserted into the chromosome of the E. coli K-12 bioconjugate producing strain W3110 was replaced by a gene variant encoding a modified Wzy O-antigen polymerase having SEQ ID NO: 1 with the mutations shown in Table 3 by a homologous recombination-based genetic modification technique. In addition to the rfb cluster from E. coli serotype O18A, the E. coli bioconjugate producing strain W3110 further contained a nucleic acid encoding an EPA carrier protein (having SEQ ID NO: 3) and a nucleic acid encoding the oligosaccharyltransferase PglB (having SEQ ID NO: 6). The O18A polysaccharide bioconjugate was produced in shake flask cultures and extracted using osmotic shock, as previously described, for example, in WO 2020 / 191082.

[0137] Example 4: Characterization of O18-bioconjugates Periplasmic extracts from shake flask cultures of E. coli K-12 bioconjugate producing strain W3110 containing the Wzy variant (with the mutations shown in Table 3) induced for O18A polysaccharide bioconjugate expression were subjected to capillary electrophoresis immunoassay using an O18A specific monoclonal antibody for immunodetection of the bioconjugate. Relative quantification was performed for each sample based on the signal intensity of the peaks corresponding to sEPA, monoglycosylated EPA and higher forms of glycosylated EPA (di- / tri- / tetraglycosylated EPA) as determined by MW relative to the total product signal (Table 3).

[0138] Table 3. Wild-type and mutant Wzy O antigen polymerases with amino acid substitutions at one or more of positions 199, 377, and 395 of SEQ ID NO:1 as indicated, and their effect on the glycosylation pattern of the O18 bioconjugate. Variants resulting in improved glycosylation patterns are shown in bold.

[0139] [Table 3]

[0140] Example 5: Production of O18 bioconjugates and ExPEC9V and ExPEC10V multivalent products with immunogenic activity by new production strains The new strain for producing the O18A bioconjugate was used to produce the O18A bioconjugate, the O18A bioconjugate was extracted from the periplasm after osmotic shock and subjected to chromatography to remove host cell proteins, and the extracted bioconjugate was used to prepare a pharmaceutical composition comprising the O18A bioconjugate as a drug substance essentially as described in WO 2020 / 191082 (Example 8 therein).

[0141] The novel O18A drug substance was also mixed with other bioconjugate drug substances including E. coli O-antigens O1A, O2, O4(glc+), O6A, O15, O16, O25B and O75, and possibly also O8, to obtain multivalent (9-valent, respectively 10-valent) formulations. These formulations, when administered to rabbits, induced antibodies against the E. coli serotypes whose O-antigens were present in the composition. Thus, it was demonstrated that the bioconjugates and compositions (obtained) according to the (method of) the present invention are suitable for inducing an immune response against E. coli.

Claims

1. (a) a carrier protein comprising at least one glycosylation consensus sequence; (b) the E. coli O18 rfb locus; and (c) an oligosaccharyltransferase for the transfer of oligosaccharides to an N-glycosylation site on a carrier protein; and A gram-negative bacterial host cell comprising: The cell comprises a polypeptide having Wzy O-antigen polymerase activity, the polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1, the amino acid sequence being i) an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:1; ii) a threonine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and a valine at a position corresponding to position 395 of SEQ ID NO:1; iii) a threonine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:1; iv) an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and a valine at a position corresponding to position 395 of SEQ ID NO:1, or v) an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a methionine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:1 A gram-negative bacterial host cell comprising:

2. The polypeptide having Wzy O-antigen polymerase activity comprises the amino acid sequence of SEQ ID NO:1, wherein the amino acid sequence is i) an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:1; ii) a threonine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and a valine at a position corresponding to position 395 of SEQ ID NO:1; iii) a threonine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:1; iv) an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and a valine at a position corresponding to position 395 of SEQ ID NO:1, or v) an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a methionine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:1 The host cell of claim 1 except that it comprises:

3. 3. The host cell of claim 1 or 2, wherein the polypeptide having Wzy O-antigen polymerase activity comprises an isoleucine at a position corresponding to position 199 of SEQ ID NO:1, a lysine at a position corresponding to position 377 of SEQ ID NO:1, and an alanine at a position corresponding to position 395 of SEQ ID NO:

1.

4. The host cell according to any one of claims 1 to 3, wherein a polynucleotide or vector encoding a polypeptide having Wzy O-antigen polymerase activity is integrated into the genome of the host cell.

5. The host cell according to any one of claims 1 to 4, wherein the host cell is an Escherichia coli host cell.

6. The host cell according to claim 5, wherein the host cell is an E. coli K-12 strain, preferably a W3110 strain.

7. a) the oligosaccharyltransferase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:6; b) the carrier protein comprises SEQ ID NO:3; c) the E. coli O18 rfb locus is the rfb locus of an E. coli strain having serotype O18A; The host cell according to any one of claims 1 to 6, which is at least one of the following:

8. The host cell according to any one of claims 1 to 7, wherein the oligosaccharyltransferase comprises the amino acid sequence of SEQ ID NO:

6.

9. The host cell according to any one of claims 1 to 8, wherein the host cell comprises an E. coli O18 rfb locus having a nucleotide sequence encoding a Wzy O-antigen polymerase that has Wzy O-antigen polymerase activity and that encodes a polypeptide having a sequence according to any one of claims 1 to 3.

10. A method for producing a bioconjugate of an E. coli O18 antigenic polysaccharide conjugated to a carrier protein, comprising culturing a host cell according to any one of claims 1 to 9 to produce said bioconjugate.

11. The method of claim 10, further comprising recovering the bioconjugate.

12. 12. The method of claim 11, further comprising formulating the recovered bioconjugate into a pharmaceutical composition.

13. 13. The method of claim 12, further comprising adding one or more additional bioconjugates of E. coli O-antigen polysaccharide conjugated to a carrier protein to the pharmaceutical composition to obtain a multivalent bioconjugate composition.

14. 14. The method of claim 13, wherein the one or more additional bioconjugates comprise at least one O-antigen polysaccharide selected from the group consisting of E. coli serotypes O1, O2, O4, O6, O8, O15, O16, O25, and O75.

15. 20. The multivalent bioconjugate composition, (i) a bioconjugate of the E. coli O18A antigen polysaccharide conjugated to a carrier protein; (ii) a bioconjugate of the E. coli O1A antigen polysaccharide conjugated to a carrier protein; (iii) a bioconjugate of the E. coli O2 antigen polysaccharide conjugated to a carrier protein; (iv) a bioconjugate of E. coli glucosylated O4 antigen polysaccharide conjugated to a carrier protein; (v) a bioconjugate of the E. coli O6A antigen polysaccharide conjugated to a carrier protein; (vi) a bioconjugate of the E. coli O15 antigen polysaccharide conjugated to a carrier protein; (vii) a bioconjugate of the E. coli O16 antigen polysaccharide conjugated to a carrier protein; (viii) a bioconjugate of the E. coli O25B antigen polysaccharide conjugated to a carrier protein, and (ix) Bioconjugates of E. coli O75 antigen polysaccharide conjugated to a carrier protein Including, The method of claim 14, wherein the carrier protein in each bioconjugate preferably comprises SEQ ID NO:

3.

16. 20. The multivalent bioconjugate composition, (x) a bioconjugate of the E. coli O8 antigen polysaccharide conjugated to a carrier protein; The method of claim 15, wherein the carrier protein preferably comprises SEQ ID NO:3.